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.

健康的突触功能依赖于线粒体的充足存在。支持长期增强（LTP）的活动依赖性过程对线粒体，事实上，线粒体功能障碍与许多神经精神疾病有关，包括尽管支持树突状线粒体重组的机制是自闭症和精神分裂症。目前尚不清楚，影响线粒体分布的关键过程是线粒体裂变。因此，有两个核心问题。剩下的问题是 LTP 诱导是否会影响线粒体分裂，以及线粒体分裂是否会影响线粒体分裂。 LTP 所需的线粒体裂变由 GTP 酶（动力相关蛋白 1 (Drp1)）调节。有趣的是，CaMK1α 和钙调磷酸酶均受到磷酸化的严格调节，两者都是 LTP 诱导所必需的。还通过调节 Drp1 磷酸化来增加 Drp1 向线粒体的募集和线粒体分裂。然而，尽管有这些迹象，LTP 和线粒体裂变之间的联系仍然未知。出于好奇，我使用化学诱导 LTP (chemLTP) 的方案来测试 LTP 诱导是否增加我的初步数据显示，线粒体裂变发生了快速而短暂的爆发。 chemLTP 刺激，除了树突棘形态的稳健和持续的典型变化之外。总之，这些观察使我认识到 LTP 需要增加树突状线粒体通过直接调节 Drp1 功能进行裂变我将使用多种共焦和超分辨率武器库。显微镜、分子扰动和生物化学技术来确定 LTP、 Drp1 功能和线粒体裂变通过解决三个具体目标首先，我将确定是否是 LTP。需要增加树突状线粒体裂变，我将通过以下方式确认并扩展我的初步发现。通过阻断 NDMAR 来测试裂变增加是否依赖于 NMDAR 来测试是否诱导。 LTP 需要裂变，我将通过表达 Drp1 显性失活突变体来阻止裂变，并测量 chemLTP 刺激后树突棘、突触和线粒体分裂的特性其次，我会。确定 chemLTP 刺激是否会改变线粒体上 Drp1 寡聚体的募集或流动性使用共聚焦显微镜粒子追踪，我的初步数据表明 Drp1 斑点变得尖锐。在诱导裂变之前移动性较差，我将测量 chemLTP 刺激对这种移动性的影响。测量线粒体上分离的 Drp1 分子的迁移率在 LTP 期间是否发生改变，我将使用单个第三，我将确定是否存在LTP。诱导需要通过使用磷酸特异性抗体的蛋白质印迹对 Drp1 进行磷酸化，并且通过在磷酸模拟物和磷酸无效 Drp1 存在的情况下测量裂变和树突棘特性总之，这些目标将极大地扩展我们对神经元线粒体裂变的理解，并且将阐明线粒体裂变和突触增强之间尚未探索的关系。