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.     

Review: Parkinson's disease: from synaptic loss to connectome dysfunction

Parkinson's disease (PD) is a common neurodegenerative disorder with prominent loss of nigro‐striatal dopaminergic neurons. The resultant dopamine (DA) deficiency underlies the onset of typical motor symptoms (MS). Nonetheless, individuals affected by PD usually show a plethora of nonmotor symptoms (NMS), part of which may precede the onset of motor signs. Besides DA neuron degeneration, a key neuropathological alteration in the PD brain is Lewy pathology. This is characterized by abnormal intraneuronal (Lewy bodies) and intraneuritic (Lewy neurites) deposits of fibrillary aggregates mainly composed of α‐synuclein. Lewy pathology has been hypothesized to progress in a stereotypical pattern over the course of PD and α‐synuclein mutations and multiplications have been found to cause monogenic forms of the disease, thus raising the question as to whether this protein is pathogenic in this disorder. Findings showing that the majority of α‐synuclein aggregates in PD are located at presynapses and this underlies the onset of synaptic and axonal degeneration, coupled to the fact that functional connectivity changes correlate with disease progression, strengthen this idea. Indeed, by altering the proper action of key molecules involved in the control of neurotransmitter release and re‐cycling as well as synaptic and structural plasticity, α‐synuclein deposition may crucially impair axonal trafficking, resulting in a series of noxious events, whose pressure may inevitably degenerate into neuronal damage and death. Here, we provide a timely overview of the molecular features of synaptic loss in PD and disclose their possible translation into clinical symptoms through functional disconnection.     

Immune system responses in Parkinson's disease: Early and dynamic

The neuropathological hallmarks of Parkinson's disease (PD) are the degeneration and death of dopamine‐producing neurons in the ventral midbrain, the widespread intraneuronal aggregation of alpha‐synuclein (α) in Lewy bodies and neurites, neuroinflammation, and gliosis. Signs of microglia activation in the PD brain postmortem as well as during disease development revealed by neuroimaging, implicate immune responses in the pathophysiology of the disease. Intensive research during the last two decades has advanced our understanding of the role of these responses in the disease process, yet many questions remain unanswered. A transformative finding in the field has been the confirmation that in vivo microglia are able to respond directly to pathological a‐syn aggregates but also to neuronal dysfunction due to intraneuronal a‐syn toxicity well in advance of neuronal death. In addition, clinical research and disease models have revealed the involvement of both the innate and adaptive immune systems. Indeed, the data suggest that PD leads not only to a microglia response, but also to a cellular and humoral peripheral immune response. Together, these findings compel us to consider a more holistic view of the immunological processes associated with the disease. Central and peripheral immune responses aimed at maintaining neuronal health will ultimately have consequences on neuronal survival. We will review here the most significant findings that have contributed to the current understanding of the immune response in PD, which is proposed to occur early, involve peripheral and brain immune cells, evolve as neuronal dysfunction progresses, and is likely to influence disease progression.     

How does parkinson's disease begin? Perspectives on neuroanatomical pathways,prions, and histology

Parkinson's disease (PD) is a multisystem disorder with involvement of the peripheral nervous system. Misfolding and aggregation of α‐synuclein is central to the pathogenesis of PD, and it has been postulated that the disease may originate in olfactory and gastrointestinal nerve terminals. The prion‐like behavior of α‐synuclein has been convincingly demonstrated in vitro and in animal models of PD. Lewy‐type pathology have been detected in peripheral organs many years prior to PD diagnosis, and 2 independent studies have now suggested that truncal vagotomy may be protective against the disorder. Other lines of evidence are difficult to reconcile with a peripheral onset of PD, most importantly the relative scarcity of post mortem cases with isolated gastrointestinal α‐synuclein pathology without concomitant CNS pathology. This Scientific Perspectives article revisits some important topics with implications for the dual‐hit hypothesis. An account of the neuroanatomical pathways necessary for stereotypical α‐synuclein spreading is presented. Parallels to the existing knowledge on true prion disorders, including Creutzfeld‐Jakob disease, are examined. Finally, the vagotomy studies and the somewhat inconsistent findings in the growing literature on peripheral α‐synuclein pathology are discussed. It is concluded that the dual‐hit hypothesis remains a potential explanation for PD pathogenesis, but several issues need to be resolved before more firm conclusions can be drawn. © 2017 International Parkinson and Movement Disorder Society     

