(P25-021-24) Palmitoleate Protects Against Zika Virus-Induced Endoplasmic Reticulum Stress and Apoptosis in Neuronal Cells

Objectives: Zika virus (ZIKV) infection during pregnancy leads to the development of major fetal complications such as microcephaly and congenital Zika syndrome affecting fetuses. We had earlier demonstrated that palmitoleate an omega-7 monosaturated fatty acid protects ZIKV-induced apoptosis in placental trophoblasts. In the present study, we hypothesize that palmitoleate prevents ZIKV infection-induced ER stress and apoptosis in neuronal cells.

Methods: Recombinant MR766 or PRVABC59 strains of ZIKV was used to infect the SH-SY5Y with 0.1-1 MOI at different hours of post-infection time points. Similarly, primary fetal cortical neuron was isolated from hSTAT2KI mice and used for the ZIKV infection. Palmitoleate with 100 μM and 200 μM was prepared using 1% BSA and used as a treatment. Apoptosis was measured by using DAPI staining, and caspase 3/7 activity. ER stress markers, mitochondrial pro-apoptotic and anti-apoptotic markers, and the expression levels of viral envelope proteins were measured through qRT-PCR and immunoblot analysis. Additionally, plaque assay was used to determine the infectious viral titer.

Results: ZIKV infection to neuronal cells showed increased levels of pro-apoptotic markers like cleaved-PARP, cleaved caspase-3, Bim, and Puma, whereas decreased the levels of anti-apoptotic markers. Further, we observed activation of three arms of ER stress pathway with ZIKV infection. Treatment of palmitoleate drastically decreased ZIKV infection-induced increase in percent apoptotic nuclei, caspase 3/7 activity, and cleaved PARP protein expression. After infection, palmitoleate supplementation attenuated ZIKV-induced ER stress activation as evidenced by decreased levels of phosphorylated forms of inositol-requiring enzyme 1α (pIRE1α) and eukaryotic initiation factor 2 alpha (p-eIF2a); as well as decreased expressions of cleaved ATF6 (p50ATF6), spliced X-box associated protein 1 and C/EBP homologous (sXBP1) protein compared to ZIKV infection alone. Treatment of palmitoleate also significantly decreased the expression of viral envelop protein and the infectious viral titer.

Conclusions: In conclusion, we have established that palmitoleate protects against ZIKV-induced ER stress and apoptosis in neuronal cells.

Funding Sources: The project was supported by the Child Health Research Institute, University of Nebraska Medical Center.