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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.

Tetraspanin CD151 is increasingly implicated as a multifaceted mediator of cancer development and progression. Here we investigated the role of CD151 in breast cancer in the context of the Wnt oncogenic activation. Our data showed that removal of one or both of CD151 alleles in the MMTV-Wnt1 model significantly decreased the tumor-free survival of mice from 34 weeks on average to 22 weeks and 18 weeks, respectively. This effect coincided with an accelerated tumor growth and an increased number of Ki-67+ proliferative cells. Mechanistically, the CD151-deficient tumors were largely ER+, and exhibited hyperactivation of the Wnt pathway as reflected by a marked upregulation in β-catenin and Cyclin D1, and their target genes. In addition, E-cadherin displayed a cytosolic distribution and transcription factor Snail was markedly upregulated. Collectively, this data implies that CD151 suppresses the Wnt1-driven tumorigenesis, at least in part, via counteracting the epithelial-mesenchymal transition (EMT)-like program in luminal epithelial cells. Meanwhile, the proportion of tumor cells expressing CK5 or p63, the biomarkers of myoepithelial/basal cells, markedly decreased in the absence of CD151. This change was accompanied by a decreased invasiveness of tumors and their incompetence to form a long-term cell culture. Consistent with this basal cell-linked role, the CD151 downregulation impairs mammosphere formation in MCF-10A cells and the defect was rescued by re-expression of intact CD151 ORF, but not its integrin binding-defective mutant. Overall, our study suggests that CD151 is a key player in the Wnt oncogene-driven tumorigenesis and impacts breast cancer malignancy in a cell type-dependent manner.

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.

Purpose Stemming from a myriad of genetic and epigenetic alterations, triple-negative breast cancer (TNBC) is tied to poor clinical outcomes and aspires for individualized therapies. Here we investigated the therapeutic potential of co-inhibiting integrin-dependent signaling pathway and BRD4, a transcriptional and epigenetic mediator, for TNBC. Methods Two independent patient cohorts were subjected to bioinformatic and IHC examination for clinical association of candidate cancer drivers. The efficacy and biological bases for co-targeting these drivers were interrogated using cancer cell lines, a protein kinase array, chemical inhibitors, RNAi/CRISPR/Cas9 approaches, and a 4 T1-Balb/c xenograft model. Results We found that amplification of the chromosome 8q24 region occurred in nearly 20% of TNBC tumors, and that it coincided with co-upregulation or amplification of c-Myc and FAK, a key effector of integrin-dependent signaling. This co-upregulation at the mRNA or protein level correlated with a poor patient survival (p p + myeloid-derived suppressor cells in vivo. Finally, we found that JQ1 and VS-6063 cooperatively induced apoptotic cell death by altering XIAP, Bcl2/Bcl-xl and Bim levels, impairing c-Src/p130Cas-, PI3K/Akt- and RelA-associated signaling, and were linked to EMT-inducing transcription factor Snail- and Slug-dependent regulation. Conclusion Based on our results, we conclude that the BRD4/c-Myc- and integrin/FAK-dependent pathways act in concert to promote breast cancer cell survival and poor clinical outcomes. As such, they represent promising targets for a synthetic lethal-type of therapy against TNBC.

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.

Hippocampal overexpression of FK506-binding protein 12.6/1b (FKBP1b), a negative regulator of ryanodine receptor Ca2+release, reverses aging-induced memory impairment and neuronal Ca2+dysregulation. Here, we tested the hypothesis thatFKBP1balso can protect downstream transcriptional networks from aging-induced dysregulation. We gave hippocampal microinjections ofFKBP1b-expressing viral vector to male rats at either 13 months of age (long-term, LT) or 19 months of age (short-term, ST) and tested memory performance in the Morris water maze at 21 months of age. Aged rats treated ST or LT withFKBP1bsubstantially outperformed age-matched vector controls and performed similarly to each other and young controls (YCs). Transcriptional profiling in the same animals identified 2342 genes with hippocampal expression that was upregulated/downregulated in aged controls (ACs) compared with YCs (the aging effect). Of these aging-dependent genes, 876 (37%) also showed altered expression in agedFKBP1b-treated rats compared with ACs, withFKBP1brestoring expression of essentially all such genes (872/876, 99.5%) in the direction opposite the aging effect and closer to levels in YCs. This inverse relationship between the aging andFKBP1beffects suggests that the aging effects arise fromFKBP1bdeficiency. Functional category analysis revealed that genes downregulated with aging and restored byFKBP1bwere associated predominantly with diverse brain structure categories, including cytoskeleton, membrane channels, and extracellular region. Conversely, genes upregulated with aging but not restored byFKBP1bassociated primarily with glial–neuroinflammatory, ribosomal, and lysosomal categories. Immunohistochemistry confirmed aging-induced rarefaction andFKBP1b-mediated restoration of neuronal microtubular structure. Therefore, a previously unrecognized genomic network modulating diverse brain structural processes is dysregulated by aging and restored byFKBP1boverexpression.SIGNIFICANCE STATEMENTPreviously, we found that hippocampal overexpression of FK506-binding protein 12.6/1b (FKBP1b), a negative regulator of intracellular Ca2+responses, reverses both aging-related Ca2+dysregulation and cognitive impairment. Here, we tested whether hippocampalFKBP1boverexpression also counteracts aging changes in gene transcriptional networks. In addition to reducing memory deficits in aged rats,FKBP1bselectively counteracted aging-induced expression changes in 37% of aging-dependent genes, with cytoskeletal and extracellular structure categories highly associated with theFKBP1b-rescued genes. Our results indicate that, in parallel with cognitive processes, a novel transcriptional network coordinating brain structural organization is dysregulated with aging and restored byFKBP1b.

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.

Aging is the leading risk factor for idiopathic Alzheimer’s disease (AD), indicating that normal aging processes promote AD and likely are present in the neurons in which AD pathogenesis originates. In AD, neurofibrillary tangles (NFTs) appear first in entorhinal cortex, implying that aging processes in entorhinal neurons promote NFT pathogenesis. Using electrophysiology and immunohistochemistry, we find pronounced aging-related Ca2 + dysregulation in rat entorhinal neurons homologous with the human neurons in which NFTs originate. Considering that humans recapitulate many aspects of animal brain aging, these results support the hypothesis that aging-related Ca2 + dysregulation occurs in human entorhinal neurons and promotes NFT pathogenesis.

