Constitutive activity and drug functional selectivity of 5-HT2A receptors in post-mortem brain of subjects with schizophrenia

Constitutive activity and drug functional selectivity of 5-HT2A receptors in post-mortem brain of subjects with schizophrenia Itziar Muneta Arrate (cc) 2022 Itziar Muneta Arrate (cc by 4.0) This doctoral thesis has been developed thanks to the financial support of a predoctoral fellowship from the Basque Government (20182022). This work was funded by the Spanish Ministry of Science and Innovation (SAF2017-88126R), the Basque Government (IT-616-13) and the Centre of Biomedical Research in Mental Health, CIBERSAM. ABREVIATION LIST AA Araquidonic acid AC Adenylyl cyclase BB Basal binding Ca2+ Calcium ion CaMKII Calcium/calmodulin-dependent kinase II CNS Central nervous system BRET Bioluminiscence resonance energy transfer cAMP Cyclic adenosine monophosphate DA Dopamine DAG Diacylglycerol DLPFC Dorsolateral prefrontal cortex (±)DOI 2,5-dimetoxy-4-iodoamphetamine DSM Diagnostic and Statistical Manual of Mental Disorders D2R Dopamine 2 receptor EC50 Concentration that promotes half-maximal stimulatory effect Emax Maximal stimulatory effect ERK Extracellular signal-regulated kinases FDA Food and drug administration GABA γ-aminobutiric acid GPCR G-protein coupled receptor GDP Guanosine diphosphate GRK G-protein coupled kinase GTP Guanosine triphosphate 5-HT 5-Hydroxytryptamine (serotonin) 5-HT2AR Serotonin 2A receptor 5-HT2AR(-/-) Knock-out 5-HT2AR(+/+) Wild-Type GWAS Genome-wide association studies IC50 Concentration that promotes half-maximal inhibitory effect Imax Maximal inhibitory effect IP3 Inositol 1,4,5-tiphosphate LSD D-lysergic acid diethylamide MAPK Mitogen-activated-protein kinase NBS Non-specific binding NMDA N-methyl-D-aspartate PCP Phencyclidine PET Positron emission tomography PFC Prefrontal cortex PIP2 Phosphatidylinositol 4,5-biphosphate PKC Protein kinase C PLA2 Phospholipase A2 PLC Phospholipase C PMD Post-mortem delay PSD-95 Postsynaptic protein density 95 PTX Bordetella pertussis toxin RSK-2 Ribosomal S6 kinase GTPγS 5'-O-[gamma-thio]triphosphate [35S]GTPγS Sulphur 35-labelled guanosine-5´-O-(γ-thio)-triphosphate SNP Single nucleotide polimorphism SPA Scintillation proximity assay 7TM Seven transmembrane Volinanserin MDL100907 INDEX Introduction _________________________________________________ 1 1.1 Schizophrenia _____________________________________________________ 3 1.1.1 Definition _______________________________________________________ 3 1.1.2 Symptomatology _________________________________________________ 3 1.1.3 Aetiology of schizophrenia __________________________________________ 4 1.1.3.1 Genetics ______________________________________________________ 5 1.1.3.2 Environmental factors ____________________________________________ 5 1.1.3.3 Neurotransmission systems alterations in schizophrenia _________________ 7 1.1.4 Morphological brain alterations _____________________________________ 11 1.2 G-protein coupled receptors (GPCRs) _________________________________ 12 1.2.1 General aspects ________________________________________________ 12 1.2.2 The basic mechanism of GPCR signalling ____________________________ 12 1.2.3 Theory of drug receptor interaction for GPCR __________________________ 17 1.2.4 GPCRs and biased signalling ______________________________________ 22 1.2.5 Evaluation of constitutive activity, inverse agonism and functional selectivity __ 25 1.3 Serotonin 2A receptor (5-HT2AR) _____________________________________ 28 1.3.1 General aspects ________________________________________________ 28 1.3.2 Localization and function of 5-HT2AR in CNS __________________________ 29 1.3.3 5-HT2AR Structural biology ________________________________________ 33 1.3.4 5-HT2AR Signalling pathways ______________________________________ 35 1.3.5 5-HT2AR ligands _________________________________________________ 37 1.3.5.1 Agonists _____________________________________________________ 38 1.3.5.2 Antagonists ___________________________________________________ 40 1.3.6 5-HT2AR functional selectivity ______________________________________ 41 1.4 5-HT2AR and schizophrenia _________________________________________ 46 1.4.1 Genetic studies _________________________________________________ 46 1.4.2 Evaluation of 5-HT2AR density, expression and functionality in schizophrenia subjects. ___________________________________________________________ 47 1.5 Antipsychotics____________________________________________________ 52 1.5.1 First-generation of antipsychotics (Typical antipsychotics) ________________ 52 1.5.2 Second-generation of antipsychotics (Atypical antipsychotics) _____________ 53 1.5.3 Third-generation of antipsychotics ___________________________________ 56 1.5.4 New generation of antipsychotics ___________________________________ 56 Aims _______________________________________________________ 59 Subjects, Materials and Methods _______________________________ 63 3.1 Human brain samples ______________________________________________ 65 3.1.1 Subjects selection, demographic characteristics, psychiatric diagnosis and toxicological analysis _________________________________________________ 65 3.1.2 Demographic characteristics and diagnosis of subjects included in pools used for Antibody-capture [35S]GTPγS Scintillation proximity Assays (SPA) and Western Blot characterization assays _______________________________________________ 73 3.2 Animals: Transgenic mice ___________________________________________ 74 3.3 Drugs __________________________________________________________ 75 3.4 Materials ________________________________________________________ 77 3.5 Methods ________________________________________________________ 79 3.5.1 Antibody-capture [35S]GTPγS Scincillation Proximity Assay (SPA) __________ 79 3.5.1.1 Preparation of membrane-enriched fraction (P2 fraction) ________________ 80 3.5.1.2 Antibody-capture [35S]GTPγS Scincillation Proximity Assay (SPA) ________ 81 3.5.1.3 Mathematical and statistical analysis of the results ____________________ 85 3.5.2 Western Blot experiments _________________________________________ 90 3.5.2.1 Preparation of membrane enriched fraction (P2 fraction) ________________ 91 3.5.2.2 Gel electrophoresis, transference and immunodetection ________________ 92 3.5.2.3 Mathematical and statistical analysis of results _______________________ 98 Results ___________________________________________________ 101 4.1 Functional selectivity of different serotonin 5-HT2AR antagonists in post-mortem human brain _______________________________________________________ 103 4.1.1 Concentration-effect of different drugs on Gαi1and Gαq/11-protein coupling to 5HT2AR in post-mortem human PFC _____________________________________ 103 4.1.2 Evaluation of maximal effect of different drugs on Gαi1-, Gαi2-, Gαi3-, Gαo-, and Gαq/11-protein coupling to 5-HT2AR in post-mortem human PFC _______________ 107 4.1.3 Involvement of 5-HT2AR in the effect induced by different drugs on [35S]GTPγS binding to Gαi1and Gαq/11-proteins in post-mortem human PFC _______________ 109 4.1.4 Evaluation of the maximal effect of different drugs on Gαi1and Gαq/11-protein coupling to 5-HT2AR in knock-out 5-HT2AR(-/-) and wild-type 5-HT2AR(+/+) mice _____ 116 4.1.5 Summary _____________________________________________________ 121 4.2 Evaluation of the functional coupling of 5-HT2AR to Gα-protein subtypes in PFC of schizophrenia subjects, non-schizophrenia suicide subjects and matched controls by antibody-capture [35S]GTPγS scintillation proximity assay (SPA) _______________ 122 4.2.1 Basal [35S]GTPγS binding to Gαi1and Gαq/11proteins in post-mortem PFC of schizophrenia subjects, non-schizophrenia suicide subjects and matched controls 122 4.2.2 Modulation of [35S]GTPγS binding to Gαi1and Gαq/11-proteins by the 5-HT2AR inverse agonist pimavanserin in post-mortem PFC of schizophrenia subjects, nonschizophrenia suicide subjects and matched controls _______________________ 125 4.2.3 Modulation of the [35S]GTPγS binding to Gαi1and Gαq/11-proteins by the 5-HT2AR inverse agonist volinanserin in post-mortem PFC of schizophrenia subjects, nonschizophrenia suicide subjects and matched controls _______________________ 132 4.2.4 Modulation of [35S]GTPγS binding to Gαi1and Gαq/11-proteins by 5-HT2AR agonist (±)DOI in post-mortem PFC of schizophrenia subjects, non-schizophrenia suicide subjects and matched controls _________________________________________ 139 4.2.5 Relationship between effects of pimavanserin, volinanserin and (±)DOI on [35S]GTPγS binding to Gαi1and Gαq/11-proteins in post-mortem human PFC _____ 145 4.2.6 Summary _____________________________________________________ 149 4.3 Evaluation of immunoreactivity of Gα-protein subtypes in membrane enriched fractions from PFC of post-mortem brain _________________________________ 150 4.3.1 Suitability of antibodies for antibody-capture [35S]GTPγS Scintillation Proximity Assay (SPA) and Western Blot _________________________________________ 150 4.3.2 Evaluation of Gαi1and Gαq/11-protein immunoreactivity in post-mortem PFC of schizophrenia subjects, non-schizophrenia suicide subjects, and matched controls 153 4.3.3 Summary _____________________________________________________ 156 4.4 Functional selectivity of different antipsychotics in post-mortem human brain PFC. Differential G-protein biased ligand properties at the 5-HT2AR _________________ 157 4.4.1 Evaluation of maximal effect of different antipsychotic drugs on Gαi1-, Gαi2-, Gαoand Gαq/11-protein coupling to 5-HT2ARs in post-mortem human PFC ___________ 157 4.4.2 Pharmacological characterization of the effects induced by clozapine and risperidone on [35S]GTPγS binding to Gαi1and Gαq/11-proteins in post-mortem human PFC _____________________________________________________________ 167 4.4.2.1 Clozapine ___________________________________________________ 168 4.4.2.2 Risperidone _________________________________________________ 171 4.4.3 Summary _____________________________________________________ 174 Discussion ________________________________________________ 177 5.1 Evaluation of 5-HT2AR functional coupling in human post-mortem brain with different 5-HT2AR agonist / antagonist / inverse agonist drugs _________________ 181 5.2 Constitutively active 5-HT2AR in post-mortem human PFC ________________ 187 Introduction 4 cognitive symptoms. Positive symptoms are the core features of schizophrenia, and include hallucinations (most frequently auditory), disillusions and psychotic behaviour, in which contact with reality is lost. Positive symptoms tend to appear in episodes, and usually spaced in time, although some patients have residual long-term psychotic symptoms. Negative symptoms are characterized by impaired motivation, reduction in spontaneous speech and social withdrawal (Liddle, 1987). Finally, cognitive symptoms include difficulties in attention and concentration, learning, memory and executive functions (Joyce & Roiser, 2007). 1.1.3 Aetiology of schizophrenia Schizophrenia is a complex disease probably caused by multiple aetiological factors. Thus, genetic and environmental factors are known to take part in the onset and development of schizophrenia (Sullivan et al., 2012; McCutcheon et al., 2020). Moreover, a neurodevelopmental aetiological hypothesis has been proposed and associated with structural, functional and neurochemical brain changes. Those changes affect several neurotransmission systems and circuits that seem to be affected in patients with schizophrenia. Evidence points to abnormalities in dopamine (DA), serotonin (5-HT) and glutamate neurotransmitter systems in the pathology of schizophrenia. Even so, none of the hypothesis seems to be sufficient to explain the full spectrum of the disease. Introduction 5 1.1.3.1 Genetics Many epidemiological studies have consistently shown a genetic component in the development of schizophrenia, with an estimated heritability nearly 80% (Sullivan et al., 2003). In recent years, several large-scale genomic studies have allow to study the contribution of specific deoxyribonucleic acid variants and different type of risk alleles. In consequence, schizophrenia is currently considered a highly polygenic disorder. Genome-wide association studies (GWAS) have shown that multiple common variants are associated with schizophrenia (Schizophrenia Working Group of the Psychiatric Genomic Consortium, 2014). Some of the most replicated gene association involves postsynaptic density (PSD) proteins, activity-regulated cytoskeleton-associated protein, N-methyl-D-aspartate (NMDA) receptor, fragile X mental retardation protein targets as well as other neurodevelopment disorders, voltage-gated calcium (Ca2+) channels, neural cell adhesion molecule and dopamine 2 receptor (D2R) (Pardiñas et al., 2018; Trubetskoy et al., 2022). A widely accepted association of genes with schizophrenia arises in part from many structurally diverse alleles from complement component 4 genes that is related with the histocompatibility complex (Sekar et al., 2016). 1.1.3.2 Environmental factors Environmental factors are also involved in the aetiology of schizophrenia. Although studies indicated a strong genetic influence and high heritability, it has been proven that different environmental factors can trigger the disease in people who already have a genetic predisposition, a term known as “stressvulnerability” model (Os et al., 2010). The environmental factors have been related with the neurodevelopmental hypothesis of schizophrenia (Fatemi & Folsom, 2009). Numerous studies have consistently reported increased incidence of schizophrenia associated to Introduction 6 several factors that affect early neurodevelopment during pregnancy, including maternal stress, maternal infections, nutritional deficits as well as birth complications (Jones et al., 1998; McGrath et al., 2010; Brown, 2012) (Figure 1.1). Several studies have revealed increased risk for developing psychosis associated to childhood adversities (Varese et al., 2012). Furthermore, well stablished evidences show that socioeconomic factors (Allardyce & Boydell, 2006) and immigration (both first and second generations) show relationship with rates of schizophrenia (Cantor-Graae & Selten, 2005). Toxic conditions also play a role in schizophrenia. Thus, accumulating evidence pointed to association between cannabis use and psychosis (Figure 1.1). These studies suggested that early chronic exposure to cannabis is associated with a higher vulnerability for psychotic outcomes, including later schizophrenia development (Moore et al., 2007; Ibarra-Lecue et al., 2018) (Figure 1.1). Therefore, many candidate genes and environmental risk factors seem to be robustly associated with schizophrenia. Introduction 7 Figure 1.1: Schematic representation of how environmental factors can lead to psychiatric disorders, such as schizophrenia in offspring. Infection during pregnancy induces proinflammatory cytokines release and immune system activation. Genetic background, autoimmune status, and second hits during childhood and adolescence (including stress and drug abuse) combined with consequences of maternal infection increase the likelihood of offspring to develop psychiatric disorders in adulthood. Illustration originally created for this thesis by N Cordero and adapted from Estes & McAllister, 2016. 1.1.3.3 Neurotransmission systems alterations in schizophrenia Dopaminergic hypothesis of schizophrenia The most widely known theory to explain clinical symptoms and drug response of schizophrenia is the dopaminergic hypothesis, which is based on different findings. First, clinical effects of typical or first-generation antipsychotic are related with their ability to block D2R (Seeman & Lee, 1975). Second, drugs that increase DA activity, such as amphetamine, produce psychotic episodes in healthy individuals, and worsened psychosis in schizophrenic patients (Lieberman et al., 1987). Maternal immune activation due to infection Stress Drug abuse Introduction 8 Dopaminergic hypothesis argues hyperactivity of dopaminergic transmission in mesolimbic and striatal regions that would be responsible of positive symptoms (hallucinations, delusions). However, negative and cognitive symptoms shown to be resistant to antipsychotics. Thus, dopaminergic hypothesis was reformulated postulating that a prefrontal hypodopaminergia, more than hyperdopaminergia, would contribute to negative and cognitive symptoms (Davis et al., 1991; Howes & Kapur, 2009; McCutcheon et al., 2020). Dopamine receptors are G-protein coupled receptors (GPCR) that can be divided into two main types: dopamine D1 receptor family, including D1 and D5 dopamine receptors (D1R, D5R), and D2 receptor family, including D2, D3 and D4 dopamine receptors (D2R, D3R, D4R). The D2R is a main target of typical and atypical antipsychotic medication, which suggest that this receptor plays a key role in schizophrenia. In vivo neuroimaging, based on positron emission tomography (PET) and single photon emission computerized tomography techniques (SPECT), have been used to evaluate the status of D2R and D1R in schizophrenia patients. Initial studies in basal ganglia were inconsistent, with some of them reporting increased D2R density and others no differences from controls (Howes et al., 2012; Cumming et al., 2021). Elevated D2R density was suggested to be a consequence of receptor up-regulation after long-term antipsychotic medication, since drug-naïve patients did not show such PET alterations (Seeman, 2013). In vitro post-mortem binding studies corroborated the finding of enhanced striatal D2R-binding density in schizophrenia (Seeman et al., 1984; Zakzanis & Hansen, 1998). Moreover, a recent study has revealed an absence of striatal D2R hyperactivity in post-mortem brain of schizophrenia subjects (Egusquiza et al., 2021). As for D2R, in vivo observations on D1R density in prefrontal cortex (PFC) of drug-naïve schizophrenia patients have shown discrepancies (Cumming et al., 2021). In contrast to this earlier focus Introduction 9 on postsynaptic D1R and D2R dysregulation, more recent findings point towards a critical role of presynaptic dopaminergic neurotransmission system in schizophrenia. Thus, elevated presynaptic DA synthesis capacity (Howes et al., 2012; Fusar-Poli & Meyer-Lindenberg, 2013), higher synaptic DA concentration (Abi-Dargham et al., 2000; Caravaggio et al., 2015), and amphetamine-induced DA release (Howes et al., 2012; Laruelle, 1998) have been demonstrated in striatum of schizophrenia subjects. Conversely, evidence suggest a reduction of amphetamine-induce DA release in frontal cortex of schizophrenia patients (Slifstein et al., 2015), indicating a presynaptic hypodopaminergia in this brain area (Slifstein et al., 2015). Glutamatergic hypothesis of schizophrenia Glutamate is the major excitatory neurotransmitter in the brain. Glutamate interacts with selective ionotropic and metabotropic receptors. Ionotropic receptor group includes NMDA, kainate and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subtypes. Metabotropic glutamate receptors (mGluRs), which activate G-protein signal transduction, are divided in groups I (mGlu1, mGlu5), II (mGlu2, mGlu3) and III (mGlu6, mGlu4, mGlu7, mGlu8) (Nakanishi, 1992; Niswender & Conn, 2010; Muguruza et al., 2016). It is well stablished that administration of NMDA receptor antagonists, such as phencyclidine (PCP) or ketamine, can induce psychosis-like states and cognitive deficits in healthy humans. Ketamine and PCP are non-competitive glutamate NMDA antagonist (Thomson et al., 1985). This fact gave support to the hypothesis of a functional impairment of NMDA receptors in schizophrenia (Javitt & Zukin, 1991; Stone et al., 2008). Interestingly, addition of amphetamine or other dopaminergic agonist as well as NMDA receptor antagonist also reassembles psychotic symptoms, and evokes cognitive and negative symptoms, better mimicking the pathophysiolology of schizophrenia Introduction 10 (Krystal et al., 2005). For this reason, non-competitive NMDA receptor antagonist administration has been chosen as a usual animal model of schizophrenia. Multiple reports have evaluated the status of glutamate in brain of schizophrenia patients and its relationship with different spectrum of symptoms. Thus, evidence that impaired glutamatergic system might be implicated in the cognitive dysfunction in schizophrenia is currently acknowledged (Moghaddam & Javitt, 2012). Glutamate hypothesis is indeed closely related to hyperand hypoactivity of dopaminergic pathways in limbic and cortical areas, respectively. First, it has been described an hypoactivity or inhibition of NMDA receptors located in γaminobutiric acid (GABA)ergic interneurons in mesolimbic dopaminergic areas. This would impair the inhibitory tonic state, resulting in higher synthesis and release of dopamine in those areas, which could predispose to psychotic symptoms. In contrast, cognitive and negative symptoms could be related with a hypofunctionality of NMDA receptors in the cortex-brainstem projection, which could make hypoactive mesocortical dopaminergic pathways (Javiit, 2010). Serotonergic hypothesis of schizophrenia The 5-HT hypothesis of schizophrenia arose from early studies on interactions between the hallucinogenic drug D-lysergic acid diethylamide (LSD) and 5-HT (Freedman 1961). LSD and related compounds produce mental disturbances, resembling those occurring at the onset of schizophrenia, and mediate their central effects through 5-HT receptors. Moreover, psychedelic drugs have some chemical similarities with 5-HT structure. Among all the 5-HT receptor subtypes, abundant evidence indicate that the effects of hallucinogens are mediated through the serotonin 2A receptor (5- Introduction 11 HT2AR) subtype (González-Maeso et al., 2007; Geyer & Vollenweider, 2008; González-Maeso & Sealfon, 2009; Madsen et al., 2019). In relation with these facts, atypical antipsychotic drugs show high affinity for 5-HT2AR, making this target one of the most widely studied in schizophrenia (Meltzer et al., 1989). A more detailed review of 5-HT2AR physiology and involvement in schizophrenia can be found in section 1.4. 1.1.4 Morphological brain alterations PFC has long been implicated in the pathophysiology of schizophrenia, especially in negative and cognitive manifestations (Kolk & Rakic, 2022). In fact, lesions of this brain region in animals and human might produce latter impairments, as disruption in working memory, reduced impulsive choice, enhanced environmental stimuli and behavioural restrains. Thus, abnormalities in PFC have been largely associated with schizophrenia and other psychiatric diseases (Xu et al., 2019). It is well described that schizophrenia patients exhibit lateral ventricular enlargement by 25% of volume, which is accompanied by a whole brain volume reduction around 2% (Johnstone et al., 1976; Haijma et al., 2013). The ventricles size increases progressively after the onset of the illness, as whole brain grey matter is reduced over time (van Erp et al., 2016). Morphological studies in post-mortem brains of schizophrenia patients have revealed differences in cellular distribution, likely attributed to altered neuronal migration early in brain development, loss of pyramidal cells, malformed cell structure, and decreased number of GABA interneurons (Schmidt & Mirnics, 2015). Introduction 12 1.2 G-protein coupled receptors (GPCRs) 1.2.1 General aspects GPCRs, also known as seven transmembrane (7TM) receptors, form one of the largest membrane receptor subfamily of plasma membrane protein in the body. These receptors bind to diverse type of ligands, such as ions, small molecules and peptides, linking them to downstream signalling (Alexander et al., 2019). GPCRs have an enormous biomedical relevance because they are involved in diverse physiological activities, and play a crucial role in pathogenesis of disorders, being important as drug-targets. Moreover, it is estimated that approximately 35% of approved drugs target GPCRs (Hauser et al., 2017). The 7TM receptor superfamily is also termed as GPCRs because exerts effects in response to different ligands through the association with G-proteins. G-proteins are heterotrimeric guanine nucleotide binding proteins, constituted by three subunits: α, β and γ. However, GPCRs can also bind to other cytosolic adaptors, including β-arrestins, which elicit G-protein independent activation (Syrovatkina et al., 2016). G-proteins mediate the earliest step in cell response to external events, by linking cell surface receptors to intracellular signalling (Sriram & Insel, 2018). G-proteins serve as transducers or amplifiers of signals from GPCRs to intracellular effectors. 1.2.2 The basic mechanism of GPCR signalling Heterotrimeric G-proteins have a crucial role in defining the specificity and temporal characteristics of cellular responses. Upon ligand activation, there is a conformational change of GPCRs that increase their affinity for G-proteins, leading to G-protein recruitment. The interaction of a G-protein with an active receptor stimulates the exchange of guanosine diphosphate (GDP) nucleotide Introduction 13 (inactive conformation) for guanosine triphosphate (GTP) (active conformation) (Figure 1.2). In the inactive conformation, Gα subunit is bound to GDP as well as to Gβ and Gγ subunits forming a heterodimer. After receptor activation, Gα subunit bounds to GTP and dissociates from the Gβγ dimer (Gilman, 1987; Marinissen & Gutkind, 2001). Afterwards, Gβγ subunits modulate downstream cellular signalling pathways, such as adenylyl cyclase (AC), phospholipase and ion channels (Hamm, 1998). The signal terminates when the GTP is hydrolysed by the GTPase activity of Gα subunits, to GDP and phosphate, and the Gβγ complex binds to Gα, forming the inactive Gprotein (Milligan & Kostenis, 2006; Hilger et al., 2018) (Figure 1.2). Introduction 20 Agonists present affinity for a target receptor, as well as efficacy. Thus, these ligands are able to bind the receptor and subsequently produce a response. Agonists can differ in their magnitude of the receptor-produced stimulus, which leads to their characterization as full or partial agonist. A full agonist produces maximal effect, while a partial agonist exerts a submaximal response, depending on their intrinsic efficacy. In contrast, antagonists display affinity but not intrinsic efficacy, since they do not induce conformational changes in the receptor to promote response. Its impact depends on their competence to reduce the probability of an agonist, such as endogenous ligand, to bind the receptor and decreasing endogenous cellular response. On the other side, Samama et al. proposed that the presence of a ligand is not necessary to generate a cellular response or signalling (Samama et al., 1993), which is defined as constitutive activity. Thus, the constitutive activity consists on spontaneous auto-activation of the receptor, adopting a conformation able to trigger intracellular signalling despite ligand absence (Lefkowitz et al., 1993). In this sense, Costa and Herz found that ligands with negative intrinsic efficacy were able to decrease the constitutive activity (Costa & Herz, 1989; Chidiac et al., 1994; Costa & Cotecchia, 2005). These, ligands were named as inverse agonists, and could be classified as full or partial inverse agonists, according to their intrinsic efficacy. Ternary complex model is the most widely accepted GPCR signalling model (Lean et al., 1980). According to this model, receptor is in a dynamic equilibrium between inactive (R) and active (R*) conformational states. Based upon this model, neutral antagonists have identical affinities for inactive and active conformational states, whereas agonists exhibit higher affinity for the active state. Because agonists have higher affinity for the active conformation of the receptor, agonist binding stabilizes GPCR in its active state, shifting the dynamic equilibrium from R to R*. Introduction 21 The maximal effect of an agonist (efficacy) is directly dependent upon the differential affinity of ligand for inactive receptor conformation (R) versus active receptor conformation (R*). Conversely, inverse agonists exhibit higher affinity for inactive receptor state (R). Therefore, inverse agonist binding results in the stabilization of the inactive state (R), shifting the dynamic equilibrium from R* to R. However, the inverse agonist efficacy is also dependent upon the magnitude of constitutive activity (Figure 1.5). Figure 1.5: Schematic representation of agonist, antagonist and inverse agonist binding to different functional states of the GPCR. Agonist binds with high affinity to active conformation of receptor (R*), whereas inverse agonist preferentially binds and stabilizes receptor in the inactive state (R). Neutral antagonist binds with the same affinity both active and inactive states of GPCR. Illustration from Muguruza et al., 2013. According to the extended ternary complex model, a receptor is able to switch from an active to inactive state in absence of a ligand (Sammama et al., 1993). In this sense, inverse agonism is considered the pharmacological property of a drug to antagonize the action of agonists, and simultaneously decrease basal constitutive activity of receptor signalling. Consequently, demonstration of inverse agonist properties requires the existence of constitutive basal Introduction 22 activity that will be reduced by the inverse agonist. The decrease of constitutive activity must be sensitive to blockade with an antagonist, and must be absent in knock-out animals for the involved receptor (Aloyo et al., 2009). After inverse agonism scientific description, many antagonist originally thought to be “neutral” turned out to be inverse agonist ligands (Strange, 2002; Kenakin, 2004). Although these findings indicate a potential usefulness of inverse agonists, there is little information about the use of pharmacological properties of these drugs in therapeutics. For example, somatic receptor mutations leading to constitutive active receptors are a causal factor in certain diseases, such as male precocious puberty (Kosugi & Mori, 1995). Thus, inverse agonists might be beneficial here, since they would decrease receptors basal activity induced by the mutation. In this case, neutral antagonists would presumably be of little use, in absence of increased endogenous ligand levelstt (Ligt et al., 2000). Overall, the concept of inverse agonist might provide new opportunities in such screening strategies. For that purpose, functional assay describing constitutive activity are need. These assays could contribute to the identification of agonist as well as inverse agonist ligands. 