Development of isogenic iPSC cell lines expressing tauopathy-related PERK risk variants to study homeostatic control of protein levels during ER stress

点击翻译按钮获取中文摘要

Project Summary In neurodegenerative diseases, accumulation of misfolded proteins is the pathological hallmark. Levels of the toxic proteins, such as tau, correspond to cellular dysfunctions and cause neurodegeneration. Protein levels are normally carefully maintained by the balance of the synthetic and degradative pathways. However, how the steady state is orchestrated between the different pathways is not clear. One protein that is dysregulated in neurodegenerative diseases is the microtubule-binding protein tau. For example, tau levels are elevated in the Alzheimer’s disease post-mortem brain, and this correlate with pathology and clinical signs. One candidate that may be able to coordinate multiple pathways, controlling both production and degradation of tau, is PERK (protein kinase R (PKR)-like endoplasmic reticulum kinase). PERK is an endoplasmic reticulum (ER) resident protein, and is activated during ER stress. The identification of a PERK risk variant for tauopathy in multiple large GWA studies underscores the importance of understanding the ways that PERK controls protein homeostasis in health and in disease. Using human induced pluripotent stem cell (iPSC) lines carrying a PERK risk variant (PERK B) associated with tauopathies, the authors made the discovery that ER stress leads to an elevation in tau levels and neuronal death. PERK B contains three coding-region SNPs resulting in nonsynonymous missense mutations at amino acid positions 136, 166 and 704. It was found that PERK B is a functional hypomorph; its kinase activity is reduced via the S704A mutation. Additionally, during ER stress triggered by tunicamycin, tau protein levels, instead of being reduced, are increased in the homozygous risk variant (PERK B/B) compared to the control (PERK A/A). These data suggest an alteration in tau protein homeostasis caused by PERK B, but the involvement of other genes could not be ruled out, because the PERK B/B and PERK A/A iPSC lines were not isogenic. This proposal seeks to test the hypothesis that PERK B dysregulates protein homeostasis, via changes in protein production and/or clearance. Aim 1 proposes to generate isogenic lines from iPSC carrying PERK A/A to B/B and B/B to A/A. The mechanisms of tau homeostatic dysregulation during ER stress will be investigated by performing protein translation poly- ribosome and SUnSET assay and protein degradation cycloheximide chase assay. Trazodone, a PERK activator which is proposed as a therapeutic agent for neurodegeneration, will be tested to determine its effect on PERK A/A and B/B genotypes. The results of this proposal will provide improved lines and cell model system to examine regulation of homeostasis of tau and proteins in general during ER stress. This proposal is suitable for the R03 mechanism, for the results will provide the support for the preparation of a future R01 submission.