PASK-regulated genes and proteins as therapeutic targets for ALS and FTD - Project Summary/Abstract Regulation of autophagy and the nutrient-sensing kinases mTOR and AMPK are not only important in cancer, but have garnered much recent attention in neurodegeneration. The Per–Arnt–Sim domain kinase (PASK) function both up- and downstream of mTOR. Our studies in spinocerebellar ataxia type 2 (SCA2) revealed that PASK is overabundant upon ATXN2 mutation. In yeast, Pask directly phosphorylates the ATXN2 homolog Prp1. This was an intriguing connection to our work as we had previously found that mTOR is dysregulated upon ATXN2 mutation. Our preliminary data support a feedforward control mechanism of PASK in SCA2. When ATXN2 is mutated, PASK levels rise further limiting autophagy. In our previous work we showed that STAU1 is also elevated with ATXN2 mutation leading to enhanced translation of mTOR mRNA, which in turn is known to activate PASK. In preliminary data we have shown that targeting PASK by RNAi or with the PASK- specific inhibitor BioE-115 in cultured cells, or genetically in SCA2 mice significantly reduced STAU1 protein levels, improved autophagic flux and levels of Purkinje cell marker proteins, effectively mimicking ASO knockdown of ATXN2. We hypothesize that the kinase activity of PASK regulates activity in the ATXN2 – STAU1 pathway modulating TDP-43 pathology. We further hypothesize that that targeting PASK is an effective way to normalizing TDP-43 proteinopathy, including amyotrophic lateral sclerosis (ALS) / frontotemporal dementia (FTD) and the Alzheimer’s disease-like dementia disorder limbic age related TDP-43 encephalopathy (LATE). The overall objective of our study is to understand PASK in neurodegeneration and to generate proof- of-concept for targeting PASK for treating SCA2 and TDP-43 proteinopathies to support future therapeutic development. Three specific aims are proposed: In Aim 1 we will use a proteomic approach to identify phosphorylated peptides of the ATXN2 protein. We will also investigate isogenic SCA2 and TDP43 mutant patient iPSC cortical neurons to produce transcriptome and proteomic analysis to identify differentially regulated genes, proteins and pathways dependent on BioE-1115. In Aim 2 we perform two genetic interaction studies to demonstrate modification of motor and molecular autopagy phenotypes in SCA2 (ATXN2-Q127) and Prp-TDP43-Q331K transgenic mice haploinsufficient for Pask. In Aim 3 we will demonstrate modification of molecular phenotypes of Prp-TDP43-Q331K mice treated with BioE-1115, and also conduct a proof-of-concept study showing effectiveness of an ASO targeting Pask in modification of both motor and molecular phenotypes of Prp-TDP43-Q331K mice. Our expectation is to demonstrate that ATXN2 is phosphorylated, that its phosphorylation is modulated by BioE-1115, and to demonstrate PASK as a therapeutic target for SCA2, ALS, FTD and possibly other AD/ADRDs.
