Mitochondrial Calcium Sensing via TUSC2 in Aging and Cognitive Function - PROJECT SUMMARY Significance: Mitochondrial dysfunction is a central driver of neurodegeneration and aging-related cognitive decline, yet mechanisms linking calcium dysregulation to neuronal failure remain poorly understood. TUSC2 (Tumor Suppressor Candidate 2), a small mitochondrial protein with a conserved calcium-binding motif, has emerged as a potential regulator of the mitochondrial permeability transition pore (mPTP), a key determinant of mitochondrial integrity, neuronal survival, and synaptic plasticity. However, its role in mPTP regulation in the brain remains unexplored. Innovation: This project introduces a previously untested concept that TUSC2 modulates mPTP opening by interacting with OSCP (ATP5PO), a key regulatory subunit of ATP synthase and a core component of the mPTP, in a calcium-dependent manner. AI-assisted 3D modeling predicts that TUSC2 binds OSCP through a β-sheet hairpin and that both proteins participate in coordinating a Ca²⁺ ion, thereby preventing excessive association of OSCP with cyclophilin D (CypD). Loss of TUSC2 is expected to destabilize calcium-regulated OSCP/CypD- mediated control of the mPTP, promoting sustained pore opening, mitochondrial dysfunction, and cognitive decline. This work reveals a previously unknown mechanistic link between calcium signaling, mitochondrial permeability, and brain aging. Approach: We will combine CRISPR-engineered Tusc2-mutant neuronal lines and a validated Tusc2 knockout (KO) mouse model of accelerated aging to test this hypothesis. • Aim 1: Define TUSC2’s role in mPTP regulation using Tusc2-deficient and WT neuronal cultures and hippocampal slices. We will assess Ca²⁺-dependent TUSC2–OSCP interactions, pore opening (calcein– Co²⁺ assay, swelling), membrane potential (TMRE), and cytosolic Ca²⁺ dynamics (Fura-2). Cross-linking, XL-MS, and pull-down assays with recombinant proteins will validate direct binding. • Aim 2: Determine functional outcomes of TUSC2 loss on cognition and synaptic plasticity (LTP) in Tusc2 KO mice and evaluate the restorative effects of REQORSA, a TUSC2-expressing nanoparticle therapy (Genprex). Expected Outcomes and Impact: This high-risk, high-reward study will reveal how TUSC2 regulates mitochondrial permeability, calcium signaling, and neuroprotection. The findings will identify new targets for treating age-related cognitive decline and other neurodegenerative disorders while providing mechanistic groundwork for future R01-level studies. Investigators and Environment: The research team integrates complementary expertise in calcium signaling, mitochondrial biology, proteomics, and electrophysiology. Feasibility is ensured by established Tusc2 KO models, CRISPR-engineered neurons, and validated assays for mPTP, Ca²⁺ imaging, and LTP recordings.
