Neuronal α-synuclein toxicity is the key driver of neurodegeneration in multiple system atrophy

Abstract

Multiple system atrophy (MSA) is a rare, rapidly progressing neurodegenerative disorder often misdiagnosed as Parkinson's disease (PD). Although both conditions share some clinical features, MSA is distinct in its pathological hallmark: oligodendroglial cytoplasmic α-synuclein (α-Syn) inclusions, known as glial cytoplasmic inclusions. These glial cytoplasmic inclusions are pathognomonic for MSA, but they do not lead to significant oligodendroglial cell loss. Instead, MSA is characterized by a substantially greater loss of non-dopaminergic neurons in the nigrostriatal and olivopontocerebellar systems compared with PD. This widespread neuronal degeneration, which is not seen to the same extent in PD, plays a crucial role in the clinical presentation of MSA and is important to consider if PD is to be redefined as a neuronal α-Syn disease. It also raises the question of differences in the potential toxicity of lesions in MSA and the underlying cause of neuronal death in MSA. By combining an N-terminus α-Syn antibody that reveals more α-Syn pathology and super-resolution microscopy, we identified α-Syn fibrils in MSA neurons penetrating the nucleus from the cytoplasm, leading to nuclear destruction and neuronal death. Our data indicate an early invasion of neuronal nuclei by α-Syn pathology in MSA, precipitating rapid nuclear envelope destruction, as observed through significant structural damage, including the loss of Lamin integrity. Although the progression of α-Syn pathology from the cytoplasm to the nucleus might be similar in oligodendroglia and neurons, the aggregation state of the α-Syn proteoforms involved differs because proteolytic resistance of α-Syn inclusions is significantly higher in neurons, and the nucleus is destroyed. We describe the progressive impact of α-Syn nuclear pathology on MSA neurons and show that this is a more detrimental and rapid pathology driving neurodegeneration. Our data suggest that oligodendroglial inclusions contain more soluble, less toxic α-Syn proteoforms, consistent with two distinct α-Syn filaments in MSA. We propose renaming MSA as a neuronal nuclear and oligodendroglial α-synucleinopathy to reflect these two distinct pathologies better.

Keywords: multiple system atrophy; neuronal inclusions; neuronal synucleinopathy; nuclear inclusions; oligodendroglial inclusions; α-synuclein.

Figure 1

Oligodendroglial and neuronal α-Syn inclusions in MSA brains. (A) Oligodendroglia (p25α/TPPP) with dense filamentous α-Syn (N-terminus). (B) Oligodendroglial α-Syn inclusions labelled with N-terminus (yellow), pS129 (magenta) and C-terminus α-Syn (white). (C) Neuron (MAP2) with thread-like α-Syn filaments (N-terminus). (D) Neuronal α-Syn inclusions labelled with N-terminus (yellow), pS129 (magenta) and C-terminus α-Syn (white). α-Syn = α-synuclein; MSA = multiple system atrophy.

Figure 2

Cytoplasmic and nuclear oligodendroglial α-Syn inclusions in MSA brains, with distinct intranuclear filamentous α-Syn penetration. [A(i and ii)] 3D render (i) and corresponding single-channel/single-plane orthogonal representations (ii) of α-Syn filaments penetrating through the nuclear envelope (Lamin, white-arrow/cross-hairs) into the nuclear space. [B(i)] Representative confocal acquisitions of the differential oligodendroglial inclusions. The oligodendrocyte on the left (cyan arrow) contains both p25α/TPPP and pathological α-Syn; the nucleus of this cell is also relatively large, indicating nuclear swelling. The oligodendrocyte in the middle (magenta arrow) has an enlarged nucleus largely devoid of both p25α/TPPP and pathological α-Syn. However, residual p25α/TPPP and α-Syn puncta are evident within the intranuclear space. Neuron (MAP2) added for spatial context. [B(ii)] 3D render of the same inclusions displayed in (i), highlighting the presence of intranuclear p25α/TPPP and α-Syn puncta. [C(i–iv)] 3D renders displaying the top view (i and ii) and bottom view (iii and iv) of intranuclear α-Syn pathology in a oligodendrocyte. The nucleus has been rendered opaque in i and iii and transparent in ii and iv to highlight the intranuclear localization of this α-Syn inclusion. α-Syn = α-synuclein; MSA = multiple system atrophy.

