Date of Award
Open Access Dissertation
Rekha C. Patel
Early onset primary dystonia 16 (DYT16) is a subtype of the neuromuscular movement disorder, dystonia. The resultant phenotypes of DYT16 consist of both agonist and antagonist muscles firing simultaneously, compromised posture and gait, as well as chronic repetitive movements. DYT16 has been shown to result from mutations in the protein activator of PKR, PACT. Under conditions of viral infection, ER stress, oxidative stress, and serum starvation, PKR phosphorylates eIF2α resulting in the attenuation of general protein synthesis. Transient eIF2α phosphorylation is favorable for cell survival, however, when prolonged leads to apoptosis. In the absence of stress, both PACT and PKR are in an inhibitory heterodimeric complex with TRBP thus preventing PKR’s activation. In response to stress stimuli, PKR is activated through the disassociation of these inhibitory heterodimers which promotes the formation of PACT homodimers, a critical intermediate for activating PKR, and PACT-PKR heterodimers that result in PKR activation. Here we describe how DYT16 mutations dysregulate eIF2α stress response signaling through PKR.
Our results indicate that a dominantly inherited DYT16 frameshift mutation truncating PACT within its first functional motif ablates PACT’s ability to bind dsRNA and interact with PKR. We then describe how this truncated protein not only retains its ability to interact with TRBP and PACT but is also capable of dissociating PACT-TRBP heterodimers. Furthermore, when expressing an N- vi terminally tagged mCherry-FS fusion protein in mammalian cells we observe the accumulation of cytosolic protein aggregates, the induction of apoptosis through activation of caspases 3/7, and dysregulation in eIF2α stress signaling kinetics.
We also describe the effect 3 recessive (C77S, C213F, C213R) and 2 dominant (N102S, T34S) DYT16 point mutations have on the biochemical properties of PACT. Our results reveal that PACT with dominant mutations interact more efficiently with PKR and all DYT16 mutations have an increased capacity to homodimerize resulting in their enhanced ability to activate PKR relative to wild type PACT. We then describe how DYT16 patient lymphoblasts form stronger PACT-PKR interactions, have high basal levels of active PKR, have dysregulated eIF2α stress response signaling, and heightened sensitivity to ER stress relative to control cells. We further show that the increased sensitivity to ER stress can be partially rescued by disrupting PACT-PKR interactions.
Finally, we investigate a novel DYT16 mouse model resulting from a frameshift mutation, Prkralear-5J (referred to as lear-5J), in the mouse homolog of PACT. We demonstrate that although the mRNA is being targeted for nonsense mediated decay, we still detect residual mRNA and the truncated lear-5J protein in the mouse brain, however, not in mouse embryonic fibroblasts. We show that lear-5J protein retains its ability to interact with PKR but is a less efficient activator PKR. We also identified defects in the development of the folia within the cerebellum and reduced levels of p-eIF2α in DYT16 mice. Finally, we further demonstrate dramatic defects in the arborization of the Purkinje neuron layer within the cerebellum of these mice.
Burnett, S. B.(2020). Dysregulation of the Integrated Stress Response in Early Onset Dystonia (DYT16) Due to Mutations in Pact. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/5864