Up-Regulation of PKR Signaling Pathway by Ethanol Displays an Age of Onset-Dependent Relationship

Abstract

Background: Ethanol (EtOH) neurotoxicity can result in devastating effects on brain and behavior by disrupting homeostatic signaling cascades and inducing cell death. One such mechanism involves double-stranded RNA activated protein kinase (PKR), a primary regulator of protein translation and cell viability in the presence of a virus or other external stimuli. EtOH-mediated up-regulation of interferon-gamma (IFN-γ; the oxidative stress-inducible regulator of PKR), PKR, and its target, p53, are still being fully elucidated.

Methods: Using Western blot analysis, immunofluorescence, and linear regression analyses, changes in the IFN-γ-PKR-p53 pathway following chronic EtOH treatment in the frontal cortex of rodents were examined. The role of PKR on cell viability was also assessed in EtOH-treated cells using PKR overexpression vector and PKR inhibitor (PKRI).

Results: In rats chronically fed EtOH, PKR, phosphorylated PKR (p-PKR), IFN-γ, and p53 were significantly increased following chronic EtOH exposure. Linear regression revealed a significant correlation between IFN-γ and p-PKR protein levels, as well as p-PKR expression and age of EtOH exposure. Overexpression of PKR resulted in greater cell death, while use of PKRI enhanced cell viability in EtOH-treated cells.

Conclusions: Chronic EtOH exposure activates the IFN-γ-PKR-p53 pathway in the frontal cortex of rodents. p-PKR expression is greater in brains of rodents exposed to EtOH at earlier ages compared to later life, suggesting a mechanism by which young brains could be more susceptible to EtOH-related brain injury. PKR and p-PKR were also colocalized in neurons and astrocytes of rats. This study provides additional insight into biochemical mechanisms underlying alcohol use disorder related neuropathology and warrants further investigation of PKR as a potential pharmacotherapeutic target to combat EtOH-related neurotoxicity, loss of protein translation and brain injury.

Keywords: Double-Stranded RNA Activated Protein Kinase; Ethanol-Induced Cell Death and Age; IFNγ-PKR-p53 Pathway and Age; Interferon-Gamma; p53.

Figure 1. Chronic ethanol (EtOH) exposure increased the expression of PKR and p-PKR in the frontal cortex of rats

Rats were fed EtOH or control liquid diets over 28 days and the protein expression of PKR and p-PKR were evaluated. (A) Representative immunoblot depicting PKR, p-PKR, and actin bands from control and EtOH-treated rats. (B) Expression of PKR was significantly increased in EtOH-treated rats compared to EtOH-preferring control rats. (C) Expression of p-PKR was significantly increased in EtOH-treated rats compared to EtOH-preferring control rats. Data presented as mean±SEM; *p<.05.

Figure 2. Interferon-gamma and p53 are increased by chronic ethanol (EtOH) exposure in the frontal cortex of rats

Rats were fed EtOH or control liquid diets over 28 days and the protein expression of IFNγ and p53 were evaluated. (A) Expression of IFNγ was significantly elevated in EtOH-treated rats compared to control rats. (B) The protein expression of IFNγ is positively correlated with p-PKR (C) Expression of p53 was significantly elevated in EtOH-treated rats compared to control rats. Upper Panels in A and C: Representative western blot showing immunolabeling in control and EtOH-treated rats. Lower Panels in A and C: Quantitative graph of average optical density ratios in the EtOH-preferring control (n=9) and EtOH-fed (n=9) groups. Data presented as mean±SEM; *p<.05.

Figure 3. Cellular and subcellular localization of PKR and p-PKR in neurons and astrocytes

Representative photomicrographs obtained from the pre-frontal cortex (PFC) to demonstrate the cellular distribution of PKR/p-PKR (red) immunoreactivity. The cell nucleus was counterstained by DAPI (blue). The astrocytes were labeled by GFAP immunoreactivity (IR) (green) and neurons were labeled by NeuN IR (magenta). (A) PKR IR distribution in PFC of rats fed with control diet depicting PKR localization to the cell nucleus; (B) PKR IR distribution in PFC of EtOH-treated rats depicting enhanced PKR localization to both nuclear and cytosolic compartments compared to control rats; (C) p-PKR IR distribution in PFC of rats fed with EtOH diet depicting p-PKR localization to the nucleus of neurons and both cytosol and nucleus in astrocytes.

Figure 4. Phosphorylated PKR protein expression is correlated with age of onset of chronic ethanol (EtOH) exposure in rodents

Control mice and EtOH-treated mice were assessed for p-PKR expression at various ages. Mice were exposed to EtOH beginning at 1, 4, or 9 months and were fed EtOH according to the Lieber-DeCarli diet for 28 days. p-PKR expression was measured thereafter and was negatively correlated with age of onset of EtOH exposure, with the youngest mice (1 mo. old) having the greatest p-PKR expression, which decreased with age at the time of EtOH exposure. Control mice also showed a negative correlation with respect to age and p-PKR expression. *p < 0.05 vs. control

Figure 5. PKR is involved in ethanol (EtOH)-induced cell death

SH-SY5Y and U-1242MG cells were exposed to EtOH for 2 days. Subgroups of EtOH-exposed cells were transfected to overexpress PKR, or treated with a PKRI for the duration of EtOH exposure. (A) EtOH decreases cell viability in both SH-SY5Y and U-1242 cells, but concomitant PKRI administration significantly alleviates loss of cell viability induced by EtOH. (B) SH-SY5Y cells which overexpress PKR result in a significant loss of cell viability compared to EtOH-treated cells. pCMV empty vector was used as a transfection control vector. (C) Similarly, U-1242MG cells which overexpress PKR result in a significant loss of cell viability compared to EtOH-treated cells. pCMV empty vector was used as a transfection control vector. All data presented as mean±SEM; p*<.05; **p<.01; #p<.001.

Figure 6. Proposed ethanol (EtOH)-induced PKR signaling

The accumulation of EtOH-induced reactive oxygen species (ROS) elevates IFN-γ expression which drives the transcription of PKR at IFN-γ Activating Sequence (GAS) elements. IFN-γ also phosphorylates PKR through direct mRNA binding to the regulatory subunit. PKR transcription can also be controlled by Sp1-like transcription factors through Sp1 binding sites. Activated p-PKR can translocate to the nucleus and also induces phosphorylation of eukaryotic translation initiation factor 2α, which inhibits protein translation, leading to cell death. pPKR also increases p53 which causes increased oxidative stress and cell death.