Determining how a single-residue PKR mutation found in patients with familial dystonia causes aberrant PKR activation

Project Summary/Abstract Protein Kinase R (PKR) is a ubiquitously expressed serine/threonine kinase that recognizes double stranded RNA (dsRNA). Upon binding viral dsRNA, PKR homodimerizes and autophosphorylates, activating PKR Kinase activity. Phosphorylated PKR will then go on to phosphorylate various targets, including the well-characterized eIF2α, to initiate translational shutdown in the cell. This translational shutdown is important for limiting viral replication and spread. Intriguingly, active PKR can also be detected in the absence of viral dsRNA in a variety of different neurologic diseases, including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. However, the underlying molecular mechanisms for PKR activation in neurodegenerative diseases are incompletely understood. Recently, several mutations, all located in the dsRNA binding domain of PKR, have been associated with early onset generalized dystonia, a neurologic disease characterized by an uncontrollable writhing motion. This suggests that abnormal PKR activity can play a causal role in neural disease. Additionally, despite the ubiquitous expression of mutant PKR, disease was restricted exclusively to the nervous system. Notably, a single-residue glycine-to-arginine mutation (G130R mutation) was found in several independent families. The G130R mutation was associated with a mild increase in PKR activation in sampled patient fibroblasts following PKR stimulation with poly I:C (a viral dsRNA mimetic). By expressing wildtype PKR (PKRWT) or PKR harboring the G130R mutation (PKRG130R) in human cells, we have found that expression of PKRG130R, but not PKRWT, leads to PKR phosphorylation in the absence of other stimuli, apparently indicating a gain-of-function mutation. The goal of this proposal is to determine the molecular mechanism of how G130R causes PKR overactivation. Our group recently found that neurons intrinsically express high levels of long dsRNA structures, and that these self-dsRNAs can activate PKR. We hypothesize that the PKRG130R mutation is an activating mutation that enhances PKR binding affinity for self- dsRNAs in neurons. Therefore, autoinflammatory PKR reactions against self-dsRNAs may be an early event that causes neurodegeneration. Specifically, our aims are: 1. Determine the mechanism of PKRG130R activation by both dsRNA affinity as well as protein-protein interactions, 2. Determine the identity and properties of the specific transcripts that bind mutant PKRG130R compared to PKRWT, and 3. Utilize human embryonic stem cell technology to determine which cell types PKRG130R is overactivated in, and what features cause PKRG130R to be overactivated in those cell types. We believe that identifying the mechanism of PKRG130R activation may provide insight on PKR dysregulation in neurologic disease states. Understanding how PKR activity is controlled by self- dsRNAs could uncover specific RNA species or RNA associated pathways for targeted therapy to modulate PKR activation and could be applied to a host of neural diseases that share PKR overactivation as a common feature.