International Journal of Molecular Sciences Article TRPV2: A Key Player in Myelination Disorders of the Central Nervous System Jennifer Enrich-Bengoa 1,2,† , Gemma Manich 2,3,† , Tony Valente 2,3,4 , Paula Sanchez-Molina 2,3 , Beatriz Almolda 2,3 , Carme Solà 5 , Josep Saura 6 , Berta González 2,3 , Bernardo Castellano 2,3 and Alex Perálvarez-Marín 1,2, * 1 Biophysics Unit, Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Catalonia, Spain; jennifer.enrich@uab.cat 2 Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Catalonia, Spain; gemma.manich@uab.cat (G.M.); tony.valente@uab.cat (T.V.); paula.sanchez@uab.cat (P.S.-M.); beatriz.almolda@uab.cat (B.A.); berta.gonzalez@uab.cat (B.G.); bernardo.castellano@uab.cat (B.C.) 3 Medical Histology Unit, Department of Cell Biology, Physiology and Immunology, School of Medicine, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Catalonia, Spain 4 Research Group on Methodology, Methods, Models and Outcomes of Health and Social Sciences (M3O), Experimental Sciences and Methodological Department, Faculty of Health Sciences and Welfare, University of Vic-Central University of Catalonia (UVic-UCC), 08500 Vic, Catalonia, Spain 5 Department of Cerebral Ischemia and Neurodegeneration, Institut D’Investigacions Biomèdiques de Barcelona-Consejo Superior de Investigaciones Científicas (CSIC), Institut D’Investigacions Biomèdiques August-Pi i Sunyer (IDIBAPS), 08036 Barcelona, Catalonia, Spain; carme.sola@iibb.csic.es 6 Biochemistry and Molecular Biology Unit, School of Medicine, Institut D’Investigacions Biomèdiques  August-Pi i Sunyer (IDIBAPS), University of Barcelona, 08036 Barcelona, Catalonia, Spain; josepsaura@ub.edu  * Correspondence: alex.peralvarez@uab.cat; Tel.: +34-93-581-4504 Citation: Enrich-Bengoa, J.; Manich, † These authors contribute equally to this work. G.; Valente, T.; Sanchez-Molina, P.; Almolda, B.; Solà, C.; Saura, J.; Abstract: Transient potential receptor vanilloid 2 (TRPV2) is widely expressed through the nervous González, B.; Castellano, B.; system and specifically found in neuronal subpopulations and some glial cells. TRPV2 is known to Perálvarez-Marín, A. TRPV2: A Key be sensitized by methionine oxidation, which results from inflammation. Here we aim to characterize Player in Myelination Disorders of the expression and regulation of TRPV2 in myelination pathologies, such as hypomyelination and the Central Nervous System. Int. J. demyelination. We validated the interaction between TRPV2 and its putative interactor Opalin, an Mol. Sci. 2022, 23, 3617. https:// oligodendrocyte marker, in mixed glial cultures under pro- and anti-inflammatory conditions. Then, doi.org/10.3390/ijms23073617 we characterized TRPV2 time-course expression in experimental animal models of hypomyelina- Academic Editors: tion (jimpy mice) and de-/remyelination (cuprizone intoxication and experimental autoimmune Antonio Ferrer-Montiel encephalomyelitis (EAE)). TRPV2 showed upregulation associated with remyelination, inflammation and Antonio Felipe in cuprizone and EAE models, and downregulation in hypomyelinated jimpy mice. TRPV2 expres- Received: 3 March 2022 sion was altered in human samples of multiple sclerosis (MS) patients. Additionally, we analyzed the Accepted: 22 March 2022 expression of methionine sulfoxide reductase A (MSRA), an enzyme that reduces oxidated methion- Published: 25 March 2022 ines in TRPV2, which we found increased in inflammatory conditions. These results suggest that Publisher’s Note: MDPI stays neutral TRPV2 may be a key player in myelination in accordance with the recapitulation hypothesis, and that with regard to jurisdictional claims in it may become an interesting clinical target in the treatment of demyelination disorders. published maps and institutional affil- iations. Keywords: transient potential receptor vanilloid 2; Opalin; oxidative stress; myelination; multiple sclerosis; recapitulation theory Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article Transient potential receptor vanilloid 2 (TRPV2) is a thermo-, mechano- and lipid distributed under the terms and sensor non-selective cation channel, and is a member of the superfamily of TRP chan- conditions of the Creative