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.