Cross-examining candidate genes implicated in multiple system atrophy

Multiple system atrophy (MSA) is a predominantly sporadic, adult-onset, fatal neurodegenerative disease of unknown etiology. MSA is characterized by autonomic failure, levodopa-unresponsive parkinsonism, cerebellar ataxia and pyramidal signs in any combination. MSA belongs to a group of neurodegenerative disorders termed α-synucleinopathies, which also include Parkinson's disease and dementia with Lewy bodies. Their common pathological feature is the occurrence of abnormal α-synuclein positive inclusions in neurons or glial cells. In MSA, the main cell type presenting aggregates composed of α-synuclein are oligodendroglial cells . This pathological hallmark, also called glial cytoplasmic inclusions (GCIs) , is associated with progressive and profound neuronal loss in various regions of the brain. The development of animal models of MSA is justified by the limited understanding of the mechanisms of neurodegeneration and GCIs formation, which is paralleled by a lack of therapeutic strategies. Two main types of rodent models have been generated to replicate different features of MSA neuropathology. On one hand, neurotoxin-based models have been produced to reproduce neuronal loss in substantia nigra pars compacta and striatum. On the other hand, transgenic mouse models with overexpression of α-synuclein in oligodendroglia have been used to reproduce GCIs-related pathology. This chapter gives an overview of the atypical Parkinson's syndrome MSA and summarizes the currently available MSA animal models and their relevance for pre-clinical testing of disease-modifying therapies.

Chapter 19 - Etiology, Pathology, and Pathogenesis

Certain genetic defects in LRRK2 and parkin are pathogenic for Parkinson's disease (PD) and both proteins deposit in the characteristic Lewy bodies. LRRK2 is thought to be involved in the early initiation of Lewy bodies. The involvement of LRRK2 and parkin in the similar cellular deposition of fibrillar alpha-synuclein in glial cytoplasmic inclusions (GCI) in multiple system atrophy (MSA) has not yet been assessed. To determine whether LRRK2 and parkin may be similarly associated with the abnormal deposition of alpha-synuclein in MSA GCI, paraffin-embedded sections from the basal ganglia of 12 patients with MSA, 4 with PD and 4 controls were immunostained for LRRK2, parkin, alpha-synuclein and oligodendroglial proteins using triple labelling procedures. The severity of neuronal loss was graded and the proportion of abnormally enlarged oligodendroglia containing different combinations of proteins assessed in 80-100 cells per case. Parkin immunoreactivity was observed in only a small proportion of GCI. In contrast, LRRK2 was found in most of the enlarged oligodendroglia in MSA and colocalised with the majority of alpha-synuclein-immunopositive GCI. Degrading myelin sheaths containing LRRK2-immunoreactivity were also observed, showing an association with one of the earliest oligodendroglial abnormalities observed in MSA. The proportion of LRRK2-immunopositive GCI was negatively associated with an increase in neuronal loss and alpha-synuclein-immunopositive dystrophic axons. Our results indicate that an increase in LRRK2 expression occurs early in association with myelin degradation and GCI formation, and that a reduction in LRRK2 expression in oligodendroglia is associated with increased neuronal loss in MSA.

Multiple system atrophy is a sporadic, adult-onset neurodegenerative disease of unknown etiology. The condition may be unique among neurodegenerative diseases by the prominent, if not primary, role played by the oligodendroglial cell in the pathogenetic process. Recent developments in our understanding of multiple system atrophy have included the detection of glial cytoplasmic inclusions and alpha-synuclein accumulation in these inclusions. The latter finding links multiple system atrophy as an "alpha-synucleinopathy" to Parkinson's disease and dementia with Lewy bodies. This article reviews recent important findings of potential relevance to the pathogenesis of multiple system atrophy. We also speculate on areas in which further advances may be made to progress our understanding of this devastating condition.

Cardiac sympathetic denervation without Lewy bodies in a case of multiple system atrophy.

