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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

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.

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.

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.

Although glucocorticoids (GCs) are known to exert numerous effects in the hippocampus, their chronic regulatory functions remain poorly understood. Moreover, evidence is inconsistent regarding the long-standing hypothesis that chronic GC exposure promotes brain aging/Alzheimer disease. Here, we adrenalectomized male F344 rats at 15 months of age, maintained them for 3 months with implanted corticosterone (CORT) pellets producing low or intermediate (glucocorticoid receptor–activating) blood levels of CORT, and performed microarray/pathway analyses in hippocampal CA1. We defined the chronic GC-dependent transcriptome as 393 genes that exhibited differential expression between intermediate and low CORT groups. Short-term CORT (4 days) did not recapitulate this transcriptome. Functional processes/pathways overrepresented by chronic CORT–up-regulated genes included learning/plasticity, differentiation, glucose metabolism, and cholesterol biosynthesis, whereas processes overrepresented by CORT–down-regulated genes included inflammatory/immune/glial responses and extracellular structure. These profiles indicate that GCs chronically activate neuronal/metabolic processes while coordinately repressing a glial axis of reactivity/inflammation. We then compared the GC transcriptome with a previously defined hippocampal aging transcriptome, revealing a high proportion of common genes. Although CORT and aging moved expression of some common genes in the same direction, the majority were shifted in opposite directions by CORT and aging (eg, glial inflammatory genes down-regulated by CORT are up-regulated with aging). These results contradict the hypothesis that GCs simply promote brain aging and also suggest that the opposite direction shifts during aging reflect resistance to CORT regulation. Therefore, we propose a new model in which aging-related GC resistance develops in some target pathways, whereas GC overstimulation develops in others, together generating much of the brain aging phenotype.

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.

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that threatens to reach epidemic proportions as our population ages. Although much research has examined molecular pathways associated with AD, relatively few such studies have focused on the disease’s critical early stages. In a prior microarray study we correlated gene expression in hippocampus with degree of Alzheimer’s disease and found close associations between upregulation of apparent glial transcription factor/epigenetic/tumor suppressor genes and incipient AD. The results suggested a new model in which AD pathology spreads along myelinated axons (Blalock et al., 2004). However, the microarray analyses were performed on RNA extracted from frozen hand-dissected hippocampal CA1 tissue blocks containing both gray and white matter, limiting the confidence with which transcriptional changes in gray matter could be distinguished from those in white matter. Here, we used laser capture microdissection (LCM) to exclude major white matter tracts while selectively collecting CA1 hippocampal gray matter from formalin-fixed, paraffin-embedded (FFPE) hippocampal sections of the same subjects assessed in our prior study. Microarray analyses of this gray matter-enriched tissue revealed many transcriptional changes similar to those seen in our past study and in studies by others, particularly for downregulated neuron-related genes. Additionally, the present analyses identified several previously undetected pathway alterations, including downregulation of molecules that stabilize ryanodine receptor Ca2+ release and upregulation of vasculature development. Conversely, we found a striking paucity of the upregulated changes in the putative glial and growth-related genes that had been strongly overrepresented in the prior mixed-tissue study. We conclude that FFPE tissue can be a reliable resource for microarray studies of brain tissue, that upregulation of growth-related epigenetic/transcription factors during incipient AD is predominantly localized in and around white matter (supporting our prior findings and model), and that novel alterations in vascular and ryanodine receptor-related pathways in gray matter are closely associated with incipient AD.

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.

