I. Ca²⁺ HYPOTHESIS OF AGING

It is now more than two decades since the first proposal of a “Ca²⁺ hypothesis of aging” [1]. In its mature formulation, the hypothesis aimed to provide a working hypothesis for explaining not only the aging process but also Alzheimer’s disease (AD). Drawing on many neurophysiological processes and mechanisms, the hypothesis contained six interrelated key elements [2], providing an extremely wide explanatory blanket. The exposition of these elements is worth repeating not only for historical reasons and to illustrate the breath of the proposal, but also because it still represents the source of some misunderstandings.

The first element of Khachaturian’s construct was simply that the cellular mechanisms that participate in Ca²⁺ homeostasis also play an important role in the process of aging and neurodegeneration. Consequently, altered Ca²⁺ homeostasis can account for a number of age-related changes associated with either aging or AD. The second element was that normal aging and the process of neurodegeneration, as in AD, must be seen as part of a continuum of processes associated with development and restructuring of the nervous system throughout life. Thus, the morphological changes in AD, such as dendritic pruning and loss of synapses and neurons, involve the same mechanisms that are responsible for neuroplasticity in the developing or mature brain. As an extension of the previous point, the third element of the hypothesis concentrated on the plasticity of the nervous system architecture seen, at any time point, as a balance between growth/regeneration and decaying/degeneration processes. In many of these processes the intracellular Ca²⁺ ([Ca²⁺]_i) plays a crucial role and, from this perspective, the neuronal dysfunction of the aged or the AD brain results from an imbalance between the two types of opposing processes, an imbalance that can result from a breakdown of the homeostatic mechanisms regulating [Ca²⁺]_i. The next, rather contentious element dealt with the temporal dimension of the aging/neurodegenerative processes and proposed that the functional product of the perturbation in [Ca²⁺]_i homeostasis and time is a constant. Thus, small changes in [Ca²⁺]_i (as those resulting, for example, from a less efficient Ca²⁺ extrusion pump, or an enhanced Ca²⁺ channel activity) exerted over a long period of time would have the same functional or morphological effects as a massive, but acute insult. It is clear now that the actions of Ca²⁺ in different temporal domains (i.e., over milliseconds, seconds, minutes, or days) generate very different types of responses and functional outcomes. A fifth element took into account the cellular effects of Ca²⁺ signaling and proposed that Ca²⁺-mediated processes are an important part of the final common pathway that leads to neuronal dysfunction and cell death. The model presented at this point an extremely broad and flexible perspective, and almost any element along the path leading from the triggers of Ca²⁺ dysregulation to various Ca²⁺ homeostatic mechanisms to, eventually, any of the Ca²⁺-dependent processes could fit the bill of explaining the involvement of Ca²⁺ in the process of aging and neurodegeneration. This point was even further expanded by the sixth element, which proposed that both the age-related and the AD-associated changes are not single factorial events but could be initiated by a wide variety of instances, acting single or in combination, simultaneous or sequentially, over a long period of time.

Underlying this hypothesis were two powerful ideas that reflected the predominant views of the time. One was that the difference between aging and neurodegeneration is just a matter of degree, both existing along a continuous path. The other idea was that intracellular Ca²⁺ signaling is one of the most important transducing processes involved in a wide range of cellular processes [3, 4]. Linking the two was the concept of Ca²⁺ regulation of neuronal death through the process of excitotoxicity. The term “excitotoxicity” was first coined by Olney in 1970 to describe the excessive activation of glutamate receptors that follows neuronal deprivation of oxygen and/or nutrients and resulting in neuronal death [5]. The subsequent demonstration in 1987 of the involvement of Ca²⁺ in mediating the excitotoxic outcome [6] strengthened the “Ca²⁺ hypothesis of aging.”

