Regulation of Cortico-Thalamic JNK1/2 and ERK1/2 MAPKs and Apoptosis-Related Signaling Pathways in PDYN Gene-Deficient Mice Following Acute and Chronic Mild Stress

Citation: Yáñez-Gómez, F.; Ramos-Miguel, A.; García-Sevilla, J.A.; Manzanares, J.; Femenía, T. Regulation of Cortico-Thalamic JNK1/2 and ERK1/2 MAPKs and Apoptosis-Related Signaling Pathways in PDYN Gene-Deficient Mice Following Acute and Chronic Mild Stress. Int. J. Mol. Sci. 2023,24, 2303. https://doi.org/10.3390/ ijms24032303 Academic Editor: Gatien Moriceau Received: 4 December 2022 Revised: 13 January 2023 Accepted: 19 January 2023 Published: 24 January 2023 Copyright: © 2023 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/). International Journal of Molecular Sciences Article Regulation of Cortico-Thalamic JNK1/2 and ERK1/2 MAPKs and Apoptosis-Related Signaling Pathways in PDYN Gene-Deficient Mice Following Acute and Chronic Mild Stress Fernando Yáñez-Gómez 1,2 , Alfredo Ramos-Miguel 3,4,* , Jesús A. García-Sevilla 1, Jorge Manzanares 5,6,7 and Teresa Femenía5,6 1Laboratorio de Neurofarmacología, IUNICS, Universitat de les Illes Balears, Crta. Valldemossa km 7.5, 07122 Palma de Mallorca, Spain 2Health Research Institute of the Balearic Islands (IdISBa), 07120 Palma de Mallorca, Spain 3Department of Pharmacology, University of the Basque Country (UPV/EHU), Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Barrio Sarriena S/N, 48940 Leioa, Spain 4BioCruces Bizkaia Health Research Institute, Plaza de Cruces 12, 48903 Barakaldo, Spain 5Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Avda. de Ramón y Cajal s/n, San Juan de Alicante, 03550 Alicante, Spain 6Redes de Investigación Cooperativa Orientada a Resultados en Salud (RICORS), Red de Investigación en Atención Primaria de Adicciones (RIAPAd), Instituto de Salud Carlos III, MICINN and FEDER, 28029 Madrid, Spain 7Instituto de Investigación Sanitaria y Biomédica de Alicante (ISABIAL), 03010 Alicante, Spain *Correspondence: alfr[email protected] Abstract: The crosstalk between the opioidergic system and mitogen-activated protein kinases (MAPKs) has a critical role in mediating stress-induced behaviors related to the pathophysiology of anxiety. The present study evaluated the basal status and stress-induced alterations of corticothalamic MAPKs and other cell fate-related signaling pathways potentially underlying the anxiogenic endophenotype of PDYN gene-deficient mice. Compared to littermates, PDYN knockout (KO) mice had lower cortical and or thalamic amounts of the phospho-activated MAPKs c-Jun N-terminal kinase (JNK1/2) and extracellular signal-regulated kinase (ERK1/2). Similarly, PDYN-KO animals displayed reduced cortico-thalamic densities of total and phosphorylated (at Ser191) species of the cell fate regulator Fas-associated protein with death domain (FADD) without alterations in the Fas receptor. Exposure to acute restraint and chronic mild stress stimuli induced the robust stimulation of JNK1/2 and ERK1/2 MAPKs, FADD, and Akt-mTOR pathways, without apparent increases in apoptotic rates. Interestingly, PDYN deficiency prevented stress-induced JNK1/2 and FADD but not ERK1/2 or Akt-mTOR hyperactivations. These findings suggest that cortico-thalamic MAPKand FADD-dependent neuroplasticity might be altered in PDYN-KO mice. In addition, the results also indicate that the PDYN gene (and hence dynorphin release) may be required to stimulate JNK1/2 and FADD (but not ERK1/2 or Akt/mTOR) pathways under environmental stress conditions. Keywords: prodynorphin; JNK; ERK; chronic mild stress; apoptosis; phosphorylation 1. Introduction Dynorphins are endogenous opioid peptides that bind with high affinity and show significant potency at both kappa (KOR) and delta (DOR) opioid receptors [ 1 ]. The distribution of dynorphin peptides and prodynorphin (PDYN) gene expression in the brain and spinal cord suggest their involvement in a large number of conditions, including stress and anxiety [ 2 – 5 ]. For example, dynorphins are co-released with corticosterone into the portal circulation modulating the release of ACTH from the anterior pituitary [ 6 , 7 ]. Additionally, PDYN-derived peptide amounts are elevated in the thalamus and pituitary following Int. J. Mol. Sci. 2023,24, 2303. https://doi.org/10.3390/ijms24032303 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023,24, 2303 2 of 17 particular forms of stress stimuli, whereas they are decreased in the pituitary after forced swimming-induced immobility [8–10]. At least four prior studies used PDYN-deficient mice to evaluate the role of dynorphins in anxiety. The first study [ 11 ] showed that PDYN gene deletion was associated with an enhanced response to stress stimuli, supporting an anxiolytic effect of dynorphins. In agreement, a second study using animals of the same germinal line [ 12 ] showed that anxious-like behaviors following PDYN gene deletion were accompanied by the overexpression of stress-related genes (i.e., proopiomelanocortin and corticotrophin-releasing factor genes) and reduced anxiolytic efficacy of benzodiazepines, probably