Excitotoxicity and Mitochondrial Dysfunction Underlie Age-Dependent Ischemic White Matter Injury

Abstract

The central nervous system white matter is damaged during an ischemic stroke and therapeutic strategies derived from experimental studies focused exclusively on young adults and gray matter have been unsuccessful in the more clinically relevant aging population. The risk for stroke increases with age and the white matter inherently becomes more susceptible to injury as a function of age. Age-related changes in the molecular architecture of white matter determine the principal injury mechanisms and the functional outcome. A prominent increase in the main plasma membrane Na⁺-dependent glutamate transporter, GLT-1/EAAT2, together with increased extracellular glutamate levels may reflect an increased need for glutamate signaling in the aging white matter to maintain its function. Mitochondria exhibit intricate dynamics to efficiently buffer Ca²⁺, to produce sufficient ATP, and to effectively scavenge reactive oxygen species (ROS) in response to excitotoxicity to sustain axon function. Aging exacerbates mitochondrial fusion, leading to progressive alterations in mitochondrial dynamics and function, presumably to effectively buffer increased Ca²⁺ load and ROS production. Interestingly, these adaptive adjustments become detrimental under ischemic conditions, leading to increased and early glutamate release and a rapid exhaustion of mitochondrial capacity to sustain energy status of axons. Consequently, protective interventions in young white matter become injurious or ineffective to promote recovery in aging white matter after an ischemic episode. An age-specific understanding of the mechanisms of injury processes in white matter is vital in order to design dynamic therapeutic approaches for stroke victims.

8.1. Introduction

Human brain comprises equal percentages of gray and white matter by volume, which means that injuries sustained after a stroke in human predictably involve almost always both white and gray matter portions of the brain. The risk for ischemic stroke increases considerably with age. In addition to vascular factors, aging results in global changes, predominantly in white matter, such that the tissue becomes intrinsically more vulnerable to ischemic injury. Experimental stroke studies commonly use rodent brain, which constitutes little white matter by volume compared to gray matter. Subsequently, after an ischemic attack, the resultant injury in rodent brain is mainly due to neuronal loss with minimal or no contribution from white matter. Therefore it is plausible that the efficacy of neuroprotective drugs identified in experimental stroke models using young healthy male rodents is a poor predictor of clinical outcome and have so far failed to successfully translate to human trials (Del Zoppo 1995, 1998; Dirnagl et al. 1999)

An ideal stroke therapeutic must preserve neurons together with their axons and glial cells to gain optimal functional recovery. Neurons without functional axons simply cannot relay the information. Furthermore, the concept of neuroprotection must consider age, given that the mechanisms of ischemia change in such a way that those cytoprotective treatments in young brain may be ineffective or even become harmful for aging brain. Therefore, interventions to promote recovery following a stroke attack must target both portions of the brain, challenging the conventional idea that a single injury mechanism mediates the effects of ischemia in gray and white matter and that the injury mechanisms remain identical across life span in all age groups.

Over the last decade a number of molecular mechanisms by which glial cells and axons die or survive after an ischemic episode have been unraveled. In this chapter I focus on age-dependent structural and functional changes in white matter, which presumably evolved to protect the brain against the consequences of aging, but inadvertently made the tissue more vulnerable in the face of an ischemic attack. In particular, I concentrate on the age-dependent changes in energy status and glutamate homeostasis to test the hypothesis that increased excitotoxicity and impaired mitochondrial dynamics underlie the increased vulnerability of aging white matter to ischemia.

