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• W2977771295 abstract "Article30 September 2019Open Access Source DataTransparent process Disruption of Sema3A/Plexin-A1 inhibitory signalling in oligodendrocytes as a therapeutic strategy to promote remyelination Fabien Binamé Fabien Binamé INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Lucas D Pham-Van Lucas D Pham-Van INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Caroline Spenlé Caroline Spenlé INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Valérie Jolivel Valérie Jolivel INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Dafni Birmpili Dafni Birmpili INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Lionel A Meyer Lionel A Meyer INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Laurent Jacob Laurent Jacob INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Laurence Meyer Laurence Meyer INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Ayikoé G Mensah-Nyagan Ayikoé G Mensah-Nyagan INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Chrystelle Po Chrystelle Po Institut de Physique Biologique, Faculté de Médecine, Strasbourg University, Strasbourg, France Search for more papers by this author Michaël Van der Heyden Michaël Van der Heyden INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Guy Roussel Guy Roussel INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Dominique Bagnard Corresponding Author Dominique Bagnard [email protected] orcid.org/0000-0002-5261-902X INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Fabien Binamé Fabien Binamé INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Lucas D Pham-Van Lucas D Pham-Van INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Caroline Spenlé Caroline Spenlé INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Valérie Jolivel Valérie Jolivel INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Dafni Birmpili Dafni Birmpili INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Lionel A Meyer Lionel A Meyer INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Laurent Jacob Laurent Jacob INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Laurence Meyer Laurence Meyer INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Ayikoé G Mensah-Nyagan Ayikoé G Mensah-Nyagan INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Chrystelle Po Chrystelle Po Institut de Physique Biologique, Faculté de Médecine, Strasbourg University, Strasbourg, France Search for more papers by this author Michaël Van der Heyden Michaël Van der Heyden INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Guy Roussel Guy Roussel INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Dominique Bagnard Corresponding Author Dominique Bagnard [email protected] orcid.org/0000-0002-5261-902X INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Search for more papers by this author Author Information Fabien Binamé1,‡, Lucas D Pham-Van1,‡, Caroline Spenlé1, Valérie Jolivel1, Dafni Birmpili1, Lionel A Meyer1, Laurent Jacob1, Laurence Meyer1, Ayikoé G Mensah-Nyagan1, Chrystelle Po2, Michaël Van der Heyden1, Guy Roussel1 and Dominique Bagnard *,1 1INSERM U1119 Biopathology of Myelin, Neuroprotection, Therapeutic Strategy, Labex Medalis, Fédération de Médecine Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France 2Institut de Physique Biologique, Faculté de Médecine, Strasbourg University, Strasbourg, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33-3-68-85-71-50; E-mail: [email protected] EMBO Mol Med (2019)11:e10378https://doi.org/10.15252/emmm.201910378 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Current treatments in multiple sclerosis (MS) are modulating the inflammatory component of the disease, but no drugs are currently available to repair lesions. Our study identifies in MS patients the overexpression of Plexin-A1, the signalling receptor of the oligodendrocyte inhibitor Semaphorin 3A. Using a novel type of peptidic antagonist, we showed the possibility to counteract the Sema3A inhibitory effect on oligodendrocyte migration and differentiation in vitro when antagonizing Plexin-A1. The use of this compound in vivo demonstrated a myelin protective effect as shown with DTI-MRI and confirmed at the histological level in the mouse