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Victoria Koontz, Sayan Ghosh, Nadezda A Stepicheva, Haitao Liu, PENG SHANG, Olivia Chowdhury, Anastasiia Strizhakova, Rachel Daley, Stacey L Hose, J. Samuel Zigler, Meysam Yazdankhah, Debasish Sinha; The differential role of distinct astrocyte populations in Persistent Fetal Vasculature (PFV) disease. Invest. Ophthalmol. Vis. Sci. 2022;63(7):266 – F0311.
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© ARVO (1962-2015); The Authors (2016-present)
PFV is a rare disease with limited treatment options, where the hyaloid artery does not regress normally after birth, potentially causing blindness in an otherwise normal child. We have shown that astrocytes in Nuc1 mutant (spontaneous mutation in Cryba1 gene that encodes for βA3/A1-crystallin) rats abnormally migrate and ensheath the hyaloid artery, likely due to abnormal lysosomal function and activated EGFR/mTORC1 signaling. Different astrocyte populations with anti/pro-inflammatory properties have been identified in human diseases and are linked to protective/neurodegenerative changes, respectively. Herein, we evaluated the importance of different astrocyte populations in our rat model, determining the roles of their underlying signaling pathways in the pathogenesis of PFV disease.
We performed scRNAseq analysis on optic nerve astrocytes from Nuc1 and WT rats. Western blot analysis evaluated the levels of mTOR and inflammatory signaling proteins. Astrocyte cell migration (wound healing assay), proliferation (Ki-67 expression) and phagocytosis (pHrodo internalization assay) were also assessed. βA1-mCherry construct was generated to overexpress the protein in astrocytes in vitro.
scRNAseq analysis revealed two distinct astrocyte populations- astrocyte clusters 1 and 2 (AC1 and 2) in Nuc1 rats, but not in WT. AC1 showed increased expression of mTORC1 components like Raptor and downregulation of mTORC2 components like Rictor, with no increase in the levels of inflammatory genes. However, AC2 showed activation of both mTORC1/2 components and inflammatory mediators like CXCL14 and IL-2a. Interestingly, Lrp2 gene, which is linked with astrocyte activation in Parkinson’s disease, as well as glial and cancer cell activation/migration, also was upregulated in AC2 but not in AC1 cells. These results were confirmed by western blots and the changes were rescued upon βA1-crystallin overexpression in AC2 cells. Induction of cell migration/proliferation and a reduction in phagocytic activity, known phenotypic changes during astrocyte activation in PFV disease were significant in AC2 cells and were rescued by βA1-crystallin overexpression.
We have identified a specific population of astrocytes which might be responsible for the pathogenesis of PFV disease: overexpression of βA1-crystallin in these astrocytes could have therapeutic importance for treating PFV disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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