8.2. White Matter Structure and Function are Integrated

The white matter architecture is distinct from gray matter and is unexpectedly complex. Axons with variable amounts of myelin extend through oligodendrocytes, astrocytes, microglia, and precursor cells to establish a vital network between neurons. A variety of signaling mechanisms and molecules complement and contribute to white matter organization to ensure high fidelity communication between neurons. Among those voltage-gated Na⁺ and K⁺ channels, Na⁺–Ca²⁺ exchangers (NCX), Na⁺–K⁺ ATPase pumps, and axonal mitochondria are essential to maintain axon excitability (Fig. 8.1). The cellular and molecular components of white matter are individually under attack during ischemia, while they maintain their intricate glia-to-glia and glia-to-axon interactions. Therefore, the structural composition of white matter, together with a concerted interaction among glial cells and axons, dictates pathophysiological mechanisms of ischemic white matter injury. Consequently, it is challenging to experimentally dissect injury mechanisms following a complex injury such as ischemia in white matter. Furthermore, there is a growing sense that the mechanisms of white matter injury vary from one white matter tract to another (Tekkök and Goldberg 2001; Tekkok et al. 2007), presumably due to the fact that white matter structure conforms to its specific function. However, the reasons for regional differences in white matter injury are not yet understood at cellular levels. The existence of regionally diverse astrocytes and/or oligodendrocytes, the degree of expression or subunit composition of glutamate receptors that contribute to the injury process (Gallo and Russell 1995; Brand-Schieber and Werner 2003a, b; Tekkok et al. 2007), the extent of myelination (Olivares et al. 2001) and axonal specialization for the function and the type of injury such as ischemia vs. anoxia (Tekkok and Ransom 2004; Baltan 2009) and region- and cell-specific expression patterns of Class 1 histone deacetylases (HDACs) (Baltan et al. 2011a, 2013) may underlie these regional differences. Interestingly, among all, glutamate homeostasis of white matter involving glutamate receptors and glutamate transporters (Fig. 8.1), expressed primarily by astrocytes as well as other glial cells in a region-specific manner, attests to the sophisticated signaling mechanisms among white matter components to adapt to their regional function, but inevitably determines the vulnerability of different white matter tracts to ischemia. Therefore, to decipher axon-glia glutamate signaling and the mismatch of this signaling system, leading to excitotoxicity during ischemia, is key to prevent or restore young and aging white matter function.

Fig. 8.1.

White matter function and structure are integrated. (a) Evoked compound action potentials (CAP) recorded across young optic nerve illustrate (1) control, (2) after 60 min oxygen and glucose deprivation (OGD), and (3) recovery traces. Scale bar = 1 mV, 1 ms. (b) molecular and cellular architecture of white matter determines function and response to injury. See legend for symbols. AMPAR, α-amino-3-hydroxy-5-methyl-isoxazole propionate receptors; EAATs, excitatory amino acid transporters; NMDAR, N-methyl d -aspartate receptor

8.3. Aging Alters the Mechanisms of Ischemic White Matter Injury

Although the immediate effect of oxygen and glucose deprivation (OGD) is deregulation of ionic homeostasis due to ATP depletion, this pathway is completely reversible (Fig. 8.2) (Tekkok et al. 2007). On the other hand the critical level of Na⁺ overload that mediates the reversal of the Na⁺ -dependent glutamate transporters to accumulate glutamate dictates the irreversible injury (Fig. 8.2). Furthermore, convergence of the ionic pathway and the excitotoxic pathway concomitantly activates the oxidative injury by activating glutamate/cysteine system, (Oka et al. 1993) leading to mitochondrial dysfunction (Baltan et al. 2011b). Sequential merging of these three pathways is the basis for the demise of white matter during ischemia.

Fig. 8.2.