cuprizone model of induced demyelination/remyelination. This effect correlated with locomotor performances fully preserved in chronically treated animals. The administration of the peptide also showed protective effects, leading to a reduced severity of demyelination in the context of experimental autoimmune encephalitis (EAE). Hence, the disruption of the inhibitory microenvironmental molecular barriers allows normal myelinating cells to exert their spontaneous remyelinating capacity. This opens unprecedented therapeutic opportunity for patients suffering a disease for which no curative options are yet available. Synopsis Sema3A is a repulsive guidance molecule known to impact the migration of several cell types including oligodendrocytes and their progenitors (OPC). In the context of multiple sclerosis (MS), Sema3A is creating a molecular barrier that prevents OPC from entering lesion sites thereby precluding remyelination. The Sema3A receptor Plexin-A1 is overexpressed in MS patients and is upregulated in animal models of MS. Silencing of Plexin-A1 in vitro cancels Sema3A inhibitory effects on oligodendrocytes migration and differentiation. The Plexin-A1 transmembrane domain targeting inhibitory peptide efficiently blocks Sema3A-induced effects without side effect in vivo. The chronic administration of the peptide in two different mouse models of MS induced beneficial therapeutic effects exemplified by improved myelin content and functional locomotor recovery. Introduction The efficient propagation of electrical nerve impulses requires the establishment of myelin sheaths around axons (Baumann & Pham-Dinh, 2001). Multiple sclerosis (MS) is a disease affecting nerve conduction as a consequence of myelin damages. Current treatments of MS are not curative but only fighting the associated inflammation without repairing myelin. The myelination process is orchestrated by integrated cellular and molecular interactions unravelling potential therapeutic strategies for remyelination (Mi et al, 2007; Abu-Rub & Miller, 2018). During development, precursors of oligodendrocytes (OL), the myelinating cells of the central nervous system (CNS), use guidance cues to migrate towards their target neuronal cells. Strikingly, this process is not restricted to developmental phases but also occurs to a certain extent in the adult brain. This is also the case in CNS-demyelinating diseases in which partial spontaneous remyelination closely mimicking developmental myelination is observed. Combinations of soluble factors and membrane-bound cues are considered to create a specific molecular environment dictating the precise recognition process leading to the myelination of axons (Piaton et al, 2010). Among these factors regulating the early phase of myelination, members of the Semaphorin family have been shown to regulate OL precursor cell migration in the optic nerve (Tsai & Miller, 2002) and inhibit adult OL process outgrowth (Ricard et al, 2001). Strikingly, an analysis of human multiple sclerosis sample tissues and the use of an experimental model of demyelination revealed a clear spatio-temporal regulation of Sema3A expression, thereby strengthening the idea of a role of Semaphorins in myelination (Williams et al, 2007). However, little is known about the expression of Semaphorin receptors in this context. This is indeed a crucial issue because the biological functions of Semaphorins are intimately linked to the composition of a receptor complex associating various partners in order to trigger appropriate growth-promoting or growth-inhibiting effects of Semaphorins (Derijck et al, 2010). Thus, we decided to investigate the expression of Plexin-A1, one of the major Sema3A-transducing receptors so far essentially described as a transducer of Sema3A inhibiting signal in neurons (Püschel, 2002) or for its role in the immune system (O'Connor & Ting, 2008). We found that Plexin-A1 is overexpressed in the white matter of MS patients. Moreover, in mouse, only few CNP-positive OL expressed Plexin-A1. However, we observed a sevenfold increase in CNP-positive OL in animals exhibiting cuprizone-induced lesions. This was concomitant to local deposition of Sema3A in the demyelinated regions. Moreover, in vitro studies showed that blocking