The Functional Role Of DDT And HnRNPA1 In The Pathogenesis Of Alzheimer’s Disease

Background and Objective:AD is the most common cause of dementia among people aged 65 and older. It is featured with progressive neuron death and loss of memory, thinking and language skills, and behavioral changes. Pathologically, Two types of abnormal lesions, extracellular beta-amyloid(Aβ) plaques and intracellular neurofibrillary tangles, are hallmarks of AD. Although early-onset familial AD case has a clear genetic cause, the vast majority of late-onset AD is likely caused by the combination of several genetic and environmental risk factors. More and more studies have identified that multiple environmental risik factors, such as exposure to pesticides, solvents, metals or prescription medication, are associated with the pathogenisis of AD. Among these, pesticides seem to be one of important environmental contributors of late-onset AD. One study reported that, compared with control participants, AD patients have approximately a 4-fold higher serum level of dichlorodiphenyldichloroethylene(DDE), which is a metabolite of the organochlorine pesticide dichlorodiphenyltrichloroethane(DDT). These findings were also recently replicated in a much larger study involving more than 600 subjects.In fact, the World Health Organization recommended the use of indoor spraying of DDT to kill malaria-carrying mosquitoes. Due to its high stability, DDT is still found in nature and people continue to be exposed from the environment and the imported food from countries where DDT is still sprayed. However, it is unclear what molecular mechanism might be mediating the effect of DDT on AD. Given the emerging connection between DDT and AD, the identification of a pathway linking them may offer a new avenue for the treatment of AD.Except environment risk factors, people found some new genetic risk factors involved in AD pathogenisis recently. Several studies identify multiple RNA-binding proteins are invovled in neurodegenerative disease. Among these, members of the heterogeneous nuclear ribonucleoproteins hnRNP A/B family, namely hnRNP A1 and A2/B1 which are among the most abundant nuclear proteins. Increasing lines of investigation suggested important roles for hnRNP A1 proteins in neurodegenerative disease. Specifically, pathogenic mutations in PrLDs of hnRNPA1 was defined in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and a case of familial ALS. Furthermore, cortices of sporadic AD patients presented selective loss of the hnRNPA1, but this loss was not found in Parkinson’s disease and epilepsy. However, it is unclear what molecular mechanism might be mediating the effect of hnRNPA1 on AD. The identification of the function of hnRNPA1 and a pathway linking hnRNPA1 and AD may offer a new avenue for the treatment of AD.Base on these knowledge, we employed molecular biology method to identify the functional role of DDT and hnRNPA1 in the pathogenesis of Alzheimer’s disease. We provided the basis for the development of AD therapies.Part I:Objective:To identify the functional role of DDT in AD with molecular biology method.Methods:(1) H4-AβPPswe cells were treated with DDT and then Aβ levels in the media and levels of proteins associated with Aβ synthesis were measured by western blot.(2) H4-AβPPswe cells were treated with DDT and mRNA levels of genes associated with Aβ synthesis, clearance and degradation were measured by qRT-PCR.(3) To further understand the mechanism underlying the change of Aβ metabolism by DDT, we added synthetic Aβ to evaluate the ability of Aβ clearance. The impact of DDT on Aβ clearance was measured by western blot. We also identify the role of some critical proteins by overexpression system.(4) We collected the cell-free conditioned medium from H4 cells treated with DDT and synthetic Aβ was spiked into the cell-free conditioned media. We evaluated the impact of DDT on Aβ degradation by western blot. The molecular mechanism was demonstrated by IDE activity assay and inhibition of IDE by insulin.Results:(1) DDT dramatically increased the levels of secreted Aβ, compared to the negative control, DMSO. DDT significantly increased AβPP, BACE1 levels. DDT significantly induced AβPP and BACE1 expression in SH-SY5 Y cells.