京都大学 / 新制・課程博士 / 博士(工学) / 甲第23218号 / 工博第4862号 / 新制||工||1759(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 田中 庸裕, 教授 跡見 晴幸, 准教授 菅瀬 謙治, 教授 梶 弘典 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Background: Primary progressive aphasia (PPA) is a clinical dementia syndrome characterized by impairments in language. The presence of Alzheimer disease (AD) neuropathology has been observed in approximately 40% of PPA cases. Cross-sectional and longitudinal features of cortical atrophy in PPA are emerging but less is known about the integrity of subcortical structures, particularly the thalamus. As a major relay station in the brain, the thalamus is implicated in language functioning given its reciprocal connections with perisylvian regions in the cortex. High-dimensional brain mapping was used to characterize thalamic morphology in individuals with and without non-semantic PPA. Further, shape differences were compared between PPA participants with suspected AD pathology (PPAAβ +) and those without suspected AD pathology (PPAAβ -) as determined by amyloid PET scans. The relationship between shape and specific language deficits were also investigated. Method: Thalamic integrity was examined in 57 PPA participants relative to cognitively healthy controls (N=44) with similar demographics. MR scans were acquired using high-resolution T1-weighted MPRAGE volumes following the ADNI protocol. Thalamic shape features were estimated using Large Deformation Diffeomorphic Metric Mapping. Thalamic nuclei of interest included mediodorsal, pulvinar, and anterior regions. General linear models compared differences in thalamic shape between groups. Pearson models characterized relationships between thalamic nuclei and language function. Results: After controlling for whole brain volume, thalamic volume did not differ between groups [F(1, 99)=0.80, p=0.80]. However, PPA participants exhibited significant bilateral inward shape deformation in dorsal and ventral regions that extended in an anterior to posterior fashion, and unilateral outward deformation in medial and lateral regions only in the left thalamus relative to controls [F(9, 91)=5.75, p<0.001, Wilk's Λ=0.64]. There were no shape differences between PPAAβ + and PPAAβ – groups. Pearson models revealed significant correlations between confrontation naming and shape deformation in the left pulvinar (r=0.59, p<0.01) and left anterior (r=0.55, p<0.01) thalamic nuclei for the PPAAβ + group only, such that lower language scores reflected greater localized volume loss. Conclusions: In the absence of volumetric differences, shape measures were able to capture unique aspects of localized morphologic differences in PPA that corresponded to worse naming performance only in those with suspected AD pathology. Thalamic changes appear to be a contributing and unrecognized component to the presentation and language characterization of PPA.

