ATH434 Reduces α-Synuclein-Related Neurodegeneration in a Murine Model of Multiple System Atrophy.

BACKGROUND: Multiple system atrophy (MSA) is a fatal neurodegenerative disorder characterized by aggregated α-synuclein (α-syn) in oligodendrocytes and accompanied by striatonigral and olivopontocerebellar degeneration and motor symptoms. Key features of MSA are replicated in the PLP-α-syn transgenic mouse, including progressive striatonigral degeneration and motor deterioration. There are currently no approved treatments for MSA. ATH434 is a novel, orally bioavailable brain penetrant small molecule inhibitor of α-syn aggregation. OBJECTIVES: To characterize ATH434 for disease modification in a mouse model of MSA. METHODS: Six-month-old PLP-α-syn mice (MSA mice) were ATH434-treated (ATH434 in food) or untreated (normal food) for 6 months. Motor behavior and numbers of nigral and striatal neurons were evaluated. α-syn aggregates and oligomers were quantified by immunohistochemical and western blot analyses. Microglial activation and neuroinflammation were assessed by histological and molecular analyses. Ferric iron in the Substantia nigra was evaluated with the Perls method. RESULTS: ATH434-treated mice demonstrated preservation of motor performance in MSA mice that was associated with neuroprotection of nigral and striatal neurons. The rescue of the phenotype correlated with the reduction of α-syn inclusions and oligomers in animals receiving ATH434. ATH434-treated mice exhibited significantly increased lysosomal activity of microglia without increased pro-inflammatory markers, suggesting a role in α-syn clearing. ATH434-treatment was associated with lower intracellular nigral iron levels. CONCLUSIONS: Our findings demonstrate the beneficial disease-modifying effect of ATH434 in oligodendroglial α-synucleinopathy on both the motor phenotype and neurodegenerative pathology in the PLP-α-syn transgenic mouse and support the development of ATH434 for MSA. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Shared Genetics of Multiple System Atrophy and Inflammatory Bowel Disease.

BACKGROUND: Multiple system atrophy (MSA) is a rare neurodegenerative disease characterized by intracellular accumulations of α-synuclein and nerve cell loss in striatonigral and olivopontocerebellar structures. Epidemiological and clinical studies have reported potential involvement of autoimmune mechanisms in MSA pathogenesis. However, genetic etiology of this interaction remains unknown. We aimed to investigate genetic overlap between MSA and 7 autoimmune diseases and to identify shared genetic loci. METHODS: Genome-wide association study summary statistics of MSA and 7 autoimmune diseases were combined in cross-trait conjunctional false discovery rate analysis to explore overlapping genetic background. Expression of selected candidate genes was compared in transgenic MSA mice and wild-type mice. Genetic variability of candidate genes was further investigated using independent whole-exome genotyping data from large cohorts of MSA and autoimmune disease patients and healthy controls. RESULTS: We observed substantial polygenic overlap between MSA and inflammatory bowel disease and identified 3 shared genetic loci with leading variants upstream of the DENND1B and RSP04 genes, and in intron of the C7 gene. Further, the C7 gene showed significantly dysregulated expression in the degenerating midbrain of transgenic MSA mice compared with wild-type mice and had elevated burden of protein-coding variants in independent MSA and inflammatory bowel disease cohorts. CONCLUSION: Our study provides evidence of shared genetic etiology between MSA and inflammatory bowel disease with an important role of the C7 gene in both phenotypes, with the implication of immune and gut dysfunction in MSA pathophysiology. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

High-salt diet does not boost neuroinflammation and neurodegeneration in a model of α-synucleinopathy.