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.

Brain Ca2+ regulatory processes are altered during aging, disrupting neuronal, and cognitive functions. In hippocampal pyramidal neurons, the Ca2+-dependent slow afterhyperpolarization (sAHP) exhibits an increase with aging, which correlates with memory impairment. The increased sAHP results from elevated L-type Ca2+ channel activity and ryanodine receptor (RyR)-mediated Ca2+ release, but underlying molecular mechanisms are poorly understood. Previously, we found that expression of the gene encoding FK506-binding protein 12.6/1b (FKBP1b), a small immunophilin that stabilizes RyR-mediated Ca2+ release in cardiomyocytes, declines in hippocampus of aged rats and Alzheimer's disease subjects. Additionally, knockdown/disruption of hippocampal FKBP1b in young rats augments neuronal Ca2+ responses. Here, we test the hypothesis that declining FKBP1b underlies aging-related hippocampal Ca2+ dysregulation. Using microinjection of adeno-associated viral vector bearing a transgene encoding FKBP1b into the hippocampus of aged male rats, we assessed the critical prediction that overexpressing FKBP1b should reverse Ca2+-mediated manifestations of brain aging. Immunohistochemistry and qRT-PCR confirmed hippocampal FKBP1b overexpression 4–6 weeks after injection. Compared to aged vector controls, aged rats overexpressing FKBP1b showed dramatic enhancement of spatial memory, which correlated with marked reduction of sAHP magnitude. Furthermore, simultaneous electrophysiological recording and Ca2+ imaging in hippocampal neurons revealed that the sAHP reduction was associated with a decrease in parallel RyR-mediated Ca2+ transients. Thus, hippocampal FKBP1b overexpression reversed key aspects of Ca2+ dysregulation and cognitive impairment in aging rats, supporting the novel hypothesis that declining FKBP1b is a molecular mechanism underlying aging-related Ca2+ dysregulation and unhealthy brain aging and pointing to FKBP1b as a potential therapeutic target. SIGNIFICANCE STATEMENT This paper reports critical tests of a novel hypothesis that proposes a molecular mechanism of unhealthy brain aging and possibly, Alzheimer's disease. For more than 30 years, evidence has been accumulating that brain aging is associated with dysregulation of calcium in neurons. Recently, we found that FK506-binding protein 12.6/1b (FKBP1b), a small protein that regulates calcium, declines with aging in the hippocampus, a brain region important for memory. Here we used gene therapy approaches and found that raising FKBP1b reversed calcium dysregulation and memory impairment in aging rats, allowing them to perform a memory task as well as young rats. These studies identify a potential molecular mechanism of brain aging and may also have implications for treatment of Alzheimer's disease.

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.

Accumulating evidence is associating disorders of lipid and glucose metabolism, including the overlapping conditions of insulin resistance/metabolic syndrome, obesity and diabetes, with moderate cognitive impairment in normal aging and elevated risk of Alzheimer’s disease. It appears that a common feature of these conditions is deficient insulin signaling, likely affecting the brain as well as canonical peripheral target tissues. A number of studies have documented that insulin directly affects brain processes and that reduced insulin signaling results in impaired learning and memory. Several studies have also shown that deficient insulin signaling induces Ca2+ dysregulation in neurons. Because brain aging is associated with substantial Ca2+ dyshomeostasis, it has been proposed that deficient insulin signaling exacerbates or accelerates aging-related Ca2+ dyshomeostasis. However, there have been few studies examining insulin interactions with Ca2+ regulation in aging animals. We have been testing predictions of the Ca2+ dysregulation/diabetes/brain aging hypothesis and have found that insulin and insulin sensitizers (thiazolidinediones) target several hippocampal Ca2+-related processes affected by aging, including larger Ca2+ transients and Ca2+-dependent afterhyperpolarizations, and counteract the effects of aging on those processes. Thus, while additional testing is needed, the results to date are consistent with the view that effects of deficient insulin signaling on brain aging are mediated in part by neuronal Ca2+ dyshomeostasis.

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.

Thiazolidinediones (TZDs) are agonists at peroxisome proliferator-activated gamma-type (PPAR-γ) receptors and are used clinically for the treatment of type 2 diabetes where they have been shown to reestablish insulin sensitivity, improve lipid profiles, and reduce inflammation. Recent work also suggests that TZDs may be beneficial in Alzheimer’s disease (AD), ameliorating cognitive decline early in the disease process. However, there have been only a few studies identifying mechanisms through which cognitive benefits may be exerted. Starting at 10 months of age, the triple transgenic mouse model of AD (3×Tg-AD) with accelerated amyloid-β (Aβ) deposition and tau pathology was treated with the TZD pioglitazone (PIO-Actos®) at 18 mg/Kg body weight/day. After four months, PIO-treated animals showed multiple beneficial effects, including improved learning on the active avoidance task, reduced serum cholesterol, decreased hippocampal amyloid-β and tau deposits, and enhanced short- and long-term plasticity. Electrophysiological membrane properties and post-treatment blood glucose levels were unchanged by PIO. Gene microarray analyses of hippocampal tissue identified predicted transcriptional responses following TZD treatment as well as potentially novel targets of TZDs, including facilitation of estrogenic processes and decreases in glutamatergic and lipid metabolic/cholesterol dependent processes. Taken together, these results confirm prior animal studies showing that TZDs can ameliorate cognitive deficits associated with AD-related pathology, but also extend these findings by pointing to novel molecular targets in the brain.

