蠕動是一種能夠幫助食物通過胃腸道以及促進胃腸道產生能動性的類似波浪的收縮運動。它由一種叫做Cajal (ICC)間質細胞的起搏器細胞產生的慢波所控制。ICCs亦幫助由腸神經系統(ENS)到平滑肌的信息傳導。嚙齒動物和人類實驗表明，老化所導致的ICC細胞數量下降和腸神經退化與排便睏難和便秘有關。通過研究ICC和ENS在正常老化情況下和加速膽碱能神經元喪失的阿爾茲海默症(AD)老鼠模型(Tg2576)中的變化，我們對治療神經退化性疾病也許會有新的認識。本課題的目的在于，研究老化情況下正常老鼠模型及澱粉樣前體蛋白質(APP)過量表達下的AD老鼠模型的胃腸道在形態及功能上的變化。 / 六個月大的Tg2576和同齡野生型對照的全樣載片免疫組化實驗顯示， 十二指腸 (P < 0.05)和迴腸 (P < 0.01)中的腸神經細胞顯著降低，迴腸 (P < 0.001)中的GFAP陽性的腸神經膠質細胞也顯著消失。S100陽性的腸神經膠質細胞在胃竇（胃部中的起搏區域）(P < 0.05), 迴腸 (P < 0.05)和結腸 (P < 0.05)中顯著喪失。這些結果表明，在早期的AD階段，ENS已經出現變質。ICC細胞數量在六個月大的Tg2576和同齡野生型對照的所有腸胃部分並沒有顯著性差異 (P > 0.05)。同時，早期AD階段的基本蠕動節奏也並沒有發生改變。除此之外，結腸和十二指腸的GFAP/S100陽性的腸神經膠質細胞比例並沒有顯著增加，表明在早期AD階段，可能出現了炎症。 / 利用石蠟切片進行β澱粉樣蛋白免疫組化，天狼猩紅溶液化驗和硫代黃素T溶液化驗可以測試不溶的澱粉樣斑塊是否存在。結果指出在六個月大的Tg2576所有腸胃部分都觀察到澱粉樣斑塊聚集而在不同的腸胃部分聚集的程度都有所分別。除了結腸外，六個月大的野生型對照所有腸胃部分都觀察不到澱粉樣斑塊聚集。澱粉樣斑塊形成的增長可能和早期AD階段出現的腸神經細胞和腸神經膠質細胞喪失互相關聯。 / 應用電泳轉移酶標免疫印斑技術，測試六個月大的Tg2576和同齡野生型對照的迴腸和結腸中，膽碱乙酰轉移酶 (ChAT，出自興奮神經元)， 神經元型一氧化氮合酶(nNOS，出自抑制神經元)， 膠質細胞源性神經營養因子 (GDNF， 出自腸神經膠質細胞)和可溶解的β澱粉樣蛋白寡聚體的表達是否改變。和野生型對照相比，Tg2576的nNOS的表達在迴腸 (P < 0.05) 而不是結腸 (P > 0.05) 中顯著增加。而ChAT，GDNF和各β澱粉樣蛋白寡聚體 (十二聚物，九聚物和六聚物)在六個月大的Tg2576和同齡野生型對照之間並沒有顯著改變 (P > 0.05)。綜上結果表明，在早期AD階段，腸胃道中的抑制信號有所增加，但是β澱粉樣蛋白寡聚體可能不是引致腸胃道中的腸神經細胞和腸神經膠質細胞喪失的原因。 / 在腸胃道的組織學和生化實驗之後，我們利用了微電極陣列 (MEA) 系統來量度出自胃竇和迴腸的慢波信號。量度出來的主導頻率(DF)和功率分佈可以成為測量在老化的ICR老鼠和早期AD階段下腸胃道的功能有沒有變化的參數。在硝苯地平存在下，尼古丁顯著地刺激三個月大 (P < 0.05) 和 六個月大 (P < 0.05) 的ICR老鼠中胃竇和迴腸的慢波活動但未能引起十二個月大 (P > 0.05) 的ICR老鼠中的慢波活動，說明神經退化可能在十二個月的年齡開始。附加了河豚毒素的情況下，尼古丁不能再刺激三個年齡組中胃竇和迴腸的慢波活動 (P > 0.05)，由此證明了尼古丁是對腸神經細胞起作用再去激發ICC的活動。六個月大的Tg2576和同齡野生型對照之間的胃竇和迴腸的基准讀數沒有顯著分別 (P > 0.05)。然而，尼古丁顯著地增加野生型對照中胃竇和迴腸的DF和胃電過速範圍 (P < 0.05) 但是不能刺激Tg2576中胃竇和迴腸的電流活動 (P > 0.05)，示意在早期AD階段腸胃道中已經出現了腸神經細胞和/或腸神經膠質細胞喪失。 / 綜上所言，研究結果提出AD老鼠模型有形態學，生物化學和功能上的轉變。本課題提供了在研究神經退化疾病上的基礎，也支持ENS是中樞神經系統早期病變前的關口這個假設。 / Peristalsis is the wave-like contraction that moves food along the gastrointestinal (GI) tract and generates GI motility. Peristalsis is modulated by slow waves that originate from pacemaker cells called interstitial cell of Cajal (ICC). ICCs also modulate and transduce inputs from the enteric nervous system (ENS) to the smooth muscle. Recent studies in rodents and humans demonstrated that a decrease in ICC number and enteric neurodegeneration during ageing is associated with difficult bowel movements and constipation. By studying ICC and the ENS during normal aging and in a mouse model (Tg2576) of Alzheimer’s disease (AD) where cholinergic loss may be exaggerated, we may gain new perspectives on the treatment of degenerative diseases. The aim of the present study therefore, was to investigate the morphological and functional changes of the GI tract of mice during ageing and in an AD mouse model over-expressing amyloid precursor protein (APP) using an isolated tissue approach. / Whole mount immunohistochemistry of 6-month-old Tg2576 mice and their age-matched wild type (WT) controls revealed that there were significant losses of enteric neurons in the duodenum (P < 0.05) and ileum (P < 0.001), and of GFAP-positive enteric glial cells in the ileum (P < 0.001). There was also a loss of S100-positive glial cells in the antrum (pacemaker region in the stomach) (P < 0.05), ileum (P < 0.05) and colon (P < 0.05). These results indicated the alteration of the ENS during the early stages of AD. There were no differences in ICC arears of all GI regions between 6-month-old Tg2576 mice and their age-matched WT controls (P > 0.05), and there was no alteration of basal peristaltic rhythm during the early stages of AD. The non-significant increase of GFAP to S100 enteric glial cell ratio in the duodenum and colon might indicate an ongoing inflammatory process in these two GI regions during the early stages of AD. / The presence of insoluble amyloid plaques was studied using Aβ immunohistochemistry, Sirius red assay and Thioflavin-T assay on paraffin wax sections. The aggregation of amyloid plaques was observed in all the GI regions of 6-month-old Tg2576 mice and the levels of amyloid plaque varied in different regions. No amyloid plaques were found in the GI tract of 6-month-old WT animals excepting the colon. The increase in formation of amyloid plaques might be correlated to the losses of enteric neurons and enteric glial cells during the early stages of AD. / Western blot analysis was performed on frozen sections of tissues from the ileum and colon to investigate whether there were changes in choline acetyltransferase (ChAT, from excitatory neurons), neuronal nitric oxide synthase (nNOS, from inhibitory neurons), glial cell line-derived neurotrophic factor (GDNF, from enteric glia) and soluble Aβ oligomers between 6-month-old Tg2576 mice and WT controls. nNOS expression significantly increased in the ileum (P < 0.05) but not in the colon (P > 0.05) of Tg2576 mice compared with WT controls. There were no differences in the expressions of ChAT, GDNF and Aβ oligomers (docecamer, nonamer and hexamer) in the ileum and colon between Tg2576 mice and WT controls (P > 0.05). These results imply that there is an increase in the inhibitory signal in the GI tract during the early stages of AD but soluble Aβ oligomers might not be the cause of neuronal and glial losses in the GI tract. / Following histological and biochemical studies of different GI regions, slow wave signals from the antrum and ileum were measured using a microelectrode array (MEA) system. The dominant frequencies (DFs) and power distributions were measured and these served as parameters for measuring functional changes in the GI tract during ageing in ICR mice and the early stages of AD. In the presence of nifedipine, nicotine significantly stimulated the slow wave activities in the antrum and ileum of 3-month-old (P < 0.05) and 6-month-old (P < 0.05) ICR mice but failed to trigger the slow wave activities in 12-month-old (P > 0.05) ICR mice, suggesting the neurodegeneration might begin with the age between 6 and 12 months. With the addition of tetrodotoxin, nicotine failed to stimulate the slow wave activities in the antrum and ileum of three age groups (P > 0.05) and it showed that nicotine only acted on enteric neurons to trigger the ICC activities. There were no differences in the antral and ileal baseline recordings between 6-month-old Tg2576 mice and their age-matched WT controls (P > 0.05). However, nicotine significantly increased DFs and tachygastria ranges of the antrum and ileum in WT controls (P < 0.05) but failed to increase electrical activitiy of the antrum and ileum in Tg2576 mice (P > 0.05), thus suggesting a loss of neuronal and/or glial cells in the GI tract during the early stages of AD. / In conclusions, these findings suggest the mouse model for AD has morphological, biochemical and functional changes in the GI tract. The present studies provide a foundation for the investigation of degenerative diseases and support the hypothesis that the ENS may be the gateway for the early pathological changes in the central nervous system. