extfieldsofstudy: Medicine Biology

text:
Investigating the neuroprotective effect of AAV-mediated β-synuclein overexpression in a transgenic model of synucleinopathy

Parkinson’s disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases characterized by inclusions mainly composed of α-synuclein (α-syn) aggregates. The objective of this study was to investigate if β-synuclein (β-syn) overexpression could have beneficial effects by inhibiting the aggregation of α-syn. The M83 transgenic mouse is a model of synucleinopathy, which develops severe motor symptoms associated with aggregation of α-syn. M83 neonate or adult mice were injected with adeno-associated virus vectors carrying the human β-syn gene (AAVβ-syn) or green fluorescent protein gene (AAVGFP) using different injection sites. The M83 disease was - or not - accelerated using extracts of M83 brains injected with brain extract from mouse (M83) or human (MSA) origins. AAV vectors expression was confirmed using Western blot and ELISA technics. AAV mediated β-syn overexpression did not delay the disease onset or reduce the α-syn phosphorylated at serine 129 levels detected by ELISA, regardless of the AAV injection route and the inoculation of brain extracts. Instead, a proteinase-K resistant β-syn staining was detected by immunohistochemistry, specifically in sick M83 mice overexpressing β-syn after inoculation of AAVβ-syn. This study indicated for the first time that viral vector-mediated β-syn overexpression could form aggregates in a model of synucleinopathy.

Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are synucleinopathies, characterized by inclusions mainly composed of an aggregated form of α-synuclein (α-syn) in the central nervous system (CNS). As for prions, aggregated forms of α-syn propagate within the CNS during the associated neuro-degenerative diseases. This characteristic was initially suggested in humans by Braak's description of PD stages, and was confirmed later in experimental models of synucleinopathies, in particular in the M83 transgenic mouse model 1 . M83 mice express the human A53T mutated α-syn found in some familial PD forms, under the control of the mouse prion promoter 1 . These mice spontaneously develop severe motor impairment at 8-16 months of age at the homozygous state. The symptomatology is associated with the accumulation in the CNS, of a pathological form of α-syn (α-syn P ), heavily phosphorylated at serine 129 residue. We previously showed that, in M83 mice, disease onset can be accelerated by the intracerebral inoculation of brain homogenates from sick M83 mice 2 . This model was further characterized by development of an original ELISA that specifically detects, and allows to easily quantify, the pathological α-syn in sick M83 mice [3][4][5] . This in vivo model may be useful for testing novel therapeutic strategies, particularly targeting progression of the α-syn aggregation.
β-synuclein (β-syn) is another member of the synuclein family, lacking a part of the non-amyloid component, a specific region suggested to be amyloidogenic in α-syn. According to in vitro studies, unlike α-syn, β-syn alone is not able to form aggregates, but could instead interact with α-syn and reduce its capacity to aggregate 6 . These anti-aggregative features have been investigated in vivo, and some of these studies indeed reported a neuroprotective effect and reduction of α-syn inclusions after overexpression of the human β-syn mediated by DNA microinjection or lentiviral vectors in the transgenic D mouse model, overexpressing human wild-type α-syn 7,8 . Another study also suggested that crossing mice overexpressing human β-syn with M83 mice delayed the M83 disease onset and reduced the α-syn aggregation 9 . However, two recent studies by Taschenberger  which analyzed the impact of human β-syn overexpression mediated by adeno-associated viral vectors (AAV) on nigral dopaminergic neurons in rats, described a neurodegeneration with β-syn aggregates 10,11 . Here, in order to assess the effects of β-syn overexpression in the context of these recent and unexpected data suggesting that β-syn produced using AAV may be able to aggregate, we investigated the impact of human β-syn overexpression mediated by AAV on the onset of the synucleinopathy of M83 mice. Two strategies of inoculation of the AAV vector were sequentially tested: (i) an intracerebroventricular (ICV) inoculation at birth, in order to generate a widespread overexpression of the vector in the central nervous system 12 , and (ii) an inoculation of the AAV vector into the ventral tegmental area (VTA) 13 , a region of the mesencephalon in which prominent α-syn aggregation occurs in sick M83 mice 1 . Our results indicate that, regardless of the AAV inoculation strategy, continuous AAV-mediated β-syn overexpression in neurons did not delay the onset of the disease and did not modify the α-syn P levels measured by ELISA, strongly suggesting that β-syn may not protect against α-syn aggregation and propagation.

