Over 71 million Americans will be over the age of 65 by the year 2030. With this rise in adults aged 65 years and older also comes an exponential rise in the estimated number of individuals with Alzheimer’s disease (AD); this number is expected to exceed 24 million Americans by the year 2040. The number one risk factor for AD is older age; this factor is also associated with an increased risk in developing a sleep disturbance. Sleep disturbances have been associated with an increased risk of cancer, heart disease, and decline in overall health. Recent research has examined the association between sleep disturbance and risk for AD; however, these results are mixed. This project analyzed existing data from the population-based longitudinal Cache County Study on Memory and Aging (CCSMA), which included permanent residents of Cache County, Utah who were aged 65 years or older in 1995. The CCSMA consisted of 4-triennal waves and ran from 1995 until 2007; the aim of the original study was to examine antecedents of AD and other forms of dementia. In this thesis, the first study examined whether sleep disturbance was associated with increased risk in developing AD. Sleep disturbance was associated with risk of developing AD, but the results differed between males and females. Among females, endorsing sleep disturbance was associated with a 54.5% decrease in the hazards of developing AD (Hazard Ratio [HR]= .455, p = .0001) compared to females without sleep disturbance. Among males, sleep disturbance was not associated with risk of developing AD (p = 0.498). The second study evaluated if use of sleep medications was associated with increased risk of developing AD and if that association differed between males and females: males who reported use of sleep medication, regardless of having a sleep disturbance, were at increased risk of developing AD (for men without a sleep disturbance HR = 3.604; p = 0.0001). By contrast, in females, risk for developing AD varied by the presence of a sleep disturbance. Compared to the reference group (females without a sleep disturbance and no sleep medication use), females who reported a sleep disturbance and use of sleep medication were at a 35.2% reduced risk of developing AD (HR = 0.648; p = 0.011) while, those not reporting a sleep disturbance but were taking sleep medications were at 3.9 times increased risk in the hazards of developing AD (HR = 3.916; p = 0.0001). Although this study is observational in nature and therefore does not prove that the use of sleep medication is harmful, it is recommended that health care providers consider alternative, nonpharmacological approaches to treat sleep disorders in older adults. Further research is needed to examine sex differences and how they may relate to the differences associated with sleep disturbances and risk for AD.

Alzheimer’s disease is a progressive disease that impairs cognitive and functional abilities. Currently, there is no cure and it is estimated there will be 81 million cases of Alzheimer’s disease by 2040. Life for the individual with Alzheimer’s disease, and their family, changes drastically when the affected individual experiences significant impairments in cognitive or functional ability (severe dementia), is placed in a skilled nursing home facility (institutionalization), or passes away (death). Until a cure is discovered, it will be important to identify modifiable factors that influence progression to severe dementia, institutionalization, and death. Three hundred thirty-five participants who were living in the community were followed after the onset of Alzheimer’s disease and continued until they left the study or the study period ended. Participants completed neuropsychological assessments at each visit. Visits occurred as close to every 6 months as possible and the mean number of visits was 5.32 (SD = 3.46). Outcomes of interest were severe dementia, institutionalization, and death. Predictor variables were hypertension, stroke, congestive heart failure, number of prescription medications being taken, General Medical Health Rating (GMHR) score, and Charlson Comorbidity Index score. Cox Regression was utilized to identify associations with progression to the specified outcomes. GMHR score, congestive heart failure, and number of prescription medications were associated with progression to severe dementia. The number of prescription medications was also associated with nursing home placement. GMHR score, stroke, and number of prescription medications were associated with death. These findings are important because they contribute to a better understanding of how measures of medical health, certain medical conditions, and potentially their prevention or treatment may help those with Alzheimer’s disease sustain a higher quality of life.

