Identification and quantification of oligodendrocyte precursor cells in multiple system atrophy, progressive supranuclear palsy and Parkinson's disease

Abstract

Multiple system atrophy is a neurodegenerative disorder characterized pathologically by abnormal accumulations of α-synuclein in the cytoplasm of oligodendrocytes, which are termed glial cytoplasmic inclusions (GCIs). Oligodendrocytes are responsible for myelinating axons and providing neurotrophic support, but in MSA, myelin loss, axonal loss and gliosis are consistent features suggesting that GCIs play a central role in disease pathogenesis. Oligodendroglial, myelin and axonal degeneration are also features of multiple sclerosis (MS) in which recent studies have highlighted the robust remyelination capacity of the central nervous system (CNS). The cells responsible for remyelination are called oligodendroglial precursor cells (OPCs). In this study, we investigated the role of OPCs in the pathogenesis of MSA and progressive supranuclear palsy (PSP), a neurodegenerative disease in which neuropathological changes include oligodendroglial inclusions composed of microtubule-associated protein tau. Despite the lability of OPC-specific antigens, we successfully identified OPCs and demonstrated that tau and α-synuclein do not accumulate in OPCs. We also showed that the density of OPCs was increased in a white matter region of the MSA brain, which is also severely affected by GCIs and myelin degeneration. These findings raise the possibility that OPCs could be available to repair disease-associated damage in MSA, consistent with their biological function.

Figure 1

Identification and validation of NG2‐positive oligodendroglial precursor cells (OPCs).  NG2‐specific immunohistochemistry using flash‐frozen post‐mortem human tissue (A) identified cells with a stellate morphology and multiple long delicate processes (B and C) suggestive of OPCs; blood vessel staining (A, arrow head) was also evident. Double immunofluorescence staining using NG2 (green) and cell‐specific markers (red), such as Olig2 (D), glial fibrillary acidic protein (GFAP, E) and ionized calcium binding adaptor 1 (Iba‐1, F), demonstrated strong expression of nuclear Olig2 in NG2‐positive cells (D, arrow), whereas astrocytes (E) and microglia (F) were NG2‐negative. Arrow heads show NG2‐positive blood vessel staining. Immunofluorescence images represent transparent projections of confocal Z‐stacks and nuclei are labeled with DAPI (blue). Supporting Information Figure S2 shows individual fluorescent channels. Scale bars: 60 μm (A), 30 μm (B,C), 25 μm (D–F).

Figure 2

Oligodendroglial precursor cells (OPCs) do not contain inclusions in multiple system atrophy (MSA) and progressive supranuclear palsy (PSP). Double immunofluorescence labeling using NG2 (green) and disease‐associated markers α‐synuclein and tau (red) demonstrated that glial cytoplasmic inclusions in MSA (A, arrows heads) and that coiled bodies (B, arrows heads) and tufted astrocytes (C, arrow head) in PSP are NG2‐negative. Furthermore, NG2‐positive OPCs (arrows) do not accumulate α‐synuclein in MSA (A) or tau in PSP (C). NG2‐positive blood vessels (B, arrow) were also observed. Nuclei are labeled with DAPI (blue) and yellow co‐localization (A) represents autoflourescent lipofuscin pigment. These images represent transparent projections of confocal Z‐stacks. Brain regions = putamen (A), cerebellar white matter (B) and caudate (C). Scale bars = 20 μm.

Figure 3

Quantification of NG2 cells and other disease‐associated pathological variables in the cerebellar and frontal white matters of multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and control cases. Horizontal lines represent mean values and dotted lines represent median values. Kruskal–Wallis rank sum tests were used for statistical testing. Oligo = oligodendroglial, GFAP = glial fibrillary acidic protein; Iba‐1 = ionized calcium binding adaptor 1; MBP = myelin basic protein.

Figure 4

Myelin basic protein (MBP) specific immunohistochemistry. A–C represent the white matter of the cerebellum and D–F the frontal lobe regions of a representative multiple system atrophy (MSA, A and D), progressive supranuclear palsy (PSP, B and E) and control (C and F) case. The cerebellar white matter of MSA cases was characterized by moderate to severe patchiness in MBP staining (A), which was less prominent in the frontal white matter (D). The cerebellar and frontal white matters of PSP cases (B and E) and control cases (C and F) were normal in comparison. Scale bar = 50 μm.