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Background: In animal models and tissue preparations, calcium dyshomeostasis is a biomarker of aging and Alzheimer’s disease that is associated with synaptic dysfunction, neuritic pruning, and dysregulated cellular processes. It is unclear, however, whether the onset of calcium dysregulation precedes, is concurrent with, or is the product of pathological cellular events (e.g., oxidation, amyloid-β production, and neuroinflammation). Further, neuronal calcium dysregulation is not always present in animal models of amyloidogenesis, questioning its reliability as a disease biomarker. Objective: Here, we directly tested for the presence of calcium dysregulation in dorsal hippocampal neurons in male and female 5×FAD mice on a C57BL/6 genetic background using sharp electrodes coupled with Oregon-green Bapta-1 imaging. We focused on three ages that coincide with the course of amyloid deposition: 1.5, 4, and 10 months old. Methods: Outcome variables included measures of the afterhyperpolarization, short-term synaptic plasticity, and calcium kinetics during synaptic activation. Quantitative analyses of spatial learning and memory were also conducted using the Morris water maze. Main effects of sex, age, and genotype were identified on measures of electrophysiology and calcium imaging. Results: Measures of resting Oregon-green Bapta-1 fluorescence showed significant reductions in the 5×FAD group compared to controls. Deficits in spatial memory, along with increases in Aβ load, were detectable at older ages, allowing us to test for temporal associations with the onset of calcium dysregulation. Conclusion: Our results provide evidence that reduced, rather than elevated, neuronal calcium is identified in this 5×FAD model and suggests that this surprising result may be a novel biomarker of AD.

Neuronal hippocampal Ca

Background: Dysregulated signaling in neurons and astrocytes participates in pathophysiological alterations seen in the Alzheimer’s disease brain, including increases in amyloid-β, hyperphosphorylated tau, inflammation, calcium dysregulation, and oxidative stress. These are often noted prior to the development of behavioral, cognitive, and non-cognitive deficits. However, the extent to which these pathological changes function together or independently is unclear. Objective: Little is known about the temporal relationship between calcium dysregulation and oxidative stress, as some reports suggest that dysregulated calcium promotes increased formation of reactive oxygen species, while others support the opposite. Prior work has quantified several key outcome measures associated with oxidative stress in aldehyde dehydrogenase 2 knockout (Aldh2–/–) mice, a non-transgenic model of sporadic Alzheimer’s disease. Methods: Here, we tested the hypothesis that early oxidative stress can promote calcium dysregulation across aging by measuring calcium-dependent processes using electrophysiological and imaging methods and focusing on the afterhyperpolarization (AHP), synaptic activation, somatic calcium, and long-term potentiation in the Aldh2–/– mouse. Results: Our results show a significant age-related decrease in the AHP along with an increase in the slow AHP amplitude in Aldh2–/– animals. Measures of synaptic excitability were unaltered, although significant reductions in long-term potentiation maintenance were noted in the Aldh2–/– animals compared to wild-type. Conclusion: With so few changes in calcium and calcium-dependent processes in an animal model that shows significant increases in HNE adducts, Aβ, p-tau, and activated caspases across age, the current findings do not support a direct link between neuronal calcium dysregulation and uncontrolled oxidative stress.

