Browsing by Subject "Alzheimer's Disease"
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Item A DEPRESSIVE SUBGROUP OF MILD COGNITIVE IMPAIRMENT(2013-04-12) Cushing, BlairPurpose: Late life depression is a risk factor for Mild Cognitive Impairment (MCI) and Alzheimer's Disease (AD). MCI represents a transitional period between normal aging and AD and, therefore, represents a potential entry point for preventing AD. Depression is a risk factor for MCI and AD; however, identifying which MCI patients suffer depression-related cognitive impairment remains difficult. The current study sought to identify a clinically-defined depressed sub group among patients with MCI Methods: Data was analyzed from 519 participants (112 MCI) from Project FRONTIER, an ongoing epidemiological study of factors impacting rural aging and health. Depression was assessed with GDS30 and cognition was assessed using the EXIT 25 and RBANS. The sample was randomly split into the training and test sample. Five GDS items were identified in the training sample that were significantly associated with MCI status and were used to create the depressive endophenotype (DepE) of MCI. In the test sample, linear regression was used to examine the impact of depression on neuropsychological tests performance, and logistic regression was conducted to examine the risk of being diagnosed with MCI. Results: In the test sample, DepE was negatively related to RBANS scores of Immediate Memory (B=-2.22, p<0.001), Visuospatial (B=-1.11, p<0.001), Language (B=-1.03, p<0.001), Attention (B=-2.56, p<0.001), and Delayed Memory (B=-1.54, p<0.001), and higher DepE scores were related to poorer executive functioning (EXIT25; B=0.65, p=0.001). DepE scores significantly increased risk for MCI ([OR]= 2.04; 95% CI=l.54-2.69). Conclusions: These findings suggest that a depressed subgroup of MCI exists. Higher DepE scores increased risk for MCI and increased risk for poorer neuropsychological functioning across a range of domains. The DepE may identify those MCI patients who experience depression-related cognitive dysfunction, thereby detecting a selective group that may benefit from depression treatment for prevention of AD.Item BIOPHYSICAL STUDIES OF MEMBRANE TOPOLOGY OF HUMAN PRESENELIN-1(2013-04-12) Midde, KrishnaPurpose: Alzheimer's disease (AD) afflicts ~5 million Americans. Amyloid (Abeta) plaques accumulate in the brain of patients during AD leading to neurodegeneration. Presenelin-1 (PS1), a trans-membrane protein, acts as the catalytic subunit of gamma-secretase enzyme to cleave amyloid precursor protein (APP) and produce Abeta peptides. Mutations in the PS1 gene have been linked to the pathogenesis of early onset of familial AD (FAD). Recently, it has been reported that PS1 also acts as Ca2+ leak channel on the membrane of endoplasmic reticulum (ER). Currently there are divergent views in the literature on the subunit association and the correct membrane topology of PS1 protein. Considering the several physiological functions and the critical role of PS1 in the pathogenesis of AD, we sought to investigate the membrane topology of PS1 protein through various biophysical studies. Methods: Neuroblastoma (SK-N-SH) cells expressing PS1 protein with NH2-terminal tagged yellow fluorescent protein (YFP-PS1) and COOH-terminal tagged cyano fluorescent protein (PS1-CFP) were used as a model in our studies. Membrane localization and subunit association of PS1 were determined by biophysical assays. Expression and colocalization of YFP-PS1 and PS1-CFP proteins were assessed by confocal imaging. Localization of the N-terminal and C-terminal of PS1 was assessed by fluorescence correlation spectroscopy (FCS). Finally subunit aggregation of PS1 protein was determined by Forster Resonance Energy Transfer (FRET) assay. Results: When cells expressing both PS1-CFP and YFP-PS1 proteins were independently excited and imaged, their respective fluorescence overlapped suggesting co-expression and co-localization of the PS1 subunits. The diffusion coefficient of the PS1 protein in the transmembrane was the same (~0.15µm2/s) when FCS was measured in cells with and without 80% glycerol in 1XPBS indicating that the NH2 and COOH termini are facing the cytosolic side of the plasma-membrane. The quench in the fluorescence lifetime of CFP (donor) in the presence of YFP (acceptor) in FRET assay demonstrates the protein-protein interaction between PS1-CFP and YFP-PS1. Conclusions: Both