Overexpression of pyruvate dehydrogenase kinase 1 and lactate dehydrogenase A in nerve cells confers resistance to amyloid β and other toxins by decreasing mitochondrial respiration and reactive oxygen species production

Background: Aerobic glycolysis promotes resistance against Aβ toxicity. Results: Increased LDHA and PDK1 expression attenuates mitochondrial activity and confers resistance to Aβ. These proteins are down-regulated in a transgenic Alzheimer disease (AD) mouse model, and PDK1 is decreased in AD brain. Conclusion: PDK and LDHA are central mediators of Aβ resistance. Significance: Drugs that augment aerobic glycolysis may enhance brain cell survival in AD patients. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Attenuation of oxidative stress in HEK 293 cells by the TCM constituents schisanhenol, baicalein, resveratrol or crocetin and two defined mixtures

PURPOSE: Our working hypothesis is that single bioactive phytochemicals with antioxidant properties that are important constituents of Traditional Chinese Medicine (TCM) and their defined mixtures have potential as chemoprotective agents for chronic conditions characterized by oxidative and nitrosative stress, including Alzheimer’s. Here we evaluate the ability of baicalein, crocetin, trans-resveratrol or schisanhenol and two defined mixtures of these TCM phytochemicals to attenuate the toxicity resulting from exposure to cell permeant t-butyl hydroperoxide (tBPH) in wild-type and bioengineered (to express choline acetyltransferase) HEK 293 cells. METHODS: Endpoints of tBHP-initiated oxidative and nitrosative stress in both types of HEK 293 cells and its attenuation by TCM constituents and mixtures included cytotoxicity (LDH release); depletion of intracellular glutathione (GSH); formation of S-glutathionylated proteins; oxidative changes to the disulfide proteome; and real-time changes in intracellular redox status. RESULTS: At low µM concentrations, each of the TCM constituents and mixtures effectively attenuated intracellular toxicity due to exposure of HEK 293 cells to 50 or 250 µM tBHP for 30 min to 3 h. Confocal microscopy of HEK 293 cells transfected with mutated green fluorescent protein (roGFP2) showed effective attenuation of tBHP oxidation by baicalein in real time. Three redox-regulated proteins prominent in the disulfide proteome of HEK 293 cells were identified by MALDI-TOF mass spectrometry. CONCLUSIONS: We conclude that single TCM chemicals and their simple mixtures have potential for use in adjunct chemoprotective therapy. Advantages of mixtures compared to single TCM constituents include the ability to combine compounds with varying molecular mechanisms of cytoprotection for enhanced biological activity; and to combine chemicals with complementary pharmacokinetic properties to increase half-life and prolong activity in vivo

Mobility and Cognition in Seniors. Report from the 2008 Institute of Aging (CIHR) Mobility and Cognition Workshop

Background The annual Scientific Meeting of the Canadian Association on Gerontology was held on October 24 and 25, 2008 in London, Ontario. Prior to the annual meeting, mobility and cognition experts met on October 23, 2008 to engage in a pre-conference workshop. Methods Discussions during the workshop addressed novel areas of research and knowledge and research gaps pertaining to the interaction between mobility and cognition in seniors. Results Workshop presenters moved from the neuromuscular, biomechanics, and neurology of gait impairments, and falls through the role of cognition and mood on mobility regulation to the whole person in the environment. Research gaps were identified. Conclusions Despite a consensus that mobility and cognition are increasingly correlated as people age, several gaps in our understanding of mechanisms and how to assess the interaction were recognized. The gaps originally identified in 2008 are still pertinent today. Common and standardized assessments for “mobility and cognition” are still not in place in current practice. Interventions that target mobility and cognitive decline as a single entity are still lacking

The Comprehensive Assessment of Neurodegeneration and Dementia: Canadian Cohort Study.

BackgroundThe Comprehensive Assessment of Neurodegeneration and Dementia (COMPASS-ND) cohort study of the Canadian Consortium on Neurodegeneration in Aging (CCNA) is a national initiative to catalyze research on dementia, set up to support the research agendas of CCNA teams. This cross-country longitudinal cohort of 2310 deeply phenotyped subjects with various forms of dementia and mild memory loss or concerns, along with cognitively intact elderly subjects, will test hypotheses generated by these teams.MethodsThe COMPASS-ND protocol, initial grant proposal for funding, fifth semi-annual CCNA Progress Report submitted to the Canadian Institutes of Health Research December 2017, and other documents supplemented by modifications made and lessons learned after implementation were used by the authors to create the description of the study provided here.ResultsThe CCNA COMPASS-ND cohort includes participants from across Canada with various cognitive conditions associated with or at risk of neurodegenerative diseases. They will undergo a wide range of experimental, clinical, imaging, and genetic investigation to specifically address the causes, diagnosis, treatment, and prevention of these conditions in the aging population. Data derived from clinical and cognitive assessments, biospecimens, brain imaging, genetics, and brain donations will be used to test hypotheses generated by CCNA research teams and other Canadian researchers. The study is the most comprehensive and ambitious Canadian study of dementia. Initial data posting occurred in 2018, with the full cohort to be accrued by 2020.ConclusionAvailability of data from the COMPASS-ND study will provide a major stimulus for dementia research in Canada in the coming years

