There is an urgent need for the development of new therapeutic strategies for Alzheimer's disease (AD). The dual-specificity tyrosine phosphorylation-regulated kinase-1A (Dyrk1a) is a protein kinase that phosphorylates the amyloid precursor protein (APP) and tau and thus represents a link between two key proteins involved in AD pathogenesis. Furthermore, Dyrk1a is upregulated in postmortem human brains, and high levels of Dyrk1a are associated with mental retardation. Here, we sought to determine the effects of Dyrk1 inhibition on AD-like pathology developed by 3xTg-AD mice, a widely used animal model of AD. We dosed 10-month-old 3xTg-AD and nontransgenic (NonTg) mice with a Dyrk1 inhibitor (Dyrk1-inh) or vehicle for eight weeks. During the last three weeks of treatment, we tested the mice in a battery of behavioral tests. The brains were then analyzed for the pathological markers of AD. We found that chronic Dyrk1 inhibition reversed cognitive deficits in 3xTg-AD mice. These effects were associated with a reduction in amyloid-beta (Ab) and tau pathology. Mechanistically, Dyrk1 inhibition reduced APP and insoluble tau phosphorylation. The reduction in APP phosphorylation increased its turnover and decreased Ab levels. These results suggest that targeting Dyrk1 could represent a new viable therapeutic approach for AD.

Alzheimer's disease (AD), marked by a serious and progressive decline in cognitive abilities, is a severely debilitating disease that is becoming an increasing concern with our aging population. Defects in neurogenesis have been shown to exist in AD and aggravate the neuropathology and cognitive deficits associated with the disease. In this study, I aimed to characterize the cellular and molecular defects of neurogenesis in the triple transgenic mouse model of AD (3xTG). To do so, I first performed a detailed immunohistochemistry characterization using neurogenic markers that were quantified and analyzed in the hippocampus of control and 3xTG mice. This analysis not only revealed an overall decrease in the pool of neural stem and progenitor cells (NSPCs) in 3xTG brains, but also defects in proliferation, differentiation and a loss within the neuroblast, immature neuron and mature neuron populations. Subsequent immunohistochemistry analysis of two molecular targets, Hopx and LPAR1, involved in NSC maintenance and proliferation respectively, revealed their dysregulation in 3xTG brains, providing some indication of molecular defects underlying this loss. The neurosphere assay was next employed to assess cell-autonomous defects and fewer neurospheres were formed from cultured 3xTG NSPCs, suggesting a defect in NSPC pool expansion that is intrinsic to 3xTG NSPC function. Molecular characterization of these cultured NSPCs via qPCR revealed the upregulation of mitochondrial and fatty acid oxidation genes in 3xTG NSPCs, suggesting not only a dysregulation of metabolic functions, but also an acclimation to oxidative stress conditions. Interestingly, 3xTG NSPCs formed larger and more neurospheres when grown in galactose medium - which is used to simulate oxidative stress - relative to the control, confirming an adaptative response to oxidative stress conditions. Further characterization of these cellular defects and underlying molecular mechanisms can reveal novel therapeutic strategies for AD.

A Doença de Alzheimer é a causa mais freqüente de demência senil e os gastos com pacientes com demência já supera os de pacientes com câncer ou com doenças cardiovasculares. As lesões características dessa doença são as placas amilóides e os emaranhados neurofibrilares. A neuroinflamação também está presente na maioria dos pacientes com Alzheimer, e parece contribuir para o dano no tecido neuronal. Adicionalmente, estudos vêm demonstrando que pacientes com Alzheimer também apresentam alterações sistêmicas, e, dessas, a mais relatada é o estado pró-inflamatório em tecidos periféricos, permitindo que a Doença de Alzheimer seja estudada em um contexto neuroimunológico. Utilizando um modelo murino para a Doença de Alzheimer, o camundongo 3xTg-AD (que desenvolve ambas as patologias β-amilóide e tau), investigamos se aumento de mediadores inflamatórios também pode ser detectado nesse modelo, tanto no hipocampo (estrutura relevante para os sintomas da doença) como no sangue. Alterações cognitivas e comportamentais e a presença do precursor da proteína amilóide (APP) e/ ou peptídeo β-amilóide em estruturas cerebrais relevantes para a doença (córtex, hipocampo, subículo e amígdala) permitiram validar o camundongo 3xTg-AD como um modelo murino da Doença de Alzheimer. Análises da expressão de mediadores inflamatórios no hipocampo demonstraram que a presença de APP e/ ou peptídeo β-amilóide no cérebro não induz um estado pró-inflamatório no hipocampo ou no sangue, até os 12 meses de idade. Porém, a expressão de APP e/ ou peptídeo β-amilóide no cérebro parece induzir distúrbios sistêmicos, já que algumas alterações periféricas foram encontradas. Como a resposta ao LPS envolve tanto tecidos periféricos, como o Sistema Nervoso Central, avaliou-se os efeitos da administração periférica de LPS nesse camundongo, aos 12 meses de idade. A resposta inflamatória ao LPS diferiu entre camundongos Wild Type (grupo controle) e 3xTg-AD. No sangue, menor aumento de IL-6 e MCP-1 e maior aumento de IFN-γ foram encontrados nos camundongos 3xTg-AD. As conseqüências deste perfil de citocinas séricas no Sistema Nervoso Central foram distintas, dependendo da resposta avaliada: enquanto que a ativação do eixo HPA foi semelhante, a produção de citocinas inflamatórias no hipocampo foi atenuada. Portanto, no camundongo 3xTg-AD, a diferente resposta inflamatória ao LPS no sangue promoveu menor produção de mediadores inflamatórios no hipocampo. / Alzheimer\'s Disease is the most frequent cause of senil dementia and the costs with demented patients already exceeds that of patients with cancer or cardiovascular diseases. The characteristic lesions of this disease are amyloid plaques and neurofibrillary tangles. Neuroinflammation is also present in most of Alzheimer\'s patients, and seems to contribute to neuronal tissue damage. In addition, studies have demonstrated that patients with Alzheimer also display systemic alterations, and of those, the most reported is the pro-inflammatory state in peripheral tissues, allowing Alzheimer\'s Disease to be studied in a neuroimmunology context. Using a murine model of Alzheimer\'s Disease, the 3xTg-AD mice (which develops both amyloid-βand tau pathologies), we investigated whether enhancement of inflammatory mediators can also be detected in this model, in both hippocampus (a relevant structure for the symptoms of the disease) and in blood. Cognitive and behavioral alterations and the presence of amyloid precursor protein (APP) and/ or amyloid-β peptide in relevant brain structures for the disease (cortex, hippocampus, subiculum and amigdala) allowed the validation of 3xTg-AD mice as a murine model of Alzheimer\'s Disease. Analysis of inflammatory mediators expression in hippocampus demonstrated that the presence of APP and/ or amyloid-β peptide in the brain does not induce a pro-inflammatory state in hippocampus or in the blood, until 12 months of age. Nevertheless, APP and/or amyloid-β peptide expression in the brain seems to induce systemic disturbances, once peripheral alterations were detected. As LPS response includes both peripheral tissues and the Central Nervous System, we evaluated peripheral administration effects of LPS in these mice, at 12 months of age. The inflammatory response to LPS differed between Wild Type (control group) and 3xTg-AD. In the blood, smaller enhancement of IL-6 and MCP-1 and higher enhancement of IFN-γ were found in 3xTg-AD mice. The consequences of this serum cytokine profile on the Central Nervous System were distinct, depending on the response evaluated: while HPA axis activation was similar, production of inflammatory cytokines in hippocampus was attenuated. Therefore, in the 3xTg-AD mice, a different inflammatory response to LPS in blood promoted lesser inflammatory mediators production in hippocampus.

A Doença de Alzheimer é a causa mais freqüente de demência senil e os gastos com pacientes com demência já supera os de pacientes com câncer ou com doenças cardiovasculares. As lesões características dessa doença são as placas amilóides e os emaranhados neurofibrilares. A neuroinflamação também está presente na maioria dos pacientes com Alzheimer, e parece contribuir para o dano no tecido neuronal. Adicionalmente, estudos vêm demonstrando que pacientes com Alzheimer também apresentam alterações sistêmicas, e, dessas, a mais relatada é o estado pró-inflamatório em tecidos periféricos, permitindo que a Doença de Alzheimer seja estudada em um contexto neuroimunológico. Utilizando um modelo murino para a Doença de Alzheimer, o camundongo 3xTg-AD (que desenvolve ambas as patologias β-amilóide e tau), investigamos se aumento de mediadores inflamatórios também pode ser detectado nesse modelo, tanto no hipocampo (estrutura relevante para os sintomas da doença) como no sangue. Alterações cognitivas e comportamentais e a presença do precursor da proteína amilóide (APP) e/ ou peptídeo β-amilóide em estruturas cerebrais relevantes para a doença (córtex, hipocampo, subículo e amígdala) permitiram validar o camundongo 3xTg-AD como um modelo murino da Doença de Alzheimer. Análises da expressão de mediadores inflamatórios no hipocampo demonstraram que a presença de APP e/ ou peptídeo β-amilóide no cérebro não induz um estado pró-inflamatório no hipocampo ou no sangue, até os 12 meses de idade. Porém, a expressão de APP e/ ou peptídeo β-amilóide no cérebro parece induzir distúrbios sistêmicos, já que algumas alterações periféricas foram encontradas. Como a resposta ao LPS envolve tanto tecidos periféricos, como o Sistema Nervoso Central, avaliou-se os efeitos da administração periférica de LPS nesse camundongo, aos 12 meses de idade. A resposta inflamatória ao LPS diferiu entre camundongos Wild Type (grupo controle) e 3xTg-AD. No sangue, menor aumento de IL-6 e MCP-1 e maior aumento de IFN-γ foram encontrados nos camundongos 3xTg-AD. As conseqüências deste perfil de citocinas séricas no Sistema Nervoso Central foram distintas, dependendo da resposta avaliada: enquanto que a ativação do eixo HPA foi semelhante, a produção de citocinas inflamatórias no hipocampo foi atenuada. Portanto, no camundongo 3xTg-AD, a diferente resposta inflamatória ao LPS no sangue promoveu menor produção de mediadores inflamatórios no hipocampo. / Alzheimer\'s Disease is the most frequent cause of senil dementia and the costs with demented patients already exceeds that of patients with cancer or cardiovascular diseases. The characteristic lesions of this disease are amyloid plaques and neurofibrillary tangles. Neuroinflammation is also present in most of Alzheimer\'s patients, and seems to contribute to neuronal tissue damage. In addition, studies have demonstrated that patients with Alzheimer also display systemic alterations, and of those, the most reported is the pro-inflammatory state in peripheral tissues, allowing Alzheimer\'s Disease to be studied in a neuroimmunology context. Using a murine model of Alzheimer\'s Disease, the 3xTg-AD mice (which develops both amyloid-βand tau pathologies), we investigated whether enhancement of inflammatory mediators can also be detected in this model, in both hippocampus (a relevant structure for the symptoms of the disease) and in blood. Cognitive and behavioral alterations and the presence of amyloid precursor protein (APP) and/ or amyloid-β peptide in relevant brain structures for the disease (cortex, hippocampus, subiculum and amigdala) allowed the validation of 3xTg-AD mice as a murine model of Alzheimer\'s Disease. Analysis of inflammatory mediators expression in hippocampus demonstrated that the presence of APP and/ or amyloid-β peptide in the brain does not induce a pro-inflammatory state in hippocampus or in the blood, until 12 months of age. Nevertheless, APP and/or amyloid-β peptide expression in the brain seems to induce systemic disturbances, once peripheral alterations were detected. As LPS response includes both peripheral tissues and the Central Nervous System, we evaluated peripheral administration effects of LPS in these mice, at 12 months of age. The inflammatory response to LPS differed between Wild Type (control group) and 3xTg-AD. In the blood, smaller enhancement of IL-6 and MCP-1 and higher enhancement of IFN-γ were found in 3xTg-AD mice. The consequences of this serum cytokine profile on the Central Nervous System were distinct, depending on the response evaluated: while HPA axis activation was similar, production of inflammatory cytokines in hippocampus was attenuated. Therefore, in the 3xTg-AD mice, a different inflammatory response to LPS in blood promoted lesser inflammatory mediators production in hippocampus.