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Hui, Chin Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 180-200). / Abstracts also in Chinese. / PUBLICATIONS RELATED TO THE WORK IN THIS THESIS --- p.i / ABSTRACT --- p.ii / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / LIST OF ABBREVIATIONS --- p.vii / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Interstitial cells of Cajal (ICCs) as electrical pacemaker cells in GI tract --- p.1 / Chapter 1.2.1 --- ICC subtypes in GI tract --- p.2 / Chapter 1.3 --- Hypotheses of slow wave generation --- p.4 / Chapter 1.3.1 --- Mechanisms of the NSCC pacemaking hypothesis --- p.5 / Chapter 1.3.2 --- Mechanisms of the alternative hypothesis --- p.6 / Chapter 1.4 --- Involvement of ion channels in slow wave generation of ICC --- p.6 / Chapter 1.4.1 --- Calcium channels --- p.6 / Chapter 1.4.2 --- Sodium channels --- p.7 / Chapter 1.4.3 --- Potassium channels --- p.7 / Chapter 1.4.4 --- Chloride channels --- p.8 / Chapter 1.4.5 --- Non-selective cation channels --- p.8 / Chapter 1.5 --- Distribution of several types of receptors in ICC --- p.11 / Chapter 1.5.1 --- Purinergic receptors --- p.11 / Chapter 1.5.2 --- Muscarinic receptors --- p.11 / Chapter 1.5.3 --- Tachykinin receptors --- p.12 / Chapter 1.5.4 --- Vasoactive intestinal peptide receptors --- p.12 / Chapter 1.5.5 --- Serotonin receptors --- p.13 / Chapter 1.6 --- Introductions and functions of enteric nervous system --- p.15 / Chapter 1.6.1 --- Interaction amongst the central, peripheral and enteric nervous system: brain-gut axis --- p.15 / Chapter 1.6.2 --- Enteric neuronal subtypes in the GI tract --- p.15 / Chapter 1.6.2.1 --- Motor neurons --- p.16 / Chapter 1.6.2.2 --- Interneurons --- p.16 / Chapter 1.6.2.3 --- Intrinsic primary afferent neurons --- p.18 / Chapter 1.6.3 --- Enteric glial cells --- p.18 / Chapter 1.6.3.1 --- Enteric glial subtypes in the GI tract --- p.18 / Chapter 1.6.3.2 --- Communication between enteric neurons and glial cells --- p.19 / Chapter 1.6.3.3 --- Possible functions of enteric glial cells in the GI tract --- p.19 / Chapter 1.6.3.3.1 --- Secretion of neurotrophic factors --- p.20 / Chapter 1.6.3.3.2 --- Secretion of reduced glutathione --- p.20 / Chapter 1.6.3.3.3 --- Secretion of transforming growth factor-beta 1 --- p.21 / Chapter 1.7 --- Interactions amongst ICC, enteric neurons and enteric glial cells --- p.21 / Chapter 1.8 --- Gastrointestinal disorders --- p.22 / Chapter 1.8.1 --- Mechanisms for cell depletion --- p.22 / Chapter 1.8.1.1 --- Autoimmune attack --- p.22 / Chapter 1.8.1.2 --- Hyperglycaemia and diabetes mellitus --- p.24 / Chapter 1.8.1.3 --- Oxidative stress --- p.25 / Chapter 1.8.1.4 --- Ageing --- p.26 / Chapter 1.9 --- Alzheimer’s disease --- p.28 / Chapter 1.9.1 --- Genetics and pathogenesis of Alzheimer’s disease --- p.28 / Chapter 1.9.1.1 --- Aggregation of amyloid beta protein --- p.29 / Chapter 1.9.1.2 --- Genetic factors of AD --- p.29 / Chapter 1.9.1.3 --- Tau hyperphosphorylation and neurofibrillary tangles --- p.31 / Chapter 1.9.2 --- Current treatment for Alzheimer’s disease --- p.33 / Chapter 1.9.2.1 --- Symptomatic treatment --- p.33 / Chapter 1.9.2.2 --- Disease-modifying treatment --- p.34 / Chapter 1.9.2.3 --- Other potential drugs for AD treatment --- p.35 / Chapter 1.9.3 --- Possible animal models for AD investigation --- p.36 / Chapter 1.9.4 --- Possible correlations between Alzheimer’s disease and the enteric nervous system --- p.36 / Chapter 1.10 --- Aim of study --- p.37 / Chapter CHAPTER 2 --- Investigation into the morphologies of enteric nervous system and interstitial cell of Cajal in Tg2576 mice --- p.38 / Chapter 2.1 --- Introduction --- p.38 / Chapter 2.1.1 --- Molecular markers for ICC, ENC, and EGC --- p.38 / Chapter 2.1.2 --- Aims and objectives --- p.39 / Chapter 2.2 --- Materials and methods --- p.41 / Chapter 2.2.1 --- Animals --- p.41 / Chapter 2.2.2 --- Tissue preparation --- p.41 / Chapter 2.2.3 --- Immunohistochemistry --- p.42 / Chapter 2.2.4 --- Image acquisition and analysis --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- General observations --- p.44 / Chapter 2.3.2 --- Area and pattern of ICCs and the ENS in the stomach --- p.46 / Chapter 2.3.3 --- Area and pattern of ICCs and the ENS in the duodenum --- p.52 / Chapter 2.3.4 --- Area and pattern of ICCs and the ENS in the jejunum --- p.56 / Chapter 2.3.5 --- Area and pattern of ICCs and the ENS in the ileum --- p.60 / Chapter 2.3.6 --- Area and pattern of ICCs and the ENS in the colon --- p.66 / Chapter 2.4 --- Discussion --- p.70 / Chapter 2.4.1 --- Major findings --- p.70 / Chapter 2.4.2 --- Possible alterations of the ENS during AD --- p.70 / Chapter 2.4.3 --- Morphological changes of the ENS in relation to genotype --- p.71 / Chapter 2.4.4 --- Morphological changes of ICCs in relation to genotype --- p.72 / Chapter 2.4.5 --- Morphological changes of the ENS and ICCs in relation to GI regions --- p.72 / Chapter 2.4.6 --- Inflammatory conditions in different GI regions --- p.73 / Chapter 2.5 --- Conclusion --- p.74 / Chapter CHAPTER 3 --- Formation of amyloid plaques in the brain and the GI tract of Tg2576 mice --- p.75 / Chapter 3.1 --- Introduction --- p.75 / Chapter 3.1.1 --- The absence of amyloid plaques in rodents --- p.75 / Chapter 3.1.2 --- Overexpression of human APP in transgenic mice --- p.76 / Chapter 3.1.3 --- Distribution of human APP and Aβ deposition in human and transgenic mice --- p.77 / Chapter 3.1.4 --- Transgene and promoter in Tg2576 mouse --- p.77 / Chapter 3.1.5 --- Methods for Aβ plaque detection --- p.78 / Chapter 3.1.6 --- Aim and objectives --- p.78 / Chapter 3.2 --- Materials and methods --- p.80 / Chapter 3.2.1 --- Animals --- p.80 / Chapter 3.2.2 --- Tissue processing --- p.80 / Chapter 3.2.3 --- Preparation of paraffin wax blocks and slide sections --- p.81 / Chapter 3.2.4 --- Aβ immunohistochemistry --- p.82 / Chapter 3.2.5 --- Sirius red assay --- p.83 / Chapter 3.2.6 --- Thioflavin-T assay --- p.84 / Chapter 3.2.7 --- Image acquisition --- p.84 / Chapter 3.3 --- Results --- p.85 / Chapter 3.3.1 --- Aβ immunohistochemistry --- p.85 / Chapter 3.3.1.1 --- The absence of positive immunoreactivity in the brain --- p.85 / Chapter 3.3.1.2 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.85 / Chapter 3.3.2 --- Sirius red assay --- p.92 / Chapter 3.3.2.1 --- The presence of positive immunoreactivity in the brain of Tg2576 mice --- p.92 / Chapter 3.3.2.2 --- Characteristics of Sirius red staining in the GI tract --- p.92 / Chapter 3.3.2.3 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.92 / Chapter 3.3.3 --- Thioflavin-T assay --- p.98 / Chapter 3.3.3.1 --- The presence of positive immunoreactivity in the brain of Tg2576 mice --- p.98 / Chapter 3.3.3.2 --- The presence of positive immunoreactivity in the GI tract of Tg2576 mice --- p.98 / Chapter 3.4 --- Discussion --- p.104 / Chapter 3.4.1 --- The presence of a small amount of amyloid plaques in the brain of young Tg2576 mice --- p.104 / Chapter 3.4.2 --- The presence of amyloid plaques in the GI tract --- p.104 / Chapter 3.4.3 --- Plaque formation in relation to genotype --- p.105 / Chapter 3.4.4 --- Possible effects of amyloid plaques in the brain and GI tract --- p.106 / Chapter 3.5 --- Conclusion --- p.108 / Chapter CHAPTER 4 --- Expression of Aβ oligomers, ChAT, nNOS and GDNF in the GI tract of Tg2576 mice --- p.109 / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.1.1 --- Common and peripheral types of ChAT --- p.109 / Chapter 4.1.2 --- Three subtypes of NOS --- p.111 / Chapter 4.1.3 --- Functions of glial cell line-derived neurotrophic factor in the ENS --- p.112 / Chapter 4.1.4 --- Neurotoxicity of soluble Aβ peptides --- p.113 / Chapter 4.1.5 --- Aims and objectives --- p.113 / Chapter 4.2 --- Materials and methods --- p.115 / Chapter 4.2.1 --- Animals --- p.115 / Chapter 4.2.2 --- Preparation of materials --- p.115 / Chapter 4.2.3 --- Sample preparation --- p.117 / Chapter 4.2.4 --- Separating and stacking gels