Novel One-Step Immunoassays to Quantify α-Synuclein: Applications for Biomarker Development and High-Throughput Screening

J Biol Chem. 2012 Jul 27. [Epub ahead of print]

Bidinosti M, Shimshek DR, Mollenhauer B, Marcellin D, Schweizer T, Lotz GP, Schlossmacher MG, Weiss A.


 

Source

Novartis Institutes for BioMedical Research, Switzerland;

 

Abstract

Familial Parkinson's disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Forster's resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 hour protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay's suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120 percent of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Further, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knock-down increased and two which decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression, which may help guide further drug disovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding which will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of compound and genetic libraries, both of which are essential to the development of PD therapies.

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