due to the dysregulated expression of the γ 2 and β 2 subunits of the GABA A receptor. In marked contrast, two other studies [ 3 , 13 ] described an anxiolytic behavioral phenotype following PDYN gene deletion in mice. Besides the debate on the role of dynorphins in anxiety, the adaptations occurring in stress-related molecular pathways following PDYN gene deletion are poorly understood. Among the potential molecular mediators between dynorphins and the pathophysiological response to stress and anxiety, the mitogen-activated protein kinase (MAPK) intracellular signaling pathways might be sensible candidates. These highly conserved molecules are stimulated in response to a wide variety of extracellular stimuli and control a large number of cellular processes such as growth, proliferation, differentiation, motility, cell survival/apoptosis, and cellular stress [ 14 – 19 ]. Each MAPK cassette consists of three sequentially phospho-activated protein kinases (generally named MAP3K, MAP2K, and MAPK, in up-to-down-stream order) that amplify and propagate the signal [ 16 ]. Although multiple MAPK pathways have been identified in mammals, few are constitutively expressed in brain cells. These include the c-Jun N-terminal kinase 1 and 2 (JNK1/2, also known as stress-activated protein kinase or SAPK) and the extracellular signaling-regulated kinase 1 and 2 (ERK1/2) cascades. Notably, the stimulation of opioid receptors with selective peptides activates both JNK1/2 and ERK1/2 [ 20 – 22 ]. Brain MAPK pathways are also robustly stimulated by acute and chronic environmental stressors [ 23 – 25 ], which in turn regulate the activity of multiple downstream targets, some implicated in cell survival and death [ 26 ]. Furthermore, the forced-swim stress-induced activation of amygdalar ERK was abrogated in KOR knockout (KO) mice [ 27 ], and JNK deficient mice displayed blunted anxiety-like phenotypes [ 28 ]. The possibility that dynorphins may mediate stress-induced JNK1/2 and/or ERK1/2 stimulation in the brain has not been tested. On the other hand, molecules involved in apoptotic machinery are well-known mediators of stress responses in rodents [ 29 , 30 ]. Notably, cortical and thalamic amounts of key components of the extracellular apoptotic pathway, including the multifunctional complex Fas/FADD (Fas-associated protein with death domain), are strongly regulated by both benzodiazepines (and other allosteric modulators of GABA A receptors) [ 31 , 32 ] and multiple opioid ligands, including the κ -opioid receptor-selective agonist U-50488H [ 33 – 36 ]. Rather than increased apoptotic rates, the benzodiazepineand opioid-induced regulation of brain Fas/FADD signaling may promote nonapoptotic, neuroplasticity-related activities [ 37 , 38 ]. Interestingly, MAPK-ERK and the extracellular apoptotic cascade are linked via the AktPea15 (phosphoprotein enriched in astrocytes of 15 kDa) pathway. Akt-induced Pea15 phosphorylation at Ser116 switches its binding affinity from ERK1/2 to FADD, allowing the nuclear translocation of ERK1/2, and hindering apoptotic signals [ 39 – 41 ]. Of note, some studies also reported a critical role of Akt dysregulation in depression and anxiety, possibly mediated by the mTOR (mammalian target of rapamycin) cascade and the disruption of neurogenesis [42–44]. Using PDYN-KO mice and wildtype (WT) littermates, the present study tested the hypothesis that dynorphins mediate the stress-induced alteration of the MAPK-FADDAkt pathways. We first investigated the cortico-thalamic neuroadaptations occurring in PDYN gene-deficient mice, possibly affecting (1) JNK1/2 and ERK1/2 MAPKs, (2) the Fas receptor/FADD extracellular apoptotic pathway, and (3) the multifunctional kinase Akt, and its substrates Pea15 and mTOR. Next, the cortical and thalamic regulation of these Int. J. Mol. Sci. 2023,24, 2303 3 of 17 molecular pathways was evaluated in mice of both genotypes following acute restraint (ARS) or chronic mild (CMS) stress procedures. 2. Results 2.1. Effect of PDYN Gene Deletion on MAPK-JNK1/2 and ERK1/2 in Mouse Brain Compared to WT animals, brain immunodensities of total (i.e., phosphorylated + nonphosphorylated) JNK1/2, ERK1/2, and Akt kinases were not significantly altered in the cortical and thalamic samples of PDYN-KO mice (see corresponding immunoblots in Figures 1–3) and were, therefore, used as loading controls to normalize their respective phosphorylated forms. Thus, the amount of “activated kinase” in brain tissues refers to the ratio of phosphorylated to total kinase immunodensities. Overall, the separate and combined quantifications of JNK (i.e., JNK1 and JNK2) and ERK (i.e., ERK1 and ERK2) species yielded very similar outcomes (not shown). For practical reasons, the present study only reports combined (i.e., JNK1 + JNK2) kinase measures. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 18 receptor/FADD extracellular apoptotic pathway, and (3) the multifunctional kinase Akt, and its substrates Pea15 and mTOR. Next, the cortical and thalamic regulation of these molecular pathways was evaluated in mice of both genotypes following acute restraint (ARS) or chronic mild (CMS) stress procedures. 