Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 122-136.

A doença de Alzheimer (DA) é uma doença neurodegenerativa caracterizada pelo declínio progressivo da memória e de outras funções cognitivas, acometendo, sobretudo, indivíduos idosos. A anormalidade do metabolismo da proteína precursora do amilóide (APP) e a hiperfosforilação da proteína TAU são processos celulares característicos desta doença. A enzima glicogênio sintase quinase 3B (GSK3B) é altamente expressa no sistema nervoso central e apresenta grande importância na regulação da plasticidade neuronal e também nos mecanismos de sobrevivência celular. Estudos têm associado a GSK3B aos mecanismos que levam à formação das placas senis e dos emaranhados neurofibrilares na DA. Diante da dificuldade para se estabelecer com precisão o diagnóstico clínico da DA, sobretudo nas fases iniciais da doença, a identificação de biomarcadores se torna particularmente importante, principalmente em tecidos periféricos. Este trabalho avaliou, por meio de dois preparos distintos, dois possíveis candidatos a marcadores bioquímicos para a DA. Em plaquetas de pacientes com DA, comprometimento cognitivo leve (CCL) e idosos saudáveis, determinamos: (1) a razão de APP (APPr), que consiste na proporção entre fragmentos de 130kDa e 110kDa da APP; e (2) a expressão das formas fosforilada (fosfo-GSK3B) e total (total-GSK3B) da enzima GSK3B, possibilitando o cálculo da razão de GSK3B (fosfo-GSK3B/total-GSK3B). Ambas as razões foram avaliadas por Western Blot utilizando anticorpos específicos. Não observamos diferença estatisticamente significante nos valores de APPr entre os três grupos estudados (p=0,847). Para o cálculo da razão de GSK3B, foram necessárias adaptações do protocolo de preparo e análise de plaquetas, prevenindo a ativação plaquetária durante o procedimento, bem como a degradação do substrato pela ação de proteases e fosfatases presentes nesta matriz biológica; deste modo foram encontradas diferenças estatisticamente significantes entre os grupos nas médias de GSK3B total e da razão de GSK3B (p=0,05 e p=0,06 respectivamente). Não foi encontrada correlação entre a razão de APP e a razão de GSK3B. Contudo, a razão de GSK3B mostrou correlação com o desempenho em testes de memória, segundo o escore na bateria cognitiva CAMCOG. Estes resultados sugerem que a razão de GSK3B em plaquetas sinaliza algumas alterações biológicas que ocorrem na progressão do CCL para a demência na DA / Alzheimer\'s disease (AD) is a neurodegenerative disease characterized by progressive decline of memory and other cognitive functions, affecting mainly elderly. The abnormal metabolism of amyloid precursor protein (APP) and protein TAU hyperphosphorylation are cellular hallmarks of this disease. Glycogen synthase kinase 3B (GSK3B) is an enzyme highly expressed in the central nervous system and of great importance in the regulation of neuronal plasticity and also the mechanisms of cell survival. Studies have associated GSK3B with the mechanisms that lead to the formation of senile plaques and neurofibrillary tangles in AD. Given the difficulty to establish the precise clinical diagnosis of AD, especially in the early stages of the disease, identification of biomarkers is particularly important, especially in peripheral tissues. This work evaluated two candidate biochemical markers for AD, using two different preparations. We investigated the ratio between 130kDa and 110kDa APP fragments (APPr) and between phosphorylated and total GSK3B (phospho-GSK3B / total-GSK3B) in platelets of patients with AD and mild cognitive impairment (MCI), comparing their results with those from healthy older adults (controls). The expression of APP fragments and GSK3B was assessed by Western blot using specific antibodies. No statistically significant differences in APPr were found between the study groups (p=0.847). We found statistically significant differences in mean total GSK3B and GSK3B ratio across disgnostic groups (p=0.05 and p=0.06, respectively). APPr and GSK3B ratio were not correlated, but the latter parameter did correlate with the performance on memory tests, as shown by the CAMCOG sub-score. The present data indicate that platelet GSK3B ratio may indicate biological changes that occur in the MCI-AD continuum

Mutations of three genes encoding amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) have been shown to reliably result in familial early-onset Alzheimer's disease (FAD); a rare, but catastrophic, subtype of Alzheimer's disease (AD) marked by symptom emergence before age 65 as well as accelerated cognitive deterioration. The current study represents the first known meta-analysis on the association of APP, PSEN1 or PSEN2 on neurocognitive variables. A total of 278 FAD mutation-carriers (FAD-MC) and 284 cognitively healthy non-mutation-carriers (NC) across 10 independent investigations meeting inclusion criteria were chosen for the current meta-analysis (random effects design). Findings revealed an overarching trend of poorer performance by FAD-MC individuals compared to NC individuals across the majority of cognitive domains identified. Significant differences in effect sizes suggested FAD-MC individuals exhibited worse performance on measures of attention, explicit memory, fluency, primary memory, verbal, and visuospatial functioning. Findings indicative of differential sensitivity to cognitive domain impairments across FAD-MC and NC groups inform neuropsychological descriptions of individuals in preclinical phases of FAD.

