Abstract

Protein folding stress is a salient pathological feature of sporadic and familial amyotrophic lateral sclerosis (ALS). The presence of TAR DNA binding protein (TDP-43) was described as the main constituent of protein aggregates in ALS. Familial mutations in TDP-43 trigger its misslocalization from the nucleus to the cytosol, its abnormal aggregation in the cysotosol. The overexpression of ALS-linked mutant TDP-43 in transgenic mice recapitulates essential features of the human pathology provoking age-dependent paralysis and neuronal degeneration. The primary mechanism by which mutations in TDP-43 contribute to progressive motor neuron loss in ALS remains unknown. Interestingly, several studies indicate that ALS progression directly correlates with a strong stress response at the endoplasmic reticulum (ER), an essential compartment involved in protein folding and quality control mechanisms. Signs of ER stress are observed in post-mortem tissue from ALS cases and in many different animal models of the disease. In particular, the up-regulation of several ER foldases of the Protein Disulfide Isomerases family (PDIs) is observed in the disease process highlighting as a major alteration, including ERp57 and PDI its self. PDIs are key foldases and chaperones that catalyze the formation of disulfide bonds, in addition to have important roles in apoptosis, protein quality control, and cell signaling. However, no studies have addressed the actual contribution of these ER factors to ALS in vivo. We have obtained preliminary data indicating that the transient expression of mutant forms of TDP-43 triggers the upregulation of ERp57 expression in the motoneuron cell line NSC34. Co- expression of ERp57 or PDI with TDP-43 led to a dramatic decrease in the degree of its pathological aggregation. Remarkably, these TDP-43 aggregates where also sensitive to DTT treatment, suggesting a relevant role of disulfide bonds in the aggregation process. Taken together, these data suggest an interesting scenario where PDIs have protective effects against the aggregation of TDP-43. In this project we aim to define the possible contribution of ERp57 to the development of experimental ALS and its relation to TDP-43 aggregation using complementary cellular, biochemical and in vivo approaches. We hypothesized that the foldase ERp57 reduces TDP-43 aggregation, protecting against the development of experimental ALS. Our goals for a three-year project include: Specific Aim 1: To assess the impact of ERp57 on the aggregation, toxicity and subcellular distribution of mutant TDP-43 in cellular models of ALS. Specific Aim 2: To determine the possible interaction between ERp57 and TDP-43. Specific Aim 3: To determine the impact of targeting ERp57 in the development of experimental ALS. In summary, this project aims to establish the individual contribution of the chaperon ERp57 to the misfolding and neurotoxicity of mutant TDP-43 in cellular and animal models of the disease. Overall, our long-term objective is to increase our fundamental understanding of the molecular basis of ALS and to identify new therapeutic targets for this fatal disease.