Today, of the two central ideas discussed above, only the latter remains unchallenged; the issue of understanding the aging as a neurodegenerative process is very much disputed. From animal studies it is clear that the age-related changes in functional and biochemical characteristics of the cortical circuitry that might underlie the moderate cognitive decline do not involve frank neuronal death but a significant amount of synaptic restructuring [7]. Some of the reasons why, from the late 1960s to the mid-to-late 1980s, the dogma of aging centered on the idea of associated significant neuronal loss were technological (the modern unbiased, dissector method for counting neurons was introduced only in 1984 [8] and helped demonstrate significant differences between neuronal numbers in normal aging and in neurodegenerative diseases [9]; see also Chapter 4) and methodological, involving the possible inclusion in some studies of material coming from subjects already affected by neurodegenerative diseases, (cf. [10]). Thus, in its initial formulation that links dysregulation of Ca²⁺ homeostasis to neuronal loss, the “Ca²⁺ hypothesis” is not directly applicable anymore to the process of normal aging, but retains much of its explanatory power in the context of Alzheimer’s disease (see [11] for a recent review). What is undisputed, however, is the fact that intracellular Ca²⁺ signaling and its regulation are central to neuronal physiology, linking with a variety of other processes (Figure 14.1).

FIGURE 14.1

(SEE COLOR INSERT FOLLOWING PAGE 204) Scope of Ca²⁺ signaling. The figure illustrates some of the main targets of Ca²⁺ signaling, through which Ca²⁺ exerts its huge range of functional effects on cellular (neuronal) physiology [168]. For some of these (more...)

II. Ca²⁺ HOMEOSTASIS

A. CA²⁺ Entry and CA²⁺ Extrusion Pathways

At any time point, the values of intracellular free Ca²⁺, as measured routinely by Ca²⁺-sensitive fluorescent dyes with different affinities, represent the functional balance between processes that increase and those that decrease [Ca²⁺]_i (Figure 14.2). Neuronal activation usually is associated with increases in [Ca²⁺]_i. For neurons, the source for these activation-induced Ca²⁺ responses is the extracellular medium, with Ca²⁺ ions flowing along a very steep (10,000-fold) concentration gradient (at rest [Ca]_i is around 50 to 100 nM, whereas the extracellular Ca²⁺ is around 1.5 to 2 mM). This process involves a number of different types of ion channels; some are activated by the depolarization of the plasma membrane and have exclusive selectivity for Ca²⁺ ions (the voltage-operated Ca²⁺ channels, VOCCs). These channels exist as several types and are functionally divided into two categories: (1) the low voltage-activated (LVA) channels (prototype, the former T-type VOCC and current Cav3 family — for new VOCC nomenclature, see [12]), activated by smaller depolarizations of the plasma membrane; and (2) the high voltage-activated (HVA) channels. Of these HVAs, the best-known functionally is the L-type Ca²⁺ channel (current Cav1 family), but the family also comprises the N-, P-, Q-, and R-types (all part of the Cav2 family) [13]. Additional types of channels involved in generating Ca²⁺ signals in neuronal cytosol are the ones activated by specific ligands (ligand operated Ca²⁺ channels, LOCs), such as NMDA, AMPA (or, in vivo, glutamate) or ATP (purinergic P2X receptors). These are nonspecific cationic channels, with a Ca²⁺ permeability that is usually lower than that for Na⁺ (the latter ion having, nevertheless, a much lower electrochemical gradient). Another type of neuronal plasma membrane channel with Ca²⁺ permeability is represented by the second messenger-operated Ca²⁺ channels (SMOCs), with cyclic nucleotides or arachidonate as activators. Finally, a ubiquitous and ever-expanding family of nonselective plasma membrane channels is that of the transient receptor potential (TRP) channels, initially described during the study of the photoreceptor transduction process.

FIGURE 14.2

(SEE COLOR INSERT FOLLOWING PAGE 204) Diagrammatic presentation of the roles of Ca²⁺ in the synaptic transmission of information. At the presynaptic site, the major pathway of Ca²⁺ increase is the activation of the VOCCs that follows the depolarization (more...)

Recovery of resting [Ca]_i values following such activation-evoked Ca²⁺ signals, which can generate increases of [Ca]_i well in excess of 10 μM, taking into account the confined small volumes where these signals appear (synaptic boutons, dendritic spines), is an energy-dependent process involving either pumps (ATPases) or specific transporters. The pumps remove cytosolic Ca²⁺ either to the extracellular medium (the plasma membrane Ca²⁺ ATPase, PMCA) or into the endoplasmic reticulum, which is a very important Ca²⁺ storing organelle (through the sarco(endo)plasmic Ca²⁺ ATPase, SERCA). A specific transporter is the Na⁺/Ca²⁺ exchanger (NCX), which harnesses the electrochemical gradient of Na⁺. These transport systems have different thresholds for activity. While the pumps have lower transport rates, they have a higher affinity for Ca²⁺ and thus are able to respond to smaller changes in [Ca]_i and ultimately to set the basal Ca²⁺ levels.