preparation --- p.118 / Chapter 4.2.5 --- Western blot --- p.119 / Chapter 4.2.6 --- Image acquisition and analysis --- p.120 / Chapter 4.3 --- Results --- p.122 / Chapter 4.3.1 --- Increase in nNOS expression in the ileum of Tg2576 mice --- p.122 / Chapter 4.3.2 --- No changes in the expressions of Aβ oligomers, ChAT, nNOS and GDNF in the colon of Tg2576 mice --- p.122 / Chapter 4.4 --- Discussion --- p.127 / Chapter 4.4.1 --- The absence of “cholinergic hypothesis of AD in the GI tract of Tg2576 mice --- p.127 / Chapter 4.4.2 --- Increased expression of nNOS in the ileum of Tg2576 mice --- p.128 / Chapter 4.4.3 --- Neuronal and glial losses may be related to the reduced GDNF expression --- p.129 / Chapter 4.4.4 --- No relationship between the Aβ oligomers and neuronal damages in the GI tract --- p.129 / Chapter 4.5 --- Conclusion --- p.129 / Chapter CHAPTER 5 --- Microelectrode array (MEA) study on slow wave activity in the GI tract --- p.131 / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.1.1 --- Components in peristalsis-controlling unit --- p.131 / Chapter 5.1.2 --- Techniques in evaluating slow wave activity --- p.131 / Chapter 5.1.2.1 --- Patch clamp --- p.132 / Chapter 5.1.2.2 --- Calcium imaging --- p.132 / Chapter 5.1.3 --- Application of microelectrode array in evaluating slow wave activity --- p.134 / Chapter 5.1.4 --- Aims and objectives --- p.136 / Chapter 5.2 --- Methods and materials --- p.137 / Chapter 5.2.1 --- Animals --- p.137 / Chapter 5.2.2 --- Tissue preparation --- p.137 / Chapter 5.2.3 --- Electrical recordings --- p.138 / Chapter 5.2.4 --- Analysis and Statistics --- p.139 / Chapter 5.3 --- Results --- p.142 / Chapter 5.3.1 --- Experiments on ICR mice --- p.142 / Chapter 5.3.1.1 --- Nicotine stimulates the slow wave activity in the antrum in the presence of NIF but not in the presence of NIF and 500 nM TTX --- p.142 / Chapter 5.3.1.2 --- Nicotine stimulates the slow wave activity in the ileum in the presence of NIF but only partially stimulates activity in the presence of NIF and 500 nM TTX --- p.152 / Chapter 5.3.1.3 --- The use of 1 μM TTX completely blocked the nicotine stimulation in the ileum --- p.160 / Chapter 5.3.1.4 --- The dominant frequency of baseline increased in the ileum of 12-month-old ICR but not in the antrum in the presence of NIF --- p.162 / Chapter 5.3.2 --- Experiments on Tg2576 mice and their wild type controls --- p.164 / Chapter 5.3.2.1 --- No differences in both antral and ileal baseline DFs between 6- month-old non-transgenic and Tg2576 mice --- p.164 / Chapter 5.3.2.2 --- Nicotine stimulates slow wave activity in the antrum of 6-month-old wild type controls but not of Tg2576 mice --- p.164 / Chapter 5.3.2.3 --- Nicotine stimulates slow wave activity in the ileum of 6-month-old wild type controls but not of Tg2576 mice --- p.167 / Chapter 5.4 --- Discussion --- p.171 / Chapter 5.4.1 --- Pharmacological effects of nicotine in the GI tract --- p.171 / Chapter 5.4.2 --- Excitatory effects of nicotine in the slow wave activities of the stomach and ileum --- p.172 / Chapter 5.4.3 --- Changes of ICC functions and neuronal activities during ageing --- p.174 / Chapter 5.4.4 --- Enteric neurodegeneration leads to alteration in the ENS function in Tg2576 mice --- p.175 / Chapter 5.4.5 --- Conclusion --- p.176 / Chapter CHAPTER 6 --- Concluding discussion --- p.177 / REFERENCES --- p.180

Hong, Sijia. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-234). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

This study examined the effects of low to moderate doses of manganese (0, 250, or 750 _g per day from PD 1-21) on a comprehensive battery of behaviors in rats during the neonatal period, preweanling period, and in adulthood.

