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.
