Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Parkinson's disease (PD) is neuropathologically defined by Lewy bodies (LBs), primarily composed of fibrillar alpha-synuclein.
• A significant portion (approx. 90%) of alpha-synuclein in LBs is phosphorylated at Serine 129 (Ser129), contrasting with normal brains (approx. 4%).
• The reason for extensive Ser129 phosphorylation in PD pathogenesis remains unclear.

Purpose of the Study:

• To investigate the mechanism and functional role of Ser129 phosphorylation in regulating alpha-synuclein accumulation.
• To understand how cellular stress impacts Ser129 phosphorylation and subsequent protein clearance pathways.

Main Methods:

• Cellular studies using CHO, SH-SY5Y cells, and primary cortical neurons.
• Induction of mitochondrial impairment using rotenone and MPP+ to mimic cellular stress.
• Investigation of calcium ion (Ca2+) influx and its role in phosphorylation.
• Analysis of proteasomal and lysosomal degradation pathways using inhibitors (epoxomicin, chloroquine).
• In vivo studies using a rat model of alpha-synuclein overexpression (AAV-mediated).

Main Results:

• Ser129 phosphorylation of soluble alpha-synuclein was maintained at constant levels in intracellular and extracellular spaces in CHO cells.
• Mitochondrial impairment in neuronal cells increased Ser129 phosphorylation via extracellular Ca2+ influx.
• Proteasomal and lysosomal pathways were involved in clearing Ser129-phosphorylated alpha-synuclein, including insoluble forms.
• In vivo, Ser129 phosphorylation did not alter the total number of alpha-synuclein aggregates but increased phosphorylated aggregates in a mutant model.

Conclusions:

• Ser129 phosphorylation is a stress-induced response, potentially triggered by increased extracellular Ca2+.
• This phosphorylation promotes the clearance of insoluble alpha-synuclein through the proteasome, complementing lysosomal degradation.
• Ser129 phosphorylation may serve as an ineffective signal for aggregates resistant to degradation, leading to their accumulation and extensive phosphorylation.