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Project 2: Tau uptake mechanisms and neuronal excitability in FTD

项目 2：FTD 中的 Tau 摄取机制和神经元兴奋性

基本信息

批准号：
10011930
负责人：
KENNETH Stephen KOSIK
金额：
$ 29.61万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
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：
2016-09-30 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10011930
关键词：
Action Potentials Address Affect Anticodon Astrocytes Axon Back Binding CRISPR interference California Categories Cell Line Cells Cellular Stress Characteristics Collaborations Collection Computer software Cultured Cells Data Defect Dendrites Detection Devices Electrophysiology (science)Enzymes Event Functional disorder Future GAG Gene Gene Expression Genes Germ Cells Growth Harvest Heparan Sulfate Proteoglycan Human Institutes Investigation Label Lead Link Measures Mediating Methods Microarray Analysis Neurons Pathogenicity Pathologic Pathology Pathway interactions Pattern Phenotype Physics Polysaccharides Population Preparation Process RNA Resolution Rest Role Signal Transduction Small RNA Stress System Techniques Technology Testing Time Training Transfer RNA Translations analytical tool base collaborative approach design differential expression falls induced pluripotent stem cell instrumentation interest member millisecond multi-electrode arrays neuronal excitability novel programs proteostasis relating to nervous system single-cell RNA sequencing small molecule stress granule tau Proteins tau aggregation tau conformation tau mutation tool transcriptome sequencing transcriptomics uptake

项目摘要

PROJECT SUMMARY/ABSTRACT — PROJECT 2 Many unknown pathogenic steps lie between normal tau protein in a healthy neuron and the cellular dysfunction that occurs as a result of tau uptake and aggregation. The aims address two emerging facets of the pathobiology: the mechanism of tau uptake in neurons and astrocytes and the effects of tau inclusions on a set of functional cellular parameters. Tau uptake will be studied using a highly novel and highly collaborative approach called CRISPRi to identify in a nonbiased manner genes involved in tau uptake followed by functional studies, which will validate the CRISPRi hits. A more focused approach to uptake will focus on HSPGs with a GAG microarray panel to identify HSPG chain features to which tau can bind specifically. Once tau is inside the cell we ask what are the effects of tau inclusions or tau mutations in human iPSCderived neurons. We have synthesized multiple types of tau aggregates and studied their uptake conditions. A variety of techniques will be applied to pinpoint cellular defects due to the burden of tau inclusions. These techniques fall into two categories—expression sequencing and electrophysiological assessment. With regard to the former, the strength of our proposal is the use of single cell RNAseq to identify precisely gene expression changes in cells with tau inclusions compared to its neighbors that do not have tau inclusions. A further strength is the data that supports our hypothesis concerning the role of tRNA in triggering tau conformational changes that may lead to aggregation as well as an investigation of the role of cellular stress in inducing tRNA cleavage and the formation of half tRNAs known as tiRNAs. We have also hypothesized that tau can induce electrophysiologic dysfunction and over the past three years in collaboration with the physics department have built tools capable of detecting conduction deficits, alterations in the action potential at the axonal initial segment and hyerexcitability. The detection methods utilize a multielectrode array (MEA) platform, modified MEAs that are patterned to confine neuron growth and direct signaling between neuronal ensembles, analytical tools for spike trains, and an automated cell harvesting device. The delineation of the effects on tau mutations and tau inclusions on cultured cells is a cellular phenotype that is not welldescribed in the field, but will be necessary in the future for small molecule screens. Working interchangeably with several cellular systems will allow us to select the ideal context for each experimental question posed and explore multiple facets in a search for taurelated cellular phenotypes. This proposal rests on strong interactions among all the team members.

项目摘要/摘要-项目2