Role Of Nuclear Receptor TLX In Cognitive Deficits Caused By Chronic Cerebral Hypoperfusion

Objective As the common pathological mechanism of Alzheimer's Disease(AD)and Vascular Dementia(Va D),Chronic cerebral hypoperfusion(CCH)is a major risk factor for cognitive deterioration and progression of degenerative diseases.There are a lot of pathophysiological changes existed in CCH,including oxidative stress,neuroinflammation,neurogenesis damage and so on.Orphan nuclear receptor TLX regulates the proliferation of neuronal stem cells(NSCs),and is important to neurogenesis.As a transcription factor,it can regulate the expression of a variety of molecules which is relavant to neurogenesis and inflammation.TLX plays an essential role in diseases such as hypoxia,schizophrenia,stroke models.However,the role of TLX in cognitive impairment induced by CCH has not been reported.Methods(1)Stereotaxic injection of AAV2/9-CMV-r-TLX-3xflag-GFP was used to overexpress TLX in the Dentate gyrus(DG)of hippocampus.2 vessel occlusion(2VO)was used to establish CCH model.The rats were randomly assigned to three groups:Control AAV+Sham group(called Sham group hereafter),Control AAV+2VO group(2VO group),and AAV-TLX+2VO group.Rats in Control Vector groups were injected with AAV2/9-CMV-GFP by stereotaxic injection,and AAV-TLX+2VO group was injected with AAV2/9-CMV-r-TLX-3xflag-GFP by the same way.Cognitive function was evaluated by the Morris water maze(MWM)and Novel object recognition(NOR).TLX expression levels in the hippocampus of rats in the three groups were detected by q RT-PCR and Western blot assay.In addition,the transduction efficiency of AAV containing TLX and the position of stereotaxic injection were detected by GFP immunofluorescence.Rats underwent subjected to stereotaxic injection AAV2/9-CMV-r-TLX-3xflag-GFP or saline.They were euthanized 2 weeks later,and brain collection was examined immediately.(2)The immunofluorescence staining was performed to evaluate neurogenesis by labeling the newborn neuron's marker Brd U/Neu N which was administration of the Brd U(200mg/kg)in the 8^th-12^th of 2VO/Sham surgery and proliferation marker Brd U which was administration of Brd U 4h before sacrifice.The expression of mi R-9 was detected by q RT-PCR.Synaptic structure was detected by transmission electronic microscopy(TEM)and the expression levels of synaptic related proteins was examined by western blot to determine the effect of CCH on synaptic structure and plasticity.In addition,plasticity related pathway PTEN/PI3K/Akt expression levels were detected to explore the potential mechanism of TLX regulation of neurogenesis and synaptic structure in CCH rats.(3)The immunofluorescence was to assess inflammatory cells by microglia marker Iba-1 and astrocyte marker GFAP in the hippocampal DG region of rats.The expression levels of pro-inflammatory cytokines IL-1?,TNF?,IL-6 was detected by ELISA and western blot.Expression level of SIRT1 and the activation of NF-?B was tested by western blot to determine the potential regulatory mechanism of the effect of TLX overexpression on neuroinflammation in CCH rats.Results(1)GFP fluorescence showed that TLX overexpression of adeno-associated virus was successfully transfected into the hippocampal DG of rats,and successfully upregulated the decreased expression level of TLX in the hippocampus of 2VO rats,accompanied with improvement in spatial learning and memory and recognition memory of 2VO rats.(2)Overexpression of TLX significantly enhanced neurogenesis in hippocampal DG region of CCH rats,promoted neuronal generation and NSCs proliferation.The postsynaptic density thickness(PSD)and the expression of synaptic related proteins were significantly improved.TLX significantly inhibited the expression of mi R-9 and PTEN in 2VO rats,and activated the PI3K/Akt signaling pathway.(3)Overexpression of TLX significantly reduced the proliferation of hippocampal glial cells and the activation of pro-inflammatory factors in 2VO rats.In addition,TLX inhibited the nuclear translocation of NF-?B by regulating the expression of SIRT1.Conclusion TLX plays a protective role in CCH-induced cognitive dysfunction,and may improve cognitive function by modulatin PTEN/PI3K/Akt signaling pathway to regulate hippocampal neurogenesis and synaptic plasticity.In addition,TLX overexpression also significantly improved hippocampal neuroinflammation in CCH rats,which may play a cognitive protective role in regulating neuroinflammatory response through SIRT1/NF-?B signaling pathway.