Results
Human β-syn is widely expressed after intracerebroventricular injection of AAVβ-syn in neonates. To overexpress β-syn in the M83 CNS, we used self-complementary (sc)AAV9 vectors expressing human β-syn (AAVβ-syn) gene or, as a control, the green fluorescent protein (AAVGFP) gene under the control of the human synapsin1 promoter and with a post-transcriptional WPRE regulatory sequence ( Supplementary Fig. 1A).
To functionally evaluate the AAV vectors, we first inoculated AAVβ-syn in the right and left cerebral ventricles of wild-type B6C3H newborn mice (genetic background of M83 transgenic mice) to obtain a widespread expression of the transgene in the CNS 12 . One month later, using viral specific primers, vector mRNA was detected in all regions of the brain and in the spinal cord ( Supplementary Fig. 1B), as described 14 , with higher levels in rostral regions of the brain (olfactory bulbs, cortex, hippocampus, striatum) and mesencephalon. Intracerebroventricular injection of AAVβ-syn did not modify the disease of M83 mice. AAV vectors were first tested in M83 mice challenged by inoculation of brain extracts from sick M83 mice, an experimental design which is characterized by a quicker onset of the disease with a lower intragroup variability as compared to unchallenged M83 mice 2 . AAV vectors were ICV inoculated at birth and two months later challenged with different brain extracts of sick M83 mice, one derived from a second passage of a sick M83 brain extract in M83 mice (M83/M83 inoculum), and the other from a second passage of a human brain extract from a MSA patient in M83 mice (MSA/M83 inoculum) (Fig. 1A, Supplementary Table 1) 5 . M83 mice were euthanized after the detection of the first symptoms of disease, i.e balance disorders or hind limb paralysis, identified by two independent observers.
In both experiments, no delay in the onset of the disease was observed in AAVβ-syn injected mice as compared to control AAVGFP mice, regardless of the type of inoculum (M83/M83 or MSA/M83). Instead, AAVβ-syn injection significantly accelerated the onset of the disease in animals challenged with the M83/M83 extract as compared to control mice (log rank test p < 0,05) (Fig. 1B). In contrast, no difference was observed in animals challenged with the MSA/M83 extract (Fig. 1E). In each experiment, we did not detect any difference concerning the clinical symptoms of sick mice after the injection of AAVβ-syn or AAVGFP. After dissection of the brains, biochemical analyses were realized. As the vector expression was higher in the rostral regions of the brain than in other CNS regions after ICV injections, we first checked the vector expression by detecting total β-syn in the hippocampus. An ELISA test adapted from Krassnig et al. allowed to detect a significant overexpression of β-syn (both murine β-syn and human β-syn produced by the vector) in the hippocampus, confirming the continuous expression of the AAVβ-syn until the disease onset (Fig. 1C,F). We next focused on the mesencephalon, the brainstem and the spinal cord because these CNS regions are known to be strongly positive in α-syn P in sick M83 mice [3][4][5] . A significant overexpression of β-syn was only detected in the mesencephalon, the brain stem and in the spinal cord of sick M83 mice injected with AAVβ-syn challenged with M83/M83 inoculum, but not in mice challenged with the MSA/M83 inoculum (Fig. 1C,F). Since the M83 disease is associated with moderate or high α-syn P levels into different brain regions and in the spinal cord 5 , we quantified α-syn P using ELISA in order to detect a potential effect of β-syn on this biochemical marker of the disease. Our analyses did not show any significant difference in the α-syn P levels in any of the examined CNS regions after AAVβ-syn inoculation, regardless of the nature of the inoculum used to accelerate the disease (Fig. 1D,G).
To further study the levels of β-syn protein after AAV injections in M83 mice throughout the CNS, we used Western blot and ELISA which allowed to detect total β-syn in specific brain regions. Comparing total β-syn levels of 5 to 7 months old sick M83 mice inoculated at birth with AAVβ-syn or AAV GFP then challenged with the MSA/M83 inoculum (same mice as in Fig. 1E), an overexpression of β-syn protein was detected by ELISA in the olfactory bulbs, cortex, striatum, hippocampus, but not in the other regions ( Fig. 2A), consistent with the results obtained by qRT-PCR at one month after AAV inoculation ( Supplementary Fig. 1B). Western blot analyses confirmed an overexpression of β-syn protein in the hippocampus of the same mice after AAVβ-syn inoculation (Fig. 2B,C). Most of the animals inoculated with AAVβ-syn showed a β-syn overexpression detectable until the disease onset (Fig. 1C,F). Only a few animals did not show significant overexpression of β-syn detected by ELISA or by immunohistochemistry (5/48 animals, in the entire study); these animals were excluded for the study analyzing the onset of disease.
We next examined the possible effects of AAVβ-syn ICV injection on the spontaneous development of the M83 disease during aging, without any challenge to accelerate the disease (Fig. 3A). Here again, AAVβ-syn injection neither delayed the disease onset of M83 mice nor modified the α-syn P levels (Fig. 3B,D), despite a sustained expression of the transgene, at least in the hippocampus, up to the disease onset ( Fig. 3C) (ELISA data correspond to 2 mice for the treated group and 3 mice for the control group; consequently, no statistical analysis was done here). Altogether, these results indicated that AAVβ-syn injection in the ventricles resulted in a widespread expression of the transgene in the CNS, but at a moderate level, especially in the structures that are the most heavily affected by the synucleinopathy lesions. We thus considered another strategy by inoculating the AAV vectors in the ventral tegmental area (VTA), which is located into the mesencephalon, where major brain lesions and accumulation of α-syn P are detected in sick M83 mice and which is also connected to multiple brain areas 13 . β-syn is overexpressed mostly in the mesencephalon after injection of AAVβ-syn in the VTA of adult mice. In order to validate this injection protocol of AAV vectors into the VTA, two months old wild-type mice were first inoculated with AAVβ-syn in the VTA and sacrificed one month later. After dissection of the brains, vector mRNA was mostly detected in the mesencephalon, but also in the cortex, striatum, After the appearance of the first specific symptoms of M83 disease (from 3 months to 8 months after challenge), mice were sacrificed and dissected in order to perform biochemical analysis. (B) M83 disease-associated survival after the inoculation of the M83/M83 brain extract is represented (significant difference according to log rank test, p < 0.05). (C,D) Quantification of total β-syn (mouse β-syn and human β-syn produced by the vector) (C) or of pathological Ser129-phosphorylated form of α-syn (α-syn P ) (D) by ELISA in CNS regions of the same sick M83 mice groups (n = 6 for the treated and the control group). (E) M83 disease-associated survival after the inoculation of the MSA/M83 brain extract (no significant difference according to log-rank test). (F,G) Quantification of total β-syn (F) or α-syn P (G) by ELISA in CNS regions of the same sick M83 mice groups (n = 6 for the treated group and n = 5 for the control group). Hi: hippocampus, Mes: mesencephalon, BS: brain stem, CSC: cervical spinal cord, TSC: thoracic spinal cord, LSC: lumbar spinal cord. Data are shown as mean ± sd. *p < 0,05; **p < 0,01; according to Wilcoxon test. Injection of AAVβ-syn in the VTA did not modify the disease of M83 mice. The same protocol was then applied to M83 mice. After the inoculation of AAV in the VTA in two months old M83 mice, the disease was accelerated by intracerebrally inoculating the mice one month later with the M83/M83 or the MSA/M83 inoculum (Fig. 4A). No significant difference in the survival was observed between the AAVβ-syn and AAVGFP injected mice, between 3 and 8 months (Fig. 4B,E). As before, we did not detect any difference concerning the clinical symptoms of sick mice after the injection of AAVβ-syn or AAVGFP. Importantly, analyses by ELISA confirmed the strong overexpression of β-syn in the mesencephalon although no difference was detected in the levels of pathological α-syn P at the disease stage ( Fig. 4C-G). By immunohistochemistry, using an antibody targeting total β-syn (human and murine β-syn), a specific β-syn staining punctate pattern was detected in the inoculated mesencephalon, as well as in the striatum and brain stem which represent connected regions 13 , of all the sick M83 mice inoculated with AAVβ-syn (5/5), but not in sick M83 mice inoculated with AAVGFP (0/2) (Fig. 4H). Further analysis indicated that these β-syn immunoreactive dots, which are specifically detected in mice inoculated with AAVβ-syn, mostly co-localize with the presynaptic protein synaptophysin (Supplementary Fig. 2A). Co-localization of GFP with neuronal marker β-tubulin type 3 in sick M83 mice inoculated with AAVGFP also confirmed that these vectors allowed a neuronal specific expression of the transgene ( Supplementary Fig. 2B). Interestingly, the morphology of the inclusions containing α-syn phosphorylated at serine 129 detected by immunohistochemistry was not modified by β-syn overexpression ( Supplementary Fig. 3A). Furthermore, these inclusions only rarely co-localized with AAV-specific β-syn staining by immunofluorescence ( Supplementary Fig. 3B). We further biochemically characterized vectors expression after injection in the VTA in M83 mice euthanized 4 months later. Total β-syn levels were quantified by ELISA and showed a significant increase of the protein in the mesencephalon of mice injected with AAVβ-syn as well as in the hippocampus and brain stem compared to control mice injected with the AAVGFP (Fig. 5A). β-syn protein overexpression was also readily detected by Western blot in the mesencephalon of most of these mice (Fig. 5B).
The absence of protective effect was not due to an insufficient dose of AAV vector injected in the VTA. We finally asked whether the absence of any significant effect of AAVβ-syn could be due to an insufficient dose of AAV vector. We thus injected 4-times more vector in the VTA one month before the challenge  in the striatum to accelerate the disease. As before, after the appearance of the first specific symptoms of M83 disease (from 3 months to 8 months after challenge), mice were sacrificed and the CNS was dissected in order to realize biochemical analysis and immunohistochemistry /immunofluorescence studies (Fig. 4H, Supplementary  Figs 2-4). (B) M83 disease-associated survival after the inoculation of the brain extract (no significant difference according to log-rank test). (C,D) Quantification of total β-syn (C) or pathological Ser129phosphorylated form of α-syn (α-syn P ) (D) by ELISA in CNS regions of the same sick M83 mice groups (n = 5 for the treated group, n = 5 for the control group). (E) The M83 disease-associated survival after the inoculation of the MSA/M83 inoculum (no significant difference according to log-rank test). (F, G) Quantification of total β-syn (F) or α-syn P (G) by ELISA in CNS regions of the same mice groups (n = 5 for the treated group, n = 5 for the control group). (H) Immunohistochemistry pictures showing total β-syn staining (mouse β-syn and human β-syn produced by the vector) in the mesencephalon of 7 months old sick M83 mice inoculated with AAV β-syn by intra-cerebral inoculation of the M83/M83 inoculum. This AAV dose allowed to significantly increase the overexpression of β-syn at the disease stage (p < 0,05) (Fig. 5C,D). However, the M83 disease was still not delayed by β-syn overexpression, but instead, as previously observed after ICV injection of AAV (Fig. 1B), the disease appeared significantly earlier in the AAVβ-syn injected mice as compared to control AAVGFP animals (p < 0,001) (Fig. 5E). As in previous experiments, no impact was found on the α-syn P levels even if β-syn was significantly increased at the stage of clinical signs in all the CNS regions (Fig. 5D,F). By immunohistochemistry, as with the low dose of AAV, a specific β-syn staining punctate pattern was detected in the inoculated mesencephalon, as well as in the striatum and brain stem in two sick M83 mice inoculated with AAVβ-syn, but not in one mouse inoculated with AAVGFP (data not shown).
AAV-mediated overexpression of β-syn might have formed aggregates of β-syn. Proteinase K (PK)-resistant β-syn aggregates have been previously described after inoculation of AAV vectors expressing human β-syn in the substantia nigra of rats 10,11 . We thus analyzed by immunohistochemistry the brain of several sick M83 mice after inoculation of AAVs in the VTA (Fig. 6). After PK digestion, total β-syn antibody revealed a punctate pattern specifically detected after the inoculation of AAVβ-syn, in the mesencephalon of 7/7 sick M83 mice (5 mice injected with low dose of AAV, of which 2 were challenged with M83/M83 inoculum and 3 with MSA/M83 inoculum, as well as 2 mice injected with high dose of AAV and challenged with M83/M83 inoculum). This punctate pattern was also detected in the striatum and the brain stem where the AAV is also expressed, but not in the cerebellum where the AAV is less expressed (Supplementary Fig. 4). A few cell bodies were also stained with total β-syn antibody after PK digestion, particularly in the mesencephalon of these mice (data not shown). Even if diffuse, PK resistant staining was also detected in the hippocampal region of two sick M83 mice injected with AAVβ-syn and not challenged with brain extract inoculation, but not in one sick M83 control mouse (data not shown). These results confirm that overexpression of β-syn resulted in PK-resistant β-syn staining which could be detected independently of the vector dose, the inoculation site or the age of inoculation. Interestingly, Western blot detection of β-syn in the brain homogenates from sick M83 mice injected with high dose of AAVβ-syn showed the appearance of an additional band, consistent with a dimer, that is not visible in the homogenates from sick M83 mice injected or not with AAVGFP (even after long exposure of the blot) ( Supplementary Fig. 5).

