Review: Novel treatment strategies targeting alpha‐synuclein in multiple system atrophy as a model of synucleinopathy

Neurodegenerative disorders with alpha‐synuclein (α‐syn) accumulation (synucleinopathies) include Parkinson's disease (PD), PD dementia, dementia with Lewy bodies and multiple system atrophy (MSA). Due to the involvement of toxic α‐syn aggregates in the molecular origin of these disorders, developing effective therapies targeting α‐syn is a priority as a disease‐modifying alternative to current symptomatic treatments. Importantly, the clinical and pathological attributes of MSA make this disorder an excellent candidate as a synucleinopathy model for accelerated drug development. Recent therapeutic strategies targeting α‐syn in in vivo and in vitro models of MSA, as well as in clinical trials, have been focused on the pathological mechanisms of α‐syn synthesis, aggregation, clearance, and/or cell‐to‐cell propagation of its neurotoxic conformers. Here we summarize the most relevant approaches in this direction, with emphasis on their potential as general synucleinopathy modifiers, and enumerate research areas for potential improvement in MSA drug discovery.     

Viral‐mediated oligodendroglial alpha‐synuclein expression models multiple system atrophy

Background : MSA is a fatal neurodegenerative disorder characterized by a combination of autonomic dysfunction, cerebellar ataxia, and l ‐dopa unresponsive parkinsonism. The hallmark of MSA is the accumulation of α‐synuclein, forming cytoplasmic inclusions in oligodendrocytes. Adeno‐associated viruses allow efficient targeting of disease‐associated genes in selected cellular ensembles and have proven efficient for the neuronal overexpression of α‐synuclein in the substantia nigra in the context of PD. Objectives : We aimed to develop viral‐based models of MSA. Methods : Chimeric viral vectors expressing either human wild‐type α‐synuclein or green fluorescent protein under the control of mouse myelin basic protein were injected in the striatum of rats and monkeys. Rats underwent a longitudinal motor assessment before histopathological analysis at 3 and 6 months. Results : Injection of vectors expressing α‐synuclein in the striatum resulted in >80% oligodendroglial selectivity in rats and >60% in monkeys. Rats developed progressive motor deficits that were l ‐dopa unresponsive when assessed at 6 months. Significant loss of dopaminergic neurons occurred at 3 months, further progressing at 6 months, together with a loss of striatal neurons. Prominent α‐synuclein accumulation, including phosphorylated and proteinase‐K–resistant α‐synuclein, was detected in the striatum and substantia nigra. Conclusions : Viral‐mediated oligodendroglial expression of α‐synuclein allows replicating some of the key features of MSA. This flexible strategy can be used to investigate, in several species, how α‐synuclein accumulation in selected oligodendroglial populations contributes to the pathophysiology of MSA and offers a new framework for preclinical validation of therapeutic strategies. © 2017 International Parkinson and Movement Disorder Society     

Perirhinal accumulation of neuronal alpha‐synuclein in a multiple system atrophy patient with dementia

We report the case of a 79‐year‐old Japanese woman who developed cerebellar ataxia followed by rigidity, dysautonomia and cognitive disorders, and was thus clinically diagnosed as having possible MSA with dementia. Neuropathological findings demonstrated not only olivopontocerebellar and striatonigral degeneration with frequent glial cytoplasmic inclusions (GCIs), but also degenerative changes in the parahippocampal region, accentuated in the anterior portion of perirhinal cortex, where neuronal cytoplasmic inclusions (NCIs) and NFTs were numerous while GCIs were limited. NCIs were frequent in the deep layer, whereas NFTs were more frequent in superficial cortical layers. Other hippocampal subregions including subiculum, dentate fascia and cornu ammonis were minimally involved. NCIs in the perirhinal cortex showed intense argyrophilia with the Campbell‐Switzer silver impregnation method, but not argyrophilic with the Gallyas method. Most neuronal alpha‐synuclein aggregates in dendrosomatic fraction formed globular/tadpole‐like, and ultrastructurally comprised granular‐coated fine fibrils 12–24 nm in diameter. To the best of our knowledge, alpha‐synuclein‐related neuronal pathology localized in the perirhinal region without hippocampal involvement has not been previously reported in MSA, and may provide clues to elucidate how neuronal pathology evolves in the hippocampal/parahippocampal regions in MSA, particularly in cases with dementia.     