Indiana University-Purdue University Indianapolis (IUPUI) / Prodromal Alzheimer’s disease (AD) has recently been identified as a disease state where pathophysiological changes may progress despite the absence of significant clinical symptoms. Yet, the specific processes of neural dysfunction occurring during this preclinical phase remain unclear. Resting state fMRI (RS-fMRI) in combination with brain connectomic measurements may be able to provide ways to measure subtle connectivity changes in different neurological disease states. For instance, RS-fMRI scans allow us to determine functionally connected yet spatially distinct brain regions that can then be separated into resting-state networks (RSNs). More recently, the exploration of RSNs in disease states have proved promising since they have been reliably altered when compared to a control population. By using brain connectomic approaches to assess functional connectivity we can evaluate the human connectome from a different and more global perspective to help us better understand and detect prodromal neurodegenerative disease states.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence of neuritic plaques consisting of extracellular amyloid-beta (Aβ) and neurofibrillary tangles comprised of hyperphosphorylated microtubule associated tau. Aβ is produced following the cleavage of amyloid precursor protein (APP) by the enzyme BACE1. Transcription factors (TFs) are proteins involved in the regulation of gene transcription. Expression levels of some TFs are perturbed in AD. SP1 binding sites on both the APP and BACE1 promoters implicate its potential role in AD. Aβ peptide itself mediates activation of cyclindependent kinase 5 (CDK5), an enzyme which phosphorylates the FOXO (Forkhead Box) TFs. In order to study mechanisms of TF regulation of Aβ production in human models, neuronally differentiated cells as well as a primary human neurosphere culture were used to test the effects of TF-modulating compounds. Our hypothesis is that by targeting relevant TFs via pharmacological inhibitors in human cells, BACE1 activity or APP expression will decrease and Aβ production will be reduced as a result. To test the involvement of TFs in the regulation of APP, we treated several mammalian cells lines and post-mitotic human neuronal cells with roscovitine, mithramycin A (MTM), MTM analogs (MTM-SDK, MTM-SK), and tolfenamic acid (TA). MTM and TA treatment of neurons differentially activated several TFs implicated in AD. Treatment of differentiated neurospheres with MTM led to a significant decrease in APP and SP1 expression along with Aβ40 levels. Epigenetic mechanisms involve alteration of the binding affinity between DNA and transcription factors. We predict that modulation of these TFs may be influenced by epigenetic modifications. To test the effects of drugs on epigenetic markers, histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity was measured. MTM-SDK significantly decreased DNMT activity in differentiated neuroblastoma cells, this may enhance or decrease the ability of SP1 to bind to target DNA and affect transcription of BACE1 or APP. Targeting TF activity is a novel means to manipulate the amyloid pathway. Compounds modifying TF binding to sites on the BACE1 or APP promoters may provide a means to limit the production of amyloid-beta and slow the symptoms of AD.

Chronic stress and elevated glucocorticoids (GCs), the major stress hormones, are risk factors for Alzheimer’s disease (AD) and promote AD pathomechanisms in animal models. These include overproduction of synaptotoxic amyloid-β (Aβ) peptides and intraneuronal accumulation of microtubule-associated protein Tau. Tau accumulation is linked to downregulation of the small GTPase Rab35, which mediates Tau degradation via the endolysosomal pathway. Whether Rab35 is also involved in stress/GC-induced Aβ overproduction remains an open question. Here, I find that hippocampal Rab35 levels are decreased not only by stress/GCs, but also by aging, another AD risk factor. Moreover, I show that Rab35 negatively regulates Aβ production by sorting amyloid precursor protein (APP) and β-secretase (BACE1) out of the endosomal network, where they interact to produce Aβ. Interestingly, Rab35 coordinates distinct intracellular trafficking events for BACE1 and APP, mediated by its effectors OCRL and ACAP2, respectively. Additionally, I show that Rab35 overexpression prevents the amyloidogenic trafficking of APP and BACE1 induced by GCs. Finally, I begin to investigate how GCs and/or Rab35 affect the intercellular spread of Aβ and Tau through exosomes. I describe methods for purifying exosomes and measuring their secretion from neurons, astrocytes, and microglial cells in order to determine the effects of stress/GCs and Rab35 on this process. These studies identify Rab35 as a key regulator of Alzheimer’s disease-related protein trafficking, and suggest that its downregulation contributes to stress- and AD-related pathomechanisms.

No treatment that would completely stop the progression of Alzheimer's disease has not been found yet. Recently used tacrine showed good results in the treatment of Alzheimer's disease, however long-term use led to chronic hepatotoxicity due to its metabolites. This master thesis deals with the compound 7-phenoxytacrine, one of the promising tacrine derivatives, which is one of the candidates for potential use in the therapy of Alzheimer's disease. Due to the formation of hepatotoxic metabolites of tacrine after the biotransformation in human liver, it appears necessary to identify the emerging metabolites of 7-phenoxytacrine molecule. Within this master's thesis in vitro biotransformation study of 7-phenoxytacrine using human liver microsomes was performed. High performance liquid chromatography with tandem mass spectrometry was used to determine the parent substance and the seventeen 7-phenoxytacrine metabolites. The analytical method showed the formation of six monohydroxylated and eleven dehydroxylated metabolites of 7-phenoxytacrine. Thus, we concluded that hydroxylation is the major metabolic reaction after in vitro microsomal biotransformation. In addition to the identification of metabolites, a quantification and microsomal stability study, including the determination of the amount of...