An increase in L-type voltage-gated calcium channel (LTCC) current is a prominent biomarker of brain aging and is believed to contribute to cognitive decline and vulnerability to neuropathologies. Studies examining age-related changes in LTCCs have focused primarily on males, although estrogen (17β-estradiol, E2) affects calcium-dependent activities associated with cognition. Therefore, to better understand brain aging in females, the effects of chronic E2 replacement on LTCC current activity in hippocampal neurons of young and aged ovariectomized rats were determined. The zipper slice preparation was used to expose cornu ammonis 1 (CA1) pyramidal neurons for recording LTCC currents using the cell-attached patch-clamp technique. We found that an age-related increase in LTCC current in neurons from control animals was prevented by E2 treatment. In addition,in situhybridization revealed that within stratum pyramidale of the CA1 area, mRNA expression of the Cav1.2 LTCC subunit, but not the Cav1.3 subunit, was decreased in aged E2-treated rats. Thus, the reported benefits of E2 on cognition and neuronal health may be attributed, at least in part, to its age-related decrease in LTCC current.

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.

Delayed excitotoxic neuronal death after insult from exposure to high glutamate concentrations appears important in several CNS disorders. Although delayed excitotoxicity is known to depend on NMDA receptor (NMDAR) activity and Ca2+elevation, the electrophysiological mechanisms underlying postinsult persistence of NMDAR activation are not well understood. Membrane depolarization and nonspecific cationic current in the postinsult period were reported previously, but were not sensitive to NMDAR antagonists. Here, we analyzed mechanisms of the postinsult period using parallel current- and voltage-clamp recording and Ca2+imaging in primary hippocampal cultured neurons. We also compared more vulnerable older neurons [about 22 days in vitro (DIV)] to more resistant younger (about 15 DIV) neurons, to identify processes selectively associated with cell death in older neurons. During exposure to a modest glutamate insult (20 μM, 5 min), similar degrees of Ca2+elevation, membrane depolarization, action potential block, and increased inward current occurred in younger and older neurons. However, after glutamate withdrawal, these processes recovered rapidly in younger but not in older neurons. The latter also exhibited a concurrent postinsult increase in spontaneous miniature excitatory postsynaptic currents, reflecting glutamate release. Importantly, postinsult NMDAR antagonist administration reversed all of these persisting responses in older cells. Conversely, repolarization of the membrane by voltage clamp immediately after glutamate exposure reversed the NMDAR-dependent Ca2+elevation. Together, these data suggest that, in vulnerable neurons, excitotoxic insult induces a sustained positive feedback loop between NMDAR-dependent current and depolarization-mediated glutamate release, which persists after withdrawal of exogenous glutamate and drives Ca2+elevation and delayed excitotoxicity.

Understanding the cellular mechanisms that characterize the functional changes of the aged brain is an ongoing and formidable challenge for the neuroscience community. Evidence now links changes in Ca(2+) influx and homeostasis with perturbations induced by the aging process in the function of the main intracellular organelles involved in Ca(2+) regulation: the endoplasmic reticulum and mitochondria. New perspectives are also offered by recent gene microarray studies, illustrating the multifactorial nature of the aging process.

The pathogenesis of incipient Alzheimer's disease (AD) has been resistant to analysis because of the complexity of AD and the overlap of its early-stage markers with normal aging. Gene microarrays provide new tools for addressing complexity because they allow overviews of the simultaneous activity of multiple cellular pathways. However, microarray data interpretation is often hindered by low statistical power, high false positives or false negatives, and by uncertain relevance to functional endpoints. Here, we analyzed hippocampal gene expression of nine control and 22 AD subjects of varying severity on 31 separate microarrays. We then tested the correlation of each gene's expression with MiniMental Status Examination (MMSE) and neurofibrillary tangle (NFT) scores across all 31 subjects regardless of diagnosis. These well powered tests revealed a major transcriptional response comprising thousands of genes significantly correlated with AD markers. Several hundred of these genes were also correlated with AD markers across only control and incipient AD subjects (MMSE > 20). Biological process categories associated with incipient AD-correlated genes were identified statistically ( ease program) and revealed up-regulation of many transcription factor/signaling genes regulating proliferation and differentiation, including tumor suppressors, oligodendrocyte growth factors, and protein kinase A modulators. In addition, up-regulation of adhesion, apoptosis, lipid metabolism, and initial inflammation processes occurred, and down-regulation of protein folding/metabolism/transport and some energy metabolism and signaling pathways took place. These findings suggest a new model of AD pathogenesis in which a genomically orchestrated up-regulation of tumor suppressor-mediated differentiation and involution processes induces the spread of pathology along myelinated axons.

