| Alzheimer’s disease(AD)is a progressively neurodegenerative disorder,characterized by cognitive deficits and personality changes.The increasing prevalence of AD is severely threatening the health of the elderly.Unfortunately,there is no efficient treatment towards this disease.Therefore,it is urgent to clarify the nuanced pathological mechanism of AD toguide novel AD treatment strategies and drug development.In recent years,the development of new drugs for AD treatment has been repeatedly frustrated.The main reason is the extreme complexity caused by the entanglement of multiple pathological factors in the pathogenesis of AD.The complexity of AD pathogenesis is mainly caused by the harmful Aβ-Tau cascade reaction between senile plaques formed by neurotoxic β-amyloid protein(Aβ)and neurofibrillary tangles formed by the hyperphosphorylation of Tau protein.Moreover,the crosstalk between neurons and microglia that are affected by the above-mentioned pathological factors synergistically promotes AD development.Notably,with in-depth research on the pathogenesis of AD,it has been reported that Aβ-mediated neuroinflammation as the main link of the cross pathological response between micrglia and neurons is not only one of the early pathologies of AD,but also akey step to initiate the Aβ-Tau cascade reaction.Therefore,the design and screening of anti-AD lead compounds targeting neuroinflammation will have important guiding significance for development of anti-AD drugs.5HT2A receptor(5HT2AR)is a subtype of 5HT2 receptor belonging to the serotonin receptor family.As a type of G protein-coupled receptor(GPCR)primarily coupling to Gaq signal transduction pathway,5HT2AR expresses widely throughout the central nervous system and participates in varied brain functions,such as cognitive learning and emotional control.It was reported that continued long-term treatment with antipsychotics(5HT2AR antagonist)may reduce the rate of dementia in AD patients.Notably,5HT2AR antagonist Pimavanserin presented positive top-line results in Phase 3 HARMONY study at clinical trials against AD,indicating the great potential of 5HT2AR antagonismin the treatment of AD.Aβ and Tau pathology as two potent pathogenic factors in AD patients are main causes of oxidative stress and neuroinflammation in neurons and microglia.Recently,many studies showed that microglial NLRP3 inflammasome activation is an important bridge linking Aβ deposition and tauopathy,but the underlying mechanism of Aβ-mediated NLRP3 inflammasome activation was not clear.Voltage-gated potassium channel Kv2.1 highly expresses in central nervous systemand greatly participates in(about 60%)the regualtion of outward potassium current of neurons and glial cells.Notably,it has been reported that the oxidation of Kv2.1 was increased in the brain of AD patients and the expression of Kv2.1 was upregulated in the brain of 3×Tg AD mice,suggesting that Kv2.1 may involve in the development of AD.Objective:The thesis aims to discover novel anti-AD drug lead compounds targeting neuroinflammation and intensively investigate the underlying mechanism.Methods:Based on the drug development strategy of "new usage for old drug",we attempted to find novel 5HT2AR antagonist and Kv2.1 inhibitor from the lab in-house commercial FDA approved drug library.5HT2AR antagonist and Kv2.1 inhibitor detection platform were established based on the detection of calcium ion current and potassium ion current.Western blot,RT-PCRand ELISA assays were used to evaluate the anti-AD effect of the active compounds at the cellular level,and behavioral tests including Morris water maze,novel object recognition and Y maze tests against APP/PS1 and 5×FAD AD transgenic mice were proformed to evaluate the anti-AD effect of the active compounds at the animal level.The underlying pharmacology mechanism of the active compounds were investigated by using si-RNA treatment and AAV injection against cell or animal AD model.Results:We determined that Desloratadine(DLT)as a novel 5HT2AR antagonist effectively ameliorated cognitive impairment in APP/PS1 mice by improving microglial dysfunction.Briefly,DLT reduced amyloid plaque deposition by promoting microglial Aβ phagocytosis and degradation functions,and suppressed innate immune response by polarizing microglia to an anti-inflammatory phenotype.DLT stimulated autophagy process and repressed neuroinflammation through regulating 5HT2AR/cAMP/PKA/CREB/Sirt1 pathway and activated GR nuclear translocation to upregulate the transcriptions of phagocytic receptors TLR2/4 in response to microglial phagocytosis stimulation.Considering the pathological polymorphism of AD,combined administration of DLT and clinical drug cholinesterase inhibitor Huperzine-A(Hup-A)was performed to synergistically treat AD.Experimental results indicated that,compared with DLT or Hup-A treatment alone,the combined administration of DLT and Hup-A can synergistically protected neurons against apoptosis and inhibited neuroinflammation,and finally synergistically improved the cognitive dysfunction of AD sporadic model rats.We identified that Drofenine(Dfe)was a novel Kv2.1 inhibitor and the underlying mechanism of Aβ-mediated NLRP3 inflammasome activation was investigated profoundly by using Dfe as a molecular probe.We found that Aβoligomer(o-Aβ)activated NLRP3 inflammasome through inducing Kv2.1-dependent potassium leakage and mediated subsequent Tau hyperphosphorylation in neurons.Furthermore,we confirmed that Dfe functioned as a Kv2.1 inhibitor and effectively suppressed o-Aβ-induced microglial NLRP3 inflammasome activation and neuronal Tau hyperphosphorylation through suppressing microglial potassium leakage,thereby blocking the occurrence of the Aβ-Tau cascade reaction,and eventually improved the cognitive impairment of 5×FAD AD model mice.Conclusion:We found that 5HT2AR antagonist DLT and Kv2.1 inhibitor Dfe improved cognitive dysfunction of AD model mice by suppressing microglial neuroinflammation.All results provided theoretical support and potential target information for anti-AD drug development strategies,highlighted the potential of 5HT2AR and Kv2.1 as targets for AD treatmentand provide valuable lead compound structures for the discovery of novel anti-AD drugs. |