1.2.4 GPCRs and biased signalling As mentioned above, GPCRs can signal simultaneously through parallel pathways such as heterotrimeric G-proteins, β-arrestins and GRKs. In this context, several ligands have been described to differ in their ability to engage the different signalling pathways coupled to the respective GPCR, a term called biased signalling or functional selectivity (Azzi et al., 2003; Galandrin et al., 2007; Perez & Karnik, 2005; Kenakin, 2011; Kenakin, 2012; Smith et al., 2018). Biased agonists are theoretically able to stabilize different receptor conformations, exhibiting different affinities for the multiple signal transducers of the GPCR. They produce, as consequence, different cellular responses. In Introduction 23 contrast, other ligands can equally activate all signalling pathways. These drugs are termed balanced agonists or un-biased ligands. Interestingly, activating or inhibiting specific signalling cascades represents a new approach to effect selectivity, which could yield to improved therapeutically effective drugs, with lower side effects. In this way, the search for ligands for a particular signalling pathway, rather than selective for specific receptors, is one of the main challenges of drug development nowadays (Komatsu et al., 2019). The best well-known example of biased signalling is present in opioids pharmacology (Che et al., 2021). Biased signalling involves a differential activation between Gαi-proteins and β-arrestins. There is evidence indicating that therapeutic effect of μ-opioid receptor agonists, including analgesia, is Gαi-protein mediated, while adverse effects, such as respiratory depression and constipation, are more related to β-arrestin recruitment (Bohn et al., 1999). In this sense, several opioid ligands were developed in order to find G-protein biased ligands with improved therapeutic effects. Recently, TRV130 (oliceridine) demonstrated in vitro functional selectivity for Gα-proteins, and showed to be safe for management of pain according to a phase III clinical trial (Singla et al., 2019). However, other studies pointed out that biased opioid ligands development is controversial and that further research is needed studying each drug’s different signalling pathways (Gillis et al., 2020; Kliewer et al., 2020). Another aspect to be considered for drug development is the small change in ligand structure that can result in large changes in functional selectivity profiles (Shonberg et al., 2014). For example, LSD and lisuride evoke distinct in vitro cellular signalling and in vivo responses (González-Maeso et al., 2003, González-Maeso et al., 2007), despite the structural features and shared affinity for the 5-HT2AR. Moreover, risperidone and its active metabolite paliperidone are atypical antipsychotics, which only differ in a single hydroxyl Introduction 24 group. They show different pharmacological profiles that influence in their functional selectivity profile (Clarke et al., 2013). These facts highlight support the importance of deep structure-functional selectivity relation studies (Berg & Clarke, 2018). Although many studies have shed light on ligand bias profiles of different compounds, most of the studies are limited to few signalling pathways, such as G-proteins versus β-arrestins. However, many other possibilities are feasible, and all signalling pathways should be considered when characterizing the individual ligand efficacy (Figure 1.6). Introduction 25 Figure 1.6: Schematic representation of biased agonism on GPCR. Hypothetical and simplified example of μ-opioid receptor biased agonism. G-protein signalling pathway undergoes therapeutical effect while β-arrestins cause side effects. Direct interaction of unbiased ligands would results in activation of both signalling pathways, with therapeutic and side effects. However, functional or biased agonists favour preferentially one of the signal cascades, like G-proteins, promoting therapeutic effects over side effects. 1.2.5 Evaluation of constitutive activity, inverse agonism and functional selectivity GPCR implication in different diseases has increased the number of studies analysing different ligands profile to target these receptors. Because of their high relevance, several assays have been developed with a view to characterize the functional profile of different drugs. Classical functional assays measured downstream messengers, such as Ca2+ release, IP3 accumulation or cAMP production, in order to determine the functional profile of a drug after binding a GPCR. Advantages of measuring second messengers are that unmodified receptors, in native tissues, can be Introduction 26 studied. These assays have been broadly used for better comparison between different publications. Even if these techniques are widely used in assays with substantial amplification, in several assays both full and partial agonist could reach the same maximal response (Smith et al., 2018). Moreover, it is not clear the second messenger attribution to specific G-protein subtypes. Therefore, a nearest functional quantification of drug-receptor interaction was seen as necessary. A direct evaluation of GPCR activation can be made by measuring the stimulation or inhibition of guanine nucleotide exchange on G-protein receptors using radiolabelled GTP analogues. This approach is called sulphur 35labelled guanosine-5´-O-(γ-thio)-triphosphate ([35S]GTPγS) binding assay. Measurement of G-protein activation is the functional consequence of receptor occupancy as earliest event, and it is not subjected to signal amplification (González-Maeso et al., 2000; Harrison & Traynor, 2003). Conventional [35S]GTPγS binding assays are only limited to the measurement of GPCR coupling to Gαi/o-proteins; probably due to their higher rates of nucleotide exchange and constitutive activity (Seifert & Wenzel-Seifert, 2002). Nevertheless, different assays have been developed in order to measure different Gα-protein responses such as [35S]GTPγS binding immunoprecipitation assay using magnetic beads or scintillation proximity assays (SPA) (Diez-Alarcia et al., 2021b). This assay combines classical [35S]GTPγS binding assay with immunoprecipitation. This technique can be applied for studying receptor of interest in cell cultures and in native tissues. Moreover, this assay allows the evaluation of constitutive activity using pharmacological tools, as inverse agonists, in native tissue (Diez-Alarcia et al., 2021b). Nevertheless, this [35S]GTPγS binding assay combined with immunoprecipitation is currently limited to Gα-proteins, while β-arrestinmediated response is not possible to be studied as yet. Introduction 27 In addition to previous assays, fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays, which are technologies to detect protein-protein interaction and dynamic conformational changes have been developed for directly monitoring conformational changes in GPCRs, Gproteins, and β-arrestins (Angers et al., 2000; Galés et al., 2006; Zhou et al., 2021; Wright & Bouvier, 2021). These assays are suitable for monitoring live cell events in real time, and display adaptability to high-throughput screening. Despite these advantages, the introduction of fluorescent donor and acceptor molecules needs to be considered as a factor that could influence the observed outcomes (Pottie & Stove, 2022). The use of BRET is only suitable for in vitro cell culture systems, whereas in live animals, the application is limited to studies in superficial locations. This assay is not suitable for the study of postmortem tissue right now (Drinovec et al., 2012). Introduction 28 1.3 Serotonin 2A receptor (5-HT2AR) 1.3.1 General aspects 5-HT regulates a wide range of physiological processes in the central nervous system (CNS), including memory, perception, cognition, emotion, mood and consciousness (Berger et al., 2009). Dysfunction of 5-HT system has been implicated in numerous psychiatric disorders (Hoyer, 2020). Therefore, pharmacological manipulation of 5-HT system has therapeutic potential. Mammalian 5-HT receptors are now classified into 14 structurally and pharmacologically distinct subtypes (Hannon & Hoyer, 2008), which are divided into seven main families (5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, 5HT7 receptors), based on operational (drug related characteristics), transductional and structural characteristics (Figure 1.7). At a structural level, only 5-HT3 receptor is a ligand-gated ion channel (Maricq et al., 1991), whilst the rest of 5-HT receptors are members of GPCR superfamily (Kroeze & Roth, 1998). The 5-HT2 receptors are among the most widely studied 5-HT receptors. The 5-HT2 receptor family is divided in three different receptors, termed as 5-HT2AR 5-HT2BR and 5-HT2CR, with 46-50% sequence homology (Hoyer et al., 2002). Moreover, transmembrane domains of 5-HT2AR and 5-HT2CR share 80% sequence homology and very close pharmacological profiles (Boess & Martin, 1994). Thus, development of selective drugs for each receptor is a fundamental challenge due to the similar binding pockets. At present, it is not easy matter to discriminate pharmacologically 5-HT2 receptor family members, due to the lack of truly selective 5-HT2AR or 5-HT2CR ligands. Radioligand binding techniques were initially used for characterization of 5-HT receptors in mammalian brain homogenates. During the course of early investigations using radioligands, two classes of 5-HT binding sites were described. High affinity sites for [3H]5-HT corresponded to the 5-HT1 receptor Introduction 29 subtype, while low affinity sites were designated as 5-HT2 receptor subtype. The discovery of [3H]ketanserin as a reasonably selective ligand for 5-HT2AR (Leysen et al., 1982) turned to be a huge advance, and greatly potentiated the investigation of 5-HT2AR (Leysen et al., 1982). However, ketanserin also shows moderate affinity for 5-HT2CR, among other receptors (Choudhary et al., 1992). During the last years, new selective 5-HT2AR ligands have been subsequently developed for the evaluation of 5-HT2AR, such as [18F]altanserin, [3H]MDL100907 (L´Estrade et al., 2018) and [11C]Cimbi-36 (Ettrup et al., 2014). Sequence analysis of 5-HT2AR coding regions disclosed a high overall genetic conservation across species. The exception is a non-conserved change, most notably at residue 242, corresponding to a serine in humans and an alanine in rodents. It has been described that this change might influence in affinity and efficacy of a variety of 5-HT2AR agonist (López-Giménez & González-Maeso, 2018; Kim et al., 2020; Slocum et al., 2021). 1.3.2 Localization and function of 5-HT2AR in CNS 5-HT2AR and 5-HT2CR are both expressed in CNS. Peripherally, 5-HT2AR is found in platelets, vascular smooth muscle cells and ocular tissue (Leysen, 2004). Rather, 5-HT2BR is primarily found in periphery and, specifically, in human heart cardiac valves (Bonaventure et al., 2005) (Figure 1.7). Introduction 36 2006). The AA release seems to be mediated by a complex mechanism that involves RhoA signalling, MAPK and extracellular signal-regulated kinases (ERK). All of them are depending on Gα-proteins, but different from Gαq/11proteins, such as Gα12/13and Gαi/o-protein subtypes (Kurrasch-Orbaugh et al., 2003a; Kurrasch-Orbaugh et al., 2003b). Several studies revealed that the 5HT2AR has ability to trigger the activation of Gαi/o-proteins, a coupling phenomenon that underlies hallucinogenic actions caused by psychedelic drugs (González-Maeso et al., 2007). Additionally, several phosphorylated downstream proteins have been identified by phosphoproteomic studies after 5HT2AR activation (Karaki et al., 2014). This includes ERKs, ribosomal S6 kinase (RSK-2) (Strachan et al., 2008) and arrestins (Schmid et al., 2008; Schmid & Bohn, 2010). Furthermore, 5-HT2AR interaction with a large number of scaffolding proteins has been described, including postsynaptic density protein 95 (PSD-95) and other PSD95/discs large/zonula-occludens 1 (PDZ)-domain-containing proteins (Xia Gray et al., 2003; Bécamel et al., 2004; Abbas et al., 2009), caveolin-1 (Bhatnagar et al., 2004; Sommer et al., 2009), and microtubule associated protein A1 (MAP1A) (Sheffler et al., 2006). Specifically, 5-HT2AR interaction with PSD-95, caveolin-1, RSK-2 and β-arrestin2 have been described to be essential for modulation of the functional signalling. Introduction 37 Figure 1.10: Illustration of the main intracellular signal transduction pathways associated with 5-HT2AR. 5-HT2AR signalling involves the stimulation of Gαq/11-proteins, which promotes PLCmediated catalysis of PIP2 to IP3 and DAG hydrolysis, thus activating PKC and elevating cytosolic Ca2+. However, 5-HT2AR also activates other signalling pathways. For example, it mediates AA release, presumably through the activation of PLA2, mediated by a complex mechanism involving Rho and p38. All those mechanisms are depending on Gα-proteins, different from Gαq/11, such as Gα12/13. 5-HT is suggested to activate 5-HT2AR and assemble proteins, such as β-arrestin2, Src and Akt. In contrast, hallucinogenic 5-HT2AR agonists do not require this complex recruitment, and elicit differential gene regulation via Gαi/o-protein subtypes. Illustration fom Ibarra-Lecue et al., 2021. 1.3.5 5-HT2AR ligands 5-HT2AR binding drugs belong to structurally diverse chemical classes: indolealkylamines, phenylalkylamines, arylpiperazines, alkylpiperidines, alkylpiperazines and polycyclic/tricyclic agents, among others (Westkaermper & Glennon, 2002). 5-HT2AR drugs (either agonists, antagonists or inverse agonists) represent some of the most important drugs in neuropsychiatry, Introduction 38 including atypical or second-generation antipsychotics, psychedelics and antidepressant drugs (Barnes et al., 2021). Therefore, many efforts have focused on development of 5-HT2AR selective ligands. However, many of the ligands that bind 5HT2AR also bind 5-HT2CR, with similar affinities, given the sequence homology found between the transmembrane portions of both receptors. 1.3.5.1 Agonists 5-HT2AR agonists and partial agonists have traditionally been divided into three structural groups: ergolines (LSD, lisuride and pergolide), indolealkylamines (psilocin) (both included in indoleamines group) and phenylalkylamines (mescaline, 2,5-dimetoxy-4-iodoamphetamine ((±)DOI)) (Halberstandt & Geyer, 2011; Nichols, 2016). The physiological ligand 5-HT is a non-selective agonist that binds to all 5-HT receptors. In 5-HT2 receptor family the affinity order for this molecule is 5-HT2BR > 5-HT2AR > 5-HT2CR (Baxter et al., 1995). Some of the 5-HT2AR agonist ligands display hallucinogenic effects in humans (LSD, psilocybin, psilocin and mescaline). In this sense, classic hallucinogens or serotonergic psychedelics are described as substances capable of altering thoughts, perception, and mood, via activation of 5-HT2AR (Glennon et al., 1984; Vollenweider et al.,1998; González-Maeso et al., 2007; Halberstadt, 2015; Madsen et al., 2019). Despite this effects has been reported to be mediated by 5-HT2AR, classic hallucinogens are not selective and bind other receptors. In this sense, phenylalkylamine hallucinogens are selective for 5-HT2 receptors, including 5HT2AR, 5HT2BR and 5-HT2CR. Among the different molecules, (±)DOI has been described as a potent but non-specific ligand for 5-HT2AR and 5-HT2CR. (±)DOI has often been the agonist of choice for ex vivo/in vivo studies probing 5-HT2AR-mediated functions (Nelson et al., 1999; Introduction 39 Pigott et al., 2012; Canal et al., 2013). [35S]GTPγS experiments in human postmortem brain and 5-HT2AR knock-out animals confirmed that (±)DOI behaves as 5-HT2AR and 5-HT2CR partial agonist (Diez-Alarcia et al., 2019; Garcia-Bea et al., 2019; Muneta-Arrate et al., 2020). During the last years, a new class of ligands have been developed with higher affinity on 5HT2AR, based on N-benzylphenethylamine (NBOMe) scaffold such as n-(2-methoxybenzyl)-2,5-dimethoxy-4-bromophenyletylamine (25BNBOMe, Cimbi-36) and 25-CN-NBOH (Jensen et al., 2020). These molecules are suitable tools for PET imaging and pharmacological studies. 25-CN-NBOH and Cimbi-36 show partial agonist activity, exhibiting higher affinity for 5-HT2AR vs 5-HT2CR and 5-HT2BR (Hansen et al., 2014; Jensen et al., 2017). On the other hand, indolealkylamines like psilocin (the active metabolite of psilocybine) and ergolines, like LSD, are relatively non-selective 5-HT receptors ligands, displaying moderate to high affinity for 5-HT1 and 5-HT2 receptors (Roth, 2007; Nichols, 2016; Wacker et al., 2017). Moreover, LSD binds with high affinity to other 5-HT receptors, but also to dopamine receptors (Halbertadt & Geyer, 2011; Borroto-Escuela et al., 2014). Head-twitch response (a rapid side-to-side movement of head) represent the most widely used rodent response to evaluate hallucinogenic effects induced by psychedelics through 5-HT2AR. This is considered a critical test for in vivo evaluation of functional response to psychedelics. Several studies using selective antagonist and 5HT2AR knock-out animals have demonstrated that head-twitch response is very selective of 5-HT2AR, and is limited to psychedelics (González-Maeso et al., 2007; Canal & Morgan, 2012). However, chemically closely related 5-HT2AR agonist like lisuride, ergotamine and pergolide (González-Maeso et al., 2003; González-Maeso et al., 2007) does not induce this behaviour. Lisuride and pergolide are termed nonhallucinogenic 5-HT2AR agonists. Those compounds, as other ergolines, present a very complex polypharmacological profile, showing affinity for Introduction 40 several aminergic receptors, including serotonergic and dopaminergic receptors (Halberstadt & Geyer, 2011). Moreover, lisuride and pergolide are considered antiparkinsonian drugs, due to their high affinity for dopamine receptors and 5-HT1AR (Langtry & Clissold, 1990; Marona-Lewicka et al., 2002). 1.3.5.2 Antagonists One of the largest and selective classes of 5-HT2AR antagonist are the Nalkylpiperidones, being ketanserin the most widely used for years. Ketanserin is selective for 5-HT2AR vs 5-HT2CR (15-80 fold) and 5-HT2BR (500-1000 fold) (Jerman et al., 2001; Knight et al., 2004; Diez-Alarcia et al 2019). Chemically, ritanserin is close to ketanserin, is highly potent, relatively selective and long acting 5-HT2AR antagonist, but is also described as inverse agonist (Bonhaus et al., 1995). Another 5-HT2AR antagonist, chemically related to ketanserin, is the benzoylperidine altanserin, that has been described as potent and selective 5-HT2AR antagonist with a 20-fold greater affinity for human 5-HT2AR versus human 5-HT2CR (Tan et al., 1999). However, inverse agonist properties on 5-HT2AR have been previously reported for altanserin (Aloyo et al., 2009; Diez-Alarcia et al., 2019). Other 5-HT2AR-selective (or preferring) ligands have been developed such as MDL100907, also known as volinanserin, and MDL-11,939. Volinanserin is a potent 5-HT2AR antagonist and shows 300-fold selectivity for 5-HT2AR receptor versus 5-HT2CR and other GPCRs (Sorensen et al., 1993; López-Giménez et al., 1998). Volinanserin and ritanserin were evaluated as antipsychotics for schizophrenia; however, they turn not to be successful (Jones et al., 2020). Nowadays, volinanserin is used as reference 5-HT2AR antagonist due to its high selectivity. Introduction 41 Recently, new drugs as nelotanserin and eplivanserin have been developed for insomnia treatment. These compounds show high affinity for 5-HT2AR with 20-fold selectivity versus 5-HT2CR (Rinaldi-Carmona et al., 1992; Al-Shamma et al., 2010). Pimavanserin, also known as ACP-103, is described as a highly selective drug for 5-HT2AR, lacking affinity for other receptors except 5-HT2CR (30-fold lower selectivity), and no significant activity on any other GPCR (Vanover et al., 2006; Abbas & Roth, 2008). The US Food and Drug Administration (FDA) has approved this drug for hallucinations and delusions treatment associated with Parkinson´s disease psychosis (Cummings et al., 2014). Some other 5-HT2AR antagonist, although selective for 5-HT2A/2C receptors bind with modest to high affinity to dopaminergic, histaminergic, and/or adrenergic receptors. Thus, atypical antipsychotics (e.g. risperidone, clozapine and olanzapine) and tricyclic antidepressants (e.g. amitriptyline, clomipramine, and imipramine) also bind to 5HT2AR as antagonist (Roth et al., 2004; Meltzer & Massey, 2011; Meltzer, 2012). 1.3.6 5-HT2AR functional selectivity 5-HT receptors, specifically 5-HT2AR, were among the first GPCR for which occurrence of functional selectivity was suggested (Berg et al., 1998). Several studies support that drugs with high affinity to 5-HT2AR (both agonist and antagonist) stabilize distinct receptor conformations. This fact lead to biased interactions or functional selectivity with various downstream effectors, which include the canonical Gαq/11-protein and non-canonical signalling (LópezGiménez & González-Maeso, 2018). In this sense, biased agonism has been proposed to explain the fact that hallucinogenic and non-hallucinogenic drugs activate the same population of cortical 5-HT2AR, but they differ in Gα-protein regulation, transcriptome fingerprints, electrophysiological responses, as well Introduction 42 as, behavioural states (González-Maeso et al., 2003; González-Maeso et al., 2007; Karaki et al., 2014; Banerjee & Vaidya, 2020). One of the first evidence of functional selectivity arose from the finding of Berg and co-workers, regarding ability of this receptor to signal not only via Gαqdependent PLC activation, but also via PLA2 (Berg et al., 1998). Specifically, PLC-dependent IP increase and AA release, via PLA2 activation, were measured to demonstrate the difference of efficacies depending on which signal transduction pathway was activated. According to those results, 5-HT preferentially activated PLC-IP pathway, whereas LSD favoured PLA2-AA pathway (Berg et al., 1998; Martí-Solano et al., 2015). Hallucinogenic and non-hallucinogenic 5-HT2AR agonists also differentially influence gene expression patterns (González-Maeso et al., 2003; GonzálezMaeso et al., 2007). This approach were tested in cells and mouse somatosensory cortex, showing different transcriptome fingerprint between hallucinogenic and non-hallucinogenic 5-HT2AR agonists. Thus, both hallucinogenic and non-hallucinogenic drugs induced c-fos expression. However, transcripts egr-1 and erg-2 were activated by hallucinogens-like (±)DOI and LSD, but expression of these two genes was unaffected by nonhallucinogenic agonists (lisuride and ergotamine) (Figure 1.11). These results also indicated that all 5-HT2AR agonists activated 5-HT2AR coupled to PLC, whereas hallucinogenic-dependent response involved PTX-sensitive heterotrimeric Gαi/o proteins. Notably this hypothesis was validated by quantitative phosphoproteomic approach (Karaki et al., 2014). This hypothesis was strongly supported by comparison between functional selectivity profile of the hallucinogenic drug (±)DOI with the non-hallucinogenic drug pergolide in post-mortem human brain cortex. Both 5-HT2AR agonists induced Gαq/11protein activation, while Gαi1-protein stimulation was only limited to the hallucinogenic drug (±)DOI (Muneta-Arrate et al., 2020). In conclusion, hallucinogenic agonists, such as LSD and psilocybin, promote activation of Introduction 43 canonical Gαq/11-protein cascade as well as a Gαi/o protein-mediated signalling pathway. In contrast, chemically analogous 5-HT2AR agonists lacking of hallucinogenic properties, like lisuride, ergotamine and pergolide, only stimulate Gαq/11-protein-dependent pathway. Other study revealed a distinct signalling signature between hallucinogenic and non-hallucinogenic 5-HT2AR agonists. The authors noted higher levels of phospho-PLC, pERK, pCaMII, pCREB as well as higher levels of IP and DAG production after receptor stimulation with (±)DOI than those observed with lisuride (Banerjee & Vaidya, 2020). Additionally, 5-HT2AR activates other signal transduction cascades, such as βarrestin, besides G-protein mediated PLC-β pathway, in a ligand-dependent manner. 