Gene expression microarrays provide a powerful new tool for studying complex processes such as brain aging. However, inferences from microarray data are often hindered by multiple comparisons, small sample sizes, and uncertain relationships to functional endpoints. Here we sought gene expression correlates of aging-dependent cognitive decline, using statistical profiling of gene microarrays in well powered groups of young, mid-aged, and aged rats (n= 10 per group). Animals were trained on two memory tasks, and the hippocampal CA1 region of each was analyzed on an individual microarray (one chip per animal). Aging- and cognition-related genes were identified by testing each gene by ANOVA (for aging effects) and then by Pearson's test (correlating expression with memory). Genes identified by this algorithm were associated with several phenomena known to be aging-dependent, including inflammation, oxidative stress, altered protein processing, and decreased mitochondrial function, but also with multiple processes not previously linked to functional brain aging. These novel processes included downregulated early response signaling, biosynthesis and activity-regulated synaptogenesis, and upregulated myelin turnover, cholesterol synthesis, lipid and monoamine metabolism, iron utilization, structural reorganization, and intracellular Ca(2+) release pathways. Multiple transcriptional regulators and cytokines also were identified. Although most gene expression changes began by mid-life, cognition was not clearly impaired until late life. Collectively, these results suggest a new integrative model of brain aging in which genomic alterations in early adulthood initiate interacting cascades of decreased signaling and synaptic plasticity in neurons, extracellular changes, and increased myelin turnover-fueled inflammation in glia that cumulatively induce aging-related cognitive impairment.

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.

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.

Considerable evidence supports a Ca2+dysregulation hypothesis of brain aging and Alzheimer's disease. However, it is still not known whether (1) intracellular [Ca2+]iis altered in aged brain neurons during synaptically activated neuronal activity; (2) altered [Ca2+]iis directly correlated with impaired neuronal plasticity; or (3) the previously observed age-related increase in L-type voltage-sensitive Ca2+channel (L-VSCC) density in hippocampal neurons is sufficient to impair synaptic plasticity. Here, we used confocal microscopy to image [Ca2+]iin single CA1 neurons in hippocampal slices of young-adult and aged rats during repetitive synaptic activation. Simultaneously, we recorded intracellular EPSP frequency facilitation (FF), a form of short-term synaptic plasticity that is impaired with aging and inversely correlated with cognitive function. Resting [Ca2+]idid not differ clearly with age. Greater elevation of somatic [Ca2+]iand greater depression of FF developed in aged neurons during 20 sec trains of 7 Hz synaptic activation, but only if the activation triggered repetitive action potentials for several seconds. Elevated [Ca2+]iand FF also were negatively correlated in individual aged neurons. In addition, the selective L-VSCC agonist Bay K8644 increased the afterhyperpolarization and mimicked the depressive effects of aging on FF in young-adult neurons. Thus, during physiologically relevant firing patterns in aging neurons, postsynaptic Ca2+elevation is closely associated with altered neuronal plasticity. Moreover, selectively increasing postsynaptic L-VSCC activity, as occurs in aging, negatively regulated a form of short-term plasticity that enhances synaptic throughput. Together, the results elucidate novel processes that may contribute to impaired cognitive function in aging.

The sections in this article are: 1 Glucocorticoids in Relation to Stress and Aging 1.1 Association of Glucocorticoids with Brain Aging 1.2 Experimental Interventions in Glucocorticoid Actions 1.3 Caloric Restriction 1.4 Possible Trophic Actions of Glucocorticoids 1.5 Studies in Humans 2 Modulation of the Adrenal Axis 2.1 Basic Regulatory Mechanisms: Systemic, Receptor, and Genomic Levels 2.2 Adrenocorticoid Receptors: Effects of Stress and Aging 3 Cellular Mechanisms of Neuronal Toxicity of Glucocorticoids 3.1 Excitotoxicity and Glucose Metabolism 3.2 Ion Channels and Calcium Homeostasis 4 Conclusion

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.

