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alpha-Synuclein: a Modulator During Inflammatory CNS Demyelination

alpha-Synuclein: a Modulator During Inflammatory CNS Demyelination Neuroinflammation and demyelination are hallmarks of several neurological disorders such as multiple sclerosis and multiple system atrophy. To better understand the underlying mechanisms of de- and regeneration in respective diseases, it is critical to identify factors modulating these processes. One candidate factor is alpha-Synuclein (aSyn), which is known to be involved in the pathology of various neurodegenerative diseases. Recently, we have shown that aSyn is involved in the modulation of peripheral immune responses during acute neuroinflammatory processes. In the present study, the effect of aSyn deficiency on de- and regenerative events in the CNS was analyzed by using two different demyelinating animal models: chronic MOG -induced 35–55 experimental autoimmune encephalomyelitis (EAE) and the cuprizone model. Histopathological analysis of spinal cord cross sections 8 weeks after EAE induction revealed a significant reduction of CNS inflammation accompanied by decreased myelin loss during late-stage inflammatory demyelination in aSyn-deficient mice. In contrast, after cuprizone-induced demyelination or remyelination following withdrawal of cuprizone, myelination and neuroinflammatory patterns were not affected by aSyn deficiency. These data provide further evidence for aSyn as regulator of peripheral immune responses under neuroinflammatory conditions, thereby also modulating degenerative events in late-stage demyelinating disease. . . . . Keywords Neuroinflammation Demyelination alpha-Synuclein Experimental autoimmune encephalomyelitis Cuprizone Introduction sclerosis (MS) (Compston and Coles 2008; Fanciulli and Wenning 2015). Physiologically, compensating repair mecha- Demyelination in combination with neuroinflammatory re- nisms exist which sustain myelin regeneration. While in some sponses is a common feature of several neurological disorders MS patients these processes frequently occur, others only show including multiple system atrophy (MSA) and multiple sparse remyelination (Patrikios et al. 2006). The reasons for this regeneration failure still remain unclear. Up to date, no thera- Jürgen Winkler and Ralf A. Linker contributed equally to this work. peutic options exist to cure these diseases or to halt disease progression. For the development of such treatment strategies, * Jürgen Winkler it is essential to identify factors which are involved in de- and Juergen.Winkler@uk-erlangen.de remyelinating processes as well as oligodendrocyte cell damage and death. In rodent models, it has been shown that Department of Molecular Neurology, University Hospital Erlangen, remyelination critically depends on the presence of oligoden- Friedrich-Alexander-University Erlangen-Nürnberg, drocyte precursor cells (OPCs) (Sim et al. 2002). In the central Schwabachanlage 6, 91054 Erlangen, Germany nervous system (CNS), OPCs are recruited to demyelinated Department of Neurology, University Hospital Erlangen, areas and give rise to myelinating oligodendrocytes upon acti- Friedrich-Alexander-University Erlangen-Nürnberg, vation (Franklin and ffrench-Constant 2017). Current concepts Erlangen, Germany to explain the failure of remyelination include insufficient re- Department of Neurology, University of Regensburg, cruitment of OPCs to the demyelinated region or the inhibition Regensburg, Germany of maturation into oligodendrocytes (Sim et al. 2002; Institute of Radiology, Preclinical Imaging Platform Erlangen Kuhlmann et al. 2008). So far, various extrinsic and intrinsic (PIPE), University Hospital Erlangen, factors modulating oligodendrocyte maturation have been de- Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany scribed (French et al. 2009; Hughes et al. 2013;Heetal. 2016). J Mol Neurosci (2020) 70:1038–1049 1039 Recent studies using in vitro and in vivo MSA models suggest Experimental Autoimmune Encephalomyelitis alpha-Synuclein (aSyn) as a candidate factor inhibiting OPC +/+ maturation (Ettle et al. 2014; May et al. 2014). aSyn is For active EAE induction, 10–14-week-old aSyn and −/− expressed in axons, neuritic processes, and presynaptic termi- aSyn mice received 200 μgMOG and 200 μgcom- 35–55 nals of neurons, being important for vesicle transport. In addi- plete Freund’s adjuvant (CFA) subcutaneously. Pertussis toxin tion, aSyn protein levels have also been detected in glia and (200 ng) was applied intraperitoneally at the day of immuni- hematopoietic cells (Maroteaux et al. 1988; Barbour et al. zation and 48 h later. Mice were daily weighed and scored for 2008). Pathological aSyn aggregations are associated with neu- clinical signs using a 10-point scale as described previously rodegenerative disorders, in particular Parkinson’s disease, de- (Linker et al. 2002). mentia with Lewy bodies, and MSA, also summarized as synucleinopathies (Spillantini et al. 1998). In the latter, aSyn Cuprizone Model accumulates in glial cytoplasmic inclusions (GCI) in oligoden- drocytes being considered pathological hallmark and associat- Toxic demyelination was induced by feeding 8–10-week-old ed with widespread demyelination (Papp et al. 1989). +/+ −/− aSyn and aSyn mice a diet containing 0.2% cuprizone Additionally, degenerative features are often accompanied by mixed into ground standard rodent chow for 5 weeks. During distinct neuroinflammatory processes in these this period, mice were closely monitored for weight loss as synucleinopathies (Lim et al. 2016; Hoffmann et al. 2019). well as abnormalities and trained for Rotarod analysis. After Interestingly, aSyn immunoreactivity was also reported in glial 5 weeks, motor coordination skills were assessed by an accel- cells in MS lesions and in spinal cord lesions of mice induced erating Rotarod test. To initialize remyelination, after 5 weeks, with experimental autoimmune encephalomyelitis (EAE), a cuprizone was removed from the diet and mice received common animal model of MS (Papadopoulos et al. 2006;Lu ground standard rodent chow for 3 or 7 days, respectively. et al. 2009). Recently, we identified aSyn as regulator of pe- At the end of de- and remyelination experiments, mice were ripheral immune responses in neuroinflammation (Ettle et al. sacrificed to dissect the brain for histological analysis. 2016b). Considering these findings, we studied the role of C57BL/6N wildtype mice fed with normal chow served as aSyn during late-stage neuroinflammatory demyelination and naïve controls and are termed “naïve wt.” in the context of toxin-induced de- as well as remyelination. For this purpose, demyelination was induced in aSyn-deficient −/− Rotarod Test mice (aSyn ) and littermate control animals with endogenous +/+ aSyn expression (aSyn ) by using two different demyelin- Motor performance of mice treated with cuprizone was eval- ation models. More precisely, autoimmune-mediated late-stage uated by performing an accelerating Rotarod test. The Rotarod demyelination was examined with the help of myelin oligo- consisted of a 30-mm diameter rod rotating about its long axis. dendrocyte glycoprotein (MOG) –EAE and toxin-induced 35–55 Over the course of 180 s, the Rotarod accelerated from 4 to 40 demyelination was studied in the cuprizone model. rounds per minute (rpm). The latency of each mouse to fall Furthermore, regenerative events were assessed at two differ- was recorded in two consecutive trials with a 30-min resting ent time points after stopping cuprizone administration. phase in the home cage between the trials. One week prior to the accelerating test, mice were trained on the rod rotating with constant speed at 12 rpm for 120 s. Material and Methods Animal Experiments In Vivo Magnetic Resonance Imaging −/− aSyn mice were maintained on a C57BL/6N background In vivo magnetic resonance imaging (MRI) was performed +/+ −/− for more than ten generations (Abeliovich et al. 2000). with aSyn and aSyn mice before and after cuprizone +/+ aSyn littermates with endogenous aSyn expression served administration. In inhalation anesthesia (1.5% isoflurane), as controls. All mice were kept under standard animal hous- heads of mice were fixed in a mouse brain surface array coil ing conditions with a 12-h day/night cycle and free access to and scanned on a preclinical ultra-high field MRI system (7 food and water. All applicable international, national, and/or Tesla ClinScan 70/30, Bruker). During the entire imaging pro- institutional guidelines for the care and use of animals were cedure, respiration of mice was monitored and kept constant, followed. Animal experiments were performed in accor- while the body temperature was stabilized with a heating cir- dance with the German laws of animal protection and were culator bath. The imaging protocol consisted of a 3D T2- approved by the local ethics committee (Government of weighted turbo spin echo sequence (repetition time (TR), Unterfranken, Bavaria, Germany, ref. # 55.2-2532-2-395 3690 s; echo time (TE), 40 s; voxel size, 0.082 × 0.082 × and # 55.2-2532-2-450). 