Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice

International Journal of Molecular Sciences Article Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice Rubén Pavia-Collado 1,2,3, Valentín Cóppola-Segovia 4, Lluís Miquel-Rio 1,2,3, Diana Alarcón-Aris 1,2, Raquel Rodríguez-Aller 5,6, María Torres-López 1,2 , Verónica Paz 1,2,3, Esther Ruiz-Bronchal 1,2,3, Leticia Campa 1,2,3, Francesc Artigas 1,2,3, Andrés Montefeltro 6,7, Raquel Revilla 6,7 and Analia Bortolozzi 1,2,3,*   Citation: Pavia-Collado, R.; Cóppola-Segovia, V.; Miquel-Rio, L.; Alarcón-Aris, D.; Rodríguez-Aller, R.; Torres-López, M.; Paz, V.; Ruiz-Bronchal, E.; Campa, L.; Artigas, F.; et al. Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice. Int. J. Mol. Sci. 2021,22, 2939. https://doi.org/10.3390/ijms22062939 Academic Editor: Salvador F. Aliño Received: 9 February 2021 Accepted: 11 March 2021 Published: 13 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Institut d’Investigacions Biomèdiques de Barcelona (IIBB), Spanish National Research Council (CSIC), 08036 Barcelona, Spain; r[email protected] (R.P.-C.); [email protected] (L.M.-R.); [email protected] (D.A.-A.); maria.torr[email protected] (M.T.-L.); [email protected] (V.P.); esther[email protected] (E.R.-B.); [email protected] (L.C.); [email protected] (F.A.) 2Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain 3Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), ISCIII, 28029 Madrid, Spain 4Laboratory of Neurobiology and Redox Pathology, Department of Basic Pathology, Federal University of Paraná(UFPR), Curitiba 81531-980, Brazil; [email protected] 5CHU de Quebec Research Center, Axe Neurosciences. Department of Molecular Medicine, Faculty of Medicine, UniversitéLaval, Quebec City, QC G1V 4G2, Canada; raquel.rodriguez-aller[email protected] 6CERVO Brain Research Centre, Quebec City, QC G1J 2G3, Canada; [email protected] (A.M.); [email protected] (R.R.) 7n-Life Therapeutics, S.L., 18100 Granada, Spain *Correspondence: [email protected] Abstract: α -Synuclein ( α -Syn) protein is involved in the pathogenesis of Parkinson’s disease (PD). Point mutations and multiplications of the α -Syn, which encodes the SNCA gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if delivered to the key affected neurons. Several experimental strategies for PD have been developed in recent years using oligonucleotide therapeutics. However, some of them have failed or even caused neuronal toxicity. One limiting step in the success of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations. Previously, we developed an indatraline-conjugated antisense oligonucleotide (IND-1233-ASO), that selectively reduces α -Syn synthesis in midbrain monoamine neurons of mice, and nonhuman primates. Here, we extended these observations using a transgenic male mouse strain carrying both A30P and A53T mutant human α -Syn (A30P*A53T* α -Syn). We found that A30P*A53T* α -Syn mice at 4–5 months of age showed 3.5-fold increases in human α -Syn expression in dopamine (DA) and norepinephrine (NE) neurons of the substantia nigra pars compacta (SNc) and locus coeruleus (LC), respectively, compared with mouse α -Syn levels. In parallel, transgenic mice exhibited altered nigrostriatal DA neurotransmission, motor alterations, and an anxiety-like phenotype. Intracerebroventricular IND1233-ASO administration (100 µ g/day, 28 days) prevented the α -Syn synthesis and accumulation in the SNc and LC, and recovered DA neurotransmission, although it did not reverse the behavioral phenotype. Therefore, the present therapeutic strategy based on a conjugated ASO could be used for the selective inhibition of α -Syn expression in PD-vulnerable monoamine neurons, showing the benefit of the optimization of ASO molecules as a disease modifying therapy for PD and related α-synucleinopathies. Keywords: α -synuclein; antisense oligonucleotide; dopamine neurotransmission; double mutant A30P*A53T*; motor deficits; Parkinson’s disease; transgenic mouse model Int. J. Mol. Sci. 2021,22, 2939. https://doi.org/10.3390/ijms22062939 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021,22, 2939 2 of 20 1. Introduction In the elderly population, Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease [ 1 , 2 ]. The illness is characterized by a variety of motor dysfunctions including difficulties in initiating movements, bradykinesia, rigidity, and resting tremor. These signs result from a reduction in striatal dopamine (DA) neurotransmission, which accompanies progressive degeneration of DA neurons in the substantia nigra pars compacta (SNc) [ 3 – 6 ]. However, PD is also characterized by a premotor phase associated with the development of neuropsychiatric symptoms, cognitive deficits, among others, involving dysfunctions of non-DA neurons, e.g., serotonin (5-HT) and norepinephrine (NE) neurons, which precedes the development of motor symptoms [ 7 , 8 ]. Although the causes of PD are still poorly understood, genetic studies have identified two highly penetrant mutations, A30P and A53T, in α -synuclein ( α -Syn), which encodes SNCA gene associated with autosomal dominant inheritance of the PD [ 9 , 10 ]. α - Syn is a highly conserved protein made up of 140 amino acid residues that is predominantly expressed in neurons, and is abundantly localized in presynaptic terminals, which plays a significant role in the regulation of neurotransmitter release, synaptic function, and neuroplasticity [11–13]. Accumulation of α -Syn in intracytoplasmic inclusions called Lewy bodies is a neuropathological hallmark of PD. Therefore, one approach to modelling the disease is to modify the mouse genome in order to over-express wild-type human α -Syn (hα -Syn), as well as the PD-linked α -Syn mutants A53T, A30P, and E46K, or even combinations of them [ 14 – 19 ]. Indeed, Richfield et al. [ 20 ] introduced a transgenic mouse model carrying a double mutant hα -Syn gene with both A30P and A53T point mutations under the tyrosine hydroxylase (TH) promoter (A30P*A53T* α -Syn). Aged A30P*A53T* α -Syn mice (13–23 months) successfully recapitulated many important features of α-synucleinopathy, including increased α -Syn expression, which adversely induced an age-dependent DA neurodegeneration and a decrease in the DA concentration in the striatal tissue, leading to deficits in motor coordination. Moreover, A30P*A53T* α -Syn mice showed swollen, dystrophic TH + neurites in the SNc, as well as accumulation of oligomeric α -Syn forms at 12 months, supporting the proposal that A30P*A53T*α-Syn mice may be used as a model to test new PD-modifying therapies that reduce α-Syn expression/accumulation [21,22]. In the last decade, remarkable advances in the development of oligonucleotide therapies aimed at inhibiting α -Syn synthesis have been made [ 23 ]. Gene