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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: J Neurosci Res. 2014 Nov 25;93(4):623–632. doi: 10.1002/jnr.23517

Ischemic Tolerance in an In Vivo Model of Glutamate “Preconditioning”

Yomna Badawi 1,2, Ranu Pal 2, Dongwei Hui 2, Elias K Michaelis 1,2,*, Honglian Shi 1,2,*
PMCID: PMC4329082  NIHMSID: NIHMS640438  PMID: 25421886

Abstract

Ischemia initiates a complicated biochemical cascade of events that triggers neuronal death. In this study, we focused on glutamate –mediated neuronal tolerance to ischemia-reperfusion. We employed an animal model of life-long excess release of glutamate, the glutamate dehydrogenase 1 transgenic (Tg) mouse, as a model of in vivo “glutamate preconditioning”. Nine- and 22-month old Tg and wild type (wt) mice were subjected to 90 min of middle cerebral artery occlusion followed by 24 hr reperfusion. The Tg mice suffered significantly reduced infarction and edema volume, compared with their wt counterparts. We further analyzed proteasomal activity, level of ubiquitin immunostaining, and MAP2A expression to understand the mechanism of neuroprotection observed in the Tg Mice. We found that in the absence of ischemia, the Tg mice exhibited higher activity of the 20S and 26S proteasomes while there were no significant differences in the level of hippocampal ubiquitin immunostaining between wt and Tg mice. A surprising observation was that of a significant increase in MAP2A expression in neurons of the Tg hippocampus following ischemia-reperfusion, compared with that in wt hippocampus. The results suggest that increased proteasome activity and MAP2A synthesis and transport might account for the effectiveness of glutamate preconditioning against ischemia-reperfusion.

Keywords: stroke, ubiquitin, MAP2, aging, hippocampus

INTRODUCTION

Stroke results in a rapid cessation of blood flow that compromises energy metabolism in affected brain tissues and leads to large increases in glutamate release (Benveniste et al., 1984). Glutamate is ubiquitously distributed in the brain, is present in concentrations that are higher than those of any other amino acid (Fonnum, 1984), and functions as the principal excitatory neurotransmitter in the central nervous system (CNS). Previous studies have demonstrated that blocking ionotropic glutamate receptors significantly reduces ischemic damage (Sims & Muyderman, 2010). This is attributed to the triggering by the released glutamate of an intracellular excitotoxic cascade involving an excessive accumulation of calcium (Ca2+) and activation of downstream pathways that cause cell death. Therefore, impeding the development of excitotoxicity in animal experimental stroke has been a frequently targeted mechanism in the development of neuroprotective stroke treatments (O’Collins et al., 2006; Minnerup et al., 2012). The effect of over 20 anti-excitatory drugs has been evaluated in over 270 preclinical studies, yet none of them have been shown to be effective in the treatment of stroke in humans (O’Collins et al., 2006).

There are increased efforts to identify potential strategies to protect neurons following ischemic stroke. A current focus is on understanding the protective mechanisms that induce tolerance following ischemic preconditioning. Current knowledge suggests that preconditioning with a brief period of ischemia is an effective approach to decrease neuronal death after a more severe ischemic episode (Kirino, 2002). The same concept can also be applied to excitotoxicity where preconditioning with a mild glutamate-induced stress can promote a tolerant state that reduces the injury caused by a subsequent, more severe glutamate exposure. The effect of glutamate preconditioning in the development of resistance to a subsequent ischemic insult has been assessed previously, although such preconditioning was produced in in vitro, not in vivo, preparations through the addition of exogenous glutamate or glutamate analogs, such as receptor agonists or antagonists. For example, the death of cortical neurons in primary culture following in vitro ischemia is prevented by pre-exposure of the cells to N-methyl-D-aspartate (NMDA), a selective glutamate NMDA receptor agonist (Lin et al., 2008). In other studies, the ischemic preconditioning of primary hippocampal neurons was disrupted following exposure to the NMDA receptor inhibitor MK-801 (Mabuchi et al., 2001), and the ischemic preconditioning of co-cultures of neurons and glial cells was suppressed following exposure to the NMDA receptor inhibitor 3-((D)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (Grabb & Choi, 1999). Both of these studies provided indirect evidence of glutamate involvement in the process of ischemic preconditioning. However, there are no studies that have examined the role of endogenous, moderate excess synaptic glutamate release and subsequent receptor activation in vivo on ischemia-induced neuronal damage.

In the present study, our main objective was to determine whether moderate excess synaptic release of endogenous glutamate throughout the lifespan of an organism, could lead to increased ischemic tolerance, i.e., a preconditioning-like state, and thus protect neurons from ischemia in an in vivo model. We used transgenic (Tg) mice that overexpress the gene for glutamate dehydrogenase 1 (Glud1) only in neurons, not in glial cells, of the CNS. These Tg mice are characterized by an increase in GLUD1 enzyme levels and activity (Bao et al. 2009; Hascup et al. 2011), relatively modest, yet significant, increases in brain glutamate concentrations (approximately 10–15% above wild-type littermates) throughout the lifespan (Bao et al. 2009; Choi et al. 2014), and a moderate increase (30–40% higher levels than normal) in depolarization-induced synaptic glutamate release (Bao et al. 2009; Michaelis et al. 2011). Unlike other models of excessive accumulation of extracellular glutamate (from 150% to approximately 30-fold increases in glutamate levels) because of null mutations of glial glutamate transporters or of regulators of such transporters (Rothstein et al. 1996; Tanaka et al. 1997; Zeng et al. 2007), the Glud1 Tg mice do not suffer from massive neuronal damage in the brain or an early death. Thus, the Glud1 Tg mice offer a potentially good model for probing the effect of moderately increased, pulsatile, chronic release of endogenous glutamate by neurons on the development of resistance to an ischemic episode in adult and aged mice.