AIM: Pre-clinical studies in models of multiple sclerosis and other inflammatory disorders suggest that high-salt diet may induce activation of the immune system and potentiate inflammation. However, high-salt diet constitutes a common non-pharmacological intervention to treat autonomic problems in synucleinopathies such as Parkinson's disease and multiple system atrophy. Since neuroinflammation plays an important pathogenic role in these neurodegenerative disorders, we asked here whether high-salt diet may aggravate the disease phenotype in a transgenic model of multiple system atrophy. METHODS: Nine-month-old PLP-hαSyn and matched wildtype mice received normal or high-salt diet for a period of 3 months. Behavioral, histological, and molecular analyses were performed to evaluate the effect of high-salt diet on motor decline, neuroinflammation, neurodegeneration, and α-synuclein accumulation in these mice. RESULTS: Brain subregion-specific molecular and histological analyses showed no deleterious effects of high-salt diet on the level of microglial activation. Moreover, neuroinflammation-related cytokines and chemokines, T cell recruitment or astrogliosis were unaffected by high-salt diet exposure. Behavioral testing showed no effect of diet on motor decline. High-salt diet was not related to the deterioration of neurodegeneration or α-synuclein accumulation in PLP-hαSyn mice. CONCLUSIONS: Here, we demonstrate that high-salt diet does not aggravate neuroinflammation and neurodegeneration in PLP-hαSyn mice. Our findings discard a deleterious pro-neuroinflammatory effect of high-salt diet in multiple system atrophy.

Signs of Chronic Hypoxia Suggest a Novel Pathophysiological Event in α-Synucleinopathies.

BACKGROUND: Multiple system atrophy (MSA) and Parkinson's disease (PD) patients develop respiratory and cardiovascular disturbances including obstructive sleep apnea, orthostatic hypotension, and nocturnal stridor. We hypothesized that, associated with these respiratory and cardiovascular disturbances, hypoxic events may occur in MSA and PD brains that may play a role in disease progression. The objective of this study was to evaluate the presence of hypoxia in nonneurological controls and PD and MSA patients. METHODS: Molecular levels of hypoxia markers were measured in postmortem brain tissue from controls and PD and MSA cases. RESULTS: MSA brain showed signs of chronic hypoxia characterized by the significant accumulation of the hypoxic marker HIF2α as compared to PD patients and controls. We detected no differences between MSA subtypes. Signs of hypoxia were also observed in PD patients with a clinical presentation similar to the MSA cases. CONCLUSIONS: The results obtained from this study suggest a new alternative pathway associated with α-synucleinopathies that may contribute to the pathogenesis of these disorders. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Anle138b modulates α-synuclein oligomerization and prevents motor decline and neurodegeneration in a mouse model of multiple system atrophy.

BACKGROUND: MSA is a fatal neurodegenerative disease characterized by autonomic failure and severe motor impairment. Its main pathological hallmark is the accumulation of α-synuclein in oligodendrocytes, leading to glial and neuronal dysfunction and neurodegeneration. These features are recapitulated in the PLP-hαSyn mouse model expressing human α-synuclein in oligodendrocytes. At present, there is no effective disease-modifying therapy. Previous experiments have shown that the aggregation inhibitor, anle138b, reduces neurodegeneration and behavioral deficits in mouse models of other proteinopathies. OBJECTIVES: To test the therapeutic potential of anle138b in a mouse model of MSA. METHODS: Two-month-old PLP-hαSyn mice were fed over a period of 4 months with pellets containing anle138b at two different doses (0.6 and 2 g/kg) and compared to healthy controls and PLP-hαSyn mice fed with placebo pellets. At the end of the treatment, behavioral and histological analyses were performed. RESULTS: We observed a reversal of motor function to healthy control levels when PLP-hαSyn mice were treated with both doses of anle138b. Histological and molecular analyses showed a significant reduction in α-synuclein oligomers and glial cytoplasmic inclusions in animals fed with anle138b compared to nontreated mice. These animals also present preservation of dopaminergic neurons and reduction in microglial activation in SN correlating with the α-synuclein reduction observed. CONCLUSIONS: Anle138b reduces α-synuclein accumulation in PLP-hαSyn mice, leading to neuroprotection, reduction of microglial activation, and preservation of motor function supporting the use of anle138b in a future clinical trial for MSA. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Prolyl hydroxylase-dependent modulation of eukaryotic elongation factor 2 activity and protein translation under acute hypoxia.

