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Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.

ABSTRACT: ?-synuclein (?S) is a small protein that self-aggregates into ?-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of ?S oligomers and fibrils has been long debated and more recent data are suggesting that both species can induce neurodegeneration. However while most of these data are based on differences in structure between oligomer and aggregates, often preassembled in vitro, the in vivo situation might be more complex and subcellular locations where ?S species accumulate, rather than their conformation, might contribute to enhanced toxicity. In line with this observation, we have shown that ?S oligomers and aggregates are associated with the endoplasmic reticulum/microsomes (ER/M) membrane in vivo and how accumulation of soluble ?S oligomers at the ER/M level precedes neuronal degeneration in a mouse model of ?-synucleinopathies. In this paper we took a further step, investigating the biochemical and functional features of ?S species associated with the ER/M membrane. We found that by comparison with non-microsomal associated ?S (P10), the ER/M-associated ?S pool is a unique population of oligomers and aggregates with specific biochemical traits such as increased aggregation, N- and C-terminal truncations and phosphorylation at serine 129. Moreover, when administered to murine primary neurons, ER/M-associated ?S species isolated from diseased A53T human ?S transgenic mice induced neuronal changes in a time- and dose-dependent manner. In fact the addition of small amounts of ER/M-associated ?S species from diseased mice to primary cultures induced the formation of beads-like structures or strings of fibrous ?S aggregates along the neurites, occasionally covering the entire process or localizing at the soma level. By comparison treatment with P10 fractions from the same diseased mice resulted in the formation of scarce and small puncta only when administered at high amount. Moreover, increasing the amount of P100/M fractions obtained from diseased and, more surprisingly, from presymptomatic mice induced a significant level of neuronal death that was prevented when neurons were treated with ER/M fractions immunodepleted of ?S high molecular weight (HMW) species. These data provide the first evidence of the existence of two different populations of ?S HMW species in vivo, putting the spotlight on the association to ER/M membrane as a necessary step for the acquisition of ?S toxic features.

SUBMITTER: Colla E

PROVIDER: S-EPMC6705388 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.

Colla Emanuela E Panattoni Giulia G Ricci Alessio A Rizzi Caterina C Rota Lucia L Carucci Nicola N Valvano Verdiana V Gobbo Francesco F Capsoni Simona S Lee Michael K MK Cattaneo Antonino A

Neurobiology of disease 20171212

α-synuclein (αS) is a small protein that self-aggregates into α-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of αS oligomers and fibrils has been long debated and more recent data are suggesting that both species can induce neurodegeneration. However while most of these data are based on differences in structure between oligomer and aggregates, often preassembled ...[more]

PMID: 29246724

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Project description:BACKGROUND: Alpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis. Abnormal accumulation of alpha-synuclein aggregates in dopaminergic neurons of the substantia nigra is diagnostic of sporadic Parkinson's disease, and mutations in the protein are linked to early onset forms of the disease. The folded conformation of the protein varies depending upon its environment and other factors that are poorly understood. When bound to phospholipid membranes, alpha-synuclein adopts a helical conformation that mediates specific interactions with other proteins. RESULTS: To investigate the role of the helical domain in transport and localization of alpha-synuclein, eGFP-tagged constructs were transfected into rat primary hippocampal neurons at 7 DIV. A series of constructs were analyzed in which each individual exon was deleted, for comparison to previous studies of lipid affinity and alpha-helix content. A53T and A30P substitutions, representing Parkinson's disease-associated variants, were analyzed as well. Single exon deletions within the lipid-binding N-terminal domain of alpha-synuclein (exons 2, 3, and 4) partially disrupted its presynaptic localization at 17-21 DIV, resulting in increased diffuse labeling of axons. Similar results were obtained for A30P, which exhibits decreased lipid binding, but not A53T. To examine whether differences in presynaptic enrichment were related to deficiencies in transport velocity, transport was visualized via live cell microscopy. Tagged alpha-synuclein migrated at a rate of 1.85 +/- 0.09 mum/s, consistent with previous reports, and single exon deletion mutants migrated at similar rates, as did A30P. Deletion of the entire N-terminal lipid-binding domain (Delta234GFP) did not significantly alter rates of particle movement, but decreased the number of moving particles. Only the A53TGFP mutant exhibited a significant decrease in transport velocity as compared to ASGFP. CONCLUSIONS: These results support the hypothesis that presynaptic localization involves a mechanism that requires helical conformation and lipid binding. Conversely, the rate of axonal transport is not determined by lipid affinity and is not sufficient to account for differences in presynaptic localization of alpha-synuclein-eGFP variants.

Project description:Alpha-synuclein pathology is associated with dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson's patients. Working across human and mouse models, we investigated mechanisms by which the accumulation of soluble α-synuclein oligomers leads to neurodegeneration. Biochemical analysis of the midbrain of α-synuclein overexpressing BAC-transgenic male and female mice revealed age- and region-dependent mitochondrial dysfunction and accumulation of damaged proteins downstream of the RE1 Silencing Transcription Factor (REST). Vulnerable SN dopaminergic neurons displayed low REST levels compared with neighboring protected SN GABAergic neurons, which correlated with the accumulation of α-synuclein oligomers and disrupted mitochondrial morphology. Consistent with a protective role, REST levels were reduced in patient induced pluripotent stem cell-derived dopaminergic neurons carrying the SNCA-Triplication mutation, which accumulated α-synuclein oligomers and mitochondrial damage, and displayed REST target gene dysregulation. Furthermore, CRISPR-mediated REST KO induced mitochondrial dysfunction and impaired mitophagy in vitro Conversely, REST overexpression attenuated mitochondrial toxicity and mitochondrial morphology disruption through the transcription factor PGC-1α. Finally, decreased α-synuclein oligomer accumulation and mitochondrial dysfunction in mice correlated with nuclear REST and PGC-1α in protected SN GABAergic neurons compared with vulnerable dopaminergic neurons. Our findings show that increased levels of α-synuclein oligomers cause dopaminergic neuronal-specific dysfunction through mitochondrial toxicity, which can be attenuated by REST in an early model of Parkinsonian pathology. These findings highlight REST as a mediator of dopaminergic vulnerability in PD.SIGNIFICANCE STATEMENT Understanding early Parkinsonian pathophysiology through studies of advanced preclinical models is fundamental to the translation of disease-modifying therapies. Here we show disease-relevant levels of α-synuclein expression in mice leads to accumulation of α-synuclein oligomers in the absence of overt aggregation, and mitochondrial dysfunction in dopaminergic neurons lacking the RE1 Silencing Transcription Factor. Our findings identify the mechanism of action of RE1 Silencing Transcription Factor and PGC-1α as mediators of dopaminergic vulnerability in α-synuclein BAC-transgenic mice and induced pluripotent stem cell-derived dopaminergic cultures, highlighting their potential as therapeutic targets.

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Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.

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Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.

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