III. EFFECTS OF AGING ON Ca²⁺ HOMEOSTATIC SYSTEMS

There are relatively few direct experimental data analyzing and describing the putative age-related changes of the various Ca²⁺ homeostatic mechanisms. Overall, they tend to show dysfunction but, as discussed at length elsewhere, the interpretation is rendered difficult by the use of a variety of cellular preparations and by the heterogeneity of neuronal composition and ratio of glia to neurons in various regions of the brain [32].

D. Effects of Aging on Intracellular CA²⁺ Compartments

1. Endoplasmic Reticulum

The endoplasmic reticulum (ER) is an important intracellular Ca²⁺ store and, depending on its refill status, can act either as a “Ca²⁺ sink,” buffering through SERCA pump-mediated Ca²⁺ reuptake into the ER, or as a “Ca²⁺ source,” through a process that involves Ca²⁺-induced Ca²⁺ release (CICR; [15]). When discussing the role of ER Ca²⁺ stores in neuronal physiology, a number of issues must be addressed, including:

• The morphology of the ER and its distribution is not uniform throughout the neuron. It is particularly patchy in the dendritic territory and is influenced by the morphology of the spine and the age of the preparation [84]. Thus, in adult animals, less than 25% of the thin spines contain SER, whereas almost 90% of the larger mushroom spines have SER. When all types of spines are analyzed, SER is present only in 50% of the spines (slightly more in the young preparations: 58% in immature brains vs. 48% in the adult spines). The important implication of these observations is that in about half of the spines, where synaptic activity presumably is still taking place, the entry of Ca²⁺ is buffered through mechanisms that do not involve ER Ca²⁺ reuptake. Where present, however, the ER is ideally adapted for Ca²⁺ uptake as its surface-to-ratio area is very large, with the SER occupying only about 3% of the volume but up to 40% of the surface of the spine territory.
• In addressing the issue of the participation of the CICR in the control and modulation of the Ca²⁺ synaptic response, there appear to be differences between the various types of CNS neurons, particularly the cerebellar Purkinje neurons and the hippocampal pyramidal neurons, in respect to the mechanisms of release of Ca²⁺ from intracellular ER stores. In Purkinje neurons, the main ER Ca²⁺ release pathway is the InsP₃ receptor. As demonstrated in 1998 by two independent, but almost simultaneous, studies [85, 86], parallel fiber stimulation activates postsynaptically, even in the absence of membrane depolarization, a Ca²⁺ signal mediated by the activation of the mGluR1 receptors and which is able to trigger LTD. For the hippocampal neurons, the picture is more complex. For a start, the distribution of the Ca²⁺ release channels on the ER varies with the localization for the CA1 neurons; there are no InsP₃ receptors, only RynR, in the spines, whereas in the dendritic shaft both receptors are present [87]. Furthermore, whereas some studies asserted a significant role for the Ca²⁺-induced Ca²⁺ release (CICR) process in postsynaptic Ca²⁺ signaling [88], others failed to detect a significant contribution [89].
• Intracellular Ca²⁺ stores can serve as “sinks” or “sources,” and the role played by the ER Ca²⁺ stores depends to a significant degree on their filling status. It was shown in earlier studies that Ca²⁺ responses that involve mobilization from intracellular stores were significantly enhanced by depolarization protocols that allowed their filling [90, 91], indicating that the stores are functionally deplete and require constant uptake, and that the ER can play a dominant role as a Ca²⁺ buffering organelle following a Ca²⁺ challenge, acting on a much longer time-scale than the mitochondria [92]. This action explains why inhibition of the ER Ca²⁺ reuptake system can result in amplification of the Ca²⁺ signals [93].

