Alzheimer’s disease (AD) is a very complex disorder and the most common form of dementia. The two pathological hallmarks of AD are extracellular amyloid-β (Aβ) plaques in cerebral cortex, and intraneuronal neurofibrillary tangles. In the early stages of the disease it can be difficult to accurately diagnose AD, as it is difficult to distinguish from normal signs of aging. There is thus a need for sensitive non-invasive tools, able to detect pathophysiological biomarker changes. One such approach is molecular imaging of Aβ plaque load in brain, using PET (positron emission tomography) ligands. We have developed and characterized two novel Aβ plaque neuroimaging PET ligands, AZD2184 and AZD4694. The 2-pyridylbenzothiazole derivate AZD2184, is a 11C-labeled PET ligand with a higher signal-to-background ratio compared to the widely used PET ligand PIB, a 11C-labeled phenylbenzothiazole based tool. This makes it possible to detect smaller changes in Aβ plaque deposition load, and therefore theoretically, also earlier diagnosis. A drawback with 11C-labeled PET ligands is the relatively short half-life. To meet the need for PET ligands with a longer half-life, we developed the pyridylbenzofuran derivate [18F]AZD4694. Although development of fluorinated radioligands is challenging due to the lipophilic nature of aromatic fluorine, we successfully developed a 18F-labeled PET ligand with a signal-to-background ratio matching PIB, the most widely used 11C-labeled PET ligand in clinical use. 3H-labeled derivates of AZD2184, AZD4694, and PIB, showed lower binding specificity towards Aβ plaques containing ApoE. The ApoE genotype per se did not significantly affect ligand binding, instead, the amount of ApoE incorporated to the Aβ plaques appears to be of importance for the binding characteristics of these amyloid PET ligands. Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid precursor protein (APP) to Aβ peptides, making BACE1 inhibition an attractive therapeutic target in AD. We developed and characterized three novel BACE1 inhibitors, AZD3839, AZ-4217, and AZD3293. AZD3839 and AZ-4217 contains an amidine group which interacts with the catalytic aspartases Asp-32 and Asp-228 of BACE1, effectively inhibiting the enzyme. All three compounds are potent and selective inhibitors of human BACE1, with in vitro potency demonstrated in several cellular models, including primary cortical neurons. All three compound exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in several species, and two of the compounds (AZD3839 and AZD3293) were progressed into clinical trials. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Submitted.</p>

Alzheimer’s disease (AD) is the most common cause of dementia, accounting for around 60-70% of cases. AD encompasses large-scale neuronal loss, resulting in progressive memory and other cognitive decline. Presently, there is no cure for dementia and in light of the ageing population demographic, this represents a clear unmet medical and socioeconomic challenge Worldwide. Much of the current AD research focuses on studying the brain once hallmark amyloid plaque and neurofibrillary tangle pathologies have presented. However their appearance is extremely end stage and to date, any therapeutic interventions aimed at alleviating them having failed to halt symptoms progression. It may therefore be beneficial to look for earlier changes, with metabolic and oxidative stress events as well as reduced cerebral metabolism thought to occur early on in disease progression. Evidence from rare, familial AD cases suggests a causative role for A in AD pathogenesis. For this reason, the enzyme beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), the rate-limiting step in A production is currently of great therapeutic interest. With the prevailing view being that reducing BACE1 levels will be beneficial in AD, there remains a need to better understand the physiological roles of BACE1 to avoid potential side effects of BACE1 inhibition. Herein is presented data showing that, in agreement with the previous literature, BACE1 is fundamentally regulated by cell stress. Notably, both acute and prolonged bouts of oxidative and metabolic stress result in significant increases in BACE1 and APP protein expression. These changes also result in a shift in APP metabolism, with amyloidogenic processing of APP predominating during times of stress. It has also been shown that chronic elevation of BACE1 and/or manipulation of APP processing can alter cellular glucose uptake and use. These changes were determined through the use of radiolabelled substrate uptake and oxidation as well as extracellular flux assays. These data highlighted a fundamental shift in cellular metabolism, with aerobic glycolysis being utilised over oxidative metabolism of glucose. These changes were later shown to come as a result of metabolic lesions, which acted to impair substrate delivery to the electron transport chain of the mitochondria. Taken together, these data show that overexpression of the AD-associated protein BACE1 phenocopies a number of the earliest detectable changes observed in the brains of people who later develop AD. Finally, these data highlighted the potential importance of a number of novel pathways (Sirtuin, AMP-activated protein kinase, and peroxisome proliferator-activated receptor- coactivator signalling) that may underlie these changes and offer therapeutic avenues for earlier and more targeted treatment to halt AD progression.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22689号 / 医博第4633号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 伊佐 正, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