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     

Co‐expression of C‐terminal truncated alpha‐synuclein enhances full‐length alpha‐synuclein‐induced pathology

Lewy bodies, which are a pathological hallmark of Parkinson’s disease, contain insoluble polymers of alpha‐synuclein (αsyn). Among the different modifications that can promote the formation of toxic αsyn species, C‐terminal truncation is among the most abundant alterations in patients with Parkinson’s disease. In vitro, C‐terminal truncated αsyn aggregates faster and sub‐stoichiometric amounts of C‐terminal truncated αsyn promote aggregation of the full‐length αsyn (αsynFL) and induce neuronal toxicity. To address in vivo the putative stimulation of αsyn‐induced pathology by the presence of truncated αsyn, we used recombinant adeno‐associated virus to express either αsynFL or a C‐terminal truncated αsyn (1‐110) in rats. We adjusted the recombinant adeno‐associated virus vector concentrations so that either protein alone led to only mild to moderate axonal pathology in the terminals of nigrostriatal dopamine neurons without frank cell loss. When these two forms of αsyn were co‐expressed at these pre‐determined levels, it resulted in a more aggressive pathology in fiber terminals as well as dopaminergic cell loss in the substantia nigra. Using an antibody that did not detect the C‐terminal truncated αsyn (1‐110) but only αsynFL, we demonstrated that the co‐expressed truncated protein promoted the progressive accumulation of αsynFL and formation of larger pathological accumulations. Moreover, in the co‐expression group, three of the eight animals showed apomorphine‐induced turning, suggesting prominent post‐synaptic alterations due to impairments in the dopamine release, whereas the mild pathology induced by either form alone did not cause motor abnormalities. Taken together these data suggest that C‐terminal truncated αsyn can interact with and exacerbate the formation of pathological accumulations containing αsynFL in vivo.     

Localization of CHMP2B‐immunoreactivity in the brainstem of Lewy body disease

Alpha‐synuclein (αS) is one of the major constituents of Lewy bodies (LBs). Several lines of evidence suggest that the autophagy‐lysosome pathway (ALP) is involved in the removal of αS. We have previously reported that granulovacuolar degeneration (GVD) in neurons involved a subunit of the endosomal sorting complexes required for transport (ESCRT). In this study, we examined the association between alpha‐synucleinopathy and autophagy through immunohistochemical analysis of charged multivesicular body protein 2B (CHMP2B), a component of the ESCRT‐pathway. We examined the brainstems of 17 patients with Parkinson's disease (PD), incidental Lewy body disease (ILBD), multiple system atrophy (MSA), and Alzheimer's disease (AD) immunohistochemically using antibodies against phosphorylated αS (pαS), phosphorylated tau and CHMP2B. LBs and a proportion of glial cytoplasmic inclusions (GCIs) were immunopositive for pαS and CHMP2B. Neurons containing CHMP2B‐immunoreactive granules were detected in PD and ILBD, but not in MSA and AD brains. CHMP2B immunoreactivity was increased in the dorsal motor nucleus of the vagus nerve (DMNX) in PD and ILBD brains, relative to that in MSA and AD. These findings indicate that the ESCRT‐pathway is implicated in the formation of αS inclusions, especially in PD and ILBD.     