The hypothesis that we are testing is that chronic exposure of neurons to moderate levels of synaptically released glutamate would activate protective pathways that reduce damage after a stroke. Both gene ontology and pathway analyses of whole genome expression differences between Glud1 Tg and wt mice have identified many genes that are related to glutamate and intracellular signaling as being over-expressed in the Tg mice (Wang et al. 2010; Wang et al. 2014). Some of these genes might lead to the development of resistance to ischemia. Our results demonstrated that the Glud1 Tg mice were more resistant to ischemia/reperfusion, compared with wt mice. In order to ascertain the potential molecular mechanisms for the observed resistance to ischemia in the Glud1 Tg mice, we explored the effect of glutamate hyperactivity on the proteolytic activity of the 20S and 26S proteasomes, and of glutamate hyperactivity and ischemia on the levels of ubiquitinated proteins. Proteasomal activity is decreased and ubiquitinated proteins accumulate following middle cerebral artery occlusion (MCAO) (Alves-Rodrigues et al. 1998; Keller et al. 2000a; Keller et al. 2000b), while intact proteasome activity is related to rapid ischemic tolerance (Meller et al. 2008). We hypothesized that in the Glud1 Tg mice, the proteasome activity might be elevated and less affected by ischemia, thus providing relative neuroprotection from an ischemic episode. This hypothesis was based in part on the observed increases in the genomic expression of ubiquitin proteasome system (UPS) enzymes in Glud1 Tg compared with wt mice (Wang et al. 2010; Wang et al. 2014).

To assess the susceptibility of neurons to the injurious effects of ischemia in Tg and wt mice, we examined the immune-labeling of neuronal cell bodies and dendrites by antibodies to microtubule-associated protein-2A (MAP2A). Acute glutamate- or NMDA-induced neuronal damage manifests as dendrite varicosities in neurons labeled with anti-MAP2A antibodies (Hoskison et al. 2007; Ikegaya et al. 2001). We previously showed that labeling with anti-MAP2A antibodies shows discontinuities in dendrites of 9-month old Glud1 Tg mice but without evidence of varicosities (Bao et al. 2009).

MATERIALS AND METHODS

Generation of Glud1 Tg mice

All experiments were conducted with the approval of the University of Kansas Institutional Animal Care and Use Committee. The Glud1 Tg mice were generated as described (Bao et al., 2009). Briefly, linearized DNA containing the cDNA of mouse GLUD1 was injected into fertilized C57BL6/SJL hybrid mouse oocytes. The cDNA was placed under the control of the Nse (neuron-specific enolase) promoter (Bao et al., 2009). The animals used in the present study were male, 9-month (mo)-old Tg and wt. This age was selected as this is the age when CNS transcriptomic and structural differences between the Tg and wt are most significant (Bao et al., 2009; Wang et al., 2010; Michaelis et al., 2011; Wang et al., 2014). In studies designed to determine the effects of aging on ischemia-induced brain damage, 22-mo old mice were used.

In vivo ischemia model

Reversible occlusion of the middle cerebral artery was induced using a well-established protocol (Clark et al., 1997). The mice were anesthetized with 3% Isoflurane and oxygen and maintained with 1.5% Isoflurane, or to desired anesthetic effect, throughout the procedure. Buprenorphine was used as the analgesic and was injected pre-operatively at 0.05 mg/kg. Male mice were subjected to MCAO followed by a 24 hour (hr) period of reperfusion. At the whole-brain level, TTC (2,3,5-triphenyltetrazolium chloride monohydrate) staining was used to assess brain damage (Ito et al., 1997). Brain edema volume (Vedema) was also measured from the coronal sections that were stained by TTC by determining the volumes of both the ipsilateral (affected) hemisphere (VIpsi) and the contralateral hemisphere (Vcontra) and using the equation: Vedema =Vcontra - VIpsi (Yan et al., 2011).

Measurement of proteasomal activity

Brain tissues from 9-mo old wt and Tg mice were homogenized and 20 μg of the cell homogenate proteins were incubated with proteasome activity assay buffer in which their ability to cleave the fluorogenic peptide substrate, Succinyl-LeuLeuValTyr-7-amino-4-methly-coumarin (Suc-LLVY-AMC) was determined (Figueiredo-Pereira et al., 1994). The assay buffer used in the measurement of the 26S proteasome function consisted of 50 mM Tris (pH 7.4), 5mM MgCl2, 2 mM DTT, 2 mM ATP and Suc-LLVY-AMC (80 μM in 1% DMSO, Sigma-Aldrich). The buffer employed for the determination of 20S proteasome function contained 20 mM HEPES (pH 7.8), 0.5 mM EDTA, 0.03% SDS and 80 μM Suc-LLVY-AMC. The hydrolysis of Suc-LLVY-AMC into AMC was detected using a fluorescence plate reader at ex 380 nm and em 440 nm.

