Project Summary/Abstract: Excitatory (EX) neurons are preferentially vulnerable in aging and early Alzheimer’s disease (AD), the most common form of dementia. The molecular determinants of this selective vulnerability are unclear. We recently identified ectodermal-neural cortex 1 (ENC1), a Kelch-related actin-binding protein, as being differentially expressed in EX neurons and one of master regulator genes responsible for selective neuronal vulnerability to tau pathology in AD. In culture models, upregulation of ENC1 has been shown to be detrimental for neural survival through its downregulation of a redox sensitive transcription factor, nuclear factor erythroid 2 like 2 (NFE2L2/NRF2) and/or its downregulation of the autophagic flux pathway through interaction with phosphorylated Sequestosome 1 (SQSTM1/p62). Furthermore, a single-nucleus transcriptomic analysis shows that ENC1 is enriched in the subpopulation of EX neurons associated with AD pathology. Comparing the gene signatures of single cells isolated from donors with early AD pathology to those without AD pathology, we found that the mRNA level of ENC1 is significantly lower in EX neurons, astrocytes and oligodendrocytes, whereas it does not change significantly in other types of cells. The decreased level of ENC1 in AD may indicate vulnerable cells are trying to mitigate compromised protein homeostasis by the downregulation of aggregation-prone proteins like ENC1. Controversially, ENC1 has also been suggested to determine cognitive resilience (i.e. less vulnerable) in the aging population, and is implicated in the NRF2-dependent oxidative stress response after treatment with hydrogen peroxide. However, the exact role of ENC1 in selective neuronal vulnerability in aging and early AD remains unclear. We hypothesize that EX neuron- and astrocyte- specific downregulation of ENC1 may protect or ameliorate the degeneration of EX neurons in aging and AD through the upregulation of the NRF2-p62 autophagy pathway. To test this hypothesis, this proposal will (1) identify if manipulation of ENC1 level in primary cultured neurons and human cerebral organoids will affect the vulnerability to tau pathology, electrophysiological properties, and the NRF2-p62 autophagy pathway; (2) determine if cell-type specific knockdown of ENC1 in animal models will upregulate the NRF2-p62 autophagy pathway, and ameliorate the aging phenotype, AD pathology, and cognitive deficits; and (3) define the relationship between ENC1 and Nrf2-p62 autophagy pathway in aging and AD. Identifying the cell-autonomous (neurons) and/or cell non-autonomous (astrocytes) role of ENC1 in selective neuronal vulnerability in aging and early AD could aid in the discovery of novel drug targets that can be targeted to protect vulnerable neurons from degeneration in aging and early AD. In addition, the approaches proposed to characterize cell populations vulnerable to pathological proteins in AD can be applied to a wide range of degenerative dis...