Glioma therapy relies on induction of cytotoxicity; however, the current combination of surgery, irradiation (IR) and temozolomide (TMZ) treatment does not result in a long-term cure. Our lab previously demonstrated that a small population of glioma cells enters a transient cell cycle arrest in response to chemotherapy. Treatment with TMZ significantly decreases initial neurosphere formation; however, after a short recovery period, a small number of cells resume neurosphere formation and repopulate the culture. This recovery of neurosphere growth recapitulates the inevitable glioma recurrence in the clinic. The focus of our laboratory is to study direct-target therapies that can be combined with TMZ to inhibit neurosphere recovery. The Notch pathway is a promising target because it is involved in cell growth and survival. Here, we demonstrate that blocking the Notch pathway using gamma-secretase inhibitors (GSIs) enhances TMZ treatment. The combination of TMZ and GSI treatments targets the cells capable of recovery. TMZ + GSI treated cells do not recover and are no longer capable of self-renewal. Interestingly, recovery is inhibited when the GSI is administered 24 hrs after TMZ treatment, demonstrating a sequence-dependent mechanism. TMZ + GSI treatment also decreases tumorigenicity. When glioma cell lines were treated in vitro and implanted in NU/NU nude mice, TMZ + GSI treatment extended latency and greatly increased survival. In addition, in vivo TMZ + GSI treatment completely blocked tumor progression and resulted in the loss of a palpable tumor in 50% of mice, while none of the TMZ-only treated mice survived. TMZ + GSI treated cultures and xenografts display a senescent phenotype. Cultures treated with TMZ + GSI have decreased proliferation, but no increase in cell death. We observed an increase in the number of cells expressing senescence-associated β-galactosidase in vitro and in vivo. This demonstrates that inhibition of the Notch pathway shifts TMZ-treated cells from a transient cell cycle arrest into a permanent senescent state. Senescent cells can stimulate the innate immune system. Here we demonstrate that TMZ + GSI treatment increases phagocytosis in vitro. New therapy combinations, such as TMZ + GSI, are arising in the field of therapy-induced senescence (TIS). Overall, this data demonstrates the importance of the Notch pathway in chemoprotection and maintenance of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising target-directed therapy to decrease the rate of brain tumor recurrence by inducing senescence and tumor clearance.