Immunohistochemistry

Immunostaining and statistical analyses were performed using methods similar to those described previously (Bao et al., 2009) but with minor modifications in the initial steps of preparation of brain tissue following MCAO. Specifically, before fixation of the hemisected brain tissue (ipsilateral vs. contralateral hemispheres), 2 mm coronal sections were obtained as described above for the assessment of the effects of MCAO on brain at the macroscopic level using TTC staining. For the confocal microscopy studies, the 2 mm coronal sections were fixed and permeabilized and then subjected to rapid freezing and cryo-microtome sectioning into ~25 μm thick sections. The cryotome sections were incubated overnight in buffer containing a combination of the primary antibodies against ubiquitin (polyclonal; rabbit; 1:250; Cell Signaling) and against MAP2A (monoclonal; mouse; 1:250; Millipore). The sections were subsequently rinsed in PBS and incubated with fluorescent dye-labeled secondary antibodies (Alexa 568 goat anti-rabbit and Alexa 488 goat anti-mouse) at 37 °C for 2 hrs. Cortical and hippocampal regions were examined by confocal microscopy on a Leica SPE2 laser confocal microscope. Pixel density counts were determined using the Leica Application Suite software. The results reported are from the hippocampus as neuronal structure was frequently better preserved than in the cerebral cortex, albeit still showing extensive damage. The averaged pixel densities from each sub-layer of each region of the hippocampus, i.e., from the stratum oriens (SO), stratum pyramidale (SP) and stratum radiatum (SR) of the CA1 and CA3 regions, from 3 pairs of animals, were statistically analyzed and graphed.

Statistical analysis

Student’s t-test and one-way or three-way ANOVA were used to determine statistically significant differences employing SigmaPlot 12.5. The level of significance was set at P ≤ 0.05.

RESULTS

Lower brain infarct and edema volume in Glud1 Tg vs. wt mouse brain following MCAO

Nine-mo old male wt and Tg mice were subjected to MCAO followed by a 24 hr period of reperfusion. Using TTC staining, we assessed the extent of brain damage. The reduction of TTC by dehydrogenases in living cells leads to the formation of water-insoluble red formazan crystals, which outlines the area and volume of surviving tissue. This method can be used to determine percent infarct size (Ito et al., 1997). The results revealed that the Glud1 Tg mice had a 30% decrease in infarct volume compared to their wt cohorts (Figure 1B). Brain edema volume was measured using 2 mm coronal sections by determining the difference between the volumes of both the ipsilateral hemisphere and the contralateral hemisphere. As shown in Figure 1C, the Glud1 Tg mice had a significantly lower edema volume following MCAO than the wt mice. Together, these results support our hypothesis that chronic exposure to moderate levels of glutamate released at synapses can protect against an ischemic insult.

Figure 1. Effect of MCAO on brain tissue damage in Wt vs. Glud1 Tg mice.

Figure 1

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Brain damage was determined by TTC staining after mice were subjected to 90 min ischemia followed by 24 hr reperfusion. (A) Representative TTC staining of brain coronal sections proceeding from frontal to caudal. Sections were taken from the 1 mm position of the frontal pole and proceeded in 2 mm intervals to 5 mm. (B) Quantification of infarct volume was determined in TTC stained sections. (C) Quantification of brain edema volume estimated from TTC stained sections (n=5). Data presented as means ± SEM. *p < 0.05 Tg vs. wt mice.

Increased basal levels of proteasomal activity in the brain of Glud1 Tg vs. wt mice

Next, we attempted to define molecular components of the adaptive mechanisms that allowed the Glud1 Tg mice to be more resistant to ischemia. The ubiquitin-proteasome system (UPS) plays an essential role in ischemic tolerance (Meller et al., 2008; Meller, 2009). To identify whether differences in the UPS between Tg and wt mice might account for the observed differential sensitivity to ischemia in these two types of mice, we measured the proteasomal activities in brain tissue of the Glud1 Tg and wt cohorts. Brain tissue from 9-mo old wt and Tg mice that had not been subjected to MCAO were homogenized, and proteasomal function was assessed. An ATP-free assay buffer was used to differentiate 20S proteasomal function from the 26S proteasomal function. We found that basal proteasomal activities of both the 26S and 20S proteasome (i.e., in the absence of any prior treatment) were significantly elevated in the Glud1 Tg mice compared with those in wt mice (Figure 2).

Figure 2. Increased brain proteasomal activity in the Glud1 Tg mice.

Figure 2

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Activities of the (A) 26S and the (B) 20S proteasome were assayed by determining their ability to cleave Suc-LLVY-AMC (n=3). Data presented as means ±SEM. *p<0.05 Tg vs. wt mice.

Ubiquitin labeling in hippocampus following MCAO in Tg and wt mice

The differentially higher activity of the 26S and 20S proteasomes in Glud1 Tg vs wt mouse brain fit with the previously observed elevated gene expression of UPS enzymes in the Tg mouse hippocampus (Wang et al. 2010). Increases in UPS mRNA levels and, as shown in the present study in the proteasome activity in Glud1 Tg mice, might represent adaptive responses to increases in the levels of damaged proteins brought about by glutamate hyper-stimulation of neurons in these mice. Overall increases in the activity of the UPS might account for the delayed appearance of significant accumulation of ubiquitin protein aggregates in brain neurons of Glud1 mice that is observed around the age of 16–20 months (Bao et al., 2009). However, ischemia-reperfusion, we reasoned, may cause an increased burden in terms of damaged proteins in both wt and Tg mice (Alves-Rodrigues et al. 1998; Keller et al. 2000b). The effect of ischemia-reperfusion on ubiquitinated protein accumulation and proteasome degradation might be expected to be greater in the Tg than the wt mouse but these mice might have adapted to the increased burden of ubiquitinated proteins. We examined the levels of immunolabeled ubiquitinated proteins in brain sections from Glud1 Tg and wt mice, both under baseline conditions and following cerebral artery occlusion. In contrast to our expectation, there were no significant differences in overall immune labeling of ubiquitin or ubiquitinated proteins in neurons of either the CA1 or the CA3 region of the hippocampus of Tg and wt mice subjected to ischemia (one-way ANOVA, Holm-Sidak post-hoc pairwise comparisons, P>0.05 for all comparisons; data not shown). The same was true also for the levels of immune reactivity under baseline conditions. The lack of differential labeling between wt and Tg mice matched the observations reported previously for 9 month old Glud1 Tg and wt hippocampus (Bao et al. 2009).