5-HT and (±)DOI can differentially activate 5-HT2AR in cellular models and in vivo head-twitch response. By using mice lacking β-arrestin-2, the absence of head-twitch response as demonstrated in presence of 5-HT. Akt phosphorylation also seems to be present after activation of β-arrestin-2, whereas is absent in presence of (±)DOI. However, the hallucinogenic drug (±)DOI seems to mediate head-twitch response independent of β-arrestin-2. In conclusion, these structurally distinct agonists elicit different signal transduction and trafficking patterns upon 5-HT2AR activation (Schmid et al., 2008; Schmid & Bohn, 2010). Recently, it was suggested that head-twitch response to the hallucinogenic drug LSD was β-arrestin-2-dependent and β-arrestin-1-independent (Rodriguiz et al., 2021). However, non-hallucinogenic drugs that bind to 5HT2AR stimulated β-arrestin-2 recruitment, in contrast to previous findings (Cao et al., 2022). Therefore, implication of β-arrestin-2 in the hallucinogenic response of 5-HT2AR agonists needs further research. On the other hand, the atypical antipsychotic clozapine inhibits 5-HT2AR signalling through a G-protein-dependent mechanism. It induces receptor Introduction 44 internalization and Akt phosphorylation, regardless of receptor interaction with β-arrestin-2. Thus, 5-HT and clozapine use distinct molecular mechanisms to achieve the same 5-HT2AR mediated downstream events: Akt phosphorylation and receptor internalization. This way, Akt phosphorylation is required for clozapine-mediated effects suppression, when this is studied on schizophrenic-like behaviours induced by MK-801 and PCP administration in mice (Schmid et al., 2014) The complexity of biased signalling to elicit selective functional responses has provided an alternative avenue to develop novel therapeutics with increased clinical effect and less side effects. However, the use of biased agonism and other pharmacological properties, such as inverse agonism or antagonism, remains questionable for development of new therapies for schizophrenia and depression. Introduction 45 Figure 1.11: Schematic intracellular signalling pathways of 5-HT2AR coupling to their downstream effectors. Introduction 52 1.5 Antipsychotics Antipsychotics are used as first line medication to treat schizophrenia, and are best classified into two categories: First-generation antipsychotics or typical antipsychotics (e.g., haloperidol, chlorpromazine) and second-generation antipsychotics or atypical antipsychotics (e.g., clozapine, risperidone). Existing medication to treat schizophrenia is effective for treating positive symptoms but have little impact on negative or cognitive symptoms (Conn et al., 2008; Leucht et al., 2009). This fact usually contributes to poor functional outcome. Consequently, there is an urgent need to identify new molecular targets and to develop mechanistically novel compounds for more effective and better-tolerated antipsychotic agents that could improve the therapeutic effects and the safety profile. 1.5.1 First-generation of antipsychotics (Typical antipsychotics) The mechanism of action of typical antipsychotics is the antagonism of dopamine D2R, D3R and/or D4Rs. This finding led to the hypothesis that there is a dysregulation of dopaminergic system that corresponds to a hyperactivity of mesolimbic pathway, and a hypofunction of mesocortical pathway. Thus, blockade of D2R is associated with a reduction of dopamine and psychotic symptoms (Seeman, 1992; Marder et al., 1993). The blockade of D2R is also related to extrapyramidal side effects and hyperprolactinemia (Miyamoto et al., 2008). Haloperidol is the prototypical typical antipsychotic (Figure 1.13). Typical antipsychotics have the ability to reduce positive symptoms and risk for relapse, improving clinical outcomes for many patients with schizophrenia. However, near 30% of patients have little or no response to typical Introduction 53 antipsychotics and also null benefit for negative symptoms or cognitive impairment (Conley & Kelly, 2001; Legge et al., 2020). Figure 1.13: Chemical structure of haloperidol. 1.5.2 Second-generation of antipsychotics (Atypical antipsychotics) Second-generation or atypical antipsychotics were developed looking for reduce extrapyramidal side effects at therapeutically effective doses (Figure 1.14). Atypical antipsychotics display higher affinity for 5-HT2AR comparing to D2R family, which explains lower extrapyramidal effects (Meltzer et al., 1989; Farde et al., 1992). Hence, 5-HT2AR-D2R activity ratio has been considered to be more relevant to predict a reduce side effect liability than improved efficacy (Ebdrup et al., 2011). Unfortunately, second-generation antipsychotics have an increased risk of weight gain, and disturbances in glucose and lipid metabolism (Muench & Hamer, 2010; Weston-Green et al., 2013; Grajales et al., 2019). The discovery of clozapine contributed to the introduction of new drugs with more beneficial pharmacological profile than first-generation antipsychotics (Meltzer et al., 1989). The complex pharmacological profile of clozapine has made the task of determining its mechanism of action extremely difficult. Clozapine has reasonable affinity for a large number of receptors, including Introduction 54 several histaminergic, serotonergic, adrenergic, dopaminergic and cholinergic subtype (Coward, 1992; Nucifora et al., 2017). In addition to activity on dopamine and 5-HT2AR, clozapine is also 5-HT1AR partial agonist, which is thought to be beneficial in terms of reducing cognitive and negative symptoms. Muscarinic receptors are also affected by clozapine, by blocking M1, M2, M3 and M5 receptors (M1R, M2R, M3R, M5R), while stimulated M4 receptor (M4R). Moreover, clozapine antagonizes histamine receptors, which is related to sedation effects. Clozapine also blocks adrenergic receptors, which causes hypotension and tachycardia (Coward et al., 1992; Nucifora et al., 2017). Clozapine is also associated with an elevated risk of potentially lethal hematotoxicity (agranulocytosis and neutropenia), an adverse effect that restricts its clinical use (Alphs et al., 1991). Consequently, new secondgeneration antipsychotics were introduced, such as risperidone, olanzapine, quetiapine, among others, with an effort to reduce side effects related to blood dyscrasias. Olanzapine is a chemical analogue of clozapine with similar pharmacological properties. However, it is not associated with a risk of agranulocytosis. As expected, olanzapine presents higher affinity to 5-HT2AR than to DA receptors. It also blocks histamine, muscarinic and adrenergic receptors but is weaker comparing with clozapine. Weight gain and sedation are the most frequent side effect of olanzapine (Fulton & Goa, 1997; Leucht et al., 2013). Quetiapine acts as D1R, D2R and 5-HT2AR antagonist as well as 5-HT1AR partial agonist. Side effects induced by quetiapine are associated to α1adrenergic and histaminergic antagonism (Miodownik & Lerner, 2006). Risperidone is another atypical antipsychotic drug. Therapeutic effect of risperidone results from both D2R and 5-HT2AR antagonism, showing stronger affinity for 5-HT2AR than for D2R (Cohen, 1994). Moreover, this drug also causes α1-adrenergic and histamine receptor blockade. Risperidone is not only Introduction 55 efficient in treating positive symptoms, but also negative and cognitive disturbances. This fact makes it one of the most commonly prescribed antipsychotics (Möller, 2005; Chopko & Lindsley, 2018). Paliperidone is an active metabolite of risperidone, which acts at the same receptors range. Figure 1.14: Chemical structure of clozapine, olanzapine, quetiapine, risperidone and paliperidone. In the last years, new second-generation antipsychotics have been developed like asenapine and lurasidone (Miyamoto et al., 2012). However, the improved efficacy with respect to first-generation antipsychotics has yet to be determined. Right now, clozapine is the only drug approved for resistant schizophrenia treatment (Conley & Kelly, 2001). Introduction 56 1.5.3 Third-generation of antipsychotics Another generation of antipsychotics, such as aripiprazole, have also been developed. This group of drugs is known as third-generation of antipsychotics (Figure 1.15). Unlike other neuroleptics, third-generation antipsychotics are not D2R antagonist but D2R partial agonist (Davies et al., 2004). In high concentrations of DA, those antipsychotics compete with DA, and results in partial antagonism, leading to clinical benefits. Contrary, when DA levels are low, aripiprazole can bind to D2R and act as partial agonist. Moreover, aripiprazole also shows partial agonist properties to 5-HT1AR. Contrary to second-generation antipsychotics, aripiprazole shows higher affinity for D2R than for 5-HT2AR (Chen et al., 2022). Third-generation antipsychotics are effective in alleviating psychotic symptoms without inducing extrapyramidal side effects and hyperprolactinemia, as well as lower weight gain and metabolic liabilities (Lieberman, 2004). Figure 1.15: Chemical structure of aripiprazole. 1.5.4 New generation of antipsychotics Different efforts to find new therapies for schizophrenia based on the use of 5HT2AR antagonists, like ritanserin and volinanserin, have failed to be therapeutically useful. Due to the lack of efficacy of monotherapy with selective 5-HT2AR antagonist, different researches indicate that 5-HT2AR antagonism alone is not enough to explain the efficacy of atypical antipsychotics (Miyamoto Introduction 57 et al., 2012). Thus, D2R blockade seems to be necessary. However, a potent and selective 5-HT2AR drug, pimavanserin, was developed as a new alternative for the treatment of psychosis (Figure 1.16) (Meltzer & Roth, 2013). Pimavanserin is the first approved antipsychotic that lacks of dopaminergic affinity (Hacksell et al., 2014). Until now, pimavanserin has been tested as adjunctive therapy for schizophrenia in combination with haloperidol and risperidone (Meltzer et al., 2012), and has gained FDA approval to reduce delusions and hallucinations in Parkinson’s disease (Cummings et al., 2014). Moreover, pimavanserin has shown ability to reduce negative symptoms in schizophrenia patients (Bugarski-Kirola et al., 2022). Pimavanserin has been described as 5-HT2AR inverse agonist. However, evidence of this aspect needs to be demonstrated (Vanover et al., 2006; Nutt et al., 2017). Figure 1.16: Chemical structure of pimavanserin. It has been recently recognized that many second-generation antipsychotics are 5-HT2AR inverse agonists rather than neutral antagonists (Weiner et al., 2001). In contrast to antagonists, inverse agonists possess negative intrinsic efficacy, and can attenuate basal constitutive signalling activity. Overall, a better understanding of the mechanism of action of antipsychotics could lead to the design and development of more effective and tolerable drugs. Historically, promiscuous drugs with polypharmacological profile have been thought to be more effective for treating CNS disease, although this Introduction 58 represented presence of many severe and potentially life-threating side effects. Therefore, design of selective drugs that would interact with defined molecular targets could probably result in more effective and tolerable drugs. Hence, biased agonism alone or in combination with inverse agonism arise as alternative approaches to receptor selectivity in order to improve the expected functional responses in schizophrenia. Aims Aims 61 Despite much research has been undertaken to develop new antipsychotic drugs for treating effectively the intrinsic impairments, none of the medications currently available has stood out for long-lasting efficacy without detrimental side effects. This is partially due to poorly understood neurobiology of schizophrenia. Therefore, a deeper knowledge of schizophrenia at a neurobiological level would enable the identification of molecular, cellular and/or pathway alterations, which could become therapeutic targets for new drugs. Several findings suggest that the 5-HT2AR is involved in the molecular mechanism responsible of psychotic symptoms and their treatment. On the one side, the 5-HT2AR is responsible for the hallucinogenic nature of psychedelic drugs like LSD, psilocybin, mescaline and (±)DOI, which activate the 5-HT2AR. On the other hand, atypical antipsychotics commonly used in schizophrenia would act as antagonist or even inverse agonist of the 5-HT2AR. Different studies by in vivo PET neuroimaging and in vitro post-mortem studies have shown conflicting results about 5-HT2AR brain density in schizophrenia subjects. These conflicting reports seem to be related to the use of different radiotracers binding to different conformations of the receptor. Therefore, 5HT2AR alterations in schizophrenia seem to be more related to variations in molecular state of the receptor rather than alterations in expression levels. In this context, a full characterization of 5-HT2AR inverse agonists in human brain becomes an unmet need to clarify the functional status of this receptor in schizophrenia. The 5-HT2AR is able to activate both Gαq/11and Gαi/o-proteins depending on the drug binding properties. In this context, activation of Gαi/o-proteins by 5HT2AR agonist has been proposed as a molecular fingerprint of hallucinogenic properties. Moreover, higher stimulation of Gαi1but not Gαq/11-proteins in response to the 5-HT2AR agonist (±)DOI has been described in post-mortem PFC of subjects with schizophrenia. This finding could be interpreted as Subjects, Material and Methods 68 Table 3.1: Demographic characteristics, post-mortem conditions, cause of death and toxicological analysis of individual cases of schizophrenia subjects ( S), non-schziophrenia suicide (NSS) and their respective controls (C). Case Diagnostic Gender (M/F) Age (years) PMD(h ours) Storage (months) Cause of death Mechanism of death Brain pH Drug Blod Levels (mg/L) Brain Toxicology (ng/g) S 1 Schziophrenia F 67 22 17 Natural Cardiorespiratory failure 5.8 Negative Negative C 1 Control F 66 17 83 Accident Road accident 6.06 Negative Not performed S 2 Schizophrenia M 34 23 81 Suicide Jump from a height 6.32 Negative Cotinine 23.1 C 2 Control M 34 17 69 Accident Road Accident 6.7 Negative Negative NSS 1 Persona lity disorder M 34 7 199 Suicide Hanging Not performed Ethanol 2.7 g/L Citalopram 0.1 Oxacarbazepina 6.5 Citalopram 1579.9 Cotinine 3352.8 Norcitalopram 368.2 S 3 Schizophrenia F 53 18 98 Natural Haemorrage 6.58 Alprazolam 0.05 Paliperidone 54.2 Cotinine 503.2 Alprazolam 43.9 C 3 Control F 51 10 70 Natural Cardiorespiratory failure 6.3 Negative Negative S 4 Schizophrenia M 32 21 87 Suicide Jump from a height 6.65 Negative Cotinine 380.78 C 4 Control M 33 23 95 Accident Road accident 6.55 Negative Not performed NSS 2 Personality disorder M 33 14 192 Suicide Jump from a height Not Performed Lorazepam 0.03 Venlafaxine 0.16 Desmethylvenlafaxine 475.0 Lorazepam 1421.2 Midazolam 3.7 Olanzapine 6 S 5 Schizophrenia M 45 36 15 Accident Choking Not performed Not performed Midazolam 1801.8 Nordiazepam 1247.1 Oxazepam 50.1 C 5 Control M 44 23 98 Accident Road accident 6.45 Negative Negative NSS 3 Personality disorder M 44 9 192 Suicide Jump from a heihgt Not performed Ethanol 0.22 g/L Alprazolam 0.01 Amisulpride 1.4 Clomipramine 0.2 Reboxetine 0.09 Alprazolam 141.4 Clomipramine 3040.7 Cotinine 322.5 Reboxetine 224.2 S 6 Schizophrenia M 49 23 107 Accident Fall from a height 6.40 Negative Cotinine 391 Lorazepam 16.3 C 6 Control M 49 19 93 Accident Road accident 6.7 Negative Negative S 7 Schizophrenia M 70 20 115 Suicide Hanging Not performed Not performed Negative C 7 Control M 71 22 115 Accident Fall from a height 5.92 Negative Negative Subjects, Material and Methods 69 S 8 Schizophrenia F 74 9 118 Natural Cardiorespiratory failure Not performed Phenobarbital 9 Negative C 8 Control F 74 30 167 Accident Road accident Not performed Negative Negative S 9 Schizophrenia M 46 20 129 Suicide Jump from a height 6.41 Negative Zuclopenthixol 110.8 Cotinine 622.4 Lorazepam 25.5 C 9 Control M 46 22 115 Natural Fall 6.48 Negative Negative NSS 4 Personality disorder M 47 4 195 Suicide Jump from a height Not performed Phenytoine Negative S 10 Schizophrenia M 26 24 140 Suicide Jump from a height Not performed Diazepam 0.27 Diazepam 356.5 Nor-diazepam 856.9 Oxazepam 15.1 C 10 Control M 25 21 71 Accident Fire 6.48 Negative Negative NSS 5 Personality disorder M 27 42 253 Suicide Jump from a height Not performed Negative Cotinine 337.4 Diazepam 204.9 Nordiazepam 612 Oxazepam 65.8 S 11 Schizophrenia F 75 18 140 Natural Cardiorespiratory failure Not performed Negative Cotinine 36.76 C 11 Control F 79 24 213 Accident Road accident Not performed Negative Not performed S 12 Schizophrenia M 28 28 143 Suicide Jump from a heihgt Not performed Negative Cotinine 93.79 C12 Control M 29 13 116 Accident Fall from a height 6.44 Negative Negative NSS 6 Personality disorder M 28 5 259 Suicide Jump from a height Not performed Negative Cotinine 638.2 S 13 Schizophrenia M 25 17 145 Suicide Jump from a height Not performed Negative Cotinine 153.1 C 13 Control M 23 16 141 Accident Fall from a height Not performed Negative Negative NSS 7 Obsessivecompulsive disorder M 26 19 143 Suicide Jump from a height 6.66 Clomipramine 0.5 Fluoxetine 0.7 Fluvoxamine 0.2 Quetiapine 0.5 Fluoxetine 15441 Fluvoxamine 4094 Norfluoxetine 4433.5 Norquetiapine 386.7 Quetiapine 86 S 14 Schizophrenia M 23 13 195 Suicide Jump from a height Not performed Not performed Haloperidol 136.5 Cotinine 458.5 Quetiapine 392.5 Norquetiapine 1309.4 C 14 Control M 22 20 188 Accident Road accident Not performed Nordiazepam 0.38 Cotinine 371.6 Oxazepam 80.9 Nordiazepam 909 Subjects, Material and Methods 70 NSS 8 Personality disorder M 19 8 131 Suicide Fall from a height 6.7 Negative Cotinine 236.4 S 15 Schizophrenia F 80 32 178 Natural Shock Not performed Negative Not performed C 15 Control F 78 12 185 Natural Cardiorespiratory failure Not performed Negative Not performed S 16 Schizophrenia F 38 23 216 Suicide Jump from a height Not performed Negative Negative C 16 Control F 36 19 110 Accident Fall in front of a train 6.51 Negative Negative NSS 9 Anxiety disorder F 39 19 214 Suicide Drug overdose Not performed Nordiazepam 0.13 Ethanol 5.0 g/L Diazepam 22.4 Nordiazepam 280.7 S 17 Schizophrenia F 51 15 236 Natural Cardiorespiratory failure Not performed Buflomedil 66 Metamizol 4 Cotinine 521.5 C 17 Control F 51 38 230 Accid ent Road accident Not performed Negative Not performed S 18 Schizophrenia M 62 28 240 Suicide Jump from a height Not performed Not performed Amitriptyline 170.3 Nortryptiline 453 C 18 Control M 62 23 243 Accident Road accident Not performed Negative Not Performed NSS 10 Adaptative anxiety disorder M 62 19 271 Suicide Crushing Not performed Not performed Maprotiline 257.9 Nordiazepam 1584.9 S 19 Schizophrenia M 35 22 299 Suicide Jump from a height Not performed Negative Negative C 19 Control M 36 22 286 Accident Road accident Not performed Ethanol 1.0 g/L Not performed NSS 11 Anxiety disorder M 36 15 181 Suicide Gun shot Not performed Mirtazapine 0.08 Not Performed S 20 Schizophrenia M 49 41 292 Suicide Hanging Not performed Ethanol 0.49 g/L/ Not performed Cotinine 127 Chlorpromazine 140.7 Lorazepam 388.6 Thioridazine 6079.5 C 20 Control M 45 30 275 Accident Road accident 6.82 Ethanol 3.09 g/L Not performed NSS 12 Anxiety disorder M 46 26 319 Suicide Hanging Not performed Not performed Diazepam 91.3 Nordiazepam 175.2 S 21 Schizophrenia F 56 24 125 Natural Cardiorespiratory failure Not performed Alprazolam 0.03 Cotinine 110 Alprazolam 38 C 21 Control F 54 24 10 Accident Fall from a height 6.87 Negative Negative S 22 Schizophrenia M 50 3 173 Suicide Drowning 7.09 Nordiazepam 0.4 Amisulpride 75.9 Cotinine 89 Nordiazepam 662.8 Oxazepam 47.6 Trazodone 241.6 Subjects, Material and Methods 71 C22 Control M 50 2 15 Natural Cardiorespiratory failure 6.1 Negative Negative NSS 13 Personality disorder M 49 22 141 Suicide Fall from a height 6.35 Nordiazepam 2.8 Tiapride 5.4 Venlafaxine 1.2 Cotinine 254.1 Desmethylvenlafaxine 265.6 Oxazepam 94.7 Tiapride 973.5 Venlafaxine 2146.5 S 23 Schizophrenia M 43 17 172 Natural Cardiorespiratory failure Not performed Negative Cotinine 1003.9 Lorazepam 66 C 23 Control M 41 15 7 Natural Cardiorespiratory failure Not performed Negative Negative Subjects, Material and Methods 72 All demographic characteristics, age, PMD, storage time and pH are summarized in Table 3.2. As expected, no differences were found when compared age between the three groups (F[2,56]=2.08, p=0.1341). In the same way, no statistical differences between groups were found in either PMD (F[2,56)]=1.99, p=0.1456) or pH values (F[2,21]=0.17, p=0.8433). However, a longer storage time was observed in non-schizophrenia suicide subjects compared to controls (F[2,56]=4.66, p=0.0135) (Table 3.2). Table 3.2: Demographic and post-mortem characteristics of schizophrenia, non-schizophrenia suicide and control subjects included in the study. Group values are means±SEM. *p<0.05 vs control group (Bonferroni’s multiple comparison test). Group Gender Age (years) PMD pH Storage time (months) Schizophrenia 7 F/16 M 48±4 22±2 6.5±0.1 150±15 Non-schizophrenia suicide 1 F/12 M 38±3 16±3 6.6±0.1 207±15* Control 7 F/16 M 48±3 21±2 6.5±0.1 139±15 Subjects, Material and Methods 73 3.1.2 Demographic characteristics and diagnosis of subjects included in pools used for Antibody-capture [35S]GTPγS Scintillation proximity Assays (SPA) and Western Blot characterization assays The initial pharmacological characterization of drugs was performed with a pool of DLPFC from different control subjects not included in the rest of the study. In each membrane preparation, homogenates from six prefrontal human brain samples were used. Fifteen samples collected between years 2016 and 2019 were used for the whole study (30% men and 70% women) with a mean age of 59±6 years, PMD of 11±2 hours and storage time of 27±3 months. Absence of toxicological positive test for psychotropic drugs in blood was confirmed in subjects contributing to these pools. Subjects, Material and Methods 74 3.2 Animals: Transgenic mice The 5-HT2AR knock-out (5-HT2AR(-/-)) and wild-type (5-HT2AR(+/+)) mice were generously donated by Prof. R. Maldonado (Barcelona, Spain). Animals had been originally generated on a 129S6/SvEv background further back crossed into the inbred C57BL/6J line, following standard procedures (GonzálezMaeso et al., 2003; Weisstaub et al., 2006; González-Maeso et al., 2007; Orejarena et al., 2011). After donation, supplementary backcrosses were performed. Animals were genotyped by conventional polymerase chain reaction (PCR) and subsequent electrophoresis in our laboratory (data not shown), as described by Fiorica-Howells et al., (Fiorica-Hollowells et al., 2002). Absence of 5-HT2AR expression in 5-HT2AR(-/-) mice was confirmed by the lack of [3H]ketanserin binding (Muguruza et al., 2013) and Gαi1/Gαq/11-protein activation by (±)DOI (Garcia-Bea et al., 2019). Experiments were performed on adult (15-20 weeks old) C57BL/J6 mice. Animals were housed up to five individuals under standard laboratory conditions (22±1°C, 55±5% relative humidity, 12 h light/dark cycle and free access to standard rodent chow and water). Efforts were made to minimize the number of animal used. The experimental protocols were reviewed and approved by the Local Ethical Committee of Animal Research of the University of the Basque Country (UPV/EHU, CEEA, Ref. M20-2019-321). All experiments were carried out in accordance with the European Community Council Directive on “The Protection of Animals Used for Scientific Purpose” (European Union Directive 2010/63/UE) and Spanish Law (Royal decree 53/2013) for the care of laboratory animals. Adult mice were sacrificed by cervical dislocation, brains removed, cortex dissected and samples stored at - 80°C until assay as previously described (Diez-Alarcia et al., 2016). Subjects, Material and Methods 75 3.3 Drugs -(±)-DOI: (±)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride (SigmaAldrich; Saint Louis, Missouri, USA). -Ketanserin: 3-[2-[4-(4-Fluorobenzoyl)-1-piperidinyl]ethyl]-2,4[1H,3H]- quinazolinedione tartrate (Tocris; Bristol UK). -MDL100907 (volinanserin): (R)-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4fluorophenyl)ethyl]-4-pipidinemethanol (Sigma-Aldrich; Saint Louis, Missouri, USA). -Altanserin: 3-[2-[4-(4-Fluorobenzoyl)-1-piperidinyl]ethyl]-2,3-dihydro-2thioxo-4(1H)-quinazolinone hydrochloride hydrate (Sigma-Aldrich; Saint Louis, Missouri, USA). -Pimavanserin (ACP-103): 1-(4-Fluorobenzyl)-3-(4-isobutoxybenzyl)-1-(1methylpiperidin-4-yl)urea (Axon Medchem; Groningen, The Netherlands). -Nelotanserin: 1-(3-(4-bromo-2-methyl-2H-pyrazol-3-yl)-4-methoxyphenyl)-3- (2,4-difluorophenyl)urea (Axon Medchem; Groningen, The Netherlands). -Ritanserin: 6-[2-[4-[bis(4-fluorophenyl)methylidene]piperidin-1-yl]ethyl]-7methyl-[1,3]thiazolo[3,2-a]pyrimidin-5-one (Sigma-Aldrich; Saint Louis, Missouri, USA). -Eplivanserin: 4-[(E,3Z)-3-[2-(dimethylamino)ethoxyimino]-3-(2fluorophenyl)prop-1-enyl]phenol (Axon Medchem; Groningen, The Netherlands). -MDL-11,939: α-phenyl-1-(2-phenylethyl)-4-piperidinemethanol (Tocris; Bristol, UK). -SB 242084: 6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]- 3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride (Tocris; Bristol, UK). -Clozapine: 8-Chloro-11-(4-methyl-1-piperazinyl)-5Hdibenzo[b,e][1,4]diazepine (Tocris; Bristol, UK). -Risperidone: 3-[2-[4-(6-fluoro-1,2benzisoxazol-3-yl)-1-piperidinyl]ethyl]- 6,7,8,9-tetrahydro-2-methyl-4H-pyrido[1,2a]pyrimidin-4-one (Sigma-Aldrich; Saint Louis, Missouri, USA). Subjects, Material and Methods 76 -Aripiprazole: 7-{4-[4-(2,3-Dichlorophenyl)-1-piperazinyl]butoxy}-3,4-dihydro2(1H)-quinolinone one (Sigma-Aldrich; Saint Louis, Missouri, USA). -Olanzapine: 2-Methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3b][1,5]benzodiazepine (Sigma-Aldrich; Saint Louis, Missouri, USA). -Paliperidone: 3-[2-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]ethyl]- 6,7,8,9-tetrahydro-9-hydroxy-2-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (Tocris; Bristol, UK). -Quetiapine: 2-[2-(4-Dibenzo[b,f][1,4]thiazepin-11-yl-1piperazinyl)ethoxy]ethanol hemifumarate (Tocris; Bristol, UK). -Atropine: Endo-(±)-α-(Hydroxymethyl)benzeneacetic acid 8-methyl-8azabicyclo[3.2.1]oct-3-yl ester (Sigma-Aldrich; Saint Louis, Missouri, USA). -Phentolamine: 2-[N-(3-Hydroxyphenyl)-p-toluidinomethyl]-2-imidazolidine hydrochloride (Sigma-Aldrich; Saint Louis, Missouri, USA). -Cetirizine: [2-[4-[(4-Chlorophenyl)phenylmethyl]-1-piperazinyl]ethoxy]acetic acid dihydrochloride (Sigma-Aldrich; Saint Louis, Missouri, USA). -Raclopride: 3,5-Dichloro-N-[[(2S)-1-ethyl-2-pyrrolidinyl]methyl]-2-hydroxy-6methoxybenzamide (Tocris; Bristol UK). -Haloperidol: 4-[4-(4-Chlorophenyl)-4-hydroxypiperidino]-4′- fluorobutyrophenone (Sigma-Aldrich; Saint Louis, Missouri, USA). Subjects, Material and Methods 77 3.4 Materials Antibodies Primary monoclonal antibodies used for Antibody-capture [35S]GTPγS SPA and Western Blot experiments were purchased from Santa Cruz Biotechnology, Inc (California USA). They are detailed in section 3.5.2, where Western Blot experiment protocol is explained. For Western Blot assays, different fluorescent secondary antibodies were used, such as Alexa Fluor® 680 conjugated with goat anti-mouse, provided by Invitrogen (Oregon, USA), and IRDyeTM conjugated with anti-rabbit, provided by Rockland Immunochemical (Pennsylvania, USA). Radioactive compounds Sulfur 35 labelled guanosine-5’-O-(gamma-thio)-triphosphate ([35S]GTPγS) with specific activity of 1250 Ci/mmol was from PerkinElmer (Waltman, MA, USA). Other drugs and chemicals sources -Bio-Rad Laboratories (California, USA): Ammonium persulfate (APS), Bradford Protein Assay, 2x concentrated Laemmli sample buffer, N-N-N-N´- tetramethylethylenediamine (TEMED), pre-stained SDS-PAGE molecular weight standards. -Carlo Erba Reagents (Barcelona, Spain): Methanol. -GE Healthcare (Buckinghamsire, UK): Nitrocellulose membranes (pore size: 0.45 μm) and WhatmanTM cellulose 3 mm. -Invitrogen (Barcelona, Spain): DL-Dithiothreitol (DTT), ethylenediamine tetracetic acid (EDTA). Subjects, Material and Methods 84 Table 3.4: General protocol of [35S]GTPγS binding combined with SPA. BB: Basal binding (binding values in absence of any drug), NBS: Non-specific binding (defined as the remaining [35S]GTPγS binding in presence of 100 μM unlabelled GTPγS). In general, in order to find a selective inverse agonist, initially, different