Alzheimer�s disease (AD) is a neurodegenerative disorder, charaeterised by progressive loss of memory. It is important to understand what factors initiate the onset of AD so that effective therapeutic treatments can be developed to target the precise mechanisms that initiate this disease. Currently, synaptic dysfunction is widely believed to be the first significant alteration preceding the onset of AD, and is thought to be initiated by an intracellular accumulation of amyloid-β (Aβ), or a free radical-induced increase of oxidative stress. As Aβ levels rise during the onset of AD, a concomitant reduction of secreted amyloid precursor protein-α (sAPPα) is observed, as the two proteins exist in equilibrium. Intriguingly, the neuroprotective and neurotrophic properties of sAPPα indicate that it is intimately involved in the physiological pathways of the major hypotheses for the cause of AD, and may also be involved in the mechanisms that underlie learning and memory. Therefore, it is possible that during the onset of AD, the decrease of sAPPα may contribute to synaptic dysfunction by disrupting the mechanisms of synaptic plasticity. Long-term potentiation (LTP) is the leading experimental model for investigating the neural substrate of memory formation, and describes the molecular mechanisms that underlie an increase in the strength of synaptic transmission. The role sAPPα may play in the induction and maintenance of LTP has not previously been addressed in vivo. Therefore, the aim of this thesis was to investigate whether sAPPα affects the induction of LTP in the hippocampus of the anaesthetised rat. The present findings are the first to suggest that sAPPα may modulate the induction of LTP in vivo. Decreasing the function of endogenous sAPPα (with sAPPα-binding antibodies and a pharmacological inhibition of α-secretase) significantly reduced the magnitude of LTP induced in the dentate gyrus. Therefore, the reduction of sAPPα during AD is likely to have a detrimental impact on the mechanisms of synaptic plasticity, and by extension, learning and memory. The present investigation has also found that the application of recombinant, purified sAPPα to the rat hippocampus has an �inverted U-shaped� dose-response effect on the magnitude of LTP. Low concentrations of sAPPα significantly enhanced LTP, supporting previous findings that exogenous sAPPα can facilitate in vitro LTP and enhance memory performance in animals. On the other hand, comparatively high concentrations of sAPPα significantly decreased the magnitude of LTP. This observation is also consistent with previous findings, in which high concentrations of sAPPα have been shown to be less synaptogenic and memory enhancing than lower doses. These results are the first to suggest that sAPPα modulates in vivo synaptic plasticity, and have important implications for the development of strategies to treat AD.

In spite of impressive recent progress, the aetiopathogenesis of Alzheimer’s disease (AD) remains incompletely understood. The distinctive neuropathological features of AD, in particular the plaques and tangles, have been the particular focus of most aetiological theories. It is well accepted that AD is a multifactorial disease, with alterations to a variety of brain structures and cell types, including neurons, glia and the brain vasculature. Studies of risk factors have revealed a diversity of genetic variables that interact with health, diet and lifestyle-related factors in the causation of AD. These factors influence the structure, aggregation and function of a set of proteins that are increasingly the focus of research. The work in this thesis has focused on the pathophysiological aspects of some of these proteins in a number of cellular compartments and brain. Several assays have been established and techniques utilized in the completion of this work, including; differential detergent fractionation of brain tissue, 1D and 2D PAGE, western blotting with chemiluminescence detection, ELISA assays of Abeta 1-40 and 1-42, quantitative ECNI GCMS of o- and m-tyrosine as well as metabolites of the kynurenine pathway, quantitative MALDI-TOF assay of hemorphins and LCMSMS based proteomics, to identify proteins with altered expression levels in AD relative to control brain tissue. A variety of regional differences have been observed in the biochemistry of the AD cortex which are probably the outcome of local response variations to AD pathology. One of the most consistent threads throughout this work has been an apparent resilience of the occipital lobe relative to the other brain regions, as reflected in lower overall levels of oxidative stress and increased levels of proteins associated with metabolic processes, neuronal remodeling and stress reduction.