silencing mechanisms targeting α -Syn mRNA may reduce the intracellular protein content and stop/slow the progression of the illness. Preclinical studies in rodents and nonhuman primates have successfully shown that α -Syn can be downregulated in PD-affected brain areas after direct application of oligonucleotide therapeutics including antisense oligonucleotides (ASO), small interfering RNAs (siRNA), and microRNAs (miRNA) [ 24 – 31 ]. Irrespective of these potential hitches, a major limitation in the development of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations or cell types. In an effort to solve this problem, we successfully developed a strategy to supply in vivo oligonucleotides selectively to brainstem monoamine neurons (DA, 5-HT, and NE). This was achieved by conjugating oligonucleotides with inhibitors of monoamine transporters (MAT) showing nM affinity for MAT, such as sertraline, reboxetine, and indatraline (IND), which are exclusively expressed in monoamine neurons at high densities [ 32 – 36 ]. MAT inhibitors allow the selective accumulation of oligonucleotides in monoamine neurons after internalization in deep Rab-7-associated vesicles [33,35]. Recently, we reported that intracerebroventricular or intranasal administration of an indatraline-conjugated 1233-ASO (IND-1233-ASO) can effectively reduce α -Syn protein accumulation in the brainstem monoamine neurons of wild-type mice and nonhuman primates, without causing neurotoxicity [ 34 , 36 ]. In the present study, we extended these observations and assessed whether an IND-1233-ASO sequence designed in such a way that the target mRNA sequence displays homology with the murine, rhesus macaque, and human α -Syn is able to downregulate hα -Syn expression in DA and NE brain areas of Int. J. Mol. Sci. 2021,22, 2939 3 of 20 transgenic A30P*A53T*α-Syn mice. Furthermore, since the A30P and A53T familial point mutations in the SNCA gene are a risk factor for early-onset PD [ 9 , 10 , 37 ], we also examined the anxiety-depressive phenotype and cognitive abnormalities, as well as DA function in middle-aged mice (5 months), as these features have not been assessed in previous studies using this transgenic mouse model. 2. Results 2.1. α-Syn Expression Profile in Brain Areas of A30P*A53T*α-Syn Transgenic Mice We first examined the expression of hα -Syn and murine α -Syn (mα -Syn) mRNAs in several cortical and subcortical brain areas of non-transgenic (non-Tg) and transgenic A30P*A53T* α -Syn mice (Figure 1a and Supplemental Figure S1). Using in situ hybridization, we found that hα -Syn mRNA levels were more than 3-fold higher than mα -Syn mRNA levels selectively in the brainstem nuclei containing DA and NE cell bodies in brain areas such as the SNc, ventral tegmental area—VTA and locus coeruleus—LC (SNc/VTA: 316.9% ± 18.3%; LC: 377.5% ± 22.5%, respectively, versus mα -Syn mRNA levels) (Figure 1b) . The Student’s t-test indicated values of t= 10.52, p< 0.0001 for SNc/VTA and t= 11.91, p< 0.0001 for LC, respectively. Moreover, mα -Syn mRNA expression was unchanged, and comparable values were detected in cortical and subcortical brain areas of non-Tg and transgenic A30P*A53T* α -Syn mice (Supplemental Figure S1b). In parallel, A30P*A53T* α -Syn mice showed also significant reductions in murine γ -synuclein ( γ -Syn) mRNA expression in the SNc/VTA and LC compared with non-Tg mice (t= 2.838, p= 0.0296 ;t= 3.005, p= 0.0239 , Student’s t-test, respectively) (Figure 1c,d). The increases in hα -Syn mRNA levels in the SNc/VTA and LC were confirmed by immunohistochemistry assessment of h-α-Syn protein levels (Figure 1e). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 21 accumulation in the brainstem monoamine neurons of wild-type mice and nonhuman primates, without causing neurotoxicity [34,36]. In the present study, we extended these observations and assessed whether an IND-1233-ASO sequence designed in such a way that the target mRNA sequence displays homology with the murine, rhesus macaque, and human α-Syn is able to downregulate h-α-Syn expression in DA and NE brain areas of transgenic A30P*A53T*α-Syn mice. Furthermore, since the A30P and A53T familial point mutations in the SNCA gene are a risk factor for early-onset PD [9,10,37], we also examined the anxiety-depressive phenotype and cognitive abnormalities, as well as DA function in middle-aged mice (5 months), as these features have not been assessed in previous studies using this transgenic mouse model. 2. Results 2.1. α-Syn Expression Profile in Brain Areas of A30P*A53T*α-Syn Transgenic Mice We first examined the expression of h-α-Syn and murine α-Syn (m-α-Syn) mRNAs in several cortical and subcortical brain areas of non-transgenic (non-Tg) and transgenic A30P*A53T*α-Syn mice (Figure 1a and Supplemental Figure S1). Using in situ hybridization, we found that h-α-Syn mRNA levels were more than 3-fold higher than m-α-Syn mRNA levels selectively in the brainstem nuclei containing DA and NE cell bodies in brain areas such as the SNc, ventral tegmental area—VTA and locus coeruleus—LC (SNc/VTA: 316.9% ± 18.3%; LC: 377.5% ± 22.5%, respectively, versus m-α-Syn mRNA levels) (Figure 1b). The Student’s t-test indicated values of t = 10.52, p < 0.0001 for SNc/VTA and t = 11.91, p < 0.0001 for LC, respectively. Moreover, m-α-Syn mRNA expression was unchanged, and comparable values were detected in cortical and subcortical brain areas of non-Tg and transgenic A30P*A53T*α-Syn mice (Supplemental Figure S1b). In parallel, A30P*A53T*α-Syn mice showed also significant reductions in murine γ-synuclein (γ-Syn) mRNA expression in the SNc/VTA and LC compared with non-Tg mice (t = 2.838, p = 0.0296; t = 3.005, p = 0.0239, Student’s t-test, respectively) (Figure 1c,d). The increases in hα-Syn mRNA levels in the SNc/VTA and LC were confirmed by immunohistochemistry assessment of h-α-Syn protein levels (Figure 1e). Figure 1. Profile of α-Syn expression in monoamine brain areas of A30P*A53T*α-Syn transgenic mice. (a) Coronal brain sections showing h-α-Syn mRNA levels in SNc/VTA assessed by in situ hybridization. Yellow arrowheads indicate the brain regions quantified in b. Scale bar: 1 mm. (b) Increased h-α-Syn mRNA expression in SNc/VTA and LC of A30P*A53T*α-Syn transgenic mice compared with m-α-Syn mRNA expression levels in the same mice (n = 5 mice/group; *** p < 0.001 vs. m-α-Syn mRNA levels; Student’s t-test). (c) Coronal brain sections showing m-γ-Syn mRNA levels in SNc/VTA and LC assessed by in situ hybridization. Yellow arrowheads show the lower density of m-γ-Syn expression in Figure 1. Profile of α -Syn expression in monoamine brain areas of A30P*A53T* α -Syn transgenic mice. ( a ) Coronal brain sections showing hα -Syn mRNA levels in SNc/VTA assessed by in situ hybridization. Yellow arrowheads