Exp Neurobiol 2014; 23(4): 277-291
Published online December 31, 2014
© The Korean Society for Brain and Neural Sciences
Edith Sturm and Nadia Stefanova*
Division of Neurobiology, Department of Neurology, Innsbruck Medical University, Innsbruck A-6020, Austria
Correspondence to: *To whom correspondence should be addressed.
TEL: 43-512-50424363, FAX: 43-512-50424230
Multiple system atrophy (MSA) is a rare, late-onset and fatal neurodegenerative disease including multisystem neurodegeneration and the formation of α-synuclein containing oligodendroglial cytoplasmic inclusions (GCIs), which present the hallmark of the disease. MSA is considered to be a sporadic disease; however certain genetic aspects have been studied during the last years in order to shed light on the largely unknown etiology and pathogenesis of the disease. Epidemiological studies focused on the possible impact of environmental factors on MSA disease development. This article gives an overview on the findings from genetic and epigenetic studies on MSA and discusses the role of genetic or epigenetic factors in disease pathogenesis.
Keywords: Multiple system atrophy, α-synuclein, neurodegeneration, genetics, epigenetics
In 1969 Graham and Oppenheimer suggested the term "multiple system atrophy" (MSA) to describe and combine a set of different disorders, including olivopontocerebellar atrophy (OPCA), striatonigral degeneration (SND) and Shy-Drager syndrome . MSA is nowadays considered to be a rare, late-onset and fatal neurodegenerative disease with a largely unknown etiopathogenesis. Prevalence ranges from 1.9 to 4.9 per 100,000 inhabitants, incidence is about 0.6 per 100,000 per year or rather 3 per 100,000 per year in the population over 50 years [2, 3]. Patients show an average disease onset of 60 years (SD=9; range: 34 to 83 years), affecting males and females equally ; mean disease duration is between 7 to 9 years after clinical presentation . This movement disorder is clinically represented by atypical parkinsonism, cerebellar ataxia, pyramidal signs, and always accompanied by autonomic failure; pathologically MSA is characterized by selective wide spread neuronal cell loss, gliosis and oligodendroglial cytoplasmic inclusions (GCIs) affecting several structures of the central nervous system [3, 6, 7].
Neuronal loss in MSA affects the striatum, substantia nigra pars compacta (SNpc), cerebellum, pons, inferior olives, central autonomic nuclei and the intermediolateral column of the spinal cord . Microglial and astroglial activation (gliosis) affecting several regions of the MSA brain could partly be triggered by oligodendroglial α-synuclein pathology, but the exact pathogenic mechanisms need to be further clarified [9, 10, 11, 12]. GCIs represent the major pathological hallmark of the disease  and are mostly containing misfolded, hyperphosphorylated (affecting residue Ser129) and fibrillar α-synuclein [13, 14, 15]. GCIs also contain tau, 14-3-3 protein, LRRK2, parkin, heat shock protein family members Hsp70 and Hsc70, p25α, α-tubulin, β-tubulin, microtubule associated proteins and cycline dependent kinase 5 (cdk5) among others [16, 17, 18, 19, 20 ,21, 22, 23]. The mechanisms of GCI formation in MSA remain unclear; two hypotheses try to explain. The first suggests that active uptake of α-synuclein from neighboring neurons by oligodendroglia could take place. Whereas the second hypothesis states that there could be a selective increase of α-synuclein expression in oligodendroglial cells in MSA . Disturbed protein degradation may further contribute to the accumulation of α-synuclein in MSA oligodendrocytes . Since the etiology and pathogenesis of MSA are not completely understood, no effective therapies have been established up to date to cure MSA. In 2009, the discovery of α-synuclein gene (SNCA) variants association with an increased risk for MSA especially in Caucasians suggested an important lead in the role of genetic predisposition in MSA . Along with the fact that MSA has always been considered to be a sporadic disease , this was thought to be a great breakthrough. Since then, many studies have been performed with the aim to detect disease-causing or disease-linked genes and gene variants. Other studies focused on environmental risk factors and epigenetic mechanisms, since MSA shares common features with other neurodegenerative disorders that have proven role of epigenetic modifications in their pathogenesis . Within this article, we provide an update on recent studies concerning genetic and epigenetic factors that might be involved in MSA etiology and pathogenesis.