Context: Beta-site alpha-amyloid protein cleaving enzyme1 (BACE1) plays a key role in the pathogenesis of Alzheimer’s disease. Additional to its moderate expression in the brain, high levels of BACE1 mRNA were found in the pancreas. Murine Bace1 has been immunohistochemicaly detected at the apical pole of acinar cells within the exocrine pancreas of mice and Bace1 activity was observed in pancreatic juice. In vitro experiments revealed enteropeptidase as a putative substrate for Bace1 suggesting a role in acute pancreatitis.

GPR50, an X-linked orphan G protein-coupled receptor (GPCR), is a risk factor for bipolar disorder (BD) in female subjects. It has been shown that GPR50 plays a part in neurite outgrowth, glucocorticoid receptor signalling and leptin signalling by interacting with major factors involved in these events. Yeast two-hybrid screens have identified multiple putative GPR50 interactors involved in neurodevelopment, stress response and apoptosis, lipid and glucose metabolism, as well as regulation of NMDA receptors and GABA transmission. Among these interactors, RTN3, RTN4, SREBP2 and SNX6 are known regulators of β-secretase (BACE1), a key enzyme in Aβ generation, myelination of the central/peripheral nerve, and neurite outgrowth/synapse formation. Preliminary data indicated that GPR50 expression significantly increased endogenous BACE1 activity in HEK293 cells, so I hypothesised that there is a functional interaction between the two. In this thesis, I investigated the relationship between GPR50 and BACE1 by identifying the effects of GPR50 on BACE1 expression and function, which may provide an explanation of GPR50’s potential association with psychiatric disorders and Alzheimer’s disease. Firstly, studies on expression levels revealed that when GPR50 was over-expressed, BACE1 protein expression was up-regulated in SH-SY5Y cells, but down-regulated in HEK293 cells, suggesting a differentiated regulative system between cell lines. Then I confirmed the physical association between endogenous GPR50 and BACE1 in HEK293 cells by co-localisation and co-immunoprecipitation studies. Their putative interaction sites were located at the plasma membrane and the filopodia/lamellipodia-like structures in HEK293 cells, and at the neurites in mouse primary neuronal cells. Subcellular fractionation of adult mouse brain revealed that endogenous Gpr50 and Bace1 were co-fractionated in the presynaptic vesicles. Secondly, I showed that, in contrast to HEK293 cells, GPR50 overexpression had no effects on β-secretase activity in mouse primary cortical neurons. However, the BD-associated variant GPR50del significantly decreased β-secretase activity compared to the more common variant GPR50, and showed a trend of diminishing β-secretase activity compared to the control condition. Subcellular fractionation experiments showed that in HEK293 cells, there was an increased ratio of mature BACE1 against immature BACE1 localised in the plasma membrane fractions, indicating a role in regulation of BACE1 trafficking to one of its putative activity sites; whereas in mouse primary cortical neurons, GPR50del increased co-fractionation of immature Bace1 with endoplasmic reticulum (ER) marker calreticulin, thus potentially retarding the maturation of Bace1. Importantly, the regulative trend of GPR50/GPR50del on β-secretase activity is cell line-specific and is highly correlated to their effects on β-secretase intracellular distribution. Thirdly, I found that the mRNA levels of human GPR50 and BACE1 were negatively correlated in the dorsolateral prefrontal cortex of female subjects sampled after birth. Mouse Gpr50 and Bace1 mRNA levels were negatively correlated across the telencephalon regions, and had a trend of negative correlation across the hypothalamic regions. Co-localisation of the two proteins was detected in multiple mouse brain regions, with the strongest co-localised signals occurring in CA2 pyramidal neurons, arcuate hypothalamic nucleus and dorsomedial nucleus of the hypothalamus. Finally, preliminary experiments in Alzheimer’s disease model TgSwDI mice, suggested that the expression level of Gpr50 in layer V of the entorhinal cortex was positively correlated with Aβ deposition. Decreased Gpr50 expression was identified in the hippocampus of 9 months transgenic animals compared with age-matched controls. This indicates that Gpr50 expression might be altered in this mouse model co-ordinately with Aβ deposition. The findings in this thesis provide further evidence of GPR50’s correlation to psychiatric illnesses and its interaction with enzyme BACE1 highlights a potential link to neurodegenerative disease.