Increased MAP2A labeling in the hippocampus of the Glud1 Tg mice following MCAO

MAP2 is a microtubule-associated protein that is critical for the maintenance of normal cytoskeletal architecture in neurons and for the overall function of neurons (Yan et al., 2010). MAP2 is localized in cell bodies and dendrites and is a useful marker of surviving neurons and a probe of dendrite structure (Bao et al. 2009; Hoskison et al. 2007; Ikegaya et al. 2001). In a previous study of 9-mo old Glud1 Tg mice, it was noted that the hyperglutamatergic state in the brain of the Tg animals led to a diminution of MAP2A labeling by the anti-MAP2A antibodies, while the structure of dendrites remained relatively well preserved (Bao et al. 2009). These changes were attributed to a possible disruption of either MAP2A mRNA transport to or suppression of protein synthesis within the dendrites (Rehbein et al. 2000). In the present study, under baseline conditions, we observed a significant reduction in overall MAP2A labeling in both cell bodies and dendrites, and in both CA1 and CA3 regions of the hippocampus of the Glud1 Tg as compared with that of the wt mice (Fig. 3, 4). Remarkably, following the induction of ischemia-reperfusion, the labeling of MAP2A in neuronal cell bodies and dendrites of the Tg mice was markedly enhanced above that observed at baseline, whereas the labeling of the hippocampus neurons in wt mice was markedly reduced in both the ipsilateral and contralateral CA1 and CA3 hippocampal regions (Fig. 5, 6).

Figure 3. Immune-labeling of MAP2A in the hippocampus of Glud1 Tg and wt mice under baseline conditions.

Figure 3

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Representative immunofluorescent images showing labeling of MAP2A in the CA1 and CA3 regions of the hippocampus in a pair of 9-mo old Glud1 Tg and wt mice that were not subjected to in vivo ischemia-reperfusion. All images were obtained using identical laser intensity and fluorescence amplification. SO, stratum oriens; SP, stratum pyramidale; SR stratum radiatum. Scale bar =12 μm.

Figure 4. Quantification of MAP2A immunoreactivity in CA1 and CA3 regions of the hippocampus of Tg and wt mice under baseline conditions.

Figure 4

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The mean pixel densities (±SEM) of MAP2A immune labeling in the CA1 and CA3 regions represent the average of measurements obtained from multiple subfields from the three layers of each region of the hippocampus of 3 Tg and 3 wt mice. The data were analyzed by one-way ANOVA with post hoc analysis of pairwise comparisons (Holm-Sidak) and the significant differences between measurements are indicated.

Fig. 5. Immune labeling of MAP2A in the hippocampus of Glud1 Tg and wt mice ipsilateral to the side of MCAO.

Fig. 5

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Representative immunofluorescent images showing labeling of MAP2A in the ipsilateral CA1 and CA3 regions of the hippocampus in a pair of 9-mo old Glud1 Tg and wt mice that were subjected to in vivo ischemia-reperfusion. All images were obtained using identical laser intensity and fluorescence amplification. SO, SP, and SR, same as in figure 3. Scale bar =14 μm.

Figure 6. Quantification of MAP2A immunoreactivity in CA1 and CA3 regions of the hippocampus on the ipsilateral (A) and contralateral (B) sides to MCAO in Tg and wt mice.

Figure 6

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The mean pixel densities (±SEM) of MAP2A immune labeling in the CA1 and CA3 regions represent the average of measurements obtained from multiple subfields from the three layers of each region of the hippocampus, ipsilateral (A) or contralateral (B) to MCAO of 3 Tg and 3 wt mice. The data were analyzed by one-way ANOVA with post hoc analysis of pairwise comparisons (Holm-Sidak) and the significant differences between measurements are indicated.

Lower brain infarct and edema volume in aged Glud1 Tg mice vs wt mice

Focal ischemia leads to increases in infarct volume in aged animals (Davis et al., 1995; Sutherland et al., 1996), and the effectiveness of preconditioning in inducing protection against severe ischemia has been reported to be decreasing with advancing age (He et al., 2005). To determine whether the relative ischemia tolerance observed in our model of glutamate preconditioning, i.e., the Glud1 mice, was altered by aging, we determined brain damage (infarct and edema volumes) in 22-mo old Tg and wt mice after MCAO and reperfusion. Figure 7 shows that the Glud1 Tg mice at 22 mos of age were protected in terms of decreases in infarct and edema volumes, compared to their age-matched wt counterparts (P < 0.005). Statistical analyses showed that there were no significant differences between the 22-mo old and the 8-mo old mice of the same genotype.

Figure 7. Effect of MCAO on brain tissue damage in 22 month old Wt and Glud1 Tg mice.

Figure 7

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Brain damage was determined by TTC staining in 8-mo and 22-mo old Wt and Glud1 Tg mice after 90 min ischemia followed by 24 hr reperfusion. (A) Representative TTC staining of brain coronal sections proceeding from frontal to caudal. Sections were taken from the 1 mM position of the frontal pole and proceeded in 2 mm intervals to 5 mm. (B) Quantification of infarct volume determined by TTC stained sections. (C) Quantification of brain edema volume estimated from TTC stained sections (n=3). Data presented as means ± SEM. *p < 0.05 Tg vs. wt mice.

