Identifying Mediators of Tau-Mediated Neuronal Necroptosis Using an Innovative In Vivo CRISPR Screen

Neuronal degeneration, or the death of neurons, is a critical aspect of neurodegenerative diseases like Alzheimer’s disease (AD). However, we do not understand the mechanisms leading to this neuronal cell loss and how this process is controlled. Our laboratory has generated an innovative chimeric model for AD. In this model, transplanted stem cell-derived human neurons, when exposed to amyloid plaques, recapitulate all neuropathological features of AD. This is the first in vivo model with tau pathology, neuronal tangles, and granulovacuolar neurodegeneration. The new phospho-tau biomarkers can be measured in the blood of the model animals. We have made the seminal observation that human neurons degenerate by the necroptosis pathway, and this observation was validated using human post-mortem AD brains. We have used this model to perform a CRISPR/CAS9 screen to delete genes in the human neurons. Neurons that can survive better—for instance, because kinases that induce cell death have been deleted—will be more resilient and survive in our transplantation model. By deep sequencing, we can identify genes that provide such an advantage. We have finalized such a screen and identified more than ten interesting guide RNAs against kinases that appear to protect the neurons against necroptosis. In the current project, we aim to confirm these findings in a confirmatory screen of the hits identified in the first round. Further, we will investigate and validate those kinases confirmed in AD pathology to explore whether they affect tau and necrosome pathology, and whether they can be used as drug candidates for the downstream pathways in AD. Such drugs are essential to complement amyloid beta therapies, which slow down but do not stop the disease process.