Commons Attribution (CC BY) license (https:// nels [1]. Activation of TRPV2 is triggered by noxious heat (>52 ◦ C), but also by mechanical creativecommons.org/licenses/by/ stimuli, different chemical compounds such as probenecid [1], and by oxidative stress 4.0/). through methionine (Met) oxidation [2]. TRPV2 is ubiquitously expressed in many human Int. J. Mol. Sci. 2022, 23, 3617. https://doi.org/10.3390/ijms23073617 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 3617 2 of 24 tissues, such as skeletal muscle, heart, lung, skin, thyroid, testis, and immune tissues [1,3]. In the central nervous system (CNS), TRPV2 expression has been specifically found in medium-to-large neurons bearing myelinated fibers [4], in microglia [5], astrocytes [6] and, at the mRNA level, in oligodendrocyte precursor cells (OPCs) [7], but so far, not in oligodendrocytes. TRPV2 expression and activity, both at the plasma membrane and/or internal endosomes, has been reported to be necessary for axon and neurite outgrowth of motor and sensory neurons [8,9], and in microglia and macrophages has been consistently related to phagocytosis of cellular debris [10]. Previous work from our lab [11] showed that TRPV2 putatively interacts with impor- tant key myelin proteins (i.e., proteolipid protein 1 (PLP1), Opalin, neurotrimin (NTM) and with other proteins involved in neoplasms and CNS diseases (ABR, FGF1, KCNJ10, PEBP1, PLP1 and SCD3) [12]. Intriguingly, Opalin, a protein located in the paranodal loop and soma of oligodendrocytes [13], was one of the proteins that showed the strongest interaction with TRPV2, suggesting that TRPV2 could be related to myelination, although its expression has not been shown in oligodendrocytes [7]. Studies by Hainz et al. [14–16] demonstrated that probenecid, a pannexin-1 antagonist, but also a specific TRPV2 agonist [1], improves the outcome in two experimental mouse models of multiple sclerosis (MS): in the experimental autoimmune encephalomyelitis (EAE) model, it is capable of preventing and arresting the progression of clinical symptoms and promoting oligodendrocyte proliferation [14,15] and in the cuprizone model of demyelination/remyelination, it reduces demyelination [16]. This evidence points to a putative role of TRPV2 as an upstream key player in myelination and remyelination processes in the CNS. In this study, we characterized the TRPV2–Opalin interaction in vitro and in mixed glial cell culture to assess if TRPV2 is expressed in oligodendrocytes. TRPV2 presence in oligodendrocyte-lineage cells, in addition to neurons, astrocytes and microglia, may provide support for the importance of TRPV2 in neuron-glial cells’ [7] cross-talk in CNS myelination. In parallel, we characterized TRPV2 expression in the CNS in three different myelination murine models: the hypomyelinating jimpy mice as a pathophysiological model for Pelizaeus–Merzbacher disease (PMD) [17–19] and the cuprizone- and EAE- induced experimental mouse models. To assess the potential role of TRPV2 in human pathophysiology, we determined TRPV2 gene and protein expression in tissue samples of human MS patients. 2. Results 2.1. Opalin Interacts In Vitro with the Ankyrin-Repeat Domain (ARD) of TRPV2 Since previous results from our group indicated a possible interaction between TRPV2 and NTM, PLP1 and Opalin [11], we studied these interaction pairs in vitro. Purified full- length (FL) rat TRPV2 and rat ARD were immobilized on nitrocellulose membranes, and then rat brain protein extracts were incubated with the membranes. After immunoblotting against the selected interactors, we observed that Opalin interacts with both the whole protein and with the cytosolic ARD of TRPV2 in a stronger fashion than NTM and PLP1 protein (Figure 1A). The mode of interaction between FL and the cytosolic domain of TRPV2 and Opalin indicates intracellular/transmembrane interaction, so both proteins coexist in the same cell, such as an oligodendrocyte-like cell. Int. J. Mol. Sci. 2022, 23, 3617 3 of 25 Int. J. Mol. Sci. 2022, 23, 3617 3 of 24 Figure 1. TRPV2 interaction Figure 1. TRPV2with Opalin,with interaction NTM and PLP1 Opalin, NTMin vitro and during PLP1 basal in vitro and basal during inflammatory