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KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species.

ABSTRACT: KLK6 is a serine protease highly expressed in the nervous system. In synucleinopathies, including Parkinson disease, the levels of KLK6 inversely correlate with ?-synuclein in CSF. Recently, we suggested that recombinant KLK6 mediates the degradation of extracellular ?-synuclein directly and via a proteolytic cascade that involves unidentified metalloproteinase(s). Here, we show that recombinant and naturally secreted KLK6 can readily cleave ?-synuclein fibrils that have the potential for cell-to-cell propagation in "a prion-like mechanism". Importantly, KLK6-deficient primary cortical neurons have increased ability for ?-synuclein fibril uptake. We also demonstrate that KLK6 activates proMMP2, which in turn can cleave ?-synuclein. The repertoire of proteases activated by KLK6 in a neuronal environment was analyzed by degradomic profiling, which also identified ADAMTS19 and showed that KLK6 has a limited number of substrates indicating specific biological functions such as the regulation of ?-synuclein turnover. We generated adenoviral vectors for KLK6 delivery and demonstrated that the levels of extracellular ?-synuclein can be reduced by neuronally secreted KLK6. Our findings open the possibility to exploit KLK6 as a novel therapeutic target for Parkinson disease and other synucleinopathies.

SUBMITTER: Pampalakis G

PROVIDER: S-EPMC5362421 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species.

Pampalakis Georgios G Sykioti Vasia-Samantha VS Ximerakis Methodios M Stefanakou-Kalakou Ioanna I Melki Ronald R Vekrellis Kostas K Sotiropoulou Georgia G

Oncotarget 20170201 9

KLK6 is a serine protease highly expressed in the nervous system. In synucleinopathies, including Parkinson disease, the levels of KLK6 inversely correlate with α-synuclein in CSF. Recently, we suggested that recombinant KLK6 mediates the degradation of extracellular α-synuclein directly and via a proteolytic cascade that involves unidentified metalloproteinase(s). Here, we show that recombinant and naturally secreted KLK6 can readily cleave α-synuclein fibrils that have the potential for cell-t ...[more]

PMID: 27845893

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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

KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species.

Publications

KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species.

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