(2) Low-density lipoprotein receptor(LDLR) mRNA levels were significantly reduced by DDT.(3) Compared to the negative control DMSO group, DDT treatment group had approximately 2-fold more Aβ remained in the media. which indicates that DDT impairs Aβ clearance. DDT dramatically decreased ABCA1 and LRP1 protein levels in H4 cells. Overexpression of ABCA1, but not LRP1, can rescue the impairment of Aβ clearance caused by DDT. We also found that DDT-mediated ABCA1 reduction occurred in SH-SY5 Y cells and the immortalized apoE3 astrocytes.(4) DDT dramatically impaired the extracellular degradation of Aβ. IDE activity assay and inhibition of IDE by insulin method were employed to demonstrate DDT inhibits IDE activity indirectly.Conclusion:We have conclusion below:(1) DDT promotes Aβ synthesis and decreases Aβ clearance and degradation.(2) ABCA1 and IDE are the downstream target genes adversely affected by DDT.Our study identify the role of DDT in the pathogenesis of AD and provide insight into the molecular mechanisms by which DDT exposure may increase the risk of AD and it further supports that ABCA1 and IDE may be potential therapeutic targets.Part II:Objective:To identify the functional role of hnRNPA1 in microRNAs biogenesis and AD with molecular biology method.Methods:(1) We first transfected siRNAs against hnRNPA1 into H4 cells and expression levels of 63 miRNAs were detected by qRT-PCR. Levels of several critical proteins involved in microRNAs biogenesis were measured by western blot.(2) We measured Tau and phosphorylated Tau and BACE1 protein levels and mRNA levels in H4 cells transfected with siRNAs against hnRNPA1 by western blot and qRT-PCR respectively. To understand the mechanism, we measured the luciferase activity of PGL3-BACE1-3’UTR reporter gene by dual luciferase assay. Furthermore, Tau and BACE1 protein levels were measured by western blot in H4 cells transfected with siRNAs against Dicer and AGO2.(3) We measured Aβ levels in H4-AβPP cells transfected with siRNAs against hnRNPA1.(4) The viability of H4 cells transfected with siRNAs against hnRNPA1 was measured by alamarBlue assay.(5) The protein levels of Dicer, AGO2 and hnRNPA1 in AD patients and the control subjects were measured by western blot.Results:(1) Compared to the control siRNAs, the hnRNPA1 mRNA levels was huge decreased by these siRNAs against hnRNPA1. The result of qRT-PCR indicates that among 63 microRNAs, most microRNAs expression levels were decreased after knockdown of hnRNPA1 by siRNAs. To understand the mechanism, we measured the levels of important proteins involved in microRNAs biogenesis by western blot and found the protein levels of Dicer and AGO2 were down-regulated. To confirm this result, we employed TaqMan Array Human MicroRNA Cards to measure expression levels of approximately 500 microRNAs and found most microRNAs have lower expression levels in the group transfected with siRNAs against hnRNPA1.(2) The protein levels of Tau, phosphorylated Tau and BACE1 protein levels in H4 cells transfected with siRNAs against hnRNPA1 were elevated. Their mRNA levels were also up-regulated. To identify the mechanism, we measured the luciferase activity of PGL3-BACE1-3’UTR reporter gene by dual luciferase assay and found knockdown of hnRNPA1 increased activity of PGL3-BACE1-3’UTR reporter gene. Furthermore, we found protein levels of Tau and BACE1 were elevated after knockdown of hnRNPA1 by siRNAs.(3) Knockdown of hnRNPA1 promotes Aβ production.(4) The viability of H4 cells is decreased after knockdown of hnRNPA1 by siRNAs.(5) Compared to the control subjects, the protein levels of Dicer, AGO2 and hnRNPA1 in AD patients are significantly decreased.Conclusion:We have conclusion below:(1) HnRNPA1 plays important role in microRNAs biogenesis through regulating protein levels of Dicer and AGO2.(2) HnRNP1 is involved in AD pathogenesis by regulating Tau, BACE1 and Aβ.Our findings identify the critical role of hnRNPA1 in microRNAs biogenesis and AD pathogenesis. We expanded the knowledge of microRNAs biogenesis and provide potential therapeutic targets...