and inflammatory conditions. (A) Rat TRPV2 conditions. (A) FL Ratprotein TRPV2 FL and rat ARD protein and of ratTRPV2 ARD of were TRPV2 immobilized on membranes were immobilized on membranes and and incubatedincubated with increasing with increasing concentrations concentrations of rat protein of rat protein brain lysates brain lysates (0–10Opalin, (0–10 µg). µg). Opalin, NTMNTM and and PLP1 were PLP1 were by detected detected by immunoblot. immunoblot. (B) Immunocytochemistry (B) Immunocytochemistry in mouseinmixed mouse mixed glial glial primary primary cultures shows TRPV2 (green), Opalin (red) and DAPI (blue). TRPV2-Opalin colocalization is shown cultures shows TRPV2 (green), Opalin (red) and DAPI (blue). TRPV2-Opalin colocalization is shown as yellow signal (merge) and both proteins are co-expressed in some cells (arrowheads). Expression as yellow signal (merge) and both proteins are co-expressed in some cells (arrowheads). Expression of these proteins is mainly found in the membrane. (C) TRPV2–Opalin colocalization expressed as of these proteins is mainly Pearson’s found in coefficient in the membrane. basal (C) TRPV2–Opalin and inflammatory conditions. colocalization expressed Both proteins show a highasdegree of Pearson’s coefficient in basal(D) colocalization. andTRPV2 inflammatory conditions. colocalization Both proteins with Opalin, show expressed asa Mander’s high degree of coefficient M2 (fraction of TRPV2 signal overlapping Opalin) in basal and inflammatory conditions. colocalization. (D) TRPV2 colocalization with Opalin, expressed as Mander’s coefficient M2 (fraction Both proteins of TRPV2 signalshow a high degree overlapping of colocalization. Opalin) in basal and Results were expressed inflammatory as mean conditions. ± SEM. Both Statistical proteins show analysis of the results were performed with an unpaired Student’s t-test (p < 0.05 is statistically significant). a high degree of colocalization. Results were expressed as mean ± SEM. Statistical analysis of the Scale bar = 20 µm. results were performed with an unpaired Student’s t-test (p < 0.05 is statistically significant). Scale bar = 20 µm. 2.2. TRPV2 and Opalin Display a High Degree of Colocalization in Mixed Glial Cultures 2.2. TRPV2 andEnriched OpalininDisplay Oligodendrocytes a High Degree of Colocalization in Mixed Glial Cultures Enriched in OligodendrocytesNext, to assess the possibility that TRPV2 and Opalin co-express in the same cell type, Next, towe determined assess if they were the possibility expressed that TRPV2 in Opalin and vitro inco-express mixed glialincell thecultures containing same cell microglia, astrocytes and oligodendrocytes (Supplementary Figure S1). type, we determined if they were expressed in vitro in mixed glial cell cultures containing Co-expression microglia, astrocytes and oligodendrocytes (Supplementary Figure S1). Co-expression was determined in basal conditions and after a pro-inflammatory stimulus, since myeli- Int. J. Mol. Sci. 2022, 23, 3617 4 of 24 nation disorders are associated with inflammatory processes. Results showed that some TRPV2+ cells also expressed Opalin with a high degree of colocalization (Figure 1B), as indicated by ca. 0.8 Mander’s and Pearson’s coefficients (Figure 1C,D). After exposure to a pro-inflammatory stimulus such as lipopolysaccharide (LPS) + interferon-γ (IFN-γ), levels of colocalization remained unchanged (Figure 1C,D). 2.3. TRPV2 Expression in Microglia and Oligodendrocytes Is Regulated after Pro-Inflammatory, Anti-Inflammatory and Demyelinating Treatments Cellular expression of TRPV2 was determined with double immunocytochemical staining performed in glial mixed cultures. These cultures were composedof approximately 25% microglia, 30% astrocytes and 35% oligodendrocytes in basal conditions, and their populations varied slightly after pro-inflammatory treatment (Supplementary Figure S1). In basal conditions, TRPV2 was mainly expressed in microglia, detected by lysosomal CD68, and oligodendrocytes, identified by platelet-derived growth factor receptor alpha (PDGFRα) (Figure 2A,G), while no expression was observed in glial fibrillary acidic protein (GFAP)-labelled astrocytes (Figure 2D). In both microglia