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.

There is growing evidence that alterations in calcium (Ca2+) homeostasis may play a role in processes of brain aging and neurodegeneration. There also is evidence that some of the altered Ca2+ homeostasis in hippocampal neurons may arise from an increased density of L-type voltage sensitive Ca2+ channels (L-VSCC). In the present studies, we tested the possibility that previously observed increases in functional L-VSCC with aging might be related to up-regulated gene/mRNA expression for Ca2+ channel subunits. A significant aging-related increase in mRNA content for the alpha1D subunit of the L-type VSCC was observed in hippocampus of aged F344 rats (25 months old) relative to young (4 months old) and middle-aged animals (13 months old), as assessed by both in situ hybridization analyses (densitometry and grain density) and ribonuclease protection assay (RPA). In RPA analyses, the alpha1C subunit mRNA also showed a significant increase in 25-month-old rats. No age changes were seen in mRNA for the beta1b subunit of VSCC or for GAPDH, a standard control. The clearest increases in alpha1D mRNA expression were observed in subfield CA1, with little or no change seen in dentate gyrus. Although these results alone do not demonstrate that mRNA/gene expression changes contribute directly to changes in functional Ca2+ channels, they clearly fulfill an important prediction of that hypothesis. Therefore, these studies may have important implications for the role of gene expression in aging-dependent alterations in brain Ca2+ homeostasis.

Based on a literature implicating altered calcium homeostasis in brain aging and Alzheimer's Disease (AD) and evidence of decreased vitamin D action in AD subjects, the possibility was tested that calcitriol (1,25(OH)2 vitamin D3), the active form of vitamin D3, might reduce markers of brain aging in rats. Animals were treated 5x weekly for prolonged periods (6-12 months) with either calcitriol in doses sufficient to elevate serum calcium and phosphate (20 ng/rat), calcitonin (1.5 IU/rat) or vehicle, in three separate long-term experiments on aging rats. New stereological methods (physical disector) of cell counting were used to evaluate neuronal density, a reliable biomarker of hippocampal aging in rats. In two experiments utilizing Brown-Norway x F344 hybrid rats (BN x F344), 8 months and 12 months of chronic treatment with calcitriol resulted in a higher density of CA1 neurons in the middle regions of the hippocampus, compared to vehicle or calcitonin treatment. However, one study with aging F344 rats was terminated early because of extensive strain-specific pathology and no effect of calcitriol on neuronal density was observed. These studies suggest that, under some conditions, hormonal treatments that regulate calcium homeostasis can modulate markers of brain aging.

We measured in vivo forward flux of the creatine kinase reaction in rat forebrain in young (Y: 6 month, n = 13), mid-aged (M: 12 month, n = 7) and aged (O: 27 month, n = 10) animals using 31P magnetic resonance saturation transfer. Forward flux was reduced in the aged rats (Y: 0.42 +/- 0.08; M: 0.41 +/- 0.10; O: 0.31 +/- 0.03 s(-1) +/- SD; p = 0.008 O vs. Y). In vitro studies in a subset of the same rats showed a parallel decline in CK activity (Y: 2.16 +/- 0.40; M: 2.17 +/- 0.25; O: 1.56 +/- 0.06 IU +/- S.D.; p = 0.002 O vs. Y). The in vivo spectroscopic and in vitro biochemical measures were significantly correlated. Reduced creatine kinase activity could account for the observed decreased forward flux in aging brain. Intracellular pH, phosphocreatine/inorganic phosphate ratio, and phospocreatine/gamma-adenosine triphosphate ratio did not differ between groups. Forward flux may represent a better measure of brain energy function than relative phosphocreatine or adenosine triphosphate levels observable in vivo.

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.

