Semantic knowledge was investigated in patients diagnosed with Dementia of the Alzheimer's Type (DAT) by means of an off-line probe question battery and an on-line measurement of semantic priming in a lexical decision task (LDT) that varied the stimulus onset asynchrony (SOA). The patients' performance on the detailed probe questions showed that their semantic deficit was confined primarily to animate concepts, characterized by visual descriptive features. In the primed LDT, demented patients demonstrated increased semantic priming compared to age-matched controls. A trend was also found indicating that for both normal controls and DAT subjects the priming magnitude decreased as the SOA increased. These results are interpreted in terms of a distributed representation of semantic knowledge that is impaired in demented patients.

Alzheimer’s disease (AD) is a multifactorial disease that results in progressive neurodegeneration. Brain regions are differentially affected in AD; some are more vulnerable to degeneration than others. There is an age-dependent effect on beta-amyloid (Aβ) accumulation and neuroinflammation as disease progresses. In the TgCRND8 APP transgenic mouse model, levels of various Aβ species and cytokines were determined as a function of brain region and age. Aβ was found to accumulate in the brain prior to the sequential elevation of IL-1β and CXCL1. Levels of Aβ, IL-1β and CXCL1 were elevated in regions that are severely affected in AD patients. It has been shown for the first time in an APP transgenic model that CXCL1 elevation occurs following IL-1β elevation.

Alzheimer’s disease (AD) is a multifactorial disease that results in progressive neurodegeneration. Brain regions are differentially affected in AD; some are more vulnerable to degeneration than others. There is an age-dependent effect on beta-amyloid (Aβ) accumulation and neuroinflammation as disease progresses. In the TgCRND8 APP transgenic mouse model, levels of various Aβ species and cytokines were determined as a function of brain region and age. Aβ was found to accumulate in the brain prior to the sequential elevation of IL-1β and CXCL1. Levels of Aβ, IL-1β and CXCL1 were elevated in regions that are severely affected in AD patients. It has been shown for the first time in an APP transgenic model that CXCL1 elevation occurs following IL-1β elevation.

This thesis focuses on the regulation of the ubiquitous second messenger Ca2+ in neuronal physiology and disease. Ca2+ signalling in neurons is regulated by ion channels located in the plasma membrane, as well as in the endoplasmic reticulum (ER) and mitochondrial membranes. Ca2+ signalling is essential for numerous cellular processes, including neuronal excitability, neurotransmitter release, synaptic plasticity, and induction of cell death. Age-related disruptions in Ca2+ signalling may contribute to decline of cognitive function and motor control associated with aging. Furthermore, disruption in neuronal Ca2+ signalling is implicated in several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In this thesis, I studied neuronal Ca2+ signalling and how it is affected in neurodegenerative disease. First, I examined the role of the ER Ca2+ binding protein Calreticulin (CRT) in AD. CRT is involved in regulation of ER Ca2+ signalling and modulation of susceptibility to cell death. I found that there was an increase in the expression of CRT in in vitro and in vivo models of AD. However, increased levels of CRT did not alter susceptibility of neuronal cells to death induced by AD-related stressors. Second, I examined the role of X-Linked Inhibitor of Apoptosis Protein (XIAP) in the modulation of neuronal Ca2+ signalling. I found that overexpression of XIAP in neuronal cells modified Ca2+ signalling by decreasing Ca2+ flux through multiple plasma membrane and ER channels. These effects appear to be independent of caspase inhibition, which is one of the ways that XIAP can inhibit apoptosis. Third, I examined a compound found in green tea, L-theanine, a glutamate receptor antagonist that is protective in models of excitotoxic neuronal injury. I found that 24 hour L-theanine treatment reduces the amount of Ca2+ released from neuronal intracellular stores in response to both glutamate stimulation and passive leak through ER channels. An acute 30 minute L-theanine treatment had similar effects. In conclusion, these observations further the understanding of the regulation of Ca2+ signalling in neurons and may lead to novel therapeutic strategies in neurodegenerative disease.