Serum Hcy was higher in MSA patients when compared to healthy subjects, particularly in male patients.Serum UA was lower in MSA patients when compared healthy subjects, particularly in male patients.Serum Hcy levels were significantly positively correlated with the severity of MSA.The ROC curve for the combination of Hcy and UA showed potential diagnostic value in discriminating MSA from healthy subjects. There is evidence suggesting that inflammatory responses play a critical role in the pathogenesis of multiple system atrophy (MSA). Whether inflammatory mediators can be used as reliable biomarkers to detect the severity and progression of MSA remains largely unknown. We performed a cross-sectional study that included 47 patients with MSA and 50 healthy age-matched controls. Serum levels of homocysteine (Hcy), uric acid (UA), and C-reactive protein (CRP) were measured. These levels positively correlated with the severity of MSA, based on both motor and non-motor symptoms. Several scales were used to rate the severity of MSA, including the Unified multiple system atrophy rating scale, Parkinson's disease sleep scale, Non-motor Symptoms Scale, the Schwab & England activities of daily living scale, Webster Scale, modified Hoehn and Yahr staging scale, and the Mini-Mental State Examination. Receiver operating characteristic (ROC) curves was applied to map the diagnostic accuracy of MSA against healthy subjects. Compared with healthy subjects, we found that serum Hcy was higher, UA was lower, and CRP levels were unchanged in MSA patients. These findings were especially prominent in male patients. No significant differences of serum Hcy and UA were observed between patients of MSA and PD. Interestingly, there was a significant correlation between Hcy levels and MSA severity such as movement dysfunction, declined cognition, and cardiovascular symptoms. Additionally, the ROC curve for the combination of Hcy and UA (AUC 0.736) showed potential diagnostic value in discriminating MSA from healthy subjects. Our findings suggest that the inflammatory mediators Hcy and UA may play important roles in the pathogenesis of MSA. The measurement of serum Hcy and UA levels could then be a useful tool to accurately distinguish MSA from healthy subjects.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease clinically marked by autonomic failure and variable degrees of parkinsonism and cerebellar ataxias. The average age of onset is in the sixth decade of life with a five-year survival, although fulminant and more benign courses have been described (1). The pathologic hallmark of MSA is abundant α-synuclein-positive protein aggregates within oligodendrocytes (glial cytoplasmic inclusions, GCI) and neurons (neuronal cytoplasmic inclusions, NCI, and neuronal nuclear inclusions, NNI). The density of GCIs has been correlated with disease duration and severity of neuronal loss (2). Here, we present an unusual case of MSA clinically characterized by rapid decline followed by a 20-year disease duration in a severely affected state. At autopsy, the brain showed severe gliosis and neuronal loss in a pattern characteristic of MSA. Additionally, there were atypical Pick body-like and ring-shaped α-synuclein inclusions, most prominent in limbic regions, consistent with a recently described subtype of MSA designated "atypical MSA" (3). Four different α-synuclein antibodies highlighted only scant α-synuclein-positive pathology; however, Gallyas and Campbell-Switzer silver stains identified additional GCIs most prominent in subcortical white matter. Interestingly, many of the α-synuclein-positive limbic inclusions were Gallyas-negative, while many of the Gallyas-positive inclusions were α-synuclein-negative (4). This observation of markedly disparate α-synuclein immunohistochemical versus Gallyas reactivity of MSA inclusions is unusual and could support the possibility that MSA inclusions have variable compositions, something which has been recently suggested (5).

