Date of Award

2025

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Rekha C. Patel

Abstract

ABSTRACT Dystonia is the third most common movement disorder in the United States of America , the first two being essential tremor and Parkinson’s disease. Gaining mechanistic knowledge about pathobiology of dystonia is essential for development of effective therapies. As the only therapies currently available are invasive procedures such as deep brain stimulation (DBS) or frequent botulinum toxin injections, it is critical to understand the underlying molecular mechanisms leading to dystonia. A maladaptive integrated stress response (ISR) involving dysregulation of the eukaryotic translation initiation factor  (eIF2) mediated signaling is observed in DYT-PRKRA patient cells. Mutations in PRKRA gene, which encodes PACT cause early onset primary dystonia DYT-PRKRA, a movement disorder that disrupts coordinated muscle movements. PACT activates protein kinase R (PKR, aka EIF2AK2) by a direct interaction in response to cellular stressors to mediate phosphorylation of eIF2α. Recent research has indicated that dysregulated eIF2 signaling is a convergent mechanism in etiologically diverse, genetically inherited primary dystonias. Mice homozygous for a spontaneously arisen, recessively inherited frameshift mutation, Prkralear-5J exhibit progressive dystonia. Our work on this mouse model indicates that the truncated PACT protein inhibits PKR activation and ISR. The mice homozygous for the mutation have abnormalities in the cerebellar development as well as a severe lack of dendritic arborization of Purkinje neurons, reduced eIF2α phosphorylation and reduced expression of an ISR marker protein ATF4. These results indicate that PACT mediated regulation of PKR activity and eIF2 phosphorylation may contribute to the dystonia phenotype resulting from this mutation. We also investigated the involvement of ISR in secondary dystonia. Secondary dystonia can be triggered by antipsychotic drugs, and in this study, we investigated if ISR is induced in response to antipsychotic drugs possibly leading to drug-induced dystonia (DID). To investigate the effects of antipsychotics on ISR, a mouse neural crest-derived cell line (N2a) was used. We investigated the induction of ISR by measuring the activation of two kinases involved in ISR, PERK and PKR. Additionally, we measured the induction of eIF2 phosphorylation, and its downstream effects by measuring ATF4 and CHOP induction. The results showed that all eight antipsychotic drugs we tested induced ISR. To further characterize any involvement of the known ISR kinases, we tested the effect of C-16 (PKR inhibitor), GCN2iB (GCN2 inhibitor), GSK2656157 (PERK inhibitor) on ATF4 induction by Aripiprazole (Abilify). Our results indicate that PKR inhibitor C-16 and PERK inhibitor GSK2656157 attenuate ATF4 and CHOP expression, thereby further supporting that Aripiprazole activates PERK as well as PKR. Co-immunoprecipitation analyses showed that PKR is activated via association with its activator protein PACT after exposure to Aripiprazole. As we have previously shown that luteolin, a plant flavonoid, disrupts the interaction between PACT and PKR, we tested if luteolin can alleviate the ISR response after the antipsychotic exposure. Our results showed that luteolin is effective in suppressing the ISR and these results warrant further studies to investigate if luteolin can prevent the occurrence of DID.

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© 2025, Tricia Aleena Dulice Simon

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