SMG1 Identified as a Regulator of Parkinson’s Disease-Associated alpha-Synuclein through siRNA Screening

Adrienne Henderson-Smith,Marwan N Sabbagh,Sandra A Jacobson,Meraj Aziz,Charles H Adler,Travis Dunckley,Donald Chow,Hongwei Yin,Erika D Driver-Dunckley,Thomas G Beach,John N Caviness,Holly A Shill,Bessie Meechoovet,Jaya Padmanabhan

doi:10.1371/journal.pone.0077711

Abstract

Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129) compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous in vitro and in vivo studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson’s disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn). Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD). These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.

Highlights

The a-synuclein (SNCA) protein is intricately involved in the pathogenesis of Parkinson’s disease and other synucleinopathies, including dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)
We show that knockdown of one of these kinases, the phosphatidylinositol 3-kinase related kinase SMG1, significantly enhances expression of both p-Syn and total a-synuclein (t-Syn) levels and, further, that expression of this kinase is significantly reduced in brain samples from neuropathologically confirmed cases of Parkinson’s disease with dementia (PDD) and dementia with Lewy bodies (DLB) in brain regions known to have significantly elevated p-Syn
High-Content a-Synuclein Phosphorylation Assay To facilitate the identification of kinases and phosphatases that may be important in PD relevant pathologic phosphorylation of asynuclein protein, we developed a cell-based, high-throughput immunofluorescence assay for the rapid detection and quantitation of both total a-synuclein (t-syn) and pS129 a-synuclein (p-syn)

Summary

Introduction

The a-synuclein (SNCA) protein is intricately involved in the pathogenesis of Parkinson’s disease and other synucleinopathies, including dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Each disorder is characterized by protein aggregates containing a-synuclein protein that has been hyperphosphorylated on serine 129 (p-Syn). In PD and DLB p-Syn is found in intraneuronal inclusions called Lewy bodies and in Lewy neurites. The importance of p-Syn in the pathogenesis of synucleinopathies is further strengthened by genetic findings showing that point mutations in the SNCA gene [2,3,4] and SNCA locus duplication and triplication [5,6,7] cause autosomal dominant forms of PD that manifest with neuropathological features in common with sporadic PD, including the presence of Lewy bodies with p-Syn aggregates.

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