Proteinaceous α-synuclein-containing inclusions are found in affected brain regions in patients with Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These appear in neurons as Lewy bodies in both PD and DLB and as glial cytoplasmic inclusions (GCIs) in oligodendrocytes in MSA. The role they play in the pathology of the diseases is unknown, and relatively little is still known about their composition. By purifying the inclusions from the surrounding tissue and comprehensively analysing their protein composition, vital clues to the formation mechanism and role in the disease process may be found. In this study, Lewy bodies were purified from postmortem brain tissue from DLB cases (n = 2) and GCIs were purified from MSA cases (n = 5) using a recently improved purification method, and the purified inclusions were analysed by mass spectrometry. Twenty-one percent of the proteins found consistently in the GCIs and LBs were synaptic-vesicle related. Identified proteins included those associated with exosomes (CD9), clathrin-mediated endocytosis (clathrin, AP-2 complex, dynamin), retrograde transport (dynein, dynactin, spectrin) and synaptic vesicle fusion (synaptosomal-associated protein 25, vesicle-associated membrane protein 2, syntaxin-1). This suggests that the misfolded or excess α-synuclein may be targeted to inclusions via vesicle-mediated transport, which also explains the presence of the neuronal protein α-synuclein within GCIs.

Synucleinopathies are neurodegenerative diseases pathologically characterized by aggregates of α-synuclein protein in selectively vulnerable neurons and/or glia.1 These include neuronal Lewy bodies (LB) and Lewy neurites in Parkinson's disease (PD), dementia with LB, and PD dementia, and predominantly oligodendroglial cytoplasmic inclusions in multiple system atrophy (MSA).1 Isolated rapid eye movement sleep behavior disorder (iRBD) and pure autonomic failure (PAF) are often underpinned by α-synuclein pathology, sometimes revealing themselves as prodromal stages of the aforementioned disorders in retrospect.2, 3 The self-propagation of misfolded proteins assembling into seeds that induce normal-like proteins to misshape and aggregate was first recognized as mechanism of neurotoxicity in prion diseases. Increasing evidence supports the prion-like behavior of other proteins in neurodegeneration, including amyloid-β, tau, and α-synuclein.4 Therefore, detecting protein aggregates in easily accessible biospecimens represents a promising diagnostic avenue,5 especially in synucleinopathies, which can be difficult to clinically differentiate in early stages and lack specific positron emission tomography radiotracers. Moreover, because the prion-like process may precede their clinical onset by years or decades, iRBD and PAF are potential targets for an early biochemical diagnosis of synucleinopathies.2, 3, 5 Real-time quaking-induced conversion (RT-QuIC) is a high-sensitive amplification assay to detect the seeding activity of protein aggregates in biospecimens at the expense of a substrate (ie, the recombinant protein) under shaking conditions, which is monitored by thioflavin T fluorescence. RT-QuIC was first validated in sporadic Creutzfeldt-Jacob disease and is now included in its diagnostic criteria.6 Recently, it has also been explored in synucleinopathies.7 Following an established protocol,8 Rossi and colleagues9 performed cerebrospinal fluid α-synuclein RT-QuIC in a large cohort of (1) neuropathologically verified cases of LB-positive and LB-negative dementia, atypical parkinsonism, and LB-negative controls and (2) clinically diagnosed cases of PD, atypical parkinsonism, dementia, iRBD, and PAF. The assay accurately detected α-synuclein seeding activity across LB-related synucleinopathies (ie, PD, dementia with LB, iRBD, and PAF), with an overall sensitivity of 95.3% (100% in definite dementia with LB and iRBD) and an overall specificity of 98% against cases lacking LB on neuropathology.9 Intriguingly, negative RT-QuIC results were found in almost all MSA cases, 1 iRBD case with subsequent phenoconversion to MSA, and 1 PAF case showing normal adrenergic cardiac innervation on iodine-123 meta-iodobenzylguanidine single photon emission computed tomography, as observed in MSA.9 Finally, quantification of α-synuclein seeding activity through fluorescence analysis did not discriminate among LB-related clinical syndromes.9 Besides supporting the prion-like nature of LB-related synucleinopathies, this study demonstrates that α-synuclein RT-QuIC provides an accurate and reliable biomarker for these disorders in the largest cohort hitherto investigated.9 It also proves that RT-QuIC detects α-synuclein seeding activity in prodromal syndromes, allowing early diagnosis of synucleinopathy and identification of candidates for disease-modifying trials.9 Interpretation of negative assay results in most MSA cases is arguable. Although it might confirm that MSA and LB-related syndromes are associated with different conformational α-synuclein strains,10 further research needs to clarify whether different RT-QuIC set-ups may detect α-synuclein seeding activity in MSA, thus differentiating synucleinopathies. In conclusion, these findings endorse the inclusion of an RT-QuIC-based biomarker in diagnostic criteria for LB-related synucleinopathies and promote studies to test the α-synuclein RT-QuIC analysis of other biospecimens and its potential as a progression/prognostic tool. (1) Research Project: A. Conception, B. Organization, C. Execution; (2) Data Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript Preparation: A. Writing of the First Draft, B. Review and Critique. F.M.: 1A, 2A, 2B, 3A M.T.: 2C, 3B K.P.B.: 1A, 2C, 3B We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. The authors confirm that the approval of an institutional review board was not required for this work. The authors confirm that no patient consent was required for this work. No specific funding was received for this work. The authors declare that there are no conflicts of interest relevant to this work. F.M. is supported by the European Academy of Neurology Research Fellowship 2020. M.T. reports no disclosures. K.P.B. has received grant support from Welcome/Medical Research Council, National Institute for Health Research, Parkinsons's UK, and EU Horizon 2020. He receives royalties from publication of the Oxford Specialist Handbook Parkinson's Disease and Other Movement Disorders (Oxford University Press, 2008), Marsden's Book of Movement Disorders (Oxford University Press, 2012), and Case Studies in Movement Disorders–Common and Uncommon Presentations (Cambridge University Press, 2017). He has received honoraria/personal compensation for participating as consultant/scientific board member from Ipsen, Allergan, and Merz and honoraria for speaking at meetings and from Allergan, Ipsen, Merz, Sun Pharma, Teva, UCB Pharmaceuticals, and the American Academy of Neurology, and the International Parkinson's Disease and Movement Disorders Society.