Plexin-A1 counteracted the anti-migratory and anti-differentiation effect of Sema3A in oligodendrocytes. Hence, we showed that the administration of the Plexin-A1 antagonist peptide MTP-PlexA1 improved myelin content and locomotor activity in mice fed with cuprizone to induce demyelinated lesions or in the context of experimental autoimmune encephalomyelitis (EAE). Altogether, our results suggest a therapeutic potential of inhibiting Plexin-A1 in myelin diseases such as multiple sclerosis in which we found overexpression of Plexin-A1. Results Plexin-A1 is expressed in human oligodendrocytes Previous studies showed the expression of Sema3A in MS lesions (Williams et al, 2007) where it is supposed to contribute to the lack of remyelination. We now examined whether OL express the Sema3A signalling receptor Plexin-A1. To this end, we performed immunocytochemical staining for Plexin-A1 on a human brain tissue array. The results confirm previous data (Jacob et al, 2016), showing neuronal expression of Plexin-A1 in several CNS locations, but also demonstrate the expression in oligodendrocyte cells in the white matter (Fig 1A). The global analysis of the tissue array revealed a wide expression in several regions of the central nervous system including the cortex, striatum or the spinal cord (data not shown). To confirm the identity of Plexin-A1-expressing cells in the white matter, we performed a CNP (3′,5′-cyclic nucleotide phosphodiesterase) staining (pan-marker of oligodendrocytes) on the adjacent section of the tissue array. False colour coding of the microphotographs allowed overlay of the two sections to exemplify the co-expression of Plexin-A1 and CNP (Fig 1B). Figure 1. Expression of Plexin-A1 in the human brain A. Microphotographs showing the expression of Plexin-A1 on a section of human brain array in the cerebellum (ML: molecular layer; PC: Purkinje cell layer; GL: granular layer; WM: white matter) and in the cortex. Oligodendrocytes in the white matter are expressing Plexin-A1. Scale bar = 10 μm. B. Immunostainings of Plexin-A1 and CNP (pan-oligodendrocyte marker) were conducted on adjacent sections of a human brain tissue array. Overlay of adjacent sections with false colour coding confirms the oligodendrocytic identity of Plexin-A1-positive cells in the white matter. Arrowheads indicate examples of double-stained oligodendrocytes. Scale bar = 10 μm. Download figure Download PowerPoint Plexin-A1 is overexpressed in MS patients In order to evaluate the Plexin-A1 level of expression in the context of multiple sclerosis, we first performed data mining from published gene array profiles using the GEO platform. The analysis of four chronic plaques and two healthy controls (Han et al, 2012) revealed an averaged 4.2-fold increase in Plexin-A1 mean expression (4.7-fold increase in the median) and 3.2-fold increase in SEMA3A mean expression (2.8-fold increase in the median) in the disease condition (Appendix Fig S1). We next collected and analysed white matter samples of 11 MS patients and nine healthy controls from the Netherlands Brain Bank (see Appendix Table S1 for details). We performed a Western blot analysis to evaluate Plexin-A1 content and found a 2.3-fold increased expression in MS patients (Fig 2A and B). The proportion of MS patients exhibiting such a twofold increase in Plexin-A1 expression above the averaged expression measured in healthy controls reached 45% of the patients (Fig 2C). To further characterize this overexpression of Plexin-A1, we also determined the number of CNP-Plexin-A1-positive cells by immunocytochemistry conducted on fresh-frozen sections of the white matter samples. As seen in Fig 2D and E, we found a threefold increase in Plexin-A1-positive CNP-expressing cells in MS patients compared to healthy controls. This suggested that Plexin-A1 may represent an interesting target in the context of MS. Figure 2. Expression of Plexin-A1 in multiple sclerosis patients vs. healthy controls A–C. Plexin-A1 immunoblotting analysis of brain samples of multiple sclerosis patients (n = 11) and healthy controls (n = 9). (A) Western blot revealed with anti-Plexin-A1 and stain-free method showing full protein content. (B) Relative expression normalized with full protein content (measured