|Ph.D Student||Avraham Eyal|
|Subject||Implication of Protein Phosphorylation in the Pathogenesis|
of Parkinson's Disease
|Department||Department of Medicine||Supervisor||Professor Simone Engelender|
|Full Thesis text|
Parkinson's disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows Lewy bodies and loss of neurons, mostly in the substantia nigra. A few families have PD caused by mutations in the SNCA gene (encoding α-synuclein). α-Synuclein is also present in Lewy bodies of patients with sporadic PD. Synphilin-1 is an a-synuclein interacting protein. SIAH proteins ubiquitinate synphilin-1 and promote its degradation. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitinated inclusion bodies.
In my work I show that synphilin-1 is phosphorylated by GSK3b. Mutation analysis showed that Ser556 is a major GSK3b phosphorylation site in synphilin-1. Phosphorylation of synphilin-1 by GSK3b decreased its ubiquitination in vitro and in cells in tissue culture as well as its degradation. On the other hand, inhibition of GSK3b greatly increased ubiquitination and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant formed more inclusion bodies than wild-type synphilin-1. Moreover, inhibition of GSK3b in primary neuronal cultures decreased the levels of endogenous synphilin-1. I found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild-type synphilin-1. Activation of GSK3b during ER-stress and the specific phosphorylation of synphilin-1 by GSK3b place synphilin-1 as possible mediator of ER-stress and proteasomal dysfunction observed in PD.
Mutations in parkin are responsible for a large percent of autosomal recessive juvenile parkinsonism cases. Parkin displays E3 ubiquitin-ligase activity and protects against cell death. Therefore, understanding the regulation of parkin activities is important to determine the mechanisms involved in the dopaminergic cell death observed in PD.
In the second part of my work, I report that cyclin-dependent kinase 5 (Cdk5) phosphorylates parkin in vitro and in cells in tissue culture. I identified Ser131 as the major Cdk5 phosphorylation site. The Cdk5 phosphorylation-deficient S131A parkin mutant displayed a higher auto-ubiquitination level and increased ubiquitination activity toward its substrates synphilin-1 and p38. Additionally, the S131A parkin mutant more significantly accumulated into inclusions in human dopaminergic cells when compared to the wild-type parkin. Furthermore, S131A parkin mutant increased the formation of synphilin-1/a-synuclein inclusions, suggesting that the levels of parkin phosphorylation and ubiquitination may modulate the formation of inclusion bodies relevant to the disease. The data indicate that Cdk5 is a new regulator of parkin. Phosphorylation by Cdk5 may contribute to the accumulation of toxic parkin substrates and decrease the ability of dopaminergic cells to cope with toxic insults in PD.