indicate the brain regions quantified in b. Scale bar: 1 mm. ( b ) Increased hα -Syn mRNA expression in SNc/VTA and LC of A30P*A53T* α -Syn transgenic mice compared with mα -Syn mRNA expression levels in the same mice (n= 5 mice/group; *** p< 0.001 vs. mα -Syn mRNA levels; Student’s t-test). ( c ) Coronal brain sections showing mγ -Syn mRNA levels in SNc/VTA and LC assessed by in situ hybridization. Yellow arrowheads show the lower density of mγ -Syn expression in A30P*A53T* α -Syn mice compared with non-Tg mice. Scale bar: 1 mm. ( d ) Reduced mγ -Syn mRNA expression in SNc/VTA and LC of A30P*A53T* α -Syn transgenic mice compared with non-Tg mice (n= 4–5 mice/group; * p< 0.05, ** p< 0.01 versus non-Tg mice; Student’s t-test). ( e ) Representative photomicrographs showing accumulated levels of hα -Syn protein in SNc/VTA and LC of A30P*A53T* α -Syn transgenic mice. White frames indicate the SNc/VTA and LC brain regions. Scale bar: 200 µ m. For all figures, data represent the mean ± SEM. Abbreviations: SNc, substantia nigra compacta; VTA, ventral tegmental area; LC, locus coeruleus; 4V, 4-ventricle. See also Supplemental Figure S1. Int. J. Mol. Sci. 2021,22, 2939 4 of 20 However, unlike the aged A30P*A53T* α -Syn mice (13–23 months), the middle-aged mice (5 months) did not show a loss of tyrosine hydroxylase (TH) + cells in the SNc, VTA and LC, nor were there any changes in TH density in the caudate putamen (CPu) compared with non-Tg mice (Figure 2a,b). Likewise, no alterations were found using other DA and NE neuronal markers, including the DA transporter (DAT) and NE transporter (NET) proteins, respectively. Histochemical analysis showed that the DAT density levels in SNc/VTA and CPu result in similar levels of immunoreactivity for both phenotypes (Figure 2c,d). In addition, the NET density was comparable in the LC and medial prefrontal cortex (mPFC) of A30P*A53T* α -Syn and non-Tg mice (Figure 2e,f). Altogether, these results indicate that A30P*A53T* α -Syn overexpression, and concomitant reduction in γ -Syn levels in the SNc/VTA and LC occurred in the absence of monoamine neurodegeneration, at least in middle-aged mice. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 21 A30P*A53T*α-Syn mice compared with non-Tg mice. Scale bar: 1 mm. (d) Reduced m-γ-Syn mRNA expression in SNc/VTA and LC of A30P*A53T*α-Syn transgenic mice compared with non-Tg mice (n = 4–5 mice/group; * p < 0.05, ** p < 0.01 versus non-Tg mice; Student’s t-test). (e) Representative photomicrographs showing accumulated levels of h-α-Syn protein in SNc/VTA and LC of A30P*A53T*α-Syn transgenic mice. White frames indicate the SNc/VTA and LC brain regions. Scale bar: 200 µm. For all figures, data represent the mean ± SEM. Abbreviations: SNc, substantia nigra compacta; VTA, ventral tegmental area; LC, locus coeruleus; 4V, 4-ventricle. See also Supplemental Figure S1. However, unlike the aged A30P*A53T*α-Syn mice (13–23 months), the middle-aged mice (5 months) did not show a loss of tyrosine hydroxylase (TH)+ cells in the SNc, VTA and LC, nor were there any changes in TH density in the caudate putamen (CPu) compared with non-Tg mice (Figure 2a,b). Likewise, no alterations were found using other DA and NE neuronal markers, including the DA transporter (DAT) and NE transporter (NET) proteins, respectively. Histochemical analysis showed that the DAT density levels in SNc/VTA and CPu result in similar levels of immunoreactivity for both phenotypes (Figure 2c,d). In addition, the NET density was comparable in the LC and medial prefrontal cortex (mPFC) of A30P*A53T*α-Syn and non-Tg mice (Figure 2e,f). Altogether, these results indicate that A30P*A53T*α-Syn overexpression, and concomitant reduction in γSyn levels in the SNc/VTA and LC occurred in the absence of monoamine neurodegeneration, at least in middle-aged mice. Figure 2. Integrity of DA and NE systems of middle-aged (5 months) A30P*A53T*α-Syn transgenic mice. (a) TH-immunostained brain sections containing SNc/VTA, LC and CPu. Scale bar: 200 µm. (b) No differences in the number of TH+ neurons were found in the SNc, VTA or LC of A30P*A53T*α-Syn vs. non-Tg mice. Likewise, the density of striatal TH+ terminals was comparable between both phenotypes. (c) DAT-immunostained brain sections containing SNc/VTA and CPu. Scale bar: 200 µm. (d) No differences in DAT protein density were found in the SNc, VTA or CPu of A30P*A53T*αSyn vs. non-Tg mice. (e) Confocal images showing the NET protein density in LC and mPFC of A30P*A53T*α-Syn and non-Tg mice. Scale bar: 200 µm. (f) No differences in NET protein density were found in LC and mPFC of both phenotypes. Data are represented as mean ± SEM, n = 4–5 mice/group. Abbreviations: mPFC, medial prefrontal cortex; CPu, caudate Figure 2. Integrity of DA and NE systems of middle-aged (5 months) A30P*A53T* α -Syn transgenic mice. ( a ) THimmunostained brain sections containing SNc/VTA, LC and CPu. Scale bar: 200 µ m. ( b ) No differences in the number of TH + neurons were found in the SNc, VTA or LC of A30P*A53T* α -Syn vs. non-Tg mice. Likewise, the density of striatal TH + terminals was comparable between both phenotypes. ( c ) DAT-immunostained brain sections containing SNc/VTA and CPu. Scale bar: 200 µ m. ( d ) No differences in DAT protein density were found in the SNc, VTA or CPu of A30P*A53T* α -Syn vs. non-Tg mice. ( e ) Confocal images showing the NET protein density in LC and mPFC of A30P*A53T* α -Syn and non-Tg mice. Scale bar: 200 µ m. ( f ) No differences in NET protein density were found in LC and mPFC of both phenotypes. Data are represented as mean ± SEM, n= 4–5 mice/group. Abbreviations: mPFC, medial prefrontal cortex; CPu, caudate putamen; HPC, hippocampus; SNc, substantia nigra compacta; VTA, ventral tegmental area; DR, dorsal raphe nucleus; LC, locus coeruleus. Int. J. Mol. Sci. 2021,22, 2939 5 of 20 2.2. A30P*A53T*α-Syn Transgenic Mice Show Motor Deficits and an Anxiety-Like Phenotype To determine the functional consequences of A30P*A53T* α -Syn overexpression in SNc/VTA and LC, we performed a behavioral study assessing motor, emotional and cognitive components. Compared with non-Tg mice, A30P*A53T* α -Syn mice showed a reduced spontaneous locomotor activity as assessed in the open field test (Figure 3a,b). Differences were found between A30P*A53T* α -Syn and non-Tg mice in terms of total distance traveled, fast movements, mean speed, resting time, and vertical count. The total distance traveled and frequency of fast movements, but not slow movements, were significantly lower in A30P*A53T* α -Syn than non-Tg mice (t= 3.199, p= 0.0035; t= 2.675, p= 0.0125, Student’s t-test, respectively). Likewise, A30P*A53T* α -Syn mice exhibited a reduced mean speed (t= 3.105, p= 0.0044, Student’s t-test), and a longer resting time (t= 2.785 ,p= 0.0097, Student’s t-test) compared with non-Tg mice. Vertical counts were also significantly lower in A30P*A53T* α -Syn mice than in non-Tg mice both in the open field test (Figure 3a, t= 2.91, p= 0.0074, Student’s t-test) and cylinder test (Figure 3c, t= 2.397 , p= 0.0247 , Student’s t-test). However, A30P*A53T* α -Syn mice showed no difference compared to non-Tg mice in motor asymmetry in the cylinder test as expected (Figure 3c). