Alpha-synuclein aggregates are phosphatase resistant.
alpha-Synuclein
/ metabolism
Animals
Brain
/ metabolism
Mice, Inbred C57BL
Mice
Phosphorylation
Humans
Protein Aggregates
/ physiology
Male
Mice, Transgenic
Protein Aggregation, Pathological
/ metabolism
Alkaline Phosphatase
/ metabolism
Synucleinopathies
/ metabolism
Phosphoric Monoester Hydrolases
/ metabolism
Parkinson’s disease pathogenesis
Post-translational modifications
Postmortem interval
Protein aggregation
Journal
Acta neuropathologica communications
ISSN: 2051-5960
Titre abrégé: Acta Neuropathol Commun
Pays: England
ID NLM: 101610673
Informations de publication
Date de publication:
31 May 2024
31 May 2024
Historique:
received:
09
04
2024
accepted:
12
04
2024
medline:
1
6
2024
pubmed:
1
6
2024
entrez:
1
6
2024
Statut:
epublish
Résumé
Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.
Identifiants
pubmed: 38822421
doi: 10.1186/s40478-024-01785-0
pii: 10.1186/s40478-024-01785-0
doi:
Substances chimiques
alpha-Synuclein
0
Protein Aggregates
0
Alkaline Phosphatase
EC 3.1.3.1
Phosphoric Monoester Hydrolases
EC 3.1.3.2
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
84Subventions
Organisme : NINDS NIH HHS
ID : 1R01NS128467
Pays : United States
Organisme : NINDS NIH HHS
ID : R21NS109871
Pays : United States
Organisme : Michael J. Fox Foundation for Parkinson's Research
ID : MJFF-022480
Organisme : Michael J. Fox Foundation for Parkinson's Research
ID : ASAP-024442
Informations de copyright
© 2024. The Author(s).
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