Discussion
β-syn was previously described as a neuroprotective protein, able to inhibit the aggregation process of α-syn in vitro and having benefic effects on synucleinopathies in vivo [6][7][8][9]15 . Here, we tried to slow down a synucleinopathy in a transgenic mouse model with dramatic clinical symptoms linked to pathological α-syn aggregation. In particular, we analyzed the effects of the overexpression of β-syn mediated by an AAV vector on α-syn aggregation. We show here that the delivery of the AAVβ-syn vector via two different injection routes (intracerebroventricular or in the ventral tegmental area) in transgenic M83 mice, neither delayed the disease onset nor reduced the levels of pathological α-syn at the disease stage, despite a sustained overexpression of β-syn. A limitation of our study remains the low number of animals that were followed in each of the experimental groups, given the multiple strategies that were studied (different routes and ages of AAV inoculations, different AAV doses, AAV effects during normal aging or after intracerebral experimental challenges of two M83 sources (M83/M83 or MSA/M83) as summarized in Supplementary Table 1. This represents a statistical limitation of our study, although it is noteworthy that in the only two experimental groups in which a statistically significant difference was found between the survival of mice injected with AAVβ-syn and AAVGFP vectors, this rather suggested a deleterious effect of β-syn overexpression. Studies on larger cohorts will thus be required to confirm our results. A previous study indeed suggested that constitutive overexpression of β-syn in transgenic M83 mice by generating double transgenic mice delays the M83 disease onset by several months 9 . In our study, interestingly, M83 mice overexpressing β-syn showed a higher spontaneous activity in experiments in which open field was performed and a tendency to have more balance trouble in beam walking test (Supplementary Fig. 6). These results suggest that β-syn expression might have an impact on mice behavior without affecting the ultimate development of the disease. Importantly, in this study of Fan and colleagues, human β-syn was expressed from the pan-neuronal mouse prion promoter, which is the same promoter used to express human mutated α-syn in the M83 model. It is possible that the strategies used in our study did not provide sufficient expression of β-syn, in the CNS regions targeted by the disease. Indeed, as expected, after the injection of AAV in the VTA, we obtained a very high overexpression of β-syn in the mesencephalon in which the aggregation of α-syn is high at the disease stage, but this region may not be the region where the aggregation process of α-syn begins 16 . It should be emphasized that the major signs of the clinical disease of M83 mice are in relation with some -still poorly explained-disorders in the spinal cord 17 . In an additional series of experiments, we were unable to identify a degeneration of motor neurons in the lumbar spinal cord after the injection of preformed fibrils of human α-syn in M83 mice, as previously reported after intra-muscular injections 17 , further interrogating the cause of the appearance of symptoms ( Supplementary  Fig. 7). The ICV injection of AAV9 vector in SMNΔ7 mice, a severe model of spinal muscular atrophy, allowed to express a codon-optimized human SMN1 coding sequence in the spinal cord, which as a result, has improved significantly their survival 18 . After the ICV inoculation of AAV β-syn, the overexpression of β-syn was diffuse and lower in caudal regions of the brain and spinal cord, as described 14 , but it may have been insufficient to slow or AAV GFP and challenged with MSA/M83 inoculum. Mes: mesencephalon, BS: brain stem, CSC: cervical spinal cord, TSC: thoracic spinal cord, LSC: lumbar spinal cord. Data are shown as means ± sd. *p < 0,05; **p < 0,01 according to Wilcoxon test. Scale bar 100 µm. down the synucleinopathy process. It must however be noted that an in vitro study using AAV vectors suggests that β-syn overexpression is benefic on α-syn linked toxicity only at a low ratio of β-syn/α-syn expression 10 . Even if it was not evaluated here, another study has described transgene expression in the neuromuscular junction after AAV injection into the ventricles at birth; this may be important since the degeneration of the neuromuscular junction was also described as a possible explanation of the appearance of symptoms in M83 mice 14,17 . At the cellular level, human β-syn seems mostly localized in the presynaptic button and axons in sick M83 mice, which seems appropriate because the aggregation of α-syn was suggested to start at the presynaptic level in synucleinopathies 19,20 . Another study suggested that the benefic effect of β-syn is the result of the down regulation of the expression of α-syn, but we did not detect any decrease of α-syn expression, even in the brain regions overexpressing β-syn (data not shown). In another transgenic mouse model (D line) overexpressing human wild type α-syn, two studies reported the reduction of the synucleinopathy by either generating bigenic mice that also overexpress β-syn, or vehiculating the human β-syn transgene, under the control of cytomegalovirus (CMV) promoter, in a lentiviral vector 7,8 . As some reports indicate a downregulation of the CMV promoter in the CNS over time 21 , we chose here to use the human synapsin 1 promoter to obtain a long term expression of β-syn, which was confirmed in the present study, even in old mice. Importantly, as previously described 22 , the synapsin 1 promoter also allowed to express human β-syn specifically in neurons, where α-syn aggregates are found in the M83 model 1 . According to the study by Hashimoto et al., the neuroprotective effect of β-syn was associated to the activation of Akt, a neuroprotective protein which could be indirectly down-regulated by α-syn during the synucleinopathy process 23 . However, we did not detect any significant increase of Akt activation by Western blot in 6 months-old M83 mice inoculated with AAVβ-syn in the VTA at 2 months, and challenged one month later with the M83/M83 inoculum ( Supplementary Fig. 8). It should be noticed that such Akt changes were not consistently observed, as shown in a study with a lentiviral vector used for β-syn overexpression 24 . Surprisingly, in the present study, two experiments in mice challenged with a M83/M83 inoculum rather showed a significant acceleration of the disease after AAV β-syn injection (Figs 1B and 5E). Interestingly, several studies reported that β-syn could be implicated in the pathological process of synucleinopathies. Indeed, an axonal pathology with β-syn accumulation in axons was reported in patients with Parkinson's disease, DLB or Hallervorden-Spatz syndrome 25,26 . In addition, two mutations of β-syn, identified in DLB cases, were suggested to be responsible for α-syn aggregation and DLB 27 . Generating transgenic mice co-expressing P123H mutated β-syn with human α-syn resulted in an enhanced pathology, suggesting that mutated β-syn could potentiate α-syn aggregation 28 . Variable results were also observed after overexpression of β-syn using a lentiviral vector after inoculation into the hippocampus of transgenic mice overexpressing a mutated form of the human β-amyloid precursor protein (APP) involved in Alzheimer's disease, as assessed by examining plaque load, memory deficits, and anxiety 24 . However, it should be noted that the expression level of β-syn was relatively low in this latter study (not detectable at the protein level). Also, it is interesting to notice that a truncated form of human recombinant α-syn lacking the 71-82 residues not able to form aggregates in vitro, like β-syn 29 , accelerated the disease of a few M83 mice, after injection in the muscle or in the peritoneal cavity 17,30 . These results raise the possibility that β-syn might be able to accelerate the aggregation of α-syn in M83 mice, although this has not been reported so far. Further analyzing the effects of β-syn expression in sick M83 mice inoculated with the AAV vector in the VTA allowed detecting a punctate pattern of β-syn staining in the targeted brain region and some connected regions, only in mice inoculated with AAVβ-syn. This staining was more resistant to PK digestion than that endogenous β-syn in control mice. This result suggests that overexpressed β-syn formed aggregates which could explain why β-syn did not act as expected, i.e as an α-syn aggregation inhibitor. Landeck et al. have described the same staining of β-syn resistant to PK digestion referred as "dark punctae", two months after the inoculation of AAV5 carrying human β-syn under the control of chicken beta-actin promoter including a CMV enhancer element (CBA) in the substantia nigra of rats 11 . Before this study, Taschenberger et al. have also described the detection of PK-resistant β-syn aggregates in vivo, as early as two weeks following the inoculation of AAV2 carrying human β-syn gene in the substantia nigra of rats 10 . This time, the vector design was closer to ours, using the same promoter (human synapsin) to express human β-syn, with a WPRE sequence to enhance transgene expression. It is interesting to notice that, even if the AAV serotype used as well as the vector design can differ between studies, all these studies that used AAV to carry β-syn also reported the detection of β-syn aggregates. Furthermore, our observations in immunohistochemistry are supported biochemically in the present study, by the detection of a specific β-syn pattern in Western blot that could be a specific signature of β-syn aggregation, in mice showing overexpression of β-syn after injection of high dose of AAVβ-syn ( Supplementary Fig. 5). All these observations suggest that the neuroprotective effect of β-syn may vary according to the strategy followed to overexpress it and the animal model. For example, it would be interesting to inject AAV vectors used in this study in transgenic D mice in order to see if we obtain the same β-syn aggregates. The D line is indeed an example of transgenic mice expressing the normal human α-syn, which may thus be more representative of the common situation in humans. The interest of the M83 mouse line, which is well illustrated in our study, is the possibility to easily quantify both survival of mice, ultimately showing a major clinical disease, and levels of pathological α-syn, using an ELISA test. The A53T mutation in the human protein is however associated with a high propensity to aggregation 31 and our observations with AAV-mediated β-syn overexpression may not be representative of the situation of humans with non genetic synucleinopathies. Also, it is important to point out that even if most of neurons express the transgene in the cerebral region injected of mice in our study, there is an important variability of the expression of the transgene in transduced cells after AAV vectors injection (Supplementary Fig. 9). It might explain at least partly the difficulty to detect a benefic effect of β-syn overexpression as it was suggested to depend on a specific α-syn/β-syn ratio in cell culture 10 .
The present study suggest for the first time that β-syn overexpression could not be benefic in a synucleinopathy model. This may be due at least partly to the formation of a PK-resistant β-syn species after AAVβ-syn inoculation. According to a very recent study, a mildly acidic pH environment, found in several organelles, could induce β-syn aggregation, highlighting the complexity of β-syn fibrillation mechanisms occurring in vivo 32 . Further studies are needed to better understand how and to what extent β-syn could play a role in synucleinopathies.
However, even though we could not detect any protective effects of β-syn, our study confirms that an AAV vector is suitable for long-term overexpression of proteins into the CNS. Notably, after ICV inoculations of AAV in neonates, we confirmed a widespread expression of the protein, as this was also recently described using an α-syn AAV vector 33 . The injection of AAV in the VTA also allowed to express human β-syn in various connected regions, as suggested in a previous study using serotype 9 AAV vector carrying a lysosomal enzyme gene 13 . Our data also illustrate the robustness of the experimental model of M83 mice intracerebrally challenged by inocula containing aggregated α-syn, showing rather short and relatively uniform survival periods before the appearance of characteristics clinical signs, which can be used for the assessment of future therapeutic strategies.

