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Protein Quality Control and Neurodegeneration

The presence of unfolded proteins is suggested to be a causative factor of neurodegenerative diseases (ND). This notion is based on decades of evidence ranging from early observations of distinct protein-based macroscopic deposits in all major NDs, through years of genetic and experimental evidence including the discovery of familial risk mutations in the same aggregating proteins and evidence for their neurotoxic effects. In addition genome wide association studies revealed multiple additional mutations in protein quality control pathways that increase ND risk. Proteotoxic stress elicits intricate, organelle specific, stress responses including the Endoplasmic Reticulum and mitochondrial Unfolded Protein response (erUPR and mtUPR) and the Heat Shock Response (HSR). Activation of these pathways results in the induction of protein quality control (PQC) mechanisms as the expression of chaperones, the ubiquitin proteasomal system (UPS) and the autophagy-lysosome pathway, or the initiation of cell death in cases of unmitigated damage. External activation of these pathways was shown to have short and long term alleviating effects in cellular and animal models of ND suggesting a target for therapeutic intervention.

Illustration of the steps in a pooled screen for both growth and fluorescence based cellular phenotypes 

Research in the last two decades has provided a wealth of mechanistic understanding on how stress signaling interacts with PQC mechanisms and apoptotic pathways. Yet this understanding is based on studies using a limited number of cell models, while it is increasingly recognized that the nature of these responses is cell type specific and depends on extrinsic factors such as organismal metabolism and signaling between cells. An unprecedented ability to edit the genome together with advances in cellular reprogramming technology opens the opportunity to study protein homeostasis networks in diverse cellular contexts and investigate how they are affected by genetic variation that causes ND. 

 

One research direction in the lab is aimed at mapping the response to different proteotoxic stresses in different cell types. These can be induced either pharmacologically or genetically by introducing ND risk mutations through genome editing. We plan to combine stem cell differentiation protocols and high throughput approaches with a focus on CRISPR pooled knockout and activation screens to find novel regulators of PQC pathways and understand how those differ between cell types and between wild type cells to cells that harbour ND risk mutations.

Results of a selective sensitivity screen showing unbiased identification of regulators of the endoplasmic reticulum unfolded protein response.

Underlying mechanisms of ND risk genetics through genetic interactions

Advances in human genetics are accelerating the discovery of genetic variation associated with neurodegenerative diseases, yet elucidating the molecular mechanisms that underlie pathogenesis, even of the rare highly penetrant mutations, remains a great challenge. In the lab we plan to develop novel, unbiased, genome-wide screening paradigms in cellular and animals of neurodegenerative disease to illuminate the pathways affected by individual ND risk mutations. This will be done by identifying key modifier genes using a range of toxicity and fluorescence based phenotypes that are specific to cells that harbour ND risk mutations. Such mapping of genetic interactions between risk mutations and additional genes will help us understand the genetic networks that are affected and propose new targets for therapeutic intervention.

Mutant cell line that displays increased stress sensitivity due to accumulation of undegraded autophagosomes.

            mutant cell line                               wt cell line

Gene expression regulation during proteotoxic stress and the effect of ND risk mutations

Maintanence of a healthy and functioning proteome depends on a complex regulatory network that senses pertubations and responds by adusting protein production load through genome-wide regulation of transcription, translation and both mRNA and protein degradation. We plan to use dynamic measurements of both protein and mRNA turnover in cell and animal models of ND to understand how acute and chronic pertubations affect the ability of the cells to control genome-wide gene expression programs. 

Shalem Lab

Functional Genomics

Protein Quality Control

and Neurodegeneration