Neurodegenerative diseases are characterized by accumulation of misfolded proteins in disease-specific neural circuits, which disrupts normal neuronal functions, including axonal transport, mitochondrial bioenergetics, gene expression, and synaptic connectivity. In addition, there is compelling evidence that neuroinflammation can also initiate and/or facilitate disease progression. These results support an interconnected mechanism, in which protein misfolding and neuroinflammation synergistically promote disease progression in a feed-forward manner. Among the major neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) affects upper and lower motor neurons and is often associated with aggressive clinical courses. Although the majority of ALS cases are sporadic, ~10% are caused by mutations that affect RNA metabolism, intracellular vesicular trafficking, and protein homeostasis via the ubiquitin-proteasome pathways (proteostasis). Characterizations of these cellular functions provide critical windows to understand disease mechanism and to intervene its onset and progression. During the past funding period, we showed that misfolded proteins activate endoplasmic reticulum (ER) stress and its downstream signaling pathways, including stress-activated kinase, homeodomain interacting protein kinase 2 (HIPK2), to promote neuronal cell death. We developed highly sensitive biochemical, morphological and functional assays to show that ER stress activates HIPK2 and that HIPK2 activation can be detected in the spinal motor neurons of pre-symptomatic SOD1G93A and NEFH-tTA/tetO-hTDP-43DNLS mouse ALS models, suggesting that HIPK2 activation directly contributes to neurodegeneration. In support of this idea, loss of HIPK2 or blocking HIPK2 kinase activity significantly protects motor neurons from cell death induced by SOD1G93A or TDP-43. To broaden our understanding of HIPK2 in neurodegeneration, we further showed that HIPK2 can regulate neuronal survival and cell death via transcriptional regulation of gene expression and by regulating Parkin protein levels via proteasome-mediated pathway. In addition, we performed proteomic screens to characterize HIPK2 interactomes and identified HSPA9 (also known as Mortalin) as a HIPK2 interacting partner. Together, these results support the hypothesis that HIPK2 and its interactomes regulate a delicate balance of neuronal survival and cell death via both cell autonomous and glia-mediated mechanisms. To test this, we propose to (1) characterize the role of HSPA9 in neuronal cell death during development and in neurodegeneration, (2) delineate the mechanism of HIPK2 in neuroinflammation in ALS, and (3) perform single cell transcriptomics to elucidate the role of ER stress and neuroinflammation in SALS and C9-ALS. Collectively, results from this proposal will provide a more complete understanding of the cellular and molecular mechanisms of HIPK2 in stress-induced neurodegeneration in ALS.