Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the post-mortem deposition of amyloid-beta (Aβ) containing neuritic plaques and tau-loaded tangles. According to the amyloid hypothesis, the generation of Aβ via the cleavage of Aβ precursor protein (APP) by β-APP site-cleaving enzyme 1 (BACE1) is a causative step in the development of AD. Therefore, targeting the production and/or clearance of Aβ peptide (by Aβ-degrading enzymes such as Neprilysin) would help understand the disorder as well as serves as therapeutic potential to treat the disorder. MicroRNA are small, noncoding RNA capable of modulating protein expression by primarily targeting their 3’UTR. Therefore, identifying miRNA which target APP, BACE1 and Neprilysin (NEP) would elucidate the complicated regulatory mechanisms involved in protein turnover and provide novel drug targets. We identified miR-20b as a modulator of APP and soluble Aβ. We also identified the target site for miR-20b’s binding on the APP 3’UTR. Further, miR-20b exerts influence on neuronal morphology, likely due to its APP reduction. We also identified miR-298 as a dual regulator of APP and BACE1 and confirmed miR-298’s targeting of both 3’UTRs. We also showed that miR-298 overexpression reduced levels of both soluble Aβ40 and Aβ42 peptides. Additionally, we identified two SNPs in proximity to the MIR298 gene, which are associated with AD-related biomarkers. Based on these results, we showed miR-298 targets a specific isoform of tau by putatively binding a non-canonical target site on the MAPT 3’UTR. Finally, the insertion of the NEP 3’UTR into a reporter vector increases reporter expression; suggesting regulatory elements targeting the 3’UTR. We subsequently identified miR-216 as reducing NEP 3’UTR-mediated luciferase activity. We also measured levels of NEP protein in various mammalian tissue – such as rodent and human fetal tissue, and subsequently showed measurable Aβ levels in correlation with NEP expression. Therefore, herein, we have identified miRNA involved in the regulation of proteins implicated in the pathogenesis of AD.

No description available.

The zinc metalloprotease neprilysin (NEP) has been shown to degrade small bioactive peptides. Crystal structures of seven NEP-inhibitor complexes and biochemical characterization of NEP activity have highlighted amino acid interactions that are crucial to ligand binding. Studies also indicate that NEP is one of a select group of metalloenzymes that degrade the amyloid-beta peptide (Aß) in vivo and in situ. Accumulation of neurotoxic Aß aggregates in the brain appears to be a causative agent in the pathophysiology of Alzheimer's Disease (AD). For this reason the enzymatic degradation of Aß has been studied extensively, but little is known about specific binding interactions underlying NEP degradation of Aß. Using known crystal structures of NEP, we have conducted comparative computational studies of ligand binding that predict NEP residues Arg 102 and 110 form binding interactions specific to Aß. These interactions may provide insight for using NEP degradation of Aß in AD therapy. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry; / MS; / Thesis;