Engineering synucleinopathy‐resistant human dopaminergic neurons by CRISPR‐mediated deletion of the SNCA gene

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory‐generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host‐to‐graft transfer of α‐synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α‐synuclein, in a clinical‐grade hESC line to generate SNCA^+/? and SNCA^?/? cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α‐synuclein preformed fibrils (PFFs) to seed the formation for Lewy‐like pathology as measured by phosphorylation of serine‐129 of α‐synuclein (pS129‐αSyn). Wild‐type neurons were fully susceptible to the formation of protein aggregates positive for pS129‐αSyn, while SNCA^+/? and SNCA^?/? neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n‐mediated gene editing confers a measure of resistance to Lewy pathology.     

Autonomic dysfunction in genetic forms of synucleinopathies

The discovery of genetic links between alpha‐synuclein and PD has opened unprecedented opportunities for research into a new group of diseases, now collectively known as synucleinopathies. Autonomic dysfunction, including cardiac sympathetic denervation, has been reported in familial forms of synucleinopathies that have Lewy bodies at the core of their pathogenesis. SNCA mutations and multiplications, LRRK2 disease with Lewy bodies as well as other common, sporadic forms of idiopathic PD, MSA, pure autonomic failure, and dementia with Lewy bodies have all been associated with dysautonomia. By contrast, in familial cases of parkinsonism without Lewy bodies, such as in PARK2, the autonomic profile remains normal throughout the course of the disease. The degeneration of the central and peripheral autonomic systems in genetic as well as sporadic forms of neurodegenerative synucleinopathies correlates with the accumulation of alpha‐synuclein immunoreactive‐containing inclusions. Given that dysautonomia has a significant impact on the quality of life of sufferers and autonomic symptoms are generally treatable, a prompt diagnostic testing and treatment should be provided. Moreover, new evidence suggests that autonomic dysfunction can be used as an outcome prediction factor in some forms of synucleinopathies or premotor diagnostic markers that could be used in the future to define further research avenues. In this review, we describe the autonomic dysfunction of genetic synucleinopathies in comparison to the dysautonomia of sporadic forms of alpha‐synuclein accumulation and provide the reader with an up‐to‐date overview of the current understanding in this fast‐growing field. © 2018 International Parkinson and Movement Disorder Society     

The usual suspects,dopamine and alpha‐synuclein,conspire to cause neurodegeneration

Parkinson's disease (PD) is primarily a movement disorder driven by the loss of dopamine‐producing neurons in the substantia nigra (SN). Early identification of the oxidative properties of dopamine implicated it as a potential source of oxidative stress in PD, yet few studies have investigated dopamine neurotoxicity in vivo. The discovery of PD‐causing mutations in α‐synuclein and the presence of aggregated α‐synuclein in the hallmark Lewy body pathology of PD revealed another important player. Despite extensive efforts, the precise role of α‐synuclein aggregation in neurodegeneration remains unclear. We recently manipulated both dopamine levels and α‐synuclein expression in aged mice and found that only the combination of these 2 factors caused progressive neurodegeneration of the SN and an associated motor deficit. Dopamine modified α‐synuclein aggregation in the SN, resulting in greater abundance of α‐synuclein oligomers and unique dopamine‐induced oligomeric conformations. Furthermore, disruption of the dopamine‐α‐synuclein interaction rescued dopaminergic neurons from degeneration in transgenic Caenorhabditis elegans models. In this Perspective, we discuss these findings in the context of known α‐synuclein and dopamine biology, review the evidence for α‐synuclein oligomer toxicity and potential mechanisms, and discuss therapeutic implications. © 2019 International Parkinson and Movement Disorder Society     

Can we use peripheral tissue biopsies to diagnose Parkinson's disease? A review of the literature