2. Results 2.1. Effect of PDYN Gene Deletion on MAPK-JNK1/2 and ERK1/2 in Mouse Brain Compared to WT animals, brain immunodensities of total (i.e., phosphorylated + non-phosphorylated) JNK1/2, ERK1/2, and Akt kinases were not significantly altered in the cortical and thalamic samples of PDYN-KO mice (see corresponding immunoblots in Figures 1–3) and were, therefore, used as loading controls to normalize their respective phosphorylated forms. Thus, the amount of “activated kinase” in brain tissues refers to the ratio of phosphorylated to total kinase immunodensities. Overall, the separate and combined quantifications of JNK (i.e., JNK1 and JNK2) and ERK (i.e., ERK1 and ERK2) species yielded very similar outcomes (not shown). For practical reasons, the present study only reports combined (i.e., JNK1 + JNK2) kinase measures. In cortical samples from PDYN-KO mice, the immunodensities of activated (phospho-Thr183/Tyr185) JNK1/2 were significantly lower (−37%, p < 0.05) than those in WT controls (Figure 1A). PDYN gene deletion was also associated with robust reductions in ERK1/2 activation (phospho-Thr202/Tyr204) in both the cerebral cortex (−30%, p < 0.01) and the thalamus (−22%, p < 0.01) (Figure 1B). Figure 1. Immunodensities of activated (A) JNK1/2 and (B) ERK1/2 in cortical and thalamic brain tissue samples from WT (grey bars; n = 9) and PDYN-KO (reddish bars; n = 9) mice. Normalized enzyme activation was estimated as the ratio between phosphorylated (i.e., p-Thr183/Tyr185 JNK1/2 or p-Thr202/Tyr204 ERK1/2) to non-phosphorylated protein species. Columns are means ± SEM of each experimental group and expressed in percent change from WT animals for each brain region. * p < 0.05, and ** p < 0.01, Student’s t test. (A,B) Representative immunoblots of cortical and thalamic JNK and ERK phosphorylated and non-phosphorylated species (three different animals per group) are shown at the bottom. The indicated molecular weights (in kDaltons, kD) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards. 2.2. Effect of PDYN Gene Deletion on Fas/FADD Immunodensities in Mouse Brain As previously reported [34], the immunodetection of the Fas receptor resulted in four immunoreactive bands corresponding to the monomeric (35 kDa), glycosylated (51 and 45 kDa), and aggregate (120 kDa) Fas species. Neither the cortical nor the thalamic immunodensities of these Fas receptor forms were significantly altered in PDYN-KO mice compared to WT controls (Figure 2A). WT PDYN KO Figure 1. Yáñez-Gómez et al. THALAMUS 0 PDYN KO WT CORTEX PDYN KO WT * A Normalized density (%WT) 200 150 100 50 p-JNKT183/Y185 / t-JNK <54 <54 p-JNK1/2 t-JNK1/2 <46 <46 * THALAMUS PDYN KOWT CORTEX ** PDYN KOWT B Normalized density (%WT) 200 150 100 50 0 p-ERKT202/Y204 / t-ERK <44 p-ERK1/2 t-ERK1/2 <42 <44 <42 Figure 1. Immunodensities of activated ( A ) JNK1/2 and ( B ) ERK1/2 in cortical and thalamic brain tissue samples from WT (grey bars; n = 9) and PDYN-KO (reddish bars; n = 9) mice. Normalized enzyme activation was estimated as the ratio between phosphorylated (i.e., p-Thr183/Tyr185 JNK1/2 or p-Thr202/Tyr204 ERK1/2) to non-phosphorylated protein species. Columns are means ± SEM of each experimental group and expressed in percent change from WT animals for each brain region. *p< 0.05 , and ** p< 0.01, Student’s ttest. ( A , B ) Representative immunoblots of cortical and thalamic JNK and ERK phosphorylated and non-phosphorylated species (three different animals per group) are shown at the bottom. The indicated molecular weights (in kDaltons, kD) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards. In cortical samples from PDYN-KO mice, the immunodensities of activated (phosphoThr183/Tyr185) JNK1/2 were significantly lower ( − 37%, p< 0.05) than those in WT controls (Figure 1A). PDYN gene deletion was also associated with robust reductions in ERK1/2 activation (phospho-Thr202/Tyr204) in both the cerebral cortex ( − 30%, p< 0.01) and the thalamus (−22%, p< 0.01) (Figure 1B). 2.2. Effect of PDYN Gene Deletion on Fas/FADD Immunodensities in Mouse Brain As previously reported [ 34 ], the immunodetection of the Fas receptor resulted in four immunoreactive bands corresponding to the monomeric (35 kDa), glycosylated (51 and 45 kDa), and aggregate (120 kDa) Fas species. Neither the cortical nor the thalamic immunodensities of these Fas receptor forms were significantly altered in PDYN-KO mice compared to WT controls (Figure 2A). Int. J. Mol. Sci. 2023,24, 2303 4 of 17 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 18 Figure 2. Immunodensities of (A) Fas receptor forms, (B) dimeric and oligomeric p-Ser191 FADD species, and (C) full-length (116-kDa) and cleaved (89-kDa) PARP in cortical and thalamic brain tissue samples from WT (grey bars; n = 9) and PDYN-KO (reddish bars; n = 9) mice. Normalized protein amounts were estimated as the ratio between each immunoreactive band to β-actin. Columns are means ± SEM of each experimental group and expressed in percent change from WT