The Lhx8 downstream targeted gene Capn3 is involved in the differentiation of hippocampal NSCs into cholinergic neurons

Abstract

Abstract Background Lhx8, which is specifically expressed in the medial ganglionic eminence (MGE), is believed to be a key factor in the development of cholinergic neurons. Our previous studies have demonstrated that enhanced cholinergic neurogenesis occurs in the sub granular zone (SGZ) of the hippocampal dentate gyrus (DG) after cholinergic denervation, which is closely associated with the core transcription factor Lhx8. This study aims to explore the downstream targeted genes of Lhx8 involved in the differentiation of hippocampal neural stem cells (NSCs) into cholinergic neurons. Method Chromatin immunoprecipitation (ChIP) was applied to collect the targeted DNA fragments for Lhx8. DNA sequencing and bioinformatics analysis were performed to screen for the targeting genes related to neurogenesis. The expression of related targeting genes was verified by real-time quantitative polymerase chain reaction (PCR). Calpain 3 (Capn3) was predicted to be associated with Lhx8. The interaction of Capn3 and Lhx8 was verified using luciferase reporter gene assay, and the gain- and loss-functions of Capn3 in NSC differentiation revealed the effects of Capn3 in cholinergic neurogenesis. Results ChIP analysis revealed 71 genes that Lhx8 targets upstream of its promoter within 1 k bp, and on bioinformatics analysis, Capn3 was identified as a candidate gene that Lhx8 targets. The gain function of Capn3 in Lentivirus (LV)-Lhx8 PC12 decreased the protein level of the cholinergic neuron marker choline acetyltransferase (ChAT), while down-regulation of Capn3 in LV-Lhx8 PC12 promoted protein expression of ChAT. Luciferase assay verified that Lhx8 could target the promoter region of Capn3. In a hippocampus NSC differentiation assay, NSCs that overexpressed Capn3 had decreased differentiation into microtubule-associated protein 2 (MAP2)/ChAT-positive cholinergic neurons; however, down-regulation of Capn3 in NSCs increased the proportion of MAP2/ChAT-positive cholinergic neurons. Conclusion Capn3 may be a downstream target gene, negatively regulated by Lhx8, which can impede the cholinergic differentiation of hippocampus NSCs.