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京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第24807号 / 社医博第131号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 中山 健夫, 教授 山本 洋介, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

Agonist-stimulated phosphoinositide (PPI) hydrolysis is a major signal transduction pathway in brain. These studies investigated neurotransmitter stimulated PPI hydrolysis in postmortem human brain. Preliminary studies using rat brain suggested that moderate postmortem delay has little effect on PPI hydrolysis and that human tissue might be reliably studied for differences in receptor-PLC coupling. Studies in human brain membranes (frontal cortex) indicated that the time course for GTPγS and carbachol/GTPγS-stimulated PPI hydrolysis was linear for at least 20 min. GTPγS-stimulated [3H] inositol phosphate (InsP) formation was enhanced by carbachol (232%) and 5-Hydroxytryptamine (5HT - 147%). SAX-HPLC seperation of [3H] inositol polyphosphates indicated that the major isomer of inositol trisphosphate (InsP3) was Ins(1,4,5)P3, the expected product of PtdIns(4,5)P2 hydrolysis. Ca2+ increased PPI hydrolysis progressively from 100 nM through 50 μM and synergistically enhanced carbachol/GTPγS stimulation. Comparisons of age-matched controls with Alzheimer's patients indicated that GTPγS, carbachol/GTPγS, and 5HT/GTPγS-stimulation of PPI hydrolysis is reduced approximately 50% in membranes prepared from Alzheimer's patients. Ca2+ stimulation of PPI hydrolysis was not different between controls and Alzheimer's patients suggesting that muscarinic cholinergic and serotonergic receptors are uncoupled from PLC in Alzheimer's disease. These studies indicate that there are changes in cholinergic and serotonergic signal transduction in Alzheimer's disease. Further, this method can be used to study signal transduction events in postmortem human brain.

Alzheimer's Disease (AD) is a neurodegenerative disease that causes complications with thinking capability, memory and behavior. AD is a major public health problem among the elderly in developed and developing countries. With the growth of AD around the world, there is a need to further expand our understanding of the roles different clinical measurements can have in the diagnosis of AD. In this work, we propose a machine learning-based technique to distinguish control subjects with no cognitive impairments, AD subjects, and subjects with mild cognitive impairment (MCI), often seen as precursors of AD. We utilized several machine learning (ML) techniques and found that Gradient Boosting Decision Trees achieved the highest performance above 84% classification accuracy. Also, we determined the importance of the features (clinical biomarkers) contributing to the proposed multi-class classification system. Further investigation on the biomarkers will pave the way to introduce better treatment plan for AD patients.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease. Many researchers believe that an effective AD treatment will prevent the development of disease rather than treat the disease after a diagnosis. Therefore, the development of tools to detect AD-related pathology in early stages is an important goal. In this report, MRI-based markers of neurodegeneration are explored as biomarkers of AD. In the first chapter, the sensitivity of cross-sectional MRI biomarkers to neurodegenerative changes is evaluated in AD patients and in patients with a diagnosis of mild cognitive impairment (MCI), a prodromal stage of AD. The results in Chapter 1 suggest that cross-sectional MRI biomarkers effectively measure neurodegeneration in AD and MCI patients and are sensitive to atrophic changes in patients who convert from MCI to AD up to 1 year before clinical conversion. Chapter 2 investigates longitudinal MRI-based measures of neurodegeneration as biomarkers of AD. In Chapter 2a, measures of brain atrophy rate in a cohort of AD and MCI patients are evaluated; whereas in Chapter 2b, these measures are assessed in a pre-MCI stage, namely older adults with cognitive complaints (CC) but no significant deficits. The results from Chapter 2 suggest that dynamic MRI-based measures of neurodegeneration are sensitive biomarkers for measuring progressive atrophy associated with the development of AD. In the final chapter, a novel biomarker for AD, visual contrast sensitivity, was evaluated. The results demonstrated contrast sensitivity impairments in AD and MCI patients, as well as slightly in CC participants. Impaired contrast sensitivity was also shown to be significantly associated with known markers of AD, including cognitive impairments and temporal lobe atrophy on MRI-based measures. The results of Chapter 3 support contrast sensitivity as a potential novel biomarker for AD and suggest that future studies are warranted. Overall, the results of this report support MRI-based measures of neurodegeneration as effective biomarkers for AD, even in early clinical and preclinical disease stages. Future therapeutic trials may consider utilizing these measures to evaluate potential treatment efficacy and mechanism of action, as well as for sample enrichment with patients most likely to rapidly progress towards AD.

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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

Pebbles and Shards is a collection of personal essays based on family relationships that focus upon motherhood, responsibility, and the complexity of love and loss. The essays explore how people cope with the inevitability of loss and how they move beyond that loss to find something meaningful, perhaps even beautiful. They reflect upon success and failure in the face of loss and how, either way, life goes on, heedless of people’s desires and plans. The essays in Pebbles and Shards, while meant to stand alone, are thematically connected so that, read together, each story resonates with the others. In “Promises,” I explore the fear of watching my mother die of Alzheimer’s disease. In related essays “Frame by Frame” and “In Darkness,” I focus on my mother’s efforts to struggle with Alzheimer’s and how, as an adopted daughter, I underwent a role-reversal and became the mother figure. Other essays, such as “Heart of a Deadhead” and “Circus,” consider the mothering impulse, especially the guilt and conflict that so often accompany my desire to nurture others. In attempting to support and strengthen those who seem “weak,” I have sometimes found that my own actions and thoughts underscore a deeper weakness in myself. As a collection, Pebbles and Shards contemplates the suffering and joy that is a family

Aging and Alzheimer’s disease (AD) are associated with decreased cognitive function and neural degeneration. The basal forebrain is one of the first areas of the brain to degenerate in AD and depends on the neurotrophin brain-derived neurotrophic factor (BDNF) for survival. Loss of BDNF transport from target neurons may contribute to basal forebrain cholinergic neuron (BFCN) vulnerability in AD and aging. Oxidative stress is associated with cholinergic dysfunction and cognitive decline in aging and AD, and it is possible that oxidative stress may contribute to BDNF transport deficits in BFCNs. BFCNs are grown in microfluidic chambers that allow isolation of BFCN soma and axon terminals so transport of biotinylated and fluorescently labelled BDNF can be quantified. The objective of my research was to determine if oxidative stress induces BDNF retrograde transport deficits in BFCNs, and the mechanism behind this effect. I found that oxidative stress does reduce BDNF retrograde transport in BFCNs. Because it has previously been shown that aged BFCNs have decreased BDNF transport and downregulate the BDNF receptor TrkB, expression of both TrkB and p75NTR receptors was tested following oxidative stress using immunocytochemistry (ICC) and western blotting. This experiment showed that oxidative stress does not affect p75NTR or TrkB receptor levels. A likely alternative is that oxidative stress may lead to alterations in the transport machinery responsible for retrograde BDNF transport. I hypothesized that oxidative stress decreases retrograde axonal transport of BDNF via increased insulin-like growth factor 1 receptor (IGF1R) activity, which decreases the protein expression of the adaptor proteins BICD1 and Hook1 by inhibiting GSK3β activity via the PI3K-Akt pathway. ICC and western blotting showed that oxidative stress has no effect on either BICD1 or Hook1 levels. Future directions of this work involve further studying the involvement of the IGF1R pathway in oxidative stress, and the effect on other proteins involved in BDNF transport, including htt and DISC1. / Thesis / Master of Science (MSc)