As demonstrated by increased hippocampal insulin receptor density following learning in animal models and decreased insulin signaling, receptor density, and memory decline in aging and Alzheimer's diseases, numerous studies have emphasized the importance of insulin in learning and memory processes. This has been further supported by work showing that intranasal delivery of insulin can enhance insulin receptor signaling, alter cerebral blood flow, and improve memory recall. Additionally, inhibition of insulin receptor function or expression using molecular techniques has been associated with reduced learning. Here, we sought a different approach to increase insulin receptor activity without the need for administering the ligand. A constitutively active, modified human insulin receptor (IRβ) was delivered to the hippocampus of young (2 months) and aged (18 months) male Fischer 344 rats in vivo. The impact of increasing hippocampal insulin receptor expression was investigated using several outcome measures, including Morris water maze and ambulatory gait performance, immunofluorescence, immunohistochemistry, and Western immunoblotting. In aged animals, the IRβ construct was associated with enhanced performance on the Morris water maze task, suggesting that this receptor was able to improve memory recall. Additionally, in both age‐groups, a reduced stride length was noted in IRβ‐treated animals along with elevated hippocampal insulin receptor levels. These results provide new insights into the potential impact of increasing neuronal insulin signaling in the hippocampus of aged animals and support the efficacy of molecularly elevating insulin receptor activity in vivo in the absence of the ligand to directly study this process., To assess the impact of increasing insulin signaling on ambulatory performance, spatial memory, and receptor dynamics without the need for intranasal insulin delivery, we expressed a constitutively active insulin receptor (IRβ) in the hippocampus of Fischer 344 rats. In aged animals, IRβ was associated with improved memory performance, higher receptor levels, and increased downstream signaling. This work demonstrates the efficacy of molecularly enhancing receptor activity in vivo.

Glucose uptake in the brain is critically important to brain health. Using two widely used cell line model systems, we have found that siramesine, a lysosomotropic agent and ligand for the sigma-2 receptor, inhibits glucose uptake and decreases pools of the GLUT1 glucose transporter at the plasma membrane. Siramesine induces autophagy but also disrupts degradation of autophagy substrates, providing a potential mechanism for its action on glucose uptake. In other cell systems, many of the effects of siramesine can be suppressed by α -tocopherol, a type of vitamin E and potent antioxidant, and α -tocopherol also suppressed the effect of siramesine on glucose uptake, suggesting a role for reactive oxygen species and membrane maintenance. We have also identified a novel mechanism for siramesine in which it inhibited plasma membrane levels of GAPDH, a key protein in glycolysis which localizes to the plasma membrane in some cell types. Indeed, GAPDH inhibitors decreased glucose uptake, like siramesine, likely through an overlapping pathway with siramesine. GAPDH inhibitors induced autophagy but inhibited degradation of autophagy targets. Thus, we have identified novel mechanisms required for glucose uptake which may have important implications in disease.

The insulin receptor (IR) is a ligand-activated receptor tyrosine kinase that has a key role in metabolism, cellular survival, and proliferation. Progesterone receptor membrane component 1 (PGRMC1) promotes cellular signaling via receptor trafficking and is essential for some elements of tumor growth and metastasis. In the present study, we demonstrate that PGRMC1 coprecipitates with IR. Furthermore, we show that PGRMC1 increases plasma membrane IR levels in multiple cell lines and decreases insulin binding at the cell surface. The findings have therapeutic applications because a small-molecule PGRMC1 ligand, AG205, also decreases plasma membrane IR levels. However, PGRMC1 knockdown via short hairpin RNA expression and AG205 treatment potentiated insulin-mediated phosphorylation of the IR signaling mediator AKT. Finally, PGRMC1 also increased plasma membrane levels of two key glucose transporters, GLUT-4 and GLUT-1. Our data support a role for PGRMC1 maintaining plasma membrane pools of the receptor, modulating IR signaling and function.

Memory and cognitive decline are the product of numerous physiological changes within the aging brain. Multiple theories have focused on the oxidative, calcium, cholinergic, vascular, and inflammation hypotheses of brain aging, with recent evidence suggesting that reductions in insulin signaling may also contribute. Specifically, a reduction in insulin receptor density and mRNA levels has been implicated, however, overcoming these changes remains a challenge. While increasing insulin receptor occupation has been successful in offsetting cognitive decline, alternative molecular approaches should be considered as they could bypass the need for brain insulin delivery. Moreover, this approach may be favorable to test the impact of continued insulin receptor signaling on neuronal function. Here we used hippocampal cultures infected with lentivirus with or without IRβ, a constitutively active, truncated form of the human insulin receptor, to characterize the impact continued insulin receptor signaling on voltage-gated calcium channels. Infected cultures were harvested between DIV 13 and 17 (48 hrs after infection) for Western blot analysis on pAKT and AKT. These results were complemented with whole-cell patch-clamp recordings of individual pyramidal neurons starting 96 hrs post-infection. Results indicate that while a significant increase in neuronal pAKT/AKT ratio was seen at the time point tested, effects on voltage-gated calcium channels were not detected. These results suggest that there is a significant difference between constitutively active insulin receptors and the actions of insulin on an intact receptor, highlighting potential alternate mechanisms of neuronal insulin resistance and mode of activation.