Age‐related motor dysfunction and neuropathology in a transgenic mouse model of multiple system atrophy

Multiple system atrophy (MSA) is a neurodegenerative disorder characterized by a progressive degeneration of the striatonigral, olivo‐ponto‐cerebellar, and autonomic systems. Glial cytoplasmic inclusions (GCIs) containing alpha‐synuclein represent the hallmark of MSA and are recapitulated in mice expressing alpha‐synuclein in oligodendrocytes. To assess if oligodendroglial expression of human wild‐type alpha‐synuclein in mice (proteolipid promoter, PLP‐SYN) could be associated with age‐related deficits, PLP‐SYN and wild‐type mice were assessed for motor function, brain morphometry, striatal levels of dopamine and metabolites, dopaminergic loss, and distribution of GCIs. PLP‐SYN displayed age‐related impairments on a beam‐traversing task. MRI revealed a significantly smaller brain volume in PLP‐SYN mice at 12 months, which further decreased at 18 months together with increased volume of ventricles and cortical atrophy. The distribution of GCIs was reminiscent of MSA with a high burden in the basal ganglia. Mild dopaminergic cell loss was associated with decreased dopamine turnover at 18 months. These data indicate that PLP‐SYN mice may recapitulate some of the progressive features of MSA and deliver endpoints for the evaluation of therapeutic strategies. Synapse 68:98–106, 2014. © 2013 Wiley Periodicals, Inc.     

Reduced alpha‐synuclein in cerebrospinal fluid in synucleinopathies: Evidence from a meta‐analysis

Alpha‐synuclein plays a key role in the pathology of synucleinopathies including Parkinson's disease (PD) and multiple system atrophy (MSA). However, whether alpha‐synuclein level in cerebrospinal fluid (CSF) could distinguish synucleinopathies from progressive supranuclear palsy (PSP) is still a contentious issue. A comprehensive literature search yielded nine eligible studies. We expressed the between‐group difference of the concentration of alpha‐synuclein in CSF as the standardized mean difference. The proportion of variation attributable to heterogeneity was computed and expressed as I². Nine studies involved 537 controls, 843 PD, 130 MSA, and 98 PSP patients. The overall effect of PD on alpha‐synuclein in CSF was significantly different from normal control or disease control (standardized mean difference = –0.67, P < 0.00001). These studies were heterogeneous (I² = 40%). Alpha‐synuclein in CSF in MSA was significantly reduced relative to controls with heterogeneous studies (standardized mean difference = –0.75, P < 0.0001; I² = 62%). In contrast, no significant difference of alpha‐synuclein in CSF was observed between PSP and controls with heterogeneous studies (standardized mean difference = –0.28, P = 0.13; I² = 53%). Alpha‐synuclein in CSF was significantly reduced in synucleinopathies compared with PSP (“PD vs. PSP”: standardized mean difference = –0.38, P = 0.001; “MSA vs. PSP”: standardized mean difference = –0.66, P < 0.00001). The included studies were homogeneous (I² = 0%). Our study showed that alpha‐synuclein levels in CSF in synucleinopathies was significantly lower than in PSP. This finding provides insights into the pathophysiological difference between synucleinopathies and PSP as well as possibility of development of a tool for differential diagnosis between MSA and PSP using enzyme‐linked immunosorbent assay (ELISA) and similar methods. © 2014 International Parkinson and Movement Disorder Society     

Bladder dysfunction in a transgenic mouse model of multiple system atrophy

Multiple system atrophy (MSA) is an adult‐onset neurodegenerative disorder presenting with motor impairment and autonomic dysfunction. Urological function is altered in the majority of MSA patients, and urological symptoms often precede the motor syndrome. To date, bladder function and structure have never been investigated in MSA models. We aimed to test bladder function in a transgenic MSA mouse featuring oligodendroglial α‐synucleinopathy and define its applicability as a preclinical model to study urological failure in MSA. Experiments were performed in proteolipid protein (PLP)–human α‐synuclein (hαSyn) transgenic and control wild‐type mice. Diuresis, urodynamics, and detrusor strip contractility were assessed to characterize the urological phenotype. Bladder morphology and neuropathology of the lumbosacral intermediolateral column and the pontine micturition center (PMC) were analyzed in young and aged mice. Urodynamic analysis revealed a less efficient and unstable bladder in MSA mice with increased voiding contraction amplitude, higher frequency of nonvoiding contractions, and increased postvoid residual volume. MSA mice bladder walls showed early detrusor hypertrophy and age‐related urothelium hypertrophy. Transgenic hαSyn expression was detected in Schwann cells ensheathing the local nerve fibers in the lamina propria and muscularis of MSA bladders. Early loss of parasympathetic outflow neurons and delayed degeneration of the PMC accompanied the urological deficits in MSA mice. PLP‐hαSyn mice recapitulate major urological symptoms of human MSA that may be linked to αSyn‐related central and peripheral neuropathology and can be further used as a preclinical model to decipher pathomechanisms of MSA. © 2013 Movement Disorder Society     

