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

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. We induced cortical neuronal differentiation in H9-CRISPRa-HERV-K(HML-2) as well as H9-CRISPRa-control cells and performed total RNA sequencing (RNAseq) at day 0, day 10, day 27, day 41 and day 60 of cortical neuronal differentiation.

人类内源性逆转录病毒（human endogenous retroviral, HERV）元件的生物学功能及其与疾病的关联，目前仍未完全明晰。本研究通过调控HERV-K(HML-2)的表达水平，解析其在神经元分化过程中的生理学作用。本研究采用CRISPR基因编辑技术（CRISPR engineering）对HERV-K(HML-2)进行激活或抑制，并证实：上调HERV-K(HML-2)转录水平会对皮层神经元的发育、功能维持与生长产生不利影响。该效应具有细胞类型特异性，多巴胺能神经元未受显著影响。本研究进一步发现，在HERV-K(HML-2)转录激活后，前脑类器官形成过程中的皮层分层模式发生改变。HERV-K(HML-2)的转录激活可同时上调神经营养性酪氨酸受体激酶3（Neurotrophic Tyrosine Receptor Kinase 3, NTRK3）以及其他与神经退行性变相关基因的表达。直接对NTRK3进行转录激活，所呈现的表型与HERV-K(HML-2)激活后的表型一致。有趣的是，在HERV-K(HML-2)激活的皮层神经元中降低NTRK3的表达水平，可恢复分化皮层神经元的正常状态。综上，本研究揭示了HERV-K(HML-2)在皮层神经元分化过程中一种独特的细胞类型特异性调控机制。本研究在H9-CRISPRa-HERV-K(HML-2)与H9-CRISPRa-对照细胞中诱导皮层神经元分化，并在皮层神经元分化的第0天、第10天、第27天、第41天及第60天进行了总RNA测序（total RNA sequencing, RNAseq）。