This article considers the problems encountered in the study of basic electrophysiologlcal properties of aged animal brain cells and describes several methods that are useful for such studies. Specific methods for rat brain slice preparations are reviewed, with an emphasis on factors relevant to aging animals. Alternative approaches (acutely dissociated cells) are also considered. Methods for investigating pharmacologically isolated and defined calcium potentials and calcium currents in aged rat brain neurons also are described. These may play an important role in the brain aging process.

The glucocorticoid hypothesis of brain aging, which proposes that exposure of brain cells to glucocorticoids can increase brain cell loss during aging, is a physiological aging hypothesis that emphasizes interactions among systems. Thus, it fits well with the concepts advanced by Nathan Shock. The historical background of the hypothesis, the hypothesis and supporting data, and several modificatios are briefly reviewed here, as are recent mechanistic studies linking hippocampal glucocorticoid receptor activation to increased voltage-activated calcium influx. These new data suggest that some aspects of the impact of glucocorticoids on brain cells may increase rather than decrease with aging, and that some of the etiology of aging-related neurotoxicity may depend on gradually accumulating endocrine dysregulation of neuronal calcium homeostasis.

With aging, multiple Ca(2+)-associated electrophysiological processes exhibit increased magnitude in hippocampal pyramidal neurons, including the Ca(2+)-dependent slow afterhyperpolarization (sAHP), L-type voltage-gated Ca(2+) channel (L-VGCC) activity, Ca(2+)-induced Ca(2+) release (CICR) from ryanodine receptors (RyRs), and Ca(2+) transients. This pattern of Ca(2+) 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 Ca(2+) release. Moreover, we recently found that hippocampal Fkbp1b expression is downregulated, whereas Ryr2 and Frap1/Mtor (mammalian target of rapamycin) expression is upregulated with aging in rats. Here, we tested the hypothesis that disrupting FKBP1b function also destabilizes Ca(2+) homeostasis in hippocampal neurons and is sufficient to induce the aging phenotype of Ca(2+) dysregulation in young animals. Selective knockdown of Fkbp1b with interfering RNA in vitro (96 h) enhanced voltage-gated Ca(2+) current in cultured neurons, whereas in vivo Fkbp1b knockdown 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 knockdown in vivo was associated with upregulation of RyR2 and mTOR protein expression. Thus, disruption of FKBP1b recapitulated much of the Ca(2+)-dysregulation aging phenotype in young rat hippocampus, supporting a novel hypothesis that declining FKBP function plays a major role in unhealthy brain aging.

Openings of single L-type Ca2+ channels following repolarization to negative membrane potentials from a depolarizing step (repolarization openings, ROs) have been described previously in brain cell preparations. However, these ROs have been reported to occur only infrequently. Here we report that the frequency of ROs in cell-attached patches of cultured rat hippocampal neurons can be increased dramatically by lowering the pipette Ba2+ concentration to 20 mM from the usual 90-110 mM. This increased opening probability can last for hundreds to thousands of milliseconds following repolarization. Current-voltage analyses of open probability show that the depolarization pulse threshold for inducing ROs in 20 mM Ba2+ is -10 to 0 mV but that the probability of ROs reaches maximal levels following depolarizing pulses that approach the apparent null (equilibrium) potential for Ba2+. Comparable current-voltage curves in 110 mM Ba2+ from a more positive holding potential (-50 mV) indicate that membrane surface charge screening accounts for some, but not all, of the effect of lowering the Ba2+ concentration. Consequently, current-dependent inactivation or some other ion-dependent mechanism (e.g., ion binding inside the pore) also appears to regulate this potentially major pathway of Ca2+ entry. A high probability of ROs also can be induced under relatively physiological conditions (5-ms depolarizing steps, 2-5 mM Ca2+ in the pipette). Thus, the high open probability state at negative potentials may underlie the long Ca2+ tail currents in hippocampus that were described previously and appears to have major implications for physiological functions (e.g., the slow Ca(2+)-dependent afterhyperpolarization), particularly in brain neurons.

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.