No description available.

Basal forebrain cholinergic neurons (BFCNs) are critical for learning and memory. Profound and early BFCN degeneration is a hallmark of aging and Alzheimer’s disease (AD). BFCNs depend for their survival on the retrograde axonal transport of neurotrophins, proteins critical for neuronal function. Neurotrophins like brain derived neurotrophic factor (BDNF) and pro-nerve growth factor (proNGF) are retrogradely transported to BFCNs from their synaptic targets. In AD, neurotrophin levels are increased within BFCN target areas and reduced in the basal forebrain, implicating dysfunctional neurotrophin transport in AD pathogenesis. However, neurotrophin transport within this highly susceptible neuronal population is currently poorly understood. We began by establishing protocols for the accurate quantification of axonal transport in BFCNs using microfluidic culture. We then determined the effect of age on neurotrophin transport. BFCNs were left in culture for up to 3 weeks to model aging in vitro. BFCNs initially displayed robust neurotrophin transport, which diminished with in vitro age. We observed that the levels of proNGF receptor tropomyosin-related kinase-A (TrkA) were reduced in aged neurons. Additionally, neurotrophin transport in BFCNs derived from 3xTg-AD mice, an AD model, was also impaired. Next, we sought to determine a mechanism for these transport deficits. First, we determined that proNGF transport was solely contingent upon the levels of TrkA. We then found that elevation of oxidative stress, an established AD contributor, significantly reduced both TrkA levels and proNGF retrograde transport. TrkA levels are partially regulated by protein tyrosine phosphatase-1B (PTP1B), an enzyme whose activity is reduced by oxidation. PTP1B antagonism significantly reduced TrkA levels and proNGF retrograde transport in BFCNs. Treatment of BFCNs with PTP1B-activating antioxidants rescued TrkA levels, proNGF transport, and proNGF-mediated axonal degeneration. Our results suggest that oxidative stress contributes to BFCN degeneration in aging and AD by impairing retrograde neurotrophin transport via oxidative PTP1B-mediated TrkA loss. / Thesis / Doctor of Philosophy (PhD) / During aging and Alzheimer’s disease (AD), the connections between neurons, a type of brain cell, break down, causing memory loss. This breakdown begins in a brain area called the basal forebrain. Basal forebrain neurons rely upon the transport of nutrients along their connections with other neurons, called axons, for proper function. This transport process becomes impaired in AD. Our goal was to understand why this happens. First, we determined that axonal transport was impaired with age and in basal forebrain neurons of mice genetically predisposed to develop AD. We recreated these impairments by increasing the levels of harmful molecules called reactive oxidative species (ROS). ROS levels increase with age and become abnormally high during AD. We found that increased ROS impair axonal transport and contribute to the breakdown of basal forebrain neurons. Our work suggests that reducing ROS will help prevent the breakdown of basal forebrain neurons in AD.