Information about age-related changes in the function and activity of the ER Ca²⁺ stores is rather limited, with an overall view that the size of the caffeine-releasable ER Ca²⁺ store is reduced by aging, as shown both in granule neurons of the cerebellar slices [94] and in aged acutely dissociated basal forebrain neurons [80], but not in cultured hippocampal neurons [95]. There are several mechanisms that can explain a decrease in the size of the caffeine-sensitive Ca²⁺ stores, including a decreased efficiency of the reuptake mechanisms through the SERCA pumps or an increased Ca²⁺ leakage. When investigated directly, in acutely dissociated basal forebrain neurons, aging did not affect the rate of spontaneous depletion (i.e., leakage) but decreased the efficiency of the ER loading [96]. In contrast, in long-term (30 days in vitro) cultured hippocampal neurons, glutamate stimulation activated a sustained Ca²⁺ response that was inhibited by rynanodine, a blocker of CICR, thus indicating an increased ER Ca²⁺ leak [95]. It is not clear yet if this increased Ca²⁺ leak, in effect a sustained CICR, was due to an alteration in the function of the rynanodine receptors or was related to a possible coupling between the L-type VOCCs and the rynanodine Ca²⁺-release channels [97], similar to the coupling between these types of Ca²⁺ channels in the muscle and which is known to be affected by the aging process [98]. An interesting observation on the role of intracellular Ca²⁺ stores in alterations in the normal Ca²⁺ homeostasis in the aged neurons has been recently reported with respect to the process of hippocampal LTP induction. Release of Ca²⁺ from the intracellular stores is an important participant in LTP induction for ranges of stimulation near the threshold of induction [99, 100]; but in the aged slices, inhibition of the Ca²⁺ release from the stores by either SERCA inhibitors or by rynanodine activated, rather than inhibited, LTP [101]. The explanation proposed for this paradoxical effect takes into account another well-established effect of Ca²⁺ in the aged neurons: activation of a slow afterhyperpolarization (AHP) current [102, 103], which in turn will affect the level of synaptic depolarization required for NMDA receptor and consequent LTP induction. The fact that aging is associated with a decline in LTP and synaptic plasticity [104, 105] might indicate an increase with age of the functional release of Ca²⁺ from the intracellular stores in these hippocampal neurons.

This area of discussion, the regulation of ER Ca²⁺ stores, is probably the most important one for appreciating the functional differences between normal physiological aging and the process of neurodegeneration, particularly AD, for which significant alterations in the ER Ca²⁺ handling were described [11]. Both bulwarks of pathology in AD (i.e., the Aβ protein fragments and the presenilins) are associated with or induce alterations in Ca²⁺ homeostasis [106, 107], although is not yet clear whether the changes in Ca²⁺ homeostasis precede the protein alterations in AD or vice versa [108]. One of the best-established mechanisms through which Aβ affects [Ca²⁺]_i regulation is by the formation by the Aβ fragment of a cation-selective ion channel [109] with Ca²⁺ permeability. One effect of aggregated Aβ is the increase of the oxidative stress, a mechanisms that has been linked with increases in the resting [Ca²⁺]_i [110]. The other culprits in AD pathogenesis are the presenilins (PS), which act as essential constituents of the β-secretase complex that cleaves a number of membrane proteins, including the amyloid precursor protein (APP), the latter generating the Aβ fragments [111]. Experimental manipulations of expression of either of the two genes controlling PS (PS1 and PS2) in various animal models resulted in phenotypes with disrupted Ca²⁺ homeostasis characterized, in the main, by an ER Ca²⁺ store overload [11]. As a result, neurons expressing such mutations show increased vulnerability to glutamatergic stimulation as shown, for example, in the case of hippocampal neurons, both in slices [112] and in primary cell cultures [107]. The overloading of the internal stores appear to affect the release of Ca²⁺ mediated either by the rynanodine receptor [113] or by the InsP₃ receptor [114]. However, such drastic effects of presenilin mutations on ER Ca²⁺ homeostasis might indicate a possible physiological role, and it has been shown recently that PS-1 deficiency (−/− homozygotes) can impair the glutamate-evoked Ca²⁺ signals, although the resting [Ca²⁺]_i levels were not affected [115]. Overall, these data illustrate the differences between normal aging and AD. In the former case, ER Ca²⁺ homeostasis is largely normal, with some tendency toward a reduction in the size of the Ca²⁺ store. In contrast, in the AD models, the ER stores are overfilled and the major dysfunctional proteins in AD are interfering with cellular Ca²⁺ homeostasis.