animals for each brain region. * p < 0.05, ** p < 0.01, and *** p < 0.001, Student’s t test. (A–C) Representative immunoblots of cortical and thalamic Fas, FADD, and PARP species (three different animals per group) are shown on the right. The indicated molecular weights (in kDaltons, kDa) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards. Similar to the Fas receptor, the immunodetection of the FADD adaptor protein in mammalian brains yielded two major oligomeric species: (1) non-phosphorylated homodimers of about 51 kDa (herein named FADD), associated with its pro-apoptotic function; and (2) ~116-kDa complexes of phosphorylated FADD at Ser191 (in mice) or Ser194 (in rats and humans) (referred to as p-FADD), mainly involved in nonapoptotic activities of the FADD adaptor [36,38,45]. Compared to the WT animals, the PDYN-KO mice showed significantly lower amounts of dimeric FADD in the cortex (−29%, p < 0.001) and thalamus (−46%, p < 0.01) (Figure 2B). Similar reductions in cortical (−26%, p < 0.001) CORTEX C Normalized density (%WT) 200 150 100 50 0116-kD PARP 89-kD fragment THALAMUS 116-kD PARP 89-kD fragment 89> 116> 37> kD THALAMUS PARP β-actin 89> 116> 37> CORTEX PARP β-actin PDYN KO WT * B p-FADD CORTEX 116> p-FADD 54> 37> FADD CORTEX Normalized density (%WT) 200 150 100 50 0Dimeric FADD Oligomeric p-FADDS191 THALAMUS Dimeric FADD Oligomeric p-FADDS191 54> 116> 37> THALAMUS FADD β-actin PDYN KOWT *** *** ** * kD 120> WT PDYNKO CORTEX A Normalized density (%WT) 200 150 100 50 0 120-kD 51-kD 45-kD 35-kD 120-kD 51-kD 45-kD 35-kD THALAMUS Fas receptor species Fas receptor species Fas receptor β-actin 51> 35> THALAMUS PDYN KOWT 45> 35> 120> 51> 35> 45> CORTEX Fas receptor kD β-actin 35> β-actin Figure 2. Immunodensities of ( A ) Fas receptor forms, ( B ) dimeric and oligomeric p-Ser191 FADD species, and ( C ) full-length (116-kDa) and cleaved (89-kDa) PARP in cortical and thalamic brain tissue samples from WT (grey bars; n = 9) and PDYN-KO (reddish bars; n = 9) mice. Normalized protein amounts were estimated as the ratio between each immunoreactive band to β -actin. Columns are means ± SEM of each experimental group and expressed in percent change from WT animals for each brain region. * p< 0.05, ** p< 0.01, and *** p< 0.001, Student’s ttest. ( A – C ) Representative immunoblots of cortical and thalamic Fas, FADD, and PARP species (three different animals per group) are shown on the right. The indicated molecular weights (in kDaltons, kDa) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards. Similar to the Fas receptor, the immunodetection of the FADD adaptor protein in mammalian brains yielded two major oligomeric species: (1) non-phosphorylated homodimers of about 51 kDa (herein named FADD), associated with its pro-apoptotic function; and (2) ~116-kDa complexes of phosphorylated FADD at Ser191 (in mice) or Ser194 (in rats and humans) (referred to as p-FADD), mainly involved in nonapoptotic activities of the FADD adaptor [ 36 , 38 , 45 ]. Compared to the WT animals, the PDYN-KO mice showed significantly lower amounts of dimeric FADD in the cortex ( − 29%, p< 0.001) and thalamus ( − 46%, p< 0.01 ) (Figure 2B). Similar reductions in cortical ( − 26%, p< 0.001) and thalamic Int. J. Mol. Sci. 2023,24, 2303 5 of 17 ( − 29%, p< 0.05) p-FADD oligomers were observed in brain samples from the PDYN-KO mice (Figure 2B). To address the possibility that alterations of FADD amounts in PDYN-KO mouse brains could dysregulate effector apoptotic mechanisms, the fragmentation of poly (ADP-ribose)- polymerase-1 (PARP), a major substrate in caspase-dependent and independent apoptotic pathways, was evaluated. Following apoptotic stimuli, the ~116-kDa DNA-repairing enzyme PARP is cleaved into two primary fragments of ~89 and ~31 kDa, respectively, which can be used as an indirect measure of apoptotic events in the brain [36]. In PDYN-KO mice, cortical immunodensities of the 116and 89-kDa PARP species did not differ significantly from those in WT animals (Figure 2C). Full-length 116-kDa PARP was significantly reduced in thalamic samples from PDYN-KO animals ( − 36%, p< 0.05 ). The reduced density of full-length PARP was unlikely caused by greater apoptosis-related proteolytic cleavage of PARP, as the thalamic immunodensities of the 89 kDa PARP fragment were similar in both murine strains (Figure 2C). These observations indicate that PDYN gene deletion is not associated with abnormally increased cell death rates, at least in the adult mouse brain. 2.3. Effect of PDYN Gene Deletion on the Akt/Pea15/mTOR Pathway The immunodensity of activated (i.e., p-Ser473) Akt was also similar in the PDYNKO and WT mouse brains (Figure 3A), although a non-significant reduction trend was observed in the PDYN-KO thalamic samples. A similar trend was observed for Pea15 phosphorylation at Ser116 (a key cytosolic substrate of Akt) in cortical and thalamic samples from PDYN gene-deficient mice, compared to the WT controls. Of note, the brain amounts of total Pea15 were unaltered following PDYN gene deletion (see immunoblots in Figure 4). Under our experimental conditions, basal levels of mTOR phosphorylation at Ser2448 (another relevant downstream substrate of the Akt signaling pathway) were barely detectable in brain samples of both murine strains (see corresponding immunoblots in Figure 4). Therefore, it was not possible to provide a quantitative estimate comparing brain p-mTOR levels in PDYN-KO and WT stress-naïve mice. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 18 and thalamic (−29%, p < 0.05) p-FADD oligomers were observed in brain samples from the PDYN-KO mice (Figure 2B). To address the possibility that alterations of FADD amounts in PDYN-KO mouse brains could dysregulate effector apoptotic mechanisms, the fragmentation of poly (ADPribose)-polymerase-1 (PARP), a major substrate in caspase-dependent and independent apoptotic pathways, was evaluated. Following apoptotic stimuli, the ~116-kDa DNArepairing enzyme PARP is cleaved into two primary fragments of ~89 and ~31 kDa, respectively, which can be used as an indirect measure of apoptotic events in the brain [36]. In PDYN-KO mice, cortical immunodensities of the 116and 89-kDa PARP species did not differ significantly from those in WT animals (Figure 2C). Full-length 116-kDa PARP was significantly reduced in thalamic samples from PDYN-KO animals (−36%, p < 0.05). The reduced density of full-length PARP was unlikely caused by greater apoptosisrelated proteolytic cleavage of PARP, as the thalamic immunodensities of the 89 kDa PARP fragment were similar in both murine strains (Figure 2C). These observations indicate that PDYN gene deletion is not associated with abnormally increased cell death rates, at least in the adult mouse brain. 2.3. Effect of PDYN Gene Deletion on the Akt/Pea15/mTOR Pathway The immunodensity of activated (i.e., p-Ser473) Akt was also similar in the PDYNKO and WT mouse brains (Figure 3A), although a non-significant reduction trend was observed in the PDYN-KO thalamic samples. A similar trend was observed for Pea15 phosphorylation at Ser116 (a key cytosolic substrate of Akt) in cortical and thalamic samples from PDYN gene-deficient mice, compared to the WT controls. Of note, the brain amounts of total Pea15 were unaltered following PDYN gene deletion. Under our experimental conditions, basal levels of mTOR phosphorylation at Ser2448 (another relevant downstream substrate of the Akt signaling pathway) were barely detectable in brain samples of both murine strains (see corresponding immunoblots in Figure 4). Therefore, it was not possible to provide a quantitative estimate comparing brain p-mTOR levels in PDYN-KO and WT stress-naïve mice. Figure 3. Immunodensities of activated (A) Akt and (B) Pea15 in cortical and thalamic brain tissue samples from WT (grey bars; n = 9) and PDYN-KO (reddish bars; n = 9) mice. Normalized protein activation was estimated as the ratio between phosphorylated (i.e., p-Ser473 Akt or p-Ser116 Pea15) to non-phosphorylated protein species. Columns are means ± SEM of each experimental group and expressed in percent change from WT animals for each brain region. (A,B) Representative immunoblots of cortical and thalamic Akt and Pea15 phosphorylated and non-phosphorylated species (three different animals per group) are shown at the bottom. The indicated molecular weights (in kDaltons, kDa) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards. WT PDYN KO Figure 3. Yáñez-Gómez et al. B Normalized density (%WT) 200 150 100 50 0 CORTEX THALAMUS p-Pea15S116 / t-Pea15 <15 p-Pea15 t-Pea15 PDYN KOWT PDYN KOWT <15 kD CORTEX A Normalized density (%WT) 200 150 100 50 THALAMUS p-AktS473 / t-Akt 0 <60 p-Akt t-Akt PDYN KOWT PDYN KOWT <60 kD Figure 3. Immunodensities of activated ( A ) Akt and ( B ) Pea15 in cortical and thalamic brain tissue samples from WT (grey bars; n = 9) and PDYN-KO (reddish bars; n = 9) mice. Normalized protein activation was estimated as the ratio between phosphorylated (i.e., p-Ser473 Akt or p-Ser116 Pea15) to non-phosphorylated protein species. Columns are means ± SEM of each experimental group and expressed in percent change from WT animals for each brain region. ( A , B ) Representative immunoblots of cortical and thalamic Akt and Pea15 phosphorylated and non-phosphorylated species (three different animals per group) are shown at the bottom. The indicated molecular weights (in kDaltons, kDa) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards. Int. J. Mol. Sci. 2023,24, 2303 6 of 17 2.4. Effects of Acute and Chronic Stress on Select Signaling Pathways in Cortical and Thalamic Samples from WT and PDYN-KO Mice The potential role of the PDYN gene in mediating the stress-induced dysregulation of the studied intracellular signaling pathways was tested by comparing the effects of acute restraint (ARS) or chronic mild (CMS) stress procedures versus basal stress conditions (i.e., undisturbed mice) on cortical and thalamic densities of activated MAPKs (JNK/ERK), select proteins of the apoptotic pathway (FADD/PARP), and the phosphorylation of Akt/Pea15/mTOR signaling proteins, in both PDYN-KO mice and WT littermates [ 2 , 15 ]. All neurochemical