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Identification of N-linked glycans as specific mediators of neuronal uptake of acetylated ?-Synuclein.

ABSTRACT: Cell-to-cell transmission of toxic forms of ?-Synuclein (?S) is thought to underlie disease progression in Parkinson disease. ?S in humans is constitutively N-terminally acetylated (?Sacetyl), although the impact of this modification is relatively unexplored. Here, we report that ?Sacetyl is more effective at inducing intracellular aggregation in primary neurons than unmodified ?S (?Sun). We identify complex N-linked glycans as binding partners for ?Sacetyl and demonstrate that cellular internalization of ?Sacetyl is reduced significantly upon cleavage of extracellular N-linked glycans, but not other carbohydrates. We verify binding of ?Sacetyl to N-linked glycans in vitro, using both isolated glycans and cell-derived proteoliposomes. Finally, we identify neurexin 1?, a neuronal glycoprotein, as capable of driving glycan-dependent uptake of ?Sacetyl. Importantly, our results are specific to ?Sacetyl because ?Sun does not demonstrate sensitivity for N-linked glycans in any of our assays. Our study identifies extracellular N-linked glycans-and the glycoprotein neurexin 1? specifically-as key modulators of neuronal uptake of ?Sacetyl, drawing attention to the potential therapeutic value of ?Sacetyl-glycan interactions.

SUBMITTER: Birol M

PROVIDER: S-EPMC6599126 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Identification of N-linked glycans as specific mediators of neuronal uptake of acetylated α-Synuclein.

Birol Melissa M Wojcik Slawomir P SP Miranker Andrew D AD Rhoades Elizabeth E

PLoS biology 20190618 6

Cell-to-cell transmission of toxic forms of α-Synuclein (αS) is thought to underlie disease progression in Parkinson disease. αS in humans is constitutively N-terminally acetylated (αSacetyl), although the impact of this modification is relatively unexplored. Here, we report that αSacetyl is more effective at inducing intracellular aggregation in primary neurons than unmodified αS (αSun). We identify complex N-linked glycans as binding partners for αSacetyl and demonstrate that cellular internal ...[more]

PMID: 31211781

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Project description:BackgroundThe aggregation and spread of α-synuclein (α-Syn) protein and related neuronal toxicity are the key pathological features of Parkinson's disease (PD) and Lewy body dementia (LBD). Studies have shown that pathological species of α-Syn and tau can spread in a prion-like manner between neurons, although these two proteins have distinct pathological roles and contribute to different neurodegenerative diseases. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP1) regulates the spread of tau proteins; however, the molecular regulatory mechanisms of α-Syn uptake and spread, and whether it is also regulated by LRP1, remain poorly understood.MethodsWe established LRP1 knockout (LRP1-KO) human induced pluripotent stem cells (iPSCs) isogenic lines using a CRISPR/Cas9 strategy and generated iPSC-derived neurons (iPSNs) to test the role of LRP1 in α-Syn uptake. We treated the iPSNs with fluorescently labeled α-Syn protein and measured the internalization of α-Syn using flow cytometry. Three forms of α-Syn species were tested: monomers, oligomers, and pre-formed fibrils (PFFs). To examine whether the lysine residues of α-Syn are involved in LRP1-mediated uptake, we capped the amines of lysines on α-Syn with sulfo-NHS acetate and then measured the internalization. We also tested whether the N-terminus of α-Syn is critical for LRP1-mediated internalization. Lastly, we investigated the role of Lrp1 in regulating α-Syn spread with a neuronal Lrp1 conditional knockout (Lrp1-nKO) mouse model. We generated adeno-associated viruses (AAVs) that allowed for distinguishing the α-Syn expression versus spread and injected them into the hippocampus of six-month-old Lrp1-nKO mice and the littermate wild type (WT) controls. The spread of α-Syn was evaluated three months after the injection.ResultsWe found that the uptake of both monomeric and oligomeric α-Syn was significantly reduced in iPSNs with LRP1-KO compared with the WT controls. The uptake of α-Syn PFFs was also inhibited in LRP1-KO iPSNs, albeit to a much lesser extent compared to α-Syn monomers and oligomers. The blocking of lysine residues on α-Syn effectively decreased the uptake of α-Syn in iPSNs and the N-terminus of α-Syn was critical for LRP1-mediated α-Syn uptake. Finally, in the Lrp1-nKO mice, the spread of α-Syn was significantly reduced compared with the WT littermates.ConclusionsWe identified LRP1 as a key regulator of α-Syn neuronal uptake, as well as an important mediator of α-Syn spread in the brain. This study provides new knowledge on the physiological and pathological role of LRP1 in α-Syn trafficking and pathology, offering insight for the treatment of synucleinopathies.

Dataset Information

Identification of N-linked glycans as specific mediators of neuronal uptake of acetylated ?-Synuclein.

Publications

Identification of N-linked glycans as specific mediators of neuronal uptake of acetylated α-Synuclein.

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