α-Synuclein-induced myelination deficit defines a novel interventional target for multiple system atrophy

Abstract

Multiple system atrophy (MSA) is a rare atypical parkinsonian disorder characterized by a rapidly progressing clinical course and at present without any efficient therapy. Neuropathologically, myelin loss and neurodegeneration are associated with α-synuclein accumulation in oligodendrocytes, but underlying pathomechanisms are poorly understood. Here, we analyzed the impact of oligodendrocytic α-synuclein on the formation of myelin sheaths to define a potential interventional target for MSA. Post-mortem analyses of MSA patients and controls were performed to quantify myelin and oligodendrocyte numbers. As pre-clinical models, we used transgenic MSA mice, a myelinating stem cell-derived oligodendrocyte-neuron co-culture, and primary oligodendrocytes to determine functional consequences of oligodendrocytic α-synuclein overexpression on myelination. We detected myelin loss accompanied by preserved or even increased numbers of oligodendrocytes in post-mortem MSA brains or transgenic mouse forebrains, respectively, indicating an oligodendrocytic dysfunction in myelin formation. Corroborating this observation, overexpression of α-synuclein in primary and stem cell-derived oligodendrocytes severely impaired myelin formation, defining a novel α-synuclein-linked pathomechanism in MSA. We used the pro-myelinating activity of the muscarinic acetylcholine receptor antagonist benztropine to analyze the reversibility of the myelination deficit. Transcriptome profiling of primary pre-myelinating oligodendrocytes demonstrated that benztropine readjusts myelination-related processes such as cholesterol and membrane biogenesis, being compromised by oligodendrocytic α-synuclein. Additionally, benztropine restored the α-synuclein-induced myelination deficit of stem cell-derived oligodendrocytes. Strikingly, benztropine also ameliorated the myelin deficit in transgenic MSA mice, resulting in a prevention of neuronal cell loss. In conclusion, this study defines the α-synuclein-induced myelination deficit as a novel and crucial pathomechanism in MSA. Importantly, the reversible nature of this oligodendrocytic dysfunction opens a novel avenue for an intervention in MSA.

Keywords: Multiple system atrophy; Myelin; Oligodendrocyte progenitor cells; Oligodendrocytes; α-Synuclein.

Fig. 1

Putaminal myelin loss in multiple system atrophy (MSA) without alterations in oligodendrocyte density. a Representative Luxol Fast Blue/Periodic Acid Schiff (LFB/PAS) stained images (left) depict the putamen (encircled) of a control (upper panel) and a MSA patient (lower panel). The putamen of the MSA patient showed a reduced Luxol Fast Blue staining intensity reflecting severe loss of myelin. Scale bar: 500μm. Boxed areas within the left images are magnified showing LFB/PAS-, hematoxylin-, and α-synuclein-stained striae. Note abundant α-synuclein-positive inclusions in the putamen of the MSA patient (magnified as insert). Scale bars: 50μm, insert 10μm. b Oligodendrocytes (arrowheads; round-shaped, dense chromatin) were distinguished by morphology from astrocytes (asterisk; larger and more elongated in size, loose chromatin) in the hematoxylin stained-sections. Scale bar: 10μm. c While myelin was significantly reduced in MSA patients (gray dots) compared to controls (black dots), oligodendrocyte density was unaltered (n = 6). Data are shown as mean ± standard error of mean. T-test: ##p < 0.01, n.s. (not significant) p > 0.05. f = female.

Fig. 2

Severe myelin deficit despite increased oligodendrocyte density in mice overexpressing human α-synuclein under a myelin basic protein-promoter (MBP29). MBP29 mice (3 months of age) showed a profound myelin deficit (illustrated by gray scale images of a Luxol Fast Blue staining and quantified within the corpus callosum; scale bars: 500μm). Density of callosal oligodendrocytes (identified by Olig2; scale bar: 50μm) was increased by 1.7 fold. Data are shown as mean ± standard error of mean. T-test (n = 5): ###p < 0.001. NTG = non-transgenic mice.