Methods
Animals. M83 transgenic mice were used in this study (B6;C3H-Tg[SNCA]83Vle/J, RRID:MGI:3603036, The Jackson Laboratory, Bar Harbor, ME, USA). These mice express A53T mutated human α-syn protein and spontaneously develop severe motor impairment leading to early death 1 . Homozygous M83 mice develop characteristic motor symptoms between 8 and 16 months of life, beginning with reduced ambulation, balance disorders, partial paralysis of a hind leg and then progressing to prostration, difficulty in feeding, weight loss, hunched back and general paralysis 1 . The animals were housed per group in enriched cages in a temperature-controlled room on a 12 h light/dark cycle, and received water and food ad libitum, in our approved facilities (No. C69 387 0801) for breeding and experimental studies, in accordance with EEC Directive 86/609/EEC and French decree No. 2013-118. The experimental studies described in this article were performed in containment level 3 facilities and authorized by the ≪Comité d' éthique≫ CE2A -16, ComEth ANSES/ENVA/UPEC and by the ≪Ministère de l' enseignement supérieur, de la recherche et de l'innovation≫ (ref. . All experiments were performed in accordance with relevant guidelines and regulations. AAV vectors: Recombinant self-complementary AAV9 vectors (scAAV9) encoding human β-syn or enhanced GFP (eGFP) were produced by calcium phosphate transfection of HEK-293 cells 34 . Three plasmids were transfected simultaneously: (i) a vector plasmid containing the human gene of β-syn or eGFP under control of the neuron-specific synapsin 1 gene promoter ( Supplementary Fig. 1A) (ii) a helper plasmid pXX6 35 and (iii) a plasmid carrying rep2 and cap9 genes 36 . Vector particles were extracted and purified on an iodixanol step gradient. Titers were determined by quantitative polymerase chain reaction (qPCR) and expressed as viral genomes per milliliter (vg/mL). . Two months after the inoculation of the viral solution in neonates or one month after the inoculation of the viral solution in adults, M83 mice were inoculated with brain homogenates from a second passage of a sick M83 brain sample in M83 mice (M83/M83 inoculum) or a second passage of a patient with multiple system atrophy (MSA) brain sample in M83 mice (MSA/M83 inoculum) (same sample used in a previous publication 5 ). These homogenates were injected in the left striatum, using stereotaxic coordinates (AP: +0.14 ML: +2 DV: −2.75). Before each stereotaxic surgery (AAV or brain extract inoculation), mice have been anesthetized with a xylazine (10 mg/kg) and ketamine (100 mg/kg) mixture.
Western blot. 50 µg (for phosphorylated Akt detection) or 10 µg of proteins (for total Akt detection) or 2 µg of proteins (for GFP and β-syn detection) were separated in 12% SDS-polyacrylamide gels and electroblotted onto polyvinylidene fluoride (PvF) membranes 0,45 µm (Bio-Rad). The membranes were washed 3 times in Tris-Buffered Saline (TBS) for 5 min at room temperature (RT) under agitation and were saturated 1 h with 5% BSA in TBS 0.1% Tween20 (TBST). Membranes were incubated with rabbit antibody against phosphorylated Akt at Ser473 (ref. Immunohistochemistry/Immunofluorescence. After dissection, brain samples were fixed in 4% paraformaldehyde and paraffin embedded to be cut into serial 6 µm sections. After deparaffinization, endogeneous peroxidase activity was directly blocked with oxygenated water 3% during 5 minutes at RT. Brain sections were pretreated with a citrate solution (ref. C9999, Sigma, Saint-Quentin-Fallavier, France) with heat antigen retrieval. For the detection of α-syn phosphorylated at serine 129 by immunohistochemistry, we did an additional antigen retrieval step in which sections were treated with a 4 M solution of guanidinium thyocyanate during 20 min. After washing, sections were saturated using a blocking reagent (ref. San diego, USA) diluted at 1:100) at 4 °C overnight. For the detection phosphorylated at S129 α-syn we used a rabbit antibody (ref: ab51253, Abcam, Cambridge, GB) diluted at 1:300 in TBST for immunohistochemistry, and a mouse antibody (ref: pSyn#64, FUJIFILM Wako Pure Chemical Corporation, China) diluted at 1:1000 in TBST for immunofluorescence analysis (with the rabbit anti-β-syn antibody, Supplementary Fig. 3 PK digestion. After being deparaffinized, sections were incubated with a 10 µg/mL solution of proteinase K (ref EU0090-B, Euromedex, Souffelweyersheim, France) diluted in PBS, 10 minutes at RT (protocol adapted from 10 ). Sections were washed three times in water before blocking the endogenous peroxidase activity and pursuing the immunohistochemistry staining protocol.
Statistical analysis. Survival time was defined as the time from birth until death (Fig. 3A, exclusively) or as the time from the inoculation of the brain extract until the appearance of the first specific M83 symptoms and euthanasia of the mouse. The detection of these specific symptoms was done blindly with regard to the initial AAV (β-syn or GFP) treatment. We right-censored mice found dead without M83 disease identification. Survival times were compared using log-rank test. Concerning statistical analysis of ELISA tests results, means were compared using Wilcoxon test. The difference was significant when *p < 0,05, **p < 0,01, ***p < 0,001.


PADI2-mediated citrullination is required for efficient oligodendrocyte differentiation and myelination

Citrullination, the deimination of arginine residues into citrulline, has been implicated in the aetiology of several diseases. In multiple sclerosis (MS), citrullination is thought to be a major driver of pathology, through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show citrullination by peptidylarginine deiminase 2 (PADI2) is required for normal oligodendrocyte differentiation, myelination and motor function. We identify several targets for PADI2, including myelin-related proteins and chromatin-associated proteins, implicating PADI2 in epigenetic regulation. Accordingly, we observe that PADI2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PADI2, display motor dysfunction and decreased number of myelinated axons in the corpus callosum. We conclude that citrullination is required for oligodendrocyte lineage progression and myelination and suggest its targeted activation in the oligodendrocyte lineage might be beneficial in the context of remyelination.