Early adaptive responses to hypoxia are essential for cell survival, but their nature and underlying mechanisms are poorly known. We have studied the post-transcriptional changes in the proteome of mammalian cells elicited by acute hypoxia and found that phosphorylation of eukaryotic elongation factor 2 (eEF2), a ribosomal translocase whose phosphorylation inhibits protein synthesis, is under the precise and reversible control of O(2) tension. Upon exposure to hypoxia, phosphorylation of eEF2 at Thr(56) occurred rapidly (<15 min) and resulted in modest translational arrest, a fundamental homeostatic response to hypoxia that spares ATP and thus facilitates cell survival. Acute inhibitory eEF2 phosphorylation occurred without ATP depletion or AMP kinase activation. Furthermore, eEF2 phosphorylation was mimicked by prolyl hydroxylase (PHD) inhibition with dimethyloxalylglycine or by selective PHD2 siRNA silencing but was independent of hypoxia-inducible factor α stabilization. Moreover, overexpression of PHD2 blocked hypoxic accumulation of phosphorylated eEF2. Therefore, our findings suggest that eEF2 phosphorylation status (and, as a consequence, translation rate) is controlled by PHD2 activity. They unravel a novel pathway for cell adaptation to hypoxia that could have pathophysiologic relevance in tissue ischemia and cancer.

The Concept of α-Synuclein Strains and How Different Conformations May Explain Distinct Neurodegenerative Disorders.

In the past few years, an increasing amount of studies primarily based on experimental models have investigated the existence of distinct α-synuclein strains and their different pathological effects. This novel concept could shed light on the heterogeneous nature of α-synucleinopathies, a group of disorders that includes Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, which share as their key-molecular hallmark the abnormal aggregation of α-synuclein, a process that seems pivotal in disease pathogenesis according to experimental observations. However, the etiology of α-synucleinopathies and the initial events leading to the formation of α-synuclein aggregates remains elusive. Hence, the hypothesis that structurally distinct fibrillary assemblies of α-synuclein could have a causative role in the different disease phenotypes and explain, at least to some extent, their specific neurodegenerative, disease progression, and clinical presentation patterns is very appealing. Moreover, the presence of different α-synuclein strains might represent a potential biomarker for the diagnosis of these neurodegenerative disorders. In this regard, the recent use of super resolution techniques and protein aggregation assays has offered the possibility, on the one hand, to elucidate the conformation of α-synuclein pathogenic strains and, on the other hand, to cyclically amplify to detectable levels low amounts of α-synuclein strains in blood, cerebrospinal fluid and peripheral tissue from patients. Thus, the inclusion of these techniques could facilitate the differentiation between α-synucleinopathies, even at early stages, which is crucial for successful therapeutic intervention. This mini-review summarizes the current knowledge on α-synuclein strains and discusses its possible applications and potential benefits.

Hypoxia compromises the mitochondrial metabolism of Alzheimer's disease microglia via HIF1.

Genetic Alzheimer's disease (AD) risk factors associate with reduced defensive amyloid ß plaque-associated microglia (AßAM), but the contribution of modifiable AD risk factors to microglial dysfunction is unknown. In AD mouse models, we observe concomitant activation of the hypoxia-inducible factor 1 (HIF1) pathway and transcription of mitochondrial-related genes in AßAM, and elongation of mitochondria, a cellular response to maintain aerobic respiration under low nutrient and oxygen conditions. Overactivation of HIF1 induces microglial quiescence in cellulo, with lower mitochondrial respiration and proliferation. In vivo, overstabilization of HIF1, either genetically or by exposure to systemic hypoxia, reduces AßAM clustering and proliferation and increases Aß neuropathology. In the human AD hippocampus, upregulation of HIF1α and HIF1 target genes correlates with reduced Aß plaque microglial coverage and an increase of Aß plaque-associated neuropathology. Thus, hypoxia (a modifiable AD risk factor) hijacks microglial mitochondrial metabolism and converges with genetic susceptibility to cause AD microglial dysfunction.