Fig. 3

Impaired myelination of α-synuclein-overexpressing mouse embryonic stem cell-derived oligodendrocytes. a The experimental paradigm to analyze myelination of individual oligodendrocytes in the mouse embryonic stem cell-derived oligodendrocyte-neuron co-culture is schematically illustrated according to [25]. b Stem cell-derived oligodendrocytes expressed human α-synuclein (when transduced with MBP-SYN-IG expression vector only; lower panel) and green fluorescent protein (Gfp) (also when transduced with the MBP-IG control vector; upper panel). Expression of α-synuclein overlapped with GFP expression in MBP-SYN-IG transduced oligodendrocytes. Scale bar: 20μm. c Co-labeling with stage-specific oligodendrocytic markers revealed the specificity of myelin basic protein (Mbp) promoter-driven transgene expression for mature oligodendrocytes (positive for the early maturation marker O4 and Mbp as a marker for terminal maturation), whereas transgene expression was not detectable in platelet-derived growth factor receptor-α (Pdgfrα)-positive oligodendrocyte progenitor cells. Scale bar: 20μm. d Representative images of control (transduced with MBP-IG prior to starting the co-culture) and α-synuclein-overexpressing (transduced with MBP-SYN-IG) Mbp-positive oligodendrocytes co-cultured with beta-III-tubulin (Tuj1)-positive cortical neurons are depicted. Myelination (yellow; arrowheads) was regularly detected under control condition, whereas α-synuclein overexpression impaired myelin formation. Scale bar: 20μm. e Myelination of individual oligodendrocytes (altogether: MBP-IG: 235 cells, MBP-SYN-IG: 215 cells) in the stem cell-derived co-culture was quantified (n = 6) by calculating the ratio of Mbp/Tuj1-positive pixels over total Mbp-positive pixels and is shown as percentage. α-Synuclein-overexpressing oligodendrocytes (gray dots) formed significantly less myelin than controls (black dots). Data are shown as mean ± standard error of mean. T-test: ###p < 0.001.

Fig. 4

Benztropine restores myelin basic protein (Mbp) expression in α-synuclein-overexpressing primary oligodendrocyte progenitor cells (OPCs). a The dose-dependent effect of benztropine on Mbp expression in primary OPCs is illustrated using Western blot. Gapdh signal served as control. b Benztropine significantly increased Mbp gene expression in a range of 0.1-1.0 μM as quantified by real time PCR (n = 3-6). c Immunocytochemistry for Mbp and Olig2 illustrates the effect of 0.5 μM benztropine (BT) on OPC maturation in comparison to vehicle- (veh) treated cells. Scale bar: 20μm. d The restoration of Mbp expression in α-synuclein-overexpressing cells (EF1a-SYN-IG +veh) upon treatment with 0.5 μM benztropine (EF1a-SYN-IG +BT) to control levels (EF1a-IG +veh) is demonstrated by Western blot. Gapdh signal served as control. e Benztropine restored Mbp gene expression in α-synuclein-overexpressing OPCs as quantified by real time PCR (n = 3-6). f α-Synuclein-overexpressing cells exhibited the typical morphology of OPCs (bi-/tripolarity), whereas treatment with benztropine induced Mbp expression and formation of multiple processes as signs of advanced maturity comparable to control condition. Scale bar: 20μm. Data are shown as mean ± standard error of mean. ANOVA: *p < 0.05, ***p < 0.001. T-test: ##p < 0.01.

Fig. 5

Restored myelinogenic capacity of α-synuclein-overexpressing primary rat oligodendrocyte progenitor cells (OPCs) by benztropine. a A Venn diagram depicts differentially regulated genes revealed by RNA sequencing of primary rat OPCs (n = 3). Green circle: α-synuclein-overexpressing (SYN) versus control cells (CTRL). Blue circle: benztropine (BT) versus vehicle (veh)-treated α-synuclein-overexpressing cells. b Genes differentially regulated by both α-synuclein overexpression and benztropine treatment classified into gene ontologies (GOs) implicated among others in myelin formation (black), membranogenesis (dark gray), and maintenance of oligodendrocyte immaturity (light gray). c Expression dynamics of representative genes identified by GO term enrichment are listed. Benztropine (blue) oppositely regulated genes differentially expressed upon α-synuclein overexpression (green). Myelin formation: Mbp = myelin basic protein, Plp1 = proteolipid protein 1, Lpar1 = lysophosphatidic acid receptor 1, Aspa = aspartoacylase, Atrn = attractin, Tspn2 = tetraspanin 2; Membranogenesis: Hmgcr = 3-hydroxy-3-methylglutaryl-CoA reductase, Dhcr24 = 24-dehydrocholesterol reductase, Ank3 = ankyrin 3, Gsn = gelsolin, Ebp = emopamil binding protein (sterol isomerase), Scd1 = stearoyl-CoA desaturase-1; Maintenance of immaturity: Sox9 = sex determining region Y-box 9, Tcf7l1 = transcription factor 7-like 1, Nkx2.2 = Nk2 homeobox 2, Mt3 = metallothionein 3, Hey1 = hes-related family bHLH transcription factor with YRPW motif 1, Cst3 = cystatin C.