DISCUSSION

The key observation made in the present study was that either adult or aged Glud1 Tg mice exhibited significantly greater resistance to ischemia-reperfusion than the Wt mice of the same age. The mechanisms for such tolerance to the effects of ischemia-reperfusion are still unknown. The first mechanism considered in the present study was that of a proteasome-mediated resistance to ischemia-reperfusion.

It is known that excess glutamate can induce the formation of reactive oxygen species (ROS) (Armstead et al., 1989; Duchen, 2000; Kahlert et al., 2005; Parfenova et al., 2006) and that ROS can alter proteasome activities (Ullrich et al., 1999; Ding et al., 2003; Grune et al., 2004; Aiken et al., 2011). The proteasome proteolytic pathways represent the main mechanisms responsible for the degradation of damaged or unwanted proteins. These pathways play an important role in maintaining normal cellular homeostasis, as null mutants for the proteasome subunits result in lethal phenotypes (Heinemeyer et al., 1991; Orlowski, 1999). Protein unfolding and aggregation are also dominant pathogenic events in vulnerable ischemic neurons (Ge et al., 2007). A study by Liu et al. (2005) showed that ischemic preconditioning alleviated protein aggregation in neurons. Whether this reduction is due to a decrease in overall cell damage or the result of an enhanced ability by neurons tolerant to ischemia in eliminating the irreparably damaged proteins has yet to be elucidated. Therefore, we tested the hypothesis that chronic exposure of neurons to increased levels of synaptic glutamate release, i.e., glutamate preconditioning, might lead to an increase in proteasome activity. We observed significant increases in both the 20S and 26S baseline proteasome activities in Glud1 mouse brains as compared with those of wt mice. Ubiquitination is a key step in the degradation process of many proteins. Contrary to our hypothesis that elevated proteasomal activity would lead to decreases in ubiquitinated proteins following an episode of ischemia-reperfusion, we did not observed any difference in the level of ubiquitin immunoreactivity following ischemia-reperfusion in hippocampal neurons of the Tg, compared with those of the Wt mice. In summary, the increase of the proteasome activities but not of ubiquitination suggests that the proteasomal degradation pathways are involved in the neuroprotective effect of chronic glutamate synaptic release.

The molecular dynamics that lead to increases in proteasome activity in the Glud1 mice are not known. Following an episode of stroke, the loss of ATP impairs the activity of the 26S proteasome (Keller et al., 2000). The 26S proteasome is an ATP-dependent protease composed of a core proteinase, the 20S proteasome, and two PA700 regulatory particles (the 19S complex) on both ends (Coux et al., 1996). These subunits associate and dissociate in an ATP-dependent manner, thus conditions of decreased ATP levels would bring about the dissociation of the subunits (Tanahashi et al., 2000). Following transient forebrain ischemia, the ATP-dependent re-association of the 20S catalytic and PA700 regulatory subunits to form the 26S proteasome is severely impaired in the hippocampus (Asai et al., 2002). The increased baseline proteasome activity in Glud1 mouse brains might offer relative resistance to the ischemia-induced loss of ATP. However, the present studies did not link the protective effect of chronic glutamate synaptic release in brain to the activation of proteasome degradation of proteins.

Besides the differential activities of the proteasomes in the Tg vs. wt brains, the opposite responses of neurons to ischemia-reperfusion in terms of the MAP2A levels in Tg and wt hippocampus, was one of the most remarkable differential characteristics observed. MAPs are key regulators of neuronal morphogenesis (Popa-Wagner et al., 1999). The so-called “late MAPs”, MAP2A and MAP2B, have a profound effect on the organization of cellular microtubules (Lewis et al., 1989), provide structural stabilization during process outgrowth (Chen et al., 1992), and assist in the maintenance of proper synaptic circuitry in the mature brain (Marsden et al., 1996; Popa-Wagner et al., 1999). MAP2A is preferentially associated with dendritic processes (Binder et al., 1986). Stress conditions can alter MAP2 levels in the brain cortex and hippocampus (Yan et al., 2010), and MAP2 localization and protein levels are disrupted in response to the stress induced by high extracellular glutamate levels (Arias et al., 1997). Cerebral ischemia reduces MAP2 immunoreactivity in the hippocampus and cortex of both neonatal (Malinak & Silverstein, 1996) and adult rats (Kitagawa et al., 1989; Dawson & Hallenbeck, 1996). The most significant loss of MAP2 following ischemia is in the CA1 region in both rats (Inuzuka et al., 1990) and gerbils (Yoshimi et al., 1991). The susceptibility of MAP2 to ischemia and excitotoxicity has been attributed to the elevation of intracellular Ca2+ concentrations that lead to rapid MAP2 proteolysis by calcium-activated proteinases (Kitagawa et al., 1989). However, some have reported that MAP2 levels in the penumbra surrounding the core of the ischemic lesion, as well as in surviving neurons at the core of ischemic tissue, are increased rather than decreased (Li et al. 1998). The same is true for MAP2 levels following episodes of severe epileptic seizures in rats (Jalava et al. 2007). Thus, under conditions of increased glutamate release to the extracellular environment of neurons, i.e., ischemic penumbra and post-ictal state, certain neuronal populations respond to the stress by increasing the intracellular levels of MAP2 proteins.