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

<p> </p><p>Bakgrund: I Sverige finns det cirka 160 000 personer som är drabbade av någon form av demenssjukdom och av dessa lider ca 60 % av Alzheimers sjukdom. Cirka en halv miljon svenskar beräknas vara anhöriga till någon som drabbats av Alzheimers sjukdom. Alzheimers kallas ibland för de anhörigas sjukdom, eftersom livssituationen förändras både för den drabbade och för de anhöriga. Syfte: Att beskriva anhörigas upplevelser av att vårda personer med Alzheimers sjukdom. Metod: Studien utfördes som en allmän litteraturstudie. Resultat: I resultatet framkom att de anhöriga upplevde många känslor under sjukdomsförloppet, som till exempel oro inför framtiden och en stor sorg över sjukdomsbeskedet. Många unga anhörigas situationer upplevdes som särskilt svåra då de fortfarande var beroende av sina föräldrar. Slutsats: Som sjuksköterska är det av stor vikt att se till både den sjukes och de anhörigas situation för att i god tid kunna ge familjen det stöd och den information som de kan behöva för att orka fortsätta leva som en familj tills sjukdomens slutstadie.</p><p> </p> / <p>Background: In Sweden there are about 160 000 people who are suffering from some form of dementia and of those suffering around 60% of them has Alzheimer's disease. Approximately half a million Swedes are expected to be relatives of someone affected by Alzheimer's disease. Alzheimer's is sometimes called the relatives' disease, because it is a life-changing situation both for the victim and the family. Purpose: To describe relatives' experiences of caring for people with Alzheimer's disease. Method: The study was carried out as a general literature study. Results: The results showed that many emotions were felt by the relatives during the course of the disease, such as concern for the future and a great sadness at the diagnosis. Many young families’ situations perceived as especially difficult when they were still in need of their parents. Conclusion: As a nurse it is vital to see both the patients and the relatives’ situation in good time to give the family the support and information they might need to be able to continue to live as a family until the terminal stage.</p>

Mitochondrial respiratory chain deficiency and increased oxidative stress have been closely associated with major age-associated neurodegenerative diseases. I hypothesized that mitochondrial oxidative phosphorylation defects or elevated oxidative stress, which could arise in a stochastic manner during our normal aging process, might modulate the formation of protein aggregates or production of misfolded proteins, contributing to the initiation of these diseases. To test this hypothesis, we (i) have developed and characterized mouse and cellular models of Alzheimer's and Huntington's diseases expressing aggregate-prone pathogenic proteins, beta-amyloid and mutant huntingtin (Chapters 1 and 2), (ii) have developed mouse models that exhibit neuron-specific defects in mitochondrial oxidative phosphorylation (Chapters 2 and 3), and (iii) have evaluated the alterations in the amount of aggregate loads upon genetic and pharmacological manipulations of mitochondrial oxidative phosphorylation activities (Chapters 1 and 2). The evaluation of the impacts of mitochondrial defects on the amount of huntingtin aggregates has revealed that a defect in complex III promotes the accumulation of huntingtin aggregates via the impairment of proteasome activity (Chapter 1). On the other hand, ablation of complex IV activity in a subset of postmitotic neurons revealed that complex IV deficiency does not promote either oxidative stress or the deposition of amyloid plaques in a mouse model of Alzheimer's disease, questioning the mitochondrial origin of Alzheimer's disease (Chapter 2). However, as shown previously, the tight correlation between oxidative stress and accumulation of amyloid plaques was found. Chapter 3 involved the generation of an improved mouse model, in which mitochondrial defects can be induced in a subset of forebrain neurons (cortex, hippocampus, and striatum) in a doxycycline-dependent manner. This system relies on the regulated expression of a mitochondria-targeted restriction enzyme, PstI, which digests mitochondrial DNA and thereby impairs the activity of oxidative phosphorylation. In conclusion, our studies highlighted the disease-specific complex pathways that may modulate the accumulation of misfolded proteins during aging. Future studies employing the newly-developed mouse model may reveal a contribution of age-associated global defects of oxidative phosphorylation to oxidative stress and neurodegenerative diseases.