drugs previously described as 5-HT2AR antagonist were chosen and tested for inverse agonism properties. After that, selected inverse agonists compounds were used to investigate 5-HT2AR alterations coupling to Gα-proteins in schizophrenia subjects. In an effort to evaluate the specificity and selectivity of these drugs, different selective antagonist as well as 5-HT2AR(+/+) and 5HT2AR(-/-) mice membrane homogenates were used to block the [35S]GTPγS binding signal observed for each drug. BB Agonist Agonist + Antagonist NBS SPA incubation buffer/DMSO 24 μl 12 μl / 12 μl GTPγS / / / 12 μL GDP 12 μl 12 μl 12 μl 12 μl Antagonist / / 12 μl / Membranes 160 μl 160 μl 160 μl 160 μl 15 minutes of incubation at 30°C with gentle agitation (400 rpm) Agonist / 12 μl 12 μl / 30 minutes of incubation at 30°C with gentle agitation (400 rpm) [ 35 S]GTPγS 5 μl 5 μl 5 μl 5 μl 90 minutes of incubation at 30°C with gentle agitation (400 rpm) Detergent 20 μl 20 μl 20 μl 20 μl 30 minutes of incubation at 22°C with gentle agitation (350 rpm) Antibodies 10 μl 10 μl 10 μl 10 μl 90 minutes of incubation at 22°C with gentle agitation (350 rpm) PVT Protein A SPA BEADS 50 μl 50 μl 50 μl 50 μl 180 minutes of incubation at 22°C with gentle agitation (350 rpm) Subjects, Material and Methods 85 3.5.1.3 Mathematical and statistical analysis of the results Mathematical analysis Results obtained from the Microbeta Trilux Scintillation counter are expressed as CCPM (corrected counts per minute), which assumes the equivalency between CCPM and DPM (disintegrations counts per minute). In order to interpret the results, data from CCPM must be transformed into femtomole [35S]GTPγS bound per milligram protein (fmol/mg protein). The conversion is made considering the protein concentration used in the assay and by following the Equation 3.1. The protein concentration can influence the [35S]GTPγS binding as shown in Diez-Alarcia et al., 2021b. Therefore, protein content of the assay is measured after each experiment. fmol/ mg protein: CCPM counts/(2,22 x 1250 x [assay protein concentration mg]) Equation 3.1: Results expressed in fmol/mg protein performed in [35S]GTPγS binding combined with SPA. 1250 (Ci/mmol) corresponds to specific activity of the radioligand 35S. 2,22 is a constant for the transformation from Curie (Ci) to DMP (1 Ci=2,22x1012 DPM). Basal binding (BB) of [35S]GTPγS, is defined as [35S]GTPγS binding in absence of any exogenous drug. When an agonist or inverse agonist drugs is added to the assay the basal [35S]GTPγS binding is modulated. When the drug is an agonist, an increase of [35S]GTPγS binding on [35S]GTPγS basal binding will be observed. In contrast, inverse agonists will decrease the [35S]GTPγS basal binding. The non-specific binding (NBS) in CCPM was subtracted from all CCPM obtained for each different condition (basal binding, stimulation, inhibition,..). Thus, specific [35S]GTPγS binding values were obtained and used for further calculation and statistical analysis. Subjects, Material and Methods 86 Concentration-response curves for different drugs were performed in order to determine agonist, antagonist or inverse agonist properties of each drug. The BB, in absence of the drug, was considered as 100%, and specific binding were transformed to percentage of basal binding. Thus, stimulation or inhibition effect on the [35S]GTPγS binding in presence of the drug was defined in relation to respective basal binding. Each point of the curve was calculate with Equation 3.2 and 3.3. % Stimulation: (Stimulation-NBS)/(BB-NBS)x100 Equation 3.2: Calculation of percentage over basal binding performed in [35S]GTPγS binding combined with SPA experiments in order to determine agonist, antagonist or competition effects. NBS: Non-specific binding, BB: Basal binding. % Inhibition: 100-[(Inhibition-NBS)/(BB-NBS)x100] Equation 3.3: Calculation of percentage of basal binding performed in [35S]GTPγS binding combined with SPA experiments in order to determine inverse agonist, antagonist or competition effects. NBS: Non-specific binding. BB: Basal binding. In some cases, in order to test the effect of [35S]GTPγS binding to different Gαprotein subtypes, instead of concentration-response curves, a single concentration (10 μM) of the drug was used. This concentration was selected because it gives binding values around the maximal effects (Emax/Imax) in the concentration-response curves. This concentration was selected for one-point concentration experiments. In this case, basal binding was defined as 0% and effects were expressed as positive or negative percentage changes respect to basal conditions Pharmacological parameters of stimulation or inhibition curves of [35S]GTPγS binding, maximal stimulatory or inhibitory effects (Emax/Imax) and concentration Subjects, Material and Methods 87 of the drug that determines half maximal stimulation or inhibition (EC50/IC50) were calculated. These parameters were obtained by non-linear analysis using GraphPad PrimTM. Points were fitted to the concentration-response curve shown in Equation 3.4 and 3.5. This model assumes that concentrationresponse curve displays a standard slope, equal to a Hill slope (or slope factor) of 1.0. This is the expected slope when a ligand binds to a single receptor according to the law of mass action. E= BB+(Emax-BB)(1+10(LogEC50-Log[X])) Equation 3.4: Monophasic stimulatory concentration-response curve (standard slope). E corresponds to the effect (% Stimulation of Equation 3.2) at the X concentration, BB corresponds to the specific [35S]GTPγS binding in absence of agonist or basal binding (assumed as 100%), Emax to the maximal effect (%) and LogEC50 to the concentration of the drug that determines half maximal effect. E= BB+(Imax-BB)(1+10(LogIC50-Log[X])) Equation 3.5: Monophasic inhibitory concentration-response curve (standard slope). E corresponds to the effect (% Inhibition of Equation 3.3) at X concentration, BB corresponds to the specific [35S]GTPγS binding in absence of inverse agonist or basal binding (assumed as 100%), Imax to the maximal effect (%) and LogIC50 to the concentration of the drug that determines half maximal effect. Pharmacological parameters Emax/Imax are expressed as mean±SEM (standard error of the mean). –LogEC50/IC50 are expressed as mean±SEM. These values are used for further statistical analysis. Half-maximal stimulation or inhibition are also expressed as EC50/IC50 that corresponds to the antilogarithm of LogEC50/IC50 mean values. Concentration-response curves were simultaneously co-analysed (all the experiments of each experimental group together) by non-linear regression for Subjects, Material and Methods 88 the best fit assuming an one-site model of [35S]GTPγS binding. This way, global values for each group were obtained. Results are expressed as the best fit value±95% confidence interval. Statistical analysis Different statistical analyses were used for the suitable interpretation of the results. Initially, all data were subjected to Grubb´s test, in order to detect and reject possible outlier values among experimental groups. Whether obtained data displayed a Gaussian distribution was also tested. Results from one single-concentration experiments were analyzed by onesample Student’s t-test vs basal values, in order to evaluate presence or absence of the effect induced by drugs on basal binding. Two-tailed unpaired Student´s t-test was used to compare two different conditions. For example, to compare the effect of an agonist alone vs agonist co-incubated with an antagonist. When three different conditions comparison was needed, one-way ANOVA analysis was performed followed by Bonferroni´s post-hoc analysis. This test was used, for example, to compare the modulation of [35S]GTPγS binding between schizophrenia, nonschizophrenia suicide and control groups. In order to calculate the contribution effect of two different variables, two-way ANOVA was used followed by Bonferroni´s post-hoc analysis. This analysis was appropriate to study the effect of different drugs in various mice genotypes. Pearson´s correlation r coefficient was calculated to test possible associations between independent covariables (age, PMD and storage time) and dependent functional responses. When correlation was significant, analysis of Subjects, Material and Methods 89 covariance (ANCOVA) was performed between groups (schizophrenia, nonschizophrenia suicide and control subjects) controlling for the independent covariable. ANCOVA analyses were carried out by using InVivoStat statistical software. In all statistical evaluations, differences were considered significant at p<0.05. A complementary analysis of potential differences between groups was also performed. Thus, global results obtained from coanalysis of schizophrenia, non-schizophrenia suicide and control subjects data were subjected to further evaluation (DeLean et al., 1978; Motulsky & Ransnas, 1987). This extra analysis compared the goodness of fit of a model with and without a set of constrains by means of an F test (based on the principle of extra sum of squares). First, sets of data were analysed separately (no constrains) as described above. Overall value for the sum of squares was sum of individual values from each fit and, similarly, the numbers of degrees of freedom. Next, sets were pooled, analysed simultaneously, and constrained to share one or more common parameters (basal, Emax/Imax and LogEC50/IC50), which gave different values for the sum of squares and degrees of freedom. Analysis that permitted one or more parameters sharing, without a significant increase in the residual variance, was taken as the best fit. Statistical significance of the improvement was determined with a F test at p<0.05, and was expressed as F[DFn, DFd] where F is distribution value, DFn are degrees of freedom in numerator and DFd are degrees of freedom in denominator. Subjects, Material and Methods 90 3.5.2 Western Blot experiments Western Blot is a broadly used technique for detection and identification of proteins by means of antibodies. The process is divided into various steps: electrophoresis separation by molecular weight of proteins present in the sample; protein transfer from the gel to nitrocellulose membranes, which immobilizes proteins and make them accessible to antibodies; and exposition of nitrocellulose membranes to solution containing the antibodies that recognize and bind specifically a target protein. In the present study, firstly, Western Blot experiments were performed in order to characterize and evaluate the specificity of antibodies used in antibodycapture [35S]GTPγS Scintillation Proximity Assays (SPA) for each Gα-protein subtype. Further, immunoreactive densities of Gαi1and Gαq/11-proteins were quantified in post-mortem PFC of schizophrenia, non-schizophrenia suicide subjects and their matched control subjects. Simultaneously, density of cytoskeletal protein β-actin was also measured, as a loading control. Subjects, Material and Methods 91 Figure 3.2: Schematic representation of Western Blot assays. 1. Heating of samples at 95°C for five minutes; 2. Loading of an optimized quantity of sample onto gel; 3. Afterwards, running of gel electrophoresis at 60 V for 30 minutes and change to 140 V for 90 minutes. Proteins separate according to size, with smaller proteins migrating through the gel; 4. Assembling of transfer sandwich with the gel near the cathode (-) and the membrane near the anode (+); 5. Negatively charged proteins will migrate out of the gel onto the membrane by performing transfer at 0.3 A per tray, for 90 minutes; 6. Blocking the membrane in an appropriate blocking buffer (milk). Incubation of the membrane with primary antibody and, after washing, incubation with secondary antibody for 1 hour at room temperature. Finally, detection of image fluorescence. Illustration originally created for this thesis by J. DelaCuesta-Barrutia. 3.5.2.1 Preparation of membrane enriched fraction (P2 fraction) Membrane P2 fractions to perform Western-blot assays were prepared as previously described for SPA assays (Section 3.5.1.1). The day of experiment, 0.5 mg of P2 fraction pellets were defrosted and resuspended in 125 μl of TrisHCl 0.5 nM. These samples were combined with 119 μl 2X Laemmli sample buffer and 6 μl of β-mercaptoethanol, obtaining 2 mg/ml as a final protein content. Samples from schizophrenia, non-schizophrenia suicide and control subjects were processed in parallel on the same day. Subjects, Material and Methods 92 3.5.2.2 Gel electrophoresis, transference and immunodetection Electrophoresis in polyacrylamide gels The electrophoresis in polyacrylamide gels in denaturalizing conditions or SDS-PAGE (Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis) is the most used analytical method to separate different proteins of a sample according to their molecular weight. During the electrophoresis, proteins migrate in first place though “stacking gel”. Stacking gel was prepared with 5% of polyacrylamide, 125 mM Tris HCl, 0.1% SDS, 0.07% prostatic specific antigen (PSA) and 0.14 TEMED pH 6.8. Laemmli-prepared samples were heated at 95°C for five minutes in a Thermoblock during the preparation of “stacking gel” (Figure 3.2). Protein separation according to their molecular weight started in the “running gel”. This gel was prepared in a 10% acrylamide-bisacrylamide concentration in a solution of 0.37 M Tris HCl, 0.1% SDS, 0.07% PSA and 0.07% TEMED, pH 8.8. A comb from TeflonTM was inserted in each stacking gel in order to form 15 lanes, where the samples run. Samples were loaded on those 15-lane gels, sized 6 x 8 cm each (Figure 3.3). The loading protein and volume of samples in each line depended on the Gαprotein of interest (Table 3.5). The first line of each gel was loaded with a commercial molecular weight marker suspension (10-250 kDa, Precision Plus ProteinTM, Dual Color Standards, Bio-Rad). The rest of lines were loaded with corresponding volume of the sample. Gel loading sketch is shown in Figure 3.3. Subjects, Material and Methods 93 Figure 3.3: Gel loading sketch of a western blot experiment for antibody characterization and schizophrenia, non-schizophrenia suicide and control subjects experiments. MW: Molecular weight marker, P2: membrane-enriched fraction, C: Control subjects, SCH: Schizophrenia subjects, NSCHS: Non-schizophrenia suicide subjects. Table 3.5: Experimental conditions for Western Blot experiments with human, rat and mouse brain samples. MW (Molecular weight). Protein Loaded protein/volume MW (kDa) Blocking solution Incubation solution Primary antibody dilution Secondary antibody dilution Gαi1 30 μg/ 15 μl 40 5% nonfat dry milk Blocking solution + 0.1% Tween 20 1:200 1:10000 Gαi2 15 μg/ 7.5 μl 42 5% nonfat dry milk Blocking solution + 0.1% Tween 20 1:300 1:8000 Gαi3 15 μg/ 7.5 μl 40 5% nonfat dry milk Blocking solution + 0.1% Tween 20 1:300 1:8000 Gα o 15 μg/ 7.5 μl 45 5% nonfat dry milk Blocking solution + 0.1% Tween 20 1:500 1:8000 Gαq/11 24 μg/ 12 μl 40 5% nonfat dry milk Blocking solution + 0.1% Tween 20 1:300 1:8000 Gα s 15 μg/ 7.5 μl 41 5% nonfat dry milk Blocking solution + 0.1% Tween 20 1:500 1:8000 Results Results 103 4.1 Functional selectivity of different serotonin 5-HT2AR antagonists in post-mortem human brain 4.1.1 Concentration-effect of different drugs on Gαi1and Gαq/11-protein coupling to 5-HT2AR in post-mortem human PFC Concentration-response curves in presence of increasing concentration of different drugs were used for the functional coupling characterization of 5HT2AR to different Gα-proteins. Under this experimental condition, responses of specific [35S]GTPγS binding to Gαi1-proteins (Figure 4.1.A) to increasing concentrations of MDL-11,939 (10-10–10-4 M) or ketanserin (10-10–10-4 M) were unaltered, suggesting that these drugs act as neutral antagonists for the Gαi1protein-mediated pathway. In contrast, other drugs such as altanserin, pimavanserin, nelotanserin, ritanserin, volinanserin and eplivanserin displayed a concentration-dependent inhibitory response, which points out that these drugs were able to decrease basal constitutive activity. When the canonical Gαq/11-protein pathway of 5-HT2AR was studied (Figure 4.1.B), ketanserin increased [35S]GTPγS binding to Gαq/11-protein, displaying agonist properties. Conversely, altanserin, pimavanserin, nelotanserin and MDL-11,939 presented null effect on Gαq/11-protein pathway. Finally, ritanserin, volinanserin and eplivanserin displayed concentration-response inhibitions of [35S]GTPγS binding to the canonical pathway. Therefore, it might be concluded that only MDL-11,939 acts as neutral antagonist for Gαi1and Gαq/11-protein-coupling of 5-HT2AR in human brain cortex. In contrast, the rest of drugs displayed functional selectivity between 5HT2AR coupling to Gαi1and Gαq/11-proteins. The corresponding Emax/Imax and EC50/IC50 values of concentration-response curves for each drug are shown in Table 4.1. Results 104 Figure 4.1: Concentration-response curves of specific [35S]GTPγS binding to Gαi1and Gαq/11proteins in response to addition of different 5-HT2AR drugs. Concentration-response stimulation or inhibition of [35S]GTPγS binding to Gαi1- (Figure 4.1.A) and Gαq/11- (Figure 4.1.B) proteins by altanserin, pimavanserin, nelotanserin, ritanserin, volinanserin, ketanserin, eplivanserin and MDL-11,939 was performed in human PFC membranes. The 100% dashed line denotes the specific basal [35S]GTPγS binding to each GD-protein (BB). Each point represents the mean±SEM value from independent experiments carried out in duplicate and triplicate. Results 105 The accurate determination of efficacy is essential for ligand bias detection. Analysis of concentration-response curves measures drug efficacy and potency, which are represented by maximal inhibitory or stimulatory responses (Emax/Imax), and the concentration that promotes half-maximal effect (EC50/IC50), respectively. Pimavanserin and nelotanserin showed the highest efficacy to decrease in almost a 30% [35S]GTPγS basal binding to Gαi1-protein (Imax=-27±2% and -25±2%) as shown in Table 4.1. In the same way, nelotanserin and pimavanserin also showed highest potency (IC50=8.4±0.3 nM and 8.2±0.3 nM) (Table 4.1). None of the drugs showed intrinsic positive efficacy on the [35S]GTPγS basal binding to Gαi1-protein. However, ketanserin was able to increase the [35S]GTPγS binding to Gαq/11-protein by a 18%, with a potency of 994 nM (Table 4.1). Eplivanserin decreased the [35S]GTPγS binding to Gαq/11-protein by a 16%, which corresponded to the highest inhibitory efficacy. In contrast, eplivanserin had the lowest potency (IC50=3268 nM). In terms of potency, ritanserin showed the lowest concentration required to reach the half-maximal effect of [35S]GTPγS binding to Gαq/11-proteins (IC50= 15.2 nM) (Table 4.1). Results 106 Table 4.1: Pharmacological parameters of the concentration-response curves of the specific [35S]GTPγS binding to Gαi1and Gαq/11-proteins in response to addition of different 5-HT2AR drugs in human PFC membranes. Data are shown as mean±SEM of n independent experiments carried out in duplicate or triplicate. Emax and Imax represent, in % of basal values, the maximal stimulation and inhibition, respectively, estimated from non-linear fitting of the concentration-response curves. (-)logEC50/IC50 indicate the log of concentration (in nM) that promotes half-maximal stimulatory or inhibitory effect, and IC50/EC50 are the respective antilog values. Gαi1-protein Gαq/11-protein E max / Imax (%) (-)logEC 50 /IC5 0 IC 50 /EC50 (nM) n Emax / Imax (%) ( - )logEC50 /IC 50 IC 50 /EC50 (nM) n Altanserin -19±3 6.7±0.4 205.3 6 / / / 6 Pimavanserin -25±2 8.2±0.3 6.1 5 / / / 8 Nelotanserin -27±2 8.4±0.3 4.3 5 / / / 6 Ritanserin -17±2 7.0±0.3 108.1 5 - 7±2 7.8±0.7 15.2 5 Volinanserin -22±2 6.9±0.2 125.5 6 -10±2 6.3±0.7 530.9 6 Ketanserin / / / 5 18±2 6.0±0.3 994.1 7 Eplivanserin -24±2 7.8±0.2 17.8 7 -16±4 5.5±0.5 3268 6 MDL - 11,939 / / / 5 / / / 5 Results 107 4.1.2 Evaluation of maximal effect of different drugs on Gαi1-, Gαi2-, Gαi3-, Gαo-, and Gαq/11-protein coupling to 5-HT2AR in post-mortem human PFC In order to elucidate the effects of previously evaluated drugs on [35S]GTPγS binding, not only to Gαi1and Gαq/11-proteins but also to other PTX-sensitive Gα-proteins (inhibitory Gαi/o-proteins), a new set of experiments was performed. For these experiments, [35S]GTPγS scintillation proximity assays (SPA) coupled to immunoprecipitation with different selective antibodies against Gαi1-, Gαi2-, Gαi3-, Gαoand Gαq/11-protein subtypes were performed at one single concentration (10 μM). This concentration induces effects very close to maximal efficacy. Altanserin, pimavanserin and eplivanserin induced a statistically relevant inhibition of [35S]GTPγS binding to Gαi1-proteins, while they did not show any effect on other inhibitory Gα-proteins (Table 4.2). Under these experimental conditions, nelotanserin and ritanserin decreased [35S]GTPγS basal binding to Gαi1-, Gαi2-, Gαi3-, and Gαo-proteins (Table 4.2). Volinanserin promoted inhibition of [35S]GTPγS binding to Gαi1and Gαoproteins (Table 4.2). Ketanserin and MDL-11,939 were unable to modify [35S]GTPγS binding to inhibitory Gαi/o proteins (Table 4.2). In general, the maximal inhibitory effects were observed in Gαi1-proteins (Table 4.2). The responses of [35S]GTPγS binding to Gαq/11-proteins, after addition of different drugs, were similar to those previously observed (see 4.1.1). Thus ritanserin, volinanserin and eplivanserin induced an inhibitory effect on [35S]GTPγS binding, whereas ketanserin promoted stimulation (Table 4.2). Results 108 Table 4.2: Stimulatory or inhibitory effect induced by one single concentration (10 μM) of different drugs (altanserin, pimavanserin, nelotanserin, ritanserin, volinanserin, ketanserin, eplivanserin and MDL-11,939) on the specific [35S]GTPγS binding to inhibitory Gαi/oand Gαq/11-proteins in human PFC membranes. Data are mean±SEM of n independent experiments carried out in duplicate or triplicate, and express the percentage values related to basal [35S]GTPγS binding (0%). Data were analyzed using one sample t-test vs basal binding (*p<0.05, **p<0.01, ***p<0.001). Drug Gα i1 Gα i2 Gα i3 Gα o Gαq/11 Altanserin -12±2*** (n=12) -1±3 (n=10) 2±1 (n=11) -4±5 (n=6) 4±2 (n=12) Pimavanserin -20±3*** (n=10) -2±2 (n=10) 3±2 (n=11) -1±3 (n=5) 3±2 (n=13) Nelotanserin -22±3*** (n=9) - 17±4** (n=10) -11±4* (n=8) -6±2* (n=5) 3±3 (n=11) Ritanserin -18±2*** (n=9) -15±4* (n=6) -10±3* (n=18) -15±5* (n=7) -11±3** (n=11) Volinan s erin -16±2*** (n=9) -2±3 (n=9) -3±3 (n=9) -4±1* (n=7) -7±3* (n=10) Ketanserin -1±1 (n=5) 2±4 (n=6) -2±3 (n=7) -1±2 (n=5) 20±3** (n=7) Eplivanserin -19±5* (n=5) -2±4 (n=6) 0±5 (n=7) 5±2 (n=5) -17±3*** (n=9) MDL - 11,939 3±3 (n=7) 0±2 (n=5) -1±4 (n=5) 0±1 (n=5) 0±2 (n=7) Results 109 4.1.3 Involvement of 5-HT2AR in the effect induced by different drugs on [35S]GTPγS binding to Gαi1and Gαq/11-proteins in post-mortem human PFC The described findings in previous paragraphs suggest the involvement of 5HT2AR in the modulation of [35S]GTPγS binding to Gαi1and Gαq/11-proteins because evaluated drugs share activity on 5-HT2AR. In order to confirm the involvement of 5-HT2AR, antagonism assays with selective 5-HT2AR drugs were performed. For this purpose, altanserin, pimavanserin, nelotanserin, ritanserin, volinanserin, ketanserin, eplivanserin and MDL-11,939 were added to SPA assays at 10 μM concentration in presence of ketanserin (10 μM), volinanserin (1 μM) or MDL-11,939 (1 μM). Ketanserin (pKi=8.5), volinanserin (pKi=8.73) and MDL-11,939 (pKi=7.58) were chosen as selective antagonist against 5HT2AR vs 5HT2CR, according to previous reports (Knight et al., 2004; Bonhaus et al., 1995). The selectivity of different drugs for 5-HT2AR was analysed by repeated measures one-way ANOVA, where the drug alone was compared with the drug co-incubated with the antagonist. A further Bonferroni´s post-hoc test would delineate the specific inhibition of different antagonists. Ketanserin, volinanserin and MDL-11,939 blocked the inhibitory effects exerted by altanserin (-13±1%) (F[3,18]=13.28, p<0.0001) and pimavanserin (-15±3%) (F[3,19]=11.80, p=0.0001), on [35S]GTPγS binding to Gαi1-proteins (Tables 4.3 and 4.4) (Figure 4.2.A). Moreover, since volinanserin had unexpectedly demonstrated inhibitory effect on this activity (-12±3%), ketanserin and MDL-11,939 were also tested as potential antagonists on this effect. A full blockage of volinanserin-induced inhibition was observed (F[2,15]=9.81, p=0.0019) (Tables 4.3 and 4.4) (Figure 4.2.A), suggesting partial inverse agonism of this compound on the [35S]GTPγS binding to Gαi1proteins. Results 116 4.1.4 Evaluation of the maximal effect of different drugs on Gαi1and Gαq/11-protein coupling to 5-HT2AR in knock-out 5HT2AR(-/-) and wild-type 5-HT2AR(+/+) mice To further confirm the role of 5-HT2AR in the stimulatory or inhibitory effect exerted on [35S]GTPγS binding to Gαi1and Gαq/11-proteins, SPA experiments were performed in brain tissue of knock-out 5-HT2AR(-/-) and wild-type 5HT2AR(+/+) mice. Brain membranes were incubated with a single concentration (10 μM) of altanserin, pimavanserin, nelotanserin, ritanserin, volinanserin, ketanserin, eplivanserin and MDL-11,939 as well as specific antibodies against Gαi1- (Figure 4.3.A) and Gαq/11-proteins (Figure 4.3.B). There were not differences in basal values on [35S]GTPγS binding to Gαi1and Gαq/11-proteins between 5-HT2AR(+/+) and 5-HT2AR(-/-) mice. Thus, the basal binding values for Gαi1-proteins in wild-type mice were 3824±428 CCPM, and 3971±428 CCPM in knock-out mice (p=0.8115, t=0.24, df=14). Similarly, the basal binding values for Gαq/11-protein were 3160±329 CCPM in wild-type mice, and 3224±224 CCPM in knock-out mice (p=0.8779, t=0.16, df=16). The comparison between both genotypes in response to different drugs was performed by a repeated-measures two-way ANOVA, where conditions were drug and genotype. This analysis was performed twice, corresponding to evaluation of [35S]GTPγS binding responses for each Gα-protein. Since the effects induced by drugs are variable, it was expected to find a significant interaction drug x genotype. A further Bonferroni’s post-hoc test would delineate the specific drugs displaying differential effects between wild-type and knock-out animals. In agreement with the results in human brain samples, when wild-type mice brain cortex membranes where incubated with altanserin (-10±3%), pimavanserin (-13±2%), nelotanserin (-12±2%), ritanserin (-12±3%), volinanserin (-11±2%) and eplivanserin (-12±3%), all of them exerted an Results 117 inhibitory effect on [35S]GTPγS binding to Gαi1-proteins (Table 4.5) (Figure 4.3.A). Furthermore, ketanserin and MDL-11,939 did not change basal [35S]GTPγS binding (Table 4.5) (Figure 4.3.A).The significant inhibition exerted on [35S]GTPγS binding to Gαi1-proteins by altanserin, pimavanserin and volinanserin was absent in knock-out mice membranes (Table 4.5) (Figure 4.3.A) These results confirm involvement of 5-HT2AR in the inhibition of [35S]GTPγS binding to Gαi1-proteins. On the other hand, the inhibition exerted by nelotanserin, ritanserin and eplivanserin was also observed in knock-out mice, i.e. absence of 5-HT2AR (Table 4.5) (Figure 4.3.A). This results further support that inhibitory effects on [35S]GTPγS binding to Gαi1proteins exerted by these three drugs are not mediated by 5-HT2AR. Next, [35S]GTPγS binding to Gαq/11-proteins was studied in wild-type 5HT2AR(+/+) mice. In a similar way to results in human frontal cortex, ritanserin (- 8±1%), volinanserin (-14±2%) and eplivanserin (-10±1%) decreased [35S]GTPγS binding to Gαq/11-proteins (Table 4.5) (Figure 4.3.B). Furthermore, altanserin, pimavanserin, nelotanserin and MDL-11,939 did not modify the basal [35S]GTPγS binding of this canonical