MSA is a typically sporadic neurodegenerative disease , but rare cases exist presenting a family history of MSA [29, 30, 31, 32, 33]. The ancestry arrangement in two of those family cases - probable MSA in one German and one Japanese family - [30, 32, 33] is consistent with typical autosomal dominant inheritance. Four different Japanese families with multiple affected siblings show probable autosomal recessive inheritance . In 2012, a case of two sisters from the US was reported, presenting similar syndromes of MSA and thereby suggesting autosomal dominant inheritance . Recently, two Japanese siblings (affected with either probable MSA-C and definite MSA-P) were described. Inheritance was found to be autosomal recessive, even though no consanguineous marriage took place and no genetic mutations were identified . So far, no disease-causing and hereditable mutations have been definitely identified in MSA.
Since the pathological hallmark of MSA is represented by GCIs  containing mostly α-synuclein among other components, MSA together with Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) is considered to be an α-synucleinopathy . The fundamental role of α-synuclein in MSA pathogenesis leads to the suggestion that there might be a connection between possible
Discovered in 1997, A53T has been the first SNCA point mutation identified in families with autosomal dominant PD [35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49]. This was followed by the identification of A30P [35, 44, 50, 51] and E46K point mutations of the
In 2009, the single nucleotide polymorphism (SNP) study in SNCA by Scholz and colleagues suggested the first genetic
SNCA-linked genetic predisposition seems to play an important role in MSA but further studies are needed to identify the possible mechanisms underlying these interactions.
MSA patients show overlapping features with autosomal dominant spinocerebellar ataxias (SCAs), including prominent ataxia, dysmetria and abnormal eye movement . For this reason the investigation of possible SCA genocopies in MSA patients has been of great interest. Since 1996, many studies focused on the possible clinical overlap of SCAs and MSA [86, 87, 88, 89, 90]. Gilman et al. investigated four members of a family with SCA1 mutations presenting dominantly inheritated progressive ataxia, dystonia, autonomic dysfunction and peripheral neuropathy. Several unusual SCA1 but MSA-like symptoms were found including neurodegeneration of the basal ganglia, cerebellum, brainstem and the intermediolateral columns of the spinal cord as well as tau- and ubiquitin-positive GCIs. Yet, unusual MSA features like early disease onset, cerebellar and autonomic features in the absence of pyramidal or extrapyramidal signs were also detected. It was concluded that SCA1 gene mutations might only lead to a disorder mimicking MSA . Although cases of SCA2 were identified to present with glial cytoplasmic inclusions these were α-synuclein-negative [91, 92]. A SCA3 patient showed additional neurodegenerative disorders resembling the pathological features of cerebellar MSA (MSA-C). The patient presented α-synuclein positive GCIs and neurodegeneration in the olivopontocerebellar, striatal and pyramidal motor regions , thereby meeting the criteria for definite MSA-C diagnosis . A case-control study on Caucasian MSA patients (n=80) failed to confirm the presence of SCA3 expansions, highlighting that MSA is an autonomous disease, not related to SCA-gene mutations . Furthers studies found
Several studies on mutated genes associated with MSA-related neurodegenerative disorders failed to identify a direct genetic link to MSA. Negative findings included:
Since gliosis accounts to MSA pathogenesis , several genes involved in inflammatory processes have been investigated. Microglia activation leads to the production of cytokines, including interleukin-1-α (IL-1-α), IL-1-β, IL-6 and tumor necrosis factor-α (TNF-α), as well as chemokines such as IL-8 and the inflammatory intercellular-adhesion molecule-1 (ICAM-1) . Association with an increased risk to develop MSA was found in relation to gene polymorphisms in
Since oxidative as well as nitrative stress are implicated in the progression of α-synucleinopathies, related factors have been tested and a link to MSA was found for
Genetic variability in alcohol dehydrogenase (ADH) gene risk factors was investigated in MSA.
In conclusions, the genetic studies in MSA to date do not support the use of genetic factors like SNCA, COQ2, SCAs expansions etc. to reliably diagnose MSA. Several gene polymorphisms have been linked to an increased risk for developing MSA, but many of the findings have been contradictory dependent on the high heterogeneity of the MSA patients. Further genetic analysis involving larger patients cohorts are warranted to provide reliable information on the role of selected genes in the etiopathogenesis of MSA.