The present study tested aspects of a novel etiological/pathogenetic hypothesis of Alzheimer's disease (AD), which proposes that alterations in endocrine regulation of peripheral calcium/phosphate (Ca/PO4) homeostasis (e.g., by glucocorticoids, vitamin D, etc.) induce and/or reflect altered calcium homeostasis and neurotoxicity in brain neurons. Two key predictions of this hypothesis were tested, namely that: (1) alterations in serum Ca and/or PO4 regulation should be present before or near the onset of AD and (2) these alterations should be found consistently in subjects with unconfounded AD. Previous studies have sought evidence of changes in Ca regulation primarily late in the disease, and usually in severely demented, thin and immobile inpatients in whom Ca/PO4 measures are likely to be confounded. In the present study, only mobile, relatively healthy, adequately nourished outpatients with probable AD were selected. In comparison to age-matched controls or demented subjects with even mild indications of vascular contributions, the AD subjects were characterized by lower serum PO4 and, to a lesser degree, lower serum Ca as well as a higher chloride/PO4 ratio. On serum chemistry tests from the first stages of AD (within 1 or 3 years after the onset of cognitive symptoms), these changes in serum PO4/Ca were as pronounced as they were later in the disease. Moderately low values of either PO4, Ca, or both (below -1.0 S.D. from the control group mean) identified 74% of all AD subjects and 100% of early onset AD subjects, in comparison to only 46% of mixed/vascular dementia subjects and 31% of normal age-matched controls. Thus, the results were consistent with the predictions and may have significant etiological/pathogenetic implications. If larger tests confirm these frequencies, serum Ca/PO4 indices may also prove useful in differential diagnosis.

In the present study, the effects of long-term dietary restriction (60% of the calories in the ad lib diet, beginning at 16 weeks of age) on quantitative morphometric measures and histopathologic indications of aging have been investigated in the retina of Fischer 344 male rats. The animals were maintained by the NIA Biomarkers Program, National Center for Toxicological Research. Group size ranged from 8 to 15 rats. A gradual thinning of the outer nuclear layer (ONL) of photoreceptor nuclei occurred with aging in control ad lib groups. The restricted diet did not affect retinal aging in 18-, 21-, 26-, or 27-month-old rats, as judged by photoreceptor cell death, ONL thickness, and pattern of cell loss. Retinal thickness (RT) was unaffected by restricted diet, except in the 21-month-old group; in that group, the RT was reduced significantly in thickness as compared to ad lib animals. These results are in contrast to studies of dietary restriction on most nonneuronal markers of aging and suggest that a different mechanism may modulate at least some aspects of brain aging.

There is increasing evidence that experimental interventions that alter adrenal corticosteroid plasma concentrations can modulate aging changes in the rodent hippocampus. However, there still is very little evidence that elevation of endogenous corticosteroid levels within physiological ranges, such as occurs during chronic stress, can accelerate hippocampal aging-like changes. In addition, almost all prior intervention studies of corticosteroid effects on brain biomarkers of aging have utilized morphologic measures of aging, and it is not yet clear whether electrophysiologic biomarkers of hippocampal aging can also be accelerated by conditions that elevate corticosteroids. In the present studies, specific pathogen-free rats of three ages (4, 12, and 18 months at the start) were trained for 6 months (4 hr/d, 5 d/week) in a two-way shuttle escape task, using low intensity foot shock. This task induces “anxiety” stress, because animals receive little actual shock, but chronic training in the task has been shown to elevate plasma corticosteroids and to downregulate hippocampal corticosteroid receptors. At the end of 6 months, animals were allowed to recover for 3 weeks and were then assessed in acute, anesthetized preparations on a battery of hippocampal neurophysiological markers known to separate young from aged animals (frequency potentiation, synaptic excitability thresholds, EPSP amplitude). The brains were then fixed and sectioned for quantification of neuronal density in field CA1 (a highly consistent anatomic marker of hippocampal aging). The pattern of stress effects differed considerably across age groups. The two younger stress groups exhibited increased evidence of aging-like neurophysiologic change, but exhibited no indications of accelerated neuronal loss.(ABSTRACT TRUNCATED AT 250 WORDS)