Neurodegenerative diseases occur as a result of the breakdown and deterioration of the neurons of the central nervous system (CNS). They are commonly found in elderly people and are a major cause of morbidity and mortality, thereby imposing severe strains on the social welfare systems. Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Cholinergic deficit, senile plaque/amyloid-β peptide deposition and oxidative stress have been identified as three main pathogenic pathways which contribute to the progression of AD. The current therapeutic options cause several side-effects and have problems associated with bioavailability. Therefore, the need arises to search for new compounds from natural products with potential to treat AD. Seventeen plants were selected for this study based on their documented ethno-medicinal use in improving memory, to treat insomnia, calm agitated people, and other neurological disorders. The plants were screened for inhibition of acetylcholinesterase (AChE) using the TLC and microtiter plate method. A dose-dependent inhibition of the enzyme was observed and 4.5% of all the plants showed low (<30% inhibition) AChE inhibition. The ethyl acetate extracts of the roots of Crinum bulbispermum, Xysmalobium undulatum, Lannea schweinfurthii, Scadoxus puniceus and bulbs of Boophane disticha had the best AChE inhibition. Although the IC50 of these plant extracts were higher than that of the positive control, galanthamine (0.00053 mg/ml), they showed good AChE inhibitory activity considering they are still mixtures containing various compounds. The antioxidant activity of the plant extracts was determined by their ability to scavenge ABTS (2,2´-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) and DPPH (1,1-diphenyl-2-picryl- hydrazyl) radicals. The dichloromethane/methanol (1:1) extracts of Chamaecrista mimosoides (root), Buddleja salviifolia (whole plant), Schotia brachypetala (root and bark), water extracts of Chamaecrista mimosoides (root), Buddleja salviifolia (whole plant), Schotia brachypetala (root and bark) and methanol extracts of the roots of Crinum bulbispermum, Piper capense, Terminalia sericea, Lannea schweinfurthii and Ziziphus mucronata all showed good antioxidant activity (>50%), in both assays. B. disticha contained very promising AChE inhibition and was subjected to isolation of active compounds using thin layer chromatography, column chromatography and preparative thin layer chromatography. Two compounds, 6-hydroxycrinamine (a crinine-type alkaloid) and cycloeucalenol (a cycloartane triterpene), were isolated for the first time from the bulbs of this plant. 6-Hydroxycrinamine, and two fractions, EAM 17-21 21,22 and EAE 11 (which could not be purified further due to low yield), were found to inhibit AChE with IC50 values of 0.445 ± 0.030 mM, 0.067 ± 0.005 mg/ml and 0.122 ± 0.013 mg/ml, respectively. Cytotoxicity of the isolated compounds and two active fractions was determined on human neuroblastoma (SH-SY5Y) cells using the MTT and neutral red uptake assays. 6- hydroxycrinamine and fraction EAM 17-21 21,22 were found to be toxic with IC50 values of 54.5 μM and 21.5 μg/ml as determined by the MTT assay. The isolated compounds and fractions did not show any protective effect against cell death induced by Aβ25-35 possibly due to the poor antioxidant activity of B. disticha bulbs. Cytotoxicity was also determined for the methanol extracts of the roots of C. bulbispermum, T. sericea, L. schweinfurthii and Z. mucronata, as they contained promising antioxidant activity. C. bulbispermum was the most toxic, reducing cell viability by <40% at the highest concentration tested. Z. mucronata and L. schweinfurthii were the least toxic with IC50 values exceeding 100 μg/ml, the highest concentration tested. Three concentrations of the plant extracts that were not toxic, or presented low toxicity, were selected to evaluate their possible protective effect against cell death induced by Aβ25-35. Pretreatment with Z. mucronata and T. sericea roots showed a dose dependent inhibition of cell death caused by Aβ25-35. Pre-treatment with L. schweinfurthii roots resulted in an optimum dose for inhibition of Aβ25-35 induced cell death at 25 μg/ml, while still maintaining 80% viability. The roots of C. bulbispermum at non-toxic dose still maintained >50% viability. This study confirms the neuroprotective potential of some of the plants which had AChE inhibitory and antioxidant activity. In addition, four of the plants were shown to prevent cell death caused by Aβ25-35. These plants can serve as potential leads in developing drugs relevant to treatment of AD. Furthermore, two new compounds present in the bulbs of B. disticha were identified. Additional investigations need to be carried out by applying QSAR studies to modify the structure of the alkaloid with the aim of reducing its observed toxicity. / Thesis (PhD)--University of Pretoria, 2012. / Pharmacology / unrestricted

Accumulation of amyloid beta (Aβ) in the brain is a pathological hallmark of Alzheimer's disease (AD) and has been shown to lead to synaptic dysfunction and cognitive decline. Recent studies have indicated synapse dysfunction as an early pathology in AD, but how synaptic function is altered by Aβ remains unclear. We hypothesize that neuronal functional stability may be altered by Aβ via dysregulation of homeostatic synaptic plasticity (HSP), a negative-feedback-based regulation that serves to restrain neuronal activity within a physiological range. Here, I show that Aβ can regulate HSP in response to activity deprivation with an over scaling up of postsynaptic AMPAR expression and excitatory synaptic currents. Aβ treatment during activity deprivation increases the surface expression of both calcium-permeable (Cp), GluA2-lacking (CpAMPARs) and regular, GluA2-containing AMPARs. This in turn may make neurons more vulnerable to neuronal injury after a toxic glutamatergic challenge. Homeostatic synaptic scaling requires the PI3K/Akt signaling pathway and expression of CpAMPARs. Consistent with this, I found that blockade of either PI3K or CpAMPARs occludes over-scaling in the presence of Aβ, suggesting that the enhancement of HSP is mediated through homeostatic mechanisms. Furthermore, challenging neurons with glutamate after Aβ-mediated enhancement of HSP shows increased neuronal death. These findings provide a novel mechanism by which Aβ alters neuronal plasticity and calcium homeostasis in the brain, suggesting that the HSP pathway may be a target in clinical treatment of Alzheimer's disease.