pathway (Table 4.5) (Figure 4.3.B). Moreover, ketanserin induced a stimulatory (18±4%) response in wild-type animals, as described in human brain (Table 4.5) (Figure 4.3.B). When these drugs were studied in knock-out mice, their effect was null for volinanserin and ketanserin, indicating that their effects were mediated by 5HT2AR. In contrast, ritanserin and eplivanserin decreased [35S]GTPγS binding to Gαq/11-proteins in knock-out mice as in wild-type mice, suggesting the lack of involvement of 5-HT2AR on these effects (Table 4.5) (Figure 4.3.B). Results 118 Figure 4.3: Stimulatory or inhibitory effect induced by one single concentration (10 μM) of different drugs (altanserin, pimavanserin, nelotanserin, ritanserin, volinanserin, ketanserin, eplivanserin and MDL-11,939) on the specific [35S]GTPγS binding to Gαi1- (Figure 4.3.A) and Gαq/11- (Figure 4.3.B) proteins in brain cortex membranes of wild-type 5-HT2AR(+/+) and knockout 5-HT2AR(-/-) mice. Bars are mean±SEM of independent experiments carried out in duplicate or triplicate, and express the percentage values related to basal [35S]GTPγS binding (0%). Data were analysed using one sample t-test vs basal binding (*p<0.05, **p<0.01, ***p<0.001) and two-way ANOVA followed by Bonferroni´s post-hoc test for drug x genotype effects (#p<0.05, ###p<0.001). Results 119 Table 4.5: Numerical values of results expressed in Figure 4.3. Stimulatory or inhibitory effect induced by one single concentration (10 μM) of different drugs (altanserin, pimavanserin, nelotanserin, ritanserin, volinanserin, ketanserin, eplivanserin and MDL-11,939) on the specific [35S]GTPγS binding to Gαi/oand Gαq/11-proteins in brain cortex membranes of wildtype 5-HT2AR(+/+) and knock-out 5-HT2AR(-/-) mice. Values are mean±SEM of n independent experiments carried out in duplicate or triplicate and express the percentage values related to basal [35S]GTPγS binding (0%). Data were analysed using one sample t-test vs basal binding (*p<0.05, **p<0.01, ***p<0.001). Gα i1 -protein Gα q/11 -protein 5-HT 2A R(+/+) 5-HT 2A R(-/-) 5-HT 2A R(+/+) 5-HT 2A R(-/-) Altanserin -10±3* (n=5) 0±2 (n=6) -1±3 (n=6) -1±2 (n=7) Pimavanserin -13±2** (n=5) -2±2 (n=6) 2±2 (n=6) -4±2 (n=7) Nelotanserin -12±2** (n=5) -12±3* (n=6) -1±3 (n=5) -2±2 (n=8) Ritanserin -12±3* (n=5) -11±3** (n=6) -8±1*** (n=8) -14±3** (n=7) Volinanserin -11±2** (n=5) -1±2 (n=5) -14±2*** (n=8) -1±5 (n=8) Ketanserin -2±1 (n=5) -2±3 (n=5) 18±4* (n=5) 2±1 (n=7) Eplivanserin -11±3* (n=5) -10±2** (n=6) -10±2** (n=4) -12±3** (n=5) MDL-11,939 2±2 (n=5) 0±4 (n=6) -2±5 (n=6) 0±4 (n=6) Results 120 Table 4.6: Summary of the repeated measures two-way ANOVA analysis of the effects of different drugs on wild-type 5-HT2AR(+/+) and knock-out 5-HT2AR(-/-) mice. The results of a drug x genotype interaction suggest that the response to the drugs is variable between both mice genotypes. A further post-hoc analysis was performed to discriminate differences between wild-type and knock-out animals for each drug. Results are shown in Figures 4.3.A and 4.3.B. Significant p values are shown in bold. Gα i1 -protein Gα q/11 -protein DFn DFd F P DFn DFd F p Interaction 7 70 2.84 0.0116 7 105 7.02 <0.0001 Drug 7 70 8.25 <0.0001 7 105 20.24 <0.0001 Genotype 1 70 10.44 0.0090 1 105 2.23 0.1558 Results 121 4.1.5 Summary 5-HT2AR expressed in human PFC displays functional coupling to the canonical Gαq/11-proteins but also to other cell pathways through inhibitory Gαi/o-proteins. Among them, Gαi1-protein seems to be the preferential subtype involved in this alternative signaling pathway. The functional coupling of 5-HT2AR in human PFC shows constitutive activity because [35S]GTPγS binding to different Gα-protein subunits can be inhibited by different 5-HT2AR drugs, previously assumed to be neutral antagonist. Therefore, these compounds should be considered potential inverse agonists. The blockage of inverse agonism by selective 5-HT2AR neutral antagonists, and the absence of this pharmacological feature in 5-HT2AR(-/-) mice allow us to identify them as selective 5-HT2AR inverse agonists. Functional activity of the different drugs as inverse agonists of 5-HT2AR may be different between Gαq/11and Gαi1-protein pathways. Therefore, a biased inverse agonism is feasible for this receptor in human brain. Among the different compounds tested, pimavanserin displays higher and more selective inverse efficacy in 5-HT2AR coupling to Gαi1-proteins, lacking of activity on Gαq/11-protein activation. On the other hand, volinanserin could be considered a selective 5-HT2AR inverse agonist with inverse efficacy on the coupling to both Gαq/11and Gαi1-proteins. Therefore, these two drugs were selected as suitable tools for further studies of 5-HT2AR constitutive activity in pathological conditions. Results 122 4.2 Evaluation of the functional coupling of 5-HT2AR to Gαprotein subtypes in PFC of schizophrenia subjects, nonschizophrenia suicide subjects and matched controls by antibody-capture [35S]GTPγS scintillation proximity assay (SPA) 4.2.1 Basal [35S]GTPγS binding to Gαi1and Gαq/11proteins in post-mortem PFC of schizophrenia subjects, nonschizophrenia suicide subjects and matched controls Basal [35S]GTPγS binding to Gαi1-proteins in cortical membranes was not statistically different between schizophrenia (162±7 fmol/mg prot), nonschizophrenia suicide (175±9 fmol/mg prot) and control (159±5 fmol/mg prot) groups (F[2,55]=1.13, p=0.35) (Figure 4.4.A). Similarly, basal [35S]GTPγS binding to Gαq/11-proteins was not statistically different between schizophrenia (191±9 fmol/mg prot), non-schizophrenia suicide (198±9 fmol/mg prot) and control subjects (210±11 fmol/mg prot) (F[2,56]=0.95, p=0.39) (Figure 4.4.B). Results 123 Figure 4.4: Individual values of basal [35S]GTPγS binding to Gαi1-(A) and Gαq/11-(B) proteins in PFC membranes of schizophrenia (n=23) (red square), non-schizophrenia suicide (n=13) (blue triangle) and control subjects (n=23) (black circle). The lines represents mean±SEM values of each group. In order to evaluate the influence of variables such as age at death, sex, PMD and storage time on basal [35S]GTPγS binding to both Gα-proteins, linear correlation analyses were performed. Any of these potential variables showed correlation with [35S]GTPγS basal binding values. The absence of relationship was also observed when considered separately those three population groups (Table 4.7). Results 124 Table 4.7: Effect of age (years), PMD (hours) and storage time (months) on basal [35S]GTPγS binding to Gαi1and Gαq/11-proteins. Gαi1-protein basal binding (fmol/mg protein) Schizophrenia Nonschizophrenia suicide Control r p n r p n r p n Age (years) -0.2811 0.1939 23 -0.1690 0.5995 13 0.0150 0.9458 23 PMD (hours) 0.1012 0.6459 23 0.2500 0.4332 13 -0.0716 0.7452 23 Storage time (months) -0.0175 0.9368 23 -0.0797 0.8054 13 0.0262 0.9056 23 Gαq/11-protein basal binding (fmol/mg protein) Schizophrenia Nonschizophrenia suicide Control r p n r p n r p n Age (years) -0.2783 0.1986 23 -0.3972 0.1790 13 0.1534 0.4847 23 PMD (hours) 0.1816 0.4070 23 -0.1876 0.5403 13 -0.0565 0.7978 23 Storage time (months) -0.0823 0.7071 23 0.1802 0.5558 13 0.0778 0.7245 23 Results 125 4.2.2 Modulation of [35S]GTPγS binding to Gαi1and Gαq/11proteins by the 5-HT2AR inverse agonist pimavanserin in postmortem PFC of schizophrenia subjects, non-schizophrenia suicide subjects and matched controls Pimavanserin (10-10-10-6 M) displayed concentration-dependent inhibition curves in schizophrenia, non-schizophrenia suicide and control groups (Figure 4.5). No differences in potency of pimavanserin were observed between groups (schizophrenia: -logIC50=7.94±0.1; non-schizophrenia suicide: - logIC50=8.15±0.0; control: -logIC50=8.01±0.1) (F[2,56]=2.26, p=0.0871) (Table 4.8). The inhibitory effect of pimavanserin was higher in schizophrenia (Imax=-20±1%) than in control (Imax=-14±1%) and non-schizophrenia suicide (Imax=-14±1%) groups (F[2,56]=9.19, p=0.0004) (Table 4.8). Figure 4.5: Concentration-response curves of specific [35S]GTPγS binding to Gαi1-proteins in response to pimavanserin in human PFC membranes. The 100% dashed line denotes specific basal [35S]GTPγS binding to Gαi1-proteins (BB). Each point represents the mean±SEM value from independent experiments. See coanalysis results in the text. gure 4 5 : Concentration response curves of specific [ 35 S]GTPγS binding to Gα i1 prot ei ns i n Results 132 4.2.3 Modulation of the [35S]GTPγS binding to Gαi1and Gαq/11proteins by the 5-HT2AR inverse agonist volinanserin in postmortem PFC of schizophrenia subjects, non-schizophrenia suicide subjects and matched controls Given the absence of [35S]GTPγS binding modulation by pimavanserin, the status of 5-HT2AR coupling to Gαq/11-proteins could not be quantified by using this drug. In order to evaluate whether the enhanced constitutive activity of 5HT2AR coupling to Gαi1-proteins was selective for this pathway, or Gαq/11protein canonical pathway was also altered, another 5-HT2AR inverse agonist should be tested. For this purpose, and according to previous findings, volinanserin was chosen as the most suitable pharmacological tool. Volinanserin (10-9-10-5 M) displayed a concentration-dependent inhibition curves of [35S]GTPγS binding to Gαi1-proteins in schizophrenia, nonschizophrenia suicide and control groups (Figure 4.9). No differences in potency of volinanserin were observed between groups (schizophrenia: - logIC50=6.45±0.1; non-schizophrenia suicide: -logIC50=6.53±0.1; control: - logIC50=6.54±0.1) (F[2,56]=0.5830, p=0.5616) (Table 4.10). The inhibitory effect of volinanserin was higher in schizophrenia subjects (Imax=-18±1%) than in control (Imax=-13±1%), as well as in non-schizophrenia suicide subjects (Imax=-14±1%) (F[2,56]=9.76, p=0.0002) (Table 4.10). Results 133 Figure 4.9: Concentration-response curves of specific [35S]GTPγS binding to Gαi1-proteins in response to volinanserin in human PFC membranes. The 100% dashed line denotes specific basal [35S]GTPγS binding to Gαi1-proteins (BB). Each point represents mean±SEM value from independent experiments. Table 4.10: Pharmacological parameters of volinanserin concentration-response curves of [35S]GTPγS binding to Gαi1-proteins in PFC membranes of schizophrenia, non-schizophrenia suicide and matched control subjects. IC50 values were obtained as antilog of logIC50. Imax represents the experimental maximal inhibitory effect induced by volinanserin, and is expressed as percentage of respective basal [35S]GTPγS binding value. Values are mean±SEM of n independent subjects. Data were analysed using one-way ANOVA followed by Bonferroni’s post-hoc test (**p<0.01 vs control and non-schizophrenia group). Group Basal (fmol/ mg protein) IC50 (nM) Imax (%) n Schizophrenia 162±7 456±83 -18±1** 23 Non-schizophrenia suicide 175±9 359±67 -14±1 13 Control 159±5 341±42 -13±1 23 Results 134 An analysis of covariance (ANCOVA) was further performed to evaluate the potential influence of age at death, PMD and storage time. Sex differences were also evaluated. Any of these variables modified the results and, consistent with previous findings, differences between groups in Imax values were maintained (F[2,55]=8.13, p=0.0008). A complementary statistical comparison between groups was conducted by non-linear curve fitting coanalysis of all individual experiments. The coanalysis of concentration-response curves to volinanserin demonstrated different patterns of schizophrenia respect to control (F[3,178]=14.14, p<0.0001) and non-schizophrenia suicide groups (F[3,138]=8.09, p<0.0001) (Figure 4.9). Once statistical differences between curves were obtained, further contrasts were performed to detect whether differences were attributable to changes in Imax and/or IC50 values between groups. Thus, the curve fitting coanalysis confirmed that schizophrenia subjects showed a higher inhibitory effect of volinanserin (Imax=-18%, 95%Cl 19% to 17%) than control (Imax=-13%, 95%Cl 14% to 12%) (F[1,178]=27.77, p<0.0001) and non-schizophrenia suicide subjects (Imax=-14%, 95%Cl 16% to 13%) (F[1,138]=11.85, p<0.0001). No differences were obtained for IC50 values between schizophrenia (IC50=355. nM, 95%Cl 250.7 nM to 502.6 nM), non-schizophrenia suicide (IC50=302.2 nM, 95%Cl 186.3 nM to 477.4 nM) and control groups (IC50=313.9 nM, 95%Cl 208.8 nM to 464.6 nM). The selectivity of inhibitory effect exerted by volinanserin on [35S]GTPγS binding to Gαi1-proteins was tested by using 5-HT2AR neutral antagonist MDL11,939 at 10 μM. In schizophrenia subjects, volinanserin maximal inhibitory effect on [35S]GTPγS binding (-17±1%) was blocked by the antagonist (p<0.0001, t=12.67, df=44) (Figure 4.10). Similarly, in non-schizophrenia suicide group, the maximal inhibition promoted by volinanserin on [35S]GTPγS binding to Gαi1-proteins (-14±1%) was completely blocked by MDL-11,939 (p<0.0001, t=8.50, df=24) (Figure 4.10). As previously described (See 4.1.3), Results 135 in control group, the maximal inhibition of [35S]GTPγS binding to Gαi1-proteins induced by volinanserin (-13±1%) was blocked by MDL-11,939 (p<0.0001, t=12.23, df=44) (Figure 4.10). Figure 4.10: Antagonism of the inhibitory effect induced by a maximal concentration of volinanserin (10 μM) on specific [35S]GTPγS binding to Gαi1-proteins in PFC membranes of schizophrenia, non-schizophrenia suicide and control subjects. Volinanserin was co-incubated with the selective 5-HT2AR antagonist MDL-11,939 (10 μM). Basal values of specific [35S]GTPγS binding to Gαi1-proteins are expressed as 0%, and inhibitory effects on respective basal are shown as percentage of basal values. Each point indicates individual values. Bars represent mean±SEM of percentage values of independent experiments. Data were analysed using one sample t-test vs basal binding (****p<0.0001) and two sample t-test to compare volinanserin effects in absence or presence of MDL-11,939. (####p<0.0001). The modulation of [35S]GTPγS binding to Gαq/11-proteins by the 5-HT2AR selective drug volinanserin (10-9-10-5 M) was also tested in schizophrenia, nonschizophrenia suicide and control groups. As expected, volinanserin displayed concentration-dependent inhibition curves in schizophrenia, nonschizophrenia suicide and control groups (Figure 4.11). No differences were observed in potency between groups (schizophrenia: -logIC50=6.53±0.1; non- Results 136 schizophrenia suicide: -logIC50=6.57±0.1; control: -logIC50=6.52±0.1 (F[2,56]=1.53, p=0.6617) (Table 4.11). Likewise, the inhibitory effect of volinanserin was not different between groups (schizophrenia: Imax=-16±1%; non-schizophrenia suicide: Imax=-16±1%; control: Imax=-15±1%) (F[2,56]=0.6861, p=0.5046). Figure 4.11: Concentration-response curves of specific [35S]GTPγS binding to Gαq/11-proteins in response to volinanserin in human PFC membranes. The 100% dashed line denotes specific basal [35S]GTPγS binding to Gαq/11-proteins (BB). Each point represents mean±SEM value from independent experiments. Results 137 Table 4.11: Pharmacological parameters of volinanserin concentration-response curves of [35S]GTPγS binding to Gαq/11-proteins in PFC membranes of schizophrenia, nonschizophrenia suicide and matched control subjects. IC50 values were obtained as antilog of logIC50. Imax represents the experimental maximal stimulatory or inhibitory effect induced by volinanserin, and is expressed as percentage of respective basal [35S]GTPγS binding value. Values are mean±SEM of n independent subjects. Data were analysed using one-way ANOVA. A complementary statistical comparison between groups was conducted by non-linear curve fitting coanalysis of all individual experiments. The coanalysis of concentration-response curves to volinanserin demonstrated no different patterns between schizophrenia and control groups (F[3,178]=0.32, p=0.8138) (Figure 4.11). In the same way, there was no difference between schizophrenia and non-schizophrenia suicide groups (F[3,138]=0.77, p=0.5103) (Figure 4.11). Finally, no changes were found between control and non-schizophrenia suicide group (F[3,138]=2.17, p=0.0948) (Figure 4.11). Group Basal (fmol/ mg protein) IC50 (nM) Imax (%) n Schizophrenia 191±9 341±54 -15±1 23 Non-schizophrenia suicide 198±9 310±47 -16±1 13 Control 210±11 368±52 -15±1 23 Results 138 The selectivity of the inhibitory effect exerted by volinanserin on [35S]GTPγS binding to Gαq/11-proteins was tested by using the 5-HT2AR neutral antagonist MDL-11,939 at 10 μM. In schizophrenia subjects, volinanserin maximal inhibitory effect on [35S]GTPγS binding (-15±1%) was also blocked by antagonist (p<0.0001, t=12.49, df=44) (Figure 4.12). In non-schizophrenia suicide group, the maximal inhibition promoted by volinanserin on [35S]GTPγS binding to Gαq/11-proteins (-15±1%), was also completely blocked by MDL11,939 (p<0.0001, t=10.53, df=24) (Figure 4.12). Additionally, in control group, the maximal inhibition of [35S]GTPγS binding induced by volinanserin (-14±1%) was also blocked by MDL-11,939 (p<0.0001, t=11.73, df=44) (Figure 4.12). Figure 4.12: Antagonism of the inhibitory effect induced by a maximal concentration of volinanserin (10 μM) on specific [35S]GTPγS binding to Gαq/11-proteins in PFC membranes of schizophrenia, non-schizophrenia suicide and control subjects. Volinanserin was co-incubated with the selective 5-HT2AR antagonist MDL-11,939 (10 μM). Basal values of specific [35S]GTPγS binding to [35S]GTPγS are expressed as 0%, and inhibitory effects on the respective basal are shown as percentage of basal values. Each point indicates individual values. Bars represent mean±SEM of percentage values of independent experiments. Data were analysed using one sample t-test vs basal binding (****p<0.0001) and two sample t-test to compare volinanserin effects in absence or presence of MDL-11,939 (####p<0.0001). Results 139 4.2.4 Modulation of [35S]GTPγS binding to Gαi1and Gαq/11proteins by 5-HT2AR agonist (±)DOI in post-mortem PFC of schizophrenia subjects, non-schizophrenia suicide subjects and matched controls In order to confirm previous results (Garcia-Bea et al., 2019), the effect of 5HT2AR selective agonist (±)DOI was evaluated in schizophrenia, nonschizophrenia suicide and control groups at one single concentration (10 μM), which corresponds to a submaximal concentration (Figure 4.13). As expected, (±)DOI increased [35S]GTPγS binding to Gαi1-proteins in schizophrenia, nonschizophrenia suicide and control groups (Figure 4.13). The stimulatory effect of (±)DOI was higher in schizophrenia than in control and non-schizophrenia suicide groups (F[1,56]=7.45, p=0.0014) (Table 4.12) (Figure 4.13). Results 140 Figure 4.13: (±)DOI-induced effect on the specific [35S]GTPγS binding to Gαi1-proteins in human PFC membranes. (±)DOI was added at 10 μM concentration. Basal values of specific [35S]GTPγS binding to Gαi1-proteins are expressed as 0%, and stimulatory effects on the respective basal are shown as percentage of basal values. Each point indicates individual values. Bars represent mean±SEM of percentage values of independent experiments. Data were analysed using one sample t-test vs basal binding (****p<0.0001) and one-way ANOVA followed by Bonferroni’s post-hoc test (##p<0.01 vs control and non-schizophrenia group). Table 4.12: Pharmacological parameters of (±)DOI induced-effect at one single concentration (10 μM) on [35S]GTPγS binding to Gαi1-proteins in PFC membranes of schizophrenia, nonschizophrenia suicide and matched control subjects. Emax represents the maximal stimulatory effect induced by (±)DOI, and is expressed as percentage of respective basal [35S]GTPγS binding value. Values are mean±SEM of n independent subjects. Data were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test (**p<0.01 vs control and nonschizophrenia group). Group Basal (fmol/ mg protein) Emax (%) n Schizophrenia 162±7 15±1** 23 Non-schizophrenia suicide 175±9 9±1 13 Control 159±5 10±1 23 Results 141 An analysis of covariance (ANCOVA) was further performed to evaluate the potential influence of age at death, PMD and storage time. Sex differences were also evaluated. Any of these variables modified the results and, consistent with previous findings, the differences between groups in Imax values were maintained (F[2,52]=3.13, p<0.05). The selectivity of stimulatory effect exerted by (±)DOI on [35S]GTPγS binding to Gαi1-proteins was tested by using 5-HT2AR neutral antagonist MDL-11,939 at 10 μM. In schizophrenia subjects, (±)DOI-induced maximal stimulatory effect on [35S]GTPγS binding to Gαi1-proteins (15±1%) was blocked by the antagonist (p<0.0001, t=7.93, df=44) (Figure 4.14). In non-schizophrenia suicide group, the maximal stimulation promoted by (±) DOI on [35S]GTPγS binding to Gαi1-proteins (9±1%) was also completely blocked by MDL-11,939 (p<0.0003, t=4.17, df=24) (Figure 4.14). In control group, the maximal stimulation of [35S]GTPγS binding induced by (±)DOI (10±1%) was also antagonized by MDL-11,939 (p<0.0001, t=5.86, df=44) (Figure 4.14). References 244 chromosomal localisation of the gene. European Journal of Biochemistry, 224(2), 489–495. 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Frontiers in Cell and Developmental Biology, 9, 611443. 5-HT2A hartzailearen aktibitate konstitutiboa eta farmakoen hautakortasun funtzionala: eskizofrenikoen post-mortem garunean egindako lana Itziar Muneta Arrate LABURDUREN ZERRENDA [35S]GTPγS 35 sufrearekin markaturik dagen guanosina-5´-O-(γ-tio)- trifosfatoa 5-HT 5-Hidroxitriptamina (serotonina) 5-HT2AR Serotonina 2A hartzailea 5-HT2AR(-/-) Knock-out 5-HT2AR(+/+) Wild-Type 7TM 7 domeinuko mintz zeharreko hartzailea AA Azido arakidonikoa AC Adenilato ziklasa BB Finkapen basala Bolinanserin MDL100907 BRET Biolumineszentzia-erresonantzia bidezko energia-transferentzia Ca2+ Kaltzio ioia CaMKII Kaltzio/kalmodulinaren mendeko kinasa II cAMP Adenosina monofosfato ziklikoa D2R Dopamina 2 hartzailea DA Dopamina DAG Diazilglizerola DLPFC Aurre-garunazal dortsolaterala (±)DOI 2,5-dimetoxi-4-iodoamfetamina DSM Gaixotasun mentalen diagnosia eta estatistika eskuliburua EC50 Estimulazio efektu maximoa lortzeko % 50 kontzentrazioa Emax Estimulazio efektu maximoa ERK Zelulaz kanpoko seinaleek erregulaturiko kinasak FDA Drogen eta elikagaien administrazioak GABA Azido-aminobutirikoa GPCR G-proteinei loturiko hartzaileak GDP Guanosina difosfatoa GRK G-proteinei lotutako kinasa GTP Guanosine trifosfatoa GTPγS 5'-O-[gamma-tio]trifosfatoa GWAS Genoma-osoaren asoziazio-azterketa IC50 Inhibizio efektu maximoa lortzeko % 50 kontzentrazioa Imax Inhibizio efektu maximoa IP3 Inositol 1,4,5-trifosfatoa LSD Azido lisergikoaren D-dietilamida MAPK Aktibatutako mitogenoen proteina kinasa NBS Finkapen ez espezifikoa NMDA N-Metil-D-aspartatoa NSZ Nerbio sistema zentrala PCP Fentziklidina PET Positroien igorpen bidezko tomografia PIP2 Fosfatidilinositol 4,5-bifosfatoa PKC Proteina kinasa C PLA2 A2 fosfolipasa PLC C fosfolipasa PMD Post-mortem atzerapena PSD-95 95 dentsitate proteina postsinaptikoa PTX Bordetella pertussis toxina RSK-2 S6 kinasa erribosomala SNP nukleotido bakarreko polimorfismoa SPA Immunoprezipitazioarekin akoplatutako [35S]GTPγS finkapen teknika Sarrera Sarrera 276 Hipotesi dopaminergikoaren arabera, sintoma positiboak (haluzinazioak, eldarnioak) garuneko eremu mesolimbiko-estriatalean transmisio dopaminergikoaren hiperaktibitatearen ondorio izango lirateke. Hala ere, sintoma negatiboak eta kognitiboak antipsikotikoekiko erresistenteak dira. Hori dela eta, hipotesi dopaminergikoa birformulatu egin zen, hipodopaminergia kortikala proposatuz, eta hori sintoma negatibo eta kognitiboekin erlazionatuz (Davis et al., 1991; Howes eta Kapur 2009; McCutcheon et al., 2020). Dopamina hartzaileak G-proteinei lotutako hartzaileak (GPCR) dira. Bi familia nagusitan sailkatu daitezke: D1 dopamina-hartzaileen familia, D1 eta D5 dopamina-hartzaileak barne hartzen dituena (D1R, D5R), eta D2 hartzaileenfamilia, D2, D3 eta D4 dopamina hartzaileak barne hartzen dituena (D2R, D3R, D3R). D2Ren blokeoa antipsikotikoen ekintza mekanismo nagusia da, gehienbat antipsikotiko tipikoena. Hori horrela, hartzaile dopaminergikoak funtsezko zeregina dutela proposatu da eskizofrenian. In vivo eginiko neuroirudi azterketak, positroien igorpen bidezko tomografia (PET) eta fotoi bakarraren igorpen bidezko tomografia konputarizatua (SPECT) erabiliaz, besteak beste, eskizofrenia duten pazienteen D2R eta D1R hartzaileen egoera ebaluatzeko erabili dira. Hasierako azterketek, emaitza kontrajarriak aurkitu zituzten; batzuek D2Ren igoera adieraziz, eta beste batzuek, berriz, kontrolekin alderik ez zutela (Howes et al., 2012, Cumming et al., 2021). D2Ren dentsitate altua, erregulazio mekanismoak direla eta, tratamendu antipsikotiko kronikoaren ondorio zela iradoki zen. Aldiz, farmakorik jaso ez zuten paziente eskizofrenikoek ez zituzten D2Ren igoerak aurkeztu, hartzaileen dentsitatearen igoera eta tratamendua erlazionatzen zituena (Seeman, 2013). Aurreko emaitzak indartu egin ziren in vitro postmortem giza garunean D2Ren handipena azaldu ondoren (Seeman et al., 1984, Zakzanis eta Hansen, 1998). Gainera, duela gutxi argitaratutako azterlan baten arabera, post-mortem garun eskizofrenikoetan ez dago D2Ren aktibitatearen handipena (Eguskiza et al., 2021). D2R-rekin bezala, in vivo Sarrera 277 azterketek eskizofrenikoen aurre-garunazalean D1Rren dentsitateari buruzko emaitza kontrajarriak aurkitu zituzten, aurretik tratamendurik gabeko pazienteetan (Cumming et al., 2021). Azkenengo ikerketak, berriz, D1R eta D2R postsinaptikoen egoeran baino neurotransmisio dopaminergiko parasinpatikoa aztertzera zuzendu dira. Hala, DA sintesi presinaptikoa (Howes et al., 2012; Fusar-Poli eta Meyer-Lindenberg, 2013), DA kontzentrazio sinaptikoa (Abi-Dargham et al., 2000; Caravaggio et al., 2015) eta anfetaminak eragindako DA askapena (Howes et al. 2012; Laurelle, 1998) handiagoa frogatu da paziente eskizofrenikoen estriatumean. Bestalde, eskizofrenia duten pazienteen aurre-garunazalean anfetaminak eragindako DAren askapenaren murrizketa ere frogatu da, eta horrek hipodomapinergia kortikal presinaptikoa dagoela iradokitzen du aurre-garunazalean (Slifstein et al., 2015). Eskizofreniaren hipotesi glutamatergikoa Glutamatoa garuneko neurotransmisore kitzikatzaile nagusia da, eta hartzaile ionotropiko eta metabotropiko hartzaileekin elkarrreragiten du. Batetik, hartzaile ionotropikoen taldeak NMDA, kainatoa eta α-amino-3-hidroxi-5-metilisoxazol-4-propionato (AMPA) hartzaile azpimotak biltzen ditu. Bestetik, glutamatoaren hartzaile metabotropikoak (mGluR) G-proteinaren bidezko seinalearen transdukzioa aktibatzen dute, eta I (mGlu1, mGlu5), II (mGlu2, mGlu3) eta III (mGlu6, mGlu4, mGlu7, mGlu8) taldeetan banatzen dira (Naka, 1992; Niswender eta Conlun, 2010; Muguruza et al., 2016). Ondo ezarrita dago NMDA hartzaileen antagonisten administrazioek, hala nola fentziklidina (PCP) edo ketamina, psikosiaren antzeko egoerak eta defizit kognitiboak eragin ditzaketela gizaki osasuntsuengan. Ketamina eta PCPa NMDA hartzaileen antagonista ez-lehiakorrak dira (Thomson et al., 1985). Aurkikuntza horietan oinarrituta, NMDA hartzaileetan izaten den desorekak Sarrera 278 eskizofreniarekin erlazioa izan dezakeela uste da (Javitt eta Zukin, 1991; Stone et al., 2008). Bitxia bada ere, anfetamina edo beste agonista dopaminergiko bat, NMDA hartzaileen antagonistekin batera administratzean sintoma kognitibo eta negatiboak sortzen ditu, eskizofreniaren fisiopatologia imitatuz (Krystal et al., 2005). Hori dela eta, NMDA hartzaileen antagonista ez lehiakorren administrazioa animalia eredu gisa erabiltzen da eskizofrenia aztertzea helburu duten ikerlanetan. Horrekin lotuta, hainbat ikerketatan paziente eskizofrenikoen glutamato mailak neurtu dituzte, sintoma mota desberdinekin erlazionatzeko helburuarekin. Horrela, sistema glutamatergikoaren desorekak eskizofreniaren disfuntzio kognitiboekin erlazionatu dira (Moghaddam eta Javitt, 2012). Izan ere, aurretik azaldutako hiperdopaminergia estriatala eta hipodopaminergia kortikala hipotesi glutamatergikoarekin erlazionatu dira. Lehenengo, NMDA hartzaileen hipoaktibitatea edo inhibizioa deskribatu da eremu dopaminergiko mesolinbikoetan, azido-aminobutirikoa (GABA) askatzen duten interneuronetan. Horrek, egoera toniko inhibitzaileari kalte egingo lioke, eta, ondorioz, dopaminaren sintesi eta askapen handiagoa ekarriko luke, sintoma psikotikoak eraginez. Eta alderantziz, sintoma negatiboak NDMA hartzaileen hipofuntzioak eragindako hipoaktibitate dopaminergiko kortikalarekin erlazionatu dira (Javiit, 2010). Eskizofreniaren hipotesi serotonergikoa Eskizofreniaren hipotesi serotonergikoa azido lisergikoaren D-dietilamida (LSD) droga haluzinogenoaren eta 5-HTren (Freedman 1961) arteko interakzioei buruzko lehen ikerketetatik sortu zen. LSDak eta bestelako droga haluzinogenoek eskizofreniaren sintoma positiboen antzerakoak diren buru nahasmenak eragiten dituzte, serotonina hartzaileak aktibatuz. Gainera, droga psikodelikoen egitura kimikoa 5-HT neurotransmisorearen antzerakoa da. 5- Sarrera 279 HT hartzaile azpimota guztien artean, ebidentzia ugarik adierazten dute haluzinogenoek serotonina 2A hartzaile azpimotarekin (5-HT2AR) lotzen direla, beren eraginak sortzeko (González-Maeso et al., 2007; Geyer eta Vollenweider 2008; González-Maeso eta Sealfon, 2009; Madsen et al., 2019). Bestalde, farmako antipsikotiko atipikoek 5-HT2ARekiko afinitate handia erakusten dute, eskizofrenian gehien aztertu den itu farmakologikoa bilakatuz (Meltzer et al., 1989). 