Indiana University-Purdue University Indianapolis (IUPUI) / Methylmercury (MeHg) exposure from occupational, environmental and food sources is a significant threat to public health. MeHg poisonings in adults may result in severe psychological and neurological deficits, and in utero exposures can confer significant damage to the developing brain and impair neurobehavioral and intellectual development. Recent epidemiological and vertebrate studies suggest that MeHg exposure may contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson’s disease (PD). I have developed a novel Caenorhabditis elegans (C. elegans) model of MeHg toxicity and have shown that low, chronic exposure confers embryonic defects, developmental delays, reduction in brood size, decreased animal viability and DA neuron degeneration. Toxicant exposure results in an increase in reactive oxygen species (ROS) and the robust induction of several glutathione-S-transferases (GSTs) that are largely dependent on the PD-associated phase II antioxidant transcription factor SKN-1/Nrf2. I have also shown that SKN-1 is expressed in the DA neurons, and a reduction in SKN-1 gene expression increases MeHg-induced animal vulnerability and DA neuron degeneration. Furthermore, I incorporated a novel genome wide reverse genetic screen that identified 92 genes involved in inhibiting MeHg-induced animal death. The putative multidrug resistance protein MRP-7 was identified in the screen. I have shown that this transporter is likely expressed in DA neurons, and reduced gene expression increases cellular Hg accumulation and MeHg-associated DA neurodegeneration. My studies indicate that C. elegans is a useful genetic model to explore the molecular basis of MeHg-associated DA neurodegeneration, and may identify novel therapeutic targets to address this highly relevant health issue.

Indiana University-Purdue University Indianapolis (IUPUI) / Glutamate clearance by astrocytes is an essential part of physiological excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters (EAATs) in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1 respectively in rodents). While the inhibition of a ubiquitously-expressed serine/threonine protein kinase, the calcium/calmodulindependent kinase (CaMKII) results in diminished glutamate uptake in cultured primary rodent astrocytes, the molecular mechanism underlying this regulation is unknown. In order to delineate this mechanism, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII and overexpression of a dominant-negative version of CaMKII (Asp136Asn) reduces [3H]-glutamate uptake by EAAT1, without altering EAAT2 mediated glutamate uptake. Surprisingly, overexpression of a constitutively active autophosphorylation mutant (Thr287Asp) to increase autonomous CaMKII activity and a mutant incapable of autophosphorylation (Thr287Val) had no effect on either EAAT1 or EAAT2 mediated glutamate uptake. Pulldown of FLAGtagged glutamate transporters suggests CaMKII does not interact with EAAT1 or EAAT2. SPOTS peptide arrays and recombinant GST-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) but not an Asp (phosphomimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. In sum, this is the first report of a glutamate transporter being identified as a direct CaMKII substrate. These findings indicate that CaMKII signaling is a critical driver of homeostatic glutamate uptake by EAAT1. Aberrations in basal CaMKII activity disrupt glutamate uptake, which can perpetuate glutamate-mediated excitotoxicity and result in cellular death.