<p>Alzheimer’s disease (AD) continues to plague the healthcare community as a serious healthcare crisis. Currently there fails to be an effective FDA approved drug that can treat the underlying mechanisms of the disease. Pathologically the disease is characterized by the accumulation of neurotoxic amyloid-b(Ab) plaques within a diseased patient’s brain. These plaques are widely accepted to be generated by the sequential proteolytic cleavage of amyloid precursor protein (APP) by b-secretase (BACE1, memapsin 2) and g-secretase. Numerous biochemical markers suggest that BACE1 is a viable target for AD drug development. Since the cloning and expression of BACE1 there has been an explosion of drug development efforts that have consisted of peptidomimetic-based and non-peptide-based inhibitors. These efforts have led to 13 BACE1 drug candidates some of which have made it to advanced stages of clinical trials. Unfortunately, an effective and tolerable BACE1 drug candidate continues to be rather elusive to the medicinal chemistry community. GRL-8234 is a potent BACE1 inhibitor that has been extensively shown to be tolerable during several short-term and long-term <i>in vivo </i>studies. The scaffold of GRL-8234 provides a suitable template for further lead development. Namely the P1’ 3-methoxy-benzylamine is of particular interest due to the advantageous interactions in the flap region of BACE1 that can be achieved by incorporation of substitution at the benzylic position. In the same way, the <i>meta</i>-substituent of the benzylamine P1’ ligand was investigated to probe the hydrophobic interactions in the S1’-S2’ binding pocket. A novel class of BACE1 inhibitors containing P1’ spirocyclic benzylamine derivatives were designed, synthesized and evaluated for their inhibitory activity against BACE1. In the process of preparing the aforementioned BACE1 inhibitors a method was established to be able to incorporate the desired 3,5-difluorophenyl methyl transition state isostere by utilizing an ester-derived Ti-enolate to access optically pure <i>syn</i>- and <i>anti</i>-aldol adducts as a key step. Additionally, a novel stereoselective method was established to afford heterospirocyclic benzylamines by utilizing a diastereoselective allyl grignard addition to optically pure a-silyloxy <i>N</i>-sulfinyl ketimines as a key step. Biological evaluation of these novel BACE1 inhibitors has been fruitful and continues to be ongoing. </p>

Context: Beta-site alpha-amyloid protein cleaving enzyme1 (BACE1) plays a key role in the pathogenesis of Alzheimer’s disease. Additional to its moderate expression in the brain, high levels of BACE1 mRNA were found in the pancreas. Murine Bace1 has been immunohistochemicaly detected at the apical pole of acinar cells within the exocrine pancreas of mice and Bace1 activity was observed in pancreatic juice. In vitro experiments revealed enteropeptidase as a putative substrate for Bace1 suggesting a role in acute pancreatitis.

<p>The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in <em>Escherichia coli</em>, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys₂₇₈-Cys₄₄₃being the most important and Cys₂₁₆-Cys₄₂₀ and Cys₃₃₀-Cys₃₈₀ being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.</p>