Regulation of Drp1-dependent mitochondrial fission during synaptic potentiation

突触增强过程中 Drp1 依赖性线粒体分裂的调节

基本信息

批准号：
9536140
负责人：
Sai Sachin Divakaruni
金额：
$ 4.95万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9536140
关键词：
Actins Address Adolescent Affect Alzheimer&apos s Disease Area Autistic Disorder Biochemistry Biological Assay Brain Calcineurin Cells Chemicals Chemosensitization Complex Confocal Microscopy Curiosities Data Dendrites Dendritic Spines Disease Dominant-Negative Mutation Dynamin Endoplasmic Reticulum Event Excitatory Synapse Functional disorder Guanosine Triphosphate Phosphohydrolases Heritability Hippocampus (Brain)Huntington Disease Hydrolysis Impairment Investigation Knowledge Lead Learning Link Long-Term Potentiation Measures Memory Mental Depression Microscopy Mitochondria Mitochondrial Diseases Molecular Morphology Neurodegenerative Disorders Neurons Parkinson Disease Phospho-Specific Antibodies Phosphorylation Physiology Play Prevention approach Process Property Protein Dephosphorylation Proteins Protocols documentation Regulation Resolution Role Schizophrenia Shapes Short Interspersed Nucleotide Elements Sum Surface Synapses Synaptic Transmission Synaptic Vesicles Synaptic plasticity Techniques Testing Ursidae Family Vertebral column Western Blotting autism spectrum disorder cell motility cell type design energy balance experimental study improved insight knock-down mimetics mitochondrial dysfunction nervous system disorder neuron development neuropsychiatric disorder novel diagnostics novel therapeutics particle postsynaptic presynaptic prevent recruit single molecule synaptic function synaptogenesis

项目摘要

Healthy synapse function relies on the adequate presence of mitochondria. The heightened energy utilization of activity-dependent processes supporting long-term potentiation (LTP) places even greater a demand on mitochondria and, indeed, mitochondrial dysfunction is implicated in many neuropsychiatric disorders including autism and schizophrenia. Although the mechanisms that support dendritic mitochondrial reorganization are unclear, a key process impacting mitochondrial distribution is mitochondrial fission. Thus, two central questions that remain are whether LTP induction impacts mitochondrial fission, and whether mitochondrial fission is required for LTP. Mitochondrial fission is regulated by a GTPase, dynamin-related protein 1 (Drp1), which itself is tightly regulated by phosphorylation. Intriguingly, CaMK1α and calcineurin, both required for LTP induction, also increase Drp1 recruitment to mitochondria and mitochondrial fission by regulating Drp1 phosphorylation. Yet, despite these indications, the link between LTP and mitochondrial fission remains unknown. Driven by this curiosity, I used a protocol to chemically induce LTP (chemLTP) to test whether LTP induction increases mitochondrial fission. My preliminary data show a rapid and transient burst of mitochondrial fission after chemLTP stimulation, in addition to robust and sustained canonical changes to dendritic spine morphology. Together, these observations lead me to hypothesize that LTP requires an increase in dendritic mitochondrial fission via direct regulation of Drp1 function. I will use a diverse arsenal of confocal and super-resolution microscopy, molecular perturbations, and biochemistry techniques to determine the relationship between LTP, Drp1 function, and mitochondrial fission by addressing three specific aims. First, I will determine whether LTP requires an increase in dendritic mitochondrial fission. I will confirm and extend my preliminary findings by testing whether the fission increase is NMDAR-dependent by blocking NDMARs. To test whether induction of LTP requires fission, I will block fission by expressing a Drp1 dominant negative mutant, and will measure properties of dendritic spines, synapses, and mitochondrial fission after chemLTP stimulation. Second, I will determine whether chemLTP stimulation changes recruitment or mobility of Drp1 oligomers on mitochondria using confocal microscopy particle tracking. My preliminary data suggest that Drp1 puncta become drastically less mobile prior to inducing fission and I will measure the effect of chemLTP stimulation on this mobility. To measure whether mobility of isolated Drp1 molecules on mitochondria is altered during LTP, I will use single molecule tracking photoactivated localization microscopy (sptPALM). Third, I will determine whether LTP induction requires phosphorylation of Drp1 via Western blots with phospho-specific antibodies, and by measuring fission and dendritic spine properties in the presence of phospho-mimetic and phospho-null Drp1 constructs. In sum, these Aims will greatly extend our understanding of mitochondrial fission in neurons, and will elucidate the unexplored relationship between mitochondrial fission and synaptic potentiation.

健康的突触功能取决于线粒体的足够存在。能源利用率提高支持长期增强（LTP）的活动依赖性过程的要求更大线粒体以及线粒体功能障碍在许多神经精神疾病中隐含自闭症和精神分裂症。尽管支持树突状重组的机制是尚不清楚，影响线粒体分布的关键过程是线粒体裂变。那是两个中心问题仍然存在LTP诱导是否会影响线粒体裂变，以及线粒体裂变是否为 LTP所需。线粒体裂变由GTPase，Dynamin相关蛋白1（DRP1）调节，该蛋白本身本身受磷酸化严格调节。有趣的是，LTP诱导所必需的CAMK1α和钙调神经酶，还通过调节DRP1磷酸化来增加DRP1募集到线粒体和线粒体裂变。然而，dospite这些适应症，LTP与线粒体裂变之间的联系仍然未知。由此驱动好奇心，我使用一项方案来化学诱导LTP（ChemLTP）来测试LTP诱导是否增加线粒体裂变。我的初步数据表明，线粒体裂变的快速瞬时爆发除了对树突状脊柱形态的强大和持续的规范变化外，ChemLTP模拟。总之，这些观察结果使我假设LTP需要增加树突状线粒体通过直接调节DRP1函数进行裂变。我将使用共聚焦和超分辨率的不同武器库显微镜，分子扰动和生物化学技术，以确定LTP之间的关系 DRP1功能和线粒体裂变通过解决三个特定目的。首先，我将确定LTP是否需要树突状线粒体裂变的增加。我将通过测试裂变增加是否通过阻止NDMAR的NMDAR依赖。测试是否诱导 LTP需要裂变，我将通过表达DRP1显性负突变体来阻止裂变，并将测量 ChemLTP刺激后树突状刺，突触和线粒体裂变的特性。第二，我会的确定ChemLTP刺激是否会改变DRP1低聚物在线粒体上的募集或迁移率使用共聚焦显微镜粒子跟踪。我的初步数据表明DRP1 Puncta变得急剧诱导裂变之前的移动性较低，我将衡量ChemLTP模拟对这种迁移率的影响。到测量LTP期间线粒体上孤立的DRP1分子的迁移率是否改变，我将使用单个分子跟踪光活化定位显微镜（SPTPALM）。第三，我将确定LTP是否诱导需要通过具有磷酸特异性抗体的蛋白质印迹将DRP1磷酸化，并通过在存在磷酸化和磷酸化drp1的情况下测量裂变和树突状脊柱特性构造。总而言之，这些目标将极大地扩展我们对神经元中线粒体裂变的理解，以及将阐明线粒体裂变与突触电位之间的意外关系。