In our studies, we observed that following MCAO, MAP2A was significantly increased in the CA1 region of the Glud1 Tg mice compared to the wt littermates. This striking increase in MAP2 immunolabeling is more likely a result of increased protein levels and not an increase in the number of dendritic processes. This suggests that unlike the wt mice, the Glud1 Tg mice may have a preserved ability to synthesize some of the components required for structural repair and maintenance (Popa-Wagner et al., 1999). Since some MAP2 is synthesized in the cell body and then transported to dendrites (Okabe & Hirokawa, 1989), while a substantial amount of MAP2A is synthesized in dendrites from mRNA that has been transported to dendritic sites (Rehbein et al. 2000), it would appear that the Glud1 Tg mice have improved protein and mRNA transport processes that lead to enhanced recovery following cerebral ischemia. In more recent studies, we have observed significant increases in the levels of proteins involved in the transport of membrane bound organelles in neurons, the kinesins, in mRNA levels for these proteins, and in overall axoplasmic transport in Tg vs. wt neurons (P. Lee et al., submitted for publication). Therefore, the possibility that neurons from the hyperglutamatergic mice might recover more readily the transport of particle-bound mRNA and local synthesis of MAP2A than neurons from wt mice seems possible but has to be examined in greater detail in future studies.

Previous studies have reported that the degree of preconditioning-induced protection is significantly diminished in aged rats (Fenton et al., 2000; He et al., 2005). In humans, transient ischemic attacks (TIA) were found to be neuroprotective against ischemic strokes. However, the protective mechanisms of TIAs were not present in elderly patients over the age of 65 (Della Morte et al., 2008). To determine whether the tolerance induced by glutamate preconditioning was preserved in aged mice, we evaluated brain infarct size in 22-mo old Glud1 Tg mice after MCAO and reperfusion. We showed that glutamate preconditioning was at least as, if not more, effective in the aged (22-mo) compared with the young adult (9-mo) mice. This was consistent with the results of another study in which gerbils preconditioned with 1.5 min of ischemia before a subsequent 5-min occlusion of both carotid arteries (global ischemia), exhibited differential levels of resistance to ischemia (Dowden & Corbett, 1999). The young animals had 53–67% protection of CA1 neurons while the older animals exhibited approximately 75% protection (Dowden & Corbett, 1999). The authors attributed this difference to the decrease in density and sensitivity of NMDA receptors that occur in the aged rodent brain (Gonzales et al., 1991).

In conclusion, the phenomenon of ischemic preconditioning in the brain is well documented. In this report, for the first time, we demonstrate that in vivo increased synaptic release of glutamate can induce ischemic tolerance and maintain cell viability in young adult mice that were subjected to cerebral artery occlusion. This protection was preserved in the aged 22-mo old mice. The increase in MAP2 levels following ischemia-reperfusion in the resistant Glud1 Tg mice, may suggest an enhancement in protein synthesis and protein and mRNA transport to distal processes in these mice. Increases in MAP2A synthesis and transport under conditions of neuronal stress may be an important component of the glutamate preconditioning process that leads to ischemic tolerance and, potentially, to tolerance to other stressful stimuli.

Acknowledgments

This work was supported in part by National Institutes of Health grants NS058807 from NINDS; AG12993 and AG035982 from NIA; and AA11419 from NIAAA, and a startup fund from the University of Kansas Center for Research.