OBJECTIVES: Increasing evidence suggests that vitamin D plays a role in maintaining cognitive function and that vitamin D deficiency may accelerate age-related cognitive decline. Here, we determined if a long-term enhanced vitamin D (VitD3, cholecalciferol) diet, higher than the standard dietary level, maintains or improves cognitive function in aging male and female rats. We also examined if the high VitD3 diet affected the gut microbiome. METHODS: Beginning at 12 months of age 20 male and 20 female F344 rats were fed an AIN-93 diet containing either standard (1000 IU/kg diet) or higher (10,000 IU/kg) VitD3 for 6 months. The Morris water maze (MWM) was then used to assess learning and memory. Following the MWM, the gut microbiome from undigested chyme collected from the intestinal cecum was identified and taxonomically classified by Argonne National Laboratory using 16S rRNA sequences. ZRT Laboratory determined 25-(OH)VitD3 levels from cardiac blood. A two-way ANOVA and Tukey post-hoc was used to test for statistical significance. RESULTS: After 3 days of training the probe test on day 4 indicated that the higher VitD3 diet significantly reduced path length and latency (P = 0.01) to the digital platform in females but not males. On day 5 platform location was changed and animals received one day of reversal training. On day 8, three days after reversal training, the reversal probe indicated that higher dietary VitD3 improved performance in males but not females by significantly reducing path length and latency to the digital platform (P

Intranasal insulin is a safe and effective method for ameliorating memory deficits associated with pathological brain aging. However, the impact of different formulations and the duration of treatment on insulin’s efficacy and the cellular processes targeted by the treatment remain unclear. Here, we tested whether intranasal insulin aspart, a short-acting insulin formulation, could alleviate memory decline associated with aging and whether long-term treatment affected regulation of insulin receptors and other potential targets. Outcome variables included measures of spatial learning and memory, autoradiography and immunohistochemistry of the insulin receptor, and hippocampal microarray analyses. Aged Fischer 344 rats receiving long-term (3 months) intranasal insulin did not show significant memory enhancement on the Morris water maze task. Autoradiography results showed that long-term treatment reduced insulin binding in the thalamus but not the hippocampus. Results from hippocampal immunofluorescence revealed age-related decreases in insulin immunoreactivity that were partially offset by intranasal administration. Microarray analyses highlighted numerous insulin-sensitive genes, suggesting insulin aspart was able to enter the brain and alter hippocampal RNA expression patterns including those associated with tumor suppression. Our work provides insights into potential mechanisms of intranasal insulin and insulin resistance, and highlights the importance of treatment duration and the brain regions targeted.

Novel therapies have turned to delivering compounds to the brain using nasal sprays, bypassing the blood brain barrier, and enriching treatment options for brain aging and/or Alzheimer's disease. We conducted a series of in vivo experiments to test the impact of intranasal Apidra, a zinc-free insulin formulation, on the brain of young and aged F344 rats. Both single acute and repeated daily doses were compared to test the hypothesis that insulin could improve memory recall in aged memory-deficient animals. We quantified insulin signaling in different brain regions and at different times following delivery. We measured cerebral blood flow (CBF) using MRI and also characterized several brain metabolite levels using MR spectroscopy. We show that neither acute nor chronic Apidra improved memory or recall in young or aged animals. Within 2 hours of a single dose, increased insulin signaling was seen in ventral areas of the aged brains only. Although chronic Apidra was able to offset reduced CBF with aging, it also caused significant reductions in markers of neuronal integrity. Our data suggest that this zinc-free insulin formulation may actually hasten cognitive decline with age when used chronically.

