Introduction
Multiple system atrophy (MSA) is an adult-onset neurodegenerative disorder manifested clinically as a combination of parkinsonism, cerebellar ataxia and autonomic dysfunction. MSA is now divided into two clinical subtypes: MSA with predominant parkinsonian features (MSA-P) and MSA with predominant cerebellar dysfunction (MSA-C) [
1]. MSA is characterized pathologically by any combination of coexisting olivopontocerebellar atrophy, striatonigral degeneration and preganglionic autonomic lesions [
2]. The histological hallmark of MSA is widespread glial cytoplasmic inclusions (GCIs) in the central nervous system [
3‐
6]. These GCIs can be visualized by silver staining such as the Gallyas-Braak method [
3], and ultrastructurally they consist of granule-associated filaments 20–30 nm in diameter [
3,
4,
7]. The major component of GCIs is α-synuclein [
8], which is phosphorylated at Serine 129 [
9] and ubiquitinated [
10]. Although primary oligodendroglial pathology is the main feature of MSA [
11‐
13], accumulation of phosphorylated α-synuclein is also consistently found in the neuronal cytoplasm, processes and nuclei [
14]. Similar neuronal inclusions are found less frequently in the peripheral sympathetic ganglia [
13,
15].
Although immunoreactivity of non-phosphorylated α-synuclein has been reported in normal and neoplastic Schwann cells in the peripheral nervous system of humans [
16], accumulation of phosphorylated α-synuclein in Schwann cells of patients with MSA has not been described hitherto. Here we immunohistochemically examined the cranial and spinal nerves, peripheral ganglia and visceral autonomic nervous system of patients with MSA using antibodies against phosphorylated α-synuclein, and report for the first time that Schwann cells in these patients are also affected by filamentous aggregations of phosphorylated α-synuclein.
Discussion
In the present study, we have demonstrated for the first time that phosphorylated α-synuclein accumulates in the cytoplasm of Schwann cells in patients with MSA. These SCCIs were also immunopositive for aggregated α-synuclein, ubiquitin and p62, a ubiquitin- proteasome system-related protein. Thus, the immunohistochemical profile of SCCIs is similar to that of GCIs [
3,
7,
9,
19,
25]. Ultrastructurally, SCCIs were composed of randomly arranged, loosely packed, granule-coated fibrils, approximately 15–20 nm in diameter. Both GCIs and neuronal cytoplasmic inclusions also consisted of granule-coated fibrils, approximately 20–30 nm in diameter [
3,
4,
7,
26‐
28]. These findings indicate that Schwann cells are also involved in the disease process of MSA.
SCCIs were found in 12 of 14 patients with MSA (85.7 %) in the present study. GCIs were consistently found in the brainstem and spinal cord in all of the MSA patients. By contrast, SCCIs were not observed in the cranial or spinal nerves in three patients (cases 2, 12 and 14), two of whom had early MSA [
17,
18]. These findings suggest that the occurrence of GCIs precedes that of SCCIs in MSA.
Recently, expression of human α-synuclein has been reported in Schwann cells ensheathing the nerve fibers of the urinary bladder in a transgenic mouse model of MSA showing oligodendroglial overexpression of human α-synuclein under the control of the proteolipid protein promoter [
29]. Urodynamic analysis revealed a less efficient and unstable urinary bladder in this MSA mouse model. In human MSA, widespread occurrence of GCIs in the central nervous system is a cardinal pathological feature [
3‐
6]. Moreover, neuronal cytoplasmic and nuclear inclusions have been observed in the inferior olivary and pontine nuclei, substantia nigra, putamen and cerebral cortex in patients with MSA [
14,
28]. Filamentous aggregates of α-synuclein are also found in neurons in the sympathetic ganglia [
14,
15]. In the present study, we further demonstrated that accumulation of phosphorylated α-synuclein occurs in the neuronal cytoplasm in the dorsal root ganglia. Sural nerve biopsy from patients with MSA shows a 23 % reduction of unmyelinated fibers (sensory afferent fibers and postganglionic sympathetic fibers) [
30]. Mild degeneration of cardiac sympathetic nerves can occur in MSA [
31]. Thus, MSA is a glio-neuronal α-synucleinopathy involving the central and peripheral nervous systems.
It is noteworthy that SCCIs tend to be more frequent in the peripheral nerves associated with autonomic function, i.e. glossopharyngeal-vagus nerves, and anterior spinal nerves of the thoracic and sacral cord. The vagus nerve is a mixed cranial nerve containing axons of branchiomeric motor neurons, parasympathetic preganglionic fibers, visceral afferent fibers, and somatic sensory afferent fibers. The glossopharyngeal nerve is related closely to the vagus nerve, sharing common medullary nuclei and having similar functional components [
32]. The sympathetic ganglia receive preganglionic fibers from the intermediolateral nucleus of the spinal cord through the anterior roots of all the thoracic and the upper two lumber nerves [
32]. The sacral preganglionic parasympathetic fibers exit from the sacral cord and go to the terminal ganglia of the pelvic plexuses, as well as to the myenteric and submucosal plexuses of the descending colon and rectum [
32]. The widespread occurrence of SCCIs, at least in part, may play a role for the manifestation of a variety of autonomic symptoms in MSA.