Increase of the plasma α‐synuclein levels in patients with multiple system atrophy

Multiple system atrophy, a sporadic neurodegenerative disease, is characterized by the presence of high numbers of glial cytoplasmic inclusions mainly formed by α‐synuclein protein, which is encoded by the SNCA gene. To date, however, few studies have investigated the plasma α‐synuclein levels in patients with multiple system atrophy. We studied plasma α‐synuclein concentrations by using an enzyme‐linked immunosorbent assay in 74 patients with multiple system atrophy and 90 healthy controls. The plasma α‐synuclein levels were significantly elevated in patients who had multiple system atrophy compared with the control group (P = 0.000). In a subgroup of 48 patients who had probable multiple system atrophy, there was a weakly negative correlation between plasma α‐synuclein levels and subscores on Unified Multiple System Atrophy Rating Scale item VI (r_s = ?0.307; P = 0.034). Plasma α‐synuclein levels were elevated in patients with multiple system atrophy, and these levels may be decreased with the development of disease. © 2013 International Parkinson and Movement Disorder Society.     

Erythropoietin is neuroprotective in a transgenic mouse model of multiple system atrophy

Multiple system atrophy is a rapidly progressive neurodegenerative disorder with a markedly reduced life expectancy. Failure of symptomatic treatment raises an urgent need for disease‐modifying strategies. We have investigated the neuroprotective potential of erythropoietin in (proteolipid protein)‐α‐synuclein transgenic mice exposed to 3‐nitropropionic acid featuring multiple system atrophy‐like pathology including oligodendroglial α‐synuclein inclusions and selective neuronal degeneration. Mice were treated with erythropoietin starting before (early erythropoietin) and after (late erythropoietin) intoxication with 3‐nitropropionic acid. Nonintoxicated animals receiving erythropoietin and intoxicated animals treated with saline served as control groups. Behavioral tests included pole test, open field activity, and motor behavior scale. Immunohistochemistry for tyrosine hydroxylase and dopamine and cyclic adenosine monophosphate‐regulated phosphoprotein (DARPP‐32) was analyzed stereologically. Animals receiving erythropoietin before and after 3‐nitropropionic acid intoxication scored significantly lower on the motor behavior scale and they performed better in the pole test than controls with no significant difference between early and late erythropoietin administration. Similarly, rearing scores were worse in 3‐nitropropionic acid‐treated animals with no difference between the erythropoietin subgroups. Immunohistochemistry revealed significant attenuation of 3‐nitropropionic acid‐induced loss of tyrosine hydroxylase and DARPP‐32 positive neurons in substantia nigra pars compacta and striatum, respectively, in both erythropoietin‐treated groups without significant group difference in the substantia nigra. However, at striatal level, a significant difference between early and late erythropoietin administration was observed. In the combined (proteolipid protein)‐α‐synuclein 3‐nitropropionic acid multiple system atrophy mouse model, erythropoietin appears to rescue dopaminergic and striatal gabaergic projection neurons. This effect is associated with improved motor function. Further studies are warranted to develop erythropoietin as a potential interventional therapy in multiple system atrophy. © 2011 Movement Disorder Society     

Rifampicin reduces alpha-synuclein in a transgenic mouse model of multiple system atrophy

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by oligodendrocytic cytoplasmic inclusions containing abnormally aggregated alpha-synuclein. This aggregation has been linked to the neurodegeneration observed in MSA. Current MSA treatments are aimed at controlling symptoms rather than tackling the underlying cause of neurodegeneration. This study investigates the ability of the antibiotic rifampicin to reduce alpha-synuclein aggregation and the associated neurodegeneration in a transgenic mouse model of MSA. We report a reduction in monomeric and oligomeric alpha-synuclein and a reduction in phosphorylated alpha-synuclein (S129) upon rifampicin treatment. This reduction in alpha-synuclein aggregation was accompanied by reduced neurodegeneration. On the basis of its anti-aggregenic properties, we conclude that rifampicin may have therapeutic potential for MSA.     