2. Mitochondria

On the back of the “free radicals” theory of aging [116], and involving the effects of free radicals on mitochondrial DNA, Miquel [117] proposed the “mitochondrial theory” of aging more than two decades ago. In the intervening years, the role and participation of mitochondria in cellular Ca²⁺ homeostasis became clearer, and this relationship came to be included among the important processes that underlie the functional changes of normal brain aging [35, 118]. The role of mitochondria as regulators of the cell death mode, dictating the progression toward necrosis or apoptosis, has been well established for some time now [119] but, as discussed above, normal neuronal aging is not about cellular death and the role of apoptosis in brain aging is relatively small in contrast to the situation in neurodegenerative diseases [119–121]. Instead, the role of mitochondria as the primary site of neuronal energy production and major source of metabolically linked release of reactive oxygen and nitrogen species (ROS and RNS) is much more important for the process of cellular aging [122] (Figure 14.3). Also, it should be noted that mitochondria, through their capacity of taking up or releasing Ca²⁺, can have a dramatic sculpting effect on the cytosolic Ca²⁺ signal.

FIGURE 14.3

(SEE COLOR INSERT FOLLOWING PAGE 204) Mitochondrial physiology and mitochondrial Ca²⁺ handling. (Left panel): Central to the mitochondrial state is the electrochemical gradient established across the inner membrane. The gradient is established by the (more...)

The main drive for Ca²⁺ uptake is the steep mitochondrial membrane potential (−180 mV) generated by the activity of the electron transporters in the respiratory chain that couple electron transfer to proton extrusion (Figure 14.3). The transport of Ca²⁺ is implemented by a Ca²⁺ uniporter, which has only very recently been identified as a channel protein [123], that has a high affinity for Ca²⁺ (nanomolar range); is impermeable to K⁺ and Mg²⁺, ions that are abundant in the cytosol, and is inhibited by ruthenium red. The issue of Ca²⁺ affinity is problematic because functional studies have shown this mitochondrial Ca²⁺ uptake pathway as a low-affinity, high-capacity Ca²⁺ removal system [124]. It might then be more useful to talk about a Ca²⁺ uptake set point, at which the uptake and efflux of Ca²⁺ from the mitochondrial matrix are balanced, and above which net uptake of Ca²⁺ takes place [125]. One of the very important features of mitochondrial Ca²⁺ uptake is the potentially huge capacity of this Ca²⁺ store, a property that is based on the co-accumulation of phosphate together with Ca²⁺ to form a salt complex that is both rapidly dissociable and also osmotically inactive. In isolated mitochondria incubated in physiological buffers that mimic intracellular solutions, Ca²⁺ can reach concentrations of around 100 mM (i.e., 50 times larger than the extracellular Ca²⁺ concentrations), while maintaining a normal metabolic state [126]. More recent measurements of Ca²⁺ changes in intact cellular systems (lizard motor nerve terminals) confirmed the earlier data and showed that the accumulation of mitochondrial Ca²⁺ takes place while the concentration of free Ca²⁺ in the mitochondrial matrix ([Ca²⁺]_mito) is maintained at stable levels [127].

This maintenance of a tight control on [Ca²⁺]_mito is essential as this parameter is a sword with two edges. Moderate increases in [Ca²⁺]_mito activate three important mitochondrial enzymes (pyruvate-, isocitrate-, and 2-oxoglutarate-dehydorgenases) that are involved in the Krebs (citric acid) cycle. The consequent increase in the reduction state of the NADH/NAD system will induce an increased electron transport and thus couple a cytosolic event that generated a Ca²⁺ response to a temporary increase in ATP supply, a process called “metabolic coupling” [128]. At the other extreme, significant increases in [Ca²⁺]_mito will activate, together with other factors, the permeability transition pore (mPTP) and result in catastrophic consequences for the mitochondria [129], among which mitochondrial swelling is an early indicator. It is important to note that there is a likely tissue-specific heterogeneity in the activation of the mPTP, with liver mitochondria appearing to be more susceptible to Ca²⁺-dependent activation than brain mitochondria, at least in isolated mitochondria preparations [130].