Multiple system atrophy (MSA) is a neurodegenerative disorder characterized by parkinsonism, cerebellar dysfunction, and autonomic failure. Key pathological features of MSA include the formation of glial cytoplasmic inclusions (GCIs) in oligodendrocytes (OLs), myelin loss, and neuroinflammation. Although both inflammation and myelination are known to be critical in MSA, the roles of myelin proteins and their relationship with inflammation have often been overlooked. In this study, we injected AAV-Olig001 vectors carrying either human SNCA (AAV-hSNCA) or eGFP (AAV-eGFP) into the striatum of TgM83 transgenic mice, which express the A53T mutant form of human alpha-synuclein (αSyn), as well as into wild-type (WT) mice. We then assessed myelin protein expression and inflammatory responses. TgM83 mice injected with AAV-hSNCA exhibited demyelination, increased activation of microglia and astrocytes, and altered cytokine and chemokine profiles (including IL-1α, IL-10, IL-12(p40), CCL2, CCL3, CCL4, and CCL5), compared to both WT mice and TgM83 mice injected with AAV-eGFP. Interestingly, myelin basic protein (MBP) levels were significantly elevated around the injection site in TgM83 mice injected with AAV-hSNCA. Notably, we observed a positive correlation between MBP expression and inflammatory markers, as indicated by Iba1 and GFAP staining. These findings suggest that hSNCA overexpression is associated with increased MBP levels and enhanced inflammatory responses, implicating that MBP and myelination processes may play previously underappreciated roles in the pathogenesis of MSA.