identified SEN12333 (WAY-317538) as a full agonist with an EC50 of 1.2 microM and selective over related receptors. In previous experiments, this new alpha7 nAChR agonists has demonstrated pro-cognitive properties in both Alzheimer disease as well as schizophrenia oriented behavioural tests in rodents. Here we describe experiments aimed to evaluate the neuroprotective property of this compound in in vivo models of neurodegeneration in comparison with nicotine and to investigate the mechanisms of neuroprotection. Results: Five microL of 0.5 M quisqualic acid solution injected into the nucleus basalis magnocellularis (NBM) of rats resulted in a highly significant decrease in the number of ChAT-positive neurons, massive reactive gliosis, involving both astrocytes and microglia, and a significant increase in p38-MAPK expression. A sub-chronic treatment with 3 mg/kg SEN12333 or 0.3 mg/kg nicotine for 7 days significantly attenuated the decrease in the number of ChAT-positive neurons in this brain region demonstrating neuroprotective effects. Conclusions: These effects may be relevant for treating neurodegenerative diseases such as Alzheimer’s disease. The ability of nicotinic agonist-treatments to protect brain tissues from gliosis and inflammation will be discussed.

Calcium (Ca) spike potentials were investigated in cesium-loaded, tetrodotoxin (TTX)-treated CA1 pyramidal cells in hippocampal slices from young-mature and aged rats. The duration of single Ca spike potentials was prolonged in cells from aged rats, indicating that previously observed age-related changes in Ca-dependent mechanisms (e.g. in the K-mediated after hyperpolarization and in frequency potentiation) may result from an age-related increase of voltage-dependent Ca conductance. Since we recently found that Ca spike duration in hippocampus can be modulated strongly by a form of Ca-dependent inactivation of Ca current, spike inactivation paradigms also were examined. However, following 5- or 10-s-long depolarizing pulses, or during a 2-Hz train of elicited Ca spikes, there were no age differences in percent inactivation. These results do not support (but do not fully rule out) the possibility that impaired Ca-dependent inactivation underlies the increase in the Ca spike with aging. Conceivably, this prolongation of voltage-dependent Ca influx could have implications for our understanding of normal and abnormal brain aging.

The original glucocorticoid (GC) hypothesis of brain aging and Alzheimer's disease proposed that chronic exposure to GCs promotes hippocampal aging and AD. This proposition arose from a study correlating increasing plasma corticosterone with hippocampal astrocyte reactivity in aging rats. Numerous subsequent studies have found evidence consistent with this hypothesis, in animal models and in humans. However, several results emerged that were inconsistent with the hypothesis, highlighting the need for a more definitive test with a broader panel of biomarkers. We used microarray analyses to identify a panel of hippocampal gene expression changes that were aging-dependent, and also corticosterone-dependent. These data enabled us to test a key prediction of the GC hypothesis, namely, that the expression of most target biomarkers of brain aging should be regulated in the same direction (increased or decreased) by both GCs and aging. This prediction was decisively contradicted, as a majority of biomarker genes were regulated in opposite directions by aging and GCs, particularly inflammatory and astrocyte-specific genes. Thus, the initial hypothesis of simple positive cooperativity between GCs and aging must be rejected. Instead, our microarray data suggest that in the brain GCs and aging interact in more complex ways that depend on the cell type. Therefore, we propose a new version of the GC-brain aging hypothesis; its main premise is that aging selectively increases GC efficacy in some cell types (e.g., neurons), enhancing catabolic processes, whereas aging selectively decreases GC efficacy in other cell types (e.g., astrocytes), weakening GC anti-inflammatory activity. We also propose that changes in GC efficacy might be mediated in part by cell type specific shifts in the antagonistic balance between GC and insulin actions, which may be of relevance for Alzheimer's disease pathogenesis.

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.