Zur Zeit leiden ca. 24 Millionen Menschen auf der ganzen Welt unter Demenz, Alzheimer macht dabei 50-60% aller Demenzfälle aus. Da der Anteil der Bevölkerung, der an Demenz leidet, proportional zum Alter zunimmt und der Anteil älterer Menschen in der Gesellschaft von Jahr zu Jahr steigt, wird Alzheimer immer mehr zu einem ernstzunehmenden, gesellschaftlichen Problem. Zum Stand der heutigen Forschung ist es etabliert, dass die Aminosäure Glutamat - quantitativ einer der wichtigsten Neurotransmitter im Zentralen Nervensystem (ZNS) - toxische Konzentrationen erreichen kann wenn sie - im Zuge der Übertragung von Aktionspotentialen - nach ihrer Freisetzung nicht aus dem Synaptischen Spalt entfernt wird. Viele Studien haben gezeigt, dass in der Alzheimerschen Krankheit die Glutamataufnahme beeinträchtigt ist, was zu toxischen Konzentrationen von Glutamat und dem daraus folgenden Absterben von Neuronen führt. Der exitatorische Aminosäuretransporter 1 (EAAT1) gehört zu der Familie der Na+-abhängigen Glutamattransporter und stellt nach EAAT2 den quantitativ wichtigsten Glutamattransporter im ZNS dar. In diesem Projekt wurde eine bis dahin für den Menschen nicht bekannte EAAT1 Spleißvariante, in der Exon 3 ausgeschnitten wird, nachgewiesen. Diese Variante wurde EAAT1Δ3 genannt und stellt damit mit EAAT1Δ9 die zweite für EAAT1 nachgewiesene Spleißvariante dar. Eine auf real-time RT-PCR basierende Methode wurde entwickelt, um die Transkripte von EAAT1 wildtyp (EAAT1 wt), EAAT1Δ3 und EAAT1Δ9 zu quantifizieren. Proben aus verschiedenen Hirnarealen wurden aus einem Set von Kontrollen und Alzheimerfällen bei der Quantifizierung verwendet. Die gewählten Areale sind von der Alzheimerschen Krankheit unterschiedlich stark betroffen. Dies diente als interne Kontrolle für die durchgeführten Experimente und ermöglichte so die Differenzierung zwischen beobachteten Effekten: Nur Effekte die alleinig in von Alzheimer betroffenen Gehirnarealen auftreten, können als spezifisch für die Krankheit angesehen werden. Die Resultate diese Projektes zeigen, dass EAAT1Δ3 in sehr geringer Anzahl transkribiert wird, die nur 0.15% der EAAT1 wt Transkription entspricht. Dahingegen entspricht das EAAT1 Δ9 Transkript im Durchschnitt 26.6% des EAAT1 wt Transkripts. Es wurde nachgewiesen, dass die Transkriptionsrate aller EAAT1 Varianten in Alzheimerfällen signifikant reduziert ist (P<0.0001). Dies unterstützt die Theorie, dass bei Alzheimerfällen die EAAT1 Proteinexpression stark reduziert und der Glutamattransport, der normalerweise durch diesen Transporter gewährleistet wird, stark eingeschränkt ist. Dies wiederum resultiert in toxisch hohen Glutamatkonzentrationen und damit dem Absterben von Neuronen. Die gefundene Reduktion der EAAT1Transkription ist nicht spezifisch für Gehirnareale die von Alzheimer betroffen sind, sondern tritt in selbem Maße in nicht von Alzheimer betroffenen Gehirnarealen auf. Daraus lässt sich schließen, dass die Reduktion der EAAT1 Transkription eher ein Resultat eines in der Alzheimerschen Krankheit präsenten, grundlegenden Krankheitsmechanismus ist als deren Ursache. / Today about 24 Million people worldwide suffer from dementia, Alzheimer’s Disease accounts for approximately 50-60% of all dementia cases. As the prevalence of dementia grows with increasing age Alzheimer’s Disease becomes more and more of an issue for society as the proportion of elderly people increases from year to year. It is well established, that the amino acid glutamate - quantitatively being the most important neurotransmitter in the central nervous system (CNS) - may reach toxic concentrations if not cleared from the synaptic cleft into which it is released during transmittance of action potentials. In Alzheimer’s Disease there is strong evidence for a generally impaired glutamate uptake system which in turn is thought to result in toxic levels of the amino acid with the potential to kill off neurons. The excitatory amino acid transporter 1 (EAAT1) belongs to the family of Na+-dependent glutamate transporter and accounts together with EAAT2 for most of the glutamate uptake in the CNS. In this project a new splice variant of EAAT1, skipping exon 3 was detected in human brain samples and subsequently called EAAT1Δ3, this being the second splice variant found after the recent detection of EAAT1Δ9. A method was developed to quantify the transcript of EAAT1 wt, EAAT1Δ3 and EAAT1Δ9 by means of real-time PCR. Samples were taken from different brain areas of a set of control and AD cases. The areas chosen for examination are affected differently in Alzheimer’s Disease, this was used an internal control for the experiments done in this project as to determine whether any effect observed is specific for AD, i.e. AD affected areas or is generally seen in all areas examined. The results of this project show that EAAT1Δ3 is transcribed in very low copy numbers making up a proportion of 0.15% of EAAT1 wt whereas EAAT1Δ9 is transcribed in a considerably large proportion of EAAT1 wt of 26.6%. It was moreover found that all EAAT1 variants are transcribed at significantly lower rates (P<0.0001) in AD cases, supporting the theory that EAAT1 protein expression is reduced to a point where glutamate uptake normally mediated by this transporter is impaired. This in turn is thought to result in toxic levels glutamate accounting for neuronal loss in the disease. No area-dependent effects were found, suggesting that the reduction of EAAT1 transcription is rather a result of an underlying general mechanism present in AD. Further research will have to be done to assess the degree of EAAT1 expression in AD and whether those future findings match with the result of this project.