Dysfunction in the Sigma-1 receptor (Sig-1R) is implicated in many neurodegenerative diseases such as Alzheimer’s Disease (AD). Recently, agonists of the Sig-1R have been found to be neuroprotective in AD and provide significant improvements in symptoms. The hallmarks of AD are aggregation of amyloid-β (Aβ) plaques and development of neurofibrillary tau tangles in the brain, which are thought to be correlated with progressive neuronal cell death in AD. Aβ leads to increased endoplasmic reticulum (ER) stress, decreased autophagy, and increased apoptosis, all of which may be contributing to the neuronal cell death that is seen in AD. The Sig-1R is known to reduce ER stress, increase autophagy, and decrease apoptosis. However, as of yet there is little research on the ability of the Sig-1R to specifically reduce Aβ toxicity through these pathways. Therefore, through the use of in vitro and ex vivo models, this study examined the pathways through which activation of the Sig-1R may exert its protective effects against Aβ toxicity. Here, it is shown that activation of the Sig-1R reduces neuronal cell apoptosis in vitro, and reduces tissue death in the CA3 region of the hippocampus ex vivo. Furthermore, this reduction in cell and tissue death may be a result of reduction of ER stress and a return towards baseline levels of autophagy. Together, this research provides insight as to how the Sig-1R may be an important therapeutic target in AD through protection against apoptosis and tissue death.

Alzheimer's disease (AD) is the most prevalent form of age-associated dementia. Cholesterol dysregulation is linked with AD onset. Besides age, the most important risk factor associated with AD is the inheritance of the epsilon-4 allele of apolipoprotein E, a cholesterol transporter. In addition, while hypercholesterolemia has been shown to be an independent risk factor for AD, the nature of the cholesterol-AD link is still not clear. This gap in our understanding is partly due to a lack of knowledge about cholesterol metabolism in the central nervous system (CNS). The low-density lipoprotein receptor (LDLR) is the main receptor of apoE and a central regulator of serum cholesterol levels. Therefore, we sought to characterize the potential participation of LDLR in AD pathogenesis and/or progression. Previous reports with similar aims came to contradictory conclusions. Such studies assessed potential changes in AD in the absence of LDLR by utilizing the LDLR-/- mouse model and crossing it to AD mouse models. Initially we evaluated LDLR-/- mice as a suitable model to study AD. We found that LDLR-/- mice overexpressed a functional splice-variant of LDLR, LDLRDelta4. Moreover, its protein localized in similar regions as the LDLR did in control mice. Finally, we determined that LDLRDelta4 bound apoE, which underscores the impact of the isoform's function in the CNS. We then focused on characterizing changes to LDLR in AD models. We found that APP overexpression in cells increased LDLR mRNA and protein. APP overexpression and Abeta treatment shifted LDLR localization. An AD mouse model showed increased LDLR in hippocampus. Conversely, LDLR levels were decreased in APP-/- mice. Finally, we found that microtubules were affected in cells overexpressing APP. In conclusion, the data presented argue for the importance of LDLR-mediated regulation of cholesterol during AD progression. Also, LDLR may participate in the initial pathogenic insults leading to amyloid deposition, which make it a potential therapeutic target to treat AD. Finally, we propose that APP/Abeta overexpression disrupts microtubule formation; this alteration affects protein trafficking. One of the proteins affected is LDLR, the repercussions of which may ultimately result in cholesterol dysregulation.