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18194号 / 人健博第11号 / 新制||人健||1(附属図書館) / 31052 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 細田 公則, 教授 高桑 徹也, 教授 任 和子 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

The circadian system uses environmental cues to coordinate the plethora of physiological functions that occur with diurnal rhythmicity, with light being one of the strongest regulators of the circadian system. The suprachiasmatic nucleus (SCN) is positioned at the top of the circadian hierarchy, receiving photic input from the retina and using neuronal, humoral and endocrine signals to synchronize oscillatory patterns across virtually all organ systems. Though the circadian system is plastic to deviations from a normal light/dark (LD) cycle, there are biological limits as to the rate and degree to which the SCN can adapt to these deviations, with peripheral oscillators responding in a delayed manner to the master clock. Alzheimer’s disease (AD) has long been linked to disruptions in the circadian system, with circadian deficits generally portrayed as a consequence of disease development. Recent evidence, however, suggests that circadian disruptions may precede noticeable cognitive deficits associated with AD. Our study aims to determine whether inducible circadian disruption via exposure to our social jetlag protocol, taking advantage of manipulating light exposure to disrupt the circadian system, can exacerbate the rate and severity of AD pathology in both the AβPP/PS1 and APPNL-F/NL-F mouse models of AD. AβPP/PS1 and APPNL-F/NL-F subjected to a preliminary study at 2-4 months in which overall activity in normal LD conditions, re-entrainment to a maintained 8-hour phase advance of the LD cycle, and endogenous period in constant darkness were measured. Glucose Tolerance Test (GTT) was used to assess metabolic health. Following the preliminary study, wild type (WT; C57BL/6J) controls, AβPP/PS1 and APPNL-F/NL-F AD models were entrained to a control light/dark cycle starting at 6 weeks of age. Following 2-weeks of entrainment, half of the animals were maintained on the control schedule, and half were exposed to the social jetlag protocol, involving an 8-hour phase advance on day 5 and an 8-hour phase delay on day 7 of repeated 7-day sessions, effectively inducing chronic circadian disruption until the assigned 6 and 12-month endpoints. One month prior to the endpoint, activity measures and GTT were performed, following which the animals were all entrained to a normal 12:12 LD schedule for 3-5 weeks. Finally, animals underwent an 8-day Morris Water Maze (MWM) to assess effects of chronic disruption on AD-related cognitive decline. Animals were then sacrificed and tissues collected at Zeitgeber time (ZT) 12, time of lights off. At 2-4 months of age, preclinical stages in both AD models, APPNL-F/NL-F males displayed significantly higher fasting glucose levels and circadian period (day length). There were trending increases in overall activity levels under normal 12:12 LD conditions in both AD models over WT controls. 2-4 month females re-entrained to an 8-hour phase advance in significantly less time than males in all genotypes. AβPP/PS1 mice demonstrated hyperactivity as compared to age and sex-matched WT controls. Chronic circadian disruption dampened lights off activity in all cohorts. In all genotypes, female animals showed a higher degree of re-entrainment to the phase advancement of the lighting schedule going into the subjective weekend (day 6-7). Metabolic data as measured by glucose tolerance test on day 7 of the (social jet lag) SJL schedule indicated that disrupted animals were metabolically entrained to the day 1-5 schedule at the peak of rhythmic metabolic function, whereas control animals were at a low point in metabolic rhythms at the time of testing, indicating that circadian regulation of metabolic function was not able to adapt to the weekend phase shifts. Arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) expression in the SCN were significantly dampened as a result of chronic disruption in 12-month male AD mice. Amyloid plaque analysis indicated a severely worsened pathological phenotype in AβPP/PS1 mice as compared to age and sex-matched APPNL-F/NL-F mice. MWM data provides evidence for impaired spatial learning in both AD models that is significantly worsened by chronic jetlag exposure. Taken together, the data suggests that chronic exposure to the social jet lag schedule disrupts rhythmic behavior, metabolic function, and spatial learning significantly in both AD animal models.