Putative mechanisms of ischemic white matter injury. Ionic, excitotoxic, and oxidative stress converge in sequential order to cause irreversible injury. Glutamate release, due to reverse Na⁺-dependent transport, dictates the irreversible nature of the injury (curved arrow). Note that there are multiple mechanisms that contribute to ischemic white matter injury. AMPA/KA R, AMPA/kainate receptors. Studies assessing nitric oxide synthase (NOS) activity are currently underway (Reproduced and Modified from Baltan 2009)

Naturally, white matter components that are vulnerable to excitotoxicity and oxidative injury are the principal targets of an ischemic attack. After induction of OGD, the earliest changes are observed on oligodendrocytes as swelling of the cell body that extends to myelin processes and subsequently axonal beading occurs (Fig. 8.3a). These morphological changes are irreversible such that even after resuming oxygen and glucose for prolonged periods of time, structural disruption progresses. Meanwhile, axon function gradually diminishes, resulting in complete loss of conduction (Fig. 8.3b). Oligodendrocyte death and axon loss are the ultimate outcome following ischemia (60 min), disabling white matter function. Oligodendrocytes express a wealth of glutamate receptors such as AMPA and NMDA receptors (Fig. 8.1), and although these are critical for glia-axon signaling, the presence of these receptors also sets oligodendrocytes as a target for excitotoxic injury. Axons are disrupted secondary to oligodendrocyte death, although there is experimental evidence that axons are a potential source of glutamate (Li et al. 1999) and express glutamate receptors under myelin sheaths that are directly activated by glutamate (Ouardouz et al. 2005; Alberdi et al. 2006).

Fig. 8.3.

Oligodendrocyte cell body, myelin sheaths, and axon function are under attack during ischemia. (a) Using multidiolistic technique and a “gene gun,” gold pellets coated by a mixture of fluorescent dyes (DiI-blue, DiO-green, and DiA-red) is delivered to acute coronal slices to label white matter structures in corpus callosum. In some cases, an oligodendrocyte together with its cell body, myelin sheaths, and the myelinated axon are labeled (top image, control). Exposing slices to oxygen glucose deprivation (OGD) for 30 min causes swelling and blabbing/swelling (red arrow-heads) of oligodendrocytes cell body (10 min). This swelling of the cell body that extends to myelin processes (20 min) and subsequently axonal beading occurs (30 min). These structural changes are irreversible even after prolonged periods of reperfusion (RP) (150′ RP). (b) Under these conditions, axon function, as per evoked compound action potentials recorded, progressively decreases over time and is completely lost after (30′ OGD). Axon function fails to recover after a prolonged period of RP

Aging is the most significant risk factor for an ischemic episode. Excitotoxicity and oxidative injury are essential steps in ensuing irreversible injury; therefore, the age-dependent changes in these two pathways are of the utmost importance to decipher the reasons underlying the increased vulnerability of aging white matter to ischemia. Exposing mouse optic nerves (MONs) obtained from 1- to 12-month-old animals to OGD confirms that aging axon function is more vulnerable to ischemia (rapid loss of axon function) and recovers less in a duration-dependent manner (Fig. 8.4a). On the other hand, keeping OGD duration constant, the axon function recovers less as age increases throughout the life span (Fig. 8.4b). Glutamate levels remain constant during the first 30 min of OGD (Tekkok et al. 2007—see also Fig. 8.7a), inferring that this period mostly involves ionic changes. In agreement, axon function shows comparable recovery until 18 months of age after 30 min of OGD while prolonging the OGD duration to 45 min degrades axon recovery at every age group, implying that switching from the ionic to the excitotoxic pathway unmasks the age-related vulnerability of axon function to ischemia. In accordance, two main steps in the ionic pathway, blockade of NCX (Fig. 8.5a) or removal of extracellular Ca²⁺ (Fig. 8.5b), fail to protect aging axon function against OGD. Indeed, the absence of Ca²⁺ hinders axon function recovery, pointing to an interesting dependence of aging axon excitability on Ca²⁺ entry. In contrast, blockade of the reversal of the Na⁺-dependent glutamate transporters (Fig. 8.6a) or blockade of AMPA/kainate receptors (Fig. 8.6b) preserves aging axon function during OGD and promotes recovery comparable to young axons. Comparing spatiotemporal glutamate release patterns reveals an earlier and more robust glutamate release in aging white matter, despite efficient control of baseline glutamate levels (Fig. 8.7a). Moreover, in young white matter, glutamate levels return to baseline upon termination of OGD, while glutamate levels remain elevated after the end of OGD in aging MONs. The most prominent molecular structure leading to this enhanced excitotoxicity in aging white matter is the upregulation of GLT-1 (in human termed EAAT2) levels (red, Fig. 8.7b). The GLT-1 is mostly located on glial fibrillary acidic protein-positive (GFAP+) astrocytes in young white matter, but with aging GLT-1 is expressed on structures other than GFAP + astrocytes, presumably on oligodendrocytes based on their distinctive beads on a string appearance. Typically GLT-1 takes up glutamate with co-transport of Na⁺ (Fig. 8.8). The Na⁺–K⁺ ATPase pump is crucial to sustain excitability of axons by expelling Na⁺ for K⁺ and consuming ~50 % of ATP produced in the brain. During the ionic pathway of an ischemic attack, intracellular Na⁺ levels increase following ATP depletion and failure of the Na⁺–K⁺ ATPase pump. With increased intracellular Na⁺ and additional cell depolarization due to accumulation of extracellular K⁺, GLT-1 reverses to release glutamate and, at the termination of OGD, takes up glutamate according to readjusted Na⁺ levels. Therefore, the number of transporters determines the capacity of the tissue to transport glutamate but it is the ATP levels that indirectly dictate the direction of the GLT-1 to remove or to release glutamate. Therefore age-related increases in the number of GLT-1 that result in early and more glutamate release and sustained glutamate levels after OGD raise the question whether aging alters mitochondrial dynamics, thereby compromising white matter energy status.