ABBREVIATIONS

AMC

7-amino-4-methly-coumarin

ATP

adenosine triphosphate

CA

carbonic anhydrase

CNS

central nervous system

EAAT-2

excitatory amino acid transporter-2

Glud1

glutamate dehydrogenase gene

MAP

microtubule-associated proteins

MCAO

middle cerebral artery occlusion

mo

month

NMDA

N-methyl-D-aspartate

nse

neuron-specific enolase

ROS

reactive oxygen species

rt-PA

recombinant tissue plasminogen activator

SO

stratum oriens

SP

stratum pyramidale

SR

stratum radiatum

Tg

transgenic

TIA

transient ischemic attack

TTC

2,3,5-triphenyltetrazolium chloride monohydrate

UBE

ubiquitin-conjugating enzymes

UPS

ubiquitin proteasome system

wt

wild type

References

  1. Aiken CT, Kaake RM, Wang X, Huang L. Oxidative stress-mediated regulation of proteasome complexes. Mol Cell Proteom. 2011;10:R110. doi: 10.1074/mcp.M110.006924. 006924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arias C, Arrieta I, Massieu L, Tapia R. Neuronal damage and MAP2 changes induced by the glutamate transport inhibitor dihydrokainate and by kainate in rat hippocampus in vivo. Exp Brain Res. 1997;116:467–476. doi: 10.1007/pl00005774. [DOI] [PubMed] [Google Scholar]
  3. Alves-Rodrigues A, Gregori L, Figueiredo-Pereira ME. Ubiquitin, cellular inclusions and their role in neurodegeneration. Trend Neurosci. 1998;21:516–520. doi: 10.1016/s0166-2236(98)01276-4. [DOI] [PubMed] [Google Scholar]
  4. Armstead W, Mirro R, Leffler C, Busija D. Cerebral superoxide anion generation during seizures in newborn pigs. J Cerebr Blood Flow Metab. 1989;9:175–179. doi: 10.1038/jcbfm.1989.26. [DOI] [PubMed] [Google Scholar]
  5. Asai A, Tanahashi N, Qiu J-h, Saito N, Chi S, Kawahara N, Tanaka K, Kirino T. Selective proteasomal dysfunction in the hippocampal CA1 region after transient forebrain ischemia. J Cerebr Blood Flow Metab. 2002;22:705–710. doi: 10.1097/00004647-200206000-00009. [DOI] [PubMed] [Google Scholar]
  6. Bao X, Pal R, Hascup KN, Wang Y, Wang WT, Xu W, Hui D, Agbas A, Wang X, Michaelis ML. Transgenic expression of Glud1 (glutamate dehydrogenase 1) in neurons: in vivo model of enhanced glutamate release, altered synaptic plasticity, and selective neuronal vulnerability. J Neurosci. 2009;29:13929–13944. doi: 10.1523/JNEUROSCI.4413-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Benveniste H, Drejer J, Schousboe A, Diemer NH. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem. 1984;43:1369–1374. doi: 10.1111/j.1471-4159.1984.tb05396.x. [DOI] [PubMed] [Google Scholar]
  8. Binder LI, Frankfurter A, Rebhun LI. Differential localization of MAP-2 and tau in mammalian neurons in situ. Ann NY Acad Sci. 1986;466:145–166. doi: 10.1111/j.1749-6632.1986.tb38392.x. [DOI] [PubMed] [Google Scholar]
  9. Chen J, Kanai Y, Cowan NJ, Hirokawa N. Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons. Nature. 1992;360:674–677. doi: 10.1038/360674a0. [DOI] [PubMed] [Google Scholar]
  10. Choi IY, Lee P, Wang WT, Hui D, Wang X, Brooks WM, Michaelis EK. Metabolism changes during aging in the hippocampus and striatum of glud1 (glutamate dehydrogenase 1) transgenic mice. Neurochem Res. 2014;39:446–455. doi: 10.1007/s11064-014-1239-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clark WM, Lessov NS, Dixon MP, Eckenstein F. Monofilament intraluminal middle cerebral artery occlusion in the mouse. Neurol Res. 1997;19:641. doi: 10.1080/01616412.1997.11740874. [DOI] [PubMed] [Google Scholar]
  12. Coux O, Tanaka K, Goldberg AL. Structure and functions of the 20S and 26S proteasomes. Ann Rev Biochem. 1996;65:801–847. doi: 10.1146/annurev.bi.65.070196.004101. [DOI] [PubMed] [Google Scholar]
  13. Davis M, Mendelow AD, Perry RH, Chambers IR, James OF. Experimental stroke and neuroprotection in the aging rat brain. Stroke. 1995;26:1072–1078. doi: 10.1161/01.str.26.6.1072. [DOI] [PubMed] [Google Scholar]
  14. Dawson DA, Hallenbeck JM. Acute focal ischemia-induced alterations in MAP2 immunostaining: description of temporal changes and utilization as a marker for volumetric assessment of acute brain injury. J Cereb Blood Flow Metab. 1996;16:170–174. doi: 10.1097/00004647-199601000-00020. [DOI] [PubMed] [Google Scholar]
  15. Della Morte D, Abete P, Gallucci F, Scaglione A, D’Ambrosio D, Gargiulo G, De Rosa G, Dave KR, Lin HW, Cacciatore F. Transient ischemic attack before nonlacunar ischemic stroke in the elderly. J Stroke Cerebr Dis. 2008;17:257–262. doi: 10.1016/j.jstrokecerebrovasdis.2008.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ding Q, Reinacker K, Dimayuga E, Nukala V, Drake J, Butterfield DA, Dunn JC, Martin S, Bruce-Keller AJ, Keller JN. Role of the proteasome in protein oxidation and neural viability following low-level oxidative stress. FEBS Lett. 2003;546:228–232. doi: 10.1016/s0014-5793(03)00582-9. [DOI] [PubMed] [Google Scholar]
  17. Dowden J, Corbett D. Ischemic preconditioning in 18-to 20-month-old gerbils long-term survival with functional outcome measures. Stroke. 1999;30:1240–1246. doi: 10.1161/01.str.30.6.1240. [DOI] [PubMed] [Google Scholar]
  18. Duchen MR. Mitochondria and calcium: from cell signalling to cell death. J Physiol. 2000;529:57–68. doi: 10.1111/j.1469-7793.2000.00057.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fenton RA, Dickson EW, Meyer TE, Dobson JG., Jr Aging reduces the cardioprotective effect of ischemic preconditioning in the rat heart. J Mol Cell Cardiol. 2000;32:1371–1375. doi: 10.1006/jmcc.2000.1189. [DOI] [PubMed] [Google Scholar]
