Abberrant Posttranscriptional Regulation and Protein Degradtion of TDP-43 and FUS to Stress Respone in ALS (IN9-1.008)

Abstract

Objective: To further analyze molecular interactions and posttranscriptional regulation of TDP-43, FUS with HuR in the presence of mutant SOD1 and its impact on protein degradation. Background The genetic link of TDP-43 and FUS with familial and sporadic ALS underscores the importance of RNA binding proteins (RBP) and post-transcriptional regulation in the pathogenesis of ALS. HuR, another RBP, shares structural and functional similarities to TDP-43 and FUS. These RBPs provide an important contribution to stress response. We have observed aberrant cross-regulation and protein degradation of a network of RBPs in associated with mutant SOD1. Recent identification of UBQLN 2 mutation accentuated abnormal protein degradation in ALS pathogenesis. Design/Methods: HuR was upregulated ectopically or knocked down using siRNA in primary astrocytes or U251 cells expressing wild-type or mutant SOD1. Cells were subjected to oxidative, hypoxic and hormonal stress. TDP-43 39UTR regulation was measured by a luciferase reporter. Protein stability was analyzed by Western blot after treating cells with cycloheximide for different time intervals. Ubiquitination was quantified with Western blot and an ELISA kit. Results: Following oxidative stress, we observed cytoplasmic association and stress granules of TDP43 and FUS with HuR in G93A astrocytes. In both U251 and astrocytes, TDP-43 and FUS are downregulated by HuR silencing. In the presence of mutant SOD1, their protein half life significantly increased compared to wt cells. With stress induction, we observed accumulation of these RBPs and significantly increased ubiliquitination. Knockdown of TDP-43 and FUS had no effect on HuR protein and RNA level. There was increasing neuronal toxicity when co-cultured with those conditional medium. Conclusions: 1. The posttranscriptional regulation and protein stability of a network of RBPs may play a role in the pathogenesis of ALS. 2. ALS-associated mutant SOD1 and HuR dysfunction may block the ubiquitin proteasome pathway of these RBPs that leads to their aberrant degradation. Supported by: NIH/NINDS Grant NS057664, NS064133 and a merit review from the Department of Veterans Affairs. Disclosure: Dr. Lu has nothing to disclose. Dr. Zheng has nothing to disclose. Dr. Si has nothing to disclose. Dr. Chen has nothing to disclose. Dr. Luo has received personal compensation for activities with XenoPort as an employee. Dr. Oh has nothing to disclose. Dr. King has nothing to disclose.