Fig. 8.4.

Aging increases the vulnerability of axon function to ischemia as a function of age and injury duration. (a) Exposing optic nerves from 1- (black) and 12-month-(magenta) old mice to 30, 45, and 60 min OGD reveals that aging axons recover less as the duration of injury increases. (b) Exposing optic nerves from 1 (white)-, 6 (magenta)-, 12 (green)-, 18 (blue)-, and 24 (purple)-month-old mice to a fixed duration of injury shows that axons recover less with age over the same duration of injury. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA

Fig. 8.7.

Aging is correlated with early robust glutamate release due to upregulation of GLT-1. (a) Baseline glutamate levels are precisely controlled in young (black squares) and old axons (red squares). OGD causes early and robust glutamate release in older axons that remains sustained after the end of OGD (arrow). (b) The overlap of GLT-1 (red) and GFAP (green) labeling indicated that GLT-1 was mainly expressed in astrocytes (merged) in 1-month-old mouse optic nerves (MONs). There was a twofold increase in GLT-1 pixel intensity with age. The pattern of GFAP expression in MONs changed with age but without an increase in GFAP levels. * p < 0.05, ** p < 0.005, one-way ANOVA (Reproduced from Baltan et al. 2008)

Fig. 8.5.

Aging alters the mechanisms of ischemic white matter injury. (a) Blockade of reverse mode of Na⁺–Ca²⁺ exchanger (NCX) operation with KB-R7943 (KB-R) promotes axon function recovery after OGD (60 min) in young axons but fails to benefit aging axons after a shorter OGD (45 min). (b) Removal of extracellular Ca²⁺ drastically ameliorates ischemic injury in young axons but hampers recovery in aging axons even after 30 min of OGD (Reproduced and modified from Baltan et al. 2008)

Fig. 8.6.

Excitotoxicity underlies ischemic injury in young and old axons. (a) Blockade of the reversal of Na⁺-dependent glutamate transporters with threo-beta-benzyloxyaspartate (TBOA) or (b) AMPA/kainate receptors with 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) protects and promotes axon function recovery in young and old axons (Reproduced and modified from Baltan et al. 2008)

Fig. 8.8.

