Approximately 5 million Americans currently suffer from Alzheimer's disease (AD) a neurodegenerative disorder characterized by progressive impairment of cognitive function and emotional and sleep disturbances. This laboratory has developed cell culture and mouse models of AD, and have used these models to elucidate the biochemical and molecular events responsible for neuronal dysfunction and death in AD. --Pathogenic Mechanism of Presenilin-1 Mutations and Amyloid beta-Peptide: Mutations in the presenilin-1 (PS1) gene cause some cases of early-onset inherited AD. We have shown that PS1 mutations perturb cellular Ca(2+) homeostasis and thereby render neurons vulnerable to excitotoxicity and apoptosis. Our findings suggest that the overfilling of calcium stores represents the fundamental cellular defect underlying the alterations in calcium signaling conferred by presenilin mutations. We further discovered that PS1 mutations have an adverse effect on oligodendrocytes, the cells that wrap around axons to form myelin sheaths that insulate the axons and thereby facilitate the propagation of electrical impulses. Studies of PS1 mutant mice exposed to a demyelinating toxin showed that the increased degeneration of oligodendrocytes results in learning and memory impairment, suggesting a role for white matter damage in the pathogenesis of AD. In other studies we have identified novel functions for a protein called Numb in neurons that may be relevant to AD pathogenesis. We found that Numb isoforms containing a short phosphotyrosine-binding domain increase the vulnerability of neurons to death induced by Abeta1-42 by a mechanism involving dysregulation of cellular calcium homeostasis. --Endoplasmic Reticulum (ER) Stress Responses: We found that PS1 mutations cause a marked increase in basal protein levels of the pro-apoptotic transcription factor Gadd153. PS1 mutations increase Gadd153 protein translation without affecting mRNA levels, while decreasing levels of the anti-apoptotic protein Bcl-2. Moreover, an exaggerated Gadd153 response to ER stress (exposure to tunicamycin or thapsigargin) was observed in PS1 mutant cells. Cell death in response to ER stress was enhanced by PS1 mutations, and this endangering effect was attenuated by antisense-mediated suppression of Gadd153 production. An abnormality in the translational regulation of Gadd153 may sensitize cells to the detrimental effects of ER stress and contribute to the pathogenic actions of PS1 mutations. We identified a novel protein called Herp (homocysteine-inducible endoplasmic reticulum protein) that modifies ER calcium homeostasis. Overexpression of Herp protects cultured neural cells against ER stress-induced death by a mechanism involving stabilization of intracellular calcium levels and preservation of mitochondrial function. Our data suggest that Herp plays a critical role in maintaining the functions of ER and mitochondria during ER stress, and thereby promotes neuronal survival. --A New Mouse Model of AD: Mutations in the amyloid precursor protein (APP) and PS1 cause early-onset AD, while mutations in the microtubule-associated protein tau cause frontotemporal lobe dementia. We have developed a new approach for creating transgenic mice that express more than one mutation. We injected plasmids containing mutant APP and tau genes into one cell stage embryos of PS1 mutant knockin mice. The resulting embryos were viable and grew to adulthood with no apparent abnormalities. All three mutant genes were expressed in neurons in the brain. The mice developed age-related deposition of amyloid which occurred first within neurons in the hippocampus and cerebral cortex, with extracellular deposits of amyloid and tau pathology occurring subsequently. The APP/PS1/tau triple-mutant mice exhibit synaptic dysfunction and learning and memory impairment that occurs soon after intracellular deposits of amyloid are detected.
We aim to use these mice to study the pathogenesis of AD, and to develop novel approaches for preventing and treating AD. --Folic Acid and Homocysteine: We have found that folic acid can protect neurons from being damaged and killed in experimental models of AD. We have further discovered that folate deficiency results in elevated levels of homocysteine in the brain, and that homocysteine can damage and kill hippocampal neurons by promoting DNA damage.. Homocysteine markedly increases the vulnerability of hippocampal neurons to excitotoxic and oxidative injury in cell culture and in vivo, suggesting a mechanism by which homocysteine may contribute to the pathogenesis of neurodegenerative disorders. Our findings suggest that eating foods that contain folic acid and/or taking a folic acid supplement may reduce the risk of AD, a possibility supported by recent epidemiological studies. --Neuroprotection by Dietary Restriction: We have found that dietary restriction reduces damage to neurons and improves functional outcome in animal models of AD. PS1 mutant mice exhibit increased resistance of hippocampal neurons to excitotoxic injury and reduced memory impairment when they are maintained on a dietary restriction regimen. Interestingly, we have also found that dietary restriction can stimulat neurogenesis, the production of new neurons from neural stem cells. This suggests the possibility that dietary restriction can promote the replacement of damaged neurons. We have further discovered that dietary restriction increases production of a neuronal growth factor called BDNF (brain-derived neurotrophic factor) which appears to play an important role in the beneficial effects of dietary restriction. --Development of Drugs to Prevent Neuronal Death in Alzheimer's Disease: We have shown that supplementing the diets of rats or mice with 2-deoxy-D-glucose (2DG), a nonmetabolizable glucose analog that induces metabolic stress, increases the resistance of neurons in the brain to excitotoxic, ischemic, and oxidative injury. In other studies we synthesized the compound pifithrin-alpha and related analogs and evaluated their ability to inhibit the death protein p53 and to protect neurons. These preclinical studies demonstrate the efficacy of novel p53 inhibitor in models of Alzheimer's disease and stroke, and suggest that drugs that inhibit p53 may reduce the extent of brain damage in related human patients. We are currently working on three additional drug development projects centered on the agents creatine, uric acid and myriocin, each of which exhibits neuroprotective activities in cell culture models. Leading agents identified in cell culture screens will be tested in a novel APP/PS1/tau triple-mutant mouse model of AD --Impairment of Neurogenesis in AD: We have begun to test the hypothesis that neurogenesis is impaired in AD. The proliferation and survival of NPC in the dentate gyrus was reduced in a transgenic mouse model (APP mutant mice expressing human APP harboring the FAD Swedish mutation) of AD. Subtoxic levels of Abeta impaired the proliferation and differentiation of cultured human and rodent NPC into neurons, and higher concentrations can induce apoptosis of neuron-restricted NPC by a mechanism involving dysregulation of cellular calcium homeostasis and the activation of calpains and caspases. The proliferation and migration of NPC in the SVZ of amyloid precursor protein mutant mice, and in mice receiving an intraventricular infusion of Ab, were greatly decreased compared to control mice. Our data show that Ab can impair cortical neurogenesis, and suggest that this adverse effect of Ab contributes to the depletion of neurons and the resulting olfactory and cognitive deficits in AD.
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