Role of alpha-synuclein phosphorylation at Serine 129 in methamphetamine-induced neurotoxicity in vitro and in vivo.

The phosphorylation and aggregation of alpha-synuclein (α-Syn) play a key role in methamphetamine (METH)-induced dopaminergic neurotoxicity. The exact mechanism underlying the interaction between METH-induced neurotoxicity and α-Syn was poorly clarified. We aimed to figure out the role of serine 129 phosphorylation (pS129) of α-Syn on its aggregation and neurotoxicity in vitro and in vivo. In this study, we examined pS129 α-Syn expression in vitro and in vivo at the protein phosphorylation and genetic levels and evaluated its effect on METH-induced neurotoxicity. Here, we found that pS129 α-Syn was significantly increased after METH treatment; moreover, the neuronal α-Syn aggregation and apoptosis caused by METH exposure were significantly attenuated after inhibiting α-Syn phosphorylation. We demonstrate that pS129 α-Syn contributes to the aggregation of α-Syn, and that phosphorylated and aggregated forms of α-Syn play an important role in METH-induced neurotoxicity in dopaminergic neurons and SH-SY5Y cells, supporting a potential insight into the treatment of METH-induced neurotoxicity.

The effect of α-synuclein and Tau in methamphetamine induced neurotoxicity in vivo and in vitro.

The upregulated α-synuclein (α-syn) and Tau co-occur in methamphetamine (METH) abusers' brains. Here, we designed experiments mainly to investigate whether α-syn and Tau interact in METH exposure. We detected the expression of α-syn, total Tau, and phosphorylation of Tau at Serine 396 (pSer396 Tau) under in vitro and in vivo conditions after METH exposure to determine the co-occurrence of α-syn and Tau. We also explored the effect of α-syn or Tau on one another by silencing and knocking-out one of them in METH treatment. We found that METH increased the α-syn, total Tau, and pSer396 Tau protein level in SH-SY5Y cells, primary cultured neurons, and in mice brains. In additional, reducing α-syn level can relieve and even normalize the pSer396 Tau and total Tau overexpression after treatment of METH. Furthermore, knocking out Tau can effectively inhibit METH induced overexpression of α-syn in mice brains. Finally, knocking out α-syn or Tau can effectively reduce METH-induced neurotoxicity in mice brains. This research could provide potential therapeutic approaches targeting the vicious circle between α-syn and Tau in METH abusers and patients with neurodegenerative disorders.

The role of chaperone-mediated autophagy in neurotoxicity induced by alpha-synuclein after methamphetamine exposure.

INTRODUCTION: Chaperone-mediated autophagy (CMA) is an autophagy-lysosome pathway (ALP) that is different from the other two lysosomal pathways, namely, macroautophagy and microautophagy, and can selectively degrade cytosolic proteins in lysosomes without vesicle formation. CMA activity declines in neurodegenerative diseases such as Parkinson's disease, and similar neurotoxicity can occur after methamphetamine (METH) treatment. The relationship between CMA and METH-induced neurotoxicity is not clear. METHODS: We detected changes in the chaperone protein Hsc70 and the lysosomal surface receptor Lamp-2a after METH treatment and then regulated these two proteins by small interfering RNA and DNA plasmid transfection to investigate how CMA influences METH-induced neurotoxicity. RESULTS: We found that CMA activity is decreased after METH exposure in neurons and downregulated Lamp-2a can aggravate the neurotoxicity induced by α-Syn after METH exposure and that Hsc70 overexpression can relieve the abnormal levels of alpha-synuclein and its aggregate forms and the increase in cell apoptosis induced by METH. CONCLUSIONS: The results provide in vivo evidence for CMA plays a pivotal role in METH-induced neurotoxicity, and upregulation of Hsc70 expression significantly protects neuronal cells against METH-induced toxicity. This research may pave the way for potential therapeutic approaches targeting CMA for METH abuse and neurodegenerative disorders.

SUMOylation of Alpha-Synuclein Influences on Alpha-Synuclein Aggregation Induced by Methamphetamine.

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.