Increased GLT-1 transporter numbers lead to enhanced excitotoxicity in aging white matter. The principal Na⁺-dependent glutamate transporter, GLT-1, typically takes up glutamate with co-transport of Na⁺. During ATP depletion, due to increased intracellular Na⁺ levels, and with additional cell depolarization (not shown), the transporter reverses and releases glutamate. Therefore, the number of transporters determines the capacity of the system for the amount of glutamate that can be transported, but it is the ATP levels that determine the direction of the transporter (to take up or release glutamate). Reproduced and Modified from Baltan et al. 2013)

In addition to GLT-1, the critical members to conserve glutamate homeostasis are GLAST (a plasma membrane glutamate transporter in rodents; in human termed EAAT1), glutamate and glutamate synthetase (GS), an astrocyte-specific enzyme-converting glutamate to glutamine (Schousboe et al. 2014). White matter glutamate content increases considerably in correlation with increased GS levels with age (Baltan et al. 2013). Together with a twofold increase in GLT-1 levels in older white matter (Baltan et al. 2008), these adjustments may infer an age-related adaptive mechanism in white matter to remove and to convert excessive glutamate to glutamine in order to maintain glutamate homeostasis. These modifications raise the question whether changes in glutamate signaling underlie changes in aging white matter function.

8.4. Synergistic Interaction of Excitotoxicity with Mitochondrial Dysfunction Mediates Ischemic White Matter Injury

The balanced delivery of mitochondria to cell body, dendrites, and axons helps them serve various functions, including energy generation, regulation of Ca²⁺ homeostasis, cell death, synaptic transmission, and plasticity (Chang and Reynolds 2006). The bioenergetics of mitochondria in neurons and their role in glutamate excitotoxicity are well defined in gray matter (Nicholls et al. 2007). Mitochondrial dysfunction and excitotoxicity share common features and are thought to act synergistically by potentiating each other (Albin and Greenamyre 1992; Jacquard et al. 2006; Silva-Adaya et al. 2008). Mitochondria are dynamic organelles that travel along microtubules, using axonal transport to reach peripheral locations (Hollenbeck 2005; Hollenbeck and Saxton 2005) (Fig. 8.9). They constantly undergo fission and fusion events (Karbowski et al. 2004), and the relative rates of mitochondrial fusion and fission have been implicated in the regulation of their number, size, and shape (Mozdy and Shaw 2003; Scott et al. 2003; Chen et al. 2007). As a result, mitochondria display a cell-specific morphology; neuronal mitochondria are small and round as opposed to the longer tubular mitochondria in dendrites and axons (Fig. 8.9, arrows).

Fig. 8.9.

CFP + somal and axonal mitochondria exhibit region-specific morphology. Neuronal mitochondria are readily observed as cyan fluorescent protein positive (CFP+) structures in a transgenic Mito-mouse. CFP + mitochondria are small and round in neuronal cells bodies (yellow asterisk) labeled with MAP2 (red), but are more linear and tubular in primary dendrites (yellow arrows). Scale bar = 5 μm (Reproduced from Baltan et al. 2013)

The role of mitochondrial dynamics in white matter function, in particular the link between excitotoxicity and mitochondrial dysfunction, has been recently characterized (Baltan et al. 2011b; Baltan 2012). Functional studies correlated with advanced imaging techniques using MONs obtained from the so-called Mito-mice, i.e., a transgenic mouse [tg(Thy1-CFP/COX8A)] in which cyan fluorescent protein (CFP) is expressed in neuronal mitochondria (Misgeld et al. 2007), provided proof of principle that OGD causes a remarkable loss of CFP + mitochondria in white matter axons that can be completely reversed by blockade of AMPA/kainate receptors (Fig. 8.10a). Expectedly, an exciting correlation between CFP fluorescence and ATP levels connect excitotoxicity to impaired mitochondrial function in white matter (Fig. 8.10b). Interestingly, further support to verify the link between excitotoxicity and mitochondrial dysfunction in white matter comes from a series of experiments that tested the potential of HDAC inhibitors on white matter ischemic injury.