Targeting α‐synuclein: Therapeutic options

The discovery of the central role of α‐synuclein (αSyn) in the pathogenesis of Parkinson's disease (PD) has powered, in the last decade, the emergence of novel relevant models of this condition based on viral vector‐mediated expression of the disease‐causing protein or inoculation of toxic species of αSyn. Although the development of these powerful tools and models has provided considerable insights into the mechanisms underlying neurodegeneration in PD, it has also been translated into the expansion of the landscape of preclinical therapeutic strategies. Much attention is now brought to the proteotoxic mechanisms induced by αSyn and how to block them using strategies inspired by intrinsic cellular pathways such as the enhancement of cellular clearance by the lysosomal‐autophagic system, through proteasome‐mediated degradation or through immunization. The important effort undertaken by several laboratories and consortia to tackle these issues and identify novel targets warrants great promise for the discovery not only of neuroprotective approaches but also of restorative strategies for PD and other synucleinopathies. In this viewpoint, we summarize the latest advances in this new area of PD research and will discuss promising approaches and ongoing challenges. © 2016 International Parkinson and Movement Disorder Society     

Multiple system atrophy: α‐synuclein and neuronal degeneration

Multiple system atrophy (MSA) is a sporadic neurodegenerative disorder that encompasses olivopontocerebellar atrophy (OPCA), striatonigral degeneration (SND) and Shy–Drager syndrome (SDS). The histopathological hallmark is the formation of α‐synuclein‐positive glial cytoplasmic inclusions (GCIs) in oligodendroglia. α‐synuclein aggregation is also found in glial nuclear inclusions, neuronal cytoplasmic inclusions (NCIs), neuronal nuclear inclusions (NNIs) and dystrophic neurites. We evaluated the pathological features of 102 MSA cases, and presented the pathological spectrum of MSA and initial features of α‐synuclein accumulation. We found that 39% of the 102 cases showed equivalent SND and OPCA pathologies, 33% showed OPCA‐ and 22% showed SND‐predominant pathology, whereas 6% showed extremely mild changes. Our pathological analysis indicated that OPCA‐type was relatively more frequent and SND‐type was less frequent in Japanese MSA cases, compared to the relatively high frequency of SND‐type in Western countries, suggesting that different phenotypic patterns of MSA may exist between races. In the early stage, in addition to GCIs, NNIs and diffuse homogenous α‐synuclein staining in neuronal nuclei and cytoplasm were observed in lesions in the pontine nuclei, putamen, substantia nigra, locus ceruleus, inferior olivary nucleus, intermediolateral column of thoracic spinal cord, lower motor neurons and cortical pyramidal neurons. A subgroup of MSA cases with severe temporal atrophy showed numerous NCIs, particularly in the limbic system. These findings suggest that primary non‐fibrillar and fibrillar α‐synuclein aggregation also occur in neurons. The oligo‐myelin‐axon‐neuron complex mechanism, along with the direct involvement of neurons themselves, may synergistically accelerate the degenerative process of MSA.     

Accumulation of phosphorylated α‐synuclein in subpial and periventricular astrocytes in multiple system atrophy of long duration

The histological hallmark of multiple system atrophy (MSA) is accumulation of phosphorylated α‐synuclein in oligodendrocytes. However, it is uncertain whether phosphorylated α‐synuclein accumulates in astrocytes of MSA patients. We immunohistochemically examined the frontal and temporal lobes, basal ganglia, cerebellum, brainstem and spinal cord of patients with MSA (n = 15) and Lewy body disease (n = 20), and also in control subjects (n = 20). Accumulation of abnormally phosphorylated and aggregated α‐synuclein was found in subpial and periventricular astrocytes in six of the 15 patients with MSA (40%). The structures were confined to the subpial surface of the ventro‐lateral part of the spinal cord and brainstem, as well as the subependymal region of the lateral ventricles. They were not visualized by Gallyas‐Braak staining, and were immunonegative for ubiquitin and p62. Immunoelectron microscopy revealed that the phosphorylated α‐synuclein‐immunoreactive structures in astrocytes were non‐fibrillar and associated with granular and vesicular structures. The extent of phosphorylated α‐synuclein‐immunoreactive astrocytes was correlated with disease duration. No such structures were found in Lewy body disease or controls. Accumulation of phosphorylated α‐synuclein can occur in subpial and periventricular astrocytes in patients with MSA, especially in those with a long disease duration.     

