PROJECT SUMMARY: Selective neuronal vulnerability is a striking yet largely untapped aspect of Alzheimer’s disease (AD) -during early prodromal stages, pathological tau protein accumulates almost exclusively in excitatory neurons from layer II of the entorhinal cortex (ECII). ECII neurons are also the first to degenerate in AD. A large body of evidence indicates that AD pathological lesions increase the excitability of neurons in the hippocampal formation, while overall increased network activity accelerates lesion formation. Our recent work suggests that tau pathology and the regulation of neuronal excitability are particularly tightly linked in ECII neurons, through cell-autonomous mechanisms. It is therefore crucial to gain a comprehensive view on the mechanisms modulating electrophysiological properties of ECII neurons, as a pre-requisite to understand the preferential formation of pathological lesions in these cells. Here, we therefore propose to simultaneously characterize single ECII neurons at the electrophysiological and transcriptomic levels using Patch-Seq, in order to identify major intrinsic regulators of ECII excitability, and excitatory/inhibitory (E/I) synaptic balance, as well as their perturbation in a context akin to early AD. In Aim 1, we will correlate electrophysiological parameters with individual gene expression and with expression of gene modules that we previously found associated with tau pathology. By performing Patch-Seq both in wild-type mice and in a mouse model of amyloid accumulation, we will determine whether amyloid disrupts the ability of certain genes to regulate ECII electrophysiological properties. Independently, we also have preliminary evidence that an ECII-enriched miRNA downregulated in AD -miR-129-2- may modulate ECII E/I balance. In Aim 2 we will thus determine the contribution of miR-129-2 to the regulation of electrical properties of ECII neurons. We will inhibit miR-129-2 in vivo and subsequently profile EC to determine candidate target genes of the microRNA and perform whole-cell patch-clamp recordings in ECII neurons ex vivo. Taking advantage of the broad range of expression levels of miR-129-2 across ECII neurons, we will then perform Patch-miRNA-qPCR in ECII neurons: we will quantify miR-129-2 levels in electrophysiologically-characterized neurons. This will allow us to determine whether miR-129-2 levels correlate with certain electrophysiological features of ECII neurons. Taken together the work outlined in this proposal will identify the major molecular mechanisms by which ECII neurons cell-autonomously adjust their electrical properties, and how amyloid interferes with this process. This will lay the groundwork for future studies to determine whether key genes identified here also modulate tau accumulation and vulnerability of ECII neurons.