Fig. 8.10.

Blockade of excitotoxicity preserves CFP + axonal mitochondria and ATP levels in response to OGD. (a) OGD drastically reduced CFP fluorescence in MONs from Mito-mice and pretreatment with NBQX (30 μM) protected against this loss. Note the change in mitochondrial morphology from small and tubular to tiny and punctuated with OGD. Scale bar = 10 μm (insets = 2 μM) (b) Consistent with the preservation of CFP pixel intensity, NBQX pretreatment conserved ATP levels in MONs. *** p < 0.0001, one-way ANOVA (Reproduced in part from Baltan et al. 2011b)

Administration of pan- or Class 1 HDAC inhibitors, before or after a period of OGD, promotes functional recovery of axons and preserves white matter cellular architecture. This protection correlates with the upregulation of GLT-1, delays and reduces glutamate accumulation during OGD, preserves axonal mitochondria and oligodendrocytes, and maintains ATP levels. Because significant protection is also observed when HDAC inhibitor is added after OGD (after maximal glutamate accumulation), HDAC inhibition must have at least two distinct sites of action during the sequential course of ischemic white matter injury; one site related to glutamate accumulation (GLT-1 expression) and the other involving post-excitotoxic mechanisms (Baltan et al. 2011b).

Due to the consistent protection, whether applied before or after injury, HDAC inhibitors target excitotoxic and oxidative pathways, so it is reasonable to expect that HDAC inhibition protects aging white matter function against ischemic injury. Indeed pan- and Class 1 HDAC inhibitors confer long-lasting benefits to aging axons following acute ischemic injury. Ironically, both aging and HDAC inhibition upregulate GLT-1 expression levels twofold above young and control white matter, respectively, raising the question how a similar modification of GLT-1 levels causes increased vulnerability to ischemia in aging while providing protection against ischemia with HDAC inhibition. The explanation for this perplexing issue lies in the principle of GLT-1 function. The axon protective effect of HDAC inhibition correlates with preservation of mitochondria in aging MONs comparable to young MONs (Fig. 8.11), suggesting that conservation of mitochondrial function and ATP levels keep the pump in the forward direction and switching the potential of GLT-1 function to attenuate glutamate accumulation. Therefore an increase in GLT-1 levels acts as an important molecular switch between protection and injury. It is therefore of great interest that HDAC inhibition offers a pharmacological strategy to upregulate GLT-1, a target gene critically involved in various disease conditions including stroke (Baltan et al. 2011b), so as to effectively ameliorate excitotoxicity (Brevig et al. 2004; Wu et al. 2008; Allritz et al. 2009).

Fig. 8.11.

Increased excitotoxicity and mitochondrial dysfunction underlie increased vulnerability of aging white matter to ischemia. Mitochondria in aging axons become longer and thicker compared to young axons (images), which may hinder ATP production and drive GLT-1 in reverse mode (see Fig. 8.8). Consistent with this, there is an early and robust glutamate release in aging white matter (red) compared to optic nerves from young mice (black). Note, that the glutamate levels return to baseline in young white matter, but remain elevated in aging white matter. * p < 0.05, ** p < 0.005, one-way ANOVA (Reproduced in part from Baltan et al. 2008)

8.5. Aging Impairs White Matter Mitochondrial Dynamics

A variety of neurological diseases, as well as aging, is associated with defects in mitochondrial fusion and distribution (Karbowski and Youle 2003; Chen et al. 2007). Older neurons become more susceptible to glutamate excitotoxicity due to loss of mitochondrial membrane depolarization and increased reactive oxygen species (ROS) generation, leading to reduced energy supply (Parihar and Brewer 2007). Mitochondrial function appears to decline in older animals, presumably due to reduced ATP production. This has been demonstrated in cardiac (Lesnefsky et al. 2001), liver (Selzner et al. 2007), and brain (Toescu 2005) cells. Ion transport accounts for about 50 % of all ATP utilization and Na⁺/K⁺ ATPase activity alone is responsible for the majority of this consumption (Erecinska and Silver 1994). A loss of ATP reserve, diminishing the activity of this key enzyme with advanced age, is a plausible contributor to increased vulnerability of aging white matter to ischemia (Scavone et al. 2005).