Introduction
The conversion of a peptidylarginine into a peptidylcitrulline can introduce profound changes in the structure and function of the modified protein. This posttranslational modification, also known as protein citrullination or deimination, is calcium dependent and is mediated by the family of enzymes called peptidylarginine deiminases (PADIs) 1 . Five PADI isozymes (PADI1, 2, 3, 4 and 6) have been identified in mammals, and their expression pattern and function is tissue-specific 2 . Citrullination has been recently shown to play key roles in multiple cellular processes such as inflammatory immune responses 3 , apoptosis 4 , and regulation of pluripotency 5 . In the central nervous system (CNS), PADI2 is the most prevalent expressed PADI and occurs preferentially in oligodendrocytes (OL) and other glial cells 6 . OLs arise at postnatal stages from the differentiation of OL precursor cells (OPCs) and their most prominent function is the formation of the myelin sheath, an electrical insulating layer for the axons, essential for proper neuronal communication and thus CNS function 7 .
PADI2 has been reported to heavily citrullinate myelin binding protein (MBP), a fundamental myelin component. The ratio MBPcit/MBPtotal is high in the first four years of life and similarly in multiple sclerosis (MS), a disease where demyelination occurs 8 . Upon citrullination, MBP arginine residues lack positive charges, thereby having a reduced interaction with the negatively charged lipids. Consequently, citrullinated MBP does not form compact sheaths and the myelin remains immature or becomes unstable 9 . In accordance, transgenic mice overexpressing PADI2 in OL cells display myelin loss 10 . In light of this, PADIs, and in particular PADI2, have been thought to have a detrimental effect in MS and thus an improvement of the disease symptoms can be achieved by inhibiting their actions 11 . Indeed, treatment of a MS mouse model Experimental Autoimmune Encephalomyelitis (EAE) with 2-chloroacetamidine (2-CA), a PADI2 and -4 inhibitor leads to disease attenuation 12 . The improvement on disease progression by 2-CA treatment was not specifically assigned to the lack of PADI activity in OL lineage cells alone but to inhibition of all PADI-expressing cells, including immune cells that play an essential role in the disease development.
Interestingly, however, no changes in disease progression were observed upon EAE induction in PADI2 KO mice 13 . This could point to a possible compensation of PADI2 functions by other PADIs, or to additional functions of PADI2 in EAE disease progression. While citrullination of myelin might play an important role in MS, other proteins such as histone H3 have been found to be citrullinated in MS 14 . Thus, the role of PADI2-mediated citrullination in MS might not be confined to myelin. Furthermore, while the effects of Padi2 overexpression in OL have been characterized, its absence in OL lineage cells has not been further investigated, nor its physiological function in OL lineage cells and its significance for the myelin integrity maintenance.

Results
Padi2 expression is increased upon OL differentiation. By analyzing our single-cell RNA-Seq dataset of the OL lineage in the adult and juvenile mouse brain 15 , we identified Padi2 as the predominant Padi expressed in OLs (Supplementary Fig. 1a). Interestingly, Padi2 expression is found in OPCs, increases in committed OL precursors (COPs) and newly formed OLs (NFOLs) and peaks at more mature stages ( Supplementary Fig. 1a). Surprisingly, we did not observe expression of Padi4, which has been previously suggested to be present in myelin 14,16 . To further investigate the pattern of expression of Padi2 during OL lineage progression, we cultured OPCs isolated from postnatal day P1-P4 brains of the transgenic mouse line Pdgfra-histone 2b (H2B)-GFP 17 in which GFP expression is under the control of the endogenous Pdgfra promoter locus. GFP+ OPCs were collected with fluorescence activated cell sorting (FACS) to plates and expanded in media containing the growth factors (GFs) bFGF and PDGFAA and differentiated into OLs by removing the GFs for 2 days (Fig. 1a). Gene expression of the differentiation markers Mbp and Sox10 was upregulated and the progenitor marker Pdgfra was reduced upon GFs removal (Fig.  1b). In agreement with the single-cell RNA-seq data, Padi2 was expressed in OPCs and it was greatly enhanced upon differentiation ( Fig. 1b and Supplementary Fig. 1b). To investigate Padi2 expression in the OL lineage in vivo, we isolated OPCs and OLs from the brain of the transgenic mouse line Pdgfra-Cre-loxP-GFP 18 in which all OL lineage cells ex-press the GFP reporter (Fig. 1c). GFP+ OPCs were collected with FACS from P1-P4 pups (all GFP+ cells are OPCs since OLs are not formed yet) and GFP+ CD140-OLs were collected with FACS from juvenile (P21) and adult (P60) mice (at this stage GFP+ cells comprise all OL progeny including CD140+ OPCs). As a confirmation of the sorted populations we have assessed the expression of Pdgfra and Mbp as markers for OPCs and differentiated oligodendrocytes, respectively ( Fig. 1d). Padi2 mRNA was substantially enriched in OLs from both juvenile and adult brains when compared to postnatal OPCs (Fig. 1d). At the protein level, and in agreement with our gene expression data, we observed a continuous increase in PADI2 protein from P1 to adult in the spinal cord of wild type mice, concomitant with the increase in the OL marker MBP (Fig. 1e). Thus, PADI2 is rapidly upregulated upon OPC differentiation, suggesting a role of this citrullinating enzyme at this stage of OL lineage progression.
Reduction of PADI2 activity hinders OL differentiation. In order to investigate whether the increased expression of PADI2 upon OL differentiation has a functional significance, we inhibited PADI activity in the mouse OPC cell line Oli-neu 19 and rat primary OPCs, using Cl-amidine, a pan-inhibitor of PADI enzymes. Strikingly, Oli-neu cells failed to differentiate in the presence of this inhibitor, as observed by the lack of cell branching typical of differentiating OLs shown with CNPase immunostaining (Fig. 2a). In accordance, gene expression analysis on undifferentiated and differentiated Oli-neu cells treated with Cl-Amidine re-vealed a striking impairment on OPC differentiation. The increase of Mbp, Sox10 and Mog mRNA levels, crucial for proper OPC differentiation, was no longer observed after Cl-Amidine during differentiation (Fig. 2b). Interestingly, the expression of the transcription factor (TF) Sox9, required for OL specification 20 and downregulated upon OPC differentiation 15 , was increased upon PADI inhibition (Fig. 2b). Similar results were obtained by treating Oli-neu with another PADI-inhibitor 2-CA ( Supplementary Fig. 1c). Importantly, these effects were also observed in rat OPC primary cultures, where under differentiating conditions, the treatment with Cl-Amidine impaired differentiation as observed by the lower mRNA expression levels of the differentiation markers Mog and Mag (Fig. 2c).
To investigate whether the effect of PADI inhibitors was due to PADI2 inhibition, we specifically decreased Padi2 mRNA levels in mouse primary FACS OPCs by transfecting cells with siRNA against Padi2 (targeting exon 2). Similar to PADI inhibition, the reduction of Padi2 expression led to a decrease in the mRNA levels of Sox10 and Mog and an increase in Sox9 (Fig. 2d). Mog mRNA levels were also reduced in Padi2 knockdown conditions after 2-days of differentiation (Fig. 2d). Likewise, Padi2 knockdown in Oli-neu cells mimicked the effects of PADI inhibition (Supplementary Fig. 1d). Thus, our findings indicate that PADI2 is dra-matically increased on the onset of OL differentiation, where it contributes to the transition from an OPC state to a NFOL state.
OLs are reduced in conditional Padi2 knockout mouse lines. To investigate if the observed effects of PADI2 have an impact on OL differentiation and myelin maintenance in vivo, we generated a Padi2 conditional knock-out (cKO) mouse line, Pdgfra-Cre; RCE:loxP; Padi2-/-where Padi2 is not present in any cells derived from Pdgfra+ cells, which include the whole OL lineage. We estimated the number of CC1+ cells per area at P21, in both corpus callosum and in the dorsal funicullus of the spinal cord. In accordance to our results in Oli-neu and primary FACS OPC cultures (Fig. 2), we observed a reduction in the number of CC1+ OLs in the spinal cord of Padi2 cKO mice, compared to littermate controls (Fig. 3a, b). Nevertheless, we did not observe such an effect in the corpus callosum (Fig. 3a). This finding led us to further investigated if these OLs exhibited the same levels of OL-related genes by performing qPCR on the brains of P21 controls and Padi2 cKO. We observed that Pdgfra, Mbp and Mog mRNA were unaltered in FACS cKO GFP+ cells (Supplementary Fig. 2c), indicating that the effect of Padi2 cKO is more prominent in posterior regions of the CNS. We also generated an inducible Padi2 cKO (indKO) mouse  line, Pdgfra-CreERT; RCE:loxP; Padi2-/-, where Padi2 could be ablated from OPCs by tamoxifen treatment at early postnatal stages (Fig. 3c). Since we observed that PADI2 mainly modulates the transition between OPCs and NFOLs in vitro (Fig. 2), we depleted Padi2 specifically at the onset of OPC differentiation by injecting mice with tamoxifen at P6 and collecting the brains and spinal cord at P11 (Fig.  3c). We show a reduction, although not statistically significant (p=0.0828 for corpus callosum and p=0,0567 for spinal cord), of OLs formed upon tamoxifen-induced OPC Padi2 depletion, as assessed by the percentage of CC1+ cells out of total GFP+ in the corpus callosum and in the spinal cord of Padi2 indKO animals, compared to littermate controls (Fig.  3d). Thus, our results in two independent mouse lines where Padi2 is ablated from the OPC stage suggest that PADI2 is important for the generation of OLs in vivo.
Padi2 knockout mice display impaired motor and cognitive functions, and a decrease in the number of myelinated axons. Given the decrease in OLs upon Padi2 cKO in the OL lineage and the targeting of myelin proteins by PADI2, we investigated whether Padi2 cKO mice presented a behavioral phenotype, in comparison with controls. Interestingly, Padi2 cKO mice exhibited impairment in motor coordination and balance as assessed by decreased latency to fall in the rotarod test (Fig. 4a). Moreover, we observed similar impairments in the full Padi2 KO (Padi2 fKO), where exon 1 of Padi2 is deleted in all cells 13 . In addition, we found an increase in the number of slips on the beam test in the Padi2 cKO mice, strengthening the motor impairment phenotype (Fig. 4b). Strikingly, when testing the Padi2 cKO mice for cognitive functions, these mice performed worse in the novel object recognition test, displaying a decrease in the discrimination index that provides an indication of recognition memory (Fig. 4c). To further investigate the mechanisms underlying the impact of PADI2 on motor and cognitive functions we have analyzed the myelin structure of the Padi2 fKO mice and respective controls by Electron Microcopy (EM). We found a significant decrease in the number of myelinated axons in the corpus callosum of adult mice together with a decrease in the g-ratio of small diameter axons and an increase in the g-ratio of axons with 0.6-0.9 µm diameter ( Fig. 4d-f). Moreover, the percentage of myelinated axons was decreased in Padi2 fKO mice compared to controls (Fig. 4g). As such, our results indicate that PADI2 plays an important role for efficient OL differentiation and myelination, as evidenced by the motoric and cognitive defects occurring upon its ablation.