Soluble intermediates of the amyloid-β (Aβ) aggregation process are suggested to play a central role in the pathogenesis of Alzheimer’s disease (AD) by causing synaptic dysfunction and neuronal loss. In this thesis, soluble Aβ aggregates have been studied with a particular focus on the Aβ protofibril, which has served as the antigen for developing conformation dependent monoclonal antibodies. Antibodies generated from mice immunized with Aβ protofibrils were characterized regarding Aβ binding properties and the amino acid sequences of their antigen binding sites. A conformation dependent IgG antibody, mAb158, was further characterized and found to bind to Aβ protofibrils with a 200-fold higher affinity than to monomeric Aβ without affinity for soluble amyloid-β precursor protein (AβPP) or other amyloidogenic proteins. A sandwich enzyme-linked immunosorbent assay (ELISA) based on mAb158 was used to measure soluble Aβ protofibrils in brain extracts from AβPP-transgenic mice. Low levels of protofibrils could also be detected in human AD brain. However, positive signals generated from measurements in AD and control CSF samples were attributed to interference from heterophilic antibodies (HA), generating false positive signals by cross-binding the assay antibodies; consequently, a study on HA interference in Aβ oligomer ELISAs was initiated. A large set of plasma and CSF samples from AD and non-AD subjects were analyzed with and without measures taken to block HA interference, revealing that virtually all signals above the assay limit of detection were false and generated by HA interference. Many types of soluble Aβ aggregates have been described and suggested to impair neuron and synapse function. To investigate the soluble Aβ pool, synthetic Aβ and brain extracts from AβPP-transgenic mice and AD patients were ultracentrifuged on a density gradient to separate Aβ by size under native conditions. Four distinct gradient fractions were defined based on the appearance of synthetic Aβ in atomic force microscopy (AFM) and immunoreactivity in our protofibril specific sandwich ELISA. Interestingly, most Aβ from AD patients and AβPP-transgenic mice separated in the same fraction as toxic synthetic protofibrils.