While the opening of the mPTP would lead to a rapid, unregulated redistribution of the Ca²⁺ accumulated in the matrix back to the cytosol, mitochondria have other transport systems that effect mitochondrial Ca²⁺ release. Two main pathways have been described, with a certain tissue specificity: an Na⁺/Ca²⁺ exchange system that is predominant in the brain, heart, and skeletal muscle, whereas in the liver, kidney, and smooth muscles, Ca²⁺ efflux is predominantly Na⁺ independent [131].

In discussing the effects of mitochondria on cellular Ca²⁺ homeostasis, the spatial dimension also must be take into account because, as demonstrated mainly in non-excitable cells, mitochondria, acting on limited spatial domains (functional “micro-domains”), can have significant effects on the Ca²⁺ release from the ER. This effect also takes into account the fact that cytosolic Ca²⁺ has a biphasic effect on ER Ca²⁺ release, both at the InsP₃ and the rynanodine receptors [16, 132]. Removal of Ca²⁺ from the immediate vicinity of the ER Ca²⁺ release channels should enhance the release and amplify the signal, and indeed, in Xenopus oocytes, enhancement of mitochondrial respiration increases the amplitude and velocity of the InsP₃-generated cytosolic Ca²⁺ waves [133]. In a similar fashion, the mitochondrial Ca²⁺ uptake could influence the entry of Ca²⁺ from the extracellular medium. One path responsible for Ca²⁺ entry and sensitive to the energetic status of the mitochondria, strategically placed in the submembranar space, is the capacitative Ca²⁺ entry pathway [134], activated by the depletion of the intracellular Ca²⁺ stores and inhibited either by ER store refilling or by permeating Ca²⁺ ions. To date, the evidence for such a pathway effective in the central nervous system’s neurons is rather thin [135]. Although not yet investigated in detail, the same subplasmalemmal mitochondria also could modulate the activity of other Ca²⁺ channels that are inhibited by cytosolic Ca²⁺ increases, such as the voltage-operated Ca²⁺ channels [136]. Not only can Ca²⁺ uptake modulate neuronal function, but the mitochondrial release of Ca²⁺ can also affect neuronal physiology. Thus, tetanic stimulation of Xenopus motorneurons induces a potentiation of neurotransmitter release that is sensitive to the Ca²⁺ release from the mitochondria, but not from the ER [137].

To date, there is almost no information about the effects of aging on the biochemical properties of the mitochondrial Ca²⁺ transporters, either for uptake or for release, and this should be an important area of future research, both in the field of normal aging and for various neurodegenerative instances. However, other well-documented changes in mitochondrial status with age are affecting mitochondrial Ca²⁺ homeostasis. Of these, a chronic change in the mitochondrial membrane potential that progresses with age is one of the better-established features. Using a fluorescent dye (rhodamine 123) that equilibrates across cellular compartments as a function of the electrical potential across these membranes (Nerstian distribution), and thus accumulates preferentially in the mitochondria [138], it has been shown that in the aged preparations of isolated mitochondria, there is a population of mitochondria that is significantly depolarized [139]. Because the use of rhodamine has a number of important limitations — in particular, overloading of the mitochondrial compartment with a consequent quenching [138, 140] — other methods are available, including the use of a recording electrode sensitive to an ion with Nerstian distribution, tetraphenylphosphonium [141]. Use of such more precise techniques confirmed the chronic depolarization of aged mitochondria [142]. Because these experiments used preparations of isolated mitochondria that are susceptible to selective enrichment in viable mitochondria, and thus a potential underestimating bias, particularly for the more fragile aged cells [143], it was important to assess the situation in intact cellular preparations. Using the properties of protonophores to collapse the mitochondrial membrane potential and thus release the rhodamine accumulated in the mitochondria, it has been shown that in the aged neurons there is a significant age-dependent decrease in the amount of dye accumulated [143], in line with the idea of mitochondria in the aged cells being more depolarized. Metabolic control analysis showed, consistent with these ideas, that aged mitochondria have an increased mitochondrial proton leak, but it is difficult to assess if this observation is a cause or effect of reported mitochondrial depolarization, because in these experiments the aged mitochondria did not show a decreased membrane potential [144].

