FK506-binding protein 1b/12.6: a key to aging-related hippocampal Ca2+ dysregulation?

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.

Electrophysiological mechanisms of delayed excitotoxicity: positive feedback loop between NMDA receptor current and depolarization-mediated glutamate release.

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 Ca(2+) 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 Ca(2+) 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 microM, 5 min), similar degrees of Ca(2+) 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 Ca(2+) 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 Ca(2+) elevation and delayed excitotoxicity.

The neurobiology of aging.

Basic principles of the neurobiology of aging were reviewed within selected topic areas chosen for their potential relevance to epileptogenesis in the aging brain. The availability of National Institute on Aging-supported aged mouse and rat strains and other biological resources for studies of aging and age-associated diseases was presented, and general principles of animal use in gerontological research were discussed. Neurobiological changes during normal brain aging were compared and contrasted with neuropathological events of Alzheimer's disease (AD) and age-associated memory impairment (AAMI). Major themes addressed were the loss of synaptic connections as vulnerable neurons die and circuits deteriorate in AD, the absence of significant neuron loss but potential synaptic alteration in the same circuits in AAMI, and the effects of decreased estrogen on normal aging. The "calcium hypothesis of brain aging" was examined by a review of calcium dyshomeostasis and synaptic communication in aged hippocampus, with particular emphasis on the role of L-type voltage-gated calcium channels during normal aging. Established and potential mechanisms of hippocampal plasticity during aging were discussed, including long-term potentiation, changes in functional connectivity, and increased gap junctions, the latter possibly being related to enhanced network excitability. Lastly, application of microarray gene chip technology to aging brain studies was presented and use of the hippocampal "zipper slice" preparation to study aged neurons was described.

Harnessing the power of gene microarrays for the study of brain aging and Alzheimer's disease: statistical reliability and functional correlation.

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.

Calcineurin enhances L-type Ca(2+) channel activity in hippocampal neurons: increased effect with age in culture.

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.

Epilepsy-induced decrease of L-type Ca2+ channel activity and coordinate regulation of subunit mRNA in single neurons of rat hippocampal 'zipper' slices.

L-type voltage-sensitive Ca2+ channels (VSCCs) preferentially modulate several neuronal processes that are thought to be important in epileptogenesis, including the slow afterhyperpolarization (AHP), LTP, and trophic factor gene expression. However, little is yet known about the roles of L-type VSCCs in the epileptogenic process. Here, we used cell-attached patch recording techniques and single cell mRNA analyses to study L-type VSCCs in CA1 neurons from partially dissociated (zipper) hippocampal slices from entorhinally-kindled rats. L-type Ca2+-channel activity was reduced by >50% at 1.5-3 months after kindling. Following recording, the same single neurons were extracted and collected for mRNA analysis using a recently developed method that does not amputate major dendritic processes. Therefore, neurons contained essentially full complements of mRNA. For each collected neuron, mRNA contents for the L-type pore-forming alpha1D/Ca(v)1.3-subunit and for calmodulin were then analyzed by semiquantitative kinetic RT-PCR. L-type alpha1D-subunit mRNA was correlated with L-type Ca2+-channel activity across single cells, whereas calmodulin mRNA was not. Thus, these results appear to provide the first direct evidence at the single channel and gene expression levels that chronic expression of an identified Ca2+-channel type is modulated by epileptiform activity. Moreover, the present data suggest the hypothesis that down regulation of alpha1D-gene expression by kindling may contribute to the long-term maintenance of epileptiform activity, possibly through reduced Ca2+-dependent AHP and/or altered expression of other relevant genes.

Expression of alpha 1D subunit mRNA is correlated with L-type Ca2+ channel activity in single neurons of hippocampal "zipper" slices.

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 alpha(1D) pore-forming subunit. If so, alpha(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 alpha(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 alpha(1D), but not CaM, mRNA expression. Thus, these studies support the possibility that the level of alpha(1D) gene expression regulates the density of functional L-VSCCs.

Decreased G-protein-mediated regulation and shift in calcium channel types with age in hippocampal cultures.