1.4 atalean 5-HT2ARen fisiologia eta eskizofrenian duten inplikazioaren berrikuspen zehatzagoa aurki daiteke. 1.1.4 Garuneko alterazio morfologikoak Aurre-garunazala eskizofreniaren sintoma positibo eta kognitiboekin erlazionatu da (Kolk eta Rakic, 2022). Izan ere, garuneko eskualde horretan eman daitezkeen lesioak, animalietan eta gizakietan, epe luzerako ondorioak eragin ditzakete; hala nola lan-memoriaren alterazioa, hautapen inpultso murriztua, ingurumen-estimulu handiagoak sumatzea eta jokabidemurrizketak. Beraz, neurri handi batean aurre-garunazaleko anomaliak eskizofreniarekin eta beste gaixotasun psikiatriko batzuekin lotu dira, sintoma amankomunak dituzten heinean (Xu et al., 2019). Ondo deskribatuta dago eskizofrenia pairatzen duten pazienteen alboko bentrikuluaren %25eko zabaltzea ematen dela, garunaren bolumen osoaren %2ko murrizketarekin (Johnstone et al., 1976; Haijma et al., 2013). Bentrikuluen tamaina gutxika handitzen da gaixotasunaren hasieratik, garunaren materia grisa murriztu egiten den heinean (Erp et al., 2016). Eskizofrenia duten pazienteen garunen post-mortem azterketa morfologikoek desberdintasunak erakutsi dituzte banaketa zelularrean; ziurrenik garunaren garapen goiztiarrean emandako migrazio neuronalaren alterazioekin, zelula piramidalen galerarekin, egitura zelular deformatuarekin eta GABA interneuronen kopuruaren murrizketarekin lotuak (Schmidt eta Mirnics, 2015). Sarrera 280 1.2 G-proteinei loturiko hartzaileak (GPCR) 1.2.1 Ezaugarri orokorrak GPCR edo 7 domeinuko mintz zeharreko hartzailea (7TM), gorputzeko mintz hartzaile familia azpitalde nagusia osatzen dute. Hartzaileak ligando mota desberdinak, hala nola ioiak, molekula txikiak eta peptidoak finkatzeko edo lotzeko ahalmena dute, zelulaz kanpoko seinaleak zelularen barnera transmititzeko (Alexander et al., 2019). GPCRek garrantzi biomediko handia dute, hainbat jarduera fisiologiko zein patologiatan parte hartzen baitute. Gainera, farmakoen ekintza mekanismoa eragiteko itu nagusiak dira, merkaturatutako farmakoen %35a horrelakoa izanik (Hauser et al., 2017). 7TM hartzaileen superfamiliari GPCR ere deitzen zaie, beren ekintza Gproteinen bitartez betetzen dutelako. G-proteinak guanina nukleotidoak lotzen dituzten hiru azpi unitatez osaturik daude: α, β eta γ. Hala ere, GPCRak Gproteinekin lotzeaz gain beste proteina zitoplasmatikoetara lotu daitezke, βarrestinetara besteak beste (Syrovatkina et al., 2016). G-proteinen aktibazioa, hortaz, zelulaz kanpoko seinalea zelularen barneko erantzun fisiologikoan bilakatzeko lehen urratsa izango da (Sriram eta Insel, 2018). Modu horretan, G-proteinak transduktore edo GPCRek sorturiko anplifikadore gisa jokatzen dute, zelula barruko erantzuna sortzeko. 1.2.2 GPCRen seinaleztapenaren oinarrizko mekanismoa G-proteina heterotrimerikoek funtsezko zeregina dute erantzun zelularren espezifikotasuna eta ezaugarriak definitzeko. Ligandoen bidezko aktibazioaren ondorioz, GPCRen konformazio aldaketa gertatzen da, Gproteinekiko afinitatea areagotzen duena, eta G-proteinen erreklutamendua eragiten duena. Ligando, hartzaile eta G-proteinen arteko elkarrekintzak G- Sarrera 281 proteina heterotrimerikoaren α-azpiunitateari loturik dagoen guanosina difosfato (GDP) nukleotidoa askatzea dakar, eta guanosina trifosfato (GTP) nukleotidoarekin trukatzea sustatzen du. GDP-GTP trukeak proteina heterotrimerikoa osatzen duen azpiunitateen eta hartzaileen arteko disoziazioa eragiten du. Egoera horretan, Gα eta Gβγ azpiunitateak hainbat efektoreekin elkarrekintza izaten dute, ligandoaren erantzuna zelula barnera transduzitzeko; hala nola, adenilato ziklasa (AC) fosfolipasa eta erreten ionikoak (Gilman, 1987; Hamm, 1998; Marinissen eta Gutkind, 2001). Azpiunitate horien seinaleztapena amaitzeko, eta prozesu haori etengabe gertatzeko, G-proteinak bere forma heterotrimerikora bueltatu beharra du. Horretarako, Gα-azpiunitatearen GTPasa aktibitateak GTP nukeotidoa GDPra hidrolizatzen du, Gα-azpiunitatea Gβγ dimeroaren elkarketa ahalbidetuz, eta G-proteina inaktiboa osatuz berriro (Milligan eta Kostenis, 2006; Hilger et al., 2018) (1.2 Irudia). Sarrera 282 Irudia 1.2: GPCR/G-proteina aktibazio ereduaren irudikapen eskematikoa. Atseden-egoeran edo egoera inaktiboan, G-proteina heterotrimeroak dira, eta osaturik daude Gα azpiunitateaz (GDPrekin elkartuta) eta Gβγ azpiunitateaz (1a). Ligando baten aktibazioaren ondoren (neurotransmisore gisa), GPCRak aldaketa konformazionala sufritzen du, G-proteinetara lotzea ahalbidetzen duena (1b) eta GDP GTPaz trukatzea sustatzen du Gα azpiunitatea Gβγ azpiunitatearekin banatuz (2). Momentu horretan, G-proteina egoera aktiboan dago α eta βγ azpiunitateek zelula barneko efektoreekin elkarreragingo dute, hainbat seinaleztapen bide modulatzeko ahalmenarekin (3). Seinaleztapena amaitzen da GTP molekula GDPra hidrolizatzean, G-proteina erregulatzaileen bitartez (RGS) (4). Azkenik, konplexu heterotrimerikoa osatzen da, G-proteinak egoera inaktibora bueltatuz (5). Oliveria et al., 2019ren ilustrazio egokitua. Agonistak GPCRarekin elkartzeak, G-proteinak estimulatzen dituzten konformazio-aldaketak abiarazteaz gain, G-proteinei lotutako kinasen (GRK) bidezko hartzailearen fosforilazioa errazten dute. Fosforilazioak arrestinen erreklutamendua sustatzen du, GPCRen erregulazioa murrizteko eta Gproteinen mendeko seinaleztapena arintzeko. Horrez gain, β-arrestinen eta GGPCR/G protein activation GPCR Ligandoa α-azpiunitatea βγ-azpiunitatea RGS Proteina efektoreak (AC, GRIK, PLC) Sarrera 283 proteinekiko independienteak diren seinaleztapenak aktibatu ahal dira, aktibatutako-mitogenoen-protein-kinasen (MAPK) bidea aktibatuz (Wang et al., 2018). G-proteinekin eta β-arrestinekin bat egiteaz gain, GPCRak beste GPCRekin elkarreragin dezakete dimeroak eratzeko; baita goi-mailako oligomeroak ere, sarritan funtsezkoak direnak GPCRen funtzioa modulatzeko (Milligan et al., 2019). G-proteinak G-proteina heterotrimerikoak orokorrean lau talde nagusitan sailkatzen dira, Gα azpiunitatearen arabera: Gαs/olf, Gαi/o, Gαq/11 eta Gα12/13 (Simon et al., 1991). Hartzailearen aktibazioaren ondoren, Gα-proteina familia bakoitzak seinaleztapen-bide desberdinak aktibatzen ditu, hainbat erantzun fisiologiko eraginez (1.3 irudia). Gαs familia bi azpiunitatez osatuta dago: Gαs-proteina, zelula gehienetan espresatzen dena, eta Gαolf-proteina usaimen-neurona sentsorialetan espresatzen dena, batez ere (Weinstein et al., 2007). Gαs familiako proteinek, adenilato ziklasa (AC) estimulatzen dute, bere jarduera katalitikoa estimulatuz, eta, ondorioz, adenosina monofosfato ziklikoaren (cAMP) ekoizpena sustatzen du (Milligan eta Kostenis, 2006). Horrez gain, Gαi/o familiak AC inhibitzen du cAMP maila zelularrak murriztuz (Busnelli et al., 2013). Gαi/o familia Gαi1-, Gαi2-, Gαi3-, Gαoeta Gαz-proteina azpimota desberdinetan banatzen da. Gαi azpiunitateak zelula gehienetan espresatzen dira, garuna barne, eta %85-95 homologia partekatzen dute (Plummer et al., 2012). Gαo neuronetan espresatzen da, batez ere, eta garuneko G-proteina ugariena da (Sternweis eta Robishaw, 1984). Gαz-proteinaren espresioa ehun neuronalean eta plaketetan mugatzen da (Hultman et al., 2014). Bukatzeko, Gαi/o familiako kide Sarrera 284 guztiek (Gαz izan ezik) karboxilo terminalean ondo kontserbatutako zisteina hondakin bat dute. Eremu hori adenosina difosfatoaren (ADP) erribosilazioguneari dagokio, G-proteinen aktibazioaren inhibizioa eragiten duena, Bordetella pertussis toxina (PTX) katalizatu ostean, (Morris eta Malbon, 1999). Ondorioz, proteina familia horiek PTX-sentikor gisa sailkatu ohi dira. Gαq/11 familiako kideek fosfolipasa β (PLC-β) aktibatzeko ahalmena dute, inositol 1,4,5-trifosfatoaren (IP3) eta diazilglizerolaren (DAG) formakuntza bultzatuz, mintz plasmatikoan fosfatidilinositol 4,5-bifosfatotik (PIP2) abiatuta. IP3ak erretikulu endoplasmatikoan kaltzioa mugiarazten du, DAG proteina kinasa C (PKC) aktibatzen duen bitartean (Wettschureck et al., 2005; Wilkie et al., 2021). Gαq/11 familia Gαq-, Gα11-, Gα14eta Gα15/16 azpi-proteinez osaturik dago. Gα11eta Gαq-proteinak edonon adierazten dira, eta aminoazidoen %88an sekuentzia partekatzen dute (Wilkie et al., 1991). Aldiz, Gα14eta Gα15/16-proteinen espresioa ehun espezifikoetara mugatua dago; hala nola giltzurrunetara (Tanaka et al., 2000). Azkenik, Gα12/13-proteina familiak Rho guanina nukleotidoen aldaketa faktorea (RhoGEFs) estimulatzen du, eta bi azpi-familiatan sailkatzen da; Gα12eta Gα13-proteinak, zelula mota gehienetan espresatzen direnak (Siehler, 2009). Nahiz eta hasieran GPCR bakoitza mota bakarreko G-proteinek estimulatzeko ahalmena zuela uste uste izan, gaur egun frogatu da GPCRek G-proteina desberdinetara lotzeko ahalmena dutela, seinaleztapen bide desberdinak aktibatuz. Sarrera 285 1.3 irudia: G-proteina desberdinen seinaleztapenaren irudikapen eskematikoa. Ilustrazioa: Diez-Alarcia et al., 2016. 1.2.3 Farmako eta GPCR hartzaileen arteko elkarreraginen teoria Farmakoek GPCRengan duten eragina funtsezko bi gertaeren araberako da: Lehenik eta behin, ligandoa hartzailearekin lotu behar da, eta hori farmako bakoitzaren afinitatearen araberakoa da. Bigarrenik, GPCR eta farmakoaren arteko loturak hartzailearen konformazio aldaketa eragin dezake, seinaleztapen-sistema batekin erlazionaturik dagoena, eta horri farmakoaren eraginkortasuna deritzo (Kenakin, 2002). Afinitate farmakologikoa farmako bakoitza hartzaile espezifiko batekin elkartzeko edo finkatzeko duen gaitasuna da. Gainera, afinitatea orekadisoziazioaren konstantearen alderantzizkoari dagokio (1/KD); hau da, hartzaile kopuru osoaren %50a okupatzeko behar den farmakoaren Sarrera 292 Farmakoak garatzerako orduan, kontuan hartzeko beste alderdi bat, egitura kimikoak hautakortasun funtzionalean izan dezakeen eragina da (Shonberg et al., 2014). Adibidez, LDSak eta lisuride farmakoak in vitro eta in vivo erantzun desberdinak dituzte (González-Maeso et al., 2003, González-Maeso et al., 2007), nahiz eta egitura eta 5-HT2AR-rekiko afinitate altua partekatu. Bestalde, risperidona eta bere metabolito aktiboa, paliperidona, talde hidroxilo bakar batean bereizten dira, eta biak farmako antipsikotiko atipikoak dira. Farmako horien ezaugarri farmakologikoak desberdinak dira, eta farmakoen hautakortasun funtzionalaren ezaugarrietan du jatorria desberdintasunak (Clarke et al., 2013). Aurkikuntza guzti horiek, egitura-hautakortasun funtzionalaren arteko erlazioaren azterketa sakonaren garrantzia azalarazten dute (Berg eta Clarke, 2018). Nahiz eta ikerketa askok ligandoen hautakortasun funtzionalari buruzko informazioa argitu duten, ikerlan gehienak seinaleztapen bide jakin batzuetara mugatuta daude; hala nola G-proteinak edo β-arrestinen bideak elkarrekin alderatuz. Hala ere, beste seinaleztapen bide desberdinak ere aktibatu daitezke, eta horiek ere kontuan hartu beharko lirateke eraginkortasun ezaugarriak deskribatzerako orduan (1.6 irudia). Sarrera 293 1.6 irudia: GPCRen hautakortasun funtzionalaren irudikapen grafikoa. μ-hartzaile opiodeen adibide hipotetiko eta sinplifikatua. G-proteinen aktibazio edo seinaleztapenak eragin terapeutikoa du; β-arrestinen aktibazioak, aldiz, eragin desiragaitzak sortzen ditu. Agonismo alboraturik gabeko ligandoek bi seinaleztapen bideak aktibatuko dituzte, eragin terapeutiko nahiz eragin desiragaitzak sortuz. Aldiz, agonismo alboratua aurkezten duten agonistek seinaleztapen bide bat aktibatzeko ahalmena dute, modu hautakorrean, eragin terapeutikoak sustatuz eta eragin desiragaitzik sortu gabe. 1.2.5 Aktibitate konstitutiboaren, alderantzizko agonismoaren eta hautakortasun funtzionalaren ebaluazioa GPCRen inplikazioak hainbat gaixotasunetan handitu egin du hartzailei finkatzeko ahalmena duten ligandoak ikertzeko metodoen kopurua. Garrantzi handia dutenez, hainbat saiakuntza egin dira farmako desberdinen profil funtzionala zehazteko. Saiakuntza funtzional klasikoak bigarren mezularien mailen neurketan oinarritzen dira; hala nola Ca2+, IP3ren metatzea edo cAMP ekoizpenean, Ligando alboratua Ligando ez-alboratua β-arrestinak β-arrestinak G-proteinak G-proteinak Eragin desiragaitzak Eragin desiragaitzak Eragin terapeutikoak Eragin terapeutikoak Sarrera 294 besteak beste. Saiakuntza horiek farmako bat GPCRarekin finkatu ostean duen profil funtzionala zehazteko baliagarriak dira. Bigarren mezulariak neurtzean, aldatu gabeko hartzaileak azter daitezke jatorrizko ehunetan. Bigarren mezularien neurketan oinarritzen diren saiakerak ikerkuntza desberdinetan erabili dira, eta horrek abantaila suposatzen du, argitalpenen artean emaitzak alderatzea ahalbidetzen duelako. Bigarren mezularien neurketan oinarritzen diren teknikak asko erabiltzen diren arren, anplifikazio maila altua duten teknikak dira, eta, ondorioz, agonista partzialek eta agonista osoek desberdintzea zaila da; erantzun maximo bera izan dezaketelako (Smith et al., 2018). Gainera, ez da argi geratzen bigarren mezularien erantzuna zein G-proteina azpimotaren aktibazioaren menpekoa den. Hortaz, beharrezkoa da farmakoen eta G-proteinen arteko elkarreraginaren kuantifikazio funtzional hurbilagoa burutzea. GPCR aktibazioaren ebaluazio zuzena egin daiteke, G-proteinetan ematen den guanina nukleotidoen trukearen estimulazioa edo inhibizioa neurtuz, erradioaktiboa eta ez-hidrolizagarria den GTP molekula erabiliz. Teknika honi 35 sufrearekin markaturik dagen guanosina-5´-O-(γ-ttio)-trifosfatoaren ([35S]GTPγS) finkapen teknika esaten zaio. GPCR eta farmakoaren artean ematen den loturaren ondorioz gertatzen den G-proteinen erantzun funtzional goiztiarra neurtzen da, eta ez dago seinale-anplifikazioren menpe (GonzálezMaeso et al., 2000; Harrison eta Traynor, 2003). [35S]GTPγS finkapen teknika konbentzionala Gαi/o-proteina eta GPCRen artean ematen den akoplamendua neurtzera mugatzen dira; nukleotidoen aldaketa tasa altuena eta aktibitate konstitutibo altuena duen G-proteinen familia delako Gαi/o (Seifert eta WenzelSeifert, 2002). Hala ere, Gα-proteina espezifikoak neurtzeko saiakuntza desberdinak garatu dira; hala nola, immunoprezipitazioarekin akoplatutako [35S]GTPγS finkapen teknika (SPA) (Diez-Alarcia et al., 2021b). Teknika horrek [35S]GTPγS finkapen teknika klasikoa G-proteina bakoitzaren kontrako antigorputzekin konbinatzen ditu. Metodologia kultibo zelularretara zein Sarrera 295 jatorrizko ehunetara aplikatu daiteke, hartzaileetan aldaketarik eragin gabe. Gainera, teknika horrek aktibitate konstitutiboa neurtzea ahalbidetzen du, jatorrizko ehunean, tresna farmakologikoak (alderantzizko agonistak, adibidez) erabiliz (Diez-Alarcia et al., 2021b). Hala ere, immunoprezipitazioarekin akoplatutako [35S]GTPγS finkapen teknika (SPA), Gα-proteinetara mugaturik dago. Hori horrela, β-arrestinen erantzuna ezin da oraindik neurtu. Aurreko saiakuntzez gain, fluoreszentzia eta biolumineszentzia erresonantzia bidezko energia-transferentzian oinarritzen diren saiakuntzak garatu dira (FRET eta BRET). Proteinen arteko elkarrekintza eta konformazio-aldaketa dinamikoak detektatzeko teknologiak dira, GPCR, G-proteinak, eta βarrestinen konformazio aldaketak zuzenean neurtzea baimentzen dutenak (Zhou et al., 2021; Wright eta Bouvier, 2021). Saiakuntza hauek, zelula bizien gertaerak denbora errelean monitorizatzea ahalbidetzen dute, eta errendimendu handiko detekziora egokitzeko aukera ematen dute. Teknika horiek abantailak izan arren, detekziorako erabiltzen diren molekula fluoreszenteek lortzen diren emaitzetan izan dezaketen eragina frogatu beharko litzateke (Pottie eta Stove, 2022). BRET teknikaren erabilera mugaturik dago in vitro zelula hazkuntza sistemetara, bizirik dauden animalietan ez baita erabilgarria. Gainera, oraingoz, erresonantzia bidezko energia-transferentzian oinarritzen diren teknikak ez dira erabilgarriak postmortem ehuna aztertzeko (Drinovec et al., 2012). Sarrera 296 1.3 Serotonina 2A hartzaileak (5-HT2AR) 1.3.1 Orokortasunak Nerbio sistema zentralean (NSZ) 5-HTk hainbat prozesu fisiologikotan parte hartze du, oroimena, pertzepzioa, kognizioa, emozioak, gogo aldartea eta kontzientzia, besteak beste (Berger et al., 2009). Sistema serotonergikoaren disfuntzioa gaixotasun psikiatriko askotan inplikaturik dago (Hoyer, 2020). Hartzaile serotonergikoak egitura eta ezaugarri farmakologikoen arabera, 14 azpimota desberdinetan sailkatzen dira (Hannon eta Hoyer, 2008), eta zazpi familia nagusi osatzen dituzte (5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, 5HT7 hartzaileak) (1.7 irudia). Egiturari dagokionez, 5-HT3 hartzailea erreten ionikoa den hartzaile bakarra da (Maricq et al., 1991). Gainerako hartzaile serotonerikoak, aldiz, G-proteinetara loturiko hartzaile metabotropikoak dira (Kroeze eta Roth, 1998). 5-HT2 hartzaileak gehien ikertu diren hartzaile serotonergikoen artean daude. 5-HT2 hartzaileen familia 3 hartzaile azpimota desberdinetan banatzen da, 5HT2AR, 5-HT2BR eta 5-HT2CR, non %40-50 sekuentzia-homologia duten (Hoyer et al., 2002). Gainera, 5-HT2ARen eta 5-HT2CRen mintz arteko eremuek %80eko sekuentzia-homologia dute, eta antzekoak diren profil farmakologikoak partekatzen dituzte (Boess eta Martin, 1994). Hori dela eta, hartzaile bakoitzarentzat farmako hautakorrak garatzea funtsezko erronka da. Gaur egun, 5-HT2 hartzaileen azpimota desberdinak farmakologikoki sailkatzea nahiko zaila da, ligando hautakorrik ez dagoelako. Hasieran batean, 5-HT azpimota desberdinak sailkatzeko erradioligandoen finkapen teknikak erabili ziren. Erradioligandoekin egindako lehenengo ikerketan oinarrituta, 5-HT molekularen bi finkapen eremu desberdin zeudela deskribatu ziren. [3H]5-HT erradioligandoaren afinitate altuko gunea 5-HT1 hartzailearen azpimotari dagokio. Afinitate baxuko guneak, berriz, 5-HT2 Sarrera 297 hartzaile gisa sailkatu ziren. [3H]ketanserina erradioligando hautakorraren aurkikuntza aurrerapen zientifiko izugarria izan zen, 5-HT2ARak ikertzeko (Leysen et al., 1982). Hala ere, ketanserinak 5-HT2CRekiko afinitatea aurkezten du, beste hartzaile batzuen artean (Choudhary et al., 1992). Azken urtetan, 5-HT2ARren azterketa egiteko ligando hautakor berriak garatu dira, [18F]altanserina, [3H]MDL100907 (L´Estrade et al., 2018) eta [11C]Cimbi-36, besteak beste (Ettrup et al., 2014). 5-HT2AR-ren eremu kodifikatzaileen sekuentzia analisiek espezien artean kontserbazio genetiko handia dagoela baieztatu zuten. Hala ere, gizakion 242 hondarrean serina bat aurkitzen bada ere, karraskarietan alanina bat aurkitzen da. Aldaketa horrek 5-HT2AR agonisten afinitate eta eraginkortasunean eragina izan dezakeela proposatu da gainera (López-Giménez eta GonzálezMaeso, 2018; Kim et al., 2020; Slocum et al., 2021). 1.3.2 5-HT2AR kokapena eta funtzioa NSZean 5-HT2AR eta 5-HT2CR NSZean espresatzen dira biak, eta periferian, berriz, 5HT2AR plaketetan; muskulu-zeluletan eta begi ehunetan aurkitzen da (Leysen, 2004). Aitzitik, 5-HT2BR batez ere periferian espresatzen da; zehazki, giza bihotz balbuletan (Bonaventure et al., 2005) (1.7 irudia). Sarrera 298 1.7 irudia: Hartzaile serotonergiko azpimoten irudikapen eskematikoa (5-HT1-5-HT 7). 5-HT2 familia 3 hartzaile mota desberdinetan banatzen da (5-HT2AR, 5-HT2BR eta 5-HT2CR). 5-HT2 familiako hartzaile mota bakoitzak garunean eta bestelako ehunetan banaketa desberdina du. 5-HT2AR gehien bat espresatzen da garun azaleko V geruzako neurona piramidaletan. 5HT2CR garunean ere espresatzen da, baina hipokanpoan. Azkenik 5-HT2BR periferian, bihotzeko balbulen zeluletan dago. Ilustrazioa: Meltzer eta Roth, 2013. 5-HT2AR-ren espresioa ikertzea posible izan da erradioligandoen finkapen teknika, immunohistokimika, mikroskopia elektronikoa eta in situ hibridazio teknikei esker. Bai post-mortem erradiografiak, bai in vivo neuroirudiaren ikerketek baieztatu ahal izan dute 5-HT2ARaren espresioa giza garunean (Pazos et al., 1987; Forutan et al., 2002). Giza garunean gehientsuenat eskualde kortikaletan (batez ere, aurre-garunazalean, parietalean eta somatosentsorialean) espresatzen da. Eremu subkortikaletan eta hipokanpoan, berriz, dentsitatea baxuagoan aurkitzen da (Hoyer et al., 1986; Pazos et al., 1987; López-Giménez et al., 1997; Varnäs et al., 2004; Beliveau Garunazalaren 5. geruza Hipotalamoa Bihotz balbulen ehun interstiziala Sarrera 299 et al., 2017). Zehazkiago, post-mortem garunetan egindako ikerketa batek frogatu zuen 5-HT2AR III eta IV geruza kortikaletan, hipotalamoan, eta neurri txikiagoan, hipokanpoan eta egitura estriataletan espresatzen dela (Pazos et al., 1987). Horrez gain, garunean aurkitzen den 5-HT2ARen espresioa adinarekin murriztu egiten da (Gross-Isseroff et al., 1990; González-Maeso et al., 2008; Moses-Kolko et al., 2011; Uchida et al., 2011; Muguruza et al., 2013; Diez-Alarcia et al., 2021a). Gainera, 5-HT2AR neurona glutamatergiko kortikaletan aurkitzen da, zehazki dendrita apikaletan (Jakab eta Goldman-Rackic, 1998; Miner et al., 2003; Santana et al., 2004). Interneurona GABAergikoetan ere espresatzen da (Burnet et al., 1995). Orokorrean, 5-HT2AR sinapsi ondoko eremuetan kokatzen da, hartzaile post-sinaptiko moduan (sinapsi glutamatergikoei dagokionez) (Jakab eta Goldman-Rakic, 1998). Hala ere, 5-HT2AR hein batean eremu presinaptikoan ere kokatzen dela uste da, neurona monoaminergikoen axoietan, hain zuzen (Miner et al., 2003; Bécamel et al., 2017). Gainera, 5HT2AR astrozitoetan eta mikroglian ere identifikatu da (Krabbe et al., 2012; Martin eta Nichols, 2016). Ugaztunen garunean, 5-HT2AR espresioa handiagoa da frakzio zitosolikoetan mintz plasmatikoan baino (Cornea-Hebert et al., 1999; Eastwood et al., 2001). Mintz zitoplasmatikoan espresatzen den hartzailea G-proteinetara loturik dagoela uste da, mintzetik barneraturiko hartzailea ez bezala. 5-HT2AR Gαq/11-proteinetara lotzen da gehienbat, eta behin aktibatuta dagoela,PLC isoforma aktibatzen du, IP3k ekoizteak Ca2+ren askapena eragiten du, eta azkenik, PKC aktibatzen da (ikusi 1.3.4 atala informazio gehiagorako) (1.8 irudia). In vitro esperimentuei esker frogatu da 5-HT2ARen aktibazioaren ondorioz aurre-garunazaleko neuronen kitzikagarritasuna handitzen dela (Araneda eta Andrade, 1991). Gainera, agonisten administrazio kortikalak kitzikagarritasun neuronala eta neurotransmisoreen askapena handitzen ditu (Ashby et al., 1990; Arvanov et al., 1999). Sarrera 300 Bestelako erantzun batzuk, 5-HT2ARen bitartez ematen dira; hipertermia, hiperlokomozioa eta erantzun endokrinoak barne; hala nola kortisol, renina eta prolaktinaren jarioa areagotzea (Gudelsky et al., 1986; Barnes eta Sharp, 1999; Pytliak et al., 2011). Beste ikerketa batzuek begien kliskatzea 5HT2ARen bitartez gertatzen dela frogatu dute (Welsh et al., 1998a; Welsh et al., 1998b; Romano et al., 2000; Harvey, 2003). Aurre garunazalean espresatzen diren 5-HT2ARek haluzinogeno psikodelikoen erantzun psikotomimetikoekin erlazionatu dira. Normalean haluzinogenoak, LSD eta psilozibina besteak beste, erabiltzen dira eskizofreniaren sintoma positiboen antzerakoak diren sintomak eragiteko animalia ereduetan (Vollenweider et al., 1998; González-Maeso et al., 2007; González-Maeso eta Sealfon, 2009; Nichols, 2016). Egungo klinikan erabiltzen diren antipsikotiko atipikoen itu nagusia 5-HT2ARak dira. Klozapina, risperidona eta olanzapina, adibidez, 5-HT2ARen antagonista edo alderantzizko agonista gisa jokatzen dute (Meltzer, 1999). Sarrera 301 1.8 irudia: 5-HT2AR neurona piramidalen dendritetan kokatzen da. Droga psikodelikoek 5HT2AR aktibatzen dute, eta zelula barneko seinaleztapen bide desberdinak aktibatzen dira. Ondorioz, aktibitate neuronala areagotzen da, eta horrek ere zelula barneko bestelako seinaleztapen bideak aktibatzen ditu. Ilustrazioa: McClure-Begley eta Roth, 2022. 1.3.3 5-HT2ARen egitura biologikoa Azken hamarkadan, GPCR mota desberdinen egitura biologikoak zehaztu dira, farmakologia profil desberdineko ligandoekin konplexuak sortzen dituztenak. Horrela, ligando-hartzaileen arteko eredu farmakoforoak garatu dira, eta ondorioz, mintz zeharreko hartzaileen konformazio aldaketak deskribatu dira, egitura aktiboa eratuz eta seinaleztapen bide desberdinetan eraginez (Seyedabadi et al., 2022). Homologia-ereduek eta kristalezko egiturek, agonista eta antagonista desberdinen akoplamendu-puntuak zehazteko aukera eman dute (Mozumder et al., 2020). Hartzaileen aminoazido bakarreko mutagenesiaren bidez eta ondorengo esperimentu funtzionalei esker, G-proteinen eta GPCRen artean ematen den loturan parte hartzen Sarrera 308 vivo/ex vivo ikerketetarako agonista eredu gisa erabili da (Nelson et al., 1999; Pigott et al., 2012; Canal et al., 2013). [35S]GTPγS finkapen teknikaren bitartez, giza garun post-mortem eta 5-HT2AR kock-out animalietan egindako esperimentuetan egiaztatu zen (±)DOI farmakoak 5-HT2AR eta 5-HT2CRen agonista partzial gisa jokatzen duela (Diez-Alarcia et al., 2019; Garcia-Bea et al., 2019; Muneta-Arrate et al., 2020). Azken urteetan zehar, 5-HT2ARekiko afinitate altuagoa duten farmako mota berria garatu da, N-bentzilfenetilamina (NBOMe)ren egituran oinarrituta; hala nola n-(2-metoxibentzil-2,5-dimetoxi-4-bromofeniletilamina (25B-NBOMe, Cimbi-36) eta N-(2-hidroxibentxil)-2,5-dimetoxi-4-zianofeniletilamina (25-CNNBOH) (Jensen et al., 2020). Molekula berri horiek tresna egokiak dira PET irudi bidezko teknikan erabilitako zein azterketa farmakologikoak egiteko. 25CN-NBOH eta Cimbi-36ak agonista partzial gisa jarduten dute, 5-HT2ARekiko afinitate handiagoa erakutsiz, 5-HT2CR eta 5-HT2BRen aldean (Hansen et al., 2014; Jensen et al., 2017). Bestalde, psilozina (psilozibinaren metabolito aktiboa) eta ergolinak (LSD, adibidez) hartzaile serotonergikoen agonista ez-hautakorak dira, 5-HT1 eta 5HT2 hartzaileekiko afinitatea dutenak (Roth, 2007; Nichols, 2016; Wacker et al., 2017). Gainera, LSD farmakoa bestelako hartzaile serotonergiko zein hartzaile dopaminergikoetara lotzen da afinitate altuarekin (Halbertadt eta Geyer, 2011; Borroto-Escuela et al., 2014). Head-twitch erantzuna (alde batetik bestera ematen den buruaren mugimendu azkarra) psikodelikoen erantzun haluzinogenoa neurtzeko gehien erabiltzen den ebaluazio froga da karraskarietan. Horregatik, head-twich erantzuna psikodelikoen ekintza in vivo ebaluatzeko froga funtzional gisa sailkatzen da. Antagonista hautakorrak eta 5-HT2AR knock-out animaliak erabiliz frogatu da head-twich erantzuna 5-HT2ARen bitartez ematen dela, eta psikodelikoetara mugaturik dagoela (González-Maeso et al., 2007, Canal eta Morgan, 2012). Sarrera 309 Hala ere, kimikoki oso antzerakoak diren molekulek, (lisuride, ergotamina eta pergolide, besteak beste), ez dute head-twitch erantzuna eragiten, nahiz eta 5-HT2ARekiko afinitate altua izan (González-Maeso et al., 2003; GonzálezMaeso et al., 2007). Hori dela eta, lisuride eta pergolide 5-HT2ARen agonista ez-aluzinogeno gisa sailkatzen dira. Molekula horiek, bestelako ergolinak bezala, profil farmakologiko oso konplexua dute, eta hainbat hartzaile aminergikoekiko kidetasuna aurkezten dute, hartzaile serotonergikoak eta dopaminergikoak barne (Halberstadt eta Geyer, 2011). Gainera, lisuride eta pergolide, parkinson gaixotasunaren aurkako farmako gisa erabiltzen dira, dopamina eta 5-HT1A hartzaileekiko duten afinitate altua dela-eta (Langtry eta Clissold, 1990; Marona-Lewicka et al., 2002). 