0.5 mm; acquisition time, 4 min 37 s; slice thickness, 0.5 mm). 1040 J Mol Neurosci (2020) 70:1038–1049 Sequences were analyzed with the help of the medical im- NH OH and distilled water and fixed in 5% Na S O for 4 2 2 3 aging interaction toolkit (MITK). T2-weighted signal intensi- 3 min. Finally, sections were washed in distilled water, +/+ −/− ties were assessed in aSyn and aSyn mice before and dehydrated, and mounted. after cuprizone diet. The boundaries of the region of interest (ROI) within white matter regions of the corpus callosum Histological Evaluation were manually defined (Wu et al. 2018). To eliminate con- founding signals arising from slice-dependent signal variation For histological evaluation of EAE experiments, spinal cord during MRI acquisition, the signal intensity of the ventricle cross sections were analyzed blinded using a BX-51 light mi- within the same slice was used for normalization. To calculate croscope (Olympus). For each staining, at least five lesions T2-weighted signal intensity ratios, signal intensities were de- within each spinal cord segment (cervical, thoracic, and lum- termined in the ROI and the ventricle of each animal in the bar) were inspected. T cells, mononuclear phagocytes, astro- same layer. cytes, and oligodendrocytes were counted at × 200 magnifica- tion within the margins of a 1/16 mm grid in individual lesions (Immuno-)Histochemistry and counts were normalized to cells/mm .Analysisof demyelinated areas in the white matter of LFB and anti- Following perfusion with 4% paraformaldehyde (PFA), spinal CNPase-stained sections was performed semi-automatically cords or brains were removed and post-fixed in 4% PFA for 2– with the help of CellSens or CellP software (Olympus). 3 h. After embedding in paraffin, 4 μm thin sections were Axonal density was quantified in silver impregnated sections prepared by using a microtome. For immunohistochemistry, by counting on a 100 μm diameter grid at × 500 magnification. anti-CD3 (MCA1477, Biorad) and anti-Mac-3 (M3/84, BD To analyze brain sections in cuprizone experiments, sec- Pharmingen) antibodies were used to detect T cells and mono- tions between bregma − 1.15 and bregma − 2.15 according to nuclear phagocytes, respectively. Glial cells were stained with the Mouse Brain atlas by Franklin and Paxinos were examined anti-Olig2 (Millipore), anti-NogoA (Millipore), anti-GFAP (Franklin and Paxinos 2013). The level of demyelination in (Dako), and anti-Iba1 (20A12.1, Millipore) antibodies, respec- the corpus callosum was assessed by determination of myelin tively. Anti-MOG (8-18C5, Millipore), anti-MBP (12, Biorad), scores by two independent blinded observers as follows: 0: and anti-2′,3′-Cyclic-nucleotide 3′-phosphodiesterase (CNPase) complete demyelination, 1: 1/3 of corpus callosum myelinat- (SMI91R, BioLegend) antibodies were used as myelin markers. ed, 2: 2/3 of corpus callosum myelinated, 3: complete For chromogenic immunodetection, Vectastain Elite Avidin- myelination (Lindner et al. 2008). Biotin-Complex Kit (Vector Laboratories) was used according Number of cells was determined in three adjacent regions to the manufacturer’s instructions. Diaminobenzidine (DAB) in the median corpus callosum of each animal using a mor- was employed to visualize the conjugated peroxidase. phometric grid. Immunopositive cells with identified nucleus Luxol fast blue-periodic acid Schiff (LFB-PAS) staining (counterstaining with hematoxylin) were counted at × 200 was performed to assess the extent of demyelination. magnification and counts were normalized to cells/mm . Therefore, deparaffinized slides were incubated overnight in 0.1% Luxol fast blue (LFB) solution at 56 °C. Next day, sec- Statistical Analysis tions were rinsed in 96% ethanol and distilled water. For dif- ferentiation, sections were incubated in 1% lithium carbonate Statistical testing was performed using Graph Pad Prism. All solution for a few seconds, rinsed shortly in 70% ethanol, and ex vivo data were analyzed by one- or two-way ANOVA washed in distilled water. PAS staining was performed by followed by Tukey’s posttest or unpaired t test. EAE data were oxidizing the slides in 0.8% periodic acid solution for analyzed either by Mann-Whitney U test or logrank test (for 10 min. Afterwards, sections were stained in Schiff reagent disease incidence analysis). For statistical analysis of MRI data, for 20 min and washed in sulfite solution, followed by a 10- the two-way ANOVA test for repeated measurements was used. min washing step with running tap water. Finally, sections Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, or were dehydrated and mounted. ***p < 0.001 were considered to be statistically significant. For analysis of axonal integrity, Bielschowsky silver im- pregnation was employed. Rehydrated sections were trans- ferred into a 20% AgNO solution and incubated for 15 min Results at 37 °C. Then the solution was washed off with distilled water and 25% NH OH solution was added to the AgNO solution. aSyn Deficiency Ameliorates EAE Severity 4 3 Slides were incubated in this solution for 10 min in the dark at 37 °C and washed in 0.1% NH OH. After adding developer First, we used the EAE model to study the effect of aSyn stock solution to the AgOH solution, sections were stained for deficiency during late-stage inflammatory demyelination. +/+ −/− 4 min until axons turned black. Slides were rinsed in 0.1% Therefore, aSyn and aSyn mice were immunized with J Mol Neurosci (2020) 70:1038–1049 1041 MOG peptide and monitored for clinical signs over a and Olig2 cells were determined in demyelinated areas. 35–55 period of 8 weeks. Compared with littermate controls, aSyn- While the number of NogoA mature oligodendrocytes did deficient mice displayed an ameliorated disease course not differ between both groups, significantly more cells posi- (Fig. 1a). At the end of the observation period, they showed tive for Olig2 , a relatively broad oligodendrocytic marker, +/+ a better outcome by one score point in comparison with were detected in lesions of aSyn mice (Fig. 2g, h). +/+ aSyn mice. More precisely, they suffered from tail paraly- sis, while the control group still exhibited signs of gait ataxia aSyn Deficiency Does Not Affect Myelination Pattern and mild paralysis of hind limbs. There were no differences in After Cuprizone-Induced Demyelination the overall disease incidence or mortality rate between both groups (Fig. 1b). Next to the EAE model, the cuprizone model was employed to investigate the impact of aSyn deficiency on acute demyelin- Reduced Demyelination, CNS Inflammation, ation and remyelinating processes in the absence of peripheral and Axonal Loss in aSyn-Deficient Mice immune responses. To evoke acute demyelination in the brain +/+ −/− During Late-Stage EAE of aSyn and aSyn mice, age-matched animals were fed a diet containing 0.2% cuprizone for 5 weeks. Motor coordina- In the EAE model, disease course and severity of symptoms tion skills of mice were assessed after cuprizone treatment by are determined by the extent of CNS inflammation and spinal an accelerating Rotarod test. Both groups showed equal per- cord damage is mainly apparent in a form of demyelination formances regarding latency to stay on the accelerating rod and axonal loss. Therefore, we histologically analyzed spinal (Fig. 3a). In vivo MRI was used to detect myelin deficits in the +/+ −/− cords of aSyn and aSyn mice for these parameters corpus callosum following cuprizone treatment. After 5 weeks 8 weeks after induction of EAE. Average clinical scores at this of cuprizone diet, gray-white matter contrast was conspicu- −/− +/+ time point were 1.38 for aSyn mice and 2.5 for aSyn ously reduced. Quantification of T2-weighted signal intensity mice, respectively. Matching the disease course, immunohis- ratios of white matter and liquor revealed a 70% increase in tochemical staining for CNPase as well as LFB-PAS staining animals receiving cuprizone diet, indicating a considerable revealed a more than 2.5-fold increase in white matter demy- reduction in myelin content in the corpus callosum. +/+ elination in aSyn mice (Fig. 2a, b). Furthermore, axonal However, this method did not detect any differences in demy- +/+ −/− densities determined by Bielschowsky silver staining were elination patterns between aSyn and aSyn mice follow- +/+ reduced by 30% in lesions of aSyn mice compared with ing cuprizone exposure (Fig. 3b). To validate these findings, a −/− aSyn mice (Fig. 2c). Additionally, we detected a marked comprehensive histological analysis of brain sections was per- increase in GFAP processes and significantly higher numbers formed. In line with results obtained from the MRI measure- of GFAP cell bodies within and in direct vicinity of spinal ment, immunohistochemical staining for different myelin cord lesions in these mice indicative for astrocytic activation markers, such as MBP, CNPase, and MOG, as well as LFB- (Fig. 2d). Further examination of neuroinflammatory process- PAS staining demonstrated that 5 weeks of cuprizone treat- +/+ es revealed a pronounced inflammatory response in aSyn ment resulted in comparable reduction of myelin staining in −/− +/+ −/− mice. Overall, fewer lesions were present in aSyn mice both, aSyn and aSyn mice, in the central part of the containing a lower number of invading immune cells. More corpus callosum compared with naïve wt mice (Fig. 3c–f). + + specifically, CD3 Tcells and Mac-3 mononuclear phago- Additionally, the impact on CNS cells was studied. cytes infiltrating the CNS were reduced by 50% compared Cuprizone treatment strongly reduced the number of mature +/+ + with aSyn animals (Fig. 2e, f). To closer examine the effect NogoA oligodendrocytes compared with naïve mice of aSyn deficiency on oligodendroglia, numbers of NogoA (Fig. 3g). However, no differences were observed in the +/+ −/− Fig. 1 aSyn deficiency attenuates disease severity in late-stage EAE. a EAE incidence in aSyn (solid line) and aSyn (dashed line) mice (n = +/+ −/− Clinical course of MOG -EAE in aSyn (black dots) and aSyn 16–17 per group). p.i. post immunization. *p <0.05 35–55 (gray squares) mice (n =11–13 per group). b Kaplan-Meier plot depicting 1042 J Mol Neurosci (2020) 70:1038–1049 Fig. 2 aSyn-deficient mice exhibit less severe demyelination, axonal loss CNPase. c Bar graph depicting axonal densities (number of axons and inflammation in CNS lesions. (Immuno-)Histochemical analysis of counted on a 100 μm grid) determined by Bielschowsky silver staining. +/+ −/− + + + spinal cord cross sections of aSyn (black bars) and aSyn (gray bars) Numbers of d GFAP astrocytes, infiltrating e CD3 and f Mac-3 cells, g + + mice 8 weeks after immunization with MOG peptide. Average Olig2 and h NogoA cells were assessed by immunohistochemical 35–55 −/− clinical scores at the time point of analysis were 1.38 for aSyn mice staining. Representative pictures are shown for each staining (n =5–6 +/+ and 2.5 for aSyn mice. Percentage of demyelinated area in the white per group). **p < 0.01, ***p <0.001 matter area determined by a LFB-PAS staining or b staining with anti- + + numbers of mature NogoA oligodendrocytes or Olig2 cells aSyn Deficiency Has No Impact on Early +/+ −/− in the corpus callosum between aSyn and aSyn mice Remyelination in the Cuprizone Model (Fig. 3g, h). Following cuprizone administration, neuroinflammatory processes including activation of astro- The cuprizone model is considered a well-established tool to cytes and microglia are induced (Hiremath et al. 1998). investigate remyelination since, only few days after adminis- + + Indeed, numbers of Iba1 microglia and GFAP astrocytes tration, regenerative processes are emerging. In order to study were markedly increased by 14-fold and twofold, respectively, the effect of aSyn deficiency on early remyelination, we ex- +/+ −/− +/+ −/− in the corpus callosum of aSyn and aSyn mice treated amined aSyn and aSyn mice for their myelination pattern with cuprizone for 5 weeks compared with untreated controls. by histological analysis 3 days after terminating the cuprizone +/+ −/− However, in aSyn and aSyn mice, numbers of respective diet. In both groups, myelination scores assessed by histolog- cells were nearly equal (Fig. 3i, j). ical staining for myelin markers (MOG, MBP) and LFB J Mol Neurosci (2020) 70:1038–1049 1043 Fig. 3 aSyn deficiency does not affect acute demyelination after 5 weeks of cuprizone treatment. +/+ −/− aSyn (black bars) and aSyn (gray bars) mice received cuprizone diet for 5 weeks. a Motor coordination was tested by an accelerating Rotarod test (n = 5–6 per group). b T2-weighted (T2w) signal intensity ratios (white matter in corpus callosum and liquor) were determined by +/+ in vivo MRI in aSyn and −/− aSyn mice before (baseline) and after 5 weeks of cuprizone diet (demyelination; n =5–6per group; WM, white matter). Representative MRI images are presented with parts of the corpus callosum used for analysis shown in black frames. Demyelination patterns in the corpus callosum were assessed by (immuno-) histochemical staining with c LFB, d anti-CNPase, e anti-MBP, or f anti-MOG antibody. Numbers of CNS cells in the corpus callosum were determined by staining with g) anti-NogoA, h) anti-Olig2 i anti-GFAP, or j anti- Iba1 antibody staining (naïve wt: n =3; aSyn= 4–6 per group). **p < 0.01, ***p <0.001 1044 J Mol Neurosci (2020) 70:1038–1049 +/+ −/− −/− staining were similar in aSyn and aSyn mice (Fig. 4a–d). clearance of myelin debris and apoptotic cells in aSyn Compared with myelin analysis determined at the time point mice still remains to be elucidated. Previously, it was shown of full demyelination, MOG and MBP scores were significant- that increased levels of aSyn reduce phagocytic activity of ly higher in both groups, further indicating the presence of microglial cells and macrophages in an animal model of PD remyelination processes (Fig. 5b, d, Appendix). Analogous as well as in human cells (Gardai et al. 2013). In MSA and to the results obtained at the time point of full demyelination, its corresponding mouse models, increased levels of oligo- + + also numbers of Olig2 and NogoA oligodendroglia as well dendroglial aSyn are accompanied by astro- and + + as numbers of GFAP cells and Iba1 microglia were similar microgliosis already indicating that aSyn may play an im- +/+ −/− in aSyn and aSyn mice (Fig. 4e–h). Notably, in both portant role during neuroinflammation in synucleinopathies groups, the number of mature NogoA oligodendrocytes (Stefanova et al. 2005; Ahmed et al. 2012). Overall, results was increased by about 50% compared with the cell number from both early and late phase EAE studies support the detected in the corpus callosum after 5 weeks of cuprizone notion that aSyn is involved in the regulation of central diet, suggesting recovery of the oligodendrocyte population and peripheral inflammatory processes. after marked toxin-induced reduction (Fig. 5e, Appendix). In order to provide further evidence for a role in regulating Additionally, regenerative processes were assessed 1 week demyelinating processes in late-stage EAE, future studies in- after stopping the cuprizone diet. Similar to the observations vestigating the impact of aSyn deficiency on the peripheral made during early remyelination, aSyn deficiency did not af- versus central immune system in late-stage inflammation are fect remyelination pattern and numbers of CNS cells after required. Analogous to previous analysis in the early EAE 7 days of remyelination (Fig. 6a–h, Appendix). Matching phase, flow cytometry analysis of splenocytes or adoptive these data, performance on the Rotarod did not significantly transfer EAE experiments using T cells isolated from aSyn- +/+ −/− differ between aSyn and aSyn mice at this time point deficient mice may help to elucidate the role of aSyn during (Fig. 6i,Appendix). immune responses in more detail (Ettle et al. 2016b). Another possibility to more closely investigate underlying mechanisms is to induce active EAE in conditional knock-out mice specif- Discussion ically lacking aSyn in the central nervous system or in the peripheral immune compartment. aSyn is known to play an important role in the pathology of In the present study, evidence for an immune-mediated various neurodegenerative and neuroinflammatory disorders. mechanism of demyelination also comes from the results ob- Here we analyzed the impact of aSyn deficiency at the late tained by cuprizone-induced toxic de- and remyelination. In stage of EAE, 8 weeks after immunization with MOG this model, demyelination is induced by feeding mice with the 35–55 peptide. A comprehensive histological analysis of spinal cord chopper chelator cuprizone which selectively depletes mature sections revealed decreased numbers of CNS infiltrating T oligodendrocytes (Bénardais et al. 2013). While this treatment cells and mononuclear phagocytes in