Heparan sulfate (HS) is a highly sulfated polysaccharide expressed on the cell surface and in the extracellular matrix, interacting with a large number of proteins. HS is implicated in human diseases, including different types of cancer and amyloid diseases such as Alzheimer's disease (AD). The aims of this thesis were to gain deeper insights into AD and cancer progression by elucidating the roles of HS in amyloid-β (Aβ) pathology and hypoxia. The toxic Aβ-peptide is a key molecule in AD due to its ability to aggregate and form amyloid plaques in the brains of diseased patients. It has been reported that HS accumulates with Aβ in these amyloid plaques. We have found that HS is differentially accumulated with Aβ species within the amyloid plaques in the brains of AD patients. We also identified that the HS in the plaques originated from glial cells. Further, we investigated the role of HS in Aβ toxicity using cell models that either lack HS or express abnormal HS. The results show that cell surface HS mediates Aβ internalization and cytotoxicity. Upregulation of heparanase, an endo-glucuronidase that specifically cleaves HS chains, in human cancers increases the potential of tumor cells to metastasize. Spalax, an animal model for hypoxic tolerance, expresses high levels of heparanase. Analysis of HS from different Spalax organs revealed a high sulfation degree and an atypical domain structure, likely modulated by high heparanase expression in the organs. Cells cultured under hypoxic conditions showed a similar HS domain structure and had an increase in heparanase mRNA. We propose that hypoxia-induced heparanase expression is relevant for tumor progression, a process often associated with oxygen deficiency. Altogether, the findings in this thesis are important for future development of therapeutics aiming at interfering with HS functions in AD and cancer.

Organic nitrates, such as glyceryl trinitrate (GTN), have been used clinically for more than a century. However optimal nitrate therapy is hindered by the development of tolerance, which is associated with a desensitized response to GTN, oxidative stress, and the inactivation of aldehyde dehydrogenase 2 (ALDH2). This thesis evaluated the ALDH2 inactivation hypothesis of GTN tolerance and investigated the role of oxidative stress in GTN tolerance mediated by the lipid peroxidation product, 4-hydroxynonenal (HNE). Evidence for a direct role of ALDH2 in nitrate action was sought using a stably transfected cell line that overexpressed ALDH2, or siRNA to deplete endogenous ALDH2. Neither manipulation altered GTN-induced cGMP formation, indicating that ALDH2 does not mediate GTN bioactivation and tolerance. In a second study using an in vivo GTN tolerance model and a cell culture model of nitrate action, a marked increase in HNE adduct formation was detected in GTN-tolerant tissues, and treatment with HNE reduced the cGMP and vasodilator responses to GTN, thus mimicking GTN-tolerance. Together, the results suggest a primary role for HNE in the development of GTN tolerance, and provide the framework for a unified hypothesis that accommodates the previous findings of sulfhydryl depletion, ALDH2 inactivation and oxidative stress that are associated with nitrate tolerance. Studies have implicated oxidative stress and increased HNE formation in the pathogenesis of Alzheimer’s disease (AD). It was hypothesized that the gene deletion of ALDH2 would result in increased HNE-adduct formation leading to impaired cognitive function, and AD-like pathological changes. We observed a marked increase in HNE-adduct formation in Aldh2-/- mouse hippocampi as well as hyperphosphorylated tau, activated caspases, age-related changes in hippocampal amyloid βeta1-42 (Aβ1-42), post-synaptic density protein 95 (PSD95) and phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB) expression, endothelial dysfunction and other vascular pathologies. These data provide further evidence for the importance of HNE and oxidative stress in AD pathogenesis, and establish Aldh2-/- mice as a new, oxidative stress-based animal model of age-related cognitive impairment and AD. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-05-31 11:10:58.145

Alzheimer's disease (AD) is characterized by neuronal atrophy caused by soluble amyloid β protein (Aβ) peptide "oligomers" and a microglial-mediated inflammatory response elicited by extensive amyloid deposition in the brain. We show that CNI-1493, a tetravalent guanylhydrazone with established antiinflammatory properties, interferes with Aβ assembly and protects neuronal cells from the toxic effect of soluble Aβ oligomers. Administration of CNI-1493 to TgCRND8 mice overexpressing human amyloid precursor protein (APP) for a treatment period of 8 wk significantly reduced Aβ deposition. CNI-1493 treatment resulted in 70% reduction of amyloid plaque area in the cortex and 87% reduction in the hippocampus of these animals. Administration of CNI-1493 significantly improved memory performance in a cognition task compared with vehicle-treated mice. In vitro analysis of CNI-1493 on APP processing in an APP-overexpressing cell line revealed a significant dose-dependent decrease of total Aβ accumulation. This study indicates that the antiinflammatory agent CNI-1493 can ameliorate the pathophysiology and cognitive defects in a murine model of AD.