and oligodendrocytes, TRPV2 was highly expressed and concentrated in the cell body, without labelling in their ramifications. After the pro-inflammatory treatment with LPS, TRPV2 intensity decreased in activated microglia, but its expression was extended to all of the microglial cell body and membrane, with a punctate staining similar to lysosomal CD68 (Figure 2B). In oligodendrocytes, we observed a similar expression pattern, consisting of a decreased intensity of TRPV2 staining spread throughout PDGFRα+ cell bodies (Figure 2H). After interleukin (IL)-4 anti-inflammatory treatment, TRPV2 was found in both ramified and activated microglia phenotypes, following the expression patterns explained above (Figure 2C). Similarly, oligodendrocytes showed high TRPV2 expression in their cell bodies, as been observed in the control condition (Figure 2I). In none of the treatments did GFAP+ astrocytes show TRPV2 expression (Figure 2E,F). Quantification of total TRPV2 expression in mixed glial cultures showed a significant decrease of TRPV2 after LPS pro-inflammatory treatment (Figure 2J, p < 0.01) compared to controls. After an anti-inflammatory IL-4 treatment, TRPV2 expression tended to increase (Figure 2J, p = 0.05) when compared to controls. Oxidative stress is known to induce oxidation of specific Met residues in TRPV2 (Met528 and Met607 in rat TRPV2) [2,20], endogenously activating the channel. As an indicator of oxidative stress in the cell culture, we measured nitric oxide (NO) concentration in cell media. Indeed, NO is also interesting since it promotes TRPV2 translocation to the plasma membrane [5]. Results showed that IL-4 treated cultures presented lower levels of NO in the cell culture medium compared to the control, while LPS-treated cultures showed a significant increase of NO (Figure 2L). In an attempt to closely determine the TRPV2 response in demyelination processes, we treated quaternary glial mixed cell cultures containing myelin-binding protein (MBP)- positive mature oligodendrocytes (see cell populations in Supplementary Figure S2) with the demyelinating and apoptotic cell agent L-α-lysophosphatidylcholine (LPC), which is also a direct activator of TRPV2 [21]. TRPV2 levels were significantly decreased in LPC-treated cultures compared to their respective controls, as similarly observed in the LPS pro-inflammatory treatment (Figure 2K, p < 0.01). Determination of NO in the cell media of LPC-treated cells, as a measure of oxidative stress, demonstrated similar levels compared to the control condition (Figure 2M). 2.4. TRPV2 and Opalin Expression, but Not Methionine Sulfoxide Reductase A (MSRA) Levels, Are Importantly Reduced in the Hypomyelinating Jimpy Mutant Mice TRPV2, Opalin and MSRA protein expression was explored in spinal cord sections of control and jimpy mutant mice by immunohistochemistry. First, we determined the degree of hypomyelination in jimpy mice by staining the MBP, one of the most abundant proteins in myelin. As expected, MBP was highly expressed and homogenously distributed in the white matter (WM) of spinal cord sections of control mice (Figure 3A,a) and slightly Int. J. Mol. Sci. 2022, 23, 3617 5 of 24 Int. J. Mol. Sci. 2022, 23, 3617 5 of 25 expressed in grey matter (GM) (Figure 3a’). Jimpy mutant mice showed a clear reduction of MBP expression and myelin thickness in both WM and GM (Figure 3B,b,b’,C, p < 0.0001). Figure 2. TRPV2 expression Figure 2. in mixedinglia TRPV2 expression mixed cultures of mice glia cultures of after pro-inflammatory mice after pro-inflammatory (LPS), anti- (LPS), anti- inflammatory inflammatory (IL-4) and(IL-4) and demyelinating demyelinating (LPC) treatments. (LPC) treatments. (A–I) (A–I) Double Double immunohistochemical immunohistochemical staining allowed for the determination of whether TRPV2 was expressed in microglia (A–C), staining allowed for the determination of whether TRPV2 was expressed in microglia (A–C), astro- astrocytes (D–F) or oligodendrocyte cells (G–I). TRPV2 was highly expressed in the cell body of cytes (D–F) some or oligodendrocyte cells (G–I). microglia (full arrowheads) TRPV2 but was not all of highly them (emptyexpressed arrowheads)in the cell body in control of