YFP-PS1 and PS1-CFP chimeric proteins are expressed on the plasma membrane and intracellular membranes with NH2-terminal and COOH-terminal oriented towards the cytosolic side of the membrane. PS1 is a dynamic transmembrane protein which associates as dimer or multimer to form ERCa2+ channel and thus regulates intracellular calcium signaling.Item IN VITRO MODEL FOR ALZHEIMER'S DISEASE DRUG DISCOVERY AND DEVELOPMENT FOCUSING ON OXIDATIVE-NITROSATIVE PATHWAYS(2013-04-12) Wesp, KrystynaPurpose: The Alzheimer's Association estimates that in the absence of disease-modifying treatments the total costs of care for individuals with Alzheimer's disease by all payers will soar from about $170 billion today to more than $1 trillion in 2050. The goal of our project is to develop an in vitro model for testing drugs capable of preventing or slowing the progression of Alzheimer's disease (AD). Previous studies have demonstrated that oxidative-nitrosative stress is an important trigger for Alzheimer's disease and other neurodegenerative disorders. Different nitrosative mechanisms have been proposed for the pathology of this disease, including 3-nitrotyrosination of proteins. Nitric oxide synthase (NOS) is an enzyme that forms nitric oxide. Though important for normal brain function, nitric oxide can react with superoxide formed under oxidative stress conditions to create peroxynitrite, a damaging chemical species that reacts with tyrosine residues to produce aberrant 3-nitrotyrosinated proteins. Recent research has shown certain receptors can modulate NOS activity, but further experiments need to be done to confirm the clinical effectiveness of targeting such receptors with selective drugs as a new therapeutic approach to treating AD and other brain injury states. Our current research is aimed at establishing an in vitro model containing all the necessary elements of the oxidative-nitrosative stress signaling pathway for future drug testing in a cellular model. Methods: Towards this end we have begun to identify relevant pathway elements in a number of cell lines utilizing polymerase chain reaction (PCR) techniques to amplify RNA coding for these elements. This was achieved by culturing cells, extracting purified RNA and reverse transcribing it into DNA and then amplifying the DNA with oligonucleotide primers designed to target specific pathway components (e.g. NOS). Amplified PCR products were confirmed by size determination utilizing agarose gel DNA electrophoresis and DNA sequencing of the correctly-sized bands. Results: Three cell lines, including a neuronal and a glial cell line, have been identified so far that contain a number of the critical elements required for the in vitro model. Conclusions: The identification of a viable in vitro model will allow for further experimentation that can determine the effectiveness of new drugs acting on specific receptor targets to modulate NOS activity and thereby slow the progression of diseases with a nitrosative stress component.Item Presenilins Modulate Cellular Activity of Ryanodine Receptors(2012-12-01) Payne, Andrew J.; Peter KoulenPayne, Andrew J., Presenilins Modulate Cellular Activity of Ryanodine Receptors. Doctor of Philosophy (Biomedical Sciences), December, 2012, 160 pp., 7 tables, 39 figures, bibliography 241 titles. Ryanodine Receptors (RyRs) are large, endoplasmic reticulum (ER) intracellular calcium channels in excitable cells. RyRs are major cellular mediators of calcium-induced calcium release and crucial regulators of intracellular calcium homeostasis. Disruption of RyR function has been described in pathologies of dysregulated calcium such as Alzheimer’s disease (AD). Presenilins (PS1 and PS2) are ER transmembrane proteins expressed in the central nervous system mediating calcium homeostasis and Notch signaling, and act as the proteolytic core of γ-secretase in amyloid cleavage. Previous single channel electrophysiology studies described a direct interaction between RyR and the N-termini of presenilin 1 (PS1NTF) and presenilin 2 (PS2NTF) that resulted in differential modulation of the RyR open probability and mean Ca2+ current at the RyR single channel level. We herein tested the hypothesis that PS1NTF and PS2NTF functionally modulate RyRs in a physiologically relevant in vitro model resulting in changes to RyR-mediated intracellular calcium release. Confocal microscopy, microfluorimetry, and coimmunoprecipitation studies confirmed a physical interaction