{"doi":"10.3390/ijms25073643","title":"Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies","abstract":"Synucleinopathies are a group of central nervous system pathologies that are characterized by the intracellular accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in post-mortem specimens from human patients. Neuronal expression of α-synuclein in model organisms, including Drosophila melanogaster, has been a typical approach employed to study its physiological effects. Biochemical analysis of α-synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of α-synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for the evaluation of human α-synuclein solubility isolated from Drosophila tissues has remained elusive. Here, we tested different detergents for their ability to solubilize human α-synuclein carrying the pathological mutation A53T from the brains of aged flies. We also assessed the effect of sonication on the solubility of human α-synuclein and optimized a protocol to discriminate the relative amounts of soluble/insoluble human α-synuclein from dopaminergic neurons of the Drosophila brain. Our data established that, using a 5% SDS buffer, the three-step protocol separates cytosolic soluble, detergent-soluble and insoluble proteins in three sequential fractions according to their chemical properties. This protocol shows that sonication breaks down α-synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and thus enriching the detergent-soluble fraction of the protocol.","journal":"International Journal of Molecular Sciences","year":2024,"id":495686,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9543,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":707574,"name":"Joshua Lopez‐Scarim","orcid":"0000-0003-3290-7670","position":1,"is_corresponding":false},{"id":1335264,"name":"Ila Bagheri","orcid":"0009-0009-0624-4536","position":2,"is_corresponding":false},{"id":291627,"name":"Valerie Joers","orcid":"0000-0001-6743-6587","position":3,"is_corresponding":false},{"id":77489,"name":"Malú G. Tansey","orcid":"0000-0002-1719-4708","position":4,"is_corresponding":false},{"id":652242,"name":"Alfonso Martín‐Peña","orcid":"0000-0001-7598-0777","position":5,"is_corresponding":false},{"id":1335263,"name":"Khondamir Imomnazarov","orcid":"0009-0007-0808-7808","position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":null,"created_at":"2026-07-19T02:09:19.627139Z","pmid":"38612454","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}