  20. Figueiredo-Pereira ME, Berg KA, Wilk S. A new inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces accumulation of ubiquitin-protein conjugates in a neuronal cell. J Neurochem. 1994;63:1578–1581. doi: 10.1046/j.1471-4159.1994.63041578.x. [DOI] [PubMed] [Google Scholar]
  21. Fonnum F. Glutamate: a neurotransmitter in mammalian brain. J Neurochem. 1984;42:1–11. doi: 10.1111/j.1471-4159.1984.tb09689.x. [DOI] [PubMed] [Google Scholar]
  22. Ge P, Luo Y, Liu CL, Hu B. Protein aggregation and proteasome dysfunction after brain ischemia. Stroke. 2007;38:3230–3236. doi: 10.1161/STROKEAHA.107.487108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gonzales RA, Brown LM, Jones TW, Trent RD, Westbrook SL, Leslie SW. N-methyl-D-aspartate mediated responses decrease with age in Fischer 344 rat brain. Neurobiol Aging. 1991;12:219–225. doi: 10.1016/0197-4580(91)90100-x. [DOI] [PubMed] [Google Scholar]
  24. Grabb MC, Choi DW. Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors. J Neurosci. 1999;19:1657–1662. doi: 10.1523/JNEUROSCI.19-05-01657.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Grune T, Jung T, Merker K, Davies KJA. Decreased proteolysis caused by protein aggregates, inclusion bodies, plaques, lipofuscin, ceroid, and ‘aggresomes’ during oxidative stress, aging, and disease. Intl J Biochem Cell Biol. 2004;36:2519–2530. doi: 10.1016/j.biocel.2004.04.020. [DOI] [PubMed] [Google Scholar]
  26. Hascup KN, Bao X, Hascup ER, Hui D, Xu W, Pomerleau F, Huettl P, Michaelis ML, Michaelis EK, Gerhardt GA. Differential levels of glutamate dehydrogenase 1 (GLUD1) in Balb/c and C57BL/6 mice and the effects of overexpression of the Glud1 gene on glutamate release in striatum. ASN Neuro. 2011;3:e00057. doi: 10.1042/AN20110005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. He Z, Crook JE, Meschia JF, Brott TG, Dickson DW, McKinney M. Aging blunts ischemic-preconditioning-induced neuroprotection following transient global ischemia in rats. Curr Neurovasc Res. 2005;2:365–374. doi: 10.2174/156720205774962674. [DOI] [PubMed] [Google Scholar]
  28. Heinemeyer W, Kleinschmidt J, Saidowsky J, Escher C, Wolf D. Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival. EMBO J. 1991;10:555. doi: 10.1002/j.1460-2075.1991.tb07982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hoskison MM, Yanagawa Y, Obata K, Shuttleworth CW. Calcium-dependent NMDA-induced dendritic injury and MAP2 loss in acute hippocampal slices. Neuroscience. 2007;145:66–79. doi: 10.1016/j.neuroscience.2006.11.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ikegaya Y, Kim JA, Baba M, Iwatsubo T, Nishiyama N, Matsuki N. Rapid and reversible changes in dendrite morphology and synaptic efficacy following NMDA receptor activation: implication for a cellular defense against excitotoxicity. J Cell Sci. 2001;114:4083–4093. doi: 10.1242/jcs.114.22.4083. [DOI] [PubMed] [Google Scholar]
  31. Inuzuka T, Tamura A, Sato S, Kirino T, Yanagisawa K, Toyoshima I, Miyatake T. Changes in the concentrations of cerebral proteins following occlusion of the middle cerebral artery in rats. Stroke. 1990;21:917–922. doi: 10.1161/01.str.21.6.917. [DOI] [PubMed] [Google Scholar]
  32. Ito W, Schaarschmidt S, Klask R, Hansen S, Schafer H, Mathey D, Bhakdi S. Infarct Size Measurement by Triphenyltetrazolium Chloride StainingVersus In VivoInjection of Propidium Iodide. J Mol Cell Cardiol. 1997;29:2169–2175. doi: 10.1006/jmcc.1997.0456. [DOI] [PubMed] [Google Scholar]
  33. Jalava NS, Lopez-Picon FR, Kukko-Lukjanov TK, Holopainen IE. Changes in microtubule-associated protein-2 (MAP2) expression during development and after status epilepticus in the immature rat hippocampus. Intl J Dev Neurosci. 2007;25:121–131. doi: 10.1016/j.ijdevneu.2006.12.001. [DOI] [PubMed] [Google Scholar]
  34. Kahlert S, Zundorf G, Reiser G. Glutamate-mediated influx of extracellular Ca2+ is coupled with reactive oxygen species generation in cultured hippocampal neurons but not in astrocytes. J Neurosci Res. 2005;79:262–271. doi: 10.1002/jnr.20322. [DOI] [PubMed] [Google Scholar]
  35. Keller JN, Huang FF, Markesbery WR. Decreased levels of proteasome activity and proteasome expression in aging spinal cord. Neuroscience. 2000a;98:149–156. doi: 10.1016/s0306-4522(00)00067-1. [DOI] [PubMed] [Google Scholar]
  36. Keller JN, Huang FF, Zhu H, Yu J, Ho YS, Kindy TS. Oxidative stress-associated impairment of proteasome activity during ischemia-reperfusion injury. J Cerebr Blood Flow Metab. 2000b;20:1467–1473. doi: 10.1097/00004647-200010000-00008. [DOI] [PubMed] [Google Scholar]
  37. Kirino T. Ischemic tolerance. J Cerebr Blood Flow Metab. 2002;22:1283–1296. doi: 10.1097/01.WCB.0000040942.89393.88. [DOI] [PubMed] [Google Scholar]
  38. Kitagawa K, Matsumoto M, Niinobe M, Mikoshiba K, Hata R, Ueda H, Handa N, Fukunaga R, Isaka Y, Kimura K, et al. Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage--immunohistochemical investigation of dendritic damage. Neuroscience. 1989;31:401–411. doi: 10.1016/0306-4522(89)90383-7. [DOI] [PubMed] [Google Scholar]
  39. Lewis SA, Ivanov IE, Lee GH, Cowan NJ. Organization of microtubules in dendrites and axons is determined by a short hydrophobic zipper in microtubule-associated proteins MAP2 and tau. Nature. 1989;342:498–505. doi: 10.1038/342498a0. [DOI] [PubMed] [Google Scholar]
  40. Li Y, Jiang N, Powers C, Chopp M. Neuronal damage and plasticity identified by microtubule-associated protein 2, growth-associated protein 43, and cyclin D1 immunoreactivity after focal cerebral ischemia in rats. Stroke. 1998;29:1972–1980. doi: 10.1161/01.str.29.9.1972. [