Chemical Genetic Approach To Identify Therapeutic Targets For Alzheimer's Disease

Chapter One: The cell-based screening and identification of β-amyloid peptideinhibitorAlzheimer's disease (AD) is one of the most prevalent progressive neurodegenerative diseases, which primarily affects the elderly population. The etiology of AD remains to be elucidated. Cholinesterase inhibitors, anti inflammatory drugs, estrogen and Vitamin E are the most widely used drugs for the treatment of AD. Unfortunately, the effects of these drugs are only to ameliorate symptoms and do not achieve any permanent improvement for the disease. Traditional Chinese Medicine (TCM) contains natural product compounds that are annotated with clinical data from thousands of years of experience in China. Therefore, TCM provides a unique resource for drug screening and development for different diseases, including AD.The cell model of AD has been generated by imitating the main pathological change of the disease. It has been applied in pharmacological studies for the identification of therapeutic targets and drug development. In addition, the cell model can be used for the studies on the mechanism of action of drugs at the cellular and molecular level. Since hippocampus and cortex are the most important brain regions for learning and memory and mainly affected in the AD brain, the primary cultured hippocampal and cortical neurons are widely used for AD studies.We have used SH-SY5Y neuronal cell line for the screening of β-amyloid peptide inhibitors from TCM extracts. A number of natural compounds have been identified that could effectively block β-amyloid peptide induced neuronal cell death. Among these inhibitors, EUK4010 is the most effective compound. At 0.5 to 10 μM, EUK4010 could effectively protect SH-SY5Y cells from the toxicity of Aβ 1-42. Furthermore, the compoundcould protect primary cultured hippocampal neuron from A|3 1-42 induced neuronal degeneration at 50 uM. The morphology and nucleus assay showed that AP 1-42 could induce both neuronal apoptosis and necrosis. Interestingly, EUK4010 could alleviate A|3 1-42 induced primary cultured neuronal damage. It has been reported that activation of externalization of phosphatidylserine (PS) residues in the cell membrane appeared to be the early events of apoptosis in most systems. Annexin V, a calcium-dependent phospholipid-binding protein, has high affinity for PS, and therefore, binds to the PS exposed on the surface of apoptotic cells. We have used both Calcein AM and annexin-V to examine the neuronal apoptosis. Our results demonstrated that in primary cultured cortical neurons, 5 urn APi-42 decreased neuronal survival rate to 47.2%, in which the percentage of programmed death neurons was 65.2%. After Ap 1-42 treatment, necrosis cells and the cell proportion of programmed death were doubled. Furthermore, we found that EUK4010 could inhibit the death rate induced by Api-42. The cell survival rate increased to 63.2%, and the apoptotic neurons reduced to 36.5%. The rate of necrosis cell and programmed death cell became 1.6. These results suggested that EUK4010 may protect the neurons from death through multiple routes.To understand the molecular mechanism of AP1-42 induced neuronal degeneration, we examined the gene expression profiling in neurons treated with AP1.42. We found that GABA-A receptor pi subunit (Gabrp) and semaphorin family plexin A3 similar protein genes were up-regulated, while calcium-binding protein regucalcin (Rgn) and huntingtin binding protein optineurin (Optn) genes were down-regulated after AP1.42 treatment in rat cortical neurons. Interestingly, real-time quantitative PCR analysis demonstrated that the expression changes in some of these genes were reversed by EUK4010. Api.42 treatment increased the expression of GABA-A receptor pi subunit 5.5 fold, while EUK4010 almost completely suppressed the up-regulation of the gene expression. Moreover, the expression of semaphorin family plexin A3 similar protein gene was up-regulated by 2.5-fold after Api-42 treatment, and EUK4010 completely reversed the effect of AP1.42 and down-regulated its expression level by about 2-fold. One the other hand, EUK4010 partially reversed the down-regulation of calcium-binding protein regucalcin from 3.9-fold to 1.7-fold.Chapter Two: Gene expression profiling analysis of Ap 1-42 induced neuronaldegenerationAp accumulation in the brain is one of the most important pathogenesis in AD disease, but how AP induces the toxicity is still not clear. Our gene expression profiling analysis using microarray technology demonstrated that AP induced the neuronal degeneration through multiple pathways. A number of pathological changes, including endoplasmic reticulum stress (ERS), free radical damage, calcium overload, cell apoptosis, inflammation as well as the neuronal peptide changes, have been observed in the brains of AD patients. Our gene expression analysis results showed that AP treatment led to the changes of the genes involved in these pathological processes. In this study, we performed an oligonucleotide microarray analysis to investigate the molecular events underlying the Ap induced neuronal degeneration. The microarray contains approximately 23,000 genes and expressed sequence tags. We found that 174 genes were significantly down-regulated, while 35 genes were up-regulated in primary cultured cortical neurons treated with AP .