Abstract: Calpains are Ca2+-activated, neutral (non-lysosomal) proteases implicated in neurodegeneration following acute insults such as stroke and traumatic brain injury, as well as in neurodegenerative disorders such as Alzheimer’s disease. Calpain research in the CNS has focused almost exclusively on the classical calpains, Calpains 1 and 2. Calpain 5 (CAPN5), which lacks the penta-EF hand domain of classical calpains, is the 2nd most highly expressed calpain in the CNS. Calpain 5 has homology to other calpains in the cysteine protease domain and also has a unique C2 domain at the C-terminus. C2 domains are involved in binding to membranes and lipids. Very little is known regarding the functions and substrates of Calpain 5. The C. elegans orthologue of Calpain 5, Tra-3, regulates transcriptional activity involved in sex determination. Tra-3 is also required for neurodegeneration induced by TDP-43 toxicity and for necrotic death of sensory neurons following calcium overload. At the subcellular level, CAPN5 is found in nuclear and crude mitochondrial fractions, and associated with promeylocytic nuclear bodies. The purpose of this proposal is gain insight into the CNS functions of Calpain 5. Aim 1 will identify Calpain 5 binding partners and substrates using three complimentary techniques: tandem affinity purification; proximity dependent localization; and in vitro affinity capture, with each technique paired with mass spectrometry for protein identification. The binding partners and substrates will be evaluated under both basal and elevated intracellular calcium conditions. The role of the C2 domain in Ca2+ binding along with CAPN5 activation and localization will be evaluated in Aim 2. Aim 3 will explore the physiological functions of CAPN5, utilizing knockout cell lines generated using CRISPR/Cas-9 and conditional knockout mice. Preliminary data support the feasibility of each Aim. Together, these results will provide essential information for deciphering the CNS function(s) of CAPN5.