With aging, multiple Ca2+-associated electrophysiological processes exhibit increased magnitude in hippocampal pyramidal neurons, including the Ca2+-dependent slow afterhyperpolarization (sAHP), L-type voltage-gated Ca2+channel (L-VGCC) activity, Ca2+-induced Ca2+release (CICR) from ryanodine receptors (RyRs), and Ca2+transients. This pattern of Ca2+dysregulation correlates with reduced neuronal excitability/plasticity and impaired learning/memory and has been proposed to contribute to unhealthy brain aging and Alzheimer's disease. However, little is known about the underlying molecular mechanisms. In cardiomyocytes, FK506-binding protein 1b/12.6 (FKBP1b) binds and stabilizes RyR2 in the closed state, inhibiting RyR-mediated Ca2+release. Moreover, we recently found that hippocampalFkbp1bexpression is downregulated, whereasRyr2andFrap1/Mtor(mammalian target of rapamycin) expression is upregulated with aging in rats. Here, we tested the hypothesis that disrupting FKBP1b function also destabilizes Ca2+homeostasis in hippocampal neurons and is sufficient to induce the aging phenotype of Ca2+dysregulation in young animals. Selective knockdown ofFkbp1bwith interfering RNAin vitro(96 h) enhanced voltage-gated Ca2+current in cultured neurons, whereasin vivo Fkbp1bknockdown by microinjection of viral vector (3–4 weeks) dramatically increased the sAHP in hippocampal slice neurons from young-adult rats. Rapamycin, which displaces FKBP1b from RyRs in myocytes, similarly enhanced VGCC current and the sAHP and also increased CICR. Moreover, FKBP1b knockdownin vivowas associated with upregulation of RyR2 and mTOR protein expression. Thus, disruption of FKBP1b recapitulated much of the Ca2+-dysregulation aging phenotype in young rat hippocampus, supporting a novel hypothesis that declining FKBP function plays a major role in unhealthy brain aging.

Age-dependent metabolic syndrome (MetS) is a well established risk factor for cardiovascular disease, but it also confers major risk for impaired cognition in normal aging or Alzheimer's disease (AD). However, little is known about the specific pathways mediating MetS–brain interactions. Here, we performed the first studies quantitatively linking MetS variables to aging changes in brain genome-wide expression and mitochondrial function. In six young adult and six aging female rhesus monkeys, we analyzed gene expression in two major hippocampal subdivisions critical for memory/cognitive function [hippocampus proper, or cornu ammonis (CA), and dentate gyrus (DG)]. Genes that changed with aging [aging-related genes (ARGs)] were identified in each region. Serum variables reflecting insulin resistance and dyslipidemia were used to construct a quantitative MetS index (MSI). This MSI increased with age and correlated negatively with hippocampal mitochondrial function (state III oxidation). More than 2000 ARGs were identified in CA and/or DG, in approximately equal numbers, but substantially more ARGs in CA than in DG were correlated selectively with the MSI. Pathways represented by MSI-correlated ARGs were determined from the Gene Ontology Database and literature. In particular, upregulated CA ARGs representing glucocorticoid receptor (GR), chromatin assembly/histone acetyltransferase, and inflammatory/immune pathways were closely associated with the MSI. These results suggest a novel model in which MetS is associated with upregulation of hippocampal GR-dependent transcription and epigenetic coactivators, contributing to decreased mitochondrial function and brain energetic dysregulation. In turn, these MSI-associated neuroenergetic changes may promote inflammation, neuronal vulnerability, and risk of cognitive impairment/AD.

Previous studies have shown that inhibition of the Ca(2+)-/calmodulin-dependent protein phosphatase calcineurin (CN) blocks L-type voltage sensitive Ca(2+) channel (L-VSCC) activity in cultured hippocampal neurons. However, it is not known whether CN contributes to the increase in hippocampal L-VSCC activity that occurs with aging in at least some mammalian species. It is also unclear whether CN's necessary role in VSCC activity is simply permissive or is directly enhancing. To resolve these questions, we used partially dissociated hippocampal "zipper" slices to conduct cell-attached patch recording and RT-PCR on largely intact single neurons from young-adult, mid-aged, and aged rats. Further, we tested for direct CN enhancement of L-VSCCs using virally mediated infection of cultured neurons with an activated form of CN. Similar to previous work, L-VSCC activity was elevated in CA1 neurons of mid-aged and aged rats relative to young adults. The CN inhibitor, FK-506 (5muM) completely blocked the aging-related increase in VSCC activity, reducing the activity level in aged rat neurons to that in younger rat neurons. However, aging was not associated with an increase in neuronal CN mRNA expression, nor was CN expression correlated with VSCC activity. Delivery of activated CN to primary hippocampal cultures induced an increase in neuronal L-VSCC activity but did not elevate L-VSCC protein levels. Together, the results provide the first evidence that CN activity, but not increased expression, plays a selective and necessary role in the aging-related increase in available L-VSCCs, possibly by direct activation. Thus, these studies point to altered CN function as a novel and potentially key factor in aging-dependent neuronal Ca(2+) dysregulation.

Multiple hippocampal processes and cognitive functions change with aging or Alzheimer's disease, but the potential triggers of these aging cascades are not well understood. Here, we quantified hippocampal expression profiles and behavior across the adult lifespan to identify early aging changes and changes that coincide with subsequent onset of cognitive impairment. Well powered microarray analyses (N= 49 arrays), immunohistochemistry, and Morris spatial maze learning were used to study male F344 rats at five age points. Genes that changed with aging (by ANOVA) were assigned to one of four onset age ranges based on template pattern matching; functional pathways represented by these genes were identified statistically (Gene Ontology). In the earliest onset age range (3–6 months old), upregulation began for genes in lipid/protein catabolic and lysosomal pathways, indicating a shift in metabolic substrates, whereas downregulation began for lipid synthesis, GTP/ATP-dependent signaling, and neural development genes. By 6–9 months of age, upregulation of immune/inflammatory cytokines was pronounced. Cognitive impairment first appeared in the midlife range (9–12 months) and coincided and correlated primarily with midlife upregulation of genes associated with cholesterol trafficking (apolipoprotein E), myelinogenic, and proteolytic/major histocompatibility complex antigen-presenting pathways. Immunolabeling revealed that cholesterol trafficking proteins were substantially increased in astrocytes and that myelination increased with aging. Together, our data suggest a novel sequential model in which an early-adult metabolic shift, favoring lipid/ketone body oxidation, triggers inflammatory degradation of myelin and resultant excess cholesterol that, by midlife, activates cholesterol transport from astrocytes to remyelinating oligodendrocytes. These processes may damage structure and compete with neuronal pathways for bioenergetic resources, thereby impairing cognitive function.