late-stage EAE lesions leads to a neuroinflammatory response in the CNS character- −/− of aSyn mice, accompanied by reduced axonal and myelin ized by micro- and astrogliosis, peripheral immune cells play a loss as compared with endogenous aSyn-expressing littermate minor role (Hiremath et al. 1998; Torkildsen et al. 2009). +/+ controls (aSyn ). In a previous study, we investigated the Using this approach, we did not detect striking differences +/+ −/− effect of aSyn deficiency in the onset phase of EAE and dem- between aSyn and aSyn mice regarding de- and onstrated that aSyn is involved in the regulation of immuno- remyelinating and neuroinflammatory processes. Surprisingly, −/− logical responses. T helper 1 cells of aSyn mice showed a aSyn deficiency showed no direct impact on oligodendroglial hyperproliferative phenotype which was associated with in- cells. Based on previous findings indicating that aSyn overex- creased IL-2 expression and increased infiltration of T cells pression affects OPC maturation in vitro and in vivo, we orig- into the spinal cord (Ettle et al. 2016b). In line with these data, inally hypothesized that lack of aSyn might also show effects aSyn deficiency has been reported to promote a on oligodendrocyte lineage cells (Ettle et al. 2014, 2016a;May hyperproliferative phenotype and to affect T cell development et al. 2014). However, the models used for the present experi- (Shameli et al. 2016). Since, in the EAE model, demyelination ments might account for these observations. In the EAE model, and axonal loss is triggered by the invasion of autoreactive T demyelination and oligodendrocyte injury are triggered by cells and other peripheral immune cells into the CNS, the strong immunological responses. Considering that aSyn has present data further indicate that aSyn deficiency modulates also been described as modulator of inflammatory processes, peripheral immune responses thereby affecting demyelinat- these effects most probably conceal CNS-endogenous detri- ing processes (Constantinescu et al. 2011). Whether the re- mental events. This makes it difficult to dissect potential pri- duction of myelin loss in the phase of late-stage demyelin- mary effects of aSyn deficiency on oligodendroglial cells and ation is due to T cell exhaustion following hyperproliferation demyelination from immune-mediated effects (Gardai et al. observed in the early stages of EAE or caused by enhanced 2013; Shameli et al. 2016). Accordingly, the observed increase J Mol Neurosci (2020) 70:1038–1049 1045 1046 J Mol Neurosci (2020) 70:1038–1049 −/− +/+ Fig. 4 Early remyelinating processes are similar in aSyn and aSyn preclinical studies of MSA (Valera et al. 2017). Whether this mice. Three days after terminating the cuprizone diet, remyelination in the therapeutic strategy might also show beneficial results in +/+ −/− corpus callosum of aSyn (black bars) and aSyn (gray bars) mice was early- and late-stage EAE needs to be investigated in the histologically assessed by (immuno-) histochemical staining with a LFB/ future. PAS or antibodies against b CNPase, c MBP, or d MOG. Numbers of CNS cells were analyzed by staining with antibodies against e Olig2, f Of note, a recent study implicated beta-Synuclein (bSyn), a NogoA, g GFAP, or h Iba1 (naïve wt: n = 3; aSyn = 4–6 per group). member of the synuclein family with strong homology to **p < 0.01, ***p <0.001 aSyn, in T cell–mediated pathology. Enhanced numbers of bSyn-reactive T cells have been detected in the blood of +/+ in OPCs in lesions of aSyn mice might be rather due to chronic progressive MS patients. Moreover, it has been shown increased recruitment of OPCs triggered by pronounced in- that bSyn-specific T cells were highly pathogenic as adoptive flammation and tissue damage in these animals. Furthermore, transfer elicited heterogeneous EAE symptoms in Lewis rats cuprizone is a very strong toxin selectively affecting mature and specifically induced severe inflammation in the gray mat- oligodendrocytes, but the exact mechanism has not been ter of the brain ultimately resulting in permanent damage completely understood yet. Therefore, in this demyelination (Lodygin et al. 2019). Lately, pronounced numbers of aSyn- model, effects of aSyn deficiency on de- and remyelination reactive T cells have been detected in PD patients, and strik- processes might be overwhelmed by these strong toxic effects. ingly also in individuals suffering from MS (Sulzer et al. Despite strenuous research efforts, effective strategies to 2017; Lodygin et al. 2019). Considering that in our study prevent CNS demyelination or to induce remyelination are aSyn deficiency partly protected from late-stage demyelin- still missing and the exact mechanisms underlying autoim- ation, it will be of particular interest to determine whether mune myelin destruction are incompletely understood. This aSyn may also serve as target of CNS autoimmune reactions. emphasizes the importance to unravel factors triggering de- Importantly, the identification of CNS target antigens is im- myelination. Expression of oligodendroglial aSyn has been perative for the development of highly selective immunother- shown to be an important feature in the neuropathology of apies preventing demyelination caused by autoreactive MSA inducing a regional-specific pattern not only of demye- processes. lination but also of severe inflammation (Ishizawa et al. 2004; In summary, the data presented in this study so far indicate Ahmed et al. 2012; Ettle et al. 2016a; Hoffmann et al. 2019). that aSyn acts as modulator of peripheral autoimmune- A combined therapy of anti-inflammatory targets and immu- mediated neuroinflammatory demyelination. This further notherapy against aSyn has shown promising results in identifies aSyn as a regulator of immune responses and +/+ −/− Fig. 5 Similar de- and regenerative patterns in aSyn and aSyn mice g GFAP, or h Iba1. The time points investigated were demyelination following cuprizone treatment. Comparison of de- and remyelination (5 weeks after cuprizone treatment), 3 days after stopping the cuprizone +/+ patterns and CNS cell numbers in the corpus callosum of aSyn diet (3 days remyelination), and 7 days after termination of the cuprizone −/− (black bars) and aSyn (gray bars) mice at different time points. treatment (7 days remyelination) (aSyn = 3–5 per group and time point). Histological analysis was performed by staining with a LFB/PAS or *p <0.05, **p < 0.01, ***p <0.001 antibodies against b) MBP, c) CNPase, or d MOG, e)NogoA, f) Olig2, J Mol Neurosci (2020) 70:1038–1049 1047 Fig. 6 aSyn deficiency does not alter remyelination processes 1weekafter terminating cuprizone administration. Seven days after stopping the cuprizone diet, remyelination in the corpus +/+ callosum of aSyn (black bars) −/− and aSyn (gray bars) mice was histologically assessed by (immuno-) histochemical staining with a LFB/PAS or antibodies against b)MBP, c) CNPase, or d MOG. Numbers of CNS cells were analyzed by staining with antibodies against e)NogoA, f) Olig2, g GFAP, or h Iba1 (naïve wt: n =3;aSyn=3–5 per group). i Motor coordination skills were +/+ −/− assessed in aSyn and aSyn animals 7 days after withdrawal of cuprizone using an accelerating Rotarod test (n =5 per group). *p <0.05, **p <0.01, ***p <0.001 1048 J Mol Neurosci (2020) 70:1038–1049 Compston A, Coles A (2008) Multiple sclerosis. Lancet 372:1502–1517. renders this protein a potential bridging element between the https://doi.org/10.1016/S0140-6736(08)61620-7 peripheral immune system and the CNS in demyelinating dis- Constantinescu CS, Farooqi N, O’Brien K, Gran B (2011) Experimental eases including MS. autoimmune encephalomyelitis (EAE) as a model for multiple scle- rosis (MS). Br J Pharmacol 164:1079–1106. https://doi.org/10.1111/ Acknowledgments We thank Prof. Dr. Stephan von Hörsten and Anne- j.1476-5381.2011.01302.x Christine Plank for the assistance with the Rotarod experiments. The data Ettle B, Reiprich S, Deusser J, Schlachetzki JC, Xiang W, Prots I, Masliah are part of the PhD thesis “MAdCAM-1 and alpha-Synuclein as endog- E, Winner B, Wegner M, Winkler J (2014) Intracellular alpha- enous regulators of immune responses and tissue susceptibility in neuro- synuclein affects early maturation of primary oligodendrocyte pro- inflammation” written by Kristina Kuhbandner at the Friedrich- genitor cells. Mol Cell Neurosci 62:68–78. https://doi.org/10.1016/j. Alexander-University Erlangen-Nürnberg. mcn.2014.06.012 Ettle B, Kerman BE, Valera E, Gillmann C, Schlachetzki JC, Reiprich S, BüttnerC,Ekici AB,ReisA,WegnerM,Bäuerle T, Funding Information Open Access funding provided by Projekt DEAL. 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alpha-Synuclein: a Modulator During Inflammatory CNS Demyelination