Consistent with this axon function, when transiently challenged with OGD, it is slower to restore normal ion gradients (Fig. 8.4a), permitting pathological processes related to ion derangement to operate for longer periods; hence, reversing Na⁺-dependent glutamate transporters earlier (Fig. 8.7a), and producing more injury in older MONs (Baltan et al. 2008). Because reversal of the Na⁺-dependent transporter dictates when glutamate is released (Fig. 8.2), earlier release of glutamate implies accelerated Na⁺ overload due to decreased tolerance to energy deprivation in aging white matter. The disadvantage of compromised ATP levels in older animals is further verified by better recovery of white matter function in older animals when OGD is induced at lower temperature (Baltan et al. 2008).

Excitotoxicity and elevated Ca²⁺ induce marked changes in mitochondrial structure, slowing their motion (Rintoul et al. 2003; Barsoum et al. 2006; Chang and Reynolds 2006) and generating ROS in neurons (Nicholls et al. 2007). In young white matter, excitotoxicity due to overactivation of AMPA/kainate receptors loads mitochondria with Ca²⁺ and fission is enhanced (Fig. 8.10-insets), associated with loss of fluorescence of mitochondria genetically tagged with CFP. A Ca²⁺ overload activates neuronal NOS to produce nitric oxide and ROS, which are proposed as diffusible second messengers linking oligodendrocyte excitotoxicity to axon injury (Matute et al. 2001; Ouardouz et al. 2006). Axon function directly correlates with tissue energy reserves, since Na⁺–K⁺ ATPase activity is intimately dependent on ATP levels. As a result, OGD causes a significant reduction in ATP levels and CFP + mitochondria, which could be prevented by AMPA/kainate receptor blockade (Fig. 8.10).

Expectedly, the compromised energy status of aging white matter is intimately related to mitochondrial dynamics. Imaging axonal mitochondria in aging MONs in the mito-CFP mouse (Fig. 8.11-insets) revealed that mitochondrial shape, size, and location and response to OGD differ compared to young axons. Mitochondria in aging axons appear more abundant based on the increased levels of CFP fluorescence with longer and thicker mitochondria compared to young MONs (Fig. 8.11). The regulated process of mitochondrial fusion and fission controls the spatiotemporal properties of mitochondrial Ca²⁺ responses and the physiological and pathophysiological consequences of Ca²⁺ signals (Szabadkai and Rizzuto 2004; Szabadkai et al. 2004). By enhancing fusion or inhibiting fission, elongated mitochondria possibly absorb Ca²⁺, efficiently preventing n-NOS activation and subsequent ROS production (Cheung et al. 2007). Age-specific mitochondrial fusion (Fig. 8.11) is accompanied by a modification in the levels of mitochondrial shaping proteins such as Mfn1, Mfn2, Opa1, and Drp-1 (unpublished data). However, this age-related adaptive reorganization of mitochondria becomes detrimental under ischemic conditions. Ischemia in aging white matter further exacerbates mitochondrial fusion, presumably due to age-dependent drop in Drp-1 and age-dependent arrest of mitochondrial motility with exposure to glutamate (Chang and Reynolds 2006; Parihar and Brewer 2007). Mitochondria fuse to collectively counteract the already increased excitotoxicity and Ca²⁺ load with aging, and this age-related change in mitochondrial dynamics hinders ATP production (unpublished data). Because basal ROS generation is already elevated with aging, further increases in ROS accumulation under ischemic conditions contribute to increased vulnerability to ischemia. Conditions that ameliorated excitotoxicity in older MONs, such as blockade of AMPA/kainate receptors with 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f] quinoxaline-7-sulfonamide (NBQX) (Baltan et al. 2008) significantly preserved CFP + mitochondria and improved axon function recovery in older MONs. Therefore if excitotoxicity and mitochondrial dysfunction act synergistically to mediate injury, amelioration of excitotoxicity must preserve mitochondrial function and ATP levels, which in turn maintains the function of GLT-1 in a forward direction, providing an energy-efficient cycle to minimize glutamate accumulation.