Into the Fourth Dimension: Dysregulation of Genome Architecture in Aging and Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by synapse dysfunction and cognitive impairment. Understanding the development and progression of AD is challenging, as the disease is highly complex and multifactorial. Both environmental and genetic factors play a role in AD pathogenesis, highlighted by observations of complex DNA modifications at the single gene level, and by new evidence that also implicates changes in genome architecture in AD patients. The four-dimensional structure of chromatin in space and time is essential for context-dependent regulation of gene expression in post-mitotic neurons. Dysregulation of epigenetic processes have been observed in the aging brain and in patients with AD, though there is not yet agreement on the impact of these changes on transcription. New evidence shows that proteins involved in genome organization have altered expression and localization in the AD brain, suggesting that the genomic landscape may play a critical role in the development of AD. This review discusses the role of the chromatin organizers and epigenetic modifiers in post-mitotic cells, the aging brain, and in the development and progression of AD. How these new insights can be used to help determine disease risk and inform treatment strategies will also be discussed

Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT.

Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer's Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization

Dynamics of CHT internalization are altered by acute insulin treatment.

<p>Cells were grown for 24 h with the addition of either vehicle (water) or 20 nM insulin. To facilitate TIRF microscopy, FLAG-CHT was labeled by 20 min incubation at 37°C with AlexaFluor 555-labeled rabbit anti-FLAG antibody, then the cells were washed 3 times with HBSS at the end of this period to remove background non-specific labeling. The initial experiments tested the ability of either acute vehicle or insulin addition to alter total cell fluorescence levels related to the movement of CHT proteins to the cell surface; neither of these treatments caused significant changes in fluorescence levels. <b>Panel A.</b> Bright-field and fluorescence images of cells prior to and after KCl addition with scale bar indicating 20 μm. The imaging protocol was as follows: the culture dish was set on the stage of the TIRF microscope, either insulin or vehicle were added and imaging was started for 30–60 sec, then a small volume of 1 M KCl was applied to the area of the cells by pressure ejection and imaging was continued for a further 3–4 min. Images were captured at 150 nm depth from the coverslip, and experiments were performed at room temperature to reduce the rate of cellular trafficking events being imaged. <b>Panel B.</b> Representative traces of changes in the cellular fluorescence levels over the time course of imaging with various treatments, as indicated. The arrows indicate when KCl was applied to the cells. <b>Panel C.</b> Schematic representation indicating analysis of changes in cell fluorescence in live cells in <b>Panels D – G. Panel D.</b> Area-under-the-curve (AUC) analysis of fluorescence images calculated as the sum of fluorescence values between t1 and t3, analyzed using GraphPad Prism v.5.0. <b><i>Panel E</i>.</b> Percentage change in cell surface fluorescence levels following K⁺-depolarization levels, calculated using the formula [(f2-f1)/((f1+f2)/2)*100)]. <b>Panel F.</b> Time-to-peak value calculated as the time at point t2 minus that at point t1. <b>Panel G.</b> Time taken for cell fluorescence to return to baseline values calculated as time point t3 minus time point t2. Typically 2 or 3 regions of interest having high fluorescence corresponding to 2 to 3 individual cells were chosen for analysis per culture dish, with 4 to 5 dishes analyzed per treatment group in 2 or 3 independent experiments. Data in Panels D to G are expressed as the mean ± SEM, with statistically-significant differences assessed by unpaired Student’s <i>t</i>-test; asterisks denote <i>p</i> ≤ 0.05 compared to cells grown in control conditions (absence of 24 h treatment with insulin) and acutely stimulated with insulin.</p

Dynamics of CHT trafficking are altered by LY294002 treatment.

<p>Cells were grown for 24 h in the presence of either vehicle or 10 μM LY294002 before being imaged by TIRF microscopy. CHT proteins were fluorescently labeled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. The imaging protocol was as follows: the dish was set on the stage of the TIRF microscope and imaging was started for 30–60 sec, then a small volume of 2.5 M KCl was applied to the area of the cells by pressure ejection and imaging was continued for a further 3–4 min. Images were captured at 150 nm depth from the coverslip, and experiments were performed at room temperature. <b>Panel A.</b> Representative traces of changes in the cellular fluorescence levels over the time course of imaging with various treatments, as indicated. The arrows indicate when KCl was applied to the cells. <b>Panel B.</b> Area-under-the-curve (AUC) analysis of fluorescence images was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. <b>Panel C.</b> Percentage changes in cell surface fluorescence levels following K⁺-depolarization levels calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. <b>Panel D.</b> Time-to-peak value calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. <b>Panel E.</b> Time taken for cell fluorescence to return to baseline values calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. In these experiments, 1 to 5 regions of interest having high fluorescence corresponding to 1 to 5 individual cells were analyzed per culture dish, with 4 to 5 dishes analyzed per treatment group in 3 independent experiments. Data in Panels B to E are expressed as the mean ± SEM, with statistically-significant differences assessed by unpaired Student’s <i>t</i>-test; asterisks denote <i>p</i> ≤ 0.05.</p