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Abstract

Neuroinflammation and demyelination are hallmarks of several neurological disorders such as multiple sclerosis and multiple system atrophy. To better understand the underlying mechanisms of de- and regeneration in respective diseases, it is critical to identify factors modulating these processes. One candidate factor is alpha-Synuclein (aSyn), which is known to be involved in the pathology of various neurodegenerative diseases. Recently, we have shown that aSyn is involved in the modulation of peripheral immune responses during acute neuroinflammatory processes. In the present study, the effect of aSyn deficiency on de- and regenerative events in the CNS was analyzed by using two different demyelinating animal models: chronic MOG -induced 35–55 experimental autoimmune encephalomyelitis (EAE) and the cuprizone model. Histopathological analysis of spinal cord cross sections 8 weeks after EAE induction revealed a significant reduction of CNS inflammation accompanied by decreased myelin loss during late-stage inflammatory demyelination in aSyn-deficient mice. In contrast, after cuprizone-induced demyelination or remyelination following withdrawal of cuprizone, myelination and neuroinflammatory patterns were not affected by aSyn deficiency. These data provide further evidence for aSyn as regulator of peripheral immune responses under neuroinflammatory conditions, thereby also modulating degenerative events in late-stage demyelinating disease. . . . . Keywords Neuroinflammation Demyelination alpha-Synuclein Experimental autoimmune encephalomyelitis Cuprizone Introduction sclerosis (MS) (Compston and Coles 2008; Fanciulli and Wenning 2015). Physiologically, compensating repair mecha- Demyelination in combination with neuroinflammatory re- nisms exist which sustain myelin regeneration. While in some sponses is a common feature of several neurological disorders MS patients these processes frequently occur, others only show including multiple system atrophy (MSA) and multiple sparse remyelination (Patrikios et al. 2006). The reasons for this regeneration failure still remain unclear. Up to date, no thera- Jürgen Winkler and Ralf A. Linker contributed equally to this work. peutic options exist to cure these diseases or to halt disease progression. For the development of such treatment strategies, * Jürgen Winkler it is essential to identify factors which are involved in de- and Juergen.Winkler@uk-erlangen.de remyelinating processes as well as oligodendrocyte cell damage and death. In rodent models, it has been shown that Department of Molecular Neurology, University Hospital Erlangen, remyelination critically depends on the presence of oligoden- Friedrich-Alexander-University Erlangen-Nürnberg, drocyte precursor cells (OPCs) (Sim et al. 2002). In the central Schwabachanlage 6, 91054 Erlangen, Germany nervous system (CNS), OPCs are recruited to demyelinated Department of Neurology, University Hospital Erlangen, areas and give rise to myelinating oligodendrocytes upon acti- Friedrich-Alexander-University Erlangen-Nürnberg, vation (Franklin and ffrench-Constant 2017). Current concepts Erlangen, Germany to explain the failure of remyelination include insufficient re- Department of Neurology, University of Regensburg, cruitment of OPCs to the demyelinated region or the inhibition Regensburg, Germany of maturation into oligodendrocytes (Sim et al. 2002; Institute of Radiology, Preclinical Imaging Platform Erlangen Kuhlmann et al. 2008). So far, various extrinsic and intrinsic (PIPE), University Hospital Erlangen, factors modulating oligodendrocyte maturation have been de- Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany scribed (French et al. 2009; Hughes et al. 2013;Heetal. 2016). J Mol Neurosci (2020) 70:1038–1049 1039 Recent studies using in vitro and in vivo MSA models suggest Experimental Autoimmune Encephalomyelitis alpha-Synuclein (aSyn) as a candidate factor inhibiting OPC +/+ maturation (Ettle et al. 2014; May et al. 2014). aSyn is For active EAE induction, 10–14-week-old aSyn and −/− expressed in axons, neuritic processes, and presynaptic termi- aSyn mice received 200 μgMOG and 200 μgcom- 35–55 nals of neurons, being important for vesicle transport. In addi- plete Freund’s adjuvant (CFA) subcutaneously. Pertussis toxin tion, aSyn protein levels have also been detected in glia and (200 ng) was applied intraperitoneally at the day of immuni- hematopoietic cells (Maroteaux et al. 1988; Barbour et al. zation and 48 h later. Mice were daily weighed and scored for 2008). Pathological aSyn aggregations are associated with neu- clinical signs using a 10-point scale as described previously rodegenerative disorders, in particular Parkinson’s disease, de- (Linker et al. 2002). mentia with Lewy bodies, and MSA, also summarized as synucleinopathies (Spillantini et al. 1998). In the latter, aSyn Cuprizone Model accumulates in glial cytoplasmic inclusions (GCI) in oligoden- drocytes being considered pathological hallmark and associat- Toxic demyelination was induced by feeding 8–10-week-old ed with widespread demyelination (Papp et al. 1989). +/+ −/− aSyn and aSyn mice a diet containing 0.2% cuprizone Additionally, degenerative features are often accompanied by mixed into ground standard rodent chow for 5 weeks. During distinct neuroinflammatory processes in these this period, mice were closely monitored for weight loss as synucleinopathies (Lim et al. 2016; Hoffmann et al. 2019). well as abnormalities and trained for Rotarod analysis. After Interestingly, aSyn immunoreactivity was also reported in glial 5 weeks, motor coordination skills were assessed by an accel- cells in MS lesions and in spinal cord lesions of mice induced erating Rotarod test. To initialize remyelination, after 5 weeks, with experimental autoimmune encephalomyelitis (EAE), a cuprizone was removed from the diet and mice received common animal model of MS (Papadopoulos et al. 2006;Lu ground standard rodent chow for 3 or 7 days, respectively. et al. 2009). Recently, we identified aSyn as regulator of pe- At the end of de- and remyelination experiments, mice were ripheral immune responses in neuroinflammation (Ettle et al. sacrificed to dissect the brain for histological analysis. 2016b). Considering these findings, we studied the role of C57BL/6N wildtype mice fed with normal chow served as aSyn during late-stage neuroinflammatory demyelination and naïve controls and are termed “naïve wt.” in the context of toxin-induced de- as well as remyelination. For this purpose, demyelination was induced in aSyn-deficient −/− Rotarod Test mice (aSyn ) and littermate control animals with endogenous +/+ aSyn expression (aSyn ) by using two different demyelin- Motor performance of mice treated with cuprizone was eval- ation models. More precisely, autoimmune-mediated late-stage uated by performing an accelerating Rotarod test. The Rotarod demyelination was examined with the help of myelin oligo- consisted of a 30-mm diameter rod rotating about its long axis. dendrocyte glycoprotein (MOG) –EAE and toxin-induced 35–55 Over the course of 180 s, the Rotarod accelerated from 4 to 40 demyelination was studied in the cuprizone model. rounds per minute (rpm). The latency of each mouse to fall Furthermore, regenerative events were assessed at two differ- was recorded in two consecutive trials with a 30-min resting ent time points after stopping cuprizone administration. phase in the home cage between the trials. One week prior to the accelerating test, mice were trained on the rod rotating with constant speed at 12 rpm for 120 s. Material and Methods Animal Experiments In Vivo Magnetic Resonance Imaging −/− aSyn mice were maintained on a C57BL/6N background In vivo magnetic resonance imaging (MRI) was performed +/+ −/− for more than ten generations (Abeliovich et al. 2000). with aSyn and aSyn mice before and after cuprizone +/+ aSyn littermates with endogenous aSyn expression served administration. In inhalation anesthesia (1.5% isoflurane), as controls. All mice were kept under standard animal hous- heads of mice were fixed in a mouse brain surface array coil ing conditions with a 12-h day/night cycle and free access to and scanned on a preclinical ultra-high field MRI system (7 food and water. All applicable international, national, and/or Tesla ClinScan 70/30, Bruker). During the entire imaging pro- institutional guidelines for the care and use of animals were cedure, respiration of mice was monitored and kept constant, followed. Animal experiments were performed in accor- while the body temperature was stabilized with a heating cir- dance with the German laws of animal protection and were culator bath. The imaging protocol consisted of a 3D T2- approved by the local ethics committee (Government of weighted turbo spin echo sequence (repetition time (TR), Unterfranken, Bavaria, Germany, ref. # 55.2-2532-2-395 3690 s; echo time (TE), 40 s; voxel size, 0.082 × 0.082 × and # 55.2-2532-2-450). 