1.3.5.2 Antagonistak N-alkilpiperidonak 5-HT2ARen taldeko antagonista klase handiena eta hautakorrenen artean dago. Horietatik, ketanserina izan da urteetan zehar antagonista gisa erabili izan den farmako nagusia. Ketanserinak hautakortasun altuagoa dauka 5-HT2ARekiko 5-HT2CRekiko baino (15-80 aldiz), eta baita 5-HT2BRekin alderatuz (500-1000 aldiz) (Jerman et al., 2001; Knight et al., 2004; Diez-Alarcia et al 2019). Kimikoki, ritanserina eta ketanserina oso antzekoak dira, 5-HT2ARen antagonista potente eta hautakor zein alderantzizko agonista gisa deskribatu direnak biak (Bonhaus et al., 1995). Beste 5-HT2AR antagonista, kimikoki ketanserinarekin erlazionatua ere, altanserina da; 5-HT2ARen antagonista potente eta hautakor gisa deskribatu dena, 5-HT2ARekin 20 aldiz afinitate altuagorekin lotzen dena bestelako hartzaileekin baino (Tan et al., 1999). Hala ere, alderantzizko agonista ezaugarriak ere deskribatu zaizkio altanserinari (Aloyo et al., 2009; DiezAlarcia et al., 2019). Sarrera 310 5-HT2ARen beste ligando hautakor batzuk ere garatu dira; hala nola MDL100907, bolinanserina ere deiturikoa, eta MDL-11,939. Bolinanserina 5HT2ARen antagonista potentea da, eta 300 aldiz afinitate altuagoa du 5HT2ARekiko, 5-HT2cRekiko baino, eta baita beste hartzaileekin konparatuz (Sorensen et al., 1993; López-Giménez et al., 1998). Bolinanserina eta ritanserina antipsikotiko gisa ebaluatu ziren eskizofreniaren tratamendurako, hala ere, ez zuten arrakastarik izan, eta ondorioz beren erabilera mugatu egin zen (Jones et al., 2020). Gaur egun, bolinanserina 5-HT2ARen antagonista eredu gisa erabiltzen da, hautakortasun handia duelako. Duela gutxi, farmako berriak garatu dira loezina tratatzeko, nelotanserina eta eplibanserina, besteak beste. Konposatu berri horiek 5-HT2ARekiko afinitate altua aurkezten dute, 20 aldiz hautakortasun gehiago 5-HT2ARekiko baino (Rinaldi-Carmona et al., 1992; Al-Shamma et al., 2010). Pimabanserina, berriz, ACP-103 bezala ere ezagutzen dena, 5-HT2ARen farmako oso hautakora da, beste hartzaileekiko afinitaterik ez duena, eta 5HT2ARekiko 30 aldiz afinitate altuagoa aurkezten duena 5-HT2CRekiko baino(Vanover et al., 2006; Abbas eta Roth, 2008). Pimabanserina Ameriketatako Estatu Batuetako Drogen eta Elikagaien Administrazioak (FDA) farmako gisa onartu du Parkinson gaixotasunean gertatzen den psikosiarekin lotutako haluzinazioak eta eldarnioak tratatzeko (Cummings et al., 2014). 5-HT2ARen beste antagonista batzuk, nahiz eta 5-HT2A/2CRekiko hautakorrak izan, hartzaile dopaminergiko, histaminergiko edo/eta adrenergikoekiko afinitate ertain eta altuarekin lotzen dira. Horietaz gain, antipsikotiko atipikoak (adibidez, risperidona, klozapina eta olanzapina) eta antidepresibo triziklikoak (adibidez, amitriptilina, klomipramina eta imipramina) ere 5-HT2ARetara lotzen dira antagonista gisa (Roth et al., 2004; Meltzer eta Massey, 2011; Meltzer, 2012). Sarrera 311 1.3.6 5-HT2ARen hautakortasun funtzionala Hartzaile serotonergikoak, 5-HT2AR bereziki, hautakortasun funtzionala zuela iradoki zen lehen GPCRetakoa izan zen (Berg et al., 1998). Zenbait ikerketen arabera, 5-HT2ARen agonista zein antagonistek, hartzailearen konformazio desberdinak egonkortzen dituzte. Horren ondorioz, hautakortasun funtzionala sortzen da seinaleztapen bide desberdinen bidez; Gαq/11-proteinen seinaleztapen bideaz gain bestelako seinaleztapen bide alternatiboak aktibatuz (López-Giménez eta González-Maeso, 2018). Ildo horretan, hautakortasun funtzionala proposatu da erantzun zelular desberdina eragitearen fenomenoa azaltzeko, 5-HT2AR kortikalen populazio bera aktibatzen baitute droga haluzinogenoek, eta ez-haluzinogenoak diren farmakoek. Baina erantzunen artean desberdintasunak aztertu dira Gαproteinetan, geneen espresioan, erantzun elektrofisiologikoan eta portaera probetan (González-Maeso et al., 2003; González-Maeso et al., 2007; Karaki et al., 2014; Banerjee eta Vaidya, 2020). Hautakortasun funtzionalaren lehen ebidentzietakoa Berg eta kolaboratzaileen aurkikuntzatik sortu zen; hartzaileek Gαq/11-proteinen mendeko PLCaren aktibazioaz gain, PLA2ren aktibazioa ere gertatzen zela frogatu zutenean (Berg et al., 1998). Zehazki, PLC mendeko IP mailak eta PLA2ren aktibazioaren ondoriozko AAren askapena neurtu zuten, seinaleztapen bide bakoitza aktibatzeko eraginkortasun ahalmena neurtzeko. Emaitza horien arabera, 5-HTak PLC-IP seinaleztapen bidea aktibatzen zuen bereziki, eta LSDak, berriz, PLA2-AA bidea (Berg et al., 1998; Martí-Solano et al., 2015). Gainera, 5-HT2AR haluzinogeno eta ez-haluzionogenoek eragin diferentziala dute gene-adierazpenean (González-Maeso et al., 2003; González-Maeso et al., 2007). 5-HT2AR agonista haluzinogeno eta ez-haluzinogenoen presentzian, gene erantzun desberdina ematen dela frogatu zuten zelula eta saguen garunazal somatosentsorialean. Hori horrela, droga haluzinogenoak Sarrera 312 zein ez-haluzinogenoak c-fos genearen adierazpena eragiten dute. Aldiz, erg1 eta erg-2 geneen adierazpena soilik gertatzen da LSD eta (±)DOI agonista haluzinogenoen presentzian, baina azken bi geneen adierazpena ez da aldatzen lisuride eta ergotamina agonista ez-haluzinogenoen presentzian (1.11 irudia). Emaitza horien arabera, 5-HT2ARen agonista guztiek PLCrekin akoplaturiko 5-HT2AR aktibatzen dituzte. Haluzinogenoen mendeko erantzunak, aldiz, PTX sentikor diren Gαi/o-proteinek bideratzen dituzte. Ikuspegi berri hori, fosfoproteomika kuantitatiboan oinarritaturiko esperimentuen bitartez balioztatu zen (Karaki et al., 2014). Gainera, (±)DOI agonista haluzinogenoak eta pergolide agonista ez-haluzinogenoak hautakortasun funtzional desberdina aurkeztu zuten post-mortem giza aurregarunazalean (Muneta-Arrate et al., 2020). Bi agonistek Gαq/11-proteinen aktibazioa eragiten dute; Gαi1-proteinaren aktibazioa, berriz, (±)DOI droga haluzinogenoak soilik eragiten du (Muneta-Arrate et al., 2020). Ondorioz, agonista haluzinogenoek, LSD eta psilozibina kasu, Gαq/11proteinak zein Gαi/o-proteinak aktibatzen dituzte. Aitzitik, lisuride, ergotamina eta pergolide, 5-HT2ARen agonistak izanik, nahiz eta egitura kimiko antzekoa izan, ez dituzte ezaugarri haluzinogenoak, Gαq/11-proteinen mendeko bidea baino ez dutelako estimulatzen. Beste ikerketa batek, 5-HT2ARen agonista haluzinogenoak eta ezhaluzinogenoak seinaleztapen sinadura desberdina zutela frogatu zuen. Egileek, fosforilaturiko-PLC, pERK, pCREBII zein IP eta DAG ekoizpen maila altuagoak neurtu zituzten (±)DOIren presentzian lisuride farmakoarekin konparatuz (Banerjee eta Vaidya, 2020). Gainera, 5-HT2ARek G-proteinekiko independenteak diren beste seinaleztapen bideak aktibatzen dituzte, β-arrestinak besteak beste. 5-HTak eta (±)DOIk modu bereizgarrian aktibatu ditzakete 5-HT2ARak eredu zelularretan eta head-twich erantzuna neurtzean. β-arrestina-2 proteinarik Sarrera 313 gabeko animalietan, 5-HTak head-twitch erantzuna sortzeko ahalmena galtzen duela frogatu zuten. Badirudi, Akt-ren fosforilazioa gertatzen dela βarrestina-2ren aktibazioaren ondoren, baina animalia horietan (±)DOI farmakoaren aktibazioaren ondoren ez da Akt-ren aktibaziorik gertatzen. Hala eta guztiz ere, (±)DOI farmakoak eragindako head-twich erantzuna βarrestina-2arekiko independentea dela dirudi. Egitura kimiko desberdina duten agonistek 5-HT2AR aktibatu ondoren seinaleztapen bide desberdinak aktibatzeko ahalmena dutela ondorioztatu zen (Schmid et al., 2008; Schmid eta Bohn, 2010). Berriki iradoki zen LSD droga haluzinogenoak eragiten zuen head-twich erantzuna ere β-arrestina-2-mendekoa zela, baina β-arrestina-1rekiko independentea (Rodriguiz et al., 2021). Hala ere, beste ikerketa batean frogatu zuten agonista ez haluzinogenoek ere β-arrestina-2 erakartzeko ahalmena zutela, aurreko aurkikuntzaren emaitzen kontra (Cao et al., 2022). Beraz, βarrestina-2 eta haluzinogenoen ekintza mekanismoak ikerketa sakonagoa behar du. Bestalde, klozapina antipsikotiko atipikoak 5-HT2ARen seinaleztapena blokeatzeko ahalmena du, G-proteinen bitartez. Hartzailea zelulan barneratzea (internalizazioa) eragiten du, eta baita Akt-ren fosforilazioa, nahiz eta β-arrestina-2rekin ez elkarreragin. Beraz, 5-HT eta klozapinak mekanismo desberdina erabiltzen dute, 5-HT2ARen bitartez, seinaleztapen bide berdinak induzitzeko: Akt-ren fosforilazioa eta hartzailearen internalizazioa. Hori horrela, klozapinak Akt fosforilazioaren bitartez blokeatzen du 5-HT2AR, eta PCP edo MK-801 farmakoek eragindako sintoma psikotikoak ere blokeatzeko gai da. Hautakortasun funtzionalaren konplexutasuna bide alternatiboa izan daiteke farmako hautakorren garapenean, eragin kliniko areagotuak eta eragin desiragaitz gutxituak dituzten farmakoak garatzeko orduan. Hala ere, Sarrera 314 hautakortasun funtzionala eta bestelako ezaugarri farmakologikoak, hala nola alderantzizko agonismoa edo antagonismoa, farmako berrien mekanismo farmakologiko gisa zalantzan jarriak dira oraindik, depresioa eta eskizofrenia gaixotasunen terapia gisa. Sarrera 315 1.11 Irudia: 5-HT2ARen seinaleztapen intrazelularraren irudikapen eskematikoa Sarrera 316 1.4 5-HT2ARak eta eskizofrenia Hainbat ikerlanetan aztertu dute 5-HT2ARen eta eskizofreniaren arteko harremana. Eskizofrenian 5-HT neurotransmisorearen inplikazioa zehaztea zaila izan bada ere (Halberstadt & Geyer, 2013), ikerketa gehienek 5-HT2AR aztertu dute antipsikotiko atipikoek 5-HT2AR blokeatzeko ahalmenean oinarriturik (Meltzer et al., 1989; Miyamoto et al., 2005). Gainera, lehen adierazitakoari jarraituz, hainbat drogek, sintoma psikotikoak eragiten dituzte 5-HT2AR-en aktibazioaren ondorioz, bai gizakietan bai karraskarietan; psilozibinak, LSDak eta (±)DOIk, adibidez. 1.4.1 Ikerketa genetikoak HTR2A genearen aldaera eta eskizofreniaren arteko erlazioa hainbat ikerlanek proposatu dute. Hala, HTR2A genearen edo promotorearen mutazioak eskizofreniarekin erlazionatu izan dira, nahiz eta, horietako bat bera ere ez den populazioan modu sendoan erreplikatu. Izan ere, eskizofrenian egindako GWAS azterketek ez zuten inolako aldaketa esanguratsurik aurkitu HTR2A gene aldaeren artean (Farrell et al., 2015). Gainera, eskizofrenian aztertu diren HTR2A espresioaren aldaketak nukleotido bakarreko polimorfismoekin (SNPs) ere erlazionatu izan dira: A1438G eskualde promotorean (Ohara et 1998), His452Tyr eskualde kodifikatzailean (Ozaki et al., 1996), eta T102C lehenengo exonean (Arranz et al., 1996), besteak beste. Azterlan batzuk SPN horiek eskizofrenian parte hartzea bultzatzen duten arren, beste ikerketa batzuek kontrakoa frogatu dute. Horien artean, zenbaitek iradoki dute A-1438G polimorfismoa eskizofrenia izateko probabilitatearekin erlazionaturik dagoela (Parsons et al., 2004; Peñas-Lledo et al., 2007; Sáiz et al., 2007; Smith et al., 2013). Era berean, frogatu da A-1438G polimorfismoak eragina duela tratamendu antipsikotikoaren erantzunean ere (Yan et al., 2021). Sarrera 317 Azkenik, His452Tyr (rs6314) eskizofreniarekin zer ikusia duela frogatu zen, baina are gehiago antipsikotikoen erantzunarekin lotu dute. Polimorfismoa klozapina eta olanzapinaren erantzun kliniko aldakorrarekin erlazionatu da (Birkett et al., 2000; Olajossy-Hilkesberger et al., 2011). Gainera, HTR2A rs6314 5HT2Aren adierazpen eta funtzioan eragiten du, eta horrek eskizofreniaren endofenotipoa modulatzera laguntzen du, hala nola portaera kognitiboak eta horri lotutako aktibitate prefrontala (Blasi et al., 2013). Bestalde, T102C (rs6313) erlazionatu da populazio desberdineko eskizofrenia gaixotasunarekin, baita risperidona eta olanzapinaren antipsikotikoen erantzunarekin (Petronis et al., 2000; Maffioletti et al., 2020). Laburbilduz, goiko datuen sendotasun ezak, 5-HT2ARen funtzio, espresio eta antispikotikoen erantzunean eragina duten SPNei buruzko etorkizunean egin daitezkeen ikerketa prekliniko nahiz kliniko gehigarriak egitea gehiago justifikatzen ditu. 1.4.2 5-HT2ARen dentsitatea, espresioa eta funtzionalitatea subjektu eskizofrenikoetan Eskizofrenia duten subjektuen post-mortem garunean 5-HT2ARen RNAm ebaluatzean emaitza desberdinak argitaratu dira. Eskizofrenia duten subjektuen aurre-garunazalean, 5-HT2ARen RNAmaren adierazpenean murrizketak nahiz aldaketa ezak zituzten emaitzak argitaratu dira azken urteotan (Burnet et al., 1996; Hernandez & Sokolov, 1997; Hernandez & Sokolov, 2000; Lopez-Figueroa et al., 2004). Duela gutxi, tratamendu antipsikotikoa jaso zuten eta tratamendurik gabeko subjektuen post-mortem aurre-garunazalean 5-HT2ARen RNAm adierazpena aztertu zen. Emaitzen arabera, antipsikotikorik gabeko subjektu eskizofrenikoetan, 5-HT2ARen RNAmren adierazpena, kontrolekin konparatuz, Sarrera 324 sintomatologia positiboa murrizteko gaitasuna dute, eskizofrenia pairatzen duten pazienteen kuadro klinikoa hobetuz. Hala ere, pazienteen %30ak, gutxi gora behera, erantzun murriztua erakusten dute, edo inolako erantzunik ez antipsikotiko tipikoak ematean. Gainera, ez dute inolako onurarik eragiten sintoma negatibo edo asaldura kognitiboetan (Conley eta Kelly, 2001; Legge et al., 2020). 1.13 irudia: Haloperidolaren egitura kimikoa. 1.5.2 Bigarren belaunaldiko antipsikotikoak (antipsikotiko atipikoak) Bigarren belaunaldiko antipsikotikoak, edo antipsikotiko atipikoak, alboondorio estrapiramidalak murrizteko helburuarekin garatu ziren (1.14 irudia). Antipsikotiko atipikoek afinitate altuagoa aurkezten dute 5-HT2ARekiko, D2R familiarekin alderatuta. ezaugarri hori síntoma extrapiramidalen murrizketarekin erlazioanatua dago,eta ,beraz, 5-HT2A/D2 hartzaileen afinitate erlazioa eragin desigaitzen profila aurreikusteko eralbilgarria da (Ebdrup et al., 2011). Zoritzarrez, bigarren belaunaldiko antipsikotikoek pisu handitzea, eta glukosa zein lipidoen metabolismoan aldaketak izateko arriskua handitzen dute (Muench eta Hamer, 2010; Weston-Green et al., 2013; Grajales et al., 2019). Sarrera 325 Klozapinaren garapenak, lehen belaunaldiko farmakoekin alderatuta, perfil farmakologiko hobetua zuten farmako berrien garapenean lagundu zuen (Meltzer et al., 1989). Klozapinaren profil farmakologiko konplexua dela eta, oso zaila da farmakoaren eragin klinikoen ekintza mekanismoa zehaztea. Klozapinak hartzaile desberdinekiko afinitate esanguratsua aurkezten du; hartzaile histaminergiko, adrenergiko, dopaminergiko eta kolinergikoekiko, besteak beste (Coward, 1992; Nucifora et al., 2017). D2R eta 5-HT2ARetan eragiteaz gain, klozapina 5-HT1R agonista partziala ere bada, sintoma kognitibo eta negatiboen murrizketan inplikaturik daudenak. Hartzaile muskarinikoetan ere eragiten du; M1R, M2R, M3R eta M5R blokeatuz, M4R estimulatzen duen bitartean. Gainera, hartzaile histaminergikoak blokeatuz sedazio efektua sortzen du. Azkenik, klozapina hartzaile adrenergikoak blokeatzen ditu, hipotentsioa eta takikardia eraginez (Coward et al., 1992; Nucifora et al., 2017). Klozapina farmakoa hilgarria izan daiteke, hemototoxikotasun-arriskuaren (agranulozitosia eta neutropenia) ondorioz. Hori dela eta, bere erabilera klinikoa mugaturik aurkitzen da (Alphs et al., 1991). Hori horrela, bigarren belaunaldiko antipsikotiko berriak garatu ziren; hala nola risperidona, olanzapina eta ketiapina, odol-diskrasiekin loturiko albo-ondorioak murrizteko helburuarekin. Olanzapina klozapinaren analogo kimikoa da, antzeko ezaugarri farmakologikoak dituena, baina agranulozitosi arriskurik gabe. Espero zen moduan, olanzapinak 5-HT2ARekiko afinitate handiagoa du hartzaile dopaminergikoekiko baino. Hartzaile histaminergikoak, muskarinikoak eta adrenergikoak blokeatzen ditu, baina potentzia gutxiagorekin, klozapinren aldean. Hala ere, pisu handitzea eta sedazioa bezalako eragin desiragaitzak sortzen ditu olanzapinak (Fulton eta Goa, 1997; Leucht et al., 2013). Sarrera 326 Ketiapinak, berriz, D1R, D2R eta 5-HT2ARen antagonista zein 5-HT1ARen agonista partzial gisa jarduten du. Ketapinak eragindako albo-ondoriok antagonismo α1-adrenergiko eta histaminergikoarekin erlazionaturik daude (Miodownik eta Lerner, 2006). Risperidona ere antipsikotiko atipikoa da. Risperidonaren eragin terapeutikoa 5-HT2AR eta D2Ren blokeoan oinarritzen da, baina 5-HT2ARekiko afinitate handiagorekin (Cohen, 1994). Horrez gain, risperidona α1-adrenohartzaileak eta hartzaile histaminergikoak blokeatzen ditu. Risperidona sintoma positiboen tratamendurako eraginkorra izateaz gain, nahasmen kognitibo eta sintoma negatiboen tratamendurako ere eraginkorra da. Ondorioz, gehien preskribatzen den antipsikotikoa da (Möller, 2005; Chopko eta Lindsley, 2018). Paliperidona risperidonaren metabolito aktiboa da, eta profil farmakologiko bera duela frogatu da. Sarrera 327 1.14 Irudia: Klozapina, olanzapina, quetiapina, risperidona eta paliperidonaren egitura kimikoa. Azken urteotan, bigarren belaunaldiko antipsikotiko berriak garatu dira; hala nola asenapina eta lurasidona (Miyamoto et al., 2012). Hala ere, lehen belaunaldiko antipsikotikoen eraginkortasuna oraindik ez da hobetu. Gaur egun, eskizofrenia erresistentea tratatzeko onarturik dagoen botika bakarra klozapina da (Conley eta Kelly, 2001). 1.5.3 Hirugarren belaunaldiko antipsikotikoak Honezkero, hirugarren belaunaldiko antipsikotikoak ere garatu dira; hala nola aripiprazola (1.15 irudia). Beste neuroleptiko batzuk ez bezala, hirugarren belaunaldiko antipsikotikoak ez dira D2Ren antagonistak, D2Ren agonista partzialak baizik (Davies et al., 2004). DA kontzentrazio handietan, DArekin lehiatzen dira, eragin klinikoa lortuz. Aitzitik, DAren mailak txikiak direnean, aripiprazola D2R-rekin elkartu daiteke, eta agonista partzial gisa jardun. Klozapina Olanzapina Ketiapina Risperidona Paliperidona Sarrera 328 Gainera, aripiprazola 5-HT1Ren agonista partzial moduan aritzen da. Bigarren belaunaldiko antipsikotikoak ez bezala, aripiprazolak afinitate handiagoa du D2Rekiko 5-HT2ARekin alderatuz (Chen et al., 2022). Hirugarren belaunaldiko antipsikotikoak sintoma psikotikoak arintzeko eraginkorrak dira, albo-ondorio estrapiramidalik eta hiperprolaktinemiarik eragin gabe. Gainera, pisu handitzea eta albo ondorio metaboliko gutxiago sortzen dituzte (Lieberman, 2004). 1.15 irudia: Aripriprazolaren egitura kimikoa 1.5.4 Antipsikotikoen belaunaldi berria 5-HT2ARen blokeoan oinarritutako eskizofrenia tratatzeko farmako berriak aurkitzeko ahaleginak, ritanserina eta blonanserina kasu, ez dira terapeutikoki erabilgarriak izan. 5-HT2ARen antagonista selektiboen erabilerak monoterapian ez zuenez eraginkortasun klinikoa aurkeztu, zenbait ikerketen arabera, 5-HT2ARen antagonismoa soilik ez da nahikoa antipsikotiko atipikoen eraginkortasun klinikoa azaltzeko (Miyamoto et al., 2012). Hala, badirudi D2Ren blokeoa beharrezkoa dela eragin klinikoa lortzeko. Hala ere, pimabanserina deituriko 5-HT2ARen ligando potente eta selektiboa garatu zen psikosiaren tratamendurako alternatiba gisa (1.16 irudia) (Meltzer eta Roth, 2013). Pimabanserina afinitate dopaminergikorik ez duen onartutako lehen antipsikotikoa da (Hacksell et al., 2014). Orain arte, pimabanserina Sarrera 329 eskizofreniarako terapian lagungarri gisa probatu da, haloperidola eta risperidona farmakoekin batera (Meltzer et al., 2012). Pimabanserinak FDAren onarpena lortu du parkinsonen gaixotasunean gertatzen diren eldarnioak eta haluzinazioak murrizteko, (Cummings et al., 2014). Gainera, pimabanserinak eskizofrenia duten pazienteen sintoma negatiboak murrizteko gaitasuna duela ere frogatu da (Bugarski-Kirola et al., 2022). Pimabanserina 5-HT2ARen alderantzizko agonista gisa deskribatu den arren, beharrezkoa da alderantzizko agonismoa frogatzeko esperimentu gehiago egitea (Vanover et al., 2006; Nutt et al., 2017). 1.16 irudia: Pimabanserinaren egitura kimikoa. Berriki onartu da bigarren belaunaldiko antipsikotiko gehienak 5-HT2ARen alderantzizko agonistak direla, antagonista neutralak izan beharrean (Weiner et al., 2001). Antagonistek ez bezala, alderantzizko agonistek berezko eraginkortasun negatiboa dute, eta oinarrizko seinaleztapen-jarduera txikitu dezakete. Oro har, antipsikotikoen ekintza-mekanismoa hobeto ulertzeak sendagai eraginkorrak eta jasangarriagoak diseinatu eta garatzea ekar lezake. Historikoki, profil polifarmakologikoa duten farmako promiskuoak eraginkorrak izan dira NSZren gaixotasunak tratatzeko, nahiz eta albo ondorio larri asko Sarrera 330 eragin ditzaketen. Beraz, helburu molekular zehatzarekin elkarreragiten duten farmako selektiboen diseinuak sendagai eraginkorren eta onargarriagoen garapenean lagundu dezake. Hala balitz, agonismo alboratua edo selektiboa bakarka, zein alderantzizko agonismoarekin batera, aurkezten duten farmakoak, hartzaileen hautakortasunaren alternatiba gisa sortu ahalko lirateke, eskizofrenian espero diren erantzun funtzionalak lortzeko. Helburuak Helburuakl 333 Nahiz eta ikerketa asko egin diren sendagai antipsikotiko berriak garatzeko, gaur egun eskura dauden sendagaien artean ez da nabarmendu epe luzera eraginkortasuna duenik, albo-ondorio kaltegarririk gabe. Hori, eskizofreniaren neurobiologiari buruzko jakituria ezari zor zaio. Beraz, eskizofrenia maila neurobiologikoan sakonago ezagutuz gero, aldaketa molekularrak, zelularrak eta/edo seinaleztapen bideak identifikatu ahal izango dira, botika berrietarako itu terapeutiko berriak bilakatu daitezkenak. Zenbait aurkikuntzek aditzera ematen dutenez, 5-HT2AR sintoma psikotikoen eta horien tratamenduan eragiten duten mekanismo molekularretan inplikaturik dago. Alde batetik, psikodelikoen (LSD, psilozina, meskalina eta (±)DOI) izaera haluzinogenogenoa, 5-HT2ARen aktibazioan oinarritzen da. Bestetik, eskizofrenian erabili ohi diren antipsikotiko atipikoek 5-HT2ARen antagonista edo alderantzizko agonista gisa jokatzen dute. Neuroirudi azterketek, in vivo PET eta in vitro post-mortem proben bidez, eskizofrenia duten subjektuetan 5-HT2AR dentsitateari buruzko emaitza kontrajarriak erakutsi dituzte. Txosten kontrajarri horiek zerikusia daukate, antza, erabiltzen diren erradiotrazadore desberdinak hartzailearen konformazio desberdinetara finkatzearekin. Beraz, badirudi eskizofreniako 5HT2ARen aldaketek zerikusi gehiago dutela hartzailearen egoera molekularraren aldaketekin hartzailearen adierazpen mailaren aldaketekin baino. 5-HT2AR Gαq/11zein Gαi/o-proteinak aktibatzeko gai da, hartzailera finkatzen den farmakoaren arabera. Testuinguru horretan, ezaugarri haluzinogenoen aztarna molekularra 5-HT2ARen agonista bidezko Gαi1-proteinen aktibazioan oinarritzen da. Gainera, Gαi1-proteinen estimulazio handiagoa deskribatu da, baina ez horrela Gαq/11-proteinena; eskizofrenia duten subjektuen aurregarunazalaren post-mortem ehunean eta 5-HT2ARen (±)DOI agonistaren presentzian egin zenean. Aurkikuntza hori, 5-HT2ARen agonismo alboratu gisa interpreta daiteke, eskizofrenikoetan Gαi1-proteina bide haluzinogenoaren Subjektuak, Materialak and Metodoak 340 3.1 taula: Eskizofrenia taldearen (S), eskizofrenia ez zuten suiziden taldearen (NSS) eta kontrol taldearen (C) ezaugarri demografikoak, post-mortem egoera, heriotza -kausa eta analisi toxikologikoaren emaitzak. Kasua Diagnosia Generoa (G/E) Adina (urte) PMI (ordu) Metatzea (hilabete) Heriotzakausa Heriotzamekanismoa Garun pH Farmako maila odolean (mg/L) Garuntoxikologia (ng/g) S 1 Eskizofrenia E 67 22 17 Naturala Porrot kardiobaskularra 5,8 Negatiboa Negatiboa C 1 Kontrola E 66 17 83 Istripua Trafiko-istripua 6,06 Negatiboa Ez egina S 2 Eskizofrenia G 34 23 81 Suizidioa Alturatik salto 6,32 Negatiboa Kotinina 23,1 C 2 Kontrola G 34 17 69 Istripua Trafiko-istripua 6,7 Negatiboa Negatiboa NSS 1 Pertsonalitate asaldura G 34 7 199 Suizidioa Urkatua Ez egina Etanola 2700 Zitalopram 0,1 Oxakarbazepina 6,5 Zitalopram 1579,9 Kotinina 3352,8 Norzitalopram 368,2 S 3 Eskizofrenia E 53 18 98 Naturala Odulustea 6,58 Alprazolam 0,05 Paliperidona 54,2 Kotinina 503,2 Alprazolam 43,9 C 3 Kontrola E 51 10 70 Naturala Porrot kardiobaskularra 6,3 Negatiboa Negatiboa S 4 Eskizofrenia G 32 21 87 Suizidioa Altueratik salto 6,65 Negatiboa Kotinina 380,78 C 4 Kontrola G 33 23 95 Istripua Trafiko-istripua 6,55 Negatiboa Ez egina NSS 2 Pertsonalitate asaldura G 33 14 192 Suizidioa Altueratik salto Ez egina Lorazepam 0,03 Benlafaxina 0,16 Desmetilbenlafaxine 475,0 Lorazepam 1421,2 Midazolam 3,7 Olanzapine 6 S 5 Eskizofrenia G 45 36 15 Istripua Itota Ez egina Ez egina Midazolam 1801,8 Nordiazepam 1247,1 Oxazepam 50,1 C 5 Kontrola G 44 23 98 Istripua Trafiko-istripua 6,45 Negatiboa Negatiboa NSS 3 Pertsonalitate asaldura G 44 9 192 Suizidioa Altueratik salto Ez egina Etanola 220 Alprazolam 0,01 Amisulprida 1,4 Klomipramina 0,2 Reboxetina 0,09 Alprazolam 141,4 Klomipramina 3040,7 Kotinina 322,5 Reboxetina 224,2 S 6 Eskizofrenia G 49 23 107 Istripua Altueratik eroria 6,40 Negatiboa Kotinina 391 Lorazepam 16,3 C 6 Kontrola G 49 19 93 Istripua Trafiko-istripua 6,7 Negatiboa Negatiboa S 7 Eskizofrenia G 70 20 115 Suizidioa Urkatu Ez egina Ez egina Negatiboa C 7 Kontrola G 71 22 115 Istripua Altueratik eroria 5,92 Negatiboa Negatiboa S 8 Eskizofrenia E 74 9 118 Naturala Porrot kardiobaskularra Ez egina Fenobarbitala 9 Negatiboa Subjektuak, Materialak and Metodoak 341 C 8 Kontrola E 74 30 167 Istripua Altueratik eroria Ez egina Negatiboa Negatiboa S 9 Eskizofrenia G 46 20 129 Suizidioa Altueratik salto 6,41 Negatiboa Zuklopentixol 110,8 Kotinina 622,4 Lorazepam 25,5 C 9 Kontrola G 46 22 115 Naturala Porrot kardiobaskularra 6,48 Negatiboa Negatiboa NSS 4 Pertsonalitate asaldura G 47 4 195 Suizidioa Altueratik salto Ez egina Fenitoina Negatiboa S 10 Eskizofrenia G 26 24 140 Suizidioa Altueratik salto Ez egina Diazepam 0,27 Diazepam 356,5 Nor-diazepam 856,9 Oxazepam 15,1 C 10 Kontrola G 25 21 71 Istripua Su-istripua 6,48 Negatiboa Negatiboa NSS 5 Pertsonalitate asaldura G 27 42 253 Suizidioa Altueratik saltot Ez egina Negatiboa Kotinina 337,4 Diazepam 204,9 Nordiazepam 612 Oxazepam 65,8 S 11 Eskizofrenia E 75 18 140 Naturala Porrot kardiobaskularra Ez egina Negatiboa Kotinina 36,76 C 11 Kontrola E 79 24 213 Istripua Trafiko-istripua Ez egina Negatiboa Ez egina S 12 Eskizofrenia G 28 28 143 Suizidioa Altueratik salto Ez egina Negatiboa Kotinina 93,79 C12 Kontrola G 29 13 116 Istripua Altueratik eroria 6,44 Negatiboa Negatiboa NSS 6 Pertsonalitate asaldura G 28 5 259 Suizidioa Altueratik salto Ez egina Negatiboa Kotinina 638,2 S 13 Eskizofrenia G 25 17 145 Suizidioa Altueratik salto Ez egina Negatiboa Kotinina 153,1 C 13 Kontrola G 23 16 141 Istripua Altueratik eroria Ez egina Negatiboa Negatiboa NSS 7 Alsaldura obsesibo - konpultsiboa G 26 19 143 Suizidioa Altueratik salto 6,66 Klomipramina 0,5 Fluoxetina 0,7 Fluboxamina 0,2 Ketiapina 0,5 Fluoxetina 15441 Fluboxamina 4094 Norfluoxetina 4433,5 Norketiapina 386,7 Ketiapina 86 S 14 Eskizofrenia G 23 13 195 Suizidioa Altueratik salto Ez egina Ez egina Haloperidol 136,5 Kotinina 458,5 Ketiapina 392,5 Norketiapina 1309,4 C 14 Kontrola G 22 20 188 Istripua Trafiko-istripua Ez egina Nordiazepam 0,38 Kotinina 371,6 Oxazepam 80,9 Nordiazepam 909 NSS 8 Pertsonalitate asaldura G 19 8 131 Suizidioa Altueratik salto 6,7 Negatiboa Kotinina 236,4 S 15 Eskizofrenia E 80 32 178 Naturala Shock Ez egina Negatiboa Ez egina C 15 Kontrola E 78 12 185 Naturala Porrot kardiobaskularra Ez egina Negatiboa Ez egina S 16 Eskizofrenia E 38 23 216 Suizidioa Altueratik salto Ez egina Negatiboa Negatiboa C 16 Kontrola E 36 19 110 Istripua Trenbidera eroria 6,51 Negatiboa Negatiboa Subjektuak, Materialak and Metodoak 342 NSS 9 Antsietate asaldura E 39 19 214 Suizidioa Farmako gaindosia Ez egina Nordiazepam 0,13 Etanola 5000 Diazepam 22,4 Nordiazepam 280,7 S 17 Eskizofrenia E 51 15 236 Naturala Porrot kardiobaskularra Ez egina Buflomedil 66 Metamizol 4 Kotinina 521,5 C 17 Kontrola E 51 38 230 Istripua Trafiko-istripua Ez egina Negatiboa Ez egina S 18 Eskizofrenia G 62 28 240 Suizidioa Altueratik salto Ez egina Ez egina Amitriptilina 170,3 Nortriptilina 453 C 18 Kontrola G 62 23 243 Istripua Trafiko-istripua Ez egina Negatiboa Ez egina NSS 10 Moldaera antsietate asaldura G 62 19 271 Suizidioa Crushing Ez egina Ez egina Maprotilina 257,9 Nordiazepam 1584,9 S 19 Eskizofrenia G 35 22 299 Suizidioa Altueratik salto Ez egina Negatiboa Negatiboa C 19 Kontrola G 36 22 286 Istripua Trafiko-istripua Ez egina Etanol 1000 Ez egina NSS 11 Antsietate asaldura G 36 15 181 Suizidioa Arma tiroa Ez egina Mirtazapina 0,08 Ez egina S 20 Eskizofrenia G 49 41 292 Suizidioa Urkatua Ez egina Etanola 490 Ez egina Kotinina 127 Klorpromazina 140,7 Lorazepam 388,6 Tioridazina 6079,5 C 20 Kontrola G 45 30 275 Istripua Trafiko-istripua 6,82 Etanola 3090 Ez egina NSS 12 Antsietate asaldura G 46 26 319 Suizidioa Urkatua Ez egina Ez egina Diazepam 91,3 Nordiazepam 175,2 S 21 Eskizofrenia E 56 24 125 Naturala Porrot kardiobaskularra Ez egina Alprazolam 0,03 Kotinina 110 Alprazolam 38 C 21 Kontrola E 54 24 10 Istripua Altueratik eroria 6,87 Negatiboa Negatiboa S 22 Eskizofrenia G 50 3 173 Suizidioa Uretan itota 7,09 Nordiazepam 0,4 Amisulprida 75,9 Kotinina 89 Nordiazepam 662,8 Oxazepam 47,6 Trazodona 241,6 C22 Kontrola G 50 2 15 Naturala Porrot kardiobaskularra 6,1 Negatiboa Negatiboa NSS 13 Pertsonalitate asaldura G 49 22 141 Suizidioa Altueratik eroria 6,35 Nordiazepam 2,8 Tiaprida 5,4 Benlafaxina 1,2 Kotinina 254,1 Desmet illbenlafaxina 265,6 Oxazepam 94,7 Tiaprida 973,5 Benlafaxina 2146,5 S 23 Eskizofrenia G 43 17 172 Naturala Porrot kardioaskularra Ez egina Negatiboa Kotinina 1003,9 Lorazepam 66 C 23 Kontrola G 41 15 7 Naturala Porrot kardiobaskularra Ez egina Negatiboa Negatiboa Subjektuak, Materialak and Metodoak 343 Ezaugarri demografikoak, adina, PMD, metatze denbora eta pH-a, 3.2 taulan laburtuak daude. Konparaketa estatistikoak egin zirenean ez zen diferentziarik aurkitu, aztertutako hiru taldeen artean, adinak alderatzean (F[2,56]=2,08, p=0.1341). Era berean, ez zen diferentzia esanguratsurik aurkitu hiru taldeen artean PMD aztertu zenean (F[2,56)]=1,99, p=0,1456), ezta pH-a aztertu zenean ere (F[2,21]=0,17, p=0,8433). Hala ere, eskizofrenia taldearen laginen metatze denbora luzeagoa izan zen kontrol taldearekin eta eskizofrenia ez zuten suiziden taldearekin konparatuz (F[2,56]=4,66, p=0,0135) (3.2 taula). 