Fig. 6

Restored myelination deficit of α-synuclein-overexpressing mouse embryonic stem cell-derived oligodendrocytes by benztropine. a Representative images show that overexpression of α-synuclein (transduced with MBP-SYN-IG prior to starting the co-culture) impaired myelin formation of stem cell-derived oligodendrocytes (stained for myelin basic protein, Mbp) co-cultured with cortical neurons (beta-III-tubulin, Tuj1). Addition of benztropine enhanced myelin formation of α-synuclein-overexpressing cells (comparable to vehicle (veh)-treated control cells, which were transduced with MBP-IG). Mbp-positive pixels co-localizing with Tuj1-positive pixels were considered as myelin (shown in yellow). Scale bar: 20μm. b Myelination of individual oligodendrocytes (in total 90-110 cells per condition) was quantified (n = 3). α-Synuclein-overexpressing oligodendrocytes (gray dots) in the presence of vehicle (veh) formed significantly less myelin than controls (black dots, veh). Benztropine (BT) restored the myelination deficit of α-synuclein overexpressing oligodendrocytes. Data are shown as mean ± standard error of mean. ANOVA: ***p < 0.001. T-test: ##p < 0.01.

Fig. 7

Amelioration of the myelin deficit in mice with oligodendrocytic overexpression of α-synuclein (MBP29) upon benztropine treatment. a Callosal myelin levels of non-transgenic (NTG) and MBP29 mice treated with vehicle (veh) or benztropine (BT, 2mg/kg) were analyzed using magnetic resonance imaging. Upper panel illustrates morphology as determined by T2-weighted magnetic resonance imaging, whereas lower panel depicts T2*-weighted magnetic resonance imaging (shown as color-coded maps), which was used for myelin quantification. b MBP29 mice (n = 3) showed a reduction in myelin levels detected by increased T2*-relaxation times within the corpus callosum. Benztropine attenuated the myelin deficit in MBP29 confirmed by a reduction of T2*. c Representative pictures of a Luxol Fast Blue staining (color-coded for signal intensity: blue low, green intermediate, red high intensity), confirming the effects of α-synuclein overexpression and benztropine treatment on myelination. Scale bar: 500μm. d MBP29 mice (n = 5) exhibited a reduction in Luxol Fast Blue intensity, which was attenuated, but not completely restored by benztropine. Data are shown as mean ± standard error of mean. ANOVA: *p < 0.05, ***p < 0.001. T-test: #p < 0.05.

Fig. 8

Structural restoration of myelin by benztropine in mice overexpressing α-synuclein in oligodendrocytes (MBP29). Representative electron micrographs of the corpus callosum at low (upper panel, scale bar: 1μm) and high (lower panel, scale bar: 0.2μm) magnification depict myelin sheaths of non-transgenic (NTG) and MBP29 mice treated with either vehicle (veh) or benztropine (BT). In vehicle-treated MBP29 mice only, myelin appeared strongly reduced and disorganized. Quantification of myelinated axons and myelin layers per axon revealed a significant myelin deficit in vehicle-treated MBP29 mice, which was restored by benztropine treatment to control level. Data are shown as mean ± standard error of mean. ANOVA (n = 6): **p < 0.01. T-test: ##p < 0.01.

Fig. 9

No alteration of oligodendrocyte density by benztropine. a Oligodendrocytic cells within the corpus callosum were identified using stage specific markers (entire population: Olig2; oligodendrocyte progenitor cells: platelet-derived growth factor receptor-α (Pdgfrα)/Olig2^strong (arrowhead), mature oligodendrocytes: glutathione-s-transferase-π (Gstπ)/Olig2^weak, as well as cells staining for Olig2^strong only (asterisk)). Scale bar: 20μm. b A profound increase of oligodendrocytic cells within the corpus callosum of mice overexpressing α-synuclein in oligodendrocytes (MBP29) compared to non-transgenic controls (NTG) was observed. Scale bar: 20μm. c While MBP29 mice exhibited an almost 2-fold increased density of the entire spectrum of oligodendrocytic subpopulations, benztropine (BT) had no effect on oligodendrocytic cell density as compared to vehicle (veh)-treatment. Data are shown as mean ± standard error of mean. ANOVA (n = 5): **p < 0.01, ***p < 0.001. T-test: not significant (n.s.) p > 0.05.

Fig. 10

Prevention of motor cortical neuronal cell loss in mice overexpressing α-synuclein in oligodendrocytes by benztropine. a Representative images depict NeuN-positive neuronal cells within the motor cortex of non-transgenic controls (NTG) and α-synuclein transgenic mice (MBP29) treated with vehicle (veh) or benztropine (BT). Scale bar: 20μm. b Densities of motor cortical neurons are shown (n = 5). In vehicle-treated MBP29 mice, a significant reduction in neuronal cell density was observed. Benztropine treatment prevented the loss of cortical neurons in MBP29 mice. Data are shown as mean ± standard error of mean. ANOVA: *p < 0.05. T-test: ##p < 0.01.