some conditions, and microglia (full arrowheads) TRPV2 was spreadbut not all ofthrough afterwards them (empty the cellarrowheads) in control body of activated conditions, microglia cells, withand TRPV2 an expression pattern highly was spread afterwards coincident through the with CD68+of cell body lysosomes activated(arrows). While microglia no with cells, expression of TRPV2 was an expression found pattern in astrocytes highly coincident (empty lysosomes with CD68+ arrowheads), OPCs showed (arrows). While noeither low or absent expression of TRPV2expression in control was found in conditions (full and empty arrowheads, respectively) and increased expression throughout the cell astrocytes (empty arrowheads), OPCs showed either low or absent expression in control conditions (full and empty arrowheads, respectively) and increased expression throughout the cell body (arrows) Int. J. Mol. Sci. 2022, 23, 3617 6 of 24 and the principal soma (arrowheads) after both pro-inflammatory and anti-inflammatory treatments. (J–K) Quantification of TRPV2 protein (ng/mL) by ELISA in secondary mixed glial cultures in control conditions, and after LPS and IL-4 treatments (J) and in quaternary mixed glial cultures, containing myelin-binding protein (MBP)+ oligodendrocytes, in demyelinating conditions (K). Determination of TRPV2 showed a significant decrease in TRPV2 after LPS and LPC treatments (J, K), and an increase after IL-4 treatment (p = 0.05) compared to control conditions (J). (L,M) Quantification of NO secondary mixed glial cultures in control conditions, and after LPS and IL-4 treatments (L) and in quaternary mixed glial cultures in demyelinating conditions (M). An increase in NO concentration was observed in cell cultures after LPS treatment compared to control conditions, while a significant decrease was observed after IL-4 treatment. After LPC treatment, NO remained stable compared to the control. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison compared to the control in the LPS and IL-4 treated cultures (* p < 0.05, ** p < 0.01), and a paired Student’s t-test for LPC treatment (** p < 0.01). Scale bar (A–I) = 25 µm. In basal conditions, TRPV2 expression in GM was mainly located in the cell body of spinal cord neurons, while in WM, it was in the soma of cells, putatively oligodendrocytes (Figure 3D,d,d’). In the hypomyelinating jimpy mutant mice, TRPV2 expression was significantly reduced in both neurons and oligodendrocytes (Figure 3E,e,e’,F, p = 0.0062). We additionally evaluated Opalin expression in jimpy mice as an oligodendrocyte marker, but also as a TRPV2 interactor. Opalin is localized in WM-rich regions of the CNS and shows similar expression to the myelin marker MBP [13]. In the spinal cord of 21-day-old control mice we observed that Opalin was mainly distributed in WM, and located in cell bodies of oligodendrocytes and in the neuropil (Figure 3G,g). In GM, Opalin expression showed low expression, and was mainly located to some myelinated projections. As observed before for TRPV2 and MBP, Opalin was importantly reduced in both GM and WM (Figure 3H,h,h’) in the spinal cord of jimpy mice (p = 0.0092) in comparison with control animals (Figure 3I). Since TRPV2 is activated through Met oxidation, which can easily be oxidized by reactive oxygen species (ROS) [20], we explored the expression of the Met reducing enzyme MSRA, a counteracting enzyme that partially reverses TRPV2 endogenous activation [2]. Immunostaining showed that MSRA was found in the GM and WM of both control and jimpy mutant mice (Figure 3J,K). In control mice, this enzyme was expressed mainly in glial cells, putatively oligodendrocytes, in WM, and in glial cells and neurons in GM. Similar observations were made in jimpy mutant mice (Figure 3j,k,j’,k’). Despite an apparent reduction of MSRA in jimpy mutant mice compared to control mice (Figure 3K), the quantitative analysis did not show significant differences between them (Figure 3L). 