0.5 mm; acquisition time, 4 min 37 s; slice thickness, 0.5 mm). 1040 J Mol Neurosci (2020) 70:1038–1049 Sequences were analyzed with the help of the medical im- NH OH and distilled water and fixed in 5% Na S O for 4 2 2 3 aging interaction toolkit (MITK). T2-weighted signal intensi- 3 min. Finally, sections were washed in distilled water, +/+ −/− ties were assessed in aSyn and aSyn mice before and dehydrated, and mounted. after cuprizone diet. The boundaries of the region of interest (ROI) within white matter regions of the corpus callosum Histological Evaluation were manually defined (Wu et al. 2018). To eliminate con- founding signals arising from slice-dependent signal variation For histological evaluation of EAE experiments, spinal cord during MRI acquisition, the signal intensity of the ventricle cross sections were analyzed blinded using a BX-51 light mi- within the same slice was used for normalization. To calculate croscope (Olympus). For each staining, at least five lesions T2-weighted signal intensity ratios, signal intensities were de- within each spinal cord segment (cervical, thoracic, and lum- termined in the ROI and the ventricle of each animal in the bar) were inspected. T cells, mononuclear phagocytes, astro- same layer. cytes, and oligodendrocytes were counted at × 200 magnifica- tion within the margins of a 1/16 mm grid in individual lesions (Immuno-)Histochemistry and counts were normalized to cells/mm .Analysisof demyelinated areas in the white matter of LFB and anti- Following perfusion with 4% paraformaldehyde (PFA), spinal CNPase-stained sections was performed semi-automatically cords or brains were removed and post-fixed in 4% PFA for 2– with the help of CellSens or CellP software (Olympus). 3 h. After embedding in paraffin, 4 μm thin sections were Axonal density was quantified in silver impregnated sections prepared by using a microtome. For immunohistochemistry, by counting on a 100 μm diameter grid at × 500 magnification. anti-CD3 (MCA1477, Biorad) and anti-Mac-3 (M3/84, BD To analyze brain sections in cuprizone experiments, sec- Pharmingen) antibodies were used to detect T cells and mono- tions between bregma − 1.15 and bregma − 2.15 according to nuclear phagocytes, respectively. Glial cells were stained with the Mouse Brain atlas by Franklin and Paxinos were examined anti-Olig2 (Millipore), anti-NogoA (Millipore), anti-GFAP (Franklin and Paxinos 2013). The level of demyelination in (Dako), and anti-Iba1 (20A12.1, Millipore) antibodies, respec- the corpus callosum was assessed by determination of myelin tively. Anti-MOG (8-18C5, Millipore), anti-MBP (12, Biorad), scores by two independent blinded observers as follows: 0: and anti-2′,3′-Cyclic-nucleotide 3′-phosphodiesterase (CNPase) complete demyelination, 1: 1/3 of corpus callosum myelinat- (SMI91R, BioLegend) antibodies were used as myelin markers. ed, 2: 2/3 of corpus callosum myelinated, 3: complete For chromogenic immunodetection, Vectastain Elite Avidin- myelination (Lindner et al. 2008). Biotin-Complex Kit (Vector Laboratories) was used according Number of cells was determined in three adjacent regions to the manufacturer’s instructions. Diaminobenzidine (DAB) in the median corpus callosum of each animal using a mor- was employed to visualize the conjugated peroxidase. phometric grid. Immunopositive cells with identified nucleus Luxol fast blue-periodic acid Schiff (LFB-PAS) staining (counterstaining with hematoxylin) were counted at × 200 was performed to assess the extent of demyelination. magnification and counts were normalized to cells/mm . Therefore, deparaffinized slides were incubated overnight in 0.1% Luxol fast blue (LFB) solution at 56 °C. Next day, sec- Statistical Analysis tions were rinsed in 96% ethanol and distilled water. For dif- ferentiation, sections were incubated in 1% lithium carbonate Statistical testing was performed using Graph Pad Prism. All solution for a few seconds, rinsed shortly in 70% ethanol, and ex vivo data were analyzed by one- or two-way ANOVA washed in distilled water. PAS staining was performed by followed by Tukey’s posttest or unpaired t test. EAE data were oxidizing the slides in 0.8% periodic acid solution for analyzed either by Mann-Whitney U test or logrank test (for 10 min. Afterwards, sections were stained in Schiff reagent disease incidence analysis). For statistical analysis of MRI data, for 20 min and washed in sulfite solution, followed by a 10- the two-way ANOVA test for repeated measurements was used. min washing step with running tap water. Finally, sections Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, or were dehydrated and mounted. ***p < 0.001 were considered to be statistically significant. For analysis of axonal integrity, Bielschowsky silver im- pregnation was employed. Rehydrated sections were trans- ferred into a 20% AgNO solution and incubated for 15 min Results at 37 °C. Then the solution was washed off with distilled water and 25% NH OH solution was added to the AgNO solution. aSyn Deficiency Ameliorates EAE Severity 4 3 Slides were incubated in this solution for 10 min in the dark at 37 °C and washed in 0.1% NH OH. After adding developer First, we used the EAE model to study the effect of aSyn stock solution to the AgOH solution, sections were stained for deficiency during late-stage inflammatory demyelination. +/+ −/− 4 min until axons turned black. Slides were rinsed in 0.1% Therefore, aSyn and aSyn mice were immunized with J Mol Neurosci (2020) 70:1038–1049 1041 MOG peptide and monitored for clinical signs over a and Olig2 cells were determined in demyelinated areas. 35–55 period of 8 weeks. Compared with littermate controls, aSyn- While the number of NogoA mature oligodendrocytes did deficient mice displayed an ameliorated disease course not differ between both groups, significantly more cells posi- (Fig. 1a). At the end of the observation period, they showed tive for Olig2 , a relatively broad oligodendrocytic marker, +/+ a better outcome by one score point in comparison with were detected in lesions of aSyn mice (Fig. 2g, h). +/+ aSyn mice. More precisely, they suffered from tail paraly- sis, while the control group still exhibited signs of gait ataxia aSyn Deficiency Does Not Affect Myelination Pattern and mild paralysis of hind limbs. There were no differences in After Cuprizone-Induced Demyelination the overall disease incidence or mortality rate between both groups (Fig. 1b). Next to the EAE model, the cuprizone model was employed to investigate the impact of aSyn deficiency on acute demyelin- Reduced Demyelination, CNS Inflammation, ation and remyelinating processes in the absence of peripheral and Axonal Loss in aSyn-Deficient Mice immune responses. To evoke acute demyelination in the brain +/+ −/− During Late-Stage EAE of aSyn and aSyn mice, age-matched animals were fed a diet containing 0.2% cuprizone for 5 weeks. Motor coordina- In the EAE model, disease course and severity of symptoms tion skills of mice were assessed after cuprizone treatment by are determined by the extent of CNS inflammation and spinal an accelerating Rotarod test. Both groups showed equal per- cord damage is mainly apparent in a form of demyelination formances regarding latency to stay on the accelerating rod and axonal loss. Therefore, we histologically analyzed spinal (Fig. 3a). In vivo MRI was used to detect myelin deficits in the +/+ −/− cords of aSyn and aSyn mice for these parameters corpus callosum following cuprizone treatment. After 5 weeks 8 weeks after induction of EAE. Average clinical scores at this of cuprizone diet, gray-white matter contrast was conspicu- −/− +/+ time point were 1.38 for aSyn mice and 2.5 for aSyn ously reduced. Quantification of T2-weighted signal intensity mice, respectively. Matching the disease course, immunohis- ratios of white matter and liquor revealed a 70% increase in tochemical staining for CNPase as well as LFB-PAS staining animals receiving cuprizone diet, indicating a considerable revealed a more than 2.5-fold increase in white matter demy- reduction in myelin content in the corpus callosum. +/+ elination in aSyn mice (Fig. 2a, b). Furthermore, axonal However, this method did not detect any differences in demy- +/+ −/− densities determined