Zusammenfassung: Patienten mit Down Syndrom (DS, Trisomie 21) entwickeln im vierten Lebensjahrzehnt eine Neuropathologie, wie sie beim Morbus Alzheimer (AD) beobachtet wird. Im Gehirn dieser Patienten kommt es zur Ausbildung von senilen Plaques, neurofibrillären Veränderungen und zu einer Schädigung des cholinergen Systems. Als erstes Zeichen der beginnenden Veränderungen wird die erhöhte Konzentration und Akkumulation von sogenannten beta-A4-Peptiden gewertet. Diese Peptide, die auch den Hauptbestandteil der senilen Plaques darstellen, entstehen durch die Prozessierung eines größeren Proteins des Amyloidvorläuferproteins (amyloid precursor protein, APP). Beim Menschen wurde das APP-Gen auf einem distalen Segment des langen Arms des Chromosoms 21 lokalisiert. Das Homolog dieses evolutionär stark konservierten, syntenen Segmentes liegt bei der Maus auf dem Chromosom 16. Natürlich in wilden Mäusepopulationen auftretende Robertsonsche Translokationen ermöglichen es, Mäuse mit Trisomie 16 zu züchten. Mit Hilfe der Maus-Trisomie 16 sollte ein Modell etabliert werden, mit dem es unter in vivo Bedingungen möglich ist, die Auswirkungen der erhöhten APP-Gendosis auf die Ausbildung der bei DS und AD beobachteten neurodegenerativen Veränderungen zu untersuchen. Da trisomische Feten am Ende der Trächtigkeit absterben, wurden aus dem basalen Vorderhirn trisomischer und diploider Feten Transplantate gewonnen und in den Lateralventrikel adulter euploider Mäuse implantiert. Die Entwicklung der Transplantate wurde nach 1, 3, 6, 9 und 12 Monaten immunhistochemisch charakterisiert. Ein Antikörper gegen das Thymozytenantigen (Thy)-1.2 wurde, beruhend auf der unterschiedlichen Thy-1-Allel-Expression von Transplantat und Empfänger, zur Transplantatidentifikation genutzt. Mit Antikörpern gegen das neuronale Markerprotein PGP-9.5, gegen Cholinacetyltransferase, Parvalbumin und Glutamatdecarboxylase wurden Neuronen charakterisiert. Die immunologische Reaktion wurde mit Antikörpern gegen saures fibrilläres Gliaprotein, gegen das Makrophagenantigen F4/80, gegen CD3, gegen CD45/ B220 und mit Lycopersicon esculentum-Lektin untersucht. Für den APP- bzw. beta-A4-Peptidnachweis wurden ein Antikörper gegen den C-Terminus des APP und der Antikörper 4G8 eingesetzt. Zusätzlich wurde mit Hilfe von molekularbiologischen Techniken (Northern-Blot, Polymerase-Kettenreaktion) die APP-Expression in Trisomie 16-Feten untersucht. Mit immunhistochemischen und histochemischen Methoden wurde versucht, den Entwicklungstand des basalen Vorderhirns zum Zeitpunkt der Transplantatpräparation am Gestationstag 15 zu untersuchen. / Summary: Patients suffering from Down''s syndrome (DS, trisomy 21) develop neuropathological abnormalities similar to Alzheimer''s disease (AD) in the fourth decade of life. Amongst others, neuritic plaques, neurofibrillary abnormalities and alterations in cholinergic basal forebrain systems were observed. These sequentially occuring disturbancies are initiated by a rise in the concentration and accumulation of the beta-amyloid-peptides. The beta-amyloid-peptides are derived by proteolytic processing from a larger amyloid precursor protein (APP) and compose the majority of the material deposited in amyloid plaques. In humans, the APP gene maps to the distal segment of the long arm of chromosome 21, but in mice the homolog gene locus is on chromosome 16. The naturally occuring Robertsonian translocations in feral mice (Mus musculus sp.) allow to breed trisomy 16 mice. The aim of this study was to establish an in vivo model to investigate the consequences of increased APP gene dosage on the generation of neuropathological abnormalities typical for DS and AD using trisomy 16 mice. Since trisomy 16 mice die at the end of prenatal development, basal forebrain tissue of diploid and trisomic fetuses was prepared and transplanted into lateral ventricles of adult euploid mice. Grafts were identified immunocytochemically using an antibody against thymocyte antigen-1.2, selectively labeling graftet tissue. Antibodies against the neuronal markerprotein PGP-9.5, choline acetyltransferase, parvalbumin and glutamate decarboxylase were used to characterize grafted neurons over a period of twelve months after implantation. The immunological tissue response in the brains of acceptor mice was monitored using antibodies against glial fibrillary acidic protein (GFAP), the macrophage antigen F4/80, CD3,CD45/B220 and using the Lycopersicon esculentum lectin. To detect APP and beta-amyloid-peptides,antibodies against a C-terminal APP fragment and the antibody 4G8 were used. Additionally, the APP mRNA expression in trisomy 16 mice was followed employing Northern-blot analysis and RT-PCR. The developmental state of basal forebrain tissue to be transplanted was characterized at the time of transplantation (gestation day 15) by means of histochemistry and immunohistochemistry.

No description available.

