Resistance of naturally secreted α-synuclein to proteolysis.

Recent evidence suggests that specific extracellular α-synuclein (α-syn) strains are implicated in the progression of Parkinson's disease (PD) pathology. It is plausible that deregulation in the normal processing of secreted α-syn may be a causative risk factor for PD. To date, the degradation mechanisms involved have received very little attention. Here, we sought to investigate factors that regulate extracellular α-syn levels. We show, for the first time, that cell-secreted α-syn forms are resistant to direct proteolysis by kallikrein-related peptidase 6 (KLK6), an extracellular enzyme known to cleave recombinant α-syn. This differential susceptibility appears to be partially due to the association of secreted α-syn with lipids. We further provide evidence that secreted α-syn can be cleaved by KLK6 indirectly through activation of a secreted metalloprotease, suggestive of the involvement of a proteolytic cascade in the catabolism of secreted α-syn. Our results clearly suggest that physiological modifications affect the biochemical behavior of secreted α-syn and provide novel insights into mechanisms and potential targets for therapeutic interventions.-Ximerakis, M., Pampalakis, G., Roumeliotis, T. I., Sykioti, V.-S., Garbis, S. D., Stefanis, L., Sotiropoulou, G., Vekrellis, K. Resistance of naturally secreted α-synuclein to proteolysis.

Intrastriatal Administration of Exosome-Associated Pathological Alpha-Synuclein Is Not Sufficient by Itself to Cause Pathology Transmission.

α-Synuclein (α-syn) has been genetically and biochemically linked to the pathogenesis of Parkinson's disease (PD). There is accumulating evidence that misfolded α-syn species spread between cells in a prion-like manner and seed the aggregation of endogenous protein in the recipient cells. Exosomes have been proposed to mediate the transfer of misfolded α-syn and thus facilitate disease transmission, although the pathological mechanism remains elusive. Here, we investigated the seeding capacity of exosome-associated α-syn, in vivo. Disease-associated α-syn was present in exosome fractions isolated from transgenic A53T mouse brain. However, following intrastriatal injection of such exosomes in wild-type (wt) mice, we were not able to detect any accumulation of endogenous α-syn. In addition, recombinant fibrillar α-syn, when loaded to isolated brain exosomes, induced minor pathological α-syn brain accumulation at 7 months post injection. These data suggest that exosomes neutralize the effect of toxic α-syn species and raise additional questions on their paracrine modulatory role in disease transmission.

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

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-to-cell propagation in "a prion-like mechanism". Importantly, KLK6-deficient primary cortical neurons have increased ability for α-synuclein fibril uptake. We also demonstrate that KLK6 activates proMMP2, which in turn can cleave α-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 α-synuclein turnover. We generated adenoviral vectors for KLK6 delivery and demonstrated that the levels of extracellular α-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.