by Bielschowsky silver staining were elination patterns between aSyn and aSyn mice follow- +/+ reduced by 30% in lesions of aSyn mice compared with ing cuprizone exposure (Fig. 3b). To validate these findings, a −/− aSyn mice (Fig. 2c). Additionally, we detected a marked comprehensive histological analysis of brain sections was per- increase in GFAP processes and significantly higher numbers formed. In line with results obtained from the MRI measure- of GFAP cell bodies within and in direct vicinity of spinal ment, immunohistochemical staining for different myelin cord lesions in these mice indicative for astrocytic activation markers, such as MBP, CNPase, and MOG, as well as LFB- (Fig. 2d). Further examination of neuroinflammatory process- PAS staining demonstrated that 5 weeks of cuprizone treat- +/+ es revealed a pronounced inflammatory response in aSyn ment resulted in comparable reduction of myelin staining in −/− +/+ −/− mice. Overall, fewer lesions were present in aSyn mice both, aSyn and aSyn mice, in the central part of the containing a lower number of invading immune cells. More corpus callosum compared with naïve wt mice (Fig. 3c–f). + + specifically, CD3 Tcells and Mac-3 mononuclear phago- Additionally, the impact on CNS cells was studied. cytes infiltrating the CNS were reduced by 50% compared Cuprizone treatment strongly reduced the number of mature +/+ + with aSyn animals (Fig. 2e, f). To closer examine the effect NogoA oligodendrocytes compared with naïve mice of aSyn deficiency on oligodendroglia, numbers of NogoA (Fig. 3g). However, no differences were observed in the +/+ −/− Fig. 1 aSyn deficiency attenuates disease severity in late-stage EAE. a EAE incidence in aSyn (solid line) and aSyn (dashed line) mice (n = +/+ −/− Clinical course of MOG -EAE in aSyn (black dots) and aSyn 16–17 per group). p.i. post immunization. *p <0.05 35–55 (gray squares) mice (n =11–13 per group). b Kaplan-Meier plot depicting 1042 J Mol Neurosci (2020) 70:1038–1049 Fig. 2 aSyn-deficient mice exhibit less severe demyelination, axonal loss CNPase. c Bar graph depicting axonal densities (number of axons and inflammation in CNS lesions. (Immuno-)Histochemical analysis of counted on a 100 μm grid) determined by Bielschowsky silver staining. +/+ −/− + + + spinal cord cross sections of aSyn (black bars) and aSyn (gray bars) Numbers of d GFAP astrocytes, infiltrating e CD3 and f Mac-3 cells, g + + mice 8 weeks after immunization with MOG peptide. Average Olig2 and h NogoA cells were assessed by immunohistochemical 35–55 −/− clinical scores at the time point of analysis were 1.38 for aSyn mice staining. Representative pictures are shown for each staining (n =5–6 +/+ and 2.5 for aSyn mice. Percentage of demyelinated area in the white per group). **p < 0.01, ***p <0.001 matter area determined by a LFB-PAS staining or b staining with anti- + + numbers of mature NogoA oligodendrocytes or Olig2 cells aSyn Deficiency Has No Impact on Early +/+ −/− in the corpus callosum between aSyn and aSyn mice Remyelination in the Cuprizone Model (Fig. 3g, h). Following cuprizone administration, neuroinflammatory processes including activation of astro- The cuprizone model is considered a well-established tool to cytes and microglia are induced (Hiremath et al. 1998). investigate remyelination since, only few days after adminis- + + Indeed, numbers of Iba1 microglia and GFAP astrocytes tration, regenerative processes are emerging. In order to study were markedly increased by 14-fold and twofold, respectively, the effect of aSyn deficiency on early remyelination, we ex- +/+ −/− +/+ −/− in the corpus callosum of aSyn and aSyn mice treated amined aSyn and aSyn mice for their myelination pattern with cuprizone for 5 weeks compared with untreated controls. by histological analysis 3 days after terminating the cuprizone +/+ −/− However, in aSyn and aSyn mice, numbers of respective diet. In both groups, myelination scores assessed by histolog- cells were nearly equal (Fig. 3i, j). ical staining for myelin markers (MOG, MBP) and LFB J Mol Neurosci (2020) 70:1038–1049 1043 Fig. 3 aSyn deficiency does not affect acute demyelination after 5 weeks of cuprizone treatment. +/+ −/− aSyn (black bars) and aSyn (gray bars) mice received cuprizone diet for 5 weeks. a Motor coordination was tested by an accelerating Rotarod test (n = 5–6 per group). b T2-weighted (T2w) signal intensity ratios (white matter in corpus callosum and liquor) were determined by +/+ in vivo MRI in aSyn and −/− aSyn mice before (baseline) and after 5 weeks of cuprizone diet (demyelination; n =5–6per group; WM, white matter). Representative MRI images are presented with parts of the corpus callosum used for analysis shown in black frames. Demyelination patterns in the corpus callosum were assessed by (immuno-) histochemical staining with c LFB, d anti-CNPase, e anti-MBP, or f anti-MOG antibody. Numbers of CNS cells in the corpus callosum were determined by staining with g) anti-NogoA, h) anti-Olig2 i anti-GFAP, or j anti- Iba1 antibody staining (naïve wt: n =3; aSyn= 4–6 per group). **p < 0.01, ***p <0.001 1044 J Mol Neurosci (2020) 70:1038–1049 +/+ −/− −/− staining were similar in aSyn and aSyn mice (Fig. 4a–d). clearance of myelin debris and apoptotic cells in aSyn Compared with myelin analysis determined at the time point mice still remains to be elucidated. Previously, it was shown of full demyelination, MOG and MBP scores were significant- that increased levels of aSyn reduce phagocytic activity of ly higher in both groups, further indicating the presence of microglial cells and macrophages in an animal model of PD remyelination processes (Fig. 5b, d, Appendix). Analogous as well as in human cells (Gardai et al. 2013). In MSA and to the results obtained at the time point of full demyelination, its corresponding mouse models, increased levels of oligo- + + also numbers of Olig2 and NogoA oligodendroglia as well dendroglial aSyn are accompanied by astro- and + + as numbers of GFAP cells and Iba1 microglia were similar microgliosis already indicating that aSyn may play an im- +/+ −/− in aSyn and aSyn mice (Fig. 4e–h). Notably, in both portant role during neuroinflammation in synucleinopathies groups, the number of mature NogoA oligodendrocytes (Stefanova et al. 2005; Ahmed et al. 2012). Overall, results was increased by about 50% compared with the cell number from both early and late phase EAE studies support the detected in the corpus callosum after 5 weeks of cuprizone notion that aSyn is involved in the regulation of central diet, suggesting recovery of the oligodendrocyte population and peripheral inflammatory processes. after marked toxin-induced reduction (Fig. 5e, Appendix). In order to provide further evidence for a role in regulating Additionally, regenerative processes were assessed 1 week demyelinating processes in late-stage EAE, future studies in- after stopping the cuprizone diet. Similar to the observations vestigating the impact of aSyn deficiency on the peripheral made during early remyelination, aSyn deficiency did not af- versus central immune system in late-stage inflammation are fect remyelination pattern and numbers of CNS cells after required. Analogous to previous analysis in the early EAE 7 days of remyelination (Fig. 6a–h, Appendix). Matching phase, flow cytometry analysis of splenocytes or adoptive these data, performance on the Rotarod did not significantly transfer EAE experiments using T cells isolated from aSyn- +/+ −/− differ between aSyn and aSyn mice at this time point deficient mice may help to elucidate the role of aSyn during (Fig. 6i,Appendix). immune responses in more detail (Ettle et al. 2016b). Another possibility to more closely investigate underlying mechanisms is to induce active EAE in conditional knock-out mice specif- Discussion ically lacking aSyn in the central nervous system or in the peripheral immune compartment. aSyn is known to play an important role in the pathology of In the present study, evidence for an immune-mediated various neurodegenerative and neuroinflammatory disorders. mechanism of demyelination also comes from the results ob- Here we analyzed the impact of aSyn deficiency at the late tained by cuprizone-induced toxic de- and remyelination. In stage of EAE, 8 weeks after immunization with MOG this model, demyelination is induced by feeding mice with the 35–55 peptide. A comprehensive histological analysis of spinal cord chopper chelator cuprizone which selectively depletes mature sections revealed decreased numbers of CNS infiltrating T oligodendrocytes (Bénardais et al. 2013). While this treatment cells and mononuclear phagocytes in late-stage EAE lesions leads to