Indiana University-Purdue University Indianapolis (IUPUI) / The etiology of many neurodegenerative diseases is unknown, but a number of studies indicate that a combination of both genetic and environmental factors contribute to the progression of disease. Exposure to environmental metals, such as Mn2+, Fe2+, Cu2+, and Al3+, has been shown to increase cell death that is characteristic of neurodegenerative disorders such as AD, PD, Wilson’s disease and Menkes disease. These metals are important in numerous biological processes in the brain and their homeostasis is regulated through multiple mechanisms of transport, storage, and secretion. The vertebrate divalent metal transporter-1 (DMT-1) has been implicated in transport and homeostasis of these divalent cations. In these studies I utilize Caenorhabditis elegans (C. elegans) to show that long term exposure to Mn2+ decreases animal viability in a dose-dependent manner, and I demonstrate that C. elegans homologues to DMT-1, SMF-1, SMF-2, and SMF-3, play specific roles in divalent metal ion-induced DA neurodegeneration. I show that SMF-1 contributes to Fe2+-induced DA neuron degeneration, SMF-3 contributes to Al3+-induced DA neuron degeneration, and both SMF-2 and DAT-1 contribute to Cu2+-induced DA neuron cell death. These studies utilize C. elegans as a powerful model to characterize molecules and pathways involved in metal toxicity and metal-induced DA neuron degeneration.

Indiana University-Purdue University Indianapolis (IUPUI). / Calcium/calmodulin-dependent protein kinase II (CaMKII) is a critical modulator of synaptic function, plasticity, and learning and memory. In neurons and astrocytes, CaMKII regulates cellular excitability, cytoskeletal structure, and cell metabolism. A rapid increase in CaMKII activity is observed within the first few minutes of ischemic stroke in vivo; this calcium-dependent process is also observed following glutamate stimulation in vitro. Activation of CaMKII during pathological conditions is immediately followed by inactivation and aggregation of the kinase. The extent of CaMKII inactivation is directly correlated with the extent of neuronal damage. The studies presented here show that these fluctuations in CaMKII activity are not correlated with neuronal death; rather, they play a causal role in neuronal death. Pharmacological inhibition of CaMKII in the time immediately surrounding glutamate insult protects cultured cortical neurons from excitotoxicity. Interestingly, pharmacological inhibition of CaMKII during excitotoxic insult also prevents the aggregation and prolonged inactivation of the kinase, suggesting that CaMKII activity during excitotoxic glutamate signaling is detrimental to neuronal viability because it leads to a prolonged loss of CaMKII activity, culminating in neuronal death. In support of this, CaMKII inhibition in the absence of excitotoxic insult induces cortical neuron apoptosis by dysregulating intracellular calcium homeostasis and increasing excitatory glutamate signaling. Blockade of the NMDA-receptors and enzymatic degradation of the extracellular glutamate signal affords neuroprotection from CaMKII inhibition-induced toxicity. Co-cultures of neurons and glutamate-buffering astrocytes also exhibit this slow-induced excitotoxicity, as CaMKII inhibitors reduce glutamate uptake within the astrocytes. CaMKII inhibition also dysregulates calcium homeostasis in astrocytes and leads to increased ATP release, which was neurotoxic when applied to naïve cortical neurons. Together, these findings indicate that during aberrant calcium signaling, the activation of CaMKII is toxic because it supports aggregation and prolonged inactivation of the kinase. Without CaMKII activity, neurons and astrocytes release stores of transmitters that further exacerbate neuronal toxicity.

Biological signal processing can help us gain knowledge about biological complexity, as well as using this knowledge to engineer better systems. Three areas are identified as critical to understanding biology: 1) understanding DNA, 2) examining the overall biological function and 3) evaluating these systems in environmental (ie: turbulent) conditions. DNA is investigated for coding structure and redundancy, and a new tandem repeat region, an indicator of a neurodegenerative disease, is discovered. The linear algebraic framework can be used for further analysis and techniques. The work illustrates how signal processing is a tool to reverse engineer biological systems, and how our better understanding of biology can improve engineering designs. Then, the way a single-cell mobilizes in response to a chemical gradient, known as chemotaxis, is examined. Inspiration from receptor clustering in chemotaxis combined with a Hebbian learning method is shown to improve a gradient-source (chemical/thermal) localization algorithm. The algorithm is implemented, and its performance is evaluated in diffusive and turbulent environments. We then show that sensor cross-correlation can be used in solving chemical localization in difficult turbulent scenarios. This leads into future techniques which can be designed for gradient source tracking. These techniques pave the way for use of biologically-inspired sensor networks in chemical localization.