Evidence accumulated over more than two decades has implicated Ca2+ dysregulation in brain aging and Alzheimer's disease (AD), giving rise to the Ca2+ hypothesis of brain aging and dementia. Electrophysiological, imaging, and behavioral studies in hippocampal or cortical neurons of rodents and rabbits have revealed aging-related increases in the slow afterhyperpolarization, Ca2+ spikes and currents, Ca2+ transients, and L-type voltage-gated Ca2+ channel (L-VGCC) activity. Several of these changes have been associated with age-related deficits in learning or memory. Consequently, one version of the Ca2+ hypothesis has been that increased L-VGCC activity drives many of the other Ca2+-related biomarkers of hippocampal aging. In addition, other studies have reported aging- or AD model-related alterations in Ca2+ release from ryanodine receptors (RyR) on intracellular stores. The Ca2+-sensitive RyR channels amplify plasmalemmal Ca2+ influx by the mechanism of Ca2+-induced Ca2+ release (CICR). Considerable evidence indicates that a preferred functional link is present between L-VGCCs and RyRs which operate in series in heart and some brain cells. Here, we review studies implicating RyRs in altered Ca2+ regulation in cell toxicity, aging, and AD. A recent study from our laboratory showed that increased CICR plays a necessary role in the emergence of Ca2+-related biomarkers of aging. Consequently, we propose an expanded L-VGCC/Ca2+ hypothesis, in which aging/pathological changes occur in both L-type Ca2+ channels and RyRs, and interact to abnormally amplify Ca2+ transients. In turn, the increased transients result in dysregulation of multiple Ca2+-dependent processes and, through somewhat different pathways, in accelerated functional decline during aging and AD.

// Pengcheng Zhou 1, * , Sonia Erfani 2, * , Zeyi Liu 2, 3, * , Changhe Jia 2, 4, * , Yecang Chen 5, * , Bingwei Xu 2 , Xinyu Deng 2 , Jose E. Alfaro 1 , Li Chen 2 , Dana Napier 6 , Michael Lu 8 , Jian-An Huang 3 , Chunming Liu 7 , Olivier Thibault 2 , Rosalind Segal 1 , Binhua P. Zhou 7 , Natasha Kyprianou 9 , Craig Horbinski 6, # , Xiuwei H. Yang 2, # 1 Department of Cancer Biology and Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA 2 Department of Pharmacology and Nutritional Sciences, Markey Cancer Center and University of Kentucky, Lexington, KY, USA 3 Department of Respiratory Medicine, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, P. R. China 4 Department of Gastroenterology, Provincial People's Hospital, Zhengzhou, Henan Province, P. R. China 5 Department of Chemistry, University of Kentucky, Lexington, KY, USA 6 Department of Pathology and Laboratory Medicine, Markey Cancer Center and University of Kentucky, Lexington, KY, USA 7 Department of Molecular and Cellular Biochemistry, Markey Cancer Center and University of Kentucky, Lexington, KY, USA 8 Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL, USA 9 Department of Urology, Markey Cancer Center and University of Kentucky, Lexington, KY, USA * These authors have contributed equally to this study # Co-equal contribution Correspondence to: Xiuwei H. Yang, email: xiuwei-yang@uky.edu Craig Horbinski, email: craig.horbinski@uky.edu Keywords: glioblastoma, CD151, α3 integrin, EGFR, cell invasion and motility Received: May 23, 2015 Accepted: August 03, 2015 Published: August 13, 2015 ABSTRACT Glioblastoma, one of the most aggressive forms of brain cancer, is featured by high tumor cell motility and invasiveness, which not only fuel tumor infiltration, but also enable escape from surgical or other clinical interventions. Thus, better understanding of how these malignant traits are controlled will be key to the discovery of novel biomarkers and therapies against this deadly disease. Tetraspanin CD151 and its associated α3β1 integrin have been implicated in facilitating tumor progression across multiple cancer types. How these adhesion molecules are involved in the progression of glioblastoma, however, remains largely unclear. Here, we examined an in-house tissue microarray-based cohort of 96 patient biopsies and TCGA dataset to evaluate the clinical significance of CD151 and α3β1 integrin. Functional and signaling analyses were also conducted to understand how these molecules promote the aggressiveness of glioblastoma at molecular and cellular levels. Results from our analyses showed that CD151 and α3 integrin were significantly elevated in glioblastomas at both protein and mRNA levels, and exhibited strong inverse correlation with patient survival ( p < 0.006). These adhesion molecules also formed tight protein complexes and synergized with EGF/EGFR to accelerate tumor cell motility and invasion. Furthermore, disruption of such complexes enhanced the survival of tumor-bearing mice in a xenograft model, and impaired activation of FAK and small GTPases. Also, knockdown- or pharmacological agent-based attenuation of EGFR, FAK or Graf (ARHGAP26)/small GTPase-mediated pathways markedly mitigated the aggressiveness of glioblastoma cells. Collectively, our findings provide clinical, molecular and cellular evidence of CD151-α3β1 integrin complexes as promising prognostic biomarkers and therapeutic targets for glioblastoma.

It has been recognized for some years that a prolonged Ca 2+ elevation that is predictive of impending cell death develops in cultured neurons following excitotoxic insult. In addition, neurons exhibit enhanced sensitivity to excitotoxic insult with increasing age in culture. However, little is known about the processes that selectively regulate the post-insult Ca 2+ elevation and therefore, it remains unclear whether it is associated specifically with age-dependent toxicity. Here, we tested the hypothesis that a group I metabotropic glutamate receptor antagonist selectively modulates the prolonged Ca 2+ elevation in direct association with its protective effects against excitotoxicity. Rat hippocampal cultures of two ages (8–9 and 21–28 days in vitro ) were exposed to a 5-min glutamate insult (400 μM in younger and 10 μM in older cultures) sufficient to kill >50% of the neurons, and were treated with vehicle or the specific group I metabotropic glutamate receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), throughout and following the insult. Neuronal survival was quantified 24 h after insult. In parallel studies, neurons of similar age in culture were imaged ratiometrically with a confocal microscope during and for 60 min after the glutamate insult. A large post-insult Ca 2+ elevation was present in older but not most younger neurons. The N -methyl- D -aspartate receptor antagonist, MK-801, blocked the Ca 2+ elevation both during and following the insult. In contrast, AIDA blocked only the post-insult prolonged Ca 2+ elevation in older neurons. Moreover, AIDA was neuroprotective in older but not younger cultures. From these results we suggest that the post-insult Ca 2+ elevation is regulated differently from the Ca 2+ elevation during glutamate insult and is modulated by group I metabotropic glutamate receptors. Further, the prolonged Ca 2+ elevation appears to be directly linked to an age-dependent component of vulnerability.