The membrane density of L-type voltage-sensitive Ca(2+) channels (L-VSCCs) of rat hippocampal neurons increases over age [days in vitro (DIV)] in long-term primary cultures, apparently contributing both to spontaneous cell death and to enhanced excitotoxic vulnerability. Similar increases in L-VSCCs occur during brain aging in vivo in rat and rabbit hippocampal neurons. However, unraveling both the molecular basis and the functional implications of these age changes in VSCC density will require determining whether the other types of high-threshold VSCCs (e.g., N, P/Q, and R) also exhibit altered density and/or changes in regulation, for example, by the important G-protein-coupled, membrane-delimited inhibitory pathway. These possibilities were tested here in long-term hippocampal cultures. Pharmacologically defined whole-cell currents were corrected for cell size differences over age by normalization with whole-cell capacitance. The Ca(2+) channel current density (picoamperes per picofarad), mediated by each Ca(2+) channel type studied here (L, N, and a combined P/Q + R component), increased through 7 DIV. Thereafter, however, only L-type current density continued to increase, at least through 21 DIV. Concurrently, pertussis toxin-sensitive G-protein-coupled inhibition of non-L-type Ca(2+) channel current induced by the GABA(B) receptor agonist baclofen or by guanosine 5'-3-O-(thio)triphosphate declined dramatically with age in culture. Thus, the present studies identify selective and novel parallel mechanisms for the time-dependent alteration of Ca(2+) influx, which could importantly influence function and vulnerability during development and/or aging.

Aging changes in voltage-gated calcium currents in hippocampal CA1 neurons.

Previous current-clamp studies in rat hippocampal slice CA1 neurons have found aging-related increases in long-lasting calcium (Ca)-dependent and Ca-mediated potentials. These changes could reflect an increase in Ca influx through voltage-gated Ca channels but also could reflect a change in potassium currents. Moreover, if altered Ca influx is involved, it is nuclear whether it arises from generally increased Ca channel activity, lower threshold, or reduced inactivation. To analyze the basis for altered Ca potentials, whole-cell voltage-clamp studies of CA1 hippocampal neurons were performed in nondissociated hippocampal slices of adult (3- to 5-month-old) and aged (25- to 26-month-old) rats. An aging-related increase was found in high-threshold Ca and barium (Ba) currents, particularly in the less variable, slowly inactivating (late) current at the end of a depolarization step. Input resistance of neurons did not differ between age groups. In steady-state inactivation and repetitive-pulse protocols, inactivation of Ca and Ba currents was not reduced and, in some cases, was slightly greater in aged neurons, apparently because of larger inward current. The current blocked by nimodipine was greater in aged neurons, indicating that some of the aging increase was in L-type currents. These results indicate that whole-cell Ca currents are increased with aging in CA1 neurons, apparently attributable to greater channel activity rather than to reduced inactivation. The elevated Ca influx seems likely to play a role in impaired function and enhanced susceptibility to neurotoxic influences.

Activation of K+ channels and suppression of neuronal activity by secreted beta-amyloid-precursor protein.

The Alzheimer's beta-amyloid precursor protein (beta-APP) is widely expressed in neural cells, and in neurons secreted forms of beta-APP (sAPPs) are released from membrane-spanning holo-beta APP in an activity-dependent manner. Secreted APPs can modulate neurite outgrowth, synaptogenesis, synaptic plasticity and cell survival; a signal transduction mechanism of sAPPs may involve modulation of intracellular calcium levels ([Ca2+]i). Here we use whole-cell perforated patch and single-channel patch-clamp analysis of hippocampal neurons to demonstrate that sAPPs suppress action potentials and hyperpolarize neurons by activating high-conductance, charybdotoxin-sensitive K+ channels. Activation of K+ channels by sAPPs was mimicked by a cyclic GMP analogue and sodium nitroprusside and blocked by an antagonist of cGMP-dependent kinase and a phosphatase inhibitor, suggesting that the effect is mediated by cGMP and protein dephosphorylation. Calcium imaging studies indicate that activation of K+ channels mediates the ability of sAPPs to decrease [Ca2+]i. Modulation of neuronal excitability may be a major mechanism by which beta-APP regulates developmental and synaptic plasticity in the nervous system.

Single-channel and whole-cell studies of calcium currents in young and aged rat hippocampal slice neurons.

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.