between RyRs and PS-NTFs in human neuroblastoma SH-SY5Y cells, an in vitro AD model. Live cell fluorescent calcium imaging was used to quantify the effects of overexpression of PS1NTF or PS2NTF on Ca2+ release from RyR mediated stores. PS1NTF was found to increase RyR gating to the full open state at physiologically normal calcium concentrations. Verifying the previous electrophysiology data, PS2NTF had no effect on RyR at physiological calcium concentrations. Mutagenesis of critical cysteine residues on the PSNTFs was applied to determine the effect of specific structure-function differences between PS1 and PS2 molecules that underlie the isoform specific modulation of RyR calcium release. Mutations to PS2NTF removing disulfide bridging cysteines recapitulated PS1NTF-like regulation of RyR Ca2+ release. Our findings indicate that PS1NTF and PS2NTF bind RyRs differentially. Our results indicate a novel mechanism of intracellular calcium regulation by the PS-RyR interaction and a novel target for the treatment of AD, neurodegenerative disorders, and diseases controlled by RyR and PS functions.Item REELIN SIGNAL TRANSDUCTION PATHWAY IN APOE3 AND APOE4 TRANSGENIC MICE(2013-04-12) Dugal, MariceliePurpose: The ɛ4 allele of apolipoprotein E (APOE) has been associated with increased risk for the development of late-onset, familial and sporadic Alzheimer's disease (AD). The mechanisms underlying the increase risk of AD development conferred by the ɛ4 allele remains unclear. Reelin and its associated signal transduction pathway are involved in developmental processes, and more specifically, in regulating neuronal migration and cortical lamination in the embryotic brain. Recently, it has been determined that reelin is present in the adult brain throughout the neocortex and hippocampus suggesting a potential role in synaptic plasticity. Furthermore, studies have shown that disruption of the reelin pathway led to decreased memory, impaired long-term potentiation (LTP), and affected dendritic spine morphology. This preliminary study investigated the role of the reelin pathway as a potential mechanism underlying the functional declines associated with APOE polymorphism. Methods: Separate groups of young (7 months) male and female mice expressing human apolipoprotein E4 or E3 in glial cells) were subjected to a series of behavioral tests to measure spontaneous activity, reflexes (walking initiation, alley turn, and negative geotaxis), motor function (wire suspension, bridge walking, coordinated running), and cognitive function (spatial water maze, active avoidance). Brain regions were dissected to determine the levels of reelin and other contributors of its pathway such as dab1, fyn, AMPA and NR2A via western blot analyses. Results: ApoE3 mice took longer latencies to fall in bridge walking, wire suspension, and coordinated running tasks than their ApoE4 counterparts, most notably in males. In the active avoidance task, ApoE3 female mice took fewer trials to reach criterion in session 3 over their ApoE4 counterparts. Western blot analyses will reveal whether reelin may underlie the differences in brain function between genotype and sex. Conclusions: Our results indicate that there may be functional differences between sex as well as ApoE polymorphism. Western blot analyses will reveal whether reelin, dab1, fyn, AMPA and NR2A may underlie the differences in brain function between genotype and sex.Item The Intersection of Type 2 Diabetes and Cognitive Impairment in Mexican Americans: Insights from the Mitochondria(2018-05) Silzer, Talisa K.; Phillips, Nicole R.; Barber, Robert C.; Singh, Meharvan; Maddux, Scott D.Mitochondrial dysfunction is common in numerous complex age-related diseases. The role of mitochondrial dysfunction in cognitive impairment has yet to be studied in Mexican American populations. This population serves as a group of interest due to their high prevalence of type 2 diabetes (T2D). T2D is known to be associated with cognitive impairment (CI), suggesting that Mexican American populations may be at greater risk for CI. In this study, mtDNA copy number (mtDNACN), cell-free mtDNA (CFmtDNA) levels and mitochondrial gene expression was assessed. MtDNACN was found to be decreased in CI, while CFmtDNA was found to be elevated in T2D. Cross-condition comparisons of mitochondrial expression elucidated characteristic expression profiles perhaps associated with the co-occurrence of T2D and CI pathology.