The Regulatory Effects Of IPT On Hippocampal Neurogenesis In Adult C57Bl/6J Mice

In the adult mammalian brain, new neurons are continuouslygenerated from a proliferating population of neural progenitor/stem cellsand become incorporated into the existing neuronal circuitry via a processtermed adult neurogenesis. In most mammals, active neurogenesis occursthroughout life in the subventricular zone (SVZ) of the lateral ventricle andin the subgranular zone (SGZ) of the dentate gyms in the hippocampus.Areas outside these two regions are generally considered as'non-neurogenic', where proliferating NSCs contribute largely togliogenesis under normal conditions with minimal or no neurogenesis.However, under certain pathological circumstances, or after isolation andculture, these NSCs seem to be capable of producing both neurons andglia.Adult neurogenesis is a dynamic process influenced byenvironmental and internal signals, including hormones, neurotransmittersand growth factors, as well as various pharmacological compounds such asantidepressants. Many studies have revealed that dysregulation of adultneurognesis may contribute to the pathogenesis of neurodegenetativedisorders and psychiatric disorders. Manipulating adult neurogenesis hasbeen regarded as a potential strategy for cell replacement therapy forfunctional recovery in degenerative diseases. However, the cellular andmolecular mechanisms underlying adult neural stem cell proliferation,differentiation, and migration are largely unknown. Thus, seeking the endogenous target to regulate neurogenesis may offer prospective clinicaltherapeutic benefits for CNS diseases.Iptakalim (IPT) is a lipophilic para-amino compound with lowmolecular weight, and has been demonstrated to be a novel K-ATP channelopener (KCO) by pharmacological, electrophysiological and bio-chemicalstudies, and receptor binding tests. Our previous studies providedcompelling supports for the neuroprotective effects of iptakalim onbehavioral recovery and neural survival in various animal models such asstroke and PD, as well as in cultured cells. Pharmacological mechanisms ofneuroprotection induced by IPT involve regulating glutamatergic synaptictransmission and inhibiting microglia releasing inflammatory cytokines.Notably, as iptakalim can freely cross the blood-brain barrier and has feweffects on peripheral blood pressure, it may be a prospective agent fortreating CNS disorders. Besides the pronounced neuroprotective effectsof iptakalim, it is interesting to investigate whether iptakalim couldregulate neurogenesis, including the adult neural stem cell proliferation,survival and differentiation.The present study was undertaken to explore whether chronicadministration with iptakalim could affect hippocampalneurogenesis in adult C57Bl/6J mice.Aim: To investigate the potential benefit of IPT on hippocampalneurogensis in adult C57Bl/6J mice.Methods: 1) Western blotting and RT-PCT were taken for theidentification of Kir subunits in primary cultured ANSCs derive from adultC57Bl/6J mice. 2) [³H] thymidine incorporation was used to assess thecell proliferation rate of the cultured ANSCs. 3) IPT (10mg·kg^-1·day^-1.i.p) was administered to normal and CMS adult C57Bl/6Jmice for four weeks. The tail suspension test (TST), body weight gainand physical states were monitored to assess the antidepressive effects of IPT; The proliferation rate of the newborn cells were determined using5-bromo-2'-deoxyuridine (BrdU) immuno-histochemistry and thedifferentiation of newly formed cells was analyzed by double labeling formature neurons with BrdU and neuron specific unclear protein (NeuN) andor with BrdU and glia cells with glial fibrillary acid protein (GFAP);Western blotting was taken for analyses of the phosphorylated levels ofERK1/2 and CREB. 4) IPT (10 mg·kg^-1·day^-1.i.p) was administered toKir6.2^+/+ and Kir6.2^-/- mice for four weeks. BrdU immunohistochemistrywas used to investigate which subunit of K-ATP channel IPT targets.Results: 1) Western blotting analysis and RT-PCR showed that ANSCderived from adult C57Bl/6J mice expressed Kir6.1 subunit not Kir6.2. 2)The in vitro investigation by [³H]-thymidine incorporation assay revealedthe promotion effects of IPT on the proliferation of ANCS. 3) Chronicadministration with IPT (10 mg·kg^-1·day^-1.i.p) increased neural stem cellsproliferation in the hippocampus of adult C57Bl/6J mice. It also reversedthe reduction of hippocampal neurogenesis induced by chronic mild stress(CMS) in mice with a marked alleviation of the depressive-like symptoms;the population of surviving BrdU-positive ceils essentially matured intoneurons, but the phenotypic expression pattern remained unchangedbetween groups. 4) Chronic administration with iptakalim and fluoxetineboth markedly increased the phosphorylation of ERK1/2 and CREB in thehippocampus. 5) Kir6.2 knockout did not affect the effects of IPT topromote hippocampal neurogenesis in adult mice.Conclusion: Primary cultured adult neural stem cell derived fromadult C57Bl/6J mice express Kir6.1-compsed K-ATP channels, but notKir6.2 subunit. Activation K-ATP channels by IPT could promotehippocampal neurogenesis in adult C57Bl/6J mice via phosphoratingERK and CREB. IPT promoted neurogenesis through Kir6.1-composedK-ATP channels independently of Kit6.2 subunit. The major contributions of the present study lie in:1. Our original study reveals the fact that neuronal stem cells expressKir6.1-composed K-ATP channels, but not Kir6.2 subunits.2. Activation Kir6.1-composed K-ATP channels by IPT couldpromote adult neurogenesis via phosphorating ERK and CREB, suggestingthat IPT could be developed as a prospective therapeutic agent for CNSdiseases.3. IPT may be developed as a promising agent for neurogenesis inneurodegenertative disorders.