Integrins, a family of heterodimeric receptors for extracellular matrix, are promising therapeutic targets for ovarian cancer, particularly high-grade serous-type (HGSOC), as they drive tumor cell attachment, migration, proliferation and survival by activating focal adhesion kinase (FAK)-dependent signaling. Owing to the potential off-target effects of FAK inhibitors, disruption of the integrin signaling axis remains to be a challenge. Here, we tackled this barrier by screening for inhibitors being functionally cooperative with small-molecule VS-6063, a phase II FAK inhibitor. From this screening, JQ1, a potent inhibitor of Myc oncogenic network, emerged as the most robust collaborator. Treatment with a combination of VS-6063 and JQ1 synergistically caused an arrest of tumor cells at the G2/M phase and a decrease in the XIAP-linked cell survival. Our subsequent mechanistic analyses indicate that this functional cooperation was strongly associated with the concomitant disruption of activation or expression of FAK and c-Myc as well as their downstream signaling through the PI3K/Akt pathway. In line with these observations, we detected a strong co-amplification or upregulation at genomic or protein level for FAK and c-Myc in a large portion of primary tumors in the TCGA or a local HGSOC patient cohort. Taken together, our results suggest that the integrin–FAK signaling axis and c-Myc synergistically drive cell proliferation, survival and oncogenic potential in HGSOC. As such, our study provides key genetic, functional and signaling bases for the small-molecule-based co-targeting of these two distinct oncogenic drivers as a new line of targeted therapy against human ovarian cancer.

Vitamin D is an important calcium-regulating hormone with diverse functions in numerous tissues, including the brain. Increasing evidence suggests that vitamin D may play a role in maintaining cognitive function and that vitamin D deficiency may accelerate age-related cognitive decline. Using aging rodents, we attempted to model the range of human serum vitamin D levels, from deficient to sufficient, to test whether vitamin D could preserve or improve cognitive function with aging. For 5-6 mo, middle-aged F344 rats were fed diets containing low, medium (typical amount), or high (100, 1,000, or 10,000 international units/kg diet, respectively) vitamin D3, and hippocampal-dependent learning and memory were then tested in the Morris water maze. Rats on high vitamin D achieved the highest blood levels (in the sufficient range) and significantly outperformed low and medium groups on maze reversal, a particularly challenging task that detects more subtle changes in memory. In addition to calcium-related processes, hippocampal gene expression microarrays identified pathways pertaining to synaptic transmission, cell communication, and G protein function as being up-regulated with high vitamin D. Basal synaptic transmission also was enhanced, corroborating observed effects on gene expression and learning and memory. Our studies demonstrate a causal relationship between vitamin D status and cognitive function, and they suggest that vitamin D-mediated changes in hippocampal gene expression may improve the likelihood of successful brain aging.

L-type voltage-sensitive Ca 2+ channels (L-VSCCs) play an important role in developmental and aging processes, as well as during normal function of brain neurons. Here, we tested a prediction of the hypothesis that membrane density of functional L-VSCCs is regulated by the level of gene expression for its α 1D pore-forming subunit. If so, α 1D mRNA and L-VSCC activity should be positively correlated within individual neurons. Conventional methods of aspiration and/or acute cell dissociation used in prior single-cell studies have generally yielded variable and incomplete recovery of intracellular mRNA. Thus, quantitative relationships between channel function and expression have been difficult to define. In this study, we used the partially dissociated (“zipper”) hippocampal slice preparation as a method for collecting a single neuron's mRNA complement. This preparation, developed to expose neuronal somata for recording, also enables the extraction of a neuron with major processes largely intact. Thus, single-cell measures of gene/mRNA expression can be based on approximately the cell's full set of mRNA transcripts. In adult and aged rat hippocampal zipper slices, L-VSCC activity was first recorded in CA1 neurons in cell-attached patch mode. The same neurons were then extracted and collected for semiquantitative reverse transcriptase–PCR analysis of α 1D and calmodulin A (CaM) mRNA content. Across multiple single neurons, a significant, positive correlation was found between the rank orders of L-VSCC activity and of α 1D , but not CaM, mRNA expression. Thus, these studies support the possibility that the level of α 1D gene expression regulates the density of functional L-VSCCs.

It has been recognized for some time that the Ca(2+)-dependent slow afterhyperpolarization (sAHP) is larger in hippocampal neurons of aged compared with young animals. In addition, extensive studies since have shown that other Ca(2+)-mediated electrophysiological responses are increased in hippocampus with aging, including Ca(2+) transients, L-type voltage-gated Ca(2+) channel activity, Ca(2+) spike duration and action potential accommodation. Elevated Ca(2+)-induced Ca(2+) release from ryanodine receptors (RyRs) appears to drive amplification of the Ca(2+) responses. Components of this Ca(2+) dysregulation phenotype correlate with deficits in cognitive function and plasticity, indicating they may play critical roles in aging-related impairment of brain function. However, the molecular mechanisms underlying aging-related Ca(2+) dysregulation are not well understood. FK506-binding proteins 1a and 1b (FKBP1a/1b, also known as FKBP12/12.6) are immunophilin proteins that bind the immunosuppressant drugs FK506 and rapamycin. In muscle cells, FKBP1a/1b also bind RyRs and inhibits Ca(2+)-induced Ca(2+) release, but it is not clear whether FKBPs act similarly in brain cells. Recently, we found that selectively disrupting hippocampal FKBP1b function in young rats, either by microinjecting adeno-associated viral vectors expressing siRNA, or by treatment with rapamycin, increases the sAHP and recapitulates much of the hippocampal Ca(2+) dysregulation phenotype. Moreover, in microarray studies, we found FKBP1b gene expression was downregulated in hippocampus of aging rats and early-stage Alzheimer's disease subjects. These results suggest the novel hypothesis that declining FKBP function is a key factor in aging-related Ca(2+) dysregulation in the brain and point to potential new therapeutic targets for counteracting unhealthy brain aging.