PADI2 is present in both nucleus and cytoplasm in OL lineage cells.
To disclose the possible mechanisms of action underlying the observed in vitro and in vivo effects, we have further investigated the cellular location of Padi2 in OL lineage cells. Despite that PADI4 is the only one displaying a nuclear localization signal (NLS), PADI2 has recently been detected in the nucleus of mammary epithelial cells 21 and was described to be able to citrullinate nuclear histone H3R26 in MC7 breast cancer cells 22 . Remarkably, immuno-cytochemistry analysis showed the presence of PADI2 protein both in the nucleus and in the cytoplasm of OPCs (MBPnegative) and OLs (MBP-positive) (Fig. 5a). In order to confirm the localization of PADI2 in the nucleus, we transfected Oli-neu cells with the fusion vector Padi2-ZsGreen. We observed the presence of the green fusion protein both in the nucleus and cytoplasm of these cells (Fig. 5b), indicating that PADI2 can indeed be nuclear in OL lineage cells and might thereby regulate transcription through epigenetic mechanisms.

PADI2 citrullinates nuclear proteins.
To determine what could be the molecular targets of PADI2 in both nuclei and cytoplasm, we generated a stable Padi2 overexpressing Olineu cell line, where citrullination activity was greatly enhanced when tested in an Antibody-based assay for PADI activity (ABAP) 23 (Supplementary Figure 2a), and performed quantitative proteomics, by applying stable isotope labeling of amino acids in cell culture (SILAC) followed by mass spectrometry 24 . SILAC relies on the incorporation of a light (12C) or a heavy (13C) isotopic version of lysine into proteins of control and overexpressing Padi2 cell lines, respectively. The proteomes of fully light (L) and heavily (H) labelled cells were mixed 1:1 and the relative dissimilarities in the proteome citrullination target sites of these cell populations was determined and quantified by mass spectrometry  ( Fig. 5c). Interestingly, we observed a striking difference in the number of citrullination targets and ratios between proliferating and differentiating Oli-neu cells (Fig. 5d). In the proteome of proliferating Oli-neu, we found up to 151 citrullination sites, of which 81 were upregulated (H/L SILAC ratio higher than 1.5) with Padi2 overexpression. In contrast, up to 500 citrullination sites were identified in differentiating Oli-neu cells, of which 347 were upregulated upon Padi2 overexpression ( Fig. 5d and Supplementary Table 1). These observations indicate that OLs have an intracellular environment that is more permissive to PADI2 activity than OPCs.
We also uncovered citrullination sites arising only with Padi2 overexpression (no H/L SILAC ratio with peptides detected only in heavy proteomes; Fig. 5e and Supplementary Table 1). Consistent with our previous observations on the localization of PADI2 in the OL lineage ( Fig. 4a, b), PADI2 targets were not only cytoplasmic such as ribosomal proteins and myelin proteins (for example HSPA8, CNP and RAB1, ENO1 and EEF1A25), but also nuclear ( Fig. 5e and Supplementary Table 1). Chromatin components and modulators such as histone H1 (HIST1H1B), histone H2b (HIST1H2BA), histone H3 (HIST1H3), the histone demethylase KDM3B and the chromodomain helicase DNA binding protein 6 (CHD6), among others, were identified to be cit-rullinated by PADI2 at specific arginines (Fig. 5e). We also observed citrullination of several RNA binding proteins, consistent with the abundance or arginines in their RNA binding motifs (Fig. 5e). Gene ontology (GO) analysis indicated that proteins citrullinated by PADI2 are involved in translation, ribosomal biogenesis and nucleosome assembly both in proliferating and differentiating conditions, and RNA splicing, RNA processing and posttranscriptional regulation of gene expression in differentiating conditions ( Fig. 5f and Supplementary Table 4). Thus, PADI2 activity is not circumscribed to myelin and cytoplasmic proteins but is also relevant to nuclear processes.

PADI2 protein-protein interaction network is enriched in cytoplasmic and myelin proteins.
Our data suggests that PADI2 might be involved in different biological cell processes occurring both in the nucleus and in the cytoplasm. To further explore the PADI2 role in OL lineage cells, we identified the PADI2 protein interactors by pulling-down biotintagged PADI2. Control (expressing biotin ligase, BirA, and empty vector) and biotin-tagged Padi2 (bioPadi2) Oli-neu cell lines (expressing BirA and the biotin-tagged Padi2) were fed with 12C or 13C isotopes, respectively, immunoprecipitated with streptavidin beads and combined for further mass spectrometry analysis (Fig. 6a). As expected, PADI2 was the protein enriched the most (Fig. 6b). We uncovered up to 74 shared PADI2 protein interactors in proliferating and 2-days differentiated Oli-neu cells (Fig. 6b, c, e and Supplementary Table 2), among which nuclear proteins such as elongator protein complex 1 (ELP1). Nevertheless, the number of interactors was much lower than citrullination targets, suggesting that most of the PADI2 interactions leading to citrullination are transient and cannot be captured. Yet, GO analysis on the biological processes showed common categories  with the GO analysis of the PADI2 citrullination targets, such as translation and posttranscriptional regulation of gene expression (Fig. 6d), suggesting once again a role for PADI2 in these processes. Interestingly, one of the categories in the GO analysis, the intracellular protein transport, comprises RAN and KPNB1, two key regulators for active nuclear transport, which suggests that these proteins can be involved in the PADI2 nuclear-cytoplasmic shuttling (Supplementary Table  2). When performing GO analysis for the cellular component, we also have identified a myelin sheath component of PADI2 interactors (Fig. 6e, myelin sheath partners highlighted in red, Supplementary Table 4). Furthermore, comparison with a previously published myelin proteome 25 uncovers additional myelin proteins that interact with PADI2 such as WDR1, FASN, among others ( Fig. 6e and Supplementary Table 2). Thus, while interactions of PADI2 with nuclear proteins might be more transient, we observed several myelin proteins interacting with this enzyme.
Histone citrullination by PADI2 is associated with regulation of OL differentiation genes. Since we found PADI2 in the nucleus of OL lineage cells and identified sev-eral chromatin-associated proteins as targets of PADI2, we investigated whether citrullinated nuclear proteins, such as histones, are present in OL lineage cells, using an orthogonal technique. Indeed, western blot analysis of histone citrullination shows the presence of histone H3R26Cit and histone H3R2+8+17Cit in proliferating Oli-neu cells (Fig. 7a). Treatment with 200 µM of the pan PADI inhibitors Cl-Amidine or 60 µM of 2-CA led to a reduction on these modifications (Fig.  7a). We also observed an increase of histone H3R2+8+17Cit and Histone H3R26Cit in the Padi2 overexpressing cell lines indicating that PADI2 is mediating histone H3 citrullination and thus acts as an epigenetic regulator in OL lineage cells (Fig. 7b). While the expression of Mog and Sox9 was not altered upon Padi2 overexpression, we observed upregulation of Mbp and Cnp in differentiating cells (Fig. 7c), corroborating their downregulation upon PADI2 inhibition/Padi2 knockdown (Fig. 2). Thus, histone citrullination might be one of the mechanisms by which PADI2 regulates the transition of OPCs to NFOLs.
Padi2 knockdown results in reduced accessibility at OL differentiation genes. To further examine the possible role of PADI2 in transcriptional regulation and the chromatin structure, we investigated if the knockdown of Padi2 would affect the chromatin accessibility. We performed assay for transposase-accessible chromatin using sequencing (ATACseq) 26 to identify changes in accessibility of regulatory regions in differentiating Oli-neu upon knockdown of Padi2 with siRNAs (Fig. 7d). Interestingly, we observed a general reduction in accessibility upon knock down of Padi2 (Fig.  7d, Supplementary Fig. 2d and Supplementary Table 3). We further investigated accessibility of OL enhancers and promoters, presenting H3K27ac in Oli-neu cells (see methods) or H3K27ac/H3K4me3 in OPCs 27 . Indeed, we observed decreased chromatin accessibility in regulatory regions of genes as Pik3cd, Plin4 or Ezh1. Importantly, we also observed such reduction in accessibility in important regulators of OL myelination, including Sirt2, Mag and Mbp (Fig. 7d), and in Plp1, Sox10 and Mog, although to minor extent (Supplementary Fig. 2d). Accordingly, gene ontology analysis indicated an enrichment in regulatory regions of genes involved in lipid metabolism (Supplementary Fig. 2e and Supplementary Table 4). Nevertheless, PADI2 appears to target specific regulatory regions, since we did not find regulation of chromatin accessibility at regulatory regions of other genes modulating OL differentiation, such as Sox9, Tcf7l2, (Fig. 7d), Id1 or Olig2 (Supplementary Fig. 2d). Interestingly, we also find a cohort of genes where accessibility of their regulatory regions was instead increased by Padi2 knockdown, including Cxcl2 (Fig. 7d), Hspb8 or Gabra6. Gene ontology analysis indicated involvement in processes chemotaxis, Polycomb activity and apoptosis, among others (Supplementary Fig. 2d and Supplementary Table 4). As such, recruitment of PADI2 to specific regulatory regions in OLs regulates their accessibility, which will also ultimately modulate transcriptional output, as observed for Mog and Sox10.

