Project Summary Neurofibrillary tangle formation and neurodegeneration occur, during preclinical Alzheimer’s disease (AD), in remarkably restricted cell populations, in the layer II of the EC (ECII), at first in a subregion of EC, the transentorhinal area (TEC). The reason for this selective vulnerability pattern is still unknown. It is crucial to discover genes and pathways underlying vulnerability, because they could both yield mechanistic insight into the disease and open new therapeutic avenues. We previously compared protein-coding genes between ECII and various neuron types resistant to AD and uncovered a number of gene drivers of neuronal vulnerability by building an ECII functional network. Here, using single-nucleus RNA-sequencing, we identify a micro RNA (miRNA), miR-129-2, with a unique expression pattern that makes it a very promising candidate vulnerability gene. It is expressed at highest levels in the TEC, might be differentially expressed between mouse and humans, and is predicted to modulate AD pathology in the EC by our functional network analysis. Additionally, two of its targets are extremely relevant for AD: RBFOX1, an AD genetic susceptibility factor, and MAPT, the gene that codes for tau itself. miRNAs, by modulating expression of hundreds of genes at once can have profound effects on cells, serving both as functional and pathological hubs. Here we propose to investigate how miR-129-2 interferes with different aspects of AD pathogenesis and to demonstrate that it is protective against tau accumulation. In Aim 1, we propose to map the subregions of EC where miR-129-2 is expressed, in mouse and humans. In AD, miR-129-2 is downregulated, and we will test how early in the disease this downregulation occurs by comparing its levels in the EC in control and prodromal AD individuals. To determine why miR-129-2 decreases in AD, we will test its expression in AD-like contexts (Aβ, aging) in the mouse. In Aim 2, we will study the crosstalk between miR-129-2 and Rbfox1 and how both broadly regulate AD genes in ECII, by regulating splicing and RNA stability. We will test if miR-129-2 regulates amyloidosis and microglia activation. Lastly in Aim 3, we will ask if miR-129-2 has a role in directly curtailing tau levels. If we validate that this is the case, miR-129-2 will be an exciting therapeutic target that we could mimic in order to keep tau levels at bay in ECII neurons. Systematically silencing it in AD mouse models would also generate models with more abundant AD-like pathology in ECII neurons.