Down syndrome (DS) is a congenital disorder caused by partial or complete triplication of human chromosome 21. By age 40, nearly all individuals with DS develop amyloid beta (Aβ) plaques and tau neurofibrillary tangles, the pathological hallmarks of Alzheimer’s disease (AD). This increased susceptibility to Alzheimer’s Disease in Down syndrome (AD-DS) has primarily been attributed to an over-dosage of the amyloid precursor protein (APP), which generates neurotoxic Aβ fragments when cleaved by β- secretase. However, the complete molecular mechanisms of AD-DS are not completely understood, as trisomy of chromosome 21 can induce AD-like neuropathology independently of APP triplication. In addition to classical AD neuropathology, enlarged, APP-positive early endosomes appear early in AD-DS pathogenesis and are the first site of Aβ accumulation. In AD, these endocytic abnormalities have been linked to dysfunction of the endosomal-sorting system known as the retromer complex. Mechanistically, retromer dysfunction can influence amyloid beta production by increasing interaction of the retromer cargo APP with beta-secretase. However, recent studies have also implicated the retromer complex in the development of tau pathology, both through regulation of tau phosphorylation and degradation via lysosomes. Given that retromer dysfunction is associated with the endosomal phenotype found in AD-DS, and that the retromer system can modulate key aspects of AD-DS neuropathology, the objective of the current study is to investigate the role of the retromer complex in the development of AD-DS. We first examined the retromer system in cortices and hippocampi from human patients with DS. Retromer recognition core proteins were significantly decreased in both the hippocampi and cortices of young and aged DS subjects compared to controls. Correlative analyses showed a significant inverse relationship between recognition core proteins and levels of soluble forms of Ab 1-40 and 1-42 in both hippocampus and cortex tissue, and phosphorylated tau epitopes PHF1 and PHF13 in the cortex of the same patients. While this does not indicate causation, these correlative analyses support the hypothesis that dysregulation of the retromer system and AD-like pathology are closely related. Next, we analyzed the retromer system in euploid and trisomic induced pluripotent stem cell (iPSC) -derived neurons and observed an age-related decrease in retromer proteins and elevation of Ab 1-40, Ab 1-42 and phosphorylated tau proteins in trisomic neurons compared to euploid controls. Additionally, we found that pharmacological stabilization of the retromer complex can reduce Ab and phospho-tau in trisomic iPSC-derived neurons. While total levels of retromer proteins are unaffected, we hypothesize that retromer function improves with TPT-172 treatment, as levels of early endosome proteins decrease while autophagy and lysosomal proteins increase with treatment. Treatment of trisomic neurons with TPT-172 also led to reductions in the neuronal tau kinase CDK5 and its activator p25, providing a potential mechanistic link between tau phosphorylation and pharmacological stabilization of the retromer system. Additionally, we examined the effects of genetic overexpression of VPS35, the backbone of the retromer core, in trisomic neurons. We observed similar decreases in AD pathology measures, however, the magnitude of the effect was smaller with genetic overexpression than with pharmacological stabilization using TPT-172. We hypothesized that trisomy 21 may can some inherent instability of the retromer system, possibly due to overabundance of retromer cargo protein APP, that is better overcome by enhancing stability of the complex rather than increasing VPS35 protein level via genetic overexpression. To further explore the role of the retromer system in the AD-DS phenotype, we performed a full characterization of cognitive function and the retromer complex at 2, 5, 9 and 12 months of age in the Ts65dn mouse model of DS. While we observed accelerated aging related cognitive and pathological changes in DS mice, we did not observe any protein levels changes in the retromer complex. However, because these mice do develop endosomal dysfunction that could be indicative of retromer dysfunction, we treated mice with TPT-172 from 4 to 9 months of age and examined cognitive function, the retromer system and AD pathology measures. We observed improvements in cognitive function tests and synaptic function in Ts65dn mice receiving TPT-172, as well as reductions in tau pathology, early endosome proteins, and early endosome size. Taken together, these data demonstrate that retromer complex deficiency occurs in human DS and may contribute to the development of AD-like pathology and cognitive decline in AD-DS. Because pharmacological stabilization of the retromer system improves AD-like pathology and cognitive function in models of DS, we conclude the retromer represents a potential therapeutic target for AD-DS. / Biomedical Sciences

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