Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

Abstract

Hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN channels) are widely expressed in the central and peripheral nervous systems and organs, while their functions are not well elucidated especially in the sympathetic nerve. The present study aimed to investigate the roles of HCN channel isoforms in the differentiation of sympathetic neurons using PC12 cell as a model. PC12 cells derived from rat pheochromocytoma were cultured and induced by nerve growth factor (NGF) (25 ng/ml) to differentiate to sympathetic neuron-like cells. Sympathetic directional differentiation of PC12 cells were evaluated by expressions of growth-associated protein 43 (GAP-43) (a growth cone marker), tyrosine hydroxylase (TH) (a sympathetic neuron marker) and neurite outgrowth. Results show that the HCN channel isoforms (HCN1-4) were all expressed in PC12 cells; blocking HCN channels with ivabradine suppressed NGF-induced GAP-43 expression and neurite outgrowth; silencing the expression of HCN2 and HCN4 using silenced using small interfering RNAs (siRNA), rather than HCN1 and HCN3, restrained GAP-43 expression and neurite outgrowth, while overexpression of HCN2 and HCN4 channels with gene transfer promoted GAP-43 expression and neurite outgrowth. Patch clamp experiments show that PC12 cells exhibited resting potentials (RP) of about −65 to −70 mV, and also presented inward HCN channel currents and outward (K+) currents, but no inward voltage-gated Na+ current was induced; NGF did not significantly affect the RP but promoted the establishment of excitability as indicated by the increased ability to depolarize and repolarize in the evoked suspicious action potentials (AP). We conclude that HCN2 and HCN4 channel isoforms, but not HCN1 and HCN3, promote the differentiation of PC12 cells toward sympathetic neurons. NGF potentiates the establishment of excitability during PC12 cell differentiation.

Inhibition of Trpv4 rescues circuit and social deficits unmasked by acute inflammatory response in a Shank3 mouse model of Autism

“…3f ). To further consolidate the potential link between downregulation of Shank3 , overexpression of Trpv4 , and increased excitability, we evaluated the expression of Trpv1 and HCN1 genes which have been found as neuronal excitability regulators in other system [ 33 – 35 ]. RT-qPCR analysis showed that these genes are not altered after Shank3 downregulation (Fig.…”

Role of miRNA-mRNA Interaction in Neural Stem Cell Differentiation of Induced Pluripotent Stem Cells

“…These 513 predicted miRNA–mRNA interactions were highly enriched in nervous system development functions and the 85 mRNAs common in the significantly activated nervous system development functions ( Table 1 ) and their predicted interactions with DE miRNAs shown in Figure 4 identified several regulatory networks. The upregulated miR-10a-5p miRNA family was the second most abundant in our NSCs after miR-92a-3p and was predicted to regulate the expression of genes, PRKCI whose down-regulation promotes NOTCH signaling and NSC self-renewal [ 36 ], pro-neural genes RORB, BDNF and RNF112 [ 37 , 38 , 39 ], and HCN1 associated with differentiation of sympathetic neurons [ 40 ]. miR-10a-5p along with miR-30c-5p and miR342-3p, was also predicted to down-regulate the pro-apoptotic gene CASP3 .…”

Investigating the Regulation of Neural Differentiation and Injury in PC12 Cells Using Microstructure Topographic Cues

“…To further prove the relationship between microstructural cues and PC12 neural differentiation, the expressions of growth cone marker GAP-43 and sympathetic marker TH were used to identify the sympathetic characteristics of NGF-treated PC12 cells in different topographies, similar to previous work by Li-Ying Zhong et al [32]. According to the previous experimental results, we chose the anisotropic topography S and the isotropic topography P90.…”