3.2 taula: Ikerlanean aztertutako eskizofrenia taldearen, eskizofrenia ez zuten suiziden taldearen ezaugarri demografikoak, nahiz post-mortem ezaugarriak. Talde bakoitzaren batezbestekoa±SEM (batezbestekoaren errore estandarra): *p<0,05 vs kontrol taldea (Bonferroniren konparazio anizkoitzaren azterketa) Taldea Generoa Adina(urteak) PMD pH Metatze denbora (hilak) Eskizofrenia 7 E/16 G 48±4 22±2 6,5±0,1 150±15 Eskizofrenia ez zuten suizidak 1 E/12 G 38±3 16±3 6,6±0,1 207±15* Kontrol 7 E/16 G 48±3 21±2 6,5±0,1 139±15 Subjektuak, Materialak and Metodoak 344 3.1.2 Immunoprezipitazioarekin akoplatutako [35S]GTPγS finkapen teknika (SPA) eta Western Blot frogen karakterizaziorako erabili zen lagin multzoa osatzen zuten partaideen ezaugarri demografikoak. Farmakoen hasierako karakterizazio farmakologikoa DLPFC lagin multzo batekin egin zen, gainontzeko frogetan erabili ez zirenetatik abiatuta. Lagin multzoen mintzak prestatzeko sei subjektuen aurre-garun-azal zatiak homogeneizatu ziren. Guztira, 2016 eta 2019 bitartean jasotako hamabost lagin erabili ziren ikerlan osorako (%30 gizonezkoak eta %70 emakumezkoak), non batezbesteko adina 59±6 urtekoa zen, PMD 11±2 ordukoa eta metatze denbora 27±3 hilekoa zen. Partehartzaileen odolean egindako proba toxikologikoetan lortutako emaitzen arabera, ez zeukaten farmako psikotropikorik hil zirenean. Subjektuak, Materialak and Metodoak 345 3.2 Animaliak: Sagu transgenikoak Lan honetan erabilitako 5-HT2AR knock-out saguak (5-HT2AR(-/-)) eta jatorrizko edo Wild Type saguak (5-HT2AR(+/+)) R. Maldonado katedradunak (Bartzelona, Espainia) eskuzabaltasunez emanak izan ziren. Sagu transgenikoak lehenengo 129S6/SvEv anduian sortu ziren, eta ondoren C57BL/6J anduiarekin gurutzatu ziren, laborategiko prozedura estandarrak jarraituz (González-Maeso et al., 2003; Weisstaub et al., 2006; González-Maeso et al., 2007; Orejarena et al., 2011). Animaliak jaso ostean, gurutzatze gehigarriak egin ziren kolonia freskatzeko. Gure laborategian ohiko polimerizazio-katearen erreakzioak (PCR) eta elektroforesiak egin ziren gure saguak genotipatzeko (ez dira emaitzak sartu), aurretik argitaratutako prozedurak jarraituz (FioricaHollowells et al., 2002). 5-HT2AR(-/-) saguek 5-HT2AR espresatzen ez zutela egiaztatzeko, [3H]ketanserinarekin finkapen azterketak (Muguruza et al., 2013) eta (±)DOIk eragindako Gαi1/Gαq/11-proteinen aktibazio azterketak (GarciaBea et al., 2019) egin ziren. Horretarako, C57BL/J6 sagu heldu arrak erabili ziren (15-20 asteko adinarekin). Animaliok bosnaka taldekatu ziren kaioletan, ohikoak diren laborategiko baldintzapean (22±1°C, %55±5 hezetasun erlatiboa, 12 h argi/ilun zikloa eta askatasun osoa ohiko marraskarien janaria eta ura lortzeko). Erabilitako animalien zenbatekoa murrizteko ahalegina egin zen. Gainera, jarraitutako esperimentuen protokoloak Euskal herriko Unibertsitateko (UPV-EHU) Animaliekin egiten den Esperimentaziorako Etika Batzordetik (AEEB) onartuak izan dira (Erreferentzia: M20-2019-321). Era berean, esperimentu guztietan jarraitutako prozedurek Europar Batasuneko (European Union Directive 2010/63/UE) eta Espainiako (53/2013 errege dekretua) legediek ezartzen dituzten arauak betetzen dituzte, animalien ongizateari dagokionean. Sagu helduak lepo dislokazio bidez hil ziren, ondoren garuna atera zitzaien eta garun-azala disekzionatu (Diez-Alarcia et al., 2016). Laginak -80°Ctan gorde ziren erabiliak izan arte. Subjektuak, Materialak and Metodoak 346 3.3 Farmakoak -(±)-DOI: (±)-2,5-Dimetoxi-4-iodoamfetamina hidrokloruroa (Sigma-Aldrich; Saint Louis, Missouri, EEBB). -Ketanserina: 3-[2-[4-(4-Fluorobentzoil)-1-piperidinil]etil]-2,4[1H,3H]- quinazolinadiona tartratoa (Tocris; Bristol, Erresuma Batua). -MDL100907 (bolinanserina): (R)-(+)-α-(2,3-dimetoxifenil)-1-[2-(4fluorofenil)etil]-4-pipidin metanola (Sigma-Aldrich; Saint Louis, Missouri, USA). -Altanserina: 3-[2-[4-(4-Fluorobentzoil)-1-piperidinil]etil]-2,3-dihidro-2-tioxo4(1H)-kinazolinona hidrokloruro hidratoa (Sigma-Aldrich; Saint Louis, Missouri, EEBB). -Pimabanserina (ACP-103): 1-(4-Fluorobentzil)-3-(4-isobutoxibentzil)-1-(1metilpiperidin-4-il)urea (Axon Medchem; Groningen, Herbehereak). -Nelotanserina: 1-(3-(4-bromo-2-metil-2H-pirazol-3-il)-4-metoxifenil)-3-(2,4difluorofenil)urea (Axon Medchem; Groningen, Herbehereak). -Ritanserina: 6-[2-[4-[bis(4-fluorofenil)metilideno]piperidin-1-il]etil]-7-metil- [1,3]tiazolo[3,2-a]pirimidin-5-ona (Sigma-Aldrich; Saint Louis, Missouri, EEBB). -Eplibanserina: 4-[(E,3Z)-3-[2-(dimetilamino)etoximino]-3-(2-fluorofenil)prop1-enil]fenola (Axon Medchem; Groningen, Herbehereak). -MDL-11,939: α-fenil-1-(2-feniletil)-4-piperidin metanola (Tocris; Bristol, Erresuma Batua). -SB 242084: 6-Kloro-2,3-dihidro-5-metil-N-[6-[(2-metil-3-piridinil)oxi]-3piridinil]-1H-indol-1-carboxiamida dihidrokloruroa (Tocris; Bristol, Erresuma Batua). -Klozapina: 8-Kloro-11-(4-metil-1-piperazinil)-5H-dibentzo[b,e][1,4]diazepina (Tocris; Bristol, Erresuma Batua). -Risperidona: 3-[2-[4-(6-fluoro-1,2bentzisoxazol-3-il)-1-piperidinil]etil]- 6,7,8,9-tetrahidro-2-metil-4H-pirido[1,2a]pirimidin-4-ona (Sigma-Aldrich; Saint Louis, Missouri, EEBB). Subjektuak, Materialak and Metodoak 347 -Aripiprazola: 7-{4-[4-(2,3-Diklorofenil)-1-piperazinil]butoxi}-3,4-dihidro-2(1H)- quinolinona (Sigma-Aldrich; Saint Louis, Missouri, EEBB). -Olanzapina: 2-Metil-4-(4-metil-1-piperazinil)-10H-tieno[2,3b][1,5]bentzodiazepina (Sigma-Aldrich; Saint Louis, Missouri, EEBB). -Paliperidona: 3-[2-[4-(6-Fluoro-1,2-bentzisoxazol-3-il)-1-piperidinil]etil]- 6,7,8,9-tetrahidro-9-hidroxi-2-metil-4H-pirido[1,2-a]pirimidin-4-ona (Tocris; Bristol, Erresuma Batua). -Ketiapina: 2-[2-(4-Dibentzo[b,f][1,4]tiazepin-11-il-1-piperazinil)etoxi]etanol hemifumaratoa (Tocris; Bristol, Erresuma Batua). -Atropina: Azido endo-(±)-α-(hidroximetil)bentzeno azetikoaren 8-metil-8azabiziklo[3.2.1]okt-3-il esterra (Sigma-Aldrich; Saint Louis, Missouri, EEBB). -Fentolamina: 2-[N-(3-Hidroxifenil)-p-toluidinometil]-2-imidazolidina hidrokloruroa (Sigma-Aldrich; Saint Louis, Missouri, EEBB). -Zetirizina: Azido [2-[4-[(4-klorofenyl)fenilmetil]-1-piperazinil]etoxi]azetikoaren hidrokloruroa (Sigma-Aldrich; Saint Louis, Missouri, EEBB). -Raclopridea: 3,5-Dikloro-N-[[(2S)-1-etil-2-pirrolidinil]metil]-2-hidroxi-6metoxibentzamida (Tocris; Bristol, Erresuma Batua). -Haloperidola: 4-[4-(4-klorofenil)-4-hidroxipiperidino]-4′-fluorobutirofenona (Sigma-Aldrich; Saint Louis, Missouri, EEBB). Subjektuak, Materialak and Metodoak 348 3.4 Materialak Antigorputzak: Immunoprezipitazioarekin akoplatutako [35S]GTPγS finkapen teknika (SPA) eta Western Blot frogak egiteko erabili ziren antigorputz monoklonalak Santa Cruz Biotechnology, Inc (Kalifornia EEBB) enpresari erosi zitzaizkion. Antigorputzen ezaugarriak 3.5.2 atalean zehaztuak daude, non Western Blot esperimentuen protokoloa ere azaltzen den. Western Blot frogetan bi antigorputz sekundario fluoreszente ezberdin erabili ziren: bata gorria, anti-sagu antigorputzarekin elkartutako Alexa Fluor® 680 (Invitrogen, Oregon, EEBB), eta bestea berdea, anti-untxi antigorputzarekin elkartutako IRDyeTM 800 (Rockland Immunochemical, Pennsylvania, EEBB). Konposatu erradiaktiboak: Azufre 35az markatutako guanosina-5’-O-(gamma-tio)-trifosfatoa ([35S]GTPγS), aktibitate espezifikoa 1250 Ci/mmol zuena eta PerkinElmer laborategiari erosia (Waltman, MA, EEBB). Bestelako farmako eta konposatu kimikoak: -Bio-Rad Laboratories (California, EEBB): Amonio persulfatoa (APS), Bradford Protein Assay, Laemmli laginen tanpoia 2 aldiz kontzentratua, N-N-N-N´- tetrametiletilenediamina (TEMED), aurrez markatutako SDS-PAGE pisu molekularraren estandarrak. -Carlo Erba Erreaktiboak (Bartzelona, Espainia): Metanola. -GE Healthcare (Buckinghamsire, Erresuma Batua): Nitrozelulosa mintzak (poro tamaina: 0.45 μm) eta WhatmanTM zelulosa 3MM. -Invitrogen (Bartzelona, Espainia): DL-Ditiotreitola (DTT), azido etilenodiamino tetrazetikoa (EDTA). Subjektuak, Materialak and Metodoak 349 -National diagnostics (Atlanta, GA, EEBB): %30 akrilamida 30-%0,8 bisacrilamida. -Panreac S.A.U (Bartzelona, Espainia): Azido azetiko glaziala, azukrea eta HCl (%37). -Sigma-Aldrich® (Saint Louis, Missouri, EEBB): Behiaren serumeko albumina (BSA), 2-butanola, dimetilsulfoxidoa (DMSO), azido etileno glikol-bis(2aminoetilether)-N,N,N′,N′-tetraazetikoa (EGTA), glizina, guanosina difosfatoa (GDP), guanosina 5'-O-[gammatio]trifosfatoa (GTPγS), Igepal® CO-520, βmerkaptoetanola, MgCl2, NaCl, NaF, Na3VO4, Proteasa Inhibitzaile Cocktail, polioxietilenoa (20) sorbitan monolauratoa (TweenTM 20), sodio deoxikolatoa (SDC), sodio dodezil sulfatoa (SDS), Tris (hidroximetil)aminometano hidrokloruroa (Tris HCl). -Perkin Elmer (Waltham, MA, EEBB): 96 putzudun isoplaka eta A proteina itsatsirik duten polibiniltoluenozko (PVT) SPA bolatxoak. Subjektuak, Materialak and Metodoak 356 farmako bakoitzak [35S]GTPγS finkapenean neurtutako seinalea blokeatzea lortu zen. 3.5.1.3 Emaitzen analisi matematikoa eta estatistikoa Analisi matematiko Microbeta Trilux Scintillation counter detektagailuan lortutako emaitzak CCPM (minutuko zuzendutako kontu) gisa espresatuak daude, non CCPMak eta DPMak (minutuko desintegrazioak) baliokideak direla onartzen den. Emaitzak interpretatzeko, CCPMtan dauden datuak finkatutako [35S]GTPγS fentomol proteina miligramoko unitatetan bilakatu behar dira (fmol/mg proteina). Datuen bilakatzea egiteko, azterketan erabilitako proteina kontzentrazioa kontuan hartu behar da, eta 3.1 ekuazioa betetzen da. [35S]GTPγS finkapenean proteina kontzentrazioak eragina izan dezake Diez-Alarcia et al., 2021 artikuluan frogatu zen moduan. Horregatik froga horietan proteina edukia neurtzen da esperimentu bakoitzeko. fmol/ mg proteina: CCPM kontuak/(2,22 x 1250 x [frogako proteina kontzentrazioa mg]) 3.1 ekuazioa: [35S]GTPγS finkapena eta SPA dituzten esperimentuetan lortutako emaitzak fmol/mg proteinako. 1250 (Ci/mmol) 35S erradioligandoak berezkoa duen aktibitate espezifikoari dagokio, 2,22 balioa konstantea da Curie (Ci) unitateak DPMtan bilakatzeko. (1 Ci=2,22x1012 DPM). [35S]GTPγSren finkapen basala (BB), farmako exogenorik ezean gertatzen den [35S]GTPγSren finkapenari dagokio. Agonista edo alderantzizko agonista gehitzean [35S]GTPγSaren finkapen basala modulatua izango da. Izan ere, agonista gehitzean [35S]GTPγSaren finkapena handitzen da, jatorrizko [35S]GTPγS finkapen basalarekiko. Aldiz, alderantzizko agonista gehitzean [35S]GTPγSaren finkapen basala txikitzen da. Subjektuak, Materialak and Metodoak 357 Bestalde, finkapen ez espezifikoa (NBS) CCPMtan lortzeko, egoera esperimental bakoitzetik kenketa eginez atera zen; finkapen basala, estimulazioa, inhibizioa, etab. Hortaz, balio bakoitzari finkapen ez espezifikoa kentzean, [35S]GTPγSaren finkapen espezifikoa lortu zen; kalkuluak eta analisi estatistikoa egiteko erabiliko zena. Farmako agonista, antagonista edo alderantzizko agonisten ezaugarriak ezagutzeko, kontzentrazio-erantzun kurba bat lortu zen farmako bakoitzarentzat. Horretarako, farmako gabe lortutako finkapen basalaren balioa finkapenaren %100 gisa onartu zen. Ondoren, farmakoarentzat lortutako finkapen espezifikoa portzentai gisa bilakatu zen. Horrela, lortutako [35S]GTPγS finkapenaren estimulazioa edo inhibizioa finkapen basalaren arabera adierazi zen. Kurba osatzen duten puntu bakoitzaren kalkulurako 3.2 eta 3.3 ekuazioak erabili ziren: %Estimulazioaren: (Estimulazioa-NBS)/(BB-NBS)x100 3.2 ekuazioa: [35S]GTPγS finkapena SPArekin konbinatzean lortutako emaitzen kalkulua finkapen basalarekiko portzentai izaera agonista, antagonista edo lehiakorra deskribatzeko. NBS: Finkapen ez espezifikoa, BB: Finkapen basala. %Inhibizioaren: 100-[(Inhibizioa-NBS)/(BB-NBS)x100] 3.3 ekuazioa: [35S]GTPγS finkapena SPArekin konbinatzean lortutako emaitzen kalkulua finkapen basalarekiko portzentai izaera agonista, antagonista edo lehiakorra deskribatzeko. NBS: Finkapen ez espezifikoa, BB: Finkapen basala. Kasu batzuetan, [35S]GTPγSak Gα-proteina subtipo desberdinetara finkatzeko duen ahalmena aztertzeko, kontzentrazio-erantzunaren kurba osoa egin beharrean, kontzentrazio bakar batentzat (10 μM) azaldutako finkapena soilik aztertu zen. Kontzentrazio hori aukeratzerako orduan, finkapen maximoa Subjektuak, Materialak and Metodoak 358 (Emax/Imax) eragiteko ahalmena zuen farmakoaren kontzentrazioa hartu zen, kontzentrazio-erantzunaren kurbatik. Kasu horietan, finkapen basala %0 gisa adierazi zen, eta farmakoek eragindako efektua positibo edo negatibo gisa adierazi zen egoera basalarekiko. Estimulazioaren edo inhibizioaren parametro farmakologikoen kalkulurako GraphPad PrismTM programa informatikoaren bidez lortutako analisi ez-lineala erabili zen. Horiekin, [35S]GTPγSren finkapenarekiko efektu estimulatzaile eta inhibitzaile maximoak lortu ziren (Emax/Imax), baita horien erdiak lortzeko beharrezkoa den farmakoaren kontzentrazioak ere (EC50/IC50). Azterketetan lortutako puntu bakoitza 3.4 eta 3.5 ekuazioetan sartu ziren kontzentrazioerantzun kurba lortzeko. Eredu matematiko horrek onartutakoaren arabera, kontzentrazio-erantzunaren kurbaren malda estandarra da, Hillen maldaren balioa (edo malda faktorea) 1,0 izanik. Hori horrela, balio hori izan beharko litzateke malda, baldin eta masen ekintzaren legearen arabera, farmakoa hartzaile batera soilik finkatzen bada. E= BB+(Emax-BB)(1+10(LogEC50-Log[X])) 3.4 ekuazioa: Estimulazioaren kontzentrazio-erantzunaren kurba monofasikoa (malda estandarra duena). E erantzunari dagokio (3.2 ekuazioa estimulazio %), X kontzentrazioak eragindakoa, BB [ 35S]GTPγSren finkapen basalari dagokio, agonista ezean lortutako finkapena (100% gisa adierazita), Emax efektu estimulatzaile maximoa (%) eta LogEC50 efektu estimulatzaile maximoaren erdia lortzeko beharrezkoa den farmakoaren kontzentrazioa. Subjektuak, Materialak and Metodoak 359 E= BB+(Imax-BB)(1+10(LogIC50-Log[X])) 3.5 ekuazioa: Estimulazioaren kontzentrazio-erantzunaren kurba monofasikoa (malda estandarra duena). E erantzunari dagokio (3.3 ekuazioa estimulazio %), X kontzentrazioak eragindakoa, BB [ 35S]GTPγSren finkapen basalari dagokio, agonista ezean lortutako finkapena (100% gisa adierazita), Imax efektu estimulatzaile maximoa (%) eta LogIC50 efektu estimulatzaile maximoaren erdia lortzeko beharrezkoa den farmakoaren kontzentrazioa. Emax/Imax parametroak batezbestekoa±SEM (batezbestekoaren errore estandarra) gisa adierazten dira, eta –LogEC50/IC50 parametroak ere batezbestekoa±SEM gisa adierazten dira. Balio horiek izango dira analisi estatistikoa egiteko erabiliko direnak. Efektu estimulatzailearen edo inhibitzailearen maximoaren erdia lortzeko gai den farmakoaren kontzentrazioa EC50/IC50 gisa adierazten da. Parametro hori, LogEC50/IC50 balioen batezbestekoaren antilogaritmoa egitean lortzen da. Hori guztiaz gain, kontzentrazio-erantzunaren kurbak ko-analisi bidez ere aztertu ziren (talde bererako egindako esperimentu guztien analisia aldi berean), talde bakoitzaren balio orokorrak lortzeko. Horretarako, [35S]GTPγSaren finkapena leku bakar batera hobekien doitzen zuen erregresio ez-linealaren eredua erabili zen. Emaitzok, doikuntza onenaren balioa ± %95eko konfidantza-tartea gisa adierazi ziren. Analisi estatistikoa Lortutako emaitzen interpretaziorako hainbat analisi estatistiko egin ziren. Hasteko, lortutako balio guztiak Grubb’s test analisiaren bidez aztertu ziren, talde esperimental bakoitzean egon zitezkeen outlier balioak detektatu eta baztertzeko. Kontzentrazio puntu bakarrarekin egindako esperimentuetan lortutako emaitzak lagin bakarrerako Student’s test analisiaren bidez aztertu ziren; Subjektuak, Materialak and Metodoak 360 farmakoak finkapen basalarengan eragindako efektua ala efektu-eza aztertzeko asmoz. Bi buztanetako Student´s t-test parekatugabea erabili zen, bi egoera esperimental aztertu behar ziren bakoitzean; adibidez, agonistak eragindako efektua eta agonistak antagonistarekin batera eragindako efektuak alderatzeko. Hiru egoera desberdin alderatzeko, aldiz, bide bakarreko ANOVA analisia erabili zen, Bonferroniren post-hoc analisiarekin batera. Hori izan zen erabilitako analisia eskizofrenia taldea, eskizofrenia ez zuten suiziden taldea eta kontrol taldea alderatzeko, [35S]GTPγSaren finkapenaren modulazioa aztertzerako orduan. Bestalde, bi aldagaiek izan zezaketen eragina aztertzeko, bi bidetako ANOVA erabili zen, Bonferroniren post-hoc analisiarekin batera. Analisi hori izan zen egokiena genotipo desberdina zuten saguen taldeak alderatzeko, adibidez. Aldagai independenteen (adina, PMD eta metatze denbora) eta menpekoak diren eragin funtzionalen arteko erlazioa aztertzeko Pearsonen r korrelazio koefizientea kalkulatu zen. Korrelazio hori esanguratsua zen kasuetan, kobariantza analisia (ANCOVA) egin zen talde esperimentalen artean (eskizofrenia, eskizofrenia ez zuten suizidak eta kontrolak). ANCOVA analisiak egiteko InVivoStat software estatistikoa erabili zen. Lan honen emaitzak aztertzean, lortutako desberdintasunak estatistikoki esanguratsutzat jo ziren p<0,05 zela egiaztatu zenean. Hori guztiaz gain, analisi osagarriak ere egin ziren taldeen arteko desberdintasun potentzialak aztertzeko. Horrela, eskizofrenia, eskizofrenia ez zuten suizidak eta kontrol taldeko balioen koanalisian lortutako emaitza globalei, beste azterketa gehigarri bat egin zitzaien. (DeLean et al., 1978; Motulsky eta Ransnas, 1987) Analisi osagarri horretan, erabilitako analisi ereduen egokitasuna alderatu zen, behartze multzo bat aplikatuz F analisiaren bidez (Balioen karratuaren gehiketa printzipioan oinarrituta). Subjektuak, Materialak and Metodoak 361 Hasteko, balio multzo bakoitza banaka aztertu zen (behartu gabe). Ondoren, balioen karratuaren emaitza lortzeko, doikuntza bakoitzean lortutako bakarkako balioen gehiketa egiten da, askatasun gradu kopuruarekin ere egiten den moduan. Ondoren, balio multzoak nahastuta ere aztertzen dira, aldi berean guztiak, eta amankomunak dituzten parametro bat, edo gehiago, partekatzera behartzen zaio doitutako kurbari (balio basalak, Emax/Imax eta LogEC50/IC50). Horrela, balio ezberdinak lortzen dira balioen karratuekin eta askatasun graduekin. Parametro bat edo gehiago partekatzea baimentzen zuen analisia jo zen doikuntza egoki gisa, baldin eta bariantzen ondarra ez bazuen esanguratsuki handitzen. Egokitasunaren esaguratsutasun estatistikoa F test bidez neurtu zen, p<0,05 izanik, eta F[DFn, DFd] gisa adierazi zen; non F balioen banaketari dagokio, DFn zenbakitzaileko askatasun graduei dagokio eta DFd izendatzaileko askatasun graduei dagokio. Subjektuak, Materialak and Metodoak 362 3.5.2 Western Blot frogak Western Blot frogak luze eta zabal erabiliak izan dira proteinen detekzio eta identifikaziorako, antigorputz espezifikoak erabiliaz. Prozedura hainbat pausotan egiten da: lehenengo, elektroforesiaren bidez lagineko proteinak banatu egiten dira, pisu molekularraren arabera: ondoren, proteinak mugiezin bilakatu eta transferitu egiten dira, geletik nitrozelulosa mintzera, proteinak eskuragarri izanik antigorputzekin lotzeko. Azkenik, nitrozelulosa mintzak antigorputz espezifikoen disoluzioan sartzen dira, itu diren proteinak ezagutu eta horietara lotzeko. Ikerlan honetan, lehenengo helburua, immunoprezipitazioarekin akoplaturiko [35S]GTPγS finkapen teknikan (SPA) erabilitako antigorputzen karakterizazioa eta espezifikotasuna frogatzea izan zen, Gα-proteina azpimota bakoitzarentzat. Behin antigorputzak aztertuta, Gαi1eta Gαq/11-proteinen dentsitate immunoerreaktiboa neurtu genuen postmortem aurre-garunazal laginetan eskizofrenia taldean, eskizofrenia ez zuten suizida taldean eta beraiekin parekatutako kontrol taldean. Era berean, zitoeskeletoaren zati den β-aktina proteina ere neurtu zen, jarritako proteina kopurua kontrolpean izateko asmoz. Subjektuak, Materialak and Metodoak 363 3.2 irudia: Western Blot frogen adierazpen grafikoa. 1. Laginak 95ºCtan berotzen dira 5 minutuz; 2. Lagin kantitate optimizatua jartzen da gelean; 3. Gelen elektroforesia jartzen da abian 30 minutuz eta 60 V pean, lehenengo, eta ondoren 90 minutuz 140 V pean. Horrela, proteinen banaketa ematen da pisu molekularraren arabera, eta txikienek migrazio handiena jasango dute; 4. Transferentziarako sandwichak prestatzen dira; gela katodorantz jarririk (-) eta mintza anodorantz (+); 5. Negatiboki kargatutako proteinen migrazioa gertatzen da geletik mintzera, transferentzia 0,3 A pean eta 90 minutuz luzatzen da; 6. Mintzen finkapen ezespezifikoa eragozten da esnea daraman Blocking tanpoia erabilita. Ondoren, mintza antigorputz primarioarekin inkubatzen da, eta, garbitu ostean, antigorputz sekundarioarekin inkubatzen da, ordubetez eta giro tenperaturan. Bukatzeko, irudien fluoreszentziaren neurketa egiten da. Irudia J. DelaCuesta-Barrutiaren tesirako sortutako ilustrazioa. 3.5.2.1 Mintzetan aberastutako laginen prestaketa (P2 zatikia) Western Blot frogak egiteko erabili ziren mintzen laginen prestaketa, SPA teknika egiteko erabili zirenen antzera egin zen (3.5.1.1 atala). Esperimentua egin zen egunean, 0,5 mg zituzten P2 zatikia zuten pelletak desizoztu ostean, 125 μl Tris-HCl 0,5 nM tanpoian bereseki ziren. Lagin horiei, 119 μl 2X Laemmli lagin tanpoia eta β-mercaptoetanolaren 6 μl gehitu zitzaien, laginen proteina edukia 2 mg/ml izan zedin. Eskizofrenia zuten subjektuen, Lagina berotu 95 °Ctara Laginak elektroforesiko gelean kargatu Proteinen banaketa elektroforesiaren bitartez Immunodetekzioa Proteinen transferentzia mintzera Transferentziarako gelen prestakuntza Subjektuak, Materialak and Metodoak 364 eskizofrenia ez zuten suiziden eta kontrolen laginak aldi berean prozesatu ziren, egun berean. 3.5.2.2 Gel elektroforesia, transferentzia eta immunodetekzioa Poliakrilamida geletan egindako elektroforesia Poliakrilamida geletan egiten den elektroforesia edo SDS-PAGE (Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis), desnaturalizazio egoeran egiten da, eta lagin bateko proteinen banaketa egiteko (pisu molekularraren arabera) gehien erabiltzen den teknika da. Elektroforesian zehar, lehenengo, stacking gelean migratzen dute proteinek. Gelaren zati horren osaketa %5 poliakrilamida zen, 125 mM Tris HCl, %0,1 SDS, 0,07% antigeno prostatiko espezifikoa (PSA) eta %0,14 TEMED zuen disoluzioan (pH 6,8). Laemmli-erreaktiboarekin prestatutako laginak 5 minutuz berotu ziren 95ºCtan Thermoblock gailuan, stacking gela prestatzen zen (3.2 irudia). Laginean dauden proteinen banaketa, pisu molekularraren arabera, runnig gelera heltzen direnean hasten da. Gelaren zati horren osaketa %10 akrilamida-bisakrilamida zen 0,37 M Tris HCl, %0,1 SDS, %0,07 PSA eta %0,07 TEMED zuen disoluzioan (pH 8,8). TeflonTM orrazi bat sartu zitzaien gelei 15 hilara sortzeko, laginak jarriak ziren gelaren zatian. Horrela lortutako, 15 hilaratan banatutako gelen neurriak 6 x 8 cm izan ziren (3. irudia). Hilara bakoitzean jarritako bolumena eta proteina kantitatea aztertu beharreko Gα-proteinaren araberakoa izan zen (3.5 taula). Gel bakoitzaren lehenengo hilaran pisu molekularraren markatzaile komertziala jarri zen (10250 kDa, Precision Plus ProteinTM, Dual Color Standards, Bio-Rad). Gel bakoitzean jarraitu zen laginen eta antigorputzen karakterizazioan banaketa 3.3 irudian adierazita dago. Subjektuak, Materialak and Metodoak 365 3.3 irudia: Gel bakoitzean jarritako laginen banaketa, antigorputzen karakterizazio esperimentuetarako goikoa eta taldeen arteko azterketa egiteko behekoa (eskizofrenia, eskizofrenia ez duten suizidak eta kontrolak). MW: Pisu molekularren markatzailea, P2: mintzetan aberastutako zatikien lagina, C: Kontrol subjektua, SCH: Eskizofrenia subjektua, NSCHS: Eskizofrenia ez duten subjektu suizida. Proteina errekonbinanteak Giza P2 Sagu P2 Arratoi P2 Subjektuak, Materialak and Metodoak 372 Analisi estatistikoa Antigorputzen karakterizazioa egin zenean, lortutako dentsitateen balioekin ez zen inongo analisirik egin, helburua Gα-proteina azpimota bakoitzarekiko espezifikoak ziren ala ez aztertzea zelako. Hori horrela, pisu molekularraren arabera behar zen lekuan aztarnak ikusgai zeudenean soilik neurtu ziren. Emaitza guztiak Grubb´s test analisiaren bidez aztertuak izan ziren, outlier baliorik zegoen aztertzeko, eta baiezko kasuetan analisian baztertzeko. Emaitzek banaketa gaussiarraren arabera banatuak zeuden ere aztertu zen kasu guztietan. Gainera, kontrol taldea One-sample Student’s t-test analisiaren bidez aztertu zen pool laginarekiko, beraien arteko desberdintasun esanguratsurik ez zegoela frogatzeko, %100 balioa lortzeko orduan. Eskizofrenia taldea, eskizofrenia ez zuten suiziden taldea eta kontrol taldea alderatzeko bide bakarreko ANOVA analisia erabili zen, Bonferroniren posthoc analisiarekin batera. Taldeen arteko emaitzak desberdinak zirela adierazi zen p<0,05 zen kasuetan. Aldagai independenteen (adina, PMD eta metatze denbora) eta, GNAI1 eta GNAQ proteinen espresio mailen arteko erlazioa aztertzeko Pearsonen r korrelazio koefizientea kalkulatu zen. Korrelazio hori esanguratsua zen kasuetan, kobariantza analisia (ANCOVA) egin zen taldeen esperimentalen artean (eskizofrenia, eskizofrenia ez zuten suizidak eta kontrolak). ANCOVA analisiak egiteko InVivoStat software estatistikoa erabili zen (Mockett Media 20