Given the fact that it is still unclear whether genetics have a predisposing role in the etiology of MSA, several studies focused on the investigation of risk factors that could lead, together with genetic predisposition to disease development. Epigenetics includes transcriptional as well as post-transcriptional, reversible and hereditable changes to DNA that do not alter DNA sequence itself and regulate gene expression. The epigenetic machinery acts via different mechanisms. DNA-methylation includes the addition of a methyl group to the 5' carbon of cytosines, which are located to CpG islands in promoter regions (regions with high content of the bases cytosine and guanine) and associated with gene silencing. Histone modifications (acetylation, phosphorylation, methylation, ubiquitination or sumoylation) are carried out at the N-termini of the core histones that protrude from the nucleosome modulating gene expression and chromatin structure. RNA-mediated silencing pathways include non-coding RNAs as well as non-coding antisense RNAs, RNA interference and microRNAs (miRNAs). Together these epigenetic mechanisms form the "epigenetic landscape" being very dynamic, in contrast to irreversible genetic mutations, and can be influenced during life through environmental stimuli. Destruction of the epigenetic balance leads to several distinct diseases [142, 143]. Many disorders have already been connected to epigenetic alterations including cancer , cardiovascular diseases [145, 146], autoimmune disorders , metabolic diseases [148, 149], myopathies , learning and memory deficits [151, 152] and some neurodegenerative diseases .
There has been emerging evidence that neurodegenerative diseases are linked to exposure to chronic neurotoxic substances and other risk environmental factors resulting in a higher risk of disease development . For example the risk to develop multiple sclerosis is increased when being infected with Epstein-Barr virus and smoking cigarettes, whereas vitamin D is protective ; Alzheimer's disease is associated with lead (Pb) exposure resulting in higher amounts of β-amyloid, stress and traumatic brain injuries [156, 157, 158, 159, 160]; higher risk to develop PD was linked among others to exposure to metals (Pb and others), certain chemicals and magnetic fields .
Given that MSA is a neurodegenerative disease as well as α-synucleinopathy with unclear genetic background, MSA patients have been investigated with regard to identify the role of environmental and epigenetic factors including stress, occupational and daily habits and exposure to toxins, metals, solvents, plastic monomers or additives, as well as history of farming.
Exposures to metal dusts and fumes, plastic monomers and additives, organic solvents and pesticides have been linked significantly to a higher vulnerability of the nervous system supporting disease onset of MSA . Recently, one case report supported the role of intensive and extended contact with organic solvents as a risk factor in MSA, as reported when examining a long-term professional painter . A putative association to environmental toxins with MSA-like disorders has been shown, including inorganic mercury, methanol, carbon tetrachloride, carbon disulfide, cyanide, and manganese after heavy occupational exposure [164, 165]. Control studies confirmed the findings by detecting increased total iron levels in MSA and PD brains, but failed to detect alterations in manganese levels [166, 167, 168].
Epidemiological studies investigated the influence of farming on MSA, involving exposure to different chemicals and factors (pesticides, solvents, mycotoxins, dust, fuels, oils, fertilizers, animals) and a certain lifestyle (consummation of well water, rural living, diet and physical activity) . Especially pesticides have been continuously associated with an increased risk of developing MSA, supported by independent studies [162, 170, 171]. So far, there is only one study contradicting these findings and stating that only plant and machine operators and assemblers develop an increased risk for MSA that is increasing with time of exposure .
The influence of smoking on MSA has been investigated already in 1986 and studies showed an inverse association with MSA [170, 173, 174]. Vanacore and colleagues also investigated effects of smoking together with farming on MSA concluding that those two risk factors are not interacting with the disease development (ever-smokers compared with never-smokers) . In contrast to those findings, one MSA patient was reported recently having severe nicotine sensitivity. Interestingly, Graham and Oppenheimer described a similar case in 1969, but further studies are needed in order to investigate the prevalence of those cases in MSA [1, 175]. Regarding education, MSA patients seemed to have a lower educational level when compared to healthy controls [170, 172]. Concerning food habits, the consumption of meat has been associated with a higher risk of developing MSA. Other habits including drinking alcohol, aspirin use and fish or seafood consumption were found to be more common in healthy controls and are considered being protective factors. The same study was not able to confirm an effect of herbal tea or tropical fruits consumption, as it was suggested previously .
Until now, several risk factors have been associated with MSA. However, epidemiological studies display some limitations. Those studies are often affected by a certain recall bias (over-reporting of exposures) and selection bias (patients with severe disease are less able to participate) that influences the outcomes . Therefore, further epidemiological studies of expanded cohorts are needed to get more data on potential MSA risk factors.
It has been shown that nutrients and environmental toxins, especially metals, are able to cause DNA methylation changes, histone modifications and RNA interference . Together with findings on maternal nutrition that modulates gene expression already in the embryo supporting the possible development of later life diseases , this is pointing out the powerful influence of environmental factors on the epigenetic landscape and supporting the idea of a certain gene-environment interaction. Unfortunately not much is known about epigenetic modulations in MSA that could be provoked by environmental risk factors.