Indiana University-Purdue University Indianapolis (IUPUI) / Despite warnings about the dangers of drinking during pregnancy, little progress has been made in reducing alcohol drinking among women of childbearing age. Even after the recognition of pregnancy, 15% of women continue to drink, 3% of which admit to binge drinking. Because we cannot stop women from drinking during pregnancy, and many children with fetal alcohol spectrum disorders (FASD) are adopted, there is a significant need to develop postnatal interventions that can improve the long-term outcome of children adversely affected by prenatal alcohol exposure. This thesis aims to evaluate one promising new treatment in the rehabilitation or rescue of specific learning deficits long after the damage has occurred. The treatment evaluated herein (40µg D-NAP + 40µg D-SAL) has long been used in the prevention of the detrimental effects of long-term and binge-like alcohol exposures in rodent models of fetal alcohol syndrome and FASD. Until recently this peptide treatment had only been shown to be effective in preventing some of the consequences of alcohol exposure when administered concurrently with the prenatal alcohol exposure. A recent report by Incerti and colleagues (2010c), however, reported that these peptides could completely reverse a profound spatial learning deficit induced by one episode of a heavy binge-like alcohol exposure (5.9g.kg in a single intraperitoneal injection) on gestational day 8 (G8) in C57BL/6 mice. In that report, the peptide treatment was administered starting in late adolescence, beginning three days prior to and throughout water maze training, and the profound deficits in their alcohol-placebo group were completely eliminated in the alcohol-peptide group. There are currently no FDA-approved treatments for FASD. An effective treatment for the cognitive and behavioral dysfunctions suffered by the 1% of people born today could potentially improve the lives of millions of children and adults. The first aim of this thesis was to determine whether the peptide treatment could reverse the significant spatial learning deficits we have demonstrated in adult C57BL/6 mice given high-dose binge-like alcohol exposure (2.5 g/kg in each of two intraperitoneal injections separated by two hours) on postnatal day (P)7. When administered three days prior to and throughout water maze testing (P67-76), the peptide treatment had no effect on spatial learning. The second aim sought to determine whether the same peptide treatment could reverse water maze spatial learning deficits in G8 binge-like exposure models, as reported by Incerti et al. (2010c). For this analysis, the first study used a different binge-like alcohol exposure model that is more commonly used than that employed by the Incerti et al. (2010c) study, namely administration of 2.8g/kg in each of two intraperitoneal injections separated by four hours (Sulik et al., 1981). This model has been shown to produce high peak blood alcohol concentrations and neuroanatomical aberrations in the hippocampal formation and septal regions (Parnell et al., 2009), which have been implicated in learning and memory. Surprisingly, this G8 binge-like alcohol exposure failed to produce a spatial learning deficit, undermining the usefulness of this model in evaluating the peptide effects. In direct contrast to the outcomes of Incerti et al. (2010c), the G8 Webster alcohol exposure was also unable to produce any deficits in acquisition of spatial learning in the Morris water maze. Surprisingly, neither of the heavy binge-like alcohol exposures on G8 were able to produce spatial learning deficits in the Morris water maze. The binge-like alcohol exposure on P7 did yield the expected spatial learning deficit, but the peptide treatment was unsuccessful in recovering water maze learning. These findings fail to support oral administration of 40µg D-NAP and 40 µg D-SAL as a potential therapy for postnatal alcohol-induced spatial learning deficits in adult mice.

Indiana University-Purdue University Indianapolis (IUPUI) / Although peripheral motoneurons are phenotypically endowed with robust regenerative capacity, functional recovery is often suboptimal following peripheral nerve injury (PNI). Research to date indicates that the greatest success in achieving full functional recovery will require the use of a combinatorial approach that can simultaneously target different aspects of the post-injury response. In general, the concept of a combinatorial approach to neural repair has been established in the scientific literature but has yet to be successfully applied in the clinical situation. Emerging evidence from animal studies supports the use of electrical stimulation (ES) and testosterone as one type of combinatorial treatment after crush injury to the facial nerve (CN VII). With the facial nerve injury model, we have previously demonstrated that ES and testosterone target different stages of the regeneration process and enhance functional recovery after facial nerve crush injury. What is currently unknown, but critical to determine, is the impact of a combinatorial treatment strategy of ES and testosterone on functional recovery after crush injury to the sciatic nerve, a mixed sensory and motor spinal nerve which is one of the most serious PNI clinical problems. The results of the present study indicate that either treatment alone or in combination positively impact motor recovery. With regard to molecular effects,single and combinatorial treatments differentially alter the expression of regeneration-associated genes following sciatic nerve crush injury relative to facial nerve injury. Thus, our data indicate that not all injuries equally respond to treatment. Furthermore, the results support the importance of treatment strategy development in an injury-dependent manner and based upon the functional characteristics of spinal vs. cranial nerves.