Uncovering the molecular and cellular mechanisms underlying AD using CRISPR/Cas9-edited isogenic neural cells

Beckman Research Institute/City Of Hope, Duarte CA

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Abstract

Project Summary Alzheimer?s disease (AD) is the most common form of dementia in the elderly and there is no cure for this disease to date. The molecular and cellular mechanisms underlying AD pathogenesis remains to be elucidated, in order to develop effective therapies for this disease. Many transgenic mouse models have been generated for AD research and these models provide important insights to aid our understanding of the pathological basis of the disease. However, there are significant species differences between mouse and human neural cells, which may explain the observation that none of the animal models has captured the entire spectrum of the disease pathology, including considerable neuronal loss. Therefore, establishing human disease modeling platforms is needed to complement studies in animal models to better understand AD. Human induced pluripotent stem cells (hiPSCs) have been widely used for disease modeling and drug discovery since the development of the iPSC technology. However, the fetal-like properties of hiPSCs present a challenge to model late-onset diseases, for which cellular aging is important in disease pathology. Direct reprogramming converts one type of somatic cells into another without going through the iPSC stage that involves extensive epigenetic modifications, enabling generation of human neurons that possess key elements of cellular aging. Therefore, directly reprogrammed cells derived from patient somatic cells would allow us to model age-related, late-onset diseases, such as late-onset AD, in a human cellular platform. The objective of this proposal is to uncover molecular and cellular mechanisms underlying AD using aging- relevant cellular models derived from direct reprogramming in combination with CRISPR/Cas9 gene editing. We propose to establish cellular models for AD using astrocyte-neuron co-cultures derived from directly reprogrammed cells. While the apolipoprotein (Apo) E4 has been identified as the strongest genetic risk for late-onset AD, the C allele risk variant rs11136000 in the clusterin (CLU) gene represents the third strongest known genetic risk factor for late-onset AD. The mechanism as to how the CLU risk variant contributes to AD pathologies remains largely unknown. We hypothesize that CLU modulates late-onset AD pathologies in an ApoE isoform- and age-dependent manner. Accordingly, we propose three complementary aims to test this hypothesis. Aim 1: To derive isogenic neural cells with different APOE and CLU genotypes via CRISPR/Cas9 gene editing and direct reprogramming. Aim 2: To generate isogenic NSCs with different APOE and CLU genotypes by CRISPR/Cas9 editing. Aim 3: To define the individual and combined effects of APOE and CLU variants on AD pathogenesis using astrocyte-neuron co-cultures. The proposed studies will likely help to define the roles of the CLU risk variant in the development of age-associated AD pathological features, to uncover novel mechanisms underlying AD pathogenesis, and to design novel therapeutic strategies for AD.