Alzheimer's disease (AD) is the leading cause of dementia. Amyloid plaques in the brain remain a pathological feature of AD. These plaques are primarily composed of amyloid β-protein (Aβ). It has been postulated that glycogen synthase kinase-3β (GSK3β) activity might exert a central role in the development of AD. GSK3β activity has been implicated in tau phosphorylation, APP processing, Aβ production and neurodegeneration. Quetiapine is frequently used to treat psychoses in AD patients at the late stage and has inhibitory effects on GSK3β activity in mouse brains after acute/subchronic treatment. Therefore, the proposed hypothesis is that chronic quetiapine administration after amyloid plaque onset reduces AD like pathology and alleviates AD like behaviours in APP/PS1 transgenic mice by inhibiting GSK3β activity. APP/PS1 transgenic mice were treated with quetiapine (2.5, 5 mg/kg/day) in drinking water starting from 3.5 months of age, for a period of 8 months. One week after behaviour testing, mice were sacrificed at 12 months of age. Half of the hemispheres were rapidly frozen for immunoblot and ELISA analyses and the other half were fixed with 4% paraformaldehyde for histological analyses. Quetiapine treatment reduced amyloid plaques formation in the cortex and hippocampus of AD mice. It also improved the behavioural deficits in these mice, including attenuating impaired memory and anxiety-like phenotypes. In addition, chronic quetiapine administration inhibited GSK3β, which resulted in reduced production of Aβ in cortices and hippocampi of transgenic mice. Quetiapine treatment also significantly decreased the activation of astrocytes and attenuated synapse integrity impairment in transgenic mice. These findings suggest that early application of quetiapine can alleviate memory deficits and pathological changes in the APP/PS1 transgenic mouse model of AD, and further support that modulation of GSK3β activity by quetiapine may be a therapeutic option for AD.

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptide (Aβ), the principal component of amyloid plaques present in Alzheimer’s disease. It consists of a core complex of presenilin (PS), nicastrin, anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2) proteins. PS harbors the catalytic aspartates required for regulated intramembrane proteolysis and the paralogs (PS1 and PS2) contribute to the assembly of distinct subpopulations of γ-secretases that may fulfill distinct roles. To characterize the molecular environments of distinct γ-secretases complexes in-depth quantitative comparisons were performed on 1) wild-type PS1 and its derivative carrying point mutations known to cause heritable early-onset AD in mice, and 2) PS1- or PS2-containing γ-secretase complexes equipped with N-terminal tandem-affinity purification (TAP) tags on PS paralogs in HEK293 cells. Isobaric labeling of co-purifying peptides for quantitative mass spectrometry revealed that γ-secretase complexes interact with other protein networks, including the cellular catenin-cadherin network, the molecular machinery that targets and fuses synaptic vesicles to cellular membranes, and the H+-transporting lysosomal ATPase macro-complex. The study revealed mature γ-secretase complexes containing PS1 or mutant PS1 to be indistinguishable in their protein composition, confirmed several previously proposed γ-secretase interactors, identified many novel interactors and uncovered a subset of proteins which can engage in robust interactions with γ-secretase complexes in individual cell types but may escape detection when whole brains are used as biological source materials. Interestingly, signal peptide peptidase (SPP), a Type II TM cleaving aspartyl protease, was pre-dominantly found to co-purify with PS2-containing γ-secretase complexes and could be shown not to influence their maturation but to affect cleavage or release of cellular Aβ. A model emerged from this work that suggests PS1 and PS2 paralogs may divide up the task of handling a broad range of membrane stubs at least in part by associating with different molecular environments.

Oxidative stress is one of the major characteristics in Alzheimer’s disease, and converging evidence indicates that cysteine S-nitrosylation might be related in AD pathology. My results demonstrated exogenous S-nitrosoglutathione was able to S-nitrosylate vAChT, vMAT2, vGluT1 and vGluT2. S-nitrosylation of these vesicular transporters inhibited the uptake of [3H]acetylcholine, [3H]dopamine and [3H]glutamate respectively. APP/PS1 transgenic mice were used to investigate neurotransmission dysfunctions of Alzheimer’s disease. Global protein S-nitrosylation was increased in the 9 and 12 month APP/PS1 mice. Further investigation demonstrated an increase of vAChT and vGluT1 S-nitrosylation in frontal cortex of 6, 9 and 12 month APP/PS1 mice and an increased vAChT and vGluT1 S-nitrosylation was found in hippocampus of 3 month APP/PS1 mice. These findings together suggest that S-nitrosylation of vesicular transporters inhibits the uptake of neurotransmitters, and S-nitrosylation of vAChT and might be associated with the neurotransmission dysfunction of acetylcholine and glutamate in Alzheimer’s disease.