Discussion
The formation of myelin is a fundamental biological process that encompasses subsequent steps of OPC specification, differentiation and myelin sheath formation/wrapping of axons. Our study highlights PADI2 as an important player in these last two steps by 1) facilitating OPC differentiation and influencing the OL epigenetic landscape through histone citrullination and 2) interacting with and mediating the citrullination of several myelin protein components. While Padi2 expression in OLs and its occurrence in the myelin were described before 16 , we report for the first time its presence in OPCs and the dramatic upregulation of Padi2 in the transition from OPCs to NFOLs and its function facilitating OL lineage progression. To uncover PADI2 mechanisms of action, we further determined the citrullination targets and the binding partners of PADI2. Our data revealed new PADI2 citrullinated proteins and interactors, both located in the nuclear and cytoplasmic compartments. We also demonstrate, using 3 different Padi2 knockout mouse lines, that PADI2 is important for the OL development and motoric and cognitive behaviour. Our findings indicate that PADI2 mediated citrullination is required for efficient OL development with acting not only as a myelin modulator, through MBP citrullination, but also displaying other functions in the cytoplasm and nucleus, for instance as an epigenetic modulator. Additional mechanisms underlying PADI2 actions in OPC differentiation remain to be investigated.
By using an unbiased proteomics methodology based on SILAC followed by mass spectrometry we have revealed hundreds of novel citrullination protein targets and the respective arginine locations, some of those in core histones such as HIST1H1, HIST1H2 and HIST1H3. PADI2 mediated H3R26Cit has been shown to lead to local chromatin decondensation and targeted transcriptional activation in breast cancer cells 28 . In a similar manner, when we knockdown Padi2 we observe a general decrease in chromatin accessibility as revealed by ATAC-seq. Furthermore, upon PADIs inhibition or Padi2 knockdown we observed a decrease in the pro-differentiation and myelin genes such as Mbp, Mog and Sox10 suggesting that PADI2 might be locally enhancing transcription through histone H3 citrullination.
Interestingly, we detected HIST1H1C citrullination on R54 in our cells. This modification was previously described to induce global chromatin decondensation in iPS cells 5 and to be specifically mediated by PADI4, which suggests that there is an overlap of the substrates of PADI2 and PADI4. Moreover, H3R2+8+17Cit was also induced upon hPadi4 overexpression 5 . hPADI2 and hPADI4 were shown to exhibit different substrate preferences and hPADI4 had more pronounced substrate specificity, in both Cos-1 and HEK cells or by adding recombinant hPADIs to cell lysates 29 . Additional hPADI2 targets were disclosed by overexpressing hPADI2 in HEK cells using a different technology 30 . Although these are targets for hPADI2 in different human cell types, we observed many common targets with our mouse OL lineage cells such as ribosomal proteins and splicing factors. Thus, PADI2 might have a widespread and undetermined role in basic cellular processes such as mRNA splicing and translation.
Differentiated Oli-neu cells displayed considerably more citrullination targets, higher citrullination activity in basal conditions with the ABAP kit, and a more pronounced increase in H3R2+8+17Cit and H3R26Cit upon Padi2 overexpression suggesting that PADI2 activity can be enhanced upon differentiation. In agreement, most of PADI2 effects are more noticeable upon OPC differentiation. Of notice, PADI activity can also be context dependent, as these enzymes need a high concentration of calcium to become active, and calcium signaling has been shown to be fundamental for OPC migration, differentiation and myelination 31 . Although several citrullination targets found in proliferating and 2-days differentiated Oli-neu were common and/or showed additional citrullinated arginine in differentiation, they did not overlap completely, indicating that there is a preference for the targeted arginine between these conditions that could be due for instance, to differences in the binding partners. Likewise, not all proteins found interacting with PADI2 were citrullinated and vice versa. Nevertheless, GO analysis for the biological processes on the PADI2 interactors also reveals categories similar to the citrullination targets, such as translation and posttranscriptional regulation of gene expression reinforcing once again a putative role for PADI2 in these processes. Mice overexpressing Padi2 under the Mbp promoter display abnormal myelination with structural changes of the myelin 10 . This effect was attributed to the increased citrullination of MBP and also of histones that would ultimately cause OL apoptosis 10 . However, a striking finding in our study is the abundance of myelin component proteins within the interactors of PADI2, that might also mediate the effect of PADI2 on myelin when overexpressed 10 . Citrullination of myelin proteins is naturally occurring, and thus normal citrullination levels must certainly have a physiological function. 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP), which has an important role in the generation of cytoplasmic channels in OLs 32 , displayed PADI2-mediated citrullination of several arginines (Table S1). It is possible that hypocitrullination of CNP upon knocking out PADI2 could lead to disruption of cytoplasmic channels and ultimately to the observed defects on myelination in our Padi2 fKO mice (Fig. 7). Further investigation would be necessary to determine which citrullination targets of PADI2 in the myelin could be responsible for these defects. We found that PADI2 absence in OL lineage cells caused decreased OL cell density in spinal cord and a decrease in the number of myelinated axons in the corpus callosum of adult mice, that were reflected in motor and cognitive dysfunctions. Since Pdgfra and Padi2 can also be co-expressed in other non-CNS cells such as Schwann cells, the impairment on motor functions could also be due to the absence of PADI2 in peripheral cells. However, overexpression of Padi2 did not have any effect on the peripheral myelin 10 and the EM and OL densities analysis in the brain and spinal cord (Fig. 7) indicate a phenotype in OLs. In addition, the cognitive behavior test also supports a CNS specific PADI2 effect. Analysis of single-cell RNA-Seq data ( Supplementary Fig.  1) suggested that PADI2 is the only member of the PADI family robustly expressed in the OL lineage. Although PADI4 was identified by IHC in the myelin 16 we could not detect Padi4 mRNA in any OL lineage cell tested nor it was identified in the myelin proteome 25 . However, we did detect very low mRNA levels of Padi3 and Padi1 in Oli-neu cells and found it expressed in very few cells in OPCs/OLs collected from P90 brains (data not shown). As such, we cannot totally rule out that these PADIs have a function in OL lineage progression and that compensatory mechanisms might be operational upon PADI2 knockdown, knockout or inhibition, particularly since PADIs can have overlapping substrates. PADI2 has been long investigated in the context of MS since MBP hypercitrullination is a hallmark of the disease 33 . Our study demonstrates that PADI2-mediated citrullination is also required for OL lineage progression and myelination and uncovers several new citrullination targets in the OL lineage. Thus, targeted activation of PADI2 in the OL lineage might be beneficial in the context of remyelination in diseases as multiple sclerosis.