Knowledge of how intermolecular interactions of amyloidogenic proteins cause protein aggregation and how those interactions are affected by sequence and solution conditions is essential to our understanding of the onset of many degenerative diseases. Of particular interest is the aggregation of the amyloid-β (Aβ) peptide, linked to Alzheimer's disease, and the aggregation of the Sup35 yeast prion peptide, which resembles the mammalian prion protein (PrP) linked to spongiform encephalothopies. To facilitate the study of these important peptides, experimentalists have identified small peptide congeners of the full-length proteins that exhibit amyloidogenic behavior, including the KLVFFAE sequence of the Aβ protein, and the GNNQQNY sequence of Sup35. Reverse micelles provide an important environment for the study of protein folding and aggregation. In a reverse micelle, it is possible to observe the effects that confinement and water activity, believed to play a critical role in an in vivo cellular environment, have on protein folding, misfolding, and aggregation. We employed molecular dynamics simulations of reverse micelles as well as peptides encapsulated in reverse micelles in order to characterize the reverse micelle environment and identify fundamental principles that inform how sequence and solution environment influence protein aggregation. The peptides studied include the alanine-rich peptide AKA2 as well as the amyloidogenic KLVFFAE and GNNQQNY peptide fragments. The results of these studies suggest that substantial fluctuations in reverse micelle shape away from an idealized spherical geometry enables significant interaction between peptides and the surfactant interface. Analysis these results, including evaluation of water dynamics and calculated IR spectra of the amide I vibration of the peptides, indicate that our model of the reverse micelle is a robust one which captures essential features of this complex system. Moreover, our studies provide critical insight into the complex role played by a heterogeneous cellular environment in the earliest stages of protein aggregation and amyloid formation.

Alzheimer’s disease is characterized by the formation of two major pathological hallmarks: amyloid plaques and neurofibrillary tangles. Although there have been many studies to understand the role of microglia in Alzheimer’s disease, it is not yet known how microglia can promote disease progression while actively phagocytosing amyloid plaques or phosphorylated tau (p-tau). Through stereotaxic injection of adeno-associated virus expressing mutant P301L tau (AAV-P301L-tau) into the medial entorhinal cortex (MEC) of both wild-type (WT) and APPNL-G-F mice, we demonstrate how amyloid plaques exacerbate p-tau propagation to the granule cell layer (GCL) of the hippocampus. However, in mice receiving the colony-stimulating factor 1 receptor inhibitor (PLX5622), ~95% of microglia were depleted, which dramatically reduced p-tau propagation to the GCL. Although microglia depletion curtailed p-tau propagation, it also led to reduced plaque compaction and an increase in overall amyloid-beta (Aβ) plaque presence. Additionally, we found microglia depletion resulted in greater p-tau aggregation in dystrophic neurites surrounding amyloid plaques. We investigated neurodegenerative microglia (MGnD), which are activated in response to amyloid plaques, for their propensity to release extracellular vesicles in comparison to homeostatic microglia. We discovered that MGnD, identified by Clec7a or Mac2 staining, strongly express Tumor susceptibility gene 101 (Tsg101), which is an ESCRT-1 protein and a marker for extracellular vesicles (EVs). To further investigate EV release and MGnD, a novel lentivirus expressing fluorescent mEmerald conjugated to CD9 (mE-CD9) was constructed and injected into the MEC of both WT and APPNL-G-F mice which allowed for visualization of mE-CD9+ puncta around individual microglia. CD9 is a tetraspanin and also a marker for EVs. We observed that the number of mEmerald+ particles surrounding MGnD was three-fold higher compared to non-diseased, homeostatic microglia. Sequential injection of mE-CD9 and AAV-P301L-tau into the MEC revealed that microglia-derived EVs encapsulate pathologic p-tau, which is augmented by the MGnD phenotype. Taken together, these data provide strong evidence that MGnD exhibit increased secretion of tau-containing EVs, providing a possible mechanism for how amyloid deposition indirectly exacerbates tau propagation.

Alzheimer’s Disease is a growing public health problem, with the number of Americans suffering from the disease projected to more than double from 5.8 million today to 13.8 million in 2050. While there is still no cure for Alzheimer’s Disease, a preventative strategy may mitigate its cost to society in the future. Previous studies have shown an ameliorative effect of perinatal choline supplementation on amyloidosis in the hippocampus of APP.PS1 mice. In this study, we test the effects of perinatal choline supplementation on the APP.NLGF strain of mice – which uses a gene knock-in strategy to avoid the non-physiologic overexpression of amyloid precursor protein and better recapitulate the disease in humans. When compared to APP.NLGF mice raised on a control diet, the perinatal choline supplemented APP.NLGF mice exhibited: i) an amelioration of learning and memory deficits in 9- and 12-months old mice as measured by contextual fear conditioning, ii) reduced amyloidosis in the cortex of 9- and 12-months old mice, and iii) an age- and brain region-dependent response to perinatal choline supplementation. These results suggest that increasing the dietary intake of choline during pregnancy may protect the offspring from AD-associated cognitive decline and amyloidosis.

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