The hippocampal slice preparation has contributed greatly to analysis of the basic neurophysiology of brain neurons. In addition, because traumatic dissociative procedures are not used, the in vitro slice is particularly well suited for studies of electrophysiological properties of hippocampal neurons in young and aged rodent brain. Using the slice, we have previously observed an aging-dependent enhancement of voltage-activated Ca2+ influx using a combination of intracellular sharp electrode current-clamp and voltage-clamp techniques. The Ca2+-dependent afterhyperpolarization as well as the Ca2+ action potential were significantly larger in aged rat neurons. Using the sharp electrode switch clamp method, similar effects were found for high voltage-activated whole-cell Ca2+ currents. In order to study the mechanistic bases of these aging phenomena at the single-channel level, we have recently focused on recording cell-attached patches from neurons in the partially dissociated hippocampal slice (‘zipper’ slice). This technique, developed by Gray et al. in 1990, subjects slices to a mild enzymatic treatment resulting in the exposure of individual neurons for patch-clamp procedures. Using this technique, we are currently recording single Ca2+ channel activity in hippocampal slices from 4- to 29-month-old rats.

Neuronal Ca(2+) dyshomeostasis associated with cognitive impairment and mediated by changes in several Ca(2+) sources has been seen in animal models of both aging and diabetes. In the periphery, dysregulation of intracellular Ca(2+) signals may contribute to the development of insulin resistance. In the brain, while it is well-established that type 2 diabetes mellitus is a risk factor for the development of dementia in the elderly, it is not clear whether Ca(2+) dysregulation might also affect insulin sensitivity and glucose utilization. Here we present a combination of imaging techniques testing the disappearance of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as an indication of glycolytic activity in neurons and astrocytes. Our work shows that glucose utilization at rest is greater in neurons compared to astrocytes, and ceases upon activation in neurons with little change in astrocytes. Pretreatment of hippocampal cultures with pioglitazone, a drug used in the treatment of type 2 diabetes, significantly reduced glycolytic activity in neurons and enhanced it in astrocytes. This series of experiments, including Fura-2 and NADH imaging, provides results that are consistent with the idea that Ca(2+) levels may rapidly alter glycolytic activity, and that downstream events beyond Ca(2+) dysregulation with aging, may alter cellular metabolism in the brain.

Background Age-related cognitive deficits negatively affect quality of life and can presage serious neurodegenerative disorders. Despite sleep disruption's well-recognized negative influence on cognition, and its prevalence with age, surprisingly few studies have tested sleep's relationship to cognitive aging. Methodology We measured sleep stages in young adult and aged F344 rats during inactive (enhanced sleep) and active (enhanced wake) periods. Animals were behaviorally characterized on the Morris water maze and gene expression profiles of their parietal cortices were taken. Principal Findings Water maze performance was impaired, and inactive period deep sleep was decreased with age. However, increased deep sleep during the active period was most strongly correlated to maze performance. Transcriptional profiles were strongly associated with behavior and age, and were validated against prior studies. Bioinformatic analysis revealed increased translation and decreased myelin/neuronal pathways. Conclusions The F344 rat appears to serve as a reasonable model for some common sleep architecture and cognitive changes seen with age in humans, including the cognitively disrupting influence of active period deep sleep. Microarray analysis suggests that the processes engaged by this sleep are consistent with its function. Thus, active period deep sleep appears temporally misaligned but mechanistically intact, leading to the following: first, aged brain tissue appears capable of generating the slow waves necessary for deep sleep, albeit at a weaker intensity than in young. Second, this activity, presented during the active period, seems disruptive rather than beneficial to cognition. Third, this active period deep sleep may be a cognitively pathologic attempt to recover age-related loss of inactive period deep sleep. Finally, therapeutic strategies aimed at reducing active period deep sleep (e.g., by promoting active period wakefulness and/or inactive period deep sleep) may be highly relevant to cognitive function in the aging community.

This paper reviews evidence that brain aging and Alzheimer's disease (AD) are somehow closely related and that the hippocampus (CA1) is highly vulnerable to cell loss under both conditions. In addition, two current lines of evidence on the mechanisms of hippocampal cell loss with aging are considered, including studies of neuronal calcium dysregulation and studies of cumulative glucocorticoid (GC) neurotoxicity. Moreover, recent electrophysiological studies have shown that excess glucocorticoid activation of hippocampal neurons increases the influx of calcium through voltage-activated calcium channels. Second messenger systems may mediate the steroid modulation of calcium channels. Therefore, it is hypothesized that excess glucocorticoid activation and neuronal calcium dysregulation may be two phases of a single process that increases the susceptibility of neurons to neurodegeneration during aging and Alzheimer's disease.

At hippocampal synapses, repetitive synaptic stimulation (RSS) in the theta frequency range (3–12 Hz) is associated with robust EPSP frequency facilitation (FF) and consequently, enhanced action potential (spike) generation and throughput. A complex, synaptically induced hyperpolarization (SIHP) is also triggered by synaptic activation, and a Ca 2+ -dependent afterhyperpolarization (AHP) is triggered above spike threshold. With aging, the AHP is increased and impairs intracellular spike generation, at least in accommodation protocols. However, little is known about how these aging changes interact to affect spike generation at physiological frequencies of RSS, or if the SIHP also is modified in aging. Here we performed the first tests of the net impact of these excitatory and inhibitory aging changes on spike generation during RSS. We report that during RSS at spike threshold (1) spike throughput is well sustained at theta frequencies in young and aged neurons; (2) an interposed AHP dampens spike generation, particularly in aged neurons and at higher frequencies; (3) compared to the AHP, the SIHP does not exert an equivalent inhibitory effect on spike throughput; and (4) in contrast to the AHP, the SIHP is reduced with aging. Together, these results are consistent with a model in which the source of the hyperpolarization is important in determining hippocampal spike throughput within the theta frequency range.