with stain-free method). Data are presented as mean ± SEM (Mann–Whitney, *P = 0.0167; n = 9 Ctrl and 11 MS patients). (C) Chi-square analysis of the proportion of patients with Plexin-A1 intensity > 2× mean control intensity. D. Representative microphotographs illustrating the expression of Plexin-A1 in CNP-positive OL in healthy control or MS white matter samples (Plexin-A1: green, CNP: red). Arrowheads indicate oligodendrocytes (CNP) positive for Plexin-A1. Scale bar = 50 μm. E. Quantification of the number of the CNP/Plexin-A1-positive cells in the white matter of control (HC) or MS autopsies. Data are presented as mean ± SEM (unpaired t-test, ***P = 0.0035; n = 9 Ctrl and 11 MS patients). Source data are available online for this figure. Source Data for Figure 2 [emmm201910378-sup-0002-SDataFig2.pdf] Download figure Download PowerPoint Plexin-A1 is overexpressed in OL in experimental demyelination conditions To address whether Plexin-A1 may be involved in demyelination/remyelination conditions in the adult, we used the cuprizone model. In this model, a demyelination/remyelination process is obtained by feeding mice with 0.3% cuprizone (bis-cyclohexanone oxal-dihydrazone) for 4 weeks (acute demyelination phase) and normal diet for 2 weeks (initiation of remyelination) or 4 weeks (induction of total remyelination; see Fig 3A for representative examples). Administration of 8-week cuprizone diet induces permanent demyelination as described previously (Matsushima & Morell, 2001). We performed double immunostaining for CNP and Plexin-A1 and determined at the corpus callosum level the number of double-positive cells in the different demyelination/remyelination status (Fig 3B). Only few OL expressed Plexin-A1 in control conditions (normal diet, 5.3% of CNP-positive OL). However, we found that Plexin-A1 was significantly overexpressed in OL after 4-week cuprizone and 2-week normal diet administration (37.8% of CNP-positive OL, ANOVA, P = 0.0015; Fig 3C). Strikingly, Plexin-A1 expression was back to control level after total recovery (4-week cuprizone + 4-week normal diet), while it was not overexpressed in the 8-week cuprizone group (Fig 3C). Moreover, similar to what has been previously described in human samples of multiple sclerosis (Williams et al, 2007), we also found that Sema3A expression transiently increased in acute phases of cuprizone-induced demyelination (Fig 3D). The overexpression of Sema3A was not uniform throughout the brain but rather matched with cuprizone-induced lesion sites. This suggested that a Sema3A/Plexin-A1 signalling is reactivated in the adult in case of demyelination/remyelination process. Figure 3. Expression of Plexin-A1 and Sema3A in a model of adult demyelination–remyelinationHistological analysis of CNP/Plexin-A1-positive cells in animal receiving 4-week cuprizone diet (acute demyelination phase), 4-week cuprizone diet followed by 2-week normal diet (initiation of remyelination), 4-week cuprizone diet followed by 4-week normal diet (induction of total remyelination), and 8-week cuprizone diet (permanent demyelination). A. Representative examples of demyelination plaques seen by osmium tetroxide impregnation (OsO4). Scale bar = 100 μm. B. Corresponding CNP/Plexin-A1 double staining showing Plexin-A1 expression in OL present at the lesion site. Scale bar = 10 μm. C. Quantification of the number of CNP/Plexin-A1-positive cells in the different experimental groups (w for weeks; data are presented as mean ± SEM, n = 3 mice per group in three independent experiments, 3–5 slices analysed per animal; ANOVA, **P = 0.0015). D. The expression of Sema3A is shown in adult brain at the level of hippocampus (sagittal sections) for control animals or animals receiving 4-week cuprizone diet (acute demyelination phase), 4-week cuprizone diet followed by 2-week normal diet (initiation of remyelination), and 4-week cuprizone diet followed by 4-week normal diet (induction of total remyelination). Scale bar = 1 mm. Download figure Download PowerPoint MTP-PlexA1 cancels Sema3A repulsive effect on oligodendrocyte migration Sema3A deposit inhibits OPC recruitment into MS lesions, explained in part by Sema3A repulsive effect (Boyd et al, 2013). We first addressed the involvement of Plexin-A1 