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Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

ABSTRACT: Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.

SUBMITTER: Kunadt M

PROVIDER: S-EPMC4405286 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The most established pathognomonic protein of Parkinson's disease (PD), α-synuclein, is extensively investigated for disease diagnosis and prognosis; however, investigations into whether the free form of α-synuclein in the blood functions as a PD biomarker have not been fruitful. Extracellular vesicles (EVs) secreted from cells and present in blood transport molecules are novel platforms for biomarker identification. In blood EVs, α-synuclein originates predominantly from the brain without the interference of the blood-brain barrier. The present study investigated the role of plasma EV-borne α-synuclein as a biomarker of PD. Patients with mild to moderate stages of PD (n = 116) and individuals without PD (n = 46) were recruited to serve as the PD study group and the control group, respectively. Plasma EVs were isolated, and immunomagnetic reduction-based immunoassay was used to assess EV α-synuclein levels. Conventional statistical analysis was performed using SPSS 25.0, and p < 0.05 was considered significant. Compared with controls, we observed significantly lower plasma EV α-synuclein levels in the patients with PD (PD: 56.0 ± 3.7 fg/mL vs. control: 74.5 ± 4.3 fg/mL, p = 0.009), and the significance remained after adjustment for age and sex. Plasma EV α-synuclein levels in the patients with PD did not correlate with age, disease duration, Part I and II scores of the Unified Parkinson's Disease Rating Scale (UPDRS), or the Mini-Mental State Examination scores. However, such levels were significantly correlated with UPDRS Part III score, which assesses motor dysfunction. Furthermore, the severity of akinetic-rigidity symptoms, but not tremor, was inversely associated with plasma EV α-synuclein level. Plasma EV α-synuclein was significantly different between the control and PD group and was associated with akinetic-rigidity symptom severity in patients with PD. This study corroborates the possible diagnostic and subtyping roles of plasma EV α-synuclein in patients with PD, and it further provides a basis for this protein's clinical relevance and feasibility as a PD biomarker.

Project description:Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). Previous studies have demonstrated that METH causes alpha-synuclein (α-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of α-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of α-syn is involved in high-dose METH-induced α-syn aggregation. We measured the levels of α-syn SUMOylation and these enzymes involved in the SUMOylation cycle in SH-SY5Y human neuroblastoma cells (SH-SY5Y cells), in cultures of C57 BL/6 primary mouse neurons and in brain tissues of mice exposure to METH. We also demonstrated the effect of α-syn SUMOylation on α-syn aggregation after METH exposure by overexpressing the key enzyme of the SUMOylation cycle or silencing SUMO-1 expression in vitro. Then, we make introduced mutations in the major SUMOylation acceptor sites of α-syn by transfecting a lentivirus containing the sequence of WT α-syn or K96/102R α-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT α-syn or K96/102R α-syn into the mouse striatum. Levels of the ubiquitin-proteasome system (UPS)-related makers ubiquitin (Ub) and UbE1, as well as the autophagy-lysosome pathway (ALP)-related markers LC3, P62 and lysosomal associated membrane protein 2A (LAMP2A), were also measured in SH-SY5Y cells transfected with lentivirus and mice injected with adenovirus. The results showed that METH exposure decreases the SUMOylation level of α-syn, although the expression of α-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in α-syn SUMOylation by UBC9 overexpression relieves METH-induced α-syn overexpression and aggregation, whereas the decrease in α-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of α-syn also aggravate α-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of α-syn plays a fundamental part in α-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.

Project description:The misfolding and fibrillization of the protein, α-synuclein (αsyn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of αsyn misfolding is an important area of interest given that αsyn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate αsyn folding, there is little data pertaining to the mechanism(s) of this interaction. αSyn has previously been shown to associate with small lipid vesicles released by cells called extracellular vesicles (EVs) and it is postulated these interactions may assist in the spreading of pathological forms of this protein. Together, this presents the need for robust characterisation studies on αsyn fibrillization using biologically-derived vesicles. In this study, we comprehensively characterised the ability of lipid-rich small extracellular vesicles (sEVs) to alter the misfolding of αsyn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded αsyn were examined using a range of techniques including: Thioflavin T fluorescence, transmission electron microscopy, analytical centrifugation and western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation. We show that sEVs cause an acceleration in αsyn fibrillization and provide comprehensive evidence that this results in an increase in the abundance of mature insoluble fibrillar species. In order to elucidate the relevance of the lipid membrane to this interaction, sEV lipid membranes were modified by treatment with methanol, or a combination of methanol and sarkosyl. These treatments altered the ultrastructure of the sEVs without changing the protein cargo. Critically, these modified sEVs had a reduced ability to influence αsyn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of αsyn:EV interactions and demonstrates that sEVs are powerful modulators of αsyn fibrillization, which is mediated by the sEV membrane. In doing so, this work provides strong evidence for a role of sEVs in contributing directly to αsyn misfolding in the synucleinopathy disorders.

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Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

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