Although expression of some genes is known to change during neuronal activity or plasticity, the overall relationship of gene expression changes to memory or memory disorders is not well understood. Here, we combined extensive statistical microarray analyses with behavioral testing to comprehensively identify genes and pathways associated with aging and cognitive dysfunction. Aged rats were separated into cognitively unimpaired (AU) or impaired (AI) groups based on their Morris water maze performance relative to young-adult (Y) animals. Hippocampal gene expression was assessed in Y, AU, and AI on the fifth (last) day of maze training (5T) or 21 d posttraining (21PT) and in nontrained animals (eight groups total, one array per animal;n= 78 arrays). ANOVA and linear contrasts identified genes that differed from Y generally with aging (differed in both AU and AI) or selectively, with cognitive status (differed only in AI or AU). Altered pathways/processes were identified by overrepresentation analyses of changed genes. With general aging, there was downregulation of axonal growth, cytoskeletal assembly/transport, signaling, and lipogenic/uptake pathways, concomitant with upregulation in immune/inflammatory, lysosomal, lipid/protein degradation, cholesterol transport, transforming growth factor, and cAMP signaling pathways, primarily independent of training condition. Selectively, in AI, there was downregulation at 5T of immediate-early gene, Wnt (wingless integration site), insulin, and G-protein signaling, lipogenesis, and glucose utilization pathways, whereas Notch2 (oligodendrocyte development) and myelination pathways were upregulated, particularly at 21PT. In AU, receptor/signal transduction genes were upregulated, perhaps as compensatory responses. Immunohistochemistry confirmed and extended selected microarray results. Together, the findings suggest a new model, in which deficient neuroenergetics leads to downregulated neuronal signaling and increased glial activation, resulting in aging-related cognitive dysfunction.

Excessive glutamate (Glu) stimulation of the NMDA-R is a widely recognized trigger for Ca(2+)-mediated excitotoxicity. Primary neurons typically show a large increase in vulnerability to excitotoxicity with increasing days in vitro (DIV). This enhanced vulnerability has been associated with increased expression of the NR2B subunit or increased NMDA-R current, but the detailed age-courses of these variables in primary hippocampal neurons have not been compared in the same study. Further, it is not clear whether the NMDA-R is the only source of excess Ca(2+). Here, we used primary hippocampal neurons to examine the age dependence of the increase in excitotoxic vulnerability with changes in NMDA-R current, and subunit expression. We also tested whether L-type voltage-gated Ca(2+) channels (L-VGCCs) contribute to the enhanced vulnerability. The EC(50) for Glu toxicity decreased by approximately 10-fold between 8-9 and 14-15 DIV, changing little thereafter. Parallel experiments found that during the same period both amplitude and duration of NMDA-R current increased dramatically; this was associated with an increase in protein expression of the NR1 and NR2A subunits, but not of the NR2B subunit. Compared to MK-801, ifenprodil, a selective NR2B antagonist, was less effective in protecting older than younger neurons from Glu insult. Conversely, nimodipine, an L-VGCC antagonist, protected older but not younger neurons. Our results indicate that enhanced excitotoxic vulnerability with age in culture was associated with a substantial increase in NMDA-R current, concomitant increases in NR2A and NR1 but not NR2B subunit expression, and with apparent recruitment of L-VGCCs into the excitotoxic process.

Aging in the hippocampus of several species is characterized by alterations in multiple Ca 2+ -mediated processes, including an increase in L-type voltage-gated Ca 2+ channel (L-VGCC) current, an enhanced Ca 2+ -dependent slow afterhyperpolarization (AHP), impaired synaptic plasticity and elevated Ca 2+ transients. Previously, we found that 1α,25-dihydoxyvitamin D 3 (1,25VitD), a major Ca 2+ regulating hormone, down-regulates L-VGCC expression in cultured hippocampal neurons. Here, we tested whether in vivo treatment of aged F344 rats with 1,25VitD would reverse some of the Ca 2+ -mediated biomarkers of aging seen in hippocampal CA1 neurons. As previously reported, L-VGCC currents and the AHP were larger in aged than in young neurons. Treatment with 1,25VitD over 7 days decreased L-VGCC activity in aged rats, as well as the age-related increase in AHP amplitude and duration. In addition, reduced L-VGCC activity was correlated with reduced AHPs in the same animals. These data provide direct evidence that 1,25VitD can regulate multiple Ca 2+ -dependent processes in neurons, with particular impact on reducing age-related changes associated with Ca 2+ dysregulation. Thus, these results may have therapeutic implications and suggest that 1,25VitD, often taken to maintain bone health, may also retard some consequences of brain aging.

Age-dependent changes in multiple Ca2+-related electrophysiological processes in the hippocampus appear to be consistent biomarkers of aging, and several also correlate with cognitive decline. These findings have led to the hypothesis that a common mechanism of Ca2+dyshomeostasis underlies aspects of aging-dependent brain impairment. However, some key predictions of this view remain untested, including that multiple Ca2+-related biomarkers should emerge concurrently during aging and their onset should also precede/coincide with initial signs of cognitive decline. Moreover, blocking a putative common source of dysregulated Ca2+should eliminate aging differences. Here, we tested these predictions using combined electrophysiological, imaging, and pharmacological approaches in CA1 neurons to determine the ages of onset (across 4-, 10-, 12-, 14-, and 23-month-old F344 rats) of several established biomarkers, including the increases in the slow afterhyperpolarization, spike accommodation, and [Ca2+]irise during repetitive synaptic stimulation. In addition, we tested the hypothesis that altered Ca2+-induced Ca2+release (CICR) from ryanodine receptors, which can be triggered by L-type Ca2+channels, provides a common source of dysregulated Ca2+in aging. Results showed that multiple aging biomarkers were first detectable at about the same age (12 months of age; approximately midlife), sufficiently early to influence initial cognitive decline. Furthermore, selectively blocking CICR with ryanodine slowed the Ca2+rise during synaptic stimulation more in aged rat neurons and, notably, reduced or eliminated aging differences in the biomarkers. Thus, this study provides the first evidence that altered CICR plays a role in driving the early and simultaneous emergence in hippocampus of multiple Ca2+-related biomarkers of aging.

