Abstract

Misfolding and aggregation of SOD1 or TDP43 are associated with the occurrence of sporadic and familial forms of ALS. Recent reports suggest a critical role of protein folding stress in ALS, however the primary mechanism responsible for the progressive motoneuron loss in ALS remains unknown. Recent evidence from different laboratories, however, implicates the participation of adaptive responses to stress at the endoplasmic reticulum (ER) in the disease process, through a pathway known as the Unfolded Protein Response (UPR). Our collaborator Dr Daryl Bosco at the University of Massachusetts (UMASS) has greatly contributed to define the impact of stress responses and protein misfolding to ALS (Bosco et al., 2010, Nat Neurosci., Bosco et al., 2010, Hum Mol Genet.). In order to study the contribution of ER stress to fALS, we have targeted the expression of the transcription factor X-Box binding protein-1 (XBP1) in the nervous system by creating a conditional knockout mouse (Hetz et al., 2008 Proc. Natl. Acad. Sci). XBP1 is the master regulator of the UPR and controls the expression of multiple proteins involved in protein folding and quality control mechanisms. Unexpectedly, despite predictions that XBP1 deficiency would enhance the severity of experimental ALS, we observed that these mice were more resistant to developing the disease. XBP1-/-/SOD1 transgenic mice displayed a significant increase in their life span accompanied by decreased motoneuron apoptosis (Hetz et al., 2009 Genes & Dev). These effects were associated with reduced accumulation of mutant SOD1 oligomers due in part to increased autophagy in motoneurons, a cellular pathway involved in lysosome-mediated degradation of protein aggregates in many important neurological diseases. Similarly, in vitro experiments demonstrated that targeting XBP1 with small interfering RNA (shRNA) in a motoneuron cell line drastically decreased the generation of toxic mutant SOD1 protein aggregates, suggesting possible therapeutic benefits of targeting this pathway in ALS. In this project, we propose to further study and validate the contribution of the UPR to fALS through the development of a gene therapy strategy to manipulate XBP1 levels in the context of ALS. We have developed Adeno-Associated Viruses (AAVs) to either deliver an active form of XBP1 (termed XBP1s) or a validated shRNA construct to down-regulate the levels of this transcription factor. We recently reported that these vectors improved motor performance on a spinal cord injury model (Valenzuela et al., 2012, Cell Death Dis) and decreased protein aggregation on a Huntington's disease model p Company , Genzyme (Zuleta et al., 2012, BBRC). AAVs are currently the best choice for future gene therapy use in patients due to their (i) safety properties, (ii) efficiency of neuronal transduction, and (iii) the current methodologies for large-scale production, in addition to the fact that (iv) many clinical trials are been performed with AAVs in patients affected with neurodegenerative diseases including studies in UMASS where our collaborator Dr. Robert Brown is head of the Neurology Department. All methods and quality control assays to produce AAVs particles have been established in collaboration with the leading Biotechnology Company Genzyme at Boston, USA. In this project we plan to consolidate a network of collaboration with UMASS´s professors Bosco and Brown to develop a new gene therapy strategy to treat ALS using preclinical models. Based on our preliminary results, we plan to test the impact of XBP1 gain and lost of function by injecting AAVs locally into the spinal cord of early symptomatic ALS mouse models. Overall, our long-term objective is to increase our understanding of the molecular basis of ALS and, to apply this knowledge to test and validate at the pre-clinical level a new therapeutic strategy to treat this fatal disease and other neuromuscular disorders. Taking these antecedents, we propose: Hypothesis: Targeting the transcription factor XBP1 delays disease onset and increases life span of ALS mouse models. Specific aim 1: To develop gene therapy strategies to manipulate XBP1 expression levels in the spinal cord of ALS mouse models. We propose to use two genetic tools to manipulate XBP1 expression levels in the nervous system and test the efficacy of this local manipulation in the spinal cord of adult animals in the context of ALS pathogenesis. For this purpose, we plan to develop adeno-associated viruses (AAV) to either deliver an (i) efficient shRNA construct against XBP1 or to (ii) locally express the active XBP1s form. Specific aim 2: To determine and compare the possible therapeutic effects of manipulating XBP1 levels in two genetic models of ALS through AAV-mediated gene therapy approaches. With the two strategies developed in specific aim 1, we intend to monitor: 1. The levels of motoneuron loss in the spinal cord of mutant SOD1 and TDP43 transgenic mice. 2. Animal survival, motor performance and disease onset. 3. TDP43 and mutant SOD1 aggregation and subcellular distribution, in addition to ER stress and autophagy levels.