PROJECT SUMMARY/ABSTRACT JanusQ, LLC is a startup biotech company, spun out of Case Western Reserve University, and developing a peptide-based therapy for treatment of mitochondrial dysfunction in Alzheimer’s Disease (AD), which is eroding the memory and cognitive abilities of ~6 million Americans. The role of amyloid-β (Aβ) in AD pathogenesis is poorly understood, and therapies addressing A have not been successful in the clinic. The observation that, like many other neurodegenerative diseases, AD is linked to mitochondrial dysfunction that leads to neurotoxicity suggests a novel strategy: ATAD3A is a mitochondrial protein that spans both inner and outer membranes and is critical in cholesterol trafficking and maintaining the mitochondrial nucleoid complex. However, during the stress of AD, ATAD3A oligomerizes and binds Drp1 (the mitochondrial-fission GTPase), causing mitochondrial fragmentation and nucleoid instability. DA1, a peptide newly discovered by a JanusQ cofounder, prevents ATAD3A oligomerization and association with Drp1, reduces mitochondrial fragmentation, improves nucleoid stability, and reduces behavioral and neurological deficits in rodent models of AD and Huntington’s disease. However, little is known about the disposition and dose-dependence of DA1 in vivo. Thus, it is difficult to know whether unmodified DA1 is already suitable as a clinical candidate for the treatment of AD, or whether future work will be required to optimize the peptide to improve its pharmacological characteristics (potency, absorption, distribution, elimination). The purpose of this SBIR grant is to make that determination. The project has three aims: (1) Assess the pharmacokinetics of DA1 in vivo to test exposure and stability by developing approaches to extract DA1 from plasma and brain tissue, collecting samples at various time points after administering the peptide subcutaneously (SQ), and quantitating the peptide using tandem liquid chromatography mass spectroscopy. (2) Perform preliminary in vitro safety/toxicity assessment of DA1, focusing on discovery-phase assays, with an assessment of potential off-target interactions and immunogenicity. (3) Assess DA1 dose-response characteristics using an in vivo AD mouse model, treating APP knock-in AD mice (aged 3–9 months) initially with three DA1 doses SQ. Validation assays will assess ATAD3A oligomerization, mitochondrial bioenergetics, measures of AD pathology(e.g., Aβ accumulation, synaptic integrity, neuronal loss), and cognitive function during AD development. These results will directly address our central hypothesis that DA1 has the pharmacological properties necessary for advancement to the clinic (vs. future peptide optimization). Thus, the proposed work is the next logical step toward the long-term goal of developing a peptide therapy to improve quality of life and survival of AD patients. Such a treatment would reduce reliance on medical caregivers and thus reduce overall medical costs. In the U...