2.5. TRPV2 and MSRA Expression Are Modulated in the Experimental Mouse Models of MS: The Cuprizone and the EAE Models To investigate TRPV2 and MSRA roles in demyelination, we used two experimental models of demyelination: the cuprizone-induced model and the EAE model. The effects of cuprizone intoxication on myelination were observed after MBP immunodetection: after a 5-week treatment, a substantial demyelination of specific WM areas, such as the corpus callosum (CC), was observed compared to basal conditions (Figure 4A,B), and cuprizone withdrawal allowed CC remyelination one week after (Figure 4C), as already quantified in previous work [22]. In the CC of control mice, TRPV2 and MSRA showed very low to undetectable levels (Figure 4D,G). After a 5-week cuprizone treatment, demyelinated CC showed a slight qualitative increase in TRPV2 (Figure 4E,H) that was mainly located in cell bodies of the CC, putatively oligodendrocytes (Figure 4E). In the caudal CC area, known to be resistant to myelination, TRPV2 increase was more visible (Figure 4D–F). After one week of remyelination, TRPV2 expression was significantly increased in mature oligodendrocytes of the CC when compared to control groups (p < 0.01) (Figure 4F, inset, J,K), as we confirmed with a double immunostaining against adenomatous polyposis coli (APC), and its levels were almost duplicated compared to demyelinated animals (Figure 4L). In the case of MSRA, a significant increase of this protein was observed after demyelination Int. J. Mol. Sci. 2022, 23, 3617 7 of 24 (p < 0.01) (Figure 4H,K), since most cells in the CC expressed low levels of MSRA. Later, Int. J. Mol. Sci. 2022, 23, 3617 after one week of remyelination, MSRA levels decreased, and only some MSRA+ cells were observed in the CC (Figure 4I,K). Figure 3. TRPV2, Opalin and MSRA expression in spinal cord of wild-type (WT) and hypomyelina- tion jimpy mice. Immunohistochemical staining against MBP (A,B), TRPV2 (D,E), Opalin (G,H) and MSRA (J,K) were Figure 3. TRPV2, performed in spinalOpalin and of cord sections MSRA expression 21-day-old in spinal WT and jimpy mice, cord of wild-type (W and immunore- hypomyelination jimpy mice. Immunohistochemical staining against activity was quantified and analyzed as Area x Intensity. Results show that MBP was importantly MBP (A,B), TRPV reduced in both GM (a,b) and WM (a’,b’) of jimpy mice compared to WT (A–C). TRPV2 expression WT an Opalin (G,H) and MSRA (J,K) were performed in spinal cord sections of 21-day-old mice,inand was mainly located immunoreactivity neurons in GM (d’) and was quantified glial and analyzed cells, putatively as Area x and oligodendrocytes, Intensity. in WMResults sh MBP was importantly reduced in both GM (a,b) and WM (a’,b’) of jimpy mice compared to (d) of WT. A significant decrease in TRPV2 expression in the spinal cord of jimpy mice was observed C). TRPV2 expression was mainly located in neurons in GM (d’) and glial cells, pu compared to WT (E,F,e,e’). Opalin expression was importantly found in WM of WT mice (G,g), and oligodendrocytes, and in WM (d) of WT. A significant decrease in TRPV2 expression in th to a lesser extent also in GM (g’), but showed a marked decrease in WM and GM of jimpy mice cord of jimpy mice was observed compared to WT (E,F,e,e’). Opalin expression was imp (H–I,h,h’). MSRA wasinthe found WMonlyof molecule WT micethat didand (G,g), nottosuffer an important a lesser extent alsoreduction of but in GM (g’), its expression showed a marked in both WM (j,k)inand WMGM (j’,k’) and GMinof jimpy jimpymice (L).(H–I,h,h’). mice Results were expressed MSRA was asthe only± molecule mean SEM. Statistical that did not s analysis of the important results were performed reduction of with an unpaired its expression WM t-test Student’s in both andp GM (j,k) (** < 0.01, *** pin< jimpy (j’,k’) 0.001).mice (L) Scale bar (A–K)were = 50 expressed µm; (a–k,a’–k’) = 20±µm. as mean SEM. Statistical analysis of the results were performed with an u Student’s t-test (** p < 0.01, *** p < 0.001). Scale bar (A–K) = 50 µm; (a–k,a’–k’) = 20 µm. 2.5. TRPV2 and MSRA Expression Are Modulated in the Experimental Mouse Models o The Cuprizone and the EAE Models To investigate TRPV2 and MSRA roles in demyelination, we used two exper models of demyelination: the cuprizone-induced model and the EAE model. The of cuprizone intoxication on myelination were observed after MBP immunode after a 5-week treatment, a substantial demyelination