in Sema3A signalling by RNA interference. We obtained a significant 50% knockdown of Plexin-A1 in Oli-neu cells as seen by immunocytochemistry and RT–qPCR (Fig 4A). We used XCELLigence transwell chambers to monitor 2% serum-induced chemotactic migration of the OPC cell line Oli-neu during 8 h. Compared to control migration without Sema3A, addition of 20 ng/ml Sema3A in the lower chamber decreased migration of 37% of Oli-neu cells transfected with siRNA control. Oli-neu cells transfected with siRNA targeting Plexin-A1 reached 95% of control migration (Fig 4B). This result confirmed the requirement of Plexin-A1 to drive the inhibitory effect of Sema3A. This lower expression was indeed sufficient to significantly decrease the number of NRP1/Plexin-A1 dimers (a key step to trigger semaphoring signalling) as determined by proximity ligation assay (Fig 4C). We next used the recently developed peptidic antagonist MTP-PlexA1. This peptide blocks receptor dimerization and signalling by interfering with the transmembrane domain of Plexin-A1. It has been successfully used in vitro to antagonize Plexin-A1 signalling and cell migration, while it showed anti-tumour effect in vivo (Jacob et al, 2016). Strikingly, the addition of MTP-PlexA1 induced a similar disruption of NRP1/Plexin-A1 dimers, thereby confirming the inhibitory effect of the peptide (Fig 4D). Pre-incubation of the Oli-neu cells with MTP-PlexA1 (10−7 M) cancelled Sema3A inhibitory effect by bringing migration up to the control condition without Sema3A (Fig 4E). This effect of MTP-PlexA1 was dose-dependent with a loss of efficacy from 10−9 M. Because of the toxicity of the vehicle (LDS) alone on Oli-neu cells (data not shown), we were not able to correctly evaluate higher concentrations of the peptide. However, because a maximal effect was obtained with 10−7 M we chose this concentration to define the dose for in vivo evaluation consistently with previous studies (Jacob et al, 2016). Figure 4. Inhibition of PlexA1 rescues Sema3A negative effect on migration and differentiation A. Cells were transfected with siRNA control or siRNA PlexA1. Downregulation of Plexin-A1 was validated by immunofluorescence staining with anti-Plexin-A1 antibody and by RT–qPCR analysis of Plexin-A1 mRNA expression normalized with GAPDH (data are presented as mean ± SEM, n = 3; Mann–Whitney test, *P = 0.05). Scale bar = 10 μm. B. Oli-neu cells were used for transfilter chemotaxis in response to 2% serum in the presence of Sema3A chemorepulsive (data are presented as mean ± SEM, n = 4 independent experiments, 1-way ANOVA and Kruskal–Wallis test, *P = 0.0458). C. Proximity ligation assay analysis was performed to quantify NRP1/Plexin-A1 dimers per cell treated with indicated siRNA. Representative microphotographs illustrating the different experimental conditions (data are presented as mean ± SEM, n = 7, Mann–Whitney test, ***P = 0.0006). Scale bar = 10 μm. D. Proximity ligation assay analysis was performed to quantify NRP1/Plexin-A1 dimers per cell treated with vehicle or MTP-PlexA1. Representative microphotographs illustrating the different experimental conditions (data are presented as mean ± SEM, n = 8, Mann–Whitney test, **P < 0.0001). Scale bar = 10 μm. E. Oli-neu cells were used for transfilter chemotaxis in response to 2% serum in the presence of Sema3A chemorepulsive. Cells were pre-incubated with MTP-PlexA1 at indicated concentrations or vehicle alone. Results are expressed as a percentage of positive control migration, i.e. migration with 2% serum and without Sema3A and without chemoattractant (data are presented as mean ± SEM, n = 3 independent experiments, ANOVA and Bonferroni's multiple comparison test, **P = 0.0025, ***P = 0.0005). F. Expression of mature oligodendroglial marker MBP was analysed in murine neural stem cells (mNSCs) by RT–qPCR following 4 days of differentiation. Cells were concomitantly treated with Sema3A and MTP-PlexA1 or vehicle. Results are expressed relatively to differentiated cells without treatment (data are presented as mean ± SEM, n = 3 independent experiments, ANOVA and Kruskal–Wallis test, *P = 0.0132). Download figure Download PowerPoint MTP-PlexA1 favours oligodendrocyte differentiation in the presence of Sema3A Remyelination failure in MS lesions can result from the lack of OPC recruitment as well as inhibition of OPC differentiation into mature myelinating oligodendrocytes. Our second in vitro functional test evaluated by RT–qPCR MTP-PlexA1 ability to increase a late oligodendroglial marker (MBP) expression during neural stem cell (NSC) differentiation. Plating of multipotent NSC onto PLO (poly-L-ornithine) with low growth factor concentration induces their differentiation into oligodendroglial, astrocytic glial and neural lineage. By adding triiodothyronine hormone and ascorbic acid into differentiation medium, we favoured oligodendroglial lineage. 