From Synaptic Protein to Prion: The Long and Controversial Journey of α-Synuclein.

Since its discovery 30 years ago, α-synuclein (α-syn) has been one of the most studied proteins in the field of neuroscience. Dozens of groups worldwide have tried to reveal not only its role in the CNS but also in other organs. α-syn has been linked to several processes essential in brain homeostasis such as neurotransmitter release, synaptic function, and plasticity. However, despite the efforts made in this direction, the main function of α-syn is still unknown. Moreover, α-syn became a protein of interest for neurologists and neuroscientists when mutations in its gene were found associated with Parkinson's disease (PD) and even more when α-syn protein deposits were observed in the brain of PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) patients. At present, the abnormal accumulation of α-syn constitutes one of the pathological hallmarks of these disorders, also referred to as α-synucleinopathies, and it is used for post-mortem diagnostic criteria. Whether α-syn aggregation is cause or consequence of the pathogenic events underlying α-synucleinopathies remains unclear and under discussion. Recently, different in vitro and in vivo studies have shown the ability of pathogenic α-syn to spread between cells, not only within the CNS but also from peripheral locations such as the gut, salivary glands, and through the olfactory network into the CNS, inducing abnormal misfolding of endogenous α-syn and leading to neurodegeneration and motor and cognitive impairment in animal models. Thus, it has been suggested that α-syn should be considered a prion protein. Here we present an update of what we know about α-syn function, aggregation and spreading, and its role in neurodegeneration. We also discuss the rationale and findings supporting the hypothetical prion nature of α-syn, its weaknesses, and future perspectives for research and the development of disease-modifying therapies.

MSA: From basic mechanisms to experimental therapeutics.

Multiple system atrophy (MSA) is a rare and fatal neurodegenerative disorder characterized by rapidly progressive autonomic and motor dysfunction. Pathologically, MSA is mainly characterized by the abnormal accumulation of misfolded α-synuclein in the cytoplasm of oligodendrocytes, which plays a major role in the pathogenesis of the disease. Striatonigral degeneration and olivopontecerebellar atrophy underlie the motor syndrome, while degeneration of autonomic centers defines the autonomic failure in MSA. At present, there is no treatment that can halt or reverse its progression. However, over the last decade several studies in preclinical models and patients have helped to better understand the pathophysiological events underlying MSA. The etiology of this fatal disorder remains unclear and may be multifactorial, caused by a combination of factors which may serve as targets for novel therapeutic approaches. In this review, we summarize the current knowledge about the etiopathogenesis and neuropathology of MSA, its different preclinical models, and the main disease modifying therapies that have been used so far or that are planned for future clinical trials.

Targeting α-synuclein by PD03 AFFITOPE® and Anle138b rescues neurodegenerative pathology in a model of multiple system atrophy: clinical relevance.

BACKGROUND: Misfolded oligomeric α-synuclein plays a pivotal role in the pathogenesis of α-synucleinopathies including Parkinson's disease and multiple system atrophy, and its detection parallels activation of microglia and a loss of neurons in the substantia nigra pars compacta. Here we aimed to analyze the therapeutic efficacy of PD03, a new AFFITOPE® immunotherapy approach, either alone or in combination with Anle138b, in a PLP-α-syn mouse model. METHODS: The PLP-α-syn mice were treated with PD03 immunotherapy, Anle138b, or a combination of two. Five months after study initiation, the mice underwent behavioral testing and were sacrificed for neuropathological analysis. The treatment groups were compared to the vehicle group with regard to motor performance, nigral neuronal loss, microglial activation and α-synuclein pathology. RESULTS: The PLP-α-syn mice receiving the PD03 or Anle138b single therapy showed improvement of gait deficits and preservation of nigral dopaminergic neurons associated with the reduced α-synuclein oligomer levels and decreased microglial activation. The combined therapy with Anle138b and PD03 resulted in lower IgG binding in the brain as compared to the single immunotherapy with PD03. CONCLUSIONS: PD03 and Anle138b can selectively target oligomeric α-synuclein, resulting in attenuation of neurodegeneration in the PLP-α-syn mice. Both approaches are potential therapies that should be developed further for disease modification in α-synucleinopathies.

