Extracellular mitochondria transfer in gray and white matter for ameliorating sensorimotor and cognitive deficits after stroke Mitochondrial function is essential for maintaining cellular homeostasis in the central nervous system (CNS). It is now recognized that mitochondria are surprisingly released and transferred between cells. However, the underlying mechanisms remain poorly understood. In this proposal, we will investigate post-translational modification by O-GlcNAc as a critical element for extracellular mitochondrial functionality and the transfer- mediated blood-brain barrier recovery, oligodendrocyte protection and neural plasticity after stroke. Our pilot data suggest that (i) ER-Golgi trafficking may regulate mitochondrial protein O-GlcNAcylation, (ii) transfer of O-GlcNAcylated mitochondria may protect neurons, oligodendrocyte precursors and endothelial cells against oxygen-glucose deprivation, (iii) blockade of DJ1 O-GlcNAcylation may increase mitochondrial glycation and degradation, thus reducing the protective capacity of extracellular mitochondria, (iv) levels of mitochondrial O-GlcNAc-DJ1 may be positively correlated with functional recovery after stroke and negatively correlated with aging, (v) methods to dissect these mechanisms (molecular tools, optical imaging, electrophysiology, snRNA-seq etc) are feasible in our collaborating labs. Based on our pilot data, we hypothesize that damage associated molecular pattern (DAMP) or CD31 signals upregulate CD38 in reactive astrocytes, and CD38-driven post-translational modification by O-GlcNAc supports extracellular mitochondrial functionality that accelerates viable mitochondrial transfer-mediated neurovascular remodeling and remyelination, thus ameliorating sensorimotor deficits and post-stroke dementia after stroke. We have 3 specific aims. In Aim 1, we will dissect mechanisms of O-GlcNAc-modified mitochondrial release and transfer between astrocytes and neurons or brain endothelial cells or OPCs/oligodendrocytes. In Aim 2, we will investigate O-GlcNAcylated mitochondrial transfer in gray matter and white matter in young, older, male or female mice after focal ischemia. In Aim 3, we will use gain- and loss-of-function experiments to modify mitochondrial protein O-GlcNAcylation, and assess sensorimotor and cognitive outcomes in vivo. Our experiments will utilize molecular tools including a combination of pharmacologic activators/inhibitors, site- directed mutagenesis, and AAV-induced mitochondrial labeling to assess astrocyte-specific mitochondria transfer. Single-nuclei RNA seq will be performed to fully map transcriptomes in gray and white matter post- stroke. Translational relevance will be assessed with in vivo imaging, electrophysiology and long-term outcomes post-stroke. This project should define a novel mechanism of mitochondrial transfer in both gray and white matter, and hopefully provide new approaches to accelerate sensorimotor recovery and ameliorate post- stroke vascular-related dementia.

Project Narrative for R01 application by Hayakawa Extracellular mitochondria transfer in gray and white matter for ameliorating sensorimotor and cognitive deficits after stroke Mitochondria are important for maintaining energy homeostasis in mammalian cells, but it is now increasingly recognized that mitochondria may be surprisingly release and transfer from cell to cell. Is it possible that this mitochondrial transfer may represent a novel non-autonomous mechanism to supply energy and promote neuron-oligodendrocyte-vascular recovery after stroke? Here, we propose that post-translational modification by O-GlcNAc amplifies extracellular mitochondrial functionality and efficient mitochondrial transfer that can accelerate the blood-brain barrier repair, remyelination and improve neuroplasticity. Therefore, these mechanisms should boost sensorimotor recovery and ameliorate post-stroke dementia after focal ischemia.