Several studies have shown that a prolonged Ca 2+ elevation follows a glutamate-mediated excitotoxic insult in cultured neurons, and may be associated with impending cell death. Recently, we showed that the prolonged Ca 2+ elevation that emerges as neurons age in culture is specifically linked to an age-related increase in excitotoxic vulnerability. However, the multiple sources of Ca 2+ that contribute to Ca 2+ elevation during and after glutamate exposure are not well understood. Here, we examined the Ca 2+ sources of the age-related prolonged Ca 2+ elevation in cultured hippocampal neurons. Studies with caffeine showed that the ryanodine receptor-dependent releasable pool of Ca 2+ from intracellular stores was similar in older and younger neurons. Thapsigargin, which inhibits intracellular store refilling, did not mimic the age-related prolonged Ca 2+ elevation and, in fact, partially reduced it. Ryanodine, which blocks Ca 2+ -induced Ca 2+ -release (CICR) from stores, completely blocked the age-related prolonged Ca 2+ elevation following glutamate exposure but did not alter maximal Ca 2+ elevation during the glutamate exposure. Thus, we conclude that sustained CICR plays a selective and key role in generating the lethal, age-related, prolonged Ca 2+ elevation, and is the likely mechanism underlying age-related, enhanced vulnerability to excitotoxicity in neurons.

The expression of voltage-gated calcium (Ca2+) channel activity in brain cells is known to be important for several aspects of neuronal development. In addition, excessive Ca2+influx has been linked clearly to neurotoxicity bothin vivoandin vitro; however, the temporal relationship between the development of Ca2+channel activity and neuronal survival is not understood. Over a period spanning 28 din vitro, progressive increases in high voltage-activated whole-cell Ca2+current and L-type Ca2+channel activity were observed in cultured hippocampal neurons. On the basis of single-channel analyses, these increases seem to arise in part from a greater density of functionally available L-type Ca2+channels. An increase in mRNA for the α1subunit of L-type Ca2+channels occurred over a similar time course, which suggests that a change in gene expression may underlie the increased channel density. Parallel studies showed that hippocampal neuronal survival over 28 d was inversely related to increasing Ca2+current density. Chronic treatment of hippocampal neurons with the L-type Ca2+channel antagonist nimodipine significantly enhanced survival. Together, these results suggest that age-dependent increases in the density of Ca2+channels might contribute significantly to declining viability of hippocampal neurons. The results also are analogous to patterns seen in neurons of aged animals and therefore raise the possibility that long-term primary neuronal culture could serve as a model for some aspects of aging changes in hippocampal Ca2+channel function.

Healthy brain aging and cognitive function are promoted by exercise. The benefits of exercise are attributed to several mechanisms, many which highlight its neuroprotective role via actions that enhance neurogenesis, neuronal morphology and/or neurotrophin release. However, the brain is also composed of glial and vascular elements, and comparatively less is known regarding the effects of exercise on these components in the aging brain. Here, we show that aerobic exercise at mid-age decreased markers of unhealthy brain aging including astrocyte hypertrophy, a hallmark of brain aging. Middle-aged female mice were assigned to a sedentary group or provided a running wheel for six weeks. Exercise decreased hippocampal astrocyte and myelin markers of aging but increased VEGF, a marker of angiogenesis. Brain vascular casts revealed exercise-induced structural modifications associated with improved endothelial function in the periphery. Our results suggest that age-related astrocyte hypertrophy/reactivity and myelin dysregulation are aggravated by a sedentary lifestyle and accompanying reductions in vascular function. However, these effects appear reversible with exercise initiated at mid-age. As this period of the lifespan coincides with the appearance of multiple markers of brain aging, including initial signs of cognitive decline, it may represent a window of opportunity for intervention as the brain appears to still possess significant vascular plasticity. These results may also have particular implications for aging females who are more susceptible than males to certain risk factors which contribute to vascular aging.

Glucocorticoids (GCs) activate several biochemical/molecular processes in the hippocampus through two receptor types. In addition, GCs influence cognitive behaviors and hippocampal neural activity and can also increase the rate of aging-dependent cell loss in the hippocampus. However, the ionic mechanisms through which GCs modulate hippocampal neuronal function are not well understood. We report here direct evidence that activation of cytosolic steroid receptors, specifically of the type II GC receptor, can enhance voltage-dependent Ca2+ conductances in brain neurons. Ca2+ current was assessed by current-clamp measures of Ca2+ action potentials and by sharp electrode voltage-clamp analyses of voltage-sensitive currents in cesium-, tetrodotoxin-, and tetraethylammonium-treated CA1 neurons in hippocampal slices. Both Ca2+ action potentials and voltage-activated Ca2+ currents (N- and L-like) were increased by 2-hr exposure to the synthetic GC receptor agonist, RU 28362. This effect of RU 28362 was blocked by coincubation with cycloheximide, indicating that the GC receptor-Ca2+ channel interaction depends on de novo protein synthesis. Dysregulated calcium homeostasis is also viewed as a candidate mechanism in brain aging. Thus, present results are consistent with the hypothesis that excessive GC-receptor activation and resultant increased Ca2+ influx may be two sequential phases of a brain-aging process that results initially in impairment of function and eventually in neuronal loss.

It has been proposed that activation of a calcium-sensitive protease (calpain) is a crucial step in the induction of long-term potentiation (LTP). To test this hypothesis, we used chronic recording techniques to measure the effects of intraventricular infusion of leupeptin, a calpain inhibitor, on LTP in the hippocampus. Rats implanted bilaterally with stimulating electrodes in the Schaffer-commissural system and one recording electrode in the apical dendrites of field CA1 were fitted with osmotic mini-pumps delivering either leupeptin (20 mg/ml) or saline at a rate of 0.5 microliter/h into the lateral ventricle. Short bursts of high-frequency stimulation with the bursts delivered at 5/s were used to induce LTP in those animals which had stable responses for several days. Rats in the saline group (n = 11) exhibited an immediate LTP effect that remained in place over successive days of testing, while only 3 of 13 leupeptin treated animals showed evidence of LTP 24 h after high-frequency stimulation, and in only one of those was a sizeable effect recorded over several days. The average change in responses at the 24-h test point was +33% for the controls and +4% for the leupeptin group (P less than 0.01). The block of LTP induction was reversible, since high-frequency stimulation applied after disconnecting the pumps led to a robust LTP effect that lasted for several days in 6 of 7 animals tested. There were no detectable differences in baseline responses in the presence and absence of leupeptin.