Date of Award

Summer 2023

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Rekha C. Patel

Abstract

Luteolin is a natural flavonoid present in vegetables, fruits, and medicinal herbs that possesses anti-oxidative, anti-cancer, and anti-inflammatory properties. In this study, we investigated the effect of luteolin on the integrated stress response (ISR), which is an evolutionarily conserved intracellular signaling network essential for adapting to environmental stresses to maintain healthy cells. Dysregulated ISR is involved in the etiology of many human diseases including the movement disorder DYT-PRKRA which is caused by mutations in the PRKRA gene. PRKRA encodes for PACT, the protein activator of interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR.PACT-mediated PKR activation regulates the ISR via phosphorylation of the eukaryotic translation initiation factor eIF2α. A dysregulation of either the level or the kinetics of eIF2α phosphorylation in response to a stress signal can cause the normally pro-survival ISR to become pro-apoptotic. In DYT-PRKRA patient cells, the PRKRA mutations lead to an enhanced PACT-PKR interaction causing dysregulated of ISR and an increased sensitivity to apoptosis. We have previously identified luteolin as an inhibitor of the PACT-PKR interaction using high-throughput screening of chemical libraries. Our results presented in this study indicate that luteolin is markedly effective in disrupting the pathological PACT-PKR interactions to protect DYT-PRKRA cells against apoptosis. In addition to pinpointing how luteolin protects cells against maladaptive ISR, our research also suggests a therapeutic option for using luteolin to treat several diseases that result from dysregulated ISR.

Our results indicate that disrupting PACT-PKR interactions in DYT-PRKRA patient cells restores the normal kinetics of PKR activation and eIF2α phosphorylation to reduce CHOP induction thereby preventing apoptosis in response to ER stress. These results also indicated that although luteolin disrupts the abnormally strong PACT-PKR interactions observed in patient cells in the absence of stress, it allows for the normal stress-induced and transient PACT-PKR interaction which is essential for protective functions of ISR. Our results in demonstrate that luteolin does not disrupt the transient stress-dependent interaction between phosphorylated PACT and PKR. Thus, luteolin selectively prevents pathological PACT-PKR interactions in DYT-PRKRA patient cells in the absence of stress while preserving the normal stress-induced PACT-PKR interactions to allow for a transient PKR activation during ISR.

We also describe the CReP inhibitor, Nelfinavir, on its ability to promote cellular recovery of DYT-PRKRA lymphoblasts in response to ER stress. CReP levels are significantly elevated within DYT-PRKRA patient lymphoblasts relative to wt cells both in the absence of stress and in response to ER stress-induction. Due to the reduction in CReP and increased eIF2α phosphorylation due to Nelfinavir, we next determined an enhanced ISR activity thereby leading DYT-PRKRA cells to have increased sensitivity to ER-stress induced apoptosis. Despite Nelfinavir inducing a robust ISR, this did not cause cellular protection from ER stress-induced apoptosis. Of note, Nelfinavir induces apoptosis with wt and DYT-PRKRA cells even in the absence of ER stress-induction.

Next, we established that PKR is hyperactive and levels of eIF2α phosphorylation were increased within DYT-3 (XDP) neuronal progenitor cells (NPCs). To link the correlation between enhanced PKR activity and dysregulated eIF2α signaling to increased apoptosis, we investigated another target of active PKR, ATF3, which is a pro-apoptotic protein, to elucidate downstream effects further. We ascertained there is highly increased expression of ATF3 within XDP cells which buttresses the inference that dysfunctionality of the eIF2α axis and the ISR is a common causality that drives pathophysiology and disease progression within multiple dystonia types.

Lastly, we characterized three different PKRi compounds for their ability to promote cellular recovery of DYT-PRKRA lymphoblasts in response to ER stress. Two of the PKRi compounds, PKT-00888 and PKT-00954 did not protect DYT-PRKRA lymphoblasts from ER stress-induced apoptosis. Meanwhile, PKT-00941 did protect DYT-PRKRA cells from ER stress-induced apoptosis. Also, there were no cytotoxic effects seen with PKT-00941. PKT-00941 drastically diminishes p-PKR and p-eIF2α levels and duration in DYT-PRKRA cells. PKT-00941 significantly attenuates CHOP induction in response to ER stress in DYT-PRKRA cells. This disruption in CHOP induction highlights how PKT-00941 suppresses apoptosis thereby promoting cellular homeostasis as CHOP induction is essential for apoptosis after ER stress.

Rights

© 2023, Kenneth L. Frederick

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