datasets obtained in the subsequent immunoblotting assays in brain samples from undisturbed, and stressed WT and PDYN-KO animals were analyzed by two-way analysis of variance (TW-ANOVA), and the main result outputs were summarized in Table 1. Note that the degrees of freedom did not match up in all the analyses as group sizes varied due to the following reasons: (1) outliers were excluded from the analyses whenever detected by Grubb’s test, or (2) some samples were finished before completing the neurochemical analyses. Table 1. Results of TW-ANOVA reporting the effects of the stress procedures, PDYN gene deletion, and their interaction on cortical and thalamic immunodensities of the studied proteins. Brain Area Target Whole Model Stress Protocol Genotype Interaction df F-Ratio p-Val F-Ratio p-val F-Ratio p-Val F-Ratio p-Val Cortex p-JNK1/2 5, 42 26.6 <.001 35.3 <.001 39.6 <.001 8.38 <.001 p-ERK1/2 5, 42 29.8 <.001 139 <.001 9.62 0.003 0.12 0.820 FADD 5, 42 20.8 <.001 42.7 <.001 9.98 0.003 2.71 0.078 p-FADD 5, 41 22.5 <.001 53.0 <.001 47.4 0.035 0.83 0.444 PARP 116 kDa 5, 42 1.08 0.384 0.41 0.668 3.33 0.075 0.84 0.440 PARP 89 kDa 5, 42 0.12 0.988 0.21 0.809 0.17 0.682 0.01 0.994 p-Akt 5, 38 13.0 <.001 30.6 <.001 1.86 0.181 0.54 0.588 p-Pea15 5, 38 1.19 0.331 0.71 0.499 3.45 0.071 0.29 0.748 p-mTOR 5, 27 12.7 <.001 29.6 <.001 0.65 0.427 1.42 0.261 Thalamus p-JNK1/2 5, 42 1.20 0.324 0.38 0.686 4.68 0.036 0.15 0.861 FADD 5, 42 40.7 <.001 20.8 <.001 140 <.001 11.9 <.001 p-FADD 5, 42 6.42 <.001 4.00 0.026 24.6 <.001 0.23 0.796 p-Akt 5, 41 2.11 0.084 4.34 0.020 0.53 0.471 0.58 0.566 p-Pea15 5, 42 0.48 0.792 0.54 0.585 0.21 0.650 0.50 0.612 2.4.1. ARS and CMS Effects on Cortical Targets in WT and PDYN-KO Mice As expected, the phospho-activation of cortical JNK1/2 (6.6–7.1-fold increase, p< 0.001 ) and ERK1/2 (3.2–3.5-fold increase, p< 0.001) was dramatically enhanced in WT animals after both the ARS and CMS procedures, compared to undisturbed mice (Figure 4A,B). Interestingly, PDYN gene deletion had a different influence on cortical JNK1/2 and ERK1/2 stimulations following stress stimuli. Thus, ARSand CMS-mediated JNK1/2 activations observed in WT animals were largely prevented in PDYN-KO mice (response inhibition: 67–70% ,p< 0.001) (Figure 4A). In marked contrast, PDYN-KO animals displayed comparable p-ERK1/2 cortical levels than those in WT littermates following both ARS and CMS procedures (response inhibition: 6–8%, p> 0.05) (Figure 4B). CMS (3.8-fold, p< 0.001), but not ARS (1.6-fold, p> 0.05), markedly upregulated dimeric FADD in the cerebral cortex of WT mice (Figure 4C). Remarkably, the CMS-induced upregulation of dimeric FADD in the PDYN-KO mouse cortex (2.4-fold increase, p< 0.001 ) was significantly smaller than that in the WT mice (response inhibition: 49%, p< 0.01 ) (Figure 4C). Both ARS (3.5-fold, p< 0.001) and CMS (3.6-fold, p< 0.001) procedures dramatically increased the cortical density of oligomeric p-FADD in the WT animals, and similar upregulations were observed in the PDYN-KO mice after the same stress stimuli (3.5–4.4-fold increases, p< 0.001), as compared to their respective non-stressed controls (Figure 4D). These data suggest that the stress-induced upregulation of dimeric FADD, Int. J. Mol. Sci. 2023,24, 2303 7 of 17 but not its phosphorylation, rely (at least in part) on PDYN gene expression. Despite the massive upregulation of cortical FADD species, PARP fragmentation remained unchanged across the groups (Figure 4E), indicating that stress-induced FADD does not necessarily involve an increase in apoptotic rates. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 18 but not its phosphorylation, rely (at least in part) on PDYN gene expression. Despite the massive upregulation of cortical FADD species, PARP fragmentation remained unchanged across the groups (Figure 4E), indicating that stress-induced FADD does not necessarily involve an increase in apoptotic rates. Figure 4. Cortical immunodensities of (A) activated p-Thr183/Tyr185 JNK1/2, (B) activated pThr202/Tyr204 ERK1/2, (C) dimeric FADD, (D) oligomeric p-Ser191 FADD, (E) full-length and cleaved PARP, (F) activated p-Ser473 Akt, (G) p-Ser116 Pea15, and (H) activated p-Ser2448 mTOR in WT and PDYN-KO mice exposed to acute restraint (A, blue bars) or chronic mild (C, orange bars) 0 F Normalized density (%WT-B) 800 400 200 p-AktS473 / t-Akt p-Akt t-Akt <60 PDYN KO WT B A C B A C ** *** * *** <60 600 0 A Normalized density (%WT-B) 1500 1000 500 p-JNKT183/Y185 / t-JNK <54 p-JNK1/2 t-JNK1/2 <46 <54 <46 PDYN KO WT B A C B A C *** *** *** *** 0 B Normalized density (%WT-B) 600 400 200 p-ERKT202/Y204 / t-ERK p-ERK1/2 t-ERK1/2 <44 <42 PDYN KO WT B A C B A C *** *** *** *** <44 <42 Basal (B) Acute (A) Chronic (C) Stress protocol 0 D Normalized density (%WT-B) 800 400 200 Oligomeric p-FADDS191 p-FADD a-tubulin <116 PDYN KO WT B A C B A C *** *** *** *** <54 600 0 E Normalized density (%WT-B) 200 100 50 PARP PARP a-tubulin <116 <89 PDYN KO WT B A C B A C <54 150 116-kD 89-kD Figure 4. Yáñez-Gómez et al. 0 G Normalized density (%WT-B) 200 150 50 