AβCauses Neural Stem/Progenitor Cells Senescence And Reduces Response To Nicotinic Analogue ZY-1

Alzheimer’s disease (AD) is a progressive neurodegenerative diseasewhich impairs memory and cognitive functions. The disease is characterizedby extracellular as well as intracellular deposition of β-amyloid (Aβ) peptides,intracellular hyperphosphorylation of tau protein and neuronal loss. Thefailure of adult hippocampal neurogenesis is increasingly considered as animportant factor in the pathological correlates for memory decline in AD. Thedrugs approved by FDA have poor clinical curative effect. Therefore, researchand development of AD drugs need to consider the effect on adulthippocampal neurogenesis.ZY-1, a novel nicotinic analogue, has high affinity to α4β2nicotinicacetylcholine receptor (α4β2nAChR). In our previous studies, this patentedcompound inhibited Aβ production in a cell line test and had advantages inimproving cognitive function in the AD model. Therefore, to further explorewhether ZY-1can be a potential candidate for AD treatment, we integratedapplication of cell biology and molecular biology methods to study the effects of ZY-1on adult hippocampal neurogenesis, and explore its possiblemechanisms.Firstly we isolated and cultured neural stem/progenitor cells (NSPCs)from adult mouse hippocampus. CCK8experiment showed the at aconcentration range of0.5to1μM, ZY-1significantly increased cell viabilityin a dose-dependent manner as compared to control group. The maximalincrease in the cell viability was observed at1μM of ZY-1, which wasinhibited by0.1μM DHβE, an α4β2nAChR antagonist. The number anddiameter of ZY-1-treated neurospheres were increased compared to controlgroup. Scratch wound assay and Transwell assay showed that ZY-1significantly promoted the migration of NSPCs. Immunofluorescence andWestern blot experiments showed that ZY-1did not affect differentiation ofNSPCs into neurons or astrocytes. Further result indicates that the effect ofZY-1on in vitro adult hippocampal neurogenesis is at least partly due tomodulation of intracellular ROS levels.Preliminary in vivo experiments showed that ZY-1did not attenuate ADpathology in the DG of APP/PS1transgenic mice，such as Aβ depositions andneuronal damage. In the present study, we marked immature neuron byβ-III-tubulin, immunofluorescence experiment showed that the number ofimmature neuron was significantly reduced in APP/PS1transgenic mice, and ZY-1did not improve impaired hippocampal neurogenesis of AD mouse,either.To further investigate the reason why ZY-1failed in promotingneurogenesis, we studied the effects of local hostile microenvironment in thebrain of AD on NSPCs, and explored its possible mechanism.The NSPCs were isolated and cultured from adult hippocampus. Cellswere treated with Aβ42oligomer (2.5,5or10μM) for1,3and5days. CCK-8assay showed that Aβ42decreases NSPCs viability in dose-dependent andtime-dependent manners. Compared with control, the number and diameter ofAβ42-treated neurospheres were decreased. Morphologically, in suspensionculture conditions and monolayer cultivation conditions, these morphologicalchanges in Aβ42-induced NSPCs confirmed cellular senescence phenotype.Immunocytochemistry and Western blot showed that Aβ42suppressed NSPCsdifferentiation capacity into neurons or astrocytes. In addition, compared withcontrol group, Aβ42also increased the production of senescence-associatedbiomarkers p16and SA-β-gal. These results indicated that Aβ42acceleratesNSPCs senescence.In vivo experiments showed that compared with wild type mice, Aβ wasspecifically deposited in cerebral cortex and hippocampus, including the DGof APP/PS1transgenic mice. The number of NSPCs in the DG of APP/PS1 mice was significantly reduced with age, and to18months almostdisappeared. Compared with wild type mice, the number of senescent NSPCswas significantly increased in the DG of APP/PS1transgenic mice.Meanwhile, in the hippocampal tissue of APP/PS1transgenic mice, thesecretion of IL-6was significantly increased, and IGF-1level was reduced.The animal model study further suggestes that Aβ associates with adulthippocampal NSPCs senescence.To gain insight into the molecular mechanisms by which Aβ42inducedNSPCs senescence, we investigated whether Aβ42functional formylpeptidereceptor2(FPR2) is involved in this process. The results showed that FPR2was expressed in the DG of the hippocampus and purified NSPCs in culture,and Aβ42induced FPR2expression. The FPR2antagonist WRW4significantlyinhibited NSPCs senescence induced by Aβ42. In addition, the activation ofp38MAPK in response to the accumulation of ROS was involved in NSPCssenescence induced by Aβ42, which were inhibited by WRW4, antioxidantNAC and p38MAPK inhibitor SB203580. Our data suggest that Aβ42accelerates NSPCs senescence via FPR2-dependent activation of itsdownstream ROS-p38MAPK signaling.In conclusion, ZY-1promotes neurogenesis by modulating intracellularROS levels in vitro. Instead, ZY-1fails to improve AD-like pathological characteristics and impaired neurogenesis in vivo. The reason of why failurein vivo is that Aβ42induces senescent phenotype of NSPCs through activatingFPR2and its downstream ROS-p38MAPK pathway, limits function ofNSPCs and contributes to impaired neurogenesis.These results indicate that neurogenesis is more sensitive to local hostilemicroenvironments in the brain of AD and the research and development ofAD drugs should consider the influence of local hostile microenvironmentson neurogenesis.