Figure 3

Temporal staging framework for the maturation of oligodendroglial α-Syn inclusions in MSA brains. Representative confocal acquisitions demonstrating p25α/TPPP accumulation (magenta) and maturation of pathological α-Syn inclusions (yellow) within the oligodendroglia. Top row: Initially, no α-Syn pathology is present. However, nuclear and perinuclear p25α/TPPP is evident, having been relocated from the myelin sheath; nuclei are clearly stained (Stage 1). As additional p25α/TPPP is recruited to the perinuclear cytoplasm, pathological α-Syn accumulates within the oligodendroglial cytoplasm (Stage 2), adopting a nuclear-centric disposition (Stage 3), resulting in characteristic nuclear and cytoplasmic swelling (Stage 4). A subset of oligodendrocytes have only cytoplasmic α-Syn (Stage 2). Oligodendroglia with filamentous cytoplasmic α-Syn and distinct nuclear penetrations in an enlarged nucleus are typical as inclusions mature (Stage 5). Bottom two rows: p25α/TPPP eventually leaves the cell; however, the mature pathological filamentous α-Syn inclusions remain. The oligodendroglial nuclear envelope (Lamin) and nucleus remain structurally intact. Scale bar: 5 μm. α-Syn = α-synuclein; MSA = multiple system atrophy.

Figure 4

Cytoplasmic and nuclear neuronal α-Syn inclusions in MSA brains, with distinct intranuclear filamentous α-Syn penetration. [A(i–iii)] Neuronal filamentous cytoplasmic and intranuclear α-Syn penetrations (i), with ii showing a single-plane orthogonal cross-section and iii displaying a 3D render. [B(i and ii)] Neuronal cytoplasmic and filamentous intranuclear inclusions, with (i) and without (ii) Hoechst staining, highlight the filamentous morphology of intranuclear filaments. The cytoplasmic portion of the inclusion contains dense α-Syn filaments that encapsulate the entire nucleus. [C(i and ii)] Neuronal filamentous α-Syn inclusions showing suspected entry points (red arrowheads) where cytoplasmic α-Syn filaments penetrate the nuclear envelope [top view (i) and bottom view (ii)]. α-Syn = α-synuclein; MSA = multiple system atrophy.

Figure 5

Temporal staging framework for the maturation of neuronal α-Syn inclusions in MSA brains. A small subset of neurons have cytoplasmic α-Syn pathology only (Stage 1). Pathological neuronal α-Syn gradually accumulates in the cytoplasm and nucleus. The nuclear envelope and internal nuclear structure become structurally compromised (Stage 2) and diffuse into the cytoplasmic space (Stage 3), eventually disappearing from the cell altogether (Stages 4 and 5). In the final stages of inclusion maturation, pathological α-Syn diffuses throughout the entire cell, resulting in comprised cell membrane integrity and ultimate cell death (Stages 5 and 6). Residual porous extracellular α-Syn inclusions are commonly observed within the extracellular space (Stage 6). Scale bar: 5 μm. α-Syn = α-synuclein; MSA = multiple system atrophy.

Figure 6

In situ proteolytic digestion of oligodendroglial and neuronal inclusions in MSA brains. (A) A high neuronal inclusion proportion relative to oligodendroglial inclusions in the inferior olivary nucleus (left) and a high oligodendroglial inclusion proportion relative to neuronal inclusions in the medial reticular formation (right). Scale bar: 100 μm. (B) Oligodendroglial and neuronal inclusions before (top row, No PK) and after (bottom row, PK 37°C 45 min) in situ proteolytic digestion with PK, acquired with identical imaging parameters and 3D Cartesian coordinates. Scale bar: 5 μm. (C) Oligodendroglial α-Syn inclusions were largely digested after PK treatment, whereas neuronal inclusions remained relatively intact (P < 0.0001; n = 10). All quantifications are normalized to the pretreatment (No PK) immunolabelling values. Data are presented as the mean ± standard deviation. PK = proteinase K.

Figure 7

Proposed temporal staging for the maturation of neuronal and oligodendroglial α-Syn inclusions in multiple system atrophy. α-Syn = α-synuclein.