Evolving CRISPR-based platforms for the discovery of Alzheimer relevant neurodegenerative pathways

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Project Summary/Abstract Alzheimer's disease (AD) is a major unresolved public health problem for which there are no effective treatments or means of prevention. Monotherapies aimed at individual molecular targets that have been implicated in the disease by pathological, biochemical or genetic evidence have so far had only minimal, if any, successes in clinical trials, despite reasonable evidence for target engagement. Diverse lines of evidence suggest that AD is a heterogenous disorder with a multifactorial pathogenesis involving diverse factors, including amyloid peptides, tau, aberrant immune cell activities, and in many cases also apolipoprotein (apo) E4, the major genetic risk factor for the disease. These and possibly other factors may “conspire” to cause the degeneration of vulnerable neuronal populations through complex interactions that are difficult to predict based on current knowledge. We hypothesize that broad and unbiased screens in which many different genes are experimentally disrupted could shed light on these interactions and on the mechanisms underlying AD-related neurodegeneration in general. Such screens have already yielded tremendous insights into diverse biological functions in simple organisms. We hypothesize that the recently established CRISPR/Cas9 technology should make it possible to carry out such screens also in what is widely considered to be the most complex organ of mammalian species, the brain. However, so far, most CRISPR screens have been carried out in cancer cell lines, which probably cannot faithfully recapitulate the specialized functions and selective vulnerabilities of brain cells, which are most relevant to neurodegenerative diseases such as AD. Here we propose to adapt this methodology to enable unbiased genetic screens in brains of AD-relevant rodent models. In Aim 1, we will use CRISPR screening to identify genes that can protect cultured rat neurons against glutamate-induced neurodegeneration, a form of neurotoxicity that is of likely relevance to AD, vascular dementia, and many other brain disorders. In Aim 2, we will establish mixed neuronal/glial brain cell cultures from genetically modified mice expressing human apoE4 and human tau, treat them with A, and use this model as a platform for a large-scale CRISPR screen to identify genes that can block the neurodegeneration that results from the interaction among these AD-relevant pathogens. In Aim 3, we will use adeno-associated viral vectors to advance this technology toward in vivo screens in brains of mouse models co-expressing human A, apoE4 and tau. We will determine whether knockout of specific genes can promote neuronal survival in this AD-relevant context. Adapting this technology to perform large-scale, unbiased genetic screens in primary neurons and rodent brains will provide valuable guidance to the scientific community. Beyond that, our studies could identify new mediators of neurodegeneration and open new therapeutic avenues for the treatment of AD and related conditions.