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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., (© 2013 Published by Elsevier B.V.)

During normal brain aging, numerous alterations develop in the physiology, biochemistry and structure of neurons and glia. Aging changes occur in most brain regions and, in the hippocampus, have been linked to declining cognitive performance in both humans and animals. Age-related changes in hippocampal regions also may be harbingers of more severe decrements to come from neurodegenerative disorders such as Alzheimer's disease (AD). However, unraveling the mechanisms underlying brain aging, AD and impaired function has been difficult because of the complexity of the networks that drive these aging-related changes. Gene microarray technology allows massively parallel analysis of most genes expressed in a tissue, and therefore is an important new research tool that potentially can provide the investigative power needed to address the complexity of brain aging/neurodegenerative processes. However, along with this new analytic power, microarrays bring several major bioinformatics and resource problems that frequently hinder the optimal application of this technology. In particular, microarray analyses generate extremely large and unwieldy data sets and are subject to high false positive and false negative rates. Concerns also have been raised regarding their accuracy and uniformity. Furthermore, microarray analyses can result in long lists of altered genes, most of which may be difficult to evaluate for functional relevance. These and other problems have led to some skepticism regarding the reliability and functional usefulness of microarray data and to a general view that microarray data should be validated by an independent method. Given recent progress, however, we suggest that the major problem for current microarray research is no longer validity of expression measurements, but rather, the reliability of inferences from the data, an issue more appropriately redressed by statistical approaches than by validation with a separate method. If tested using statistically defined criteria for reliability/significance, microarray data do not appear a priori to require more independent validation than data obtained by any other method. In fact, because of added confidence from co-regulation, they may require less. In this article we also discuss our strategy of statistically correlating individual gene expression with biologically important endpoints designed to address the problem of evaluating functional relevance. We also review how work by ourselves and others with this powerful technology is leading to new insights into the complex processes of brain aging and AD, and to novel, more comprehensive models of aging-related brain change.

The Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, modulates a number of key Ca(2+) signaling pathways in neurons, and has been implicated in Ca(2+)-dependent negative feedback inactivation of N-methyl-D-aspartate receptors and voltage-sensitive Ca(2+) channels. In contrast, we report here that three mechanistically disparate calcineurin inhibitors, FK-506, cyclosporin A, and the calcineurin autoinhibitory peptide, inhibited high-voltage-activated Ca(2+) channel currents by up to 40% in cultured hippocampal neurons, suggesting that calcineurin acts to enhance Ca(2+) currents. This effect occurred with Ba(2+) or Ca(2+) as charge carrier, and with or without intracellular Ca(2+) buffered by EGTA. Ca(2+)-dependent inactivation of Ca(2+) channels was not affected by FK-506. The immunosuppressant, rapamycin, and the protein phosphatase 1/2A inhibitor, okadaic acid, did not decrease Ca(2+) channel current, showing specificity for effects on calcineurin. Blockade of L-type Ca(2+) channels with nimodipine fully negated the effect of FK-506 on Ca(2+) channel current, while blockade of N-, and P-/Q-type Ca(2+) channels enhanced FK-506-mediated inhibition of the remaining L-type-enriched current. FK-506 also inhibited substantially more Ca(2+) channel current in 4-week-old vs. 2-week-old cultures, an effect paralleled by an increase in calcineurin A mRNA levels. These studies provide the first evidence that calcineurin selectively enhances L-type Ca(2+) channel activity in neurons. Moreover, this action appears to be increased concomitantly with the well-characterized increase in L-type Ca(2+) channel availability in hippocampal neurons with age-in-culture.

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 Ca(2+)-dependent afterhyperpolarization as well as the Ca2+ action potential were significantly larger in aged rat neurons. Using the sharp electrode 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 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.

It has been recognized for some time that the Ca2+-dependent slow afterhyperpolarization (sAHP) is larger in hippocampal neurons of aged compared with young animals. In addition, extensive studies since have shown that other Ca2+-mediated electrophysiological responses are increased in hippocampus with aging, including Ca2+ transients, L-type voltage-gated Ca2+ channel activity, Ca2+ spike duration and action potential accommodation. Elevated Ca2+-induced Ca2+ release from ryanodine receptors (RyRs) appears to drive amplification of the Ca2+ responses. Components of this Ca2+ 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 Ca2+ 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 Ca2+-induced Ca2+ 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 Ca2+ 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 Ca2+ dysregulation in the brain and point to potential new therapeutic targets for counteracting unhealthy brain aging. [ABSTRACT FROM AUTHOR]