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 21 putamen; HPC, hippocampus; SNc, substantia nigra compacta; VTA, ventral tegmental area; DR, dorsal raphe nucleus; LC, locus coeruleus. 2.2. A30P*A53T*α-Syn Transgenic Mice Show Motor Deficits and an Anxiety-Like Phenotype To determine the functional consequences of A30P*A53T*α-Syn overexpression in SNc/VTA and LC, we performed a behavioral study assessing motor, emotional and cognitive components. Compared with non-Tg mice, A30P*A53T*α-Syn mice showed a reduced spontaneous locomotor activity as assessed in the open field test (Figure 3a,b). Differences were found between A30P*A53T*α-Syn and non-Tg mice in terms of total distance traveled, fast movements, mean speed, resting time, and vertical count. The total distance traveled and frequency of fast movements, but not slow movements, were significantly lower in A30P*A53T*α-Syn than non-Tg mice (t = 3.199, p = 0.0035; t = 2.675, p = 0.0125, Student’s t-test, respectively). Likewise, A30P*A53T*α-Syn mice exhibited a reduced mean speed (t = 3.105, p = 0.0044, Student’s t-test), and a longer resting time (t = 2.785, p = 0.0097, Student’s t-test) compared with non-Tg mice. Vertical counts were also significantly lower in A30P*A53T*α-Syn mice than in non-Tg mice both in the open field test (Figure 3a, t = 2.91, p = 0.0074, Student’s t-test) and cylinder test (Figure 3c, t = 2.397, p = 0.0247, Student’s t-test). However, A30P*A53T*α-Syn mice showed no difference compared to non-Tg mice in motor asymmetry in the cylinder test as expected (Figure 3c). Figure 3. Impairment of motor function and anxiety-like behaviors in A30P*A53T*α-Syn transgenic mice. (a) Comparison of spontaneous locomotor activity between 5-month-old A30P*A53T*α-Syn and non-Tg mice in the open field test. Total distance traveled, fast movements, average speed and vertical count were significantly decreased in A30P*A53T*α-Syn mice compared with non-Tg mice. In parallel, transgenic mice showed an increased resting time compared with non-Tg mice. (b) Representative locomotor activity tracking was obtained from both phenotypes. (c) No difference in motor asymmetry examined by the use of the left paw was detected. However, A30P*A53T*α-Syn mice showed a reduced vertical count in the cylinder test compared with non-Tg mice. (d) In the dark-light box test, A30P*A53T*α-Syn transgenic mice evoked an anxiety-like response compared with non-Tg mice, as shown by the shorter spent time in the lit box but the marginal effects in the latency period to entering in the light box or the number of entries. Data are the mean ± SEM, n = 10–15 mice/group. Student’s t-test, * p < 0.05, ** p < 0.01 compared with non-Tg mice. See also Supplemental Figure S2. Figure 3. Impairment of motor function and anxiety-like behaviors in A30P*A53T* α -Syn transgenic mice. ( a ) Comparison of spontaneous locomotor activity between 5-month-old A30P*A53T* α -Syn and non-Tg mice in the open field test. Total distance traveled, fast movements, average speed and vertical count were significantly decreased in A30P*A53T* α -Syn mice compared with non-Tg mice. In parallel, transgenic mice showed an increased resting time compared with non-Tg mice. ( b ) Representative locomotor activity tracking was obtained from both phenotypes. ( c ) No difference in motor asymmetry examined by the use of the left paw was detected. However, A30P*A53T* α -Syn mice showed a reduced vertical count in the cylinder test compared with non-Tg mice. ( d ) In the dark-light box test, A30P*A53T* α -Syn transgenic mice evoked an anxiety-like response compared with non-Tg mice, as shown by the shorter spent time in the lit box but the marginal effects in the latency period to entering in the light box or the number of entries. Data are the mean ± SEM, n= 10–15 mice/group. Student’s t-test, * p< 0.05, ** p< 0.01 compared with non-Tg mice. See also Supplemental Figure S2. Mice were also tested using a dark-light box paradigm, a behavioral task assessing the anxiety-like phenotype (Figure 3d). The A30P*A53T*α-Syn mice showed a significant Int. J. Mol. Sci. 2021,22, 2939 6 of 20 reduction in time spent in the light box (t= 2.162, p= 0.0404, Student’s t-test), and there were marginal effects on latency and the number of entries in the light box, suggesting an anxious phenotype compared with non-Tg mice. Nevertheless, no differences between both phenotypes were detected in the tail suspension test or in the novel object recognition, behavioral tasks assessing vulnerability/resilience to stress and short-term memory, respectively (Supplemental Figure S2). 2.3. A30P*A53T*α-Syn Overexpression in TH+Neurons Impairs DA Neurotransmission in the Nigrostriatal Pathway The impact of the mutant α -Syn transgene overexpression on the nigrostriatal DA function was examined in 5-month-old A30P*A53T* α -Syn and non-Tg mice using in vivo microdialysis procedures. A30P*A53T* α -Syn transgenic mice showed no differences in baseline extracellular concentrations of DA and its 3,4-dihidroxyphenylacetic acid (DOPAC) metabolite in CPu compared with non-Tg mice (Table 1). However, infusion of the depolarizing agent veratridine (50 µ M) by reverse dialysis increased extracellular DA in CPu to a lesser extend in A30P*A53T* α -Syn than in non-Tg mice (~2-fold versus ~6-fold, respectively) (Figure 4a). Two-way ANOVA followed by Tukey’s post hoc test analysis showed a marginal effect of group F (1,7) = 3.993, p= 0.0858, and effect of time F(15,105) = 19.85 , p< 0.0001 and a group-by-time interaction F(15,105) = 3.304, p= 0.0002. Table 1. Baseline DA and DOPAC dialysate concentrations in the CPu of mice. Mice Experimental Conditions Baseline DA Baseline DOPAC non-Tg aCSF 10.3 ±1.6 (15) 0.8 ±0.1 (15) aCSF + DMSO 1% 7.2 ±2.2 (4) 1.6 ±0.4 (4) A30P*A53T*α-Syn aCSF 8.5 ±1.2 (15) 1.1 ±0.1 (15) aCSF + DMSO 1% 6.6 ±1.7 (5) 1.8 ±0.5 (5) Extracellular DA and DOPAC levels are expressed as the fmol/20-min fraction. In the experiments involving the evaluation of the veratridine effect on extracellular DA and DOPAC levels, dimethyl-sulfoxide (DMSO) was added to the artificial cerebrospinal fluid (aCSF), respectively. Data are the mean ± SEM of the number of mice shown in parentheses. Moreover, both α -Syn and γ -Syn proteins modulate membrane distribution and the reuptake function of DAT at DA terminals [ 19 , 34 ]. Local