Cognition in multiple system atrophy: a single-center cohort study.

OBJECTIVE: Cognitive impairment in multiple system atrophy (MSA) is common, but remain poorly characterized. We evaluated cognitive and behavioral features in MSA patients and assessed between-group differences for MSA subtypes and the effect of orthostatic hypotension (OH) on cognition. METHODS: This retrospective study included 54 patients with clinical diagnosis of possible and probable MSA referred to the Department of Neurology at Medical University of Innsbruck between 2000 and 2018. Neurological work-up included comprehensive neuropsychological testing including Consortium to Establish a Registry for Alzheimer's Disease (CERAD-plus) test battery, Frontal Assessment Battery (FAB), digit span test (DST), clock drawing task (CLOX1), and Hospital Anxiety and Depression Scale (HADS-D). RESULTS: The mean MMSE score was 27.6 points. Overall, slight to moderate cognitive impairment was noted in up to 40% of patients, with predominant impairment of executive function and verbal memory. Patients with the cerebellar variant performed significantly worse than patients with the parkinsonian type (P < 0.05) in a screening of executive functions (FAB) and in phonemic verbal fluency. Depression and anxiety scores were elevated in 28% and 22% of MSA patients, respectively. Cognitive profile, depression, and anxiety levels were comparable between patients with and without OH. INTERPRETATION: Cognitive deficits are relatively frequent in MSA and primarily affect executive functions and verbal memory. Future comparative studies including Parkinson dementia, Lewy body disease, and MSA cases with and without OH are required to elucidate disease-specific cognitive profiles in these synucleinopathies and to examine the influence of cardiovascular autonomic dysfunction on cognitive function in MSA.

The molecular tweezer CLR01 reduces aggregated, pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy.

Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development.

Acute and Chronic Sustained Hypoxia Do Not Substantially Regulate Amyloid-ß Peptide Generation In Vivo.

BACKGROUND: Recent epidemiological evidence has linked hypoxia with the development of Alzheimer disease (AD). A number of in vitro and in vivo studies have reported that hypoxia can induce amyloid-ß peptide accumulation through various molecular mechanisms including the up-regulation of the amyloid-ß precursor protein, the ß-secretase Bace1, or the γγ-secretase complex components, as well as the down-regulation of Aß-degrading enzymes. OBJECTIVES: To investigate the effects of acute and chronic sustained hypoxia in Aß generation in vivo. METHODS: 2-3 month-old C57/Bl6J wild-type mice were exposed to either normoxia (21% O2) or hypoxia (9% O2) for either 4 to 72 h (acute) or 21-30 days (chronic sustained) in a hermetic chamber. Brain mRNA levels of Aß-related genes were measured by quantitative real-time PCR, whereas levels of Bace1 protein, full length AßPP, and its C-terminal fragments (C99/C88 ratio) were measured by Western blot. In addition, 8 and 14-month-old APP/PS1 transgenic mice were subjected to 9% O2 for 21 days and levels of Aß40, Aß42, full length AßPP, and soluble AßPPα (sAßPPα) were measured by ELISA or WB. RESULTS: Hypoxia (either acute or chronic sustained) did not impact the transcription of any of the Aß-related genes in young wild-type mice. A significant reduction of Bace1 protein level was noted with acute hypoxia for 16 h but did not correlate with an increased level of full length AßPP or a decreased C99/C83 ratio. Chronic sustained hypoxia did not significantly alter the levels of Bace1, full length AßPP or the C99/C83 ratio. Last, chronic sustained hypoxia did not significantly change the levels of Aß40, Aß42, full length AßPP, or sAßPPα in either young or aged APP/PS1 mice. DISCUSSION: Our results argue against a hypoxia-induced shift of AßPP proteolysis from the non-amyloidogenic to the amyloidogenic pathways. We discuss the possible methodological caveats of previous in vivo studies.