Ces dernières années, de nombreuses études ont prouvé que la protéine précurseur de l'amyloïde (APP) joue un rôle clé dans le processus de formation de la mémoire, le développement des connexions synaptiques et la régulation de la force synaptique. L’importance d’APP naît du fait que son clivage protéolytique produit le peptide bêta-amyloïde (Aβ), considéré comme l'un des facteurs cruciaux dans le développement de la maladie d'Alzheimer. Les recherches se sont donc concentrées sur Aβ plutôt que sur le domaine intracellulaire APP (APP-ICD). Récemment, il a été démontré qu’APP-ICD affecte l'induction de la plasticité synaptique, et Aβ à haute concentration est connu pour induire une dépression synaptique. Ici, nous montrons qu’APP-ICD et Aβ fonctionnent ensemble et induisent une dépression synaptique en modifiant la transmission synaptique par effet additif. L’activation de la caspase-3 clivant APP-ICD est nécessaire pour la dépression à long terme. Nous constatons que l’activation de la caspase-3 et son site de clivage d’APP-ICD, ainsi que le clivage d’APP par la gamma-sécrétase sont nécessaires à la dépression synaptique dépendante d’Aβ. La microglie assure la clairance d’Aβ et certains effets de plasticité. Nous démontrons qu’elle médie partiellement la dépression synaptique dépendante d’Aβ. Les mécanismes par lesquels APP-ICD et Aβ médient la dépression synaptique ne sont pas connus. Ici, nous discutons de pistes possibles pour la recherche future, notamment des changements dans l'homéostasie du calcium en tant que cible thérapeutique potentielle. Comprendre comment APP-ICD et Aβ travaillent ensemble pour induire une dépression synaptique aiderait à développer de meilleurs traitements pour la maladie d'Alzheimer. / In recent years, more and more evidence has proven that the amyloid precursor protein (APP) plays a key role in the process of memory formation, the development of synaptic connections, and the regulation of synaptic strength. APP rose to prominence since its proteolytic cleavage produces the amyloid-beta (Aβ) peptide, which is believed to be one of the crucial factors in the development of Alzheimer disease. Therefore, most of the research focused on Aβ, while APP intracellular domain (APP-ICD) received much less attention. In a recent study, APP-ICD was shown to affect the induction of synaptic plasticity, and Aβ at high concentration is known to induce synaptic depression. Here we show that APP-ICD works together with Aβ to induce synaptic depression, meaning they have an additive effect that changes synaptic transmission. Caspase-3 cleaves APP-ICD, and its activation is required for long-term depression. We found that the caspase-3 cleavage site of APP-ICD and caspase-3 activation are needed for Aβ-dependent synaptic depression. We also show that cleavage of APP by gamma-secretase is needed for the effect. Microglia mediate clearance of Aβ as well as some plasticity effects. We demonstrate that microglia partially mediate Aβ-dependent synaptic depression. The mechanisms of how APP-ICD and Aβ mediate synaptic depression are not known, here, we discuss possible avenues for future research, specifically changes in calcium homeostasis as a potential therapeutic target. Hence, understanding how APP-ICD and Aβ work together to induce synaptic depression would aid in developing better treatments for Alzheimer disease.

No description available.

No description available.

Nitric oxide (NO) is a neurotransmitter involved in memory processes. Currently, the only recognized physiological signalling pathway controlled by NO is the activation of guanylyl cyclase. In this thesis, we propose an alternative NO-signalling pathway that involves the Heme-regulated eukaryotic initiation factor-2a kinase (HRI) and eIF2a phosphorylation. We have found that the enzyme BACE1, a key protein in Alzheimer’s disease (AD), is controlled by this novel pathway. This pathway would be involved in the physiology of memory formation and learning processes. We have also studied how an external stress factor, the Herpes Simplex Virus 1, can disrupt this cascade leading to a pathological increase in BACE1 and amyloid ß-peptide (Aß) production. Aß aggregates forming fibrils that generate free radicals. These react with NO producing peroxynitrite, which contribute to AD progression. Since NO turns toxic when produced in a pro-oxidant environment we have also studied the effect of peroxynitrite in Stroke. / L’òxid nítric (NO) és un neurotransmissor involucrat en processos de memòria. Actualment, l’única cascada de senyalització fisiològica controlada per NO consisteix en l’activació de la guanilat ciclasa. En aquesta tesi, en proposem una d’alternativa que inclou la fosforilació de eIF2a per la Heme-regulated eukaryotic initiation factor-2a kinase (HRI). Hem mostrat com l’enzim BACE1, una proteïna clau en la malaltia d’Alzheimer (AD), és controlat per aquesta nova cascada de senyalització, que podria estar involucrada en la fisiologia de l’aprenentatge i la memòria. També hem estudiat com un factor d’estrès extern, l’ Herpes Simplex Virus 1, pot pertorbar aquesta cascada donant lloc a increments patològics en BACE1 i pèptid ß-amiloide (Aß). L’Aß agrega formant fibril·les que generen radicals lliures. Aquests reaccionen químicament amb NO produint peroxinitrit, que contribueix a la progressió de l’AD. Pel fet que l’NO esdevé tòxic quan és produït en un entorn pro-oxidant, hem estudiat també l’impacte que el peroxinitrit té en l’ictus.