Phosphorylated α‐synuclein (phosαSYN) containing inclusions in neurons (Lewy bodies, LB) and nerve terminals (Lewy neurites, LN), the pathological hallmark of Parkinson's disease (PD), are not confined to the central nervous system, but have also been reported in peripheral tissues. However, the usefulness of αSYN/phosαSYN detection in tissues accessible to biopsies as a reliable biomarker for prodromal PD remains unclear. A systematic review of studies using biopsies of skin, olfactory and gastrointestinal (GI) tissues was conducted to evaluate the sensitivity and specificity of both αSYN and phosαSYN staining in PD patients. Data analysis was hampered by the diversity of the methods used, e.g. choice of biopsy sites, tissue processing, staining protocols and evaluation of the findings. Tissue obtained from GI tract/salivary glands (13 post‐mortem, 13 in vivo studies) yielded the highest overall sensitivity and specificity compared to skin (three post‐mortem, eight in vivo studies) and olfactory mucosa/bulb (six post‐mortem studies, one in vivo study). In contrast to phosαSYN, αSYN was more consistently detectable in peripheral tissues of healthy controls. GI tract/salivary glands appear to be the most promising candidate tissue for peripheral biopsy‐taking. phosαSYN is considered as the marker of choice to delineate pathological aggregates from normal αSYN regularly found in peripheral neural tissues. However, the sensitivity and specificity of phosαSYN are not yet acceptable for using phosαSYN as a reliable peripheral biomarker for PD in clinical routine. Further refinement regarding the interpretation of the peripheral αSYN/phosαSYN burden and the phenotypical definition of peripheral LB/LN is needed to optimize screening methods for prodromal PD.     

Multiple organ involvement by alpha‐synuclein pathology in Lewy body disorders

Lewy body (LB) diseases are characterized by alpha‐synuclein (AS) aggregates in the central nervous system (CNS). Involvement of the peripheral autonomic nervous system (pANS) is increasingly recognized, although less studied. The aim of this study was to systematically analyze the distribution and severity of AS pathology in the CNS and pANS. Detailed postmortem histopathological study of brain and peripheral tissues from 28 brain bank donors (10 with Parkinson's disease [PD], 5 with dementia with LB [DLB], and 13 with non‐LB diseases including atypical parkinsonism and non‐LB dementia). AS aggregates were found in the pANS of all 15 LB disease cases (PD, DLB) in stellate and sympathetic ganglia (100%), vagus nerve (86.7%), gastrointestinal tract (86.7%), adrenal gland and/or surrounding fat (53.3%), heart (100%), and genitourinary tract (13.3%), as well as in 1 case of incidental Lewy body disease (iLBD). A craniocaudal gradient of AS burden in sympathetic chain and gastrointestinal tract was observed. DLB cases showed higher amounts of CNS AS aggregates than PD cases, but this was not the case in the pANS. No pANS AS aggregates were detected in Alzheimer's disease (AD) cases with or without CNS AS aggregates. All pathologically confirmed LB disease cases including 1 case of iLBD had AS aggregates in the pANS with a craniocaudal gradient of pathology burden in sympathetic chain and gastrointestinal tract. AS was not detected in the pANS of any AD case. These findings may help in the search of peripheral AS aggregates in vivo for the early diagnosis of PD. © 2014 International Parkinson and Movement Disorder Society     

“Preclinical” MSA in definite Creutzfeldt‐Jakob disease

Multiple system atrophy (MSA) is a sporadic alpha‐synucleinopathy clinically characterized by variable degrees of parkinsonism, cerebellar ataxia and autonomic dysfunction. The histopathological hallmark of MSA is glial cytoplasmic inclusion (GCI). It is considered to represent the earliest stage of the degenerative process in MSA and to precede neuronal degeneration. Sporadic Creutzfeldt‐Jakob disease (sCJD) is a fatal, rapidly progressive dementia generally associated with ataxia, pyramidal and extrapyramidal symptoms and myoclonus. Definite diagnosis needs neuropathological demonstration of variable degrees of spongiform degeneration of neuropil, neuronal loss, astro‐ and microgliosis, and the presence of abnormal deposits of the misfolded prion protein PrP^res. Both diseases, CJD and MSA are infrequent among neurodegenerative diseases. In the present report we describe clinical and neuropathological findings of a previously healthy 64‐year‐old woman who developed symptoms of classical CJD. At post mortem examination, the brain showed in addition to classical methionine/methionine PrP^res type 1 (MM1) sCJD changes and moderate Alzheimer‐type pathology, features of “preclinical” MSA with minimal histopathological changes. These were characterized by discrete amounts of alpha‐synuclein immunoreacive glial cytoplasmic inclusions in the striato‐nigral system, isolated intraneuronal inclusions in pigmented neurons of the substantia nigra, as well as some vermiform intranuclear inclusions. To our knowledge, this is the first report on the coexistence of definite sCJD and “minimal changes” MSA in the same patient.     