Progression of intestinal permeability changes and alpha‐synuclein expression in a mouse model of Parkinson's disease

Parkinson's disease (PD) is a multifocal degenerative disorder for which there is no cure. The majority of cases are sporadic with unknown etiology. Recent data indicate that untreated patients with de novo PD have increased colonic permeability and that both de novo and premotor patients have pathological expression of α‐synuclein (α‐syn) in their colon. Both endpoints potentially can serve as disease biomarkers and even may initiate PD events through gut‐derived, lipopolysaccharide (LPS)‐induced neuronal injury. Animal models could be ideal for interrogating the potential role of the intestines in the pathogenesis of PD; however, few current animal models of PD encompass these nonmotor features. We sought to establish a progressive model of PD that includes the gastrointestinal (GI) dysfunction present in human patients. C57/BL6 mice were systemically administered one dose of either LPS (2.5 mg/kg) or saline and were sacrificed in monthly intervals (n = 5 mice for 5 months) to create a time‐course. Small and large intestinal permeability was assessed by analyzing the urinary output of orally ingested sugar probes through capillary column gas chromatography. α‐Syn expression was assessed by counting the number of mildly, moderately, and severely affected myenteric ganglia neurons throughout the GI tract, and the counts were validated by quantitative optical density measurements. Nigrostriatal integrity was assessed by tyrosine hydroxylase immunohistochemistry stereology and densitometry. LPS caused an immediate and progressive increase in α‐syn expression in the large intestine but not in the small intestine. Intestinal permeability of the whole gut (large and small intestines) progressively increased between months 2 and 4 after LPS administration but returned to baseline levels at month 5. Selective measurements demonstrated that intestinal permeability in the small intestine remained largely intact, suggesting that gut leakiness was predominately in the large intestine. Phosphorylated serine 129‐α‐syn was identified in a subset of colonic myenteric neurons at months 4 and 5. Although these changes were observed in the absence of nigrostriatal degeneration, an abrupt but insignificant increase in brainstem α‐syn was observed that paralleled the restoration of permeability. No changes were observed over time in controls. LPS, an endotoxin used to model PD, causes sequential increases in α‐syn immunoreactivity, intestinal permeability, and pathological α‐syn accumulation in the colon in a manner similar to that observed in patients with PD. These features are observed without nigrostriatal degeneration and incorporate PD features before the motor syndrome. This allows for the potential use of this model in testing neuroprotective and disease‐modifying therapies, including intestinal‐directed therapies to fortify intestinal barrier integrity. © 2013 International Parkinson and Movement Disorder Society     

First‐in‐human assessment of PRX002, an anti–α‐synuclein monoclonal antibody,in healthy volunteers

Background : α‐Synuclein is a major component of pathologic inclusions that characterize Parkinson's disease. PRX002 is an antibody that targets α‐synuclein, and its murine parent antibody 9E4 has been shown in preclinical studies to reduce α‐synuclein pathology and to protect against cognitive and motor deteriorations and progressive neurodegeneration in human α‐synuclein transgenic mice. Methods : This first‐in‐human, randomized, double‐blind, placebo‐controlled, phase 1 study assessed the impact of PRX002 administered to 40 healthy participants in 5 ascending‐dose cohorts (n = 8/cohort) in which participants were randomly assigned to receive a single intravenous infusion of study drug (0.3, 1, 3, 10, or 30 mg/kg; n = 6/cohort) or placebo (n = 2/cohort). Results : PRX002 demonstrated favorable safety, tolerability, and pharmacokinetic profiles at all doses tested, with no immunogenicity. No serious adverse events, discontinuations as a result of adverse events, or dose‐limiting toxicities were reported. Serum PRX002 exposure was dose proportional; the average terminal half‐life across all doses was 18.2 days. A significant dose‐dependent reduction in free serum α‐synuclein (unbound to PRX002) was apparent within 1 hour after PRX002 administration, whereas total α‐synuclein (free plus bound) increased dose‐dependently, presumably because of the expected change in kinetics following antibody binding. Conclusions : This study demonstrates that serum α‐synuclein can be safely modulated in a dose‐dependent manner after single intravenous infusions of an anti–α‐synuclein antibody. These findings support continued development of PRX002, including further characterization of its safety, tolerability, pharmacokinetics, and pharmacodynamic effects in the central nervous system in patients with Parkinson's disease. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.