Multiple system atrophy (MSA) is a disorder that may manifest with reduced respiratory chemosensitivity and central sleep apnoea. Chemosensitive glutamatergic and serotonergic neurons located just beneath the ventral medullary surface, corresponding to the human arcuate nucleus (ArcN), have recently been implicated in control of automatic breathing in response to hypercapnia and hypoxia. We sought to determine whether these neurons were affected in MSA. Medullae were obtained at post-mortem from 11 patients (8 men, 3 women, age 64 +/- 3 years) with neuropathologically confirmed MSA and 11 control subjects (6 men and 5 women, age 66 +/- 4 years). Fifty micrometre sections obtained throughout the medulla were processed for vesicular glutamate transporter-2 (VGLUT-2), tryptophan-hydroxylase (TrOH), glial fibrillary acid protein (GFAP) and alpha-synuclein immunoreactivity. Cell counts, GFAP immunoreactivity and presence of glial cytoplasmic inclusions (GCIs) were assessed in the ArcN. In MSA, compared with controls, there was a marked depletion of ArcN neurons immunoreactive for either VGLUT-2 (74 +/- 21 versus 342 +/- 84 cells/section, P < 0.004) or TrOH (5 +/- 1 versus 16 +/- 2 cells/section, P < 0.001). There was also marked astrocytic gliosis and accumulation of alpha-synuclein immunoreactive GCIs in the ventral medullary surface in all cases. Our results indicate that there is severe loss of putative chemosensitive glutamatergic and serotonergic neurons as well as marked astrocytic gliosis in the ventral medullary surface in MSA. This may provide a possible morphological basis for impaired respiratory chemosensitivity and central sleep apnoea in this disorder.

A variety of neurodegenerative disorders are classified as synucleinopathies based on the presence of prominent α-synuclein pathology. These diseases include Parkinson disease (PD), dementia with Lewy bodies (with neuronal Lewy body formation) and multiple system atrophy (with glial cytoplasmic inclusions). The normal function of α-synuclein includes regulation of presynaptic vesicles. Autosomal dominant PD can be due to coding mutations or multiplications of the α-synuclein gene ( SNCA ). The coding mutations are thought to lead to a gain of function, in particular acceleration of the formation of protofibrils. Duplications and triplications of SNCA lead to autosomal dominant PD with a gene dosage effect on age of onset and clinical severity. Variants in the SNCA promoter, which lead to an upregulation of SNCA expression, are associated with an increased risk of sporadic PD. α-Synuclein is now recognised as the key component of Lewy bodies and as an important target for therapeutic intervention. A number of α-synuclein antibody therapy treatments are in development and have entered early-phase clinical development. Key Concepts α-Synuclein is deposited in the common neurodegenerative conditions, Parkinson disease (PD) and dementia with Lewy bodies, as neuronal cytoplasmic inclusions (Lewy bodies). The normal function of α-synuclein is incompletely understood but is likely to involve interaction with and regulation of synaptic vesicles. There is some evidence that α-synuclein may have a role as a cellular chaperone and in interacting with the proteasome. Mendelian coding mutations in the α-synuclein gene ( SNCA ) can lead to autosomal dominant PD and dementia with Lewy bodies (DLB). SNCA mutations lead to an enhancement of protofibril formation as well as affecting normal α-synuclein function. SNCA duplications and triplications lead to autosomal dominant PD: an increase in the transcription of normal sequence SNCA can lead to disease. Promoter variation at the SNCA is associated with PD; in vitro evidence suggests that protective promoter alleles lead to a downregulation of SNCA expression. There is now substantial evidence for cell-to-cell 'prion-like' spread of synuclein pathology in the brain. This spreading pathology represents a therapeutic target, and a number of anti-α-synuclein antibody therapies are in early-stage clinical trial development.

