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Activation of HERV-K(HML-2) limits cortical neuronal differentiation by modulating Neurotrophic Tyrosine Receptor Kinase 3 expression

ABSTRACT: The biological function and disease association of human endogenous retroviral (HERV) elements remains largely elusive. We addressed the physiological role of HERV-K(HML-2) in neuronal differentiation by manipulating HERV-K(HML-2) expression levels. We used CRISPR engineering to activate or repress HERV-K(HML-2) and demonstrate that elevated HERV-K(HML-2) transcription is detrimental for development, functionality and growth of cortical neurons. Effects are cell-type specific, as dopaminergic neurons were unaffected. We further show that layer formation is altered during forebrain organoid formation following activation of HERV-K(HML-2) transcription. HERV-K(HML-2) transcriptional activation concurrently elevated Neurotrophic Tyrosine Receptor Kinase 3 (NTRK3) expression along with other neurodegeneration-related genes. Direct transcriptional activation of NTRK3 resembled the HERV-K(HML-2) activation phenotype. Intriguingly, reduction of NTRK3 levels in HERV-K(HML-2)-activated cortical neurons restored differentiated cortical neurons. Hence, our findings unravel a unique cell type-specific mechanism of HERV-K(HML-2) during cortical neuronal differentiation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE171101 | GEO | 2021/03/30

REPOSITORIES: GEO

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Project description:Human Endogenous Retroviruses (HERVs) are exogenous viral elements that integrated into the germline millions of years ago. They comprise 8% of the human genome and play a critical role in cellular differentiation during development. HERV-K(HML-2) is the most recently integrated and actively transcribed HERV. It is found to be highly expressed in pluripotent stem cells and strongly downregulated during cellular differentiation. Moreover, expression of HERV-K in terminally differentiated neurons results in cytotoxicity. Expression of HERVs has been implicated in carcinogenesis and is suggested to confer stem cell like features important for cell growth. Atypical Teratoid / Rhabdoid Tumor (AT/RT) is a rare pediatric brain cancer that results from incomplete neuronal differentiation during embryonic development. In this paper, we investigated the cytopathic effect of Ouabain, a cardiac glycoside that induces cytotoxicity in stem cells, on AT/RT cells. We characterized four AT/RT cell lines by immunostaining and observed variable levels of stem cell, Oct4 and Pax6, and neuronal differentiation markers, TUBB3 and MAP2, as well as HERV-K envelope (env), polymerase (pol), and gag transcripts. We stained formalin-fixed brain tissues from 20 AT/RT patients and 5 unaffected controls for HERV-K ENV protein expression. 40% of AT/RT patient tissues showed ENV expression. In contrast, no ENV immunostaining was observed in all 5 control brain tissues. We used Ouabain, known to be cytotoxic to stem cells, observed up to 50% toxicity at 24 hr and 80% toxicity at 48 hr after treatment. While AT/RT primarily exist as large cell aggregates in suspension, Ouabain treatment downregulated HERV-K ENV protein and caused disruption of cell-to-cell interactions. Similarly, when AT/RT cells were transfected with a CRISPR/Cas9 construct designed to repress HERV-K promotor activity, cell aggregates separated and cell viability decreased significantly. Using a luciferase reporter construct under the control of a HERV-K promotor, we observed a significant decrease in HERV-K promotor activity at 8 to 72 hr post treatment with 0.5 uM Ouabain. Given that downregulation of HERV-K alone was sufficient to cause cytotoxicity in the cells, HERV-K expression may be essential for AT/RT growth and survival. Ouabain and other compounds that modulate HERV expression may provide an alternative or adjunctive therapeutic option for treatment of embryonal tumors.

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Activation of HERV-K(HML-2) limits cortical neuronal differentiation by modulating Neurotrophic Tyrosine Receptor Kinase 3 expression

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