p-Pea15S116 / t-Pea15 p-Pea15 t-Pea15 <15 PDYN KO WT B A C B A C <15 100 0 C Normalized density (%WT-B) 800 400 200 Dimeric FADD <54 FADD a-tubulin <54 PDYN KO WT B A C B A C *** *** ** 600 0 H Normalized density (%WT-B) 8000 4000 2000 p-mTORS2448 / t-mTOR p-mTOR t-mTOR <250 PDYN KO WT B A C B A C *** *** <250 6000 Figure 4. Cortical immunodensities of ( A ) activated p-Thr183/Tyr185 JNK1/2, ( B ) activated pThr202/Tyr204 ERK1/2, ( C ) dimeric FADD, ( D ) oligomeric p-Ser191 FADD, ( E ) full-length and cleaved PARP, ( F ) activated p-Ser473 Akt, ( G ) p-Ser116 Pea15, and ( H ) activated p-Ser2448 mTOR in WT and PDYN-KO mice exposed to acute restraint ( A , blue bars) or chronic mild ( C , orange bars) stress procedures, as compared to basal (B, grey bars) stress levels in undisturbed animals. Normalized protein amounts were estimated as the ratio between the corresponding immunoreactive band to total enzyme or α -tubulin. Columns are means ± SEM of n = 7–9 mice per experimental group and expressed in percent change from wildtype-basal (WT-B) mice. All datasets were analyzed by TWANOVA (see Table 1). * p< 0.05, ** p< 0.01, and *** p< 0.001, TW-ANOVA followed by Tukey’s post hoc test. ( A – H ) Representative immunoblots of phosphorylated and/or non-phosphorylated species of the indicated proteins are shown at the bottom. The indicated molecular weights (in kDaltons, kDa) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards . Int. J. Mol. Sci. 2023,24, 2303 8 of 17 Furthermore, Akt phosphorylation at Ser473 was similarly upregulated in cortical samples of WT (2.8–3.2-fold increase, p< 0.001) and PDYN-KO (2.9–4.1-fold increase, p< 0.001 ) following ARS and CMS exposure (Figure 4F), indicating that the PDYN genotype had no impact on the stress-mediated stimulation of cortical Akt. Surprisingly, Akt hyperactivation did not significantly influence Pea15 phosphorylation at Ser116, which remained unaltered after the stress procedures in the same cortical samples (Figure 4G). On the other hand, the cortical immunodensities of p-Ser2448 mTOR were similarly increased in both murine strains in response to ARS (263-370-fold increase, p< 0.001) and, to a lesser extent, CMS (144–171-fold increase, p> 0.05) (Figure 4H). Of note, cortical amounts of p-mTOR in mice with basal stress levels were probably below the linear range of detection, and the data provided should be taken qualitatively rather than quantitatively. 2.4.2. ARS and CMS Effects on Thalamic Targets in WT and PDYN-KO Mice In marked contrast, stress exposure did not significantly modify JNK1/2 activation in the thalamus of WT and PDYN-KO mice compared to their respective basal groups (Figure 5A). Unfortunately, ERK1/2 was not addressed in these samples due to limitations in tissue availability. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 18 Figure 5. Thalamic immunodensities of (A) activated p-Thr183/Tyr185 JNK1/2, (B) dimeric FADD, (C) oligomeric p-Ser191 FADD, (D) activated p-Ser473 Akt, and (E) p-Ser116 Pea15 in WT and PDYN-KO mice exposed to acute restraint (A, blue bars) or chronic mild (C, orange bars) stress procedures, as compared to basal (B, grey bars) stress levels in undisturbed animals. Normalized protein amounts were estimated as the ratio between the corresponding immunoreactive band to total enzyme or α-tubulin. Columns are means ± SEM of n = 7–9 mice per experimental group and expressed in percent change from wildtype-basal (WT-B) mice. All datasets were analyzed by TWANOVA (see Table 1). * p < 0.05, and *** p < 0.001, TW-ANOVA followed by Tukey’s post hoc test. (A–E) Representative immunoblots of phosphorylated and/or non-phosphorylated species of the indicated proteins are shown at the bottom. The indicated molecular weights (in kDaltons, kDa) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards. 3. Discussion The present study aimed to unmask some molecular mechanisms that may contribute to the anxiogenic endophenotype in mice lacking the PDYN gene. The results showed that, under basal conditions, PDYN-KO animals display lower brain amounts of activated JNK1/2 and ERK1/2 MAPKs and FADD protein species, which are critical components of signaling pathways controlling crucial cellular functions related to neuroplasticity and cell fate. Interestingly, the present findings further suggested that the cortical hyperactivation of JNK1/2 and cortico-thalamic FADD upregulation following stressful stimuli may be dynorphin-dependent mechanisms, as these effects were not observed in stressed PDYN-KO mice. As the hyperactivation of ERK1/2 and Akt/mTOR pathways in the same cortical samples of ARSand CMS-exposed animals was not prevented by PDYN gene deletion, the control of dynorphins over the JNK1/2 and FADD cascades might be a selective mechanism. These ideas are graphically summarized in Figure 6. Basal (B) Acute (A) Chronic (C) Stress protocol Figure 5. Yáñez-Gómez et al. 0 A Normalized density (%WT-B) 200 100 50 p-JNKT183/Y185 / t-JNK <54 p-JNK1/2 t-JNK1/2 <46 PDYN KO WT B A C B A C 150 <54 <46 0 B Normalized density (%WT-B) 400 200 100 Dimeric