It has been >20 years since studies first revealed that the brain is insulin sensitive, highlighted by the expression of insulin receptors in neurons and glia, the presence of circulating brain insulin, and even localized insulin production. Following these discoveries, evidence of decreased brain insulin receptor number and function was reported in both clinical samples and animal models of aging and Alzheimer's disease, setting the stage for the hypothesis that neuronal insulin resistance may underlie memory loss in these conditions. The development of therapeutic insulin delivery to the brain using intranasal insulin administration has been shown to improve aspects of memory or learning in both humans and animal models. However, whether this approach functions by compensating for poorly signaling insulin receptors, for reduced insulin levels in the brain, or for reduced trafficking of insulin into the brain remains unclear. Direct measures of insulin's impact on cellular physiology and metabolism in the brain have been sparse in models of Alzheimer's disease, and even fewer studies have analyzed these processes in the aged brain. Nevertheless, recent evidence supports the role of brain insulin as a mediator of glucose metabolism through several means, including altering glucose transporters. Here, we provide a review of contemporary literature on brain insulin resistance, highlight the rationale for improving memory function using intranasal insulin, and describe initial results from experiments using a molecular approach to more directly measure the impact of insulin receptor activation and signaling on glucose uptake in neurons.

Both insulin signaling disruption and Ca 2+ dysregulation are closely related to memory loss during aging and increase the vulnerability to Alzheimer’s disease (AD). In hippocampal neurons, aging-related changes in calcium regulatory pathways have been shown to lead to higher intracellular calcium levels and an increase in the Ca 2+ -dependent afterhyperpolarization (AHP), which is associated with cognitive decline. Recent studies suggest that insulin reduces the Ca 2+ -dependent AHP. Given the sensitivity of neurons to insulin and evidence that brain insulin signaling is reduced with age, insulin-mediated alterations in calcium homeostasis may underlie the beneficial actions of insulin in the brain. Indeed, increasing insulin signaling in the brain via intranasal delivery has yielded promising results such as improving memory in both clinical and animal studies. However, while several mechanisms have been proposed, few have focused on regulation on intracellular Ca 2+ . In the present study, we further examined the effects of acute insulin on calcium pathways in primary hippocampal neurons in culture. Using the whole-cell patch-clamp technique, we found that acute insulin delivery reduced voltage-gated calcium currents. Fura-2 imaging was used to also address acute insulin effects on spontaneous and depolarization-mediated Ca 2+ transients. Results indicate that insulin reduced Ca 2+ transients, which appears to have involved a reduction in ryanodine receptor function. Together, these results suggest insulin regulates pathways that control intracellular Ca 2+ which may reduce the AHP and improve memory. This may be one mechanism contributing to improved memory recall in response to intranasal insulin therapy in the clinic.

Neuroscientists studying normal brain aging, spinal cord injury, Alzheimer's disease (AD) and other neurodegenerative diseases have focused considerable effort on carefully characterizing intracellular perturbations in calcium dynamics or levels. At the cellular level, calcium is known for controlling life and death and orchestrating most events in between. For many years, intracellular calcium has been recognized as an essential ion associated with nearly all cellular functions from cell growth to degeneration. Often the emphasis is on the negative impact of calcium dysregulation and the typical worse-case-scenario leading inevitably to cell death. However, even high amplitude calcium transients, when executed acutely, can alter neuronal communication and synaptic strength in positive ways, without necessarily killing neurons. Here, we focus on the evidence that calcium has a subtle and distinctive role in shaping and controlling synaptic events that underpin neuronal communication and that these subtle changes in aging or AD may contribute to cognitive decline. We emphasize that calcium imaging in dendritic components is ultimately necessary to directly test for the presence of age- or disease-associated alterations during periods of synaptic activation.