of specific WM areas, such corpus callosum (CC), was observed compared to basal conditions (Figure 4A, cuprizone withdrawal allowed CC remyelination one week after (Figure 4C), as quantified in previous work [22]. In the CC of control mice, TRPV2 and MSRA s very low to undetectable levels (Figure 4D,G). After a 5-week cuprizone tre demyelinated CC showed a slight qualitative increase in TRPV2 (Figure 4E,H) th increased in mature oligodendrocytes of the CC when compared to control groups (p < 0.01) (Figure 4F, inset, J,K), as we confirmed with a double immunostaining against adenomatous polyposis coli (APC), and its levels were almost duplicated compared to demyelinated animals (Figure 4L). In the case of MSRA, a significant increase of this protein was observed after demyelination (p < 0.01) (Figure 4H,K), since most cells in the Int. J. Mol. Sci. 2022, 23, 3617 8 of 24 CC expressed low levels of MSRA. Later, after one week of remyelination, MSRA levels decreased, and only some MSRA+ cells were observed in the CC (Figure 4I,K). Figure 4.4.TRPV2 TRPV2andandMSRA MSRA expression in WM expression in of WMcuprizone-induced demyelination of cuprizone-induced and remyeli- demyelination and nation in mice. (A–I) Representative images of immunohistochemical staining directed remyelination in mice. (A–I) Representative images of immunohistochemical staining directed against against MBP, TRPV2 MBP, TRPV2 and MSRAand MSRA in the in the CC CC of control of control mice (A,D,G) mice (A,D,G) and cuprizone-intoxicated and cuprizone-intoxicated mice mice after after demyelination (B,E,H, 5 weeks of treatment) and during remyelination (C,F,I, 5 weeks of demyelination (B,E,H, 5 weeks of treatment) and during remyelination (C,F,I, 5 weeks of treatment + 1 week of normal diet). (A–C) MBP immunostaining shows the myelinated CC of WT mice in basal conditions (A). At 5 weeks of cuprizone treatment, the CC is largely demyelinated (B) with a myelination-resistant area (*), and after one week of normal diet, the CC is mostly remyelinated (C). Control mice show undetectable levels of TRPV2 and MSRA in the CC (D and G). After demyelina- tion, variably low and high levels of TRPV2 were observed in cell bodies (empty arrowheads and arrowheads, respectively in E,F). High levels of TRPV2 were clearly observed at remyelination (inset in F). During remyelination, most TRPV2+ cells co-expressed APC (full arrowheads in L), a marker for mature oligodendrocytes. In the CC area resistant to demyelination (* in E,F), TRPV2+ cells showed high levels of expression in both conditions. An increase in MSRA levels of expression were also observed in cell bodies after demyelination and remyelination (arrowheads in H and I). Int. J. Mol. Sci. 2022, 23, 3617 9 of 24 In demyelination, most cells expressed low levels of MSRA in the CC (inset in H), while during remyelination, a lower number of MSRA+ cells expressing higher levels of MSRA were observed (inset in I). (J–K) Quantification of TRPV2 and MSRA expression after cuprizone treatment. (J) Quan- titative analysis of TRPV2 immunoreactivity in the CC confirmed the progressive increase of TRPV2 expression in demyelinating and remyelinating conditions compared to control mice, which was statistically significant in the latter. (K) Quantitative analysis of MSRA immunoreactivity in the CC showed that the increase of MSRA expression during demyelination was statistically significant when compared to control mice. Data are shown as ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test (** p < 0.01). Scale bar (A–I) = 50 µm; (L) = 100 µm. In the EAE model, in which demyelination foci are developed in the spinal cord accompanied by clinical symptoms of paralysis, we analyzed TRPV2 expression in spinal cord sections. In control mice, TRPV2 was mainly expressed in neurons in GM and oligodendrocyte-like cells in the WM (Figure 5A). After EAE induction, in the onset of clinical symptomatology, similar patterns of TRPV2 expression were observed, but later, at the peak of clinical symptomatology, TRPV2 was mainly located in WM, in the inflamma- tory focuses and in infiltrated cells (Figure 5A). During the chronic phase of EAE, in the remission of clinical