ACKNOWLEDGEMENTS
We would like to thank Tony Jimenez-Beristain, Alessandra Nanni, Ahmad Moshref and Johnny Söderlund, and the Clinical Proteomics Mass Spectrometry at Karolinska University Hospital and Science for Life Laboratory for providing assistance in mass spectrometry and data analysis, and Science for Life Laboratory, the National Genomics Infrastructure (NGI), and Uppmax for providing assistance in massive parallel sequencing and computational infrastructure. We thank Belinda Panagel and the CMB FACS facility for all the support in FACS sorting of all cells, and Jean-Jacques Medard and Roman Chrast for help with electron microscopy. We thank Helena Francis, Iris Muller, Marjan Abbasi, Theresa Mader, Diana Hernandez and Wouter Beenker, all students that had participated and pushed forward this project. We thank P. Soriano (Mount Sinai, New York) for the Pdgfra-H2B-GFP mouse. The bioinformatics computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX, Uppsala University. A.M.

Experimental procedures
Animals. All mice were maintained under a pathogen-free environment at the animal facility. All experimental procedures were performed following the guidelines and recommendations of local animal protection legislation and were approved by the local committee for ethical experiments on laboratory animals (Stockholms Norra Djurförsöksetiska nämnd in Sweden). The following transgenic mice strains were used: Pdgfra-H2BGFP knock-in mice 17

Behavioral analysis.
For the beam balance test the fine motor coordination and balance was assessed by beam walking assay. Performance on the beam was quantified by measuring the time taken by the mouse to traverse the beam and the number of paw slips that occurred in the process. The beam apparatus consisted of a PVC plastic beam (1.2 cm wide × 100 cm long) located 50 cm above the table top on two poles. A black box was placed at the end of the beam as the finish point. Nesting material and faeces from home cages was placed in the black box to attract the mouse to the finish point. A nylon or cotton pad was placed below the beam, to cushion any falls. Mice were trained three times a day for two consecutive days until they were able to cross the beam without hesitation. No data were collected during these sessions. During the test session, the time needed to cross the beam and the number of paw slips were recorded. The beam and box were cleaned of mouse droppings and wiped with 70% ethanol before the next animal was placed on the apparatus. The novel object recognition task was conducted as described previously 34 , and was used to assess the nonspatial hippocampal memory. A mouse was presented with two similar objects during the first session, and then one of the two objects was replaced by a new object during a second session. The amount of time taken to explore the new object provided the index of recognition memory. The task consisted of habituation, training, and testing phases conducted on separate days. During habituation, mice were allowed to freely explore an empty white box (60 cm wide × 60 cm long × 47 cm high) for 5 min/day for two days. Twenty-four hours later, mice were re-habituated (familiarization session) in the same box for 10 min, with two identical objects placed inside, 5 cm away from the walls. Mice were allowed to freely investigate until they accumulated a total of 20 sec exploring the objects. Exploration and recognition of the object were determined when the nose of the animal was in contact with an object or directing nose to the object within a defined distance (<2 cm). Mice were then immediately returned to their home cage. After the familiarization session, the objects and the open field was cleaned with 70% ethanol to minimize olfactory cues. After 1 hour of familiarization session, object recognition was tested, using the same procedure as in training except that one of the familiar objects was substituted with a novel object (cleaned and free of olfactory cues). The position of the novel object (left or right) has to be randomized between each mouse and each group tested. Time spent with each object was recorded using a stopwatch by a trained observer. Mice inherently prefer to explore novel objects; thus, a preference for the novel object indicates intact memory for the familiar object. A discrimination index was calculated according to the following formula: time of novel object exploration/time of novel plus familiar object explo-ration×100. Rotarod behavioral analysis was performed for evaluation of coordination and motor learning. Mice were trained on a Rotarod (Ugo Basile, Comerio VA, Italy) at a constant speed (4 rpm), 120 s per day for 3 consecutive days. Subsequently, test day consisted of three trials on an accelerating rotarod for up to 300 s (starting at 4 rpm accelerating to a final speed of 40 rpm), with mice returned to their home cages for at least 30 min between trials. The time at which each animal fell from the rod was recorded as a measure of motor coordination and repeated measurements of speed at the time of animal falling were averaged.
Electron Microscopy. For EM analysis, deeply anesthetized C57BL/6-Padi2-KO mice (n=4 per condition) were perfused transcardially using a varistaltic pump (Manostat) with 0.1M PBS (pH 7.4) followed by cold Karnovsky's fixative (2.0% PFA/2.5% Glutaraldehyde in 0.1M Sodium Phosphate buffer). Using a McIlwain tissue chopper, coronal brain sections between -2.2 and -2.5 bregma were obtained and corpus callosum tissue was micro-dissected and postfixed overnight in 2.0% PFA/ 2.5% Glutaraldehyde in 0.1M Sodium Phosphate buffer. The samples were then processed using standard protocols employed by the Stony Brook University Microscopy Core (see below, and 35,36 ). The following day (after overnight post-fixation), an additional fixation step was carried out using 2% osmium tetroxide in 0.1M PBS (pH 7.4), after which samples were dehydrated in a graded series of ethyl alcohol. Finally, samples were embedded using Durcupan resin in between two pieces of ACLAR sheets (Electron Microscopy Sciences). Ultrathin sections of 80nm were obtained using a Lecia EM UC7 ultramicrotome and placed on formvar coated slot copper grids (Electron Microscopy Sciences). Uranyl acetate and lead citrate counterstained samples were viewed under a FEI Tecnai12 BioTwinG2 electron microscope. Digital images (minimum 10 per animal, taken at 11,000x) were acquired using an AMT XR-60 CCD Digital Camera system and compiled using Abobe Photoshop software. G-ratio was determined by dividing the mean axon diameter without myelin by the same axon diameter with myelin. A minimum of 100 healthy axons were considered per animal. To determine the size distribution of myelinated fibers in corpus callosum, diameters of all measured axons were separated into four ranges (0.3-0.6, 0.6-0.9, 0.9-1.2, & >1.2 µm). No axons with diameters smaller than 0.3 µm were considered. Data are expressed as comparison of means between the control and knock-out for each range. Percentage of myelinated axons was determined by placing a centered grid over the image using ImageJ software, and counting whether the axon at each intersection was myelinated or not. A minimum of 100 axons was counted per image, and 10 images were quantified per animal. All measurements were acquired on electron microscopy sections images using an ImageJ software. Mann-Whitney U tests were performed to assess statistical differences between control and knock out mice for both differences in g-ratio per axon diameter range and percent myelinated axons.

SILAC.
To generate LIGHT and HEAVY labeled cell lines, Oli-neu cells were grown and expanded in LIGHT (Lysine 12C) or HEAVY (Lysine 13C) media for at least five passages. The media was prepared with SILAC Advanced DMEM/F12-FLEX kit (Life Tec, MS10033) and contained 0.1% of dialyzed FBS, 10ng/ml bFGF, 10ng/ml PDGF-BB, 400ng/ml of L-thyroxine and 340ng/ml of T3. For the differentiation, growth factors and the FBS were removed. LIGHT labeled cells lines correspond to all controls, scramble overexpression control and BirA control, while HEAVY labeled cell lines comprise the overexpression of Padi2 or the biotin tagged Padi2. A control experiment in which control cells were also fed with heavy media and then mixed 1:1 with light control cells was performed in order to confirm that all proteins in heavy conditions were heavily labeled (Supplementary Fig. 2b).

PADI2 immunoprecipitation.
Proliferating and 2-days differentiated BirA controls and biotin tagged Padi2 cells were fixed fo 8min at RT in 1% formaldehyde solution by adding 1/10 volume to cell media of freshly prepared 11% formaldehyde solution (formaldehyde 11%, 0.1M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8 and 50mM Hepes pH8). Glycine was added (final 0.125M) to quench formaldehyde, rinsed twice in cold PBS and scrapped for subsequent centrifugation at 4000g for 10min at 4 • C. At this point cell pellets can be stored at -80°C. Cell pellets were then resuspended in 1mL of lysis buffer (10mM Hepes-KOH pH7.5, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycolate, 0.5% Na-lauroylsarcosinate, protease inhibitors) and sonicated. Cell suspension was centrifuged at 14000g for 10min at 4 • C to remove the debris. For the immunoprecipitation, protein concentrations were determined in every sample and equal amounts of protein were used for further streptavidin pull-down. 50µL of streptavidin beads (DB Myone Streptavidin T1, Invitrogen) per 5mg of protein were added to the protein suspensions and incubated overnight at 4 • C. Magnetic stand was used to precipitate the beads. At this step beads from BirA controls and biotin-tagged Padi2 were combined for the following washes: 4 times in wash buffer 1 (50mM Hepes pH7.6, 1mM EDTA, 0.1% Na-Deoxycolate), 2 times in wash buffer 2 (50mM Hepes pH7.6) and 2 times in wash buffer 3 (20 mM ammonium bicarbonate NH4HCO3). The beads can be stored at -80 • C in this step.

Mass Spectrometry for citrullination targets.
SDS-PAGE gel. Extracted proteins were resuspended in Laemmli Sample Buffer, and resolved on a 4-20% SDS-PAGE. The gel was stained with Coomassie blue, cut into 20