TDP-43 Identified from a Genome Wide RNAi Screen for SOD1 Regulators

Amyotrophic Lateral Sclerosis (ALS) is a late-onset, progressive neurodegenerative disease affecting motor neurons in the brain stem and spinal cord leading to loss of voluntary muscular function and ultimately, death due to respiratory failure. A subset of ALS cases are familial and associated with mutations in superoxide dismutase 1 (SOD1) that destabilize the protein and predispose it to aggregation. In spite of the fact that sporadic and familial forms of ALS share many common patho-physiological features, the mechanistic relationship between SOD1-associated and sporadic forms of the disease if any, is not well understood. To better understand any molecular connections, a cell-based protein folding assay was employed to screen a whole genome RNAi library for genes that regulate levels of soluble SOD1. Statistically significant hits that modulate SOD1 levels, when analyzed by pathway analysis revealed a highly ranked network containing TAR DNA binging protein (TDP-43), a major component of aggregates characteristic of sporadic ALS. Biochemical experiments confirmed the action of TDP-43 on SOD1. These results highlight an unexpected relationship between TDP-43 and SOD1 which may have implications in disease pathogenesis

Classification of prominent regulators of SOD1 in the RNAi screen.

<p><b>A & B</b>) Pie charts representing annotated biological process of proteins identified as regulators of SOD1. Contribution of each biological process as a percentage of total are shown. Contributions of processes that are less than 2% of the total are not shown in the figure. <b>C</b>) Effect of siRNA knockdowns of SOD1 isoforms in the screen. The MAD score values of the three SOD1 isoforms obtained in the screen are shown. The error bars represent SD.</p

Optimization of the reporter assay for high throughput format.

<p><b>A</b>) Effect of knock down of a proteasomal subunit on SOD1 levels. HeLa cells were transfected with siRNA targeting the S4 subunit (PSMC1) of the 26 S proteasome, followed by expression of the mutant SOD1 reporter plasmids. Supernatant fractions were tested for β-gal activity by measuring the fluorescence signal. The plot shows SOD1 levels as a measure of β-gal activity. <b>B</b>) HeLa cells were co-transfected with S4 siRNA or carrier and varying amounts of plasmids coding for A4VSOD1-HA-α fusion with a fixed amount of ω fragment. Cells were analyzed for fluorescence 96 hrs post transfection. The SOD1 levels measured as a function of β-gal activity are plotted against each transfection condition. Z′ factor was used to assess the assays suitability for HTS.</p

Protein interaction network map of hits from the RNAi screen.

<p>The data from the RNAi screen was processed to calculate MAD scores and was used for Ingenuity pathway analysis (IPA). The network annotated “Skeletal and Muscular System Development and Function, Tissue Morphology, Inflammatory Response" ranked at the top with the highest score and is shown in the figure. The hits that increased SOD1 levels above 3SD are shown in red and the ones that decreased the signal below −2SD are shown in green. The proteins represented by color-less nodes were below the selection criteria. Direct interactions are shown in bold lines and indirect interactions are shown in broken lines. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035818#pone.0035818.s005" target="_blank">Table S1</a> for proteins in this network)</p