Using the modified Gallyas-Braak method, GCIs were positive whereas SCCIs were stained only weakly or partially. Ultrastructurally, the constituent filaments of SCCIs (approximately 15–20 nm) appeared thinner than those of GCIs (approximately 20–30 nm) [
3,
4,
7]. Phosphorylated α-synuclein-immunoreactive filamentous inclusions are also found in oligodendrocytes and astrocytes in the brains of patients with Parkinson’s disease and dementia with Lewy bodies [
33‐
35] and are argyrophilic with the modified Gallyas-Braak method [
36], suggesting that the process of α-synuclein aggregation in glial cells may differ somewhat between the central and peripheral nervous systems.
Cranial nerves are composed of myelinated and unmyelinated fibers in various proportions [
37]. The nerve fibers of the anterior spinal nerve roots projecting to the autonomic ganglia are myelinated [
38]. Both myelinated and unmyelinated fibers in the peripheral nervous system are enveloped with Schwann cells. Although the number of samples was small, our immunoelectron microscopy examination demonstrated that inclusion-bearing Schwann cells, at least in part, ensheath the myelinated fibers. Considering that postganglionic sympathetic nerve fibers are unmyelinated [
39] and a small number of SCCIs were observed in the visceral autonomic nervous system in MSA, SCCI formation may also occur in Schwann cells ensheathing the unmyelinated fibers. Moreover, immunodeposition was also found in the outer and inner loops of Schwann cells. In the central nervous system, constituent filaments of GCIs are not evident in the outer or inner loops of oligodendrocytes in MSA [
7]. By contrast, tau- and Gallyas-positive filamentous structures are found in the outer and inner loops of oligodendrocytes in progressive supranuclear palsy and corticobasal degeneration [
39‐
41]. These findings suggest that phosphorylated α-synuclein pathology develops both in the perikarya and distal processes of Schwann cells, whereas the perikarya is chiefly involved in oligodendrocytes in MSA.
It is unclear how aggregated α-synuclein in the cytoplasm of Schwann cells interacts with the axon, myelin and Schwann cell itself. Both oligodendrocytes and Schwann cells are essential for axonal function and integrity. These enwrapping glia support axonal growth and myelination by transfer of metabolic substrates and secretion of neurotrophic factors [
42]. Glial cell line-derived neurotrophic factor (GDNF) is one of the neurotrophic factors produced by oligodendrocytes [
43] and Schwann cells [
44]. The level of GDNF is significantly decreased in the frontal white matter and cerebellum of human MSA patients and in the brain of a MSA mouse model overexpressing human α-synuclein under the control of the myelin basic protein promoter [
45]. Intraventricular infusion of GDNF improves behavioral deficits and ameliorates the neurodegenerative pathology in this MSA mouse model [
45]. GDNF induces Schwann cell migration and axonal regeneration in the peripheral nervous system [
46] and also prevents atrophy of facial motoneurons following axotomy [
47]. Liver kinase B1 (LKB1) is also a crucial regulator of the major metabolic pathway in Schwann cells, which are central to axonal stability [
48]. Deletion of LKB1 leads to energy depletion, mitochondrial dysfunction, abnormalities of lipid homeostasis and increased lactate release in Schwann cells [
48]. The loss of viability in human neuroblastoma cells overexpressing wild-type α-synuclein is associated with reduced activation of intracellular energy sensors, including LKB1 [
49]. α-Synuclein-overexpressing rat primary neurons also display lower LKB1 activity [
49]. Based on the above findings, it is likely that overexpression of α-synuclein in Schwann cells impairs the activity of neurotrophic factors, leading to axonal destabilization in peripheral nerves.
The origin of α-synuclein in SCCIs is uncertain. Immunoreactivity of non-phosphorylated α-synuclein has been reported in normal and neoplastic Schwann cells in the peripheral nervous system of humans [
16]. Therefore, it is possible to consider that overexpression of α-synuclein in Schwann cells would cause SCCI formation. As another possible mechanism, neuron-to-neuron transmission of α-synuclein fibrils through anterograde axonal transport has been demonstrated in primary cortical mouse neurons in vitro [
50]. The fact that SCCIs tended to appear more frequently in the proximal than in the distal spinal nerve roots is appropriate for anterograde transport of α-synuclein. α-Synuclein in SCCIs could be derived from neurons. Future studies will be necessary to clarify the origin of α-synuclein in MSA Schwann cells.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KN, FM and KW designed the study and analyzed data. KN, FM, KT, HK, YT, AK, HT and KW performed the pathological observations and evaluations. TK, YM and MT performed the clinical evaluations. FM, MY and KW supervised the whole process of the study. KN, FM and KW wrote the manuscript. All authors read and approved the final manuscript.