application of amphetamine (DA releaser and DAT inhibitor, 1–10 µ M) dose-dependently elevated the dialysate DA concentration in the CPu, being significantly higher in A30P*A53T* α -Syn transgenic mice compared with non-Tg mice (Figure 4b). Two-way ANOVA showed effects of group F(1,126) = 7.828 ,p= 0.0060 and time F (17,126) = 6.235, p< 0.0001, and a marginal effect of group-by-time interaction F (17,126) = 1.560, p= 0.0849. However, no significant differences were observed in the extracellular DA concentration in the CPu between both phenotypes after local application of DAT inhibitor nomifensine (1–10 µ M) (Figure 4c), suggesting that the greater DA elevation induced by amphetamine in A30P*A53T* α -Syn transgenic mice is not due to a differential expression/function of DAT. Two-way ANOVA showed an effect of time F (17,162) = 7.474, p< 0.0001, but not of group as well as a group-by-time interaction. To obtain more information on the mechanisms controlling DA neurotransmission in the nigrostriatal pathways of A30P*A53T* α -Syn mice, we also examined the effect of the DA D2 receptor agonist quinpirole on DA release. Local quinpirole infusion (10 µ M) by reverse dialysis comparably reduced the extracellular DA concentration in the CPu of non-Tg and A30P*A53T* α -Syn mice (Figure 4d). Two-way ANOVA showed an effect of time F (17,144) = 11.20, p< 0.0001, but not of group as well as a group-by-time interaction. No significant differences in extracellular DOPAC levels were observed between both phenotypes with the different pharmacological approaches used (data not shown) [19]. Int. J. Mol. Sci. 2021,22, 2939 7 of 20 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 21 Figure 4. A30P*A53T*α-Syn overexpression in TH+ neurons alters DA neurotransmission in the nigrostriatal pathway. (a) Local veratridine infusion (depolarizing agent; 50 µM) significantly increased DA release in the caudate putamen (CPu) of both mouse phenotypes. However, this effect was significant smaller in A30P*A53T*α-Syn mice than in non-Tg mice. (b) Direct application of amphetamine (DA releaser and DAT inhibitor; 1 and 10 µM) by reverse dialysis induced increases in DA release in the CPu, with the effect being significantly more pronounced in A30P*A53T*αSyn mice than in non-Tg mice. (c) However, local nomifensine infusion (DAT inhibitor; 1 and 10 µM) dose-dependently increased the extracellular DA concentration in CPu, with this effect comparable being in both phenotypes. (d) Local activation of DA D2 receptors with quinpirole (DA D2 agonist, 10 µM) similarly decreased striatal DA release in A30P*A53T*α-Syn and non-Tg mice. Data are expressed as the mean ± SEM, n = 4–6 mice/group as indicated in the parenthesis. Twoway ANOVA and Tukey’s multiple comparisons test, ** p < 0.01, *** p < 0.001 compared with nonTg mice. 2.4. ASO Therapy Reduces the Accumulation of Human α-Syn and Normalizes DA Neurotransmission Deficits but Not the Behavioral Phenotype in A30P*A53T*α-Syn Transgenic Mice Recently, we reported that IND-1233-ASO selectively reduced murine α-Syn expression in monoaminergic neurons of wild-type mice and nonhuman primates [34,36]. Likewise, the IND-1337-ASO sequence, designed specifically to target h-α-Syn in a PD-like mouse model overexpressing wild-type h-α-Syn induced by an adeno-associated viral vector, also prevented h-α-Syn accumulation in interconnected DA brain regions [36]. This specificity is due to the potent in vitro affinity and in vivo occupancy of IND for monoamine transporters [38], which allows the accumulation of the conjugated oligonucleotide in this neuronal population after intracerebroventricular or intranasal administration. Here, we extended these previous studies, and confirmed the intracellular accumulation of IND-conjugated oligonucleotides, specifically in TH+ neurons of the SNc/VTA and LC of A30P*A53T*α-Syn mice after a single intracerebroventricular application of Figure 4. A30P*A53T* α -Syn overexpression in TH + neurons alters DA neurotransmission in the nigrostriatal pathway. ( a ) Local veratridine infusion (depolarizing agent; 50 µ M) significantly increased DA release in the caudate putamen (CPu) of both mouse phenotypes. However, this effect was significant smaller in A30P*A53T* α -Syn mice than in non-Tg mice. ( b ) Direct application of amphetamine (DA releaser and DAT inhibitor; 1 and 10 µ M) by reverse dialysis induced increases in DA release in the CPu, with the effect being significantly more pronounced in A30P*A53T* α -Syn mice than in non-Tg mice. ( c ) However, local nomifensine infusion (DAT inhibitor; 1 and 10 µ M) dose-dependently increased the extracellular DA concentration in CPu, with this effect comparable being in both phenotypes. ( d ) Local activation of DA D2 receptors with quinpirole (DA D2 agonist, 10 µ M) similarly decreased striatal DA release in A30P*A53T* α -Syn and non-Tg mice. Data are expressed as the mean ± SEM, n= 4–6 mice/group as indicated in the parenthesis. Two-way ANOVA and Tukey’s multiple comparisons test, ** p< 0.01, *** p< 0.001 compared with non-Tg mice. 2.4. ASO Therapy Reduces the Accumulation of Human α-Syn and Normalizes DA Neurotransmission Deficits but Not the Behavioral Phenotype in A30P*A53T* α -Syn Transgenic Mice Recently, we reported that IND-1233-ASO selectively reduced murine α -Syn expression in monoaminergic neurons of wild-type mice and nonhuman primates [ 34 , 36 ]. Likewise, the IND-1337-ASO sequence, designed specifically to target hα -Syn in a PD-like mouse model overexpressing wild-type hα -Syn induced by an adeno-associated viral vector, also prevented hα -Syn accumulation in interconnected DA brain regions [ 36 ]. This specificity is due to the potent in vitro affinity and in vivo occupancy of IND for monoamine transporters [ 38 ], which allows the accumulation of the conjugated oligonucleotide in this neuronal population after intracerebroventricular or intranasal administration. Here, we extended these previous studies, and confirmed the intracellular accumulation of INDconjugated oligonucleotides, specifically in TH + neurons of the SNc/VTA and LC of A30P*A53T* α -Syn mice after a single intracerebroventricular application of IND-1233-ASO (100 µ g/mouse) (Figure 5a). In addition, IND-1233-ASO administration (100 µ g/day for 28 days) significantly reduced mutant α -Syn transgene expression in the SNc/VTA and LC compared with A30P*A53T* α -Syn mice treated with a vehicle (Figure 5b). The reduction in hα -Syn mRNA was ~54% and ~32% in the SNc/VTA and LC, respectively, compared to the level in A30P*A53T* α -Syn mice receiving a vehicle (Figure 5c). Two-way ANOVA showed an effect of treatment F (1,12) = 42.84, p< 0.0001, α -Syn phenotype F (1,12) = 99.71, p< 0.0001 and treatment-byα -Syn phenotype interaction F (1,12 ) = 42.84, p< 0.0001 for Int. J. Mol. Sci. 2021,22, 2939 8 of 20 SNc/VTA, as well as effects of treatment F (1,12) = 10.06, p= 0.0080 and α -Syn phenotype F (1,12) = 157.3, p< 0.0001 and a treatment-byα -Syn phenotype interaction F (1,12) = 17.47, p= 0.0013 for LC. Interestingly, IND-1233-ASO treatment did not alter mα -Syn mRNA expression (Figure 5c). The decreased hα -Syn mRNA level in the SNc/VTA and LC was accompanied by a significant decrease in the hα -Syn protein level, as assessed by immunohistochemistry (SNc/VTA: t= 3.047, p= 0.00226, LC: t= 5.812, p= 0.0011, Student’s t-test) (Figure 5d,e). Likewise, a lower number of hα -Syn + cells was found in the SNc/VTA, but not in the LC, of A30P*A53T* α -Syn mice treated with IND-1233-ASO vs. those treated with the vehicle (t= 7.163, p= 0.0004, Student’s t-test) (Figure 5e). Furthermore, hα -Syn protein accumulation also decreased in brain areas with dense DA and NE innervation, such as the CPu (t = 2.809, p= 0.0376, Student’s t-test) and mPFC (t= 2.591, p= 0.0411, Student’s t-test) (Figure 5d,e). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 21 IND-1233-ASO (100 µg/mouse) (Figure 5a). In addition, IND-1233-ASO administration (100 µg/day for 28 days) significantly reduced mutant α-Syn transgene expression in the SNc/VTA and LC compared with A30P*A53T*α-Syn mice treated with a vehicle (Figure 5b). The reduction in h-α-Syn mRNA was ∼54% and ∼32% in the SNc/VTA and LC, respectively, compared to the level in A30P*A53T*α-Syn mice receiving a vehicle (Figure 5c). Two-way ANOVA showed an effect of treatment F(1,12) = 42.84, p < 0.0001, α-Syn phenotype F(1,12) = 99.71, p < 0.0001 and treatment-byα-Syn phenotype interaction F(1,12) = 42.84, p < 0.0001 for SNc/VTA, as well as effects of treatment F(1,12) = 10.06, p = 0.0080 and α-Syn phenotype F(1,12) = 157.3, p < 0.0001 and a treatment-by-α-Syn phenotype interaction F(1,12) = 17.47, p = 0.0013 for LC. Interestingly, IND-1233-ASO treatment did not alter m-αSyn mRNA expression (Figure 5c). The decreased h-α-Syn mRNA level in the SNc/VTA and LC was accompanied by a significant decrease in the h-α-Syn protein level, as assessed by immunohistochemistry (SNc/VTA: t = 3.047, p = 0.00226, LC: t = 5.812, p = 0.0011, Student’s t-test) (Figure 5d,e). Likewise, a lower number of h-α-Syn+ cells was found in the SNc/VTA, but not in the LC, of A30P*A53T*α-Syn mice treated with IND-1233-ASO vs. those treated with the vehicle (t = 7.163, p = 0.0004, Student’s t-test) (Figure 5e). Furthermore, h-α-Syn protein accumulation also decreased in brain areas with dense DA and NE innervation, such as the CPu (t = 2.809, p = 0.0376, Student’s t-test) and mPFC (t = 2.591, p = 0.0411, Student’s t-test) (Figure 5d,e). Figure 5. Intracerebroventricular IND-1233-ASO treatment selectively inhibits the expression and accumulation of h-αSyn in interconnected DA and NE brain regions of A30P*A53T*α-Syn transgenic mice. (a) Confocal images showing the co-localization of A488-IND-1233-ASO (green) with TH+ neurons (red) in the SNc/VTA and LC. Cell nuclei were stained with DAPI (blue). Scale bar: 10 µm. (b) Coronal brain sections showing reduced h-α-Syn mRNA expression in the SNc/VTA and LC of A30P*A53T*α-Syn mice treated with IND-1233-ASO (100 µg/day for 28 days), as assessed by in situ hybridization. Yellow arrowheads show h-α-Syn mRNA expression in SNc/VTA and LC. Scale bar: 1 mm. (c) Bar graphs showing significant reductions in h-α-Syn mRNA levels, but not m-α-Syn mRNA, in SNc/VTA and LC of IND-1233-ASOtreated A30P*A53T*α-Syn mice (n = 5 mice/group; *** p < 0.001 vs. m-α-Syn mRNA levels; ## p < 0.01, ### p < 0.001 vs. vehicletreated A30P*A53T*α-Syn mice; two-way ANOVA and Tukey’s multiple comparisons test). (d) Representative photomicrographs showing a lower h-α-Syn protein density in the SNc/VTA, LC, CPu and mPFC of IND-1233-ASO-treated Figure 5. Intracerebroventricular IND-1233-ASO treatment selectively inhibits the expression and accumulation of hα - Syn in interconnected DA and NE brain regions of A30P*A53T* α -Syn transgenic mice. ( a ) Confocal images showing the co-localization of A488-IND-1233-ASO (green) with TH + neurons (red) in the SNc/VTA and LC. Cell nuclei were stained with DAPI (blue). Scale bar: 10 µ m. ( b ) Coronal brain sections showing reduced hα -Syn mRNA expression in the SNc/VTA and LC of A30P*A53T* α -Syn mice treated with IND-1233-ASO (100 µ g/day for 28 days), as assessed by in situ hybridization. Yellow arrowheads show hα -Syn mRNA expression in SNc/VTA and LC. Scale bar: 1 mm. ( c ) Bar graphs showing significant reductions in hα -Syn mRNA levels, but not mα -Syn mRNA, in SNc/VTA and LC of IND-1233ASO-treated A30P*A53T* α -Syn mice (n= 5 mice/group; *** p< 0.001 vs. mα -Syn mRNA levels; ## p< 0.01, ### p< 0.001 vs. vehicle-treated A30P*A53T* α -Syn mice; two-way ANOVA and Tukey’s multiple comparisons test). ( d ) Representative photomicrographs showing a lower hα -Syn protein density in the SNc/VTA, LC, CPu and mPFC of IND-1233-ASO-treated A30P*A53T* α -Syn mice vs. vehicle-treated A30P*A53T* α -Syn mice. White frames indicate SNc/VTA and LC brain regions. Scale bar: 200 µ m. ( e ) Bar graphs showing significant reductions in hα -Syn protein levels in the brain areas analyzed, as well as a decreased number of hα -Syn + cells in the SNc/VTA of IND-1233-ASO-treated A30P*A53T* α -Syn mice (n= 4 mice/group; * p< 0.05, ** p< 0.01 versus vehicle-treated A30P*A53T* α -Syn mice; Student’s t-test). ( f ) Representative images showing increased mγ -Syn mRNA expression in SNc/VTA and LC of A30P*A53T* α -Syn mice treated with IND-1233-ASO as assessed by in situ hybridization. Yellow arrowheads show m-γ-Syn mRNA expression in SNc/VTA and LC. Scale bar: 1 mm. ( g ) Bar graphs showing significant increases in mγ -Syn expression in the LC and a marginal effect in the SNc/VTA of IND-1233-ASO-treated A30P*A53T* α -Syn mice (n= 4 mice/group; * p< 0.05, ** p< 0.01 vs. non-Tg mice; # p< 0.05 vs. vehicle-treated A30P*A53T* α -Syn mice; one-way ANOVA). For all figures, data are the mean ± SEM. Abbreviations: mPFC, medial prefrontal cortex; CPu, caudate putamen; SNc, substantia nigra compacta; VTA, ventral tegmental area; LC, locus coeruleus. See also Supplemental Figure S3. Int. J. Mol. Sci. 2021,22, 2939 9 of 20 In parallel, the IND-1233-ASO-induced decrease in hα -Syn expression normalized γ - Syn mRNA expression in the SNc/VTA (marginal effect) and LC of A30P*A53T* α -Syn mice compared to those mice treated with a vehicle (Figure 5f,g). One-way ANOVA showed an effect of group in SNc/VTA (F (2,6) = 14.01, p= 0.0055) and LC (F (2,7) = 8.575, p= 0.0131), respectively. Remarkably, IND-1233-ASO treatment did not induce any changes in TH, DAT, and NET protein levels in the SNc/VTA, or LC or in DA/NE projection brain areas, which