The early pathogenesis of Parkinson's disease (PD) and the atypical parkinsonian disorders multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) is poorly understood, but presynaptic and axonal dysfunction are hypothesized to play a prominent role. To identify synapse- and/or axonal dysfunction as indicated by cerebrospinal fluid (CSF) biomarker profiles and their clinical correlates in early-stage PD, MSA, and PSP. Liquid chromatography mass spectrometry and enzyme-linked immunosorbent assay analyses of CSF samples from patients with early-stage PD (n=38), MSA (n=21), or PSP (n=19), and age-matched, neurologically healthy controls (n=30). Compared to controls, patients with early parkinsonian disorders had significantly reduced CSF levels of the synapse-associated proteins neuronal pentraxin-1 (NPTX1), amyloid precursor protein, and β-amyloid 42 (Aβ42), as well as the neurosecretory granin-derived proteins secretogranin-II, chromogranin-A, and secretogranin-VII. Among these, synapse-associated proteins correlated with non-motor features, while none correlated with age. CSF levels of the predominantly axonal proteins neurofilament light (NfL) and tau were elevated in patients with MSA or PSP. Reduced NPTX1 and Aβ42 distinguished PD from PSP, while elevated NfL and tau distinguished PSP and/or MSA from PD. Low CSF levels of biomarkers associated with synaptic and neurosecretory function (e.g., NPTX1) implicate age-independent synaptic dysfunction as a shared, early feature in the pathogenesis of PD, MSA, and PSP. Such biomarkers may be particularly sensitive correlates of early non-motor dysfunction. Early axonal dysfunction is more pronounced in PSP and MSA than in PD.

Parkinson’s Disease (PD) is generally considered a movement disorder characterized by loss of dopaminergic neurons in the central nervous system (CNS), most notably in the substantia nigra. The pathogenesis of the neuronal degeneration is not completely understood but is characterized by abnormal protein deposits forming characteristic neuronal inclusions, which are visible by light microscopy of autopsy samples, and termed Lewy bodies. α-Synuclein is one of the proteins found in these inclusions and has attracted interest because mutations of the gene encoding for this protein are found in familiar cases of PD. Most of the disability associated with PD is indeed the result of this CNS defect, and the treatment is aimed at increasing dopamine levels in the CNS, while avoiding as much as possible an increase in the periphery. Less widely recognized is the fact that orthostatic hypotension can occur in PD patients and can become symptomatic and disabling in a significant proportion of patients. The incidence of documented orthostatic hypotension in PD, in community-based studies, ranges from 10% to 40%. Orthostatic hypotension in PD is commonly attributed to the known adverse effects of dopaminergic drugs. However, over the last decade there is growing evidence that peripheral nerves, particularly autonomic neurons, are also involved in PD. Lewy body inclusions, containing α-synuclein, have been found in peripheral autonomic neurons in autopsy cases of otherwise classic PD.1 Microscopically, eosinophilic ubiquitin- and α-synuclein–positive Lewy Bodies and Lewy neurites are not only found in the substantia nigra and locus ceruleus but also are widespread in ganglions cells …

Recently, alpha-synuclein was shown to be a structural component of the filaments in Lewy bodies (LBs) of Parkinson's disease (PD), dementia with LBs (DLB) as well as the LB variant of Alzheimer's disease, and this suggests that alpha-synuclein could play a mechanistic role in the pathogenesis of these disorders. To determine whether alpha-synuclein is a building block of inclusions in other neurodegenerative movement disorders, we examined brains from patients with multiple system atrophy (MSA) and detected alpha-synuclein, but not beta- or gamma-synuclein, in glial cytoplasmic inclusions (GCIs) throughout the MSA brain. In MSA white matter, alpha-synuclein-positive GCIs were restricted to oligodendrocytes, and alpha-synuclein was localized to the filaments in GCIs by immunoelectron microscopy. Finally, we demonstrated that insoluble alpha-synuclein accumulated selectively in MSA white matter with alpha-synuclein-positive GCIs. Taken together with evidence that LBs contain insoluble alpha-synuclein, our data suggest that a reduction in the solubility of alpha-synuclein may induce this protein to form filaments that aggregate into cytoplasmic inclusions, which contribute to the dysfunction or death of glial cells as well as neurons in neurodegenerative disorders with different phenotypes.