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Identification of the Tau aggresome in progressive supranuclear palsy by in situ biotinylation by antibody recognition

ABSTRACT: Here, we used an unbiased proteomic approach, which combines antibody-mediated proximity-detection of co-aggregated proteins together with mass spectrometry. Biotinylation by antibody recognition (BAR) is a recently developed method, by which a primary antibody recognises the target of interest in fixed samples. A secondary antibody conjugated to horseradish peroxidase (HRP) recognises the primary antibody, and with the addition of biotin phenol and hydrogen peroxide, facilitates biotinylation of proteins in close proximity. Biotinylated proteins are then biochemically isolated and identified using mass spectrometry (MS).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

SUBMITTER: Albert Lee

LAB HEAD: Albert Lee

PROVIDER: PXD028237 | Pride | 2023-02-27

REPOSITORIES: Pride

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Project description:Parkinson’s disease (PD) is a neurodegenerative disease characterized by progressive motor symptoms and alpha-synuclein (αsyn) aggregation in the nervous system. For unclear reasons, PD patients with certain GBA mutations (GBA-PD) have a more aggressive clinical progression. Two testable hypotheses that can potentially account for this phenomenon are that GBA1 mutations promote αsyn spread or drive the generation of highly pathogenic αsyn polymorphs (i.e., strains). We tested these hypotheses by treating homozygous GBA1 D409V knockin (KI) mice with human α-syn-preformed fibrils (PFFs) and treating wild-type mice (WT) with several αsyn-PFF polymorphs amplified from brain autopsy samples collected from patients with idiopathic PD and GBA-PD patients with either homozygous or heterozygous GBA1 mutations. Robust phosphorylated-αsyn (PSER129) positive pathology was observed at the injection site (i.e., the olfactory bulb granular layer) and throughout the brain six months following PFF injection. The PFF seeding efficiency and degree of spread were similar regardless of the mouse genotype or PFF polymorphs. We found that PFFs amplified from the human brain, regardless of patient genotype, were generally more effective seeders than wholly synthetic PFFs (i.e., non-amplified); however, PFF concentration differed between these two studies, and this might also account for the observed differences. To investigate whether the molecular composition of pathology differed between different seeding conditions, we permed Biotinylation by Antibody Recognition on PSER129 (BAR-PSER129). We found that for BAR-PSER129, the endogenous PSER129 pool dominated identified interactions, and thus, very few potential interactions were explicitly identified for seeded pathology. However, we found Dctn2 interaction was shared across all PFF conditions, and Nckap1 and Ap3b2 were unique to PFFs amplified from GBA-PD brains of heterozygous mutation carriers. In conclusion, both the genotype and αsyn strain had little effect on overall seeding efficacy and global PSER129-interactions.

			Action	DRS
	190507_SR_122.msf	Msf
	190507_SR_122.raw	Raw
	190507_SR_136.msf	Msf
	190507_SR_136.raw	Raw
	190507_SR_136_neg.msf	Msf

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Identification of the Tau aggresome in progressive supranuclear palsy by in situ biotinylation by antibody recognition

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