(1) Ap treatment resulted in the down-regulation of a set of stress response genes, which may reduce the positive stress response, lower the immune defense system and lead to the neurons vulnerable to degeneration and death.(2) GABAA receptor-mediated synaptic inhibition could be an important reason for disorders of AD. Recent evidence suggested that a class of therapeutic drugs that lower cholesterol by blocking the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), also known as statins, could inhibit APproduction. We found that Apinduced the up-regulation of HMG-CoA reductase. It is unlikely that elevation of cholesterol alone could lead to AD. Our study showed that Api-42 up-regulated the expression of GABA-A receptor pi subunit. It has been reported that the receptor subunit could assemble with other GABAA receptor subunits and confer unique ligand binding properties. Most notably, the presence of the pi subunit alters the sensitivity of GABAA receptor to the endogenous steroid. These results suggested that GABAA receptor pi subunit and endogenous steroid might be involved in Ap-induced AD dementia.(3) Paraptosis is a form of programmed cell death that is distinct from apoptosis by thecriteria of morphology, biochemistry, and response to apoptosis inhibitors. Substance P and its receptor, neurokinin-1 receptor, were associated with an alternative, non-apoptotic form of programmed cell death. This cell death requires gene expression, displays a non-apoptotic morphology, and is independent of caspase activation. Microarray analysis of Apinduced cortical neurons degeneration revealed a reduced expression of a group of related genes of tachykinin and tachykinin receptors. We also observed that Aptreatment in primary cultured cortical neurons led to the down-regulations of Caspase 3, BH3 interacting (with BCL2 family) domain, apoptosis agonist (Bid3) and ubiquitin ligase (Nedd4) protein. These resutls suggested that Aptreatment could offer certain protection to the apoptotic neurons in earlier period.(4) Ap induced "traffic jam" within neurons that prevented proper transport of proteins. Our microarray analysis showed that Ap reduced the expression of a group of related genes involved in vesicle trafficking. For example, AP up-regulated the expression of microtubule-associated protein tau (Mapt) and serine/threonine protein kinase TA02 (Tao2), down-regulated expression of the PSD-95 binding protein > vesicle-associated membrane protein 2 (Vamp2) , syntaxin 5a (Stx5a), ADP-ribosylation factor-like 1 (Aril), ADP-ribosylation factor 4 (Arf4), and ADP-ribosylarginine hydrolase. Furthermore, AP could exert its effect on protein transport by regulating a variety of other genes, including Lysosomal amino acid transporter 1, Atp6b2, CRT, Scg2, and Slcl2al.(5) We found that AP treatment led to the down-regulation of many transcription factors, including ATF3, Jun D proto-oncogene, CCAAT/enhancer binding protein (C/EBP) delta (Cebpd), C/EBP gamma, EKK2 (Map3k2), Early growth response(Egrl) and Egr4.Chapter Three: The protective effect of regucalcin and tachikin receptor 3 geneson Ap 1-42 induced PC 12 cells degenerationGene expression analysis using DNA microarray showed that the senescence marker calcium-binding protein regucalcin (Rgn) and tachykinin receptor 3 (Tacr3) changed their expression levels in cultured neurons after AP^2 treatment. To investigate the effect of Rgn and Tacr 3 genes on Ap induced neuronal degeneration, we overexpressed these two genes inPC 12 cells. These two genes were cloned and inserted into mammalian expression vector to generate Rgn-pIRESneo2 and Tacr3-pIRESneo2. The eGFP-pIRES2 vector was used to observe the transient transduction efficiency. MTT assay was employed to measure the proliferation of PC 12 cells. We found that overexpression of the Rgn and Tacr3 genes rescued PC12 cells from cell death induced by lOuM and 20uM Api-42.In summary, our study showed that transient expression of the Rgn and Tacr3 genes protected the cells from the A|3 toxicity. These results suggested that Rgn and Tacr3 could play important roles in A(3 toxicity and the pathogenesis of Alzheimer's disease.