supports the specificity and safety of the IND-1233-ASO sequence (Supplemental Figure S3). Similarly, IND-1233-ASO was well tolerated during the 4 weeks of treatment, and no changes were found in daily observed behavioral variables, such as water and food consumptions, as well as body weight in A30P*A53T* α -Syn transgenic mice compared to vehicle-treated A30P*A53T* α-Syn mice (data not shown), as previously reported [34,36]. Next, we found that A30P*A53T* α -Syn transgenic mice treated with IND-1233-ASO showed normalization of DA neurotransmission in the nigrostriatal pathway, reaching extracellular DA levels similar to those detected in non-Tg mice (Figure 4a,b and 6a,b ). Local veratridine administration (50 µ M) significantly increased striatal DA release in A30P*A53T* α -Syn mice treated with IND-1233-ASO compared with transgenic mice receiving a vehicle (Figures 4a and 6b). Two-way ANOVA showed an effect of time F(15,80) = 10.23 , p< 0.0001 and a group-by-time interaction F (15,80) = 2.021, p= 0.0235, but no effect of group. Likewise, local amphetamine infusion (1–10 µ M) increased extracellular DA levels to a lesser extent in the CPu of A30P*A53T* α -Syn mice treated with IND-1233-ASO compared with those treated with vehicle (Figures 4a and 6b). Two-way ANOVA showed an effect of group F (1,72) = 7.359, p= 0.0083 and time F (17,72) = 4.231, p < 0.0001, but no group-bytime interaction. However, despite the changes in DA neurotransmission, treatment with IND-1233-ASO for 28 days was insufficient to significantly reverse the behavioral impairments observed in A30P*A53T* α -Syn mice, and the mice exhibited motor alterations and anxiety-like behavior similar to those that received the vehicle (Figure 6c,d). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 21 Figure 6. Intracerebroventricular IND-1233-ASO treatment recovers DA neurotransmission deficits, but not behavioral phenotype, in A30P*A53T*α-Syn transgenic mice. (a) Treatment timeline. A30P*A53T*α-Syn transgenic mice (4 months of age) received randomly vehicle or IND-1233-ASO (100 µg/day) into the lateral ventricle for 28 days using osmotic minipumps. In a group of mice, DA release was examined using intracerebral microdialysis at 3-4 days post-treatment, while behavioral tests were performed in an additional group at 5-6 days post-treatment. All mice were sacrificed on day 10 post-treatment. (b) Microdialysis approach using veratridine and amphetamine agents, as shown in Figure 3, confirmed the normalization of DA neurotransmission in the CPu of A30P*A53T*α-Syn mice treated with IND-1337-ASO (100 µg/day for 28 days) compared with transgenic mice treated with a vehicle. (c,d) No significant differences were detected in the open field test (c) or the dark-light box test (d) between both groups treated with IND-1337-ASO or a vehicle. Data are expressed as the mean ± SEM. Number of mice used in each procedure is indicated in parenthesis. Two-way ANOVA and Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01 compared to A30P*A53T*α-Syn mice treated with a vehicle. 3. Discussion The present study confirms and extends the results of previous studies on the benefit of a sustained therapy based on an IND-1233-ASO conjugate to reduce h-α-Syn expression selectively in early affected DA and NE brain areas –SNc/VTA and LC– in PD, as assessed in a transgenic mouse carrying both A30P and A53T mutant h-α-Syn. Remarkably, IND1233-ASO also decreased h-α-Syn protein accumulation in DA/NE projection cortical and subcortical brain areas such as mPFC and CPu in the same mice. Likewise, no signs of toxicity were found in DA and NE neurons as assessed with classical DA and NE neuronal markers, in line with previous reports using wild-type mice and elderly nonhuman primates [34,36]. Human α-Syn knockdown in monoaminergic nuclei normalized the nigrostriatal DA neurotransmission in the A30P*A53T*α-Syn transgenic mice, although the current IND-1233-ASO therapy could not significantly reverse the behavioral phenotype, perhaps due to an insufficient time/dose and/or the involvement of downstream changes in relevant brain circuits not recovered by the treatment. Therefore, the current strategy of using ligand-oligonucleotide conjugates appears to have the potential for future optimization of new ligand-conjugated oligonucleotide-based therapies to treat PD and related α-synucleinopathies. Selective reduction in h-α-Syn expression was found in the DA and NE brain regions of transgenic mice by exploiting our previous strategy, in which different oligonucleotide sequences were covalently bound to MAT inhibitors for the selective delivery of them to Figure 6. Intracerebroventricular IND-1233-ASO treatment recovers DA neurotransmission deficits, but not behavioral phenotype, in A30P*A53T* α -Syn transgenic mice. ( a ) Treatment timeline. A30P*A53T* α -Syn transgenic mice (4 months of age) received randomly vehicle or IND-1233-ASO (100 µ g/day) into the lateral ventricle for 28 days using osmotic minipumps. In a group of mice, DA release was examined using intracerebral microdialysis at 3–4 days post-treatment, while behavioral tests were performed in an additional group at 5–6 days post-treatment. All mice were sacrificed on day 10 post-treatment. ( b ) Microdialysis approach using veratridine and amphetamine agents, as shown in Figure 3, confirmed the normalization of DA neurotransmission in the CPu of A30P*A53T* α -Syn mice treated with IND-1337-ASO (100 µ g/day for 28 days) compared with transgenic mice treated with a vehicle. ( c , d ) No significant differences were detected in the open field test ( c ) or the dark-light box test ( d ) between both groups treated with IND-1337-ASO or a vehicle. Data are expressed as the mean ± SEM. Number of mice used in each procedure is indicated in parenthesis. Two-way ANOVA and Tukey’s multiple comparisons test, * p< 0.05, ** p< 0.01 compared to A30P*A53T*α-Syn mice treated with a vehicle. Int. J. Mol. Sci. 2021,22, 2939 16 of 20 were separated on a 2.6 µ m particle size C18 column (7.5 × 0.46 cm, Kinetex, Phenomenex, Torrance, CA, USA) at 28 ◦C. All reagents used were of analytical grade and were obtained from Merck (Darmstadt). DA hydrochloride, DOPAC acid, (–)-quinpirole hydrochloride, and nomifensine maleate were sourced from Sigma-Aldrich-RBI. D-amphetamine sulphate and veratridine were purchased from Tocris. To assess the local drug effects, compounds were dissolved in artificial cerebrospinal fluid (in mM: NaCl, 125; KCl, 2.5; CaCl 2 , 1.26 and MgCl 2 , 1.18) and administered by reverse dialysis at the stated concentrations (uncorrected for membrane recovery). 