Clearly, a decrease in the mitochondrial potential would result in a decrease in the electrochemical force driving the mitochondrial Ca²⁺ uptake. Using simultaneous measurements of both cytosolic Ca²⁺ and mitochondrial depolarization response, it was estimated that in the aged cerebellar neurons there was an increase in the [Ca²⁺]_i threshold required for triggering mitochondrial uptake [14, 145]. Associated with this question of the threshold for mitochondrial uptake is the issue of the size and dynamics of the mitochondrial Ca²⁺ pool. It has been shown previously that Ca²⁺ mitochondrial loading (as assessed by a protonophore-induced Ca²⁺ release) depends on the level and duration of stimulation and that this loading is dynamic, with a spontaneous discharge over a period of minutes [146]. Also, the extent of mitochondrial depolarization resulting from mitochondrial loading depends on the type of stimulus, with significant differences between KCl and glutamate [147], or even among the various types of glutamatergic agonists [148]. The existence of possible changes with age in these parameters of mitochondrial Ca²⁺ storage currently is under investigation. Some early results show, for the cerebellar granule neurons, that following glutamatergic stimulation the major determinant of the size of the mitochondrial Ca²⁺ accumulation is the cytoplasmic Ca²⁺ levels irrespective of the age of the preparations, and thus, by extension, of the level of chronic mitochondrial depolarization [149].

Neuronal stimulation means mitochondrial depolarization but our understanding of the effects of this mitochondrial depolarization on the state of the cell is evolving. In some studies, mitochondrial depolarization that follows glutamate stimulation appears to be the early event that initiates excitotoxic death [150–152]. However, other studies have reported an opposite effect, and mitochondrial depolarization preceding the glutamatergic stimulation proved a very effective neuroprotective protocol [153, 154]. Rather than being exclusive, these data, taken together, indicate that the mitochondrial Ca²⁺ — either as amount of Ca²⁺ loading or as level of free matrix Ca²⁺ — is a dynamic parameter that controls the physiological state of the cell. One target of mitochondrial Ca²⁺ is the regulation of the mPTP opening, mentioned above, and not surprisingly inhibition of the mPTP proved an effective neuroprotective strategy, effective in a variety of pathological instances but also improving the status of aged mitochondria [155].

Another potential target for increased mitochondrial Ca²⁺ is the production of ROS, which has been demonstrated to follow NMDA stimulation [156], in a manner sensitive to removal of Ca²⁺ [157]. However, the interpretation of studies reporting measurements of ROS production is problematic, because the acute, real-time detection of free radicals (mainly through the use of fluorescent dyes) is notoriously difficult because of (1) the selectivity of various dyes for different ROS species, (2) the different sites at which the ROS are produced and their lifetime, and (3) the fact that the fluorescence of the dyes could be influenced by other enzymatic and non-enzymatic processes [158]. In addition, the bioenergetic process that leads from increased matrix Ca²⁺ to increased free radicals is not well established. Electron leak, generating free radicals, occurs predominantly within complex III in the respiratory chain, during the so-called “Q cycle,” although complex I can also participate in the ROS generation process [159]. The crucial feature of this process is that it requires high mitochondrial membrane potential [160], and thus the Ca²⁺ uptake should inhibit rather than evoke an increase of ROS. One proposed explanation invokes (1) the stimulatory effect of Ca²⁺ on the citric acid cycle, mentioned above and that should accelerate the electron transfer rate; and (2) an effect of Ca²⁺ on nitric oxide (NO) generation, which in turn would inhibit the activity at the complex IV in the respiratory chain creating further favorable conditions for electron leak at complex III [161]. However, with respect to the effect of the respiration rate on the generation of ROS, a point of subtlety was made in a recent review by Nicholls [162]. The cytosolic environment is well hypoxic with respect to the values of the partial pressure of oxygen in the circulation or in the tissues, due to the increased diffusion pat, but even in these conditions oxygen, as a substrate for the final four-electron reduction to water, is in excess, and in sufficient supply. Under these conditions, the single electron reduction that generates free radicals is not simply a proportional slippage of the main electron carrier path, but rather the contrary. The generation of free radicals is inversely proportional to the rate of respiration, such that the higher the respiration rate and the lower the membrane potential, the less time will the electrons spend at the sites of leakage [162].