These results were further verified in experiments investigating the protective effects of Class I HDAC inhibitors in aging white matter (Baltan et al. 2011a, b—see above). Consequently, by delaying and reducing glutamate accumulation, HDAC inhibition interrupts the merging point of excitotoxicity to subsequent oxidative injury in aging axons such that the protective action associated with HDAC inhibition correlates with preservation of mitochondrial structure/function in axons and ATP levels (Baltan et al. 2013).

8.6. Conclusions

The main goal of this chapter is to establish the proof of principle that central nervous system white matter becomes inherently more susceptible to an ischemic attack with age and that enhanced excitotoxicity and impaired mitochondrial dynamics underlie the increased vulnerability of aging white matter to ischemia. Predictably, age-related changes in the molecular architecture of white matter dictate the predominant injury mechanisms and determine the functional outcome. Consequently, protective interventions in young white matter, such as removal of extracellular Ca²⁺, reduce functional recovery in aging axons (Fig. 8.5b). Together with the observation that blockade of reversal of NCX fails to protect function in older mice (Fig. 8.5a; Baltan et al. 2008), these results propose a diminished role for the ionic pathway with aging. On the other hand, aging causes a prominent increase in the expression pattern of glutamate, GS, and GLT-1 levels that extend to additional structures in white matter. Na⁺-dependent and Ca²⁺-dependent mechanisms, involving astrocytes, oligodendrocytes expressing EAAC1 plasma membrane glutamate transporter (Arranz et al. 2008), microglia expressing GLT-1 or perhaps axons with their vesicular glutamate transporters (Kukley et al. 2007; Ziskin et al. 2007), all may become additional sites of glutamate release with aging and contribute to increased excitotoxicity. These modifications may imply an age-related adaptive mechanism to maintain glutamate signaling and homeostasis. However, during an ischemic episode these adaptive changes act against the tissue and expedite and aggravate glutamate release (Fig. 8.7a) and expand the excitotoxic injury into the recovery period. Interestingly, AMPA/kainate receptors (Fig. 8.6b) mediate the ischemic injury across age groups, indicating that certain steps of injury are preserved irrespective of age. In young white matter, activation of either AMPA/kainate receptors loads mitochondria with Ca²⁺ and fission is enhanced due to abundant Drp-1 levels. Ca²⁺ overload activates NOS to produce NO and ROS, which are proposed as diffusible second messengers to link oligodendrocyte excitotoxicity to axon injury (Matute et al. 2001; Ouardouz et al. 2006). Mitochondria fuse to collectively counteract the already increased excitotoxicity and Ca²⁺ load with aging, and this age-related change in mitochondrial dynamics hinders ATP production and increases NO and ROS production. These challenge the Na⁺/K⁺ ATP pump to maintain axon excitability and the associated rise in Na⁺ levels switches GLT-1 to function in the reverse direction to release glutamate. The isoforms of NOS that mediate ischemic injury and their cellular expression pattern in white matter as function of age remain unknown.

Moreover, aging modifies cell- and region-specific organization of white matter expression of Class 1 HDACs and these cellular adjustments may further contribute to increased vulnerability of aging white matter to ischemia. An age-specific understanding of the mechanisms of injury processes in white matter is essential to design dynamic therapeutic approaches for stroke victims. Therefore an age-dependent reduction in mitochondrial bioenergetics may underlie the increased vulnerability of aging axons to ischemia.