100 ng/ml of Sema3A reduced by twofold the expression of MBP mRNA after 4 days of differentiation, whereas concomitant treatment with 10−7 M of MTP-PlexA1 brings back MBP mRNA expression to 1.2-fold of control condition without Sema3A (Fig 4F). MTP-PlexA1 exhibits no toxicity in vivo Because of the expression of Plexin-A1 in adult neurons (Jacob et al, 2016), we had to check whether a chronic treatment with MTP-PlexA1 could induce cognitive disabilities. We first evaluated the locomotion capability of vehicle (LDS) and MTP-PlexA1-treated animals that had received 1 μg/kg MTP-PlexA1 three times a week for 4 weeks in an open-field task (Fig 5A). No difference was found between the two groups, indicating that mice had the same exploration capacity. We then investigated mouse anxiety with an elevated plus maze (EPM) test. After quantification, groups exhibited no significant difference in open-arm exploration, demonstrating that MTP-PlexA1 has no measurable effect on plus maze-evaluated anxiety compared to vehicle (Fig 5B). Hence, we assessed the hippocampal function integrity with a spatial recognition task. Here again, the object discrimination capacity (determined through the recognition index) was identical in the two groups, thereby demonstrating no impact of MTP-PlexA1 on mouse cognitive functions (RI vehicle 0.36, RI MTP-PlexA1 0.35; Fig 5C and D). Figure 5. Cognitive toxicity assessmentMice were treated 4 weeks with vehicle or MTP-PlexA1 for behavioural tests. A. Determination of the global locomotion behaviour in the open-field task during 10 min (data are presented as mean ± SEM, n = 10 mice per group, Mann–Whitney test, n.s = not significant). B. Determination of the anxiety behaviour in the elevated plus maze with time spent in open arm during 10 min (data are presented as mean ± SEM, n = 10 mice per group, Mann–Whitney test, n.s = not significant). C, D. Spatial object recognition task measuring the novel object preference after training sessions. (C) Experimental procedure. (D) Recognition index = shifted object time/total exploration time (data are presented as mean ± SEM, n = 10 mice per group; Wilcoxon test, vehicle **P = 0.0020, MTP-PlexA1 **P = 0.0020). Download figure Download PowerPoint To further address the innocuity of MTP-PlexA1, we also performed a blood sample analysis on four vehicle and four MTP-PlexA1-treated animals. White and red cell numeration showed no difference. Kidney and hepatic functions were also equivalent in the two groups (Appendix Table S2). This lack of toxicity offered the possibility to test this administration schedule and dosing similar to what was previously performed to treat tumours (Jacob et al, 2016) in a model of demyelination to demonstrate the therapeutic potential of MTP-PlexA1. MTP-PlexA1 rescues corpus callosum myelination in demyelinating cuprizone murine model To investigate the therapeutic potential of MTP-PlexA1, we followed by MRI (DTI and T2WI) and histology the evolution of the white matter using the cuprizone-induced demyelination–remyelination mouse model (Fig 6A). Averaged food intake monitoring confirmed equal consumption of the cuprizone diets in all experimental groups (Fig 6B). The analysis of T2WI signal intensity was used to evaluate the level of inflammation (signing the existence of a toxic effect of cuprizone), whereas DRAD (radial dif" @default.
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• W2977771295 title "Disruption of Sema3A/Plexin‐A1 inhibitory signalling in oligodendrocytes as a therapeutic strategy to promote remyelination" @default.
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