symptomatology, TRPV2 expression was still found in neurons in GM, and in oligodendrocyte-like cells and inflammatory focuses in WM. However, at this time point, an important loss of intensity was found in comparison to TRPV2 levels of expres- sion observed in the peak (Figure 5A). Quantification of TRPV2 by western blot analysis (Figure 5B) confirmed the important increase of TRPV2 protein expression in the spinal cord of EAE mice at 14 days post-immunization (Figure 5B, p = 0.0014) compared to control mice. Later, at 21 and 28 days post-immunization, TRPV2 expression levels decreased and were maintained constant (Figure 5B). Evaluation of MSRA expression levels after EAE induction in mice showed an immediate significant increase when compared to the control complete Freund’s adjuvant (CFA)-injected mice (Figure 5B). Then, MSRA levels increased progressively until 21 days post-immunization (Figure 5B). At 28 days post-immunization, MSRA expression was significantly reduced when compared to 21 days post-immunization mice, and almost reverted to basal levels (CFA in Figure 5B). 2.6. MSRA and TRPV2 Gene and Protein Expression Are Modified in Frontal Cortex Samples of MS Patients Gene and protein expression of TRPV2 and MSRA was explored in frontal cortex tissue samples of healthy (n = 4) and MS subjects (n = 6). In MS subjects, a non-significant tendency to decrease of TRPV2 mRNA levels was observed when compared to healthy subjects (Figure 6A). On the contrary, MSRA mRNA expression in MS subjects was found to be significantly upregulated (p = 0.0014) when compared to healthy subjects (Figure 6B). TRPV2 and MSRA protein levels were also determined by western blot (Figure 6C). Protein levels of both MSRA and TRPV2 showed the same tendency as the mRNA expression analysis (Figure 6A–C): while a significant decrease of TRPV2 protein levels was observed in MS patients when compared to the control (p = 0.0232) (Figure 6C), MSRA protein expression levels were also significantly increased in MS patients compared to healthy subjects (p = 0.0124) (Figure 6C). Int. J. Mol. Sci. 2022, 23, 3617 10 of 25 Int. J. Mol. Sci. 2022, 23, 3617 10 of 24 A Control EAE Onset Peak Chronic TRPV2 B Trpv2 TRPV2protein protein MOG14 MOG28 MOG21 # MOG9 0.8 CFA * 0.6 Trpv2/β-Actin 0.4 80 KDa TRPV2 0.2 0.0 CFA MOG9 MOG14 MOG21 MOG28 Msra MSRAprotein protein ªªª ### 0.8 b *** 25 KDa MSRA & *** $$$ 0.6 ** Msra/β-Actin * 0.4 42 KDa β-Actin 0.2 0.0 CFA MOG9 MOG14 MOG21 MOG28 Figure5.5. TRPV2 Figure TRPV2 expression expressionin in spinal cord cord spinal samples from WT samples fromand EAE WT mice. and (A)EAEImmunohistochemi- mice. (A) cal staining of TRPV2 in spinal cord of control and EAE-induced Immunohistochemical staining of TRPV2 in spinal cord of control and EAE-induced mice in the onset, micepeak in and the in the onset, peak and in the chronic phase, during remission. In control conditions, TRPV2 chronic phase, during remission. In control conditions, TRPV2 is expressed in neurons in GM (inset, is expressed in arrows), neurons inandGMin (inset, arrows), andin oligodendrocytes inWMoligodendrocytes in WM (inset, (inset, arrowheads). After arrowheads). EAE induction, After an EAE increase in induction, an increase in TRPV2 expression is mainly located in inflammatory focuses at the peak TRPV2 expression is mainly located in inflammatory focuses at the peak clinical symptomatology in clinical symptomatology in WM (empty arrowheads). (B) MSRA and TRPV2 protein expression in WM (empty arrowheads). (B) MSRA and TRPV2 protein expression in thoracic spinal cord samples thoracic spinal cord samples from CFA and MOG mice (9, 14, 21 and 28 days post-immunization). (B)from CFA andincrease A significant MOG in mice TRPV2(9, 14, 21 and protein 28 (#, levels days post-immunization). p = 0.0014) is determined in(B) A significant MOG14 mice when increase in TRPV2 compared withprotein CFA (p levels < 0.05) p =when (#,and 0.0014) is determined compared in MOG14 with MOG9 mice (p < 0.05). when compared A significant increasewith (p CFA < 0.05) in MSRA (p < 0.05) and protein levels is observed when compared with MOG9in all groups of mice (p < 0.05). A treated with increase significant MOG when (p