The first autopsied case of diffuse Lewy body disease (DLBD): re‐examination by recent immunostaining methods

Materials from our first autopsied case of diffuse Lewy body disease (DLBD), that was originally reported in 1976, were re‐examined using recent immunohistochemical methods. Lewy pathology consisting of Lewy bodies and Lewy neurites appeared much more marked with alpha‐synuclein immunostaining than had been detected with classical stainings. This case and our other similar cases prompted us to propose the terms “Lewy body disease” in 1980 and “diffuse Lewy body disease” in 1984. We also reported in 1990 that DLBD was classified into two forms: a pure form and a common form. Based on these studies the term “dementia with Lewy bodies (DLB)” was proposed in 1996. Since 1980, we have insisted that DLB, Parkinson disease (PD), and PD with dementia (PDD) should be understood within the spectrum of Lewy body disease. This insistence has been recently accepted by the International Workshop and the International Working Group on DLB and PDD in 2005 and in 2006, respectively.     

Animal models of Parkinson's disease: Similarities and differences between the disease and models

Parkinson's disease (PD) is a progressive movement disorder characterized by resting tremor, rigidity, akinesia, and postural instability. In addition, PD is characterized by the appearance of Lewy bodies in the remaining neurons. The exact etiology for this disease is still unknown. However, genetic–environmental interaction could contribute to the pathomechanisms of PD. Indeed, seven causative genes responsible for familial PD have been identified. Since discovery of familial PD (FPD), genetic PD models have been developed. Moreover, new PD models using neurotoxins have been reported. In this review, the similarities between human PD and PD models such as genetic mice and Drosophila models are reviewed.     

α‐Synuclein abnormalities in mouse models of peroxisome biogenesis disorders

α‐Synuclein (αS) is a presynaptic protein implicated in Parkinson's disease (PD). Growing evidence implicates mitochondrial dysfunction, oxidative stress, and αS–lipid interactions in the gradual accumulation of αS in pathogenic forms and its deposition in Lewy bodies, the pathological hallmark of PD and related synucleinopathies. The peroxisomal biogenesis disorders (PBD), with Zellweger syndrome serving as the prototype of this group, are characterized by malformed and functionally impaired peroxisomes. Here we utilized the PBD mouse models Pex2–/–, Pex5–/–, and Pex13–/– to study the potential effects of peroxisomal dysfunction on αS‐related pathogenesis. We found increased αS oligomerization and phosphorylation and its increased deposition in cytoplasmic inclusions in these PBD mouse models. Furthermore, we show that αS abnormalities correlate with the altered lipid metabolism and, specifically, with accumulation of long chain, n‐6 polyunsaturated fatty acids that occurs in the PBD models. © 2009 Wiley‐Liss, Inc.     

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.     

Alpha‐synuclein polymorphisms are associated with Parkinson's disease in a Saskatchewan population

Alpha‐synuclein gene (SNCA) mutations cause familial Parkinsonism but the role of SNCA variability in idiopathic Parkinson's disease (PD) remains incompletely defined. We report a study of SNCA genetic variation in 452 idiopathic PD cases and 245 controls. SNCA copy number mutations were not associated with early‐onset disease in this population. The minor allele “G” at rs356165 was associated with increased odds of PD (P = 0.013) and genetic variation in D4S3481 (Rep1) was associated with age of disease onset (P = 0.007). There was a trend toward association between variation at rs2583988 and rapid PD progression. © 2009 Movement Disorder Society