Mechanistic Study Of The Neuroprotective Effects Of Squamosamide Derivative FLZ

Neurodegenerative diseases are a group of diseases in the central nervous system, which are characterized by chronic progressive degeneration of central nervous tissue. Major diseases include Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis, et cetra. AD and PD are the two most common neurodegenerative diseases. Neurodegenerative diseases have severe impact on the physical health and life quality of the elderly people. Along with the advent of the aging of population, there is significant meaning for the study of neurodegenerative diseases. Researchers are trying to explore the pathogenesis of those diseases, search for and identificate new drug targets, and develop effective drugs for the prevention and treatment of neurodegenerative diseases.FLZ is a synthetic novel cyclic analogue of squamosamide. Previous studies proved that FLZ had strong neuroprotective effects. It is a potential candidate compound for neurodegenerative diseases therapy. The application of clinical trials for FLZ is under reviewed by China SFDA. The main purpose of this thesis is to explore the mechanism of FLZ’s neuroprotective effects.The thesis includes the following contents. Chapter I:FLZ attenuats neuroinflammation and protects dopaminergic neurons through inhibiting Src tyrosine kinase. Chapter Ⅱ:Protective effects of FLZ on mitochondrial dysfunction induced by APP Swedish mutation (APPswe) overexpression.Chapter Ⅰ FLZ attenuates neuroinflammation and protects dopaminergic neurons through inhibiting Src tyrosine kinaseOver-activated microglia mediated neuroinflammation participates in the pathogenesis and pathological process of PD. Inhibiting over-activated microglia mediated neuroinflammation may show neuroprotective effects on neurons, and achieve PD treatment effects. The squamosamide derivative FLZ has been shown to inhibit over-activated microglia and protect dopaminergic neurons in previous studies, but the mechanism remains unclear. The purpose of the present study is to investigate the neuroprotective and anti-neuroinflammatory properties of FLZ both in the in vivo and in vitro PD models induced by lipopolysaccharide (LPS), and to identify the specific anti-neuroinflammatory mechanism of FLZ, find new target of anti-neuroinflammation.Rodent PD model induced by a single injection of LPS to the SN region of Wistar rats was used for in vivo studies. Results demonstrated that FLZ treatment significantly improved the abnormal behavior of PD model rats and showed potent efficacy in protecting dopaminergic neurons and inhibiting microglial activation. FLZ also increased dopamine (DA) content and decreased DA turnover rate in the striatum of PD model rats. Real-Time RT-PCR results suggested that FLZ could inhibit LPS induced mRNA expression of proinflammatory cytokines including TNF-a and MCP-1.Microglial cell line BV2was used to investigate the molecular mechanism of FLZ against LPS-induced neuroinflammation for in vitro studies. FLZ significantly inhibited LPS-induced BV2cells activation:FLZ inhibited LPS-induced Src kinase related inflammatory signaling pathway activation and subsequent neuclear transcription factor-κBp65(NF-κBp65) nuclear translocation, inhibiting nitric oxide (NO) and reactive oxygen species (ROS) production, decreasing the membrane translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47phox and p67p ox and the expression of NOX-2. The inhibition of Src tyrosine kinase leads to inhibition of NADPH oxidase and microglial activation. Most importantly, the inhibition of Src tyrosine kinase attenuates FLZ’s anti-inflammatory effects. Mechanistic study revealed the critical role of Src tyrosine kinase in the process of microglial activation and the neuroprotective effects of FLZ.In conclusion, the present study demonstrates that FLZ has strong neuroprotective and anti-neuroinflammatory effects. The neuroprotective effects of FLZ are closely related to the inhibition of microglial over-activation and the protection of dopaminergic neurons in the substantia nigra. Mechanistic study reveals that Src tyrosine kinase is the important target of FLZ’s anti-neuroinflammatory effects. Chapter Ⅱ Protective effects of FLZ on mitochondrial dysfunction induced by APPSwe overexpressionThe pathogenesis of AD is complex, no theory should be able to reasonably and comprehensively explain the etiology and pathogenesis of AD until now. The important role of mitochondrial dysfunction in the pathogenesis of AD is taken seriously since the mitochondrial dysfunction cascade hypothesis was put forward in2004.We propose that there may be a link between amyloid bate (Aβ)-induced mitochondrial dysfunction and brain-derived neurotrophic factor (BDNF) axonal transport trafficking based on the mitochondrial dysfunction cascade hypothesis. Abnormal amyloid precursor protein (APP) processing and Aβ production in mitochondria trigger the mitochondrial dysfunction. Mitochondrial disturbance induced by Aβ attenuates the neuronal axonal transport, which is the basis of BDNF-tyrosine protein kinase B (TrkB) signaling transduction. The axonal retrograde transport defect contributes to the pathogenesis of AD. Aβ-induced mitochondrial dysfunction and BDNF axonal transport damage is closely related to the pathogenesis of AD. Previous studies revealed that FLZ improved the behavioral disorder, learning and memory dysfunction in a variety of AD models. Preliminary mechanistic studies suggested that FLZ played neuroprotective effects by protecting the mitochondria and inhibiting neuronal apoptosis. Furthermore, FLZ could enhance BDNF signaling in the brains of AD model. The present study is to investigate the protective effects of FLZ on the mitochondrial dysfunction induced by APPSwe overexpression. Furthermore, on the basis of the preliminary studies, another aim of the present study is to further investigate APPswe overexpression related mitochondrial dysfunction impairs axonal trafficking, which ultimately damages BDNF signaling transduction.APPswe lentiviral infected primary hippocampal neurons were applied. The transgenic efficiency of lentiviral delivery systems is very high and APPSwe lentiviral infection upregulates the expression of APP protein. APPSwe overexpression significantly increases hydrogen peroxide (H2O2) production, decreases mitochondrial cytochrome oxidase activity and adenosine triphosphate (ATP) content. In addition, APPSwe overexpression upregulates the mitochondrial fission related protein DRP1expression, downregulates the levels of mitochondrial fussion related proteins OPA1and MFN1. APPSwe overexpression does cause mitochondrial dysfunction and dynamics abnormalities. FLZ inhibits APPSwe overexpression induced H2O2production and mitochondrial cytochrome oxidase activity decrease. FLZ also regulates the expression of mitochondrial fission related protein DRP1. FLZ improves the mitochondiral dysfunction induced by APPSwe overexpression.BDNF-RFP was successfully expressed in293T cells. BDNF-RFP is used to investigate the effects of APPSwe overexpression on BDNF signal. APPSwe overexpression reduces BDNF-RFP intake in primary hippocampal neurons. APPswe overexpression inhibits the phosphorylation of Akt and GSK3β which lie in the downstream of BDNF-TrkB signaling transduction, eventrally attenuates the function of BDNF signal.The present study demonstrates that APPSwe lentiviral infection attenuates BDNF-RFP intake in primary hippocampal neurons; inhibits the phosphorylation of key proteins, which is the downstream of BDNF-TrkB signalings. APPSwe overexpression induced mitochondrial dysfunction may mediated the impairment of AD related BNDF signaling transduction. FLZ protects the mitochondria through improving mitochondrial dysfunction and inhibiting mitochondrial fission related protein DRP1upregulation in APPSwe overexpressed primary hippocampal neurons.