4.8. Behavioral Testing Behavioral analyses were performed in non-Tg and A30P*A53T* α -Syn mice with intervals of 1–7 days between tests. Different behavioral paradigms were used to evaluate motor and cognitive functions as well as anxietyand depressive phenotype. All tests were performed between 10:00 and 15:00 h by an experimenter who was blinded to mouse treatments. On the test day, mice were placed in a dimly illuminated behavioral room and were left undisturbed for at least 1 h before testing [33,36]. Open field test. Motor activity was measured in four Plexiglas open field boxes (35 × 35 × 40 cm) that were indirectly illuminated (25–40 lux) to avoid reflection and shadows. The floor of the open field boxes was covered with an interchangeable opaque plastic base that was replaced for each animal. Motor activity was recorded for 15 min by a camera connected to a computer (Video-track, Viewpoint, Lyon, France) [ 62 ]. The following variables were measured: horizontal locomotor and exploratory activity, defined as the total distance moved in cm including fast/large (speed > 10.5 cm/s) and slow/short movements (speed 3–10.5 cm/s), as well as the activation time including the mean speed (cm/s) and resting time (s), and the number of rearings. Cylinder test. Mice were tested for motor asymmetry in the cylinder test. Each mouse was placed in an acrylic cylinder (diameter, 15 cm; height, 27 cm), and the total number of left and right forepaw touches performed, as well as the number of rearings in 5 min was counted. Behavioural equipment was cleaned with water after each test session to avoid olfactory cues. Dark-light box test. The apparatus consisted of two glass boxes (27 × 21 cm) with an interconnecting grey plastic tunnel (7 × 10 cm). One of these boxes was painted in black and was weakly lit with a red 25-W bulb (42 lux). The other box was lit with a 60-W desk lamp (400 lux) placed 30 cm above the box, which provided the unique laboratory illumination condition. At the beginning of the test, mice were placed individually in the middle of the dark area facing away from the opening, and were videotaped for 5 min. The following variables were recorded: (a) time spent in the lit box, (b) the latency of the initial movement from the dark to the lit box, and (c) the number of entries in the lit box. A mouse was considered to enter the new area when all four legs were in this area. The floor of each box was cleaned between the mice. Tail suspension test. Mice were suspended 30 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. Sessions were videotaped for 6 min and the immobility time was measured (Smart, Panlab, Cornellà, Spain). Novelty object recognition test. Mice were placed into an open field box of Plexiglas (40 × 35 × 16 cm) with a soured of illumination (60 lux) in the center of the box. Two objects of identical shape were used as familiar objects. During the habituation period, the mice could freely explore the open field box without objects for 10 min on two consecutive days. In the acquisition test, the two identical objects were placed separately in the center of the open field and the mice explored it for 10 min. To minimize the presence of olfactory traces, the objects and the open field were cleaned with water between each trial. Twenty-four hours after the acquisition test, one of the familiar objects was replaced with a new object, and the amount of time spend exploring both objects (familiar and novel) was recorded for Int. J. Mol. Sci. 2021,22, 2939 17 of 20 a period of 10 min. Exploration of an object was defined as pointing the nose at an object at a distance of <1 cm and/or touching it. 4.9. Statistical Analysis All values are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were performed using GraphPad Prism 8.01 (GraphPad software, Inc., San Diego, CA, USA) using the appropriate statistical tests, as indicated in each figure legend. Outlier values were identified by the Grubbs’ test (i.e., Extreme Student zed Deviate, ESD, method) using GraphPad Prism software and excluded from the analysis when applicable. Differences among means were analyzed by either 1or 2-way analysis of variance (ANOVA) or the two-tailed Student’s t-test, as appropriate. When ANOVA showed significant differences, pairwise comparisons between means were subjected to Tukey’s post hoc test or Sidak’s multiple comparisons test, as appropriate. Differences were considered significant at p< 0.05. Supplementary Materials: Supplementary materials can be found at https://www.mdpi.com/1422 -0067/22/6/2939/s1. Author Contributions: Conceptualization and supervision of the research, A.B. Design of the conjugated oligonucleotides, F.A., A.M., R.R. and A.B. Methodology: R.P.-C., D.A.-A. and R.R.-A. performed behavioral and microdialysis experiments; V.C.-S. and L.M.-R. performed immunohistochemistry experiments; M.T.-L. performed confocal microscopy experiments; R.P.-C., V.P. and E.R.-B. performed in situ hybridization experiments; L.C. performed DA and DOPAC determinations using HPLC. Writing, A.B. with all 12 authors providing imput. Funding acquisition, R.R. and A.B. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by grants SAF2016-75797-R, PID2019-105136RB-100, RetosColaboración Subprogram RTC-2015-3309-1, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; and CB/07/09/0034 Center for Networked Biomedical Research on Mental Health (CIBERSAM). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by Ethical Committee of Animal Experimentation of the University of Barcelona (protocol codes 9628/9832, approved on April 2017/November 2017, expiration date April 2023/March 2023). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study area available on request from the corresponding author. The data are not publicly available due to privacy. Acknowledgments: We thank M. Calvo and E. Coll for outstanding technical support in the confocal microscopy unit (CCiT-UB). We thank to the Coordenação de Aperfeiçõamento de Nivel Superior (CAPESPDSE: 19/2016 88881.135527/2016-01), Brazil, for their financial support via a scholarship awarded to V.C.-S. We also thank to JAE-Intro Program, CSIC (JAEINT_20_02116), Spain, for their financial support via a scholarship awarded to M.T.-L. Conflicts of Interest: R. Revilla, A. Montefeltro, F. Artigas, and A. Bortolozzi are the authors of the patent WO/2011/131693 issued for the siRNA and ASO molecules and the targeting approach related to this work. R. Revilla is board members of nLife Therapeutics S.L. A. Montefeltro is a stockholder of nLife Therapeutics S.L. The rest of the authors declare no competing interests. References 1. Braak, H.; Del Tredici, K.; Bohl, J.; Bratzke, H.; Braak, E. Pathological changes in the parahippocampal region in select nonAlzheimer’s dementias. Ann. N. Y. Acad. Sci. 2000,911, 221–239. [CrossRef] 2. Reeve, A.; Simcox, E.; Turnbull, D. 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