FADD <54 FADD a-tubulin <54 PDYN KO WT B A C B A C 300 *** *** ****** 0 C Normalized density (%WT-B) 200 100 50 Oligomeric p-FADDS191 p-FADD a-tubulin <116 * <54 150 PDYN KO WT B A C B A C 0 D Normalized density (%WT-B) p-AktS473 / t-Akt p-Akt t-Akt <60 <60 200 100 50 150 PDYN KOWT B A C B A C 0 E Normalized density (%WT-B) p-Pea15S116 / t-Pea15 p-Pea15 t-Pea15 <15 <15 200 100 50 150 PDYN KOWT B A C B A C Figure 5. Thalamic immunodensities of ( A ) activated p-Thr183/Tyr185 JNK1/2, ( B ) dimeric FADD, ( C ) oligomeric p-Ser191 FADD, ( D ) activated p-Ser473 Akt, and ( E ) p-Ser116 Pea15 in WT and PDYN-KO mice exposed to acute restraint (A, blue bars) or chronic mild (C, orange bars) stress procedures, as compared to basal (B, grey bars) stress levels in undisturbed animals. Normalized protein amounts were estimated as the ratio between the corresponding immunoreactive band to total enzyme or α -tubulin. Columns are means ± SEM of n = 7–9 mice per experimental group and expressed in percent change from wildtype-basal (WT-B) mice. All datasets were analyzed by TW-ANOVA (see Table 1). * p< 0.05, and *** p< 0.001, TW-ANOVA followed by Tukey’s post hoc test. ( A – E ) Representative immunoblots of phosphorylated and/or non-phosphorylated species of the indicated proteins are shown at the bottom. The indicated molecular weights (in kDaltons, kDa) of the immunoreactive bands were estimated from in-gel-loaded, prestained protein standards. Int. J. Mol. Sci. 2023,24, 2303 9 of 17 In thalamic samples from the WT mice, significant upregulations of dimeric FADD were quantified in ARS (+78%, p< 0.001)- and CMS (+109%, p< 0.001)-exposed mice, compared to undisturbed animals with basal stress levels (Figure 5B). Interestingly, the stress-induced upregulations of dimeric FADD reported in the WT animals were not observed in the ARSor CMS-exposed PDYN-KO mice (response inhibition: 80–88%, p< 0.001) (Figure 5B). In marked contrast, the thalamic densities of oligomeric p-FADD remained unchanged in the WT mice exposed to stress stimuli, and only CMS-exposed PDYN-KO animals had significantly lower p-FADD densities, as compared to WT littermates exposed to the same stress procedure ( − 44%, p< 0.05) (Figure 5C). However, this difference in thalamic p-FADD levels may be attributed exclusively to genotype but not the genotype × stress interaction effects (see Table 1). Unlike the observations in the cerebral cortex, the thalamic samples of the ARSand CMS-exposed animals displayed similar amounts of activated Akt to those in the basal group, regardless of the genotype (Figure 5D). Accordingly, the thalamic immunodensities of p-Ser116 Pea15 did not differ significantly across the experimental groups (Figure 5E). 3. Discussion The present study aimed to unmask some molecular mechanisms that may contribute to the anxiogenic endophenotype in mice lacking the PDYN gene. The results showed that, under basal conditions, PDYN-KO animals display lower brain amounts of activated JNK1/2 and ERK1/2 MAPKs and FADD protein species, which are critical components of signaling pathways controlling crucial cellular functions related to neuroplasticity and cell fate. Interestingly, the present findings further suggested that the cortical hyperactivation of JNK1/2 and cortico-thalamic FADD upregulation following stressful stimuli may be dynorphin-dependent mechanisms, as these effects were not observed in stressed PDYNKO mice. As the hyperactivation of ERK1/2 and Akt/mTOR pathways in the same cortical samples of ARSand CMS-exposed animals was not prevented by PDYN gene deletion, the control of dynorphins over the JNK1/2 and FADD cascades might be a selective mechanism. These ideas are graphically summarized in Figure 6. Although the role of the PDYN gene in anxiety-like behaviors remains controversial [ 1 ], the present work used a PDYN-KO murine strain displaying a greater vulnerability to developing anxiety-like behaviors [ 11 , 12 ] to investigate the possible compensatory effects occurring in the cortico-thalamic, stress-related signaling pathways. PDYN-KO mice displayed reduced basal activation of the JNK1/2 and ERK1/2 MAPKs, as compared to WT littermates. Similarly, the multifunctional adaptor of the extracellular apoptotic pathway FADD, and its oligomeric form phosphorylated at Ser116, were downregulated in the same brain samples without changes in the Fas receptor species or PARP fragmentation. In terms of the signaling pathways studied, the PDYN gene deletion had a similar impact on both the cortex and the thalamus, suggesting that the reported adaptations to PDYN gene deficiency may begin early in brain development. Perhaps, the neural adaptations occurring in another PDYN-KO strain showing an anxiolytic endophenotype [ 3 , 13 ] evolved over the generations in opposite directions than those presented here, which could explain the contrasting behaviors against stressful stimuli of the two PDYN-KO murine strains. 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Neurobiol. 2013,48, 13–21. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.