Extracellular Mitochondria Transfer in Gray and White Matter for Ameliorating Sensorimotor and Cognitive Deficits After Stroke

灰质和白质中的细胞外线粒体转移可改善中风后的感觉运动和认知缺陷

基本信息

批准号：
10571863
负责人：
Kazuhide Hayakawa
金额：
$ 50.3万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-15 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10571863
关键词：
Acceleration Aging Allografting Animals Astrocytes Blood - brain barrier anatomy Blood Vessels Brain CRISPR/Cas technology Cell Survival Cells Central Nervous System Cerebral Ischemia Cognitive Cognitive deficits Collaborations Confocal Microscopy Cyclic ADP-Ribose Data Dementia Electrophysiology (science)Elements Endothelial Cells Endothelium Energy Supply Etiology Female Glucose Golgi Apparatus Homeostasis Human In Vitro Infarction Integrins Ischemia Knowledge Label Mammalian Cell Maps Mass Spectrum Analysis Mediating Metabolism Methods Mitochondria Mitochondrial Proteins Modification Molecular Mus Neurologic Neuronal Plasticity Neurons Oligodendroglia Oligonucleotides Outcome Oxygen PARK7 gene PECAM1 gene Pathway interactions Pattern Plasmids Play Post-Translational Protein Processing Process Property Proteins Publishing Rattus Recombinants Recovery Recovery of Function Role Sensorimotor functions Signal Induction Signal Pathway Signal Transduction Site-Directed Mutagenesis Small Interfering RNA Specific qualifier value Stroke Testing Therapeutic Tissues Western Blotting angiogenesis behavioral outcome brain endothelial cell cognitive function cognitive recovery cognitive testing deprivation experimental study extracellular functional improvement gain of function glycation gray matter improved in vivo in vivo imaging inhibitor loss of function male neural growth neurogenesis neurological recovery neuroprotection neurovascular neutralizing antibody novel novel strategies novel therapeutic intervention oligodendrocyte precursor optical imaging pharmacologic post stroke post stroke dementia potential biomarker precursor cell prevent remyelination repaired response single nucleus RNA-sequencing spatiotemporal tool trafficking transcriptome white matter

项目摘要

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.

灰质和白质中的细胞外线粒体转移可改善感觉运动和中风后的认知缺陷线粒体功能对于维持中枢神经系统 (CNS) 的细胞稳态至关重要。它现在人们认识到线粒体令人惊讶地被释放并在细胞之间转移。然而，其根本机制仍知之甚少。在本提案中，我们将研究翻译后 O-GlcNAc 修饰作为细胞外线粒体功能和转移的关键元素介导中风后血脑屏障恢复、少突胶质细胞保护和神经可塑性。我们的试点数据表明 (i) ER-高尔基体运输可能调节线粒体蛋白 O-GlcNAcNAclation，(ii) O-GlcNA酰化线粒体的转移可以保护神经元、少突胶质细胞前体和内皮细胞细胞对抗氧-葡萄糖剥夺，(iii) 阻断 DJ1 O-GlcNAcNAc 可能会增加线粒体糖化和降解，从而降低细胞外线粒体的保护能力，(iv) 线粒体 O-GlcNAc-DJ1 可能与中风后功能恢复呈正相关，而与中风后功能恢复呈负相关与衰老相关，（v）剖析这些机制的方法（分子工具、光学成像、电生理学、snRNA-seq 等）在我们的合作实验室中是可行的。根据我们的试验数据，我们假设损伤相关分子模式 (DAMP) 或 CD31 信号上调反应性星形胶质细胞中的 CD38，并且 O-GlcNAc 支持 CD38 驱动的翻译后修饰细胞外线粒体功能加速可行的线粒体转移介导的神经血管重塑和髓鞘再生，从而改善中风后的感觉运动缺陷和中风后痴呆。我们有 3 个具体目标。在目标 1 中，我们将剖析 O-GlcNAc 修饰的线粒体释放机制以及星形胶质细胞和神经元或脑内皮细胞或OPC/少突胶质细胞之间的转移。在目标 2 中，我们将研究年轻、老年、男性灰质和白质中的 O-GlcNAc 酰化线粒体转移或局灶性缺血后的雌性小鼠。在目标 3 中，我们将使用功能增益和功能丧失实验来修改线粒体蛋白 O-GlcNAcylation，并评估体内感觉运动和认知结果。我们的实验将利用分子工具，包括药理激活剂/抑制剂的组合、位点定向诱变和 AAV 诱导的线粒体标记来评估星形胶质细胞特异性线粒体转移。将进行单核 RNA 测序，以完整绘制灰质和白质后的转录组图谱。中风。转化相关性将通过体内成像、电生理学和长期评估来评估中风后的结果。该项目应该定义一种新的线粒体转移机制。白质，并希望提供新的方法来加速感觉运动恢复和改善后遗症中风血管相关性痴呆。