Reduced mitochondrial buffering of voltage-gated calcium influx in aged rat basal forebrain neurons
Introduction
Changes in neuronal calcium (Ca2+) homeostasis have been implicated frequently as a mechanism of age-related neuronal pathology and degeneration [1], [2]. For instance, excessive Ca2+ influx and accumulation in mitochondria are key events in neuronal cell death [3], [4]. However, it has been recognized recently that normal aging (non-pathological) is not characterized by extensive loss of neurons [5]. Rather, it is thought that age-related cognitive deficits are mediated by more subtle changes, such as reduced synaptic plasticity or altered intracellular signaling [6], [7], [8]. Because Ca2+ controls or modulates an impressive array of intracellular processes, alterations in Ca2+ homeostasis could have a critical impact on the physiological function of neurons, and might be responsible for many of the cognitive aspects of aging [9].
Mitochondrial Ca2+ buffering is not only a feature of pathological conditions, but also is central to normal physiology and Ca2+ signaling [10], [11], [12], [13]. Uptake of Ca2+ by mitochondria is mediated by a uniporter which is activated by elevated cytoplasmic [Ca2+] and which allows Ca2+ to enter the matrix down the steep electrochemical gradient (composed mostly of a −150 to −180 mV mitochondrial membrane potential, Ψm) that is maintained by selective pumps and conductances. Most Ca2+ entering mitochondria is bound to some buffer or as a phosphate salt [14], while most remaining free Ca2+ is released shortly after uptake [15]. In neurons, mitochondrial Ca2+ release mechanisms include prominent Na+/Ca2+ exchange (reviews [16], [17]). By means of this mitochondrial Ca2+ uptake and release, the spatial and temporal profiles of intracellular Ca2+ signals can be controlled [18], thus modifying the operation of Ca2+ sensitive processes. Mitochondrial Ca2+ uptake increases the activity of several dehydrogenases [19], which drive the operation of the electron transport chain and ATP synthesis, thereby matching energy production with demand [20]. Neurons of the mammalian basal forebrain are known to be susceptible to age-related pathology and are thought to be involved also in cognitive decline associated with normal aging [21], [22], [23]. We have used acutely dissociated neurons of the rat basal forebrain (medial septum and nucleus of the diagonal band, MS/nDB) as a model for investigating age-related changes in cellular Ca2+ homeostasis. Ca2+ may enter the cytoplasm by influx through voltage- or ligand-gated channels, influx through store-operated or capacitative pathways and by release from intracellular stores. Ca2+ homeostatic mechanisms include rapid buffers that limit the peak intracellular Ca2+ concentration ([Ca2+]i) in the cytoplasm to a fraction of that expected given the Ca2+ influx. Additionally, there are slow buffering mechanisms that functionally clear Ca2+ from the cytoplasm by extrusion or storage and restore the resting [Ca2+]i.
Our previous observations led us to propose that an age-related increase in Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is compensated by a corresponding increase in rapid Ca2+ buffering that was not mediated by endoplasmic reticulum (ER) uptake [24], [25]. We sought to determine whether an age-related increase in mitochondrial Ca2+ uptake after Ca2+ influx through VGCCs could explain the increase in rapid Ca2+ buffering observed with age in these neurons. By measuring the changes in [Ca2+]i arising from a depolarizing stimulus in the absence and presence of mitochondrial Ca2+ uptake inhibitors, we show that mitochondrial buffering of Ca2+ influx through VGCCs is significantly reduced with age and that the age-related increase in rapid buffering is not attributable to mitochondria. Because mitochondrial buffering does not commence until moderately high levels of Ca2+ influx are achieved, the interaction of this mechanism with the other rapid buffers functionally results in a smaller Δ[Ca2+]i being experienced by aged neurons for a given small Ca2+ influx, but as influx increases, this difference from young neurons disappears due to the mitochondrial buffering deficit. Experiments with confocal fluorescence microscopy of the mitochondrially located potentiometric indicator JC-1 suggest that a reduced Ψm may contribute to the age-related change in mitochondrial buffering.
Section snippets
Experimental animals
Male Fischer 344 rats were purchased from Harlan (Indianapolis IN, NIA breeding colony). Animals took food and water ad libitum and were maintained on a 12 h light/dark cycle. Handling and care of the animals was in accordance with policies of Texas A&M University and the National Institute of Health. Neurons were harvested from 39 young adult rats (1–7 months, mean 2.6 month) and 17 aged rats (24–27 months, mean 25.6 month).
Acutely dissociated neurons
Individual MS/nDB neurons were obtained as described previously [24].
Control experiments
Mitochondrial uncouplers, such as CCCP, have been used frequently to investigate the involvement of mitochondria in Ca2+ buffering. CCCP is a protonophore that inserts into the mitochondrial inner membrane and allows H+ to enter down the steep electrochemical gradient, thus dissipating Ψm, which is the principal driving force for mitochondrial Ca2+ uptake. However, CCCP might depolarize the plasma membrane, acidify the cytoplasm and rapidly deplete ATP due to reversal of the ATP synthase. These
Discussion
The evidence presented here demonstrates functional alterations of in situ mitochondria in central mammalian neurons during aging. In aged rat MS/nDB neurons, these changes have a definite impact on the physiology of Ca2+ homeostasis, and by inference, on Ca2+ signaling and metabolism. Interactions between buffers and the capacity for age-related compensatory responses may underlie many of the subtle alterations in Ca2+ signaling which are thought to mediate changes observed in synaptic
Conclusion
It is now accepted that mitochondrial Ca2+ uptake and release is associated not only with pathological conditions, but also with physiological function in a number of cell types [10], [11], [12], [13]. Any age-related alterations of mitochondrial function, such as those described here, could be the critical determinants of mild cognitive decline with age [90]. As the evidence for decreased mitochondrial function with age accumulates, the critical question becomes: Can reversing or preventing
Acknowledgements
Special thanks for the technical assistance of Dr. Nancy Dawson. Supported by NIH grant AG007805.
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