IV. AGING AND NEURODEGENERATION

Many times, “aging” and “neurodegeneration” are mentioned in the same breath, as if they are two state-points along a continuum, leading from the normality of old age to the state of confusion of senile dementia. Indeed, Terry and Katzman made this explicit prediction: they started from the observation that dementia occurs when there is a loss of about 40% of neocortical synapses and combined this with their estimations of the rate of age-dependent synaptic loss, as assessed by their analyses in human postmortem samples, extrapolating the data to the current estimations of human lifespan [163]. Apart from the anecdotal evidence of many centenarians that age successfully [164], including the Guinness Book of Records’ oldest certified human being, Madame Calment, who died in 1997 at the age of over 122 years without showing any signs of clinical dementia, the set of data that underlies the provocative hypothesis mentioned above might have been corrupted, unknowingly, by the inclusion in the analysis of subclinical instances of neurodegenerative pathology. Indeed, a similar error crept in the early sets of data that led to the conclusion that normal aging is associated with significant neuronal loss in all regions of the brain, a conclusion that only recently has been disproved [10]. Even the term “neurodegeneration” is problematic, despite the obvious etymology, because the term has been used in reference to a large group of neurological diseases, with heterogeneous clinical and pathological manifestations, affecting specific, but different regions of the nervous system [165]. Another experimental observation that complicates the issues is that the pathological markers associated with various neurodegenerative diseases, such as Lewy bodies, neurofibrillary tangles (NFTs), senile plaques, or other protein depositions, can be detected in the brain of aged asymptomatic individuals [166]. These observations obviously raise the question of whether such individuals were just aging normally or were sampled at a presymptomatic stage of a neurodegenerative disease — there are, as yet, no definite answers to these issues. The study of the relationship between normal aging and neurode-generation can be significantly helped by studies on animal models, because none of the human neurodegenerative diseases appear naturally in the animals. Through genetic manipulations, various animal models have been constructed that mimic one or another of the neurodegenerative phenotypes [167]. At the same time, behavioral studies allow a more and more detailed assessment of the cognitive status of the animals, leading to the generation of sophisticated models of learning and memory and the study of the effects of aging on such processes [105].

The animal studies also allowed a very detailed analysis of the metabolic changes associated with the process of normal aging, as reviewed in this chapter. What these studies indicate is that normal neuronal aging is a physiological process, characterized primarily by a decrease in the neuronal homeostatic reserve, where this homeostatic reserve is defined as the capacity of the cells to oppose the destabilizing effects of various metabolic stressors (Figure 14.4). A detailed discussion of the evidence was presented in earlier articles [122, 149]. Briefly, this hypothesis is based on the view that central to the changes in the cellular physiology of the aged neurons is a dysfunction of the metabolic triad: Ca²⁺ – mitochondria – ROS. It also states that the functional deficits of the aged neurons become evident only in a use-dependent manner, when the metabolic demands become excessive. This view accounts also for the lack of significant levels of neuronal loss with aging. On this weakened homeostatic reserve, any significant pathological instance that requires a strong metabolic response, be it trauma, stroke, or any idiopathic neurodegenerative process, would result in a significant level of neuronal death. In this model, it is the decreased homeostatic reserve that explains the increased neuronal vulnerability with age, an increased vulnerability that manifests itself in a variety of pathological scenarios.

FIGURE 14.4

Diagrammatic presentation of the effects of changes in homeostatic reserve on the processes of aging and neurodegeneration. Cells, tissues, and organisms have an intrinsic property of fighting off the effects of various stressors, defined as the homeostatic (more...)

The fact that aging is “just” a functional state should be very encouraging. We might not be in a position to bring on immortality through engineering negligible senescence, but a better understanding of the metabolism of the aged neuron could open important avenues for therapeutic intervention that will impede or delay the possible cognitive decline of the aged.

ACKNOWLEDGMENTS

The author wishes to acknowledge the BBSRC, which provided, through the SAGE Initiative, a significant part of the financial support for the work performed in this laboratory. The author is also grateful to Professor A. Verkhratsky for discussions on various aspects of the work reported here. Siddhartha helped by providing a new perspective.

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