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.     

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.     

Review: Spreading the word: precise animal models and validated methods are vital when evaluating prion‐like behaviour of alpha‐synuclein

Synucleinopathies are characterized by abnormal proteinaceous aggregates, mainly composed of fibrillar α‐synuclein (α‐syn). It is now believed that α‐syn can form small aggregates in a restricted number of cells, that propagate to neighbouring cells and seed aggregation of endogenous α‐syn, in a ‘prion‐like manner’. This process could underlie the stereotypical progression of Lewy bodies described by Braak and colleagues across different stages of Parkinson's disease (PD). This prion‐like behaviour of α‐syn has been recently investigated in animal models of PD or multiple system atrophy (MSA). These models investigate the cell‐to‐cell transfer of α‐syn seeds, or the induction and spreading of α‐syn pathology in transgenic or wild‐type rodent brain. In this review, we first outline the involvement of α‐syn in Lewy body diseases and MSA, and discuss how ‘prion‐like’ mechanisms can contribute to disease. Thereon, we debate the relevance of animal models used to study prion‐like propagation. Finally, we review current main histological methods used to assess α‐syn pathology both in animal models and in human samples and their relevance to the disease. Specifically, we discuss using α‐syn phosphorylated at serine 129 as a marker of pathology, and the novel methods available that allow for more sensitive detection of early pathology, which has relevance for modelling synucleinopathies.     

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     

Bilateral striopallidodentate calcification (Fahr's syndrome) and multiple system atrophy in a patient with longstanding hypoparathyroidism

Recently, a family with idiopathic brain calcification was reported, in which one family member was diagnosed with multiple system atrophy (MSA) at autopsy. We report here a case showing similar neuropathological features in a patient with longstanding hypoparathyroidism. Our female patient had a history of hypoparathyroidism with hypocalcaemia and tetany since the age of 9 years. In her 50s she developed dementia and parkinsonism. She died of myocardial infarction aged 65 years. Neuropathology showed severe brain calcifications of the Fahr type in the basal ganglia, thalami, cerebral and cerebellar white matter and dentate nuclei. Additionally, there was prominent alpha‐synucleinopathy of the multiple system atrophy type (MSA). The patient has a healthy identical twin and there is no family history of hypoparathyroidism or neurological disease. Data on alpha‐synuclein accumulation in various cases of Fahr's syndrome are needed to establish the correlation between alpha‐synucleinopathy and bilateral striopallidodentate calcification.     

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.     

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.     

Microglial activation mediates neurodegeneration related to oligodendroglial alpha-synucleinopathy: implications for multiple system atrophy.

The role of microglial activation in multiple system atrophy (MSA) was investigated in a transgenic mouse model featuring oligodendroglial alpha-synuclein inclusions and loss of midbrain dopaminergic neurons by means of histopathology and morphometric analysis. Our findings demonstrate early progressive microglial activation in substantia nigra pars compacta (SNc) associated with increased expression of iNOS and correlating with dopaminergic neuronal loss. Suppression of microglial activation by early long-term minocycline treatment protected dopaminergic SNc neurons. The results suggest that oligodendroglial overexpression of alpha-synuclein may induce neuroinflammation related to nitrosive stress which is likely to contribute to neurodegeneration in MSA. Further, we detected increased toll-like receptor 4 immunoreactivity in both transgenic mice and MSA brains indicating a possible signaling pathway in MSA which needs to be further studied as a candidate target for neuroprotective interventions.     

Parkinson's disease,cortical dysfunction,and alpha‐synuclein

The ability to understand how Parkinson's disease neurodegeneration leads to cortical dysfunction will be critical for developing therapeutic advances in Parkinson's disease dementia. The overall purpose of this project was to study the small‐amplitude cortical myoclonus in Parkinson's disease as an in vivo model of focal cortical dysfunction secondary to Parkinson's disease neurodegeneration. The objectives were to test the hypothesis that cortical myoclonus in Parkinson's disease is linked to abnormal levels of α‐synuclein in the primary motor cortex and to define its relationship to various biochemical, clinical, and pathological measures. The primary motor cortex was evaluated for 11 Parkinson's disease subjects with and 8 without electrophysiologically confirmed cortical myoclonus (the Parkinson's disease + myoclonus group and the Parkinson's disease group, respectively) who had premortem movement and cognitive testing. Similarly assessed 9 controls were used for comparison. Measurements for α‐synuclein, Aβ‐42 peptide, and other biochemical measures were made in the primary motor cortex. A 36% increase in α‐synuclein was found in the motor cortex of Parkinson's disease + myoclonus cases when compared with Parkinson's disease without myoclonus. This occurred without significant differences in insoluble α‐synuclein, phosphorylated to total α‐synuclein ratio, or Aβ‐42 peptide levels. Higher total motor cortex α‐synuclein levels significantly correlated with the presence of cortical myoclonus but did not correlate with multiple clinical or pathological findings. These results suggest an association between elevated α‐synuclein and the dysfunctional physiology arising from the motor cortex in Parkinson's disease + myoclonus cases. Alzheimer's disease pathology was not associated with cortical myoclonus in Parkinson's disease. Cortical myoclonus arising from the motor cortex is a model to study cortical dysfunction in Parkinson's disease. © 2011 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.     

Tau‐positive glial cytoplasmic granules in multiple system atrophy

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease that is pathologically characterized by the filamentous aggregation of α‐synuclein. We report a case of MSA showing unusual neuropathological findings and review six autopsied cases of MSA. The patient progressively developed parkinsonism and ataxia for the 9 years prior to her death at the age of 72 years. Neuropathological examinations revealed neuronal loss restricted to the olivopontocerebellar and striatonigral region, which was more severe in the putamen. Staining with anti‐α‐synuclein antibody demonstrated widespread occurrence of glial cytoplasmic inclusions, which mainly accumulated in oligodendroglial cells and corresponded closely to the degree of disease progression. In addition, tau‐positive granules were detected within the glial cytoplasm in the neurodegenerative region, which was especially prominent in the putamen and internal capsule. Tau accumulation was also clearly recognized by staining with specific antibodies against three‐repeat or four‐repeat tau. The glia that demonstrated deposition of tau‐positive granules were distinguished from α‐synuclein‐positive oligodendroglia by double immunohistochemical staining. These characteristic glial accumulations of tau were also present in all six cases of MSA. These results indicate that tau‐positive granules in glia are common findings in MSA and that tau aggregation might be another pathway to neurodegeneration in MSA.