Drosophila insulin like peptides (DILPs) and their human homolog insulin act as messengers to control many physiological processes in the body. Fields in which insulin signaling is crucial are e.g. growth, stress responses and aging. Consequently, many diseases are caused by disturbed insulin signaling, of which diabetes is the most prominent. During the last decades the functions of insulins and their signaling pathways have been studied in detail; what remains less well understood is how the production of insulin and insulin like peptides is regulated. The family of Neprilysins (Neps) belongs to the M13-zinc ion binding metallopeptidases. Neprilysins cleave peptides that regulate a wide range of cellular processes and are therefore linked to a variety of diseases like cancer, analgesia, hypertension or Alzheimer’s disease. In the fruit fly Drosophila melanogaster, five Neprilysins are expressed; but their in vivo substrates have not yet been identified. One of the Drosophila Neprilysins, Nep4, is expressed in the CNS, in muscle tissue, in cardiac tissue and in male reproductive organs. Nep4 is expressed in two isoforms, Nep4A and Nep4B. Isoform A is composed of a short intracellular domain, a transmembrane domain and a large extracellular domain containing the catalytically active center, whereas soluble Nep4B only consists of the extracellular domain. This thesis reveals that overexpression of catalytically active Nep4A in muscle tissue leads to animals with impaired insulin expression, decreased size and weight, affected feeding behavior and reduced locomotion speed. Further phenotypes are an impaired energy metabolism and larval lethality. Knockdown of the whole enzyme or knockout of its catalytic activity also interferes with feeding and locomotion speed and, in addition, causes pupal lethality. As an explanation for the phenotypes, Nep4 mediated hydrolysis of different short neuropeptide F (sNPF) species, which were identified as novel substrates of the peptidase, is proposed. sNPF is known to regulate insulin signaling and knockdown of sNPF phenocopies the Nep4 overexpression phenotypes, which suggests that Nep4 mediated hydrolysis of sNPF regulates insulin expression in the fly. Based on these results additional regulatory peptides were identified as novel Nep4 substrates. Among them are peptides that do not only regulate insulin signaling, but also feeding behavior (Hallier et al., 2016). These findings represent good evidence that muscle bound Nep4 is key to regulate homeostasis of distinct hemolymph circulating peptide hormones. Nep4 localizing to the surface of the central nervous system is likely necessary to ensure effective ligand clearance and thus proper regulation of corresponding peptide receptors.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is a neurodegenerative disease histopathologically characterized by the coexistence of amyloid plaques and neurofibrillary tangles, mainly consisting of amyloid β peptides hyperphosphorylated tau proteins, respectively. Multiple proteins and pathways are involved in the pathogenesis of AD, including Aβ precursor protein (APP), β-site APP-cleaving enzyme (BACE1), neprilysin, endothelin converting enzyme (ECE), repressor element-1 silencing transcription factor (REST), microtubule-associated protein tau, glycogen synthase kinase, and pro-inflammatory cytokines. However, how these proteins and pathways are dysregulated and converge in AD pathogenesis remains unclear. Genetic, epigenetic and environmental factors play important roles in disease progression. MicroRNAs (miRNAs), a group of small noncoding RNAs, are important epigenetic regulators that participate in AD development. We have identified three miRNAs capable of targeting several proteins in different AD-related pathways: miR-181-5p, miR-153-3p and miR-101-3p. We tested miR-181 activity with recombinant reporter gene- MME 3’-UTR constructs. All four miR-181-5p (miR-181a, miR-181b, miR-181c and miR-181d) sequences downregulated the reporter signal. Human differentiated neural cells were transfected with miR-181d-5p mimics. miR-181d-5p treatment significantly reduced MME mRNA levels, protein levels and enzyme activity. In addition, miR-181d-5p increased tau and phosphorylated tau levels proportionally. We further demonstrate that miR-153-3p reduced REST 3’-UTR activities, mRNA and protein levels in multiple human cell lines. Moreover, we show that miR-153-3p, by knocking down REST protein, induces apoptosis in HeLa cells but not differentiated neural cells. In addition, miR-153-3p regulates neuronal differentiation in neuronal stem cells, potentially via REST knockdown. We further found that miR-153 levels were correlated with a reduced likelihood of developing AD. Last, we demonstrated that miR-101-3p reduced ECE1 and GSK3β protein levels in multiple cell lines. miR-101-3p increased REST and pro-inflammatory cytokine secretion in microglia cells. In sum, we tested the hypothesis that miRNAs can serve as the master regulator of AD pathogenesis. / 2024-07-01

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The accumulation of amyloid-ß; protein (Aß) in Alzheimer's disease (AD) is a well known pathological event. Decreasing the production or increasing the degradation of Aß; is therefore thought to serve as a potential therapeutic intervention in AD. Recent in vitro and in vivo studies have suggested that certain proteases may be involved in the catabolism of Aß; and defects in the degradation of Aß; could contribute to AD disease progression. Studies implicating the homing of monocytes to regions of CNS damage have led to the idea that it may be possible to use genetically modified monocytes to carry exogenous genes of interest into the brain or other organs for the purposes of gene therapy. To determine the time course of monocyte recruitment into the brain during the neurodegenerative damage characteristic of Alzheimer's disease, we used transplanted GFP labeled bone marrow monocytes to characterize the kinetics that peripheral monocytes display once injected into the circulation. We determined the half life of bone marrow derived monocytes after one injection into the peripheral circulation, and found this time to be 1.5 hours post injection. We also examined the effects of the APP+PS1 transgene on the recruitment of peripheral monocytes and showed that these cells are actively recruited to the brains in AD transgenic mouse models compared to non transgenic mice. As an approach to increase expression of NEP in a transgenic mouse model of AD, we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice. These monocytes were transfected with a NEP construct designed to express either a secreted form of NEP or a form which lacks any enzyme activity. Monocytes were administered through a microvascular port twice a week for two months and we observed recruitment of bone marrow-derived monocytes into the CNS. In addition, we found significant reductions in both Aß and Congo red staining in the NEP-S injected mice only. These studies show that putting monocytes together with an amyloid degrading enzyme such as neprilysin offers a powerful novel therapeutic tool for the treatment of AD.

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No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.