a neuroinflammatory response in the CNS character- −/− of aSyn mice, accompanied by reduced axonal and myelin ized by micro- and astrogliosis, peripheral immune cells play a loss as compared with endogenous aSyn-expressing littermate minor role (Hiremath et al. 1998; Torkildsen et al. 2009). +/+ controls (aSyn ). In a previous study, we investigated the Using this approach, we did not detect striking differences +/+ −/− effect of aSyn deficiency in the onset phase of EAE and dem- between aSyn and aSyn mice regarding de- and onstrated that aSyn is involved in the regulation of immuno- remyelinating and neuroinflammatory processes. Surprisingly, −/− logical responses. T helper 1 cells of aSyn mice showed a aSyn deficiency showed no direct impact on oligodendroglial hyperproliferative phenotype which was associated with in- cells. Based on previous findings indicating that aSyn overex- creased IL-2 expression and increased infiltration of T cells pression affects OPC maturation in vitro and in vivo, we orig- into the spinal cord (Ettle et al. 2016b). In line with these data, inally hypothesized that lack of aSyn might also show effects aSyn deficiency has been reported to promote a on oligodendrocyte lineage cells (Ettle et al. 2014, 2016a;May hyperproliferative phenotype and to affect T cell development et al. 2014). However, the models used for the present experi- (Shameli et al. 2016). Since, in the EAE model, demyelination ments might account for these observations. In the EAE model, and axonal loss is triggered by the invasion of autoreactive T demyelination and oligodendrocyte injury are triggered by cells and other peripheral immune cells into the CNS, the strong immunological responses. Considering that aSyn has present data further indicate that aSyn deficiency modulates also been described as modulator of inflammatory processes, peripheral immune responses thereby affecting demyelinat- these effects most probably conceal CNS-endogenous detri- ing processes (Constantinescu et al. 2011). Whether the re- mental events. This makes it difficult to dissect potential pri- duction of myelin loss in the phase of late-stage demyelin- mary effects of aSyn deficiency on oligodendroglial cells and ation is due to T cell exhaustion following hyperproliferation demyelination from immune-mediated effects (Gardai et al. observed in the early stages of EAE or caused by enhanced 2013; Shameli et al. 2016). Accordingly, the observed increase J Mol Neurosci (2020) 70:1038–1049 1045 1046 J Mol Neurosci (2020) 70:1038–1049 −/− +/+ Fig. 4 Early remyelinating processes are similar in aSyn and aSyn preclinical studies of MSA (Valera et al. 2017). Whether this mice. Three days after terminating the cuprizone diet, remyelination in the therapeutic strategy might also show beneficial results in +/+ −/− corpus callosum of aSyn (black bars) and aSyn (gray bars) mice was early- and late-stage EAE needs to be investigated in the histologically assessed by (immuno-) histochemical staining with a LFB/ future. PAS or antibodies against b CNPase, c MBP, or d MOG. Numbers of CNS cells were analyzed by staining with antibodies against e Olig2, f Of note, a recent study implicated beta-Synuclein (bSyn), a NogoA, g GFAP, or h Iba1 (naïve wt: n = 3; aSyn = 4–6 per group). member of the synuclein family with strong homology to **p < 0.01, ***p <0.001 aSyn, in T cell–mediated pathology. Enhanced numbers of bSyn-reactive T cells have been detected in the blood of +/+ in OPCs in lesions of aSyn mice might be rather due to chronic progressive MS patients. Moreover, it has been shown increased recruitment of OPCs triggered by pronounced in- that bSyn-specific T cells were highly pathogenic as adoptive flammation and tissue damage in these animals. Furthermore, transfer elicited heterogeneous EAE symptoms in Lewis rats cuprizone is a very strong toxin selectively affecting mature and specifically induced severe inflammation in the gray mat- oligodendrocytes, but the exact mechanism has not been ter of the brain ultimately resulting in permanent damage completely understood yet. Therefore, in this demyelination (Lodygin et al. 2019). Lately, pronounced numbers of aSyn- model, effects of aSyn deficiency on de- and remyelination reactive T cells have been detected in PD patients, and strik- processes might be overwhelmed by these strong toxic effects. ingly also in individuals suffering from MS (Sulzer et al. Despite strenuous research efforts, effective strategies to 2017; Lodygin et al. 2019). Considering that in our study prevent CNS demyelination or to induce remyelination are aSyn deficiency partly protected from late-stage demyelin- still missing and the exact mechanisms underlying autoim- ation, it will be of particular interest to determine whether mune myelin destruction are incompletely understood. This aSyn may also serve as target of CNS autoimmune reactions. emphasizes the importance to unravel factors triggering de- Importantly, the identification of CNS target antigens is im- myelination. Expression of oligodendroglial aSyn has been perative for the development of highly selective immunother- shown to be an important feature in the neuropathology of apies preventing demyelination caused by autoreactive MSA inducing a regional-specific pattern not only of demye- processes. lination but also of severe inflammation (Ishizawa et al. 2004; In summary, the data presented in this study so far indicate Ahmed et al. 2012; Ettle et al. 2016a; Hoffmann et al. 2019). that aSyn acts as modulator of peripheral autoimmune- A combined therapy of anti-inflammatory targets and immu- mediated neuroinflammatory demyelination. This further notherapy against aSyn has shown promising results in identifies aSyn as a regulator of immune responses and +/+ −/− Fig. 5 Similar de- and regenerative patterns in aSyn and aSyn mice g GFAP, or h Iba1. The time points investigated were demyelination following cuprizone treatment. Comparison of de- and remyelination (5 weeks after cuprizone treatment), 3 days after stopping the cuprizone +/+ patterns and CNS cell numbers in the corpus callosum of aSyn diet (3 days remyelination), and 7 days after termination of the cuprizone −/− (black bars) and aSyn (gray bars) mice at different time points. treatment (7 days remyelination) (aSyn = 3–5 per group and time point). Histological analysis was performed by staining with a LFB/PAS or *p <0.05, **p < 0.01, ***p <0.001 antibodies against b) MBP, c) CNPase, or d MOG, e)NogoA, f) Olig2, J Mol Neurosci (2020) 70:1038–1049 1047 Fig. 6 aSyn deficiency does not alter remyelination processes 1weekafter terminating cuprizone administration. Seven days after stopping the cuprizone diet, remyelination in the corpus +/+ callosum of aSyn (black bars) −/− and aSyn (gray bars) mice was histologically assessed by (immuno-) histochemical staining with a LFB/PAS or antibodies against b)MBP, c) CNPase, or d MOG. Numbers of CNS cells were analyzed by staining with antibodies against e)NogoA, f) Olig2, g GFAP, or h Iba1 (naïve wt: n =3;aSyn=3–5 per group). i Motor coordination skills were +/+ −/− assessed in aSyn and aSyn animals 7 days after withdrawal of cuprizone using an accelerating Rotarod test (n =5 per group). *p <0.05, **p <0.01, ***p <0.001 1048 J Mol Neurosci (2020) 70:1038–1049 Compston A, Coles A (2008) Multiple sclerosis. Lancet 372:1502–1517. renders this protein a potential bridging element between the https://doi.org/10.1016/S0140-6736(08)61620-7 peripheral immune system and the CNS in demyelinating dis- Constantinescu CS, Farooqi N, O’Brien K, Gran B (2011) Experimental eases including MS. autoimmune encephalomyelitis (EAE) as a model for multiple scle- rosis (MS). Br J Pharmacol 164:1079–1106. https://doi.org/10.1111/ Acknowledgments We thank Prof. Dr. Stephan von Hörsten and Anne- j.1476-5381.2011.01302.x Christine Plank for the assistance with the Rotarod experiments. The data Ettle B, Reiprich S, Deusser J, Schlachetzki JC, Xiang W, Prots I, Masliah are part of the PhD thesis “MAdCAM-1 and alpha-Synuclein as endog- E, Winner B, Wegner M, Winkler J (2014) Intracellular alpha- enous regulators of immune responses and tissue susceptibility in neuro- synuclein affects early maturation of primary oligodendrocyte pro- inflammation” written by Kristina Kuhbandner at the Friedrich- genitor cells. Mol Cell Neurosci 62:68–78. https://doi.org/10.1016/j. Alexander-University Erlangen-Nürnberg. mcn.2014.06.012 Ettle B, Kerman BE, Valera E, Gillmann C, Schlachetzki JC, Reiprich S, BüttnerC,Ekici AB,ReisA,WegnerM,Bäuerle T, Funding Information Open Access funding provided by Projekt DEAL. 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