As mentioned, the epigenetic landscape is shaped by several mechanisms. DNA methylation changes have not been reported in MSA yet. Histone modifications can include acetylation, phosphorylation, methylation, ubiquitination or sumoylation of the four core histone proteins (H2A, H2B, H3, and H4) of the nucleosome. None of these has been studied in MSA patients to our knowledge. Specifically histone acetylation is controlled by a pair of antagonistic enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs) that carry out acetylation and deacetylation at the N-termini of the nucleosomes, thereby modifying the histones. Histone deacetylation by HDACs influences gene expression, cell cycle regulation, chromatin structure and developmental events [179, 180, 181]. 18 different HDAC families are known in mammalians. The representative family members can be subdivided into zinc-dependent (HDAC1-11) and nicotinamide adenine dinucleotide (NAD+)-dependent (sirtuin 1-7) enzymes . Miki and colleagues showed that patients with PD and dementia with Lewy bodies (DLB) show a co-localization of HDAC6 with Lewy bodies and also HDAC6 co-localized with GCIs in MSA brains , indicating a putative role of HDAC6 in MSA. Despite its name, the role of HDAC6 in histone deacetylation directly is unclear. HDAC6 is unique to a certain extent, since it possesses two functional catalytic sites at the N-terminus in combination with an ubiquitin-binding domain at the C-terminus. Although shuttling between the cytoplasm and the nucleus has been proposed, HDAC6 is found mainly in the cytoplasm, which is suggesting a histone-independent function .
The very first indications of a changed epigenetic landscape in MSA were provided just recently, by studies describing an altered expression of microRNAs in MSA. MicroRNAs (miRNAs) are small non-coding RNAs that are able to regulate gene expression post-transcriptionally  and important for the survival of mature neurons and their functions . Ubhi and colleagues found an upregulation of the microRNA miR-96 in MSA patients, which is connected to a down-regulation of miR-96 target genes, including family members of the solute carrier protein family SLC1A1 and SLC6A6 . A previous genetic association of SLC1A4 with MSA is supporting this finding . Expression of miR-202 is upregulated in the cerebellum of MSA patients, consistent with reduced Oct1 protein expression. By that, miR-202 and the Oct1 pathway are thought to participate in sporadic cerebellar neurodegeneration representing a novel putative therapeutic target in MSA. The authors suggest that decreased Oct1 levels lead to higher vulnerability of neurons to oxidative stress , since this mechanism is involved in cerebellar neurodegeneration . Next to miR-202, other microRNAs show downregulation (miR-129-3p, miR-129-5p, miR-337-3p, miR-380, miR-433, miR-132, miR-410, miR-206 and miR-409-5p) or upregulation (miR-199a-5p) in the cerebellum, but those findings need to be re-evaluated and confirmed in future studies . A further study was able to identify circulating microRNAs (cimRNAs) to be differentially expressed in MSA and PD patients. MSA patients are distinguished from PD patients and healthy controls by an up-regulation of miR-24, miR-34b and miR-148b, whereas miR-339-5p is downregulated. . The role of these changes is still unclear, however similar they show definite link to neurodegeneration. Previous studies showed that miR-339-5p is expressed at low levels in mature neurons and connected to axon guiding ; miR-24 is upregulated in multiple sclerosis and myocardial ischemia [192, 193]; miR-34b is connected to Huntington's disease, PD and Dementia with Lewy Bodies [194, 195]; miR-148b is downregulated in the parietal lobe cortex and hippocampal as well as medial frontal gyrus of Alzheimer's patients [196, 197].
So far, the underlying mechanisms of MSA etiopathogenesis are still elusive. Several familial MSA cases exist, but no hereditable mutations have been found supporting hereditary disease. COQ2 mutations have been proposed to associate with familial and sporadic MSA, however this observation could not be replicated in different patient cohorts. Investigations of other genetic "hotspots" linked
Therefore, MSA could be considered a disease that is related to a complex genetic and non-genetic interplay. Interaction of those factors could also contribute to the phenotypic variability of MSA (cerebellar or parkinsonian MSA in various combinations) among different populations, as it was discussed by Ozawa and colleagues . How and whether certain risk factors contribute to MSA remains unclear, but the investigation of those factors is a key topic for promising future research initiatives. Furthermore, epigenetic disease markers may prove a potential role as novel biomarkers in the early diagnosis of MSA.