Technological developments have enabled mass spectrometry (MS) to evolve asone of the most versatile, sensitive and widely used analytical methods. Key areas ofresearch in biological MS include the development of analyte-selective MSmethodologies, along with the design of MS compatible separation technology. Analytesof interest range from small, biologically active molecules in disease progressionresearch, to macromolecules such as proteins, in proteomics investigations. Advances inthese areas are vital to maintaining the level of sophistication that has become thebenchmark for MS analyses.Mass spectrometry has found a permanent station in disease progression studies,particularly in biomarker discovery. This is especially true for Alzheimer's disease (AD),a condition marked by widespread lipid peroxidation (LPO) in the brain. The mainhypothesis of the first part of this dissertation is that LPO produces aldehydes that canpotentially be exploited as AD biomarkers. Design of novel LC-MS/MS methods forbrain aldehyde analysis is described. The methods were applied towards aldehydequantification in the hippocampus, superior and middle temporal gyrus and cerebellum ofsubjects with early AD (EAD), mild cognitive impairment (MCI) and age-matchedcontrols. Results obtained indicated elevation of neurotoxic aldehydes in MCI and EADbrain and suggested that LPO occurred early in AD. Understanding AD progression hasbecome important for developing diagnostic methods and treatments.Mass spectrometry is also the major analytical tool in proteomics, where gelelectrophoresis is dominant in pre-MS separations. The main hypothesis of the latter partof this dissertation is that exposure of microbe fermenters including Clostridiumthermocellum to an external stimulus, such as ethanol, can alter the membrane proteome.Design of novel doubled-SDS-PAGE (dSDS-PAGE) methods for membrane proteinanalysis is described, as these proteins are under-represented in standard 2D-PAGE. Thenewly developed Bicine-dSDS-PAGE offered superior separation over other methods andwas applied towards analysis of wild type and ethanol-adapted C. thermocellum cellmembranes. Significant differences in protein expression were observed. Anunderstanding of ethanol adaptation will promote the design of more ethanol-tolerantstrains. Such an outcome can have dramatic effects in the fuel industry as the trendtowards more efficient fuel development gathers momentum.

The studies described in this dissertation were performed with the goal of understandingthe function of antioxidant compounds delivered in vivo to rodents and the implication of theresults towards oxidative stress (OS)-related neurodegenerative disorders with particularemphasis on Alzheimer's disease (AD). OS has been implicated in AD and is characterized byextensive oxidative damage to protein, lipids and DNA. A major thrust of this dissertation workwas to gain insight into antioxidant properties of compounds used in the following studies andtheir efficacy as potential therapeutics for treatment of OS-related disorders.D609, a glutathione (GSH) mimetic is known to trap OH. Radicals, scavenge H2O2 andreduce the A?? (1-42)-induced OS and cytotoxicity in neurons. The present dissertation studyshowed in vivo protective effect of D609 in synaptosomes and mitochondria isolated fromgerbils against OS mediated by Fe2+/H2O2, AAPH, and A?? (1-42). Upon intraperitonial (i.p.)injection of gerbils, D609 showed protection of subsequently isolated brain moieties against OS.In vivo administration of D609 also modulates brain GSH levels and increases the activity of keyGSH-related enzymes, thereby likely provides a protection against OS.Adriamycin (ADR), a quinone-containing chemotherapeutic, is known to produce ROS inheart. Patients under treatment with ADR often show persistent changes in cognitive function(effect called as chemobrain by patients). Upon i.p. injection, ADR causes OS, increasesexpression of multidrug resistant protein-1 (MRP-1) in brain and alters GSH levels and itsrelated enzyme activities. ??-Glutamyl cysteinyl ethyl ester (GCEE) is known to increase GSHlevels in brain, in vivo. Research reported in this dissertation shows that in vivo GCEE reversesthe ADR-mediated OS in mice brain.N-acetylcysteine (NAC), a GSH precursor provides the limiting substrate cysteine inGSH synthesis. Previously, our laboratory showed increased GSH levels post i.p. injection ofNAC and reduces OS in synaptosomes treated with acrolein. The present study showed thatNAC given in drinking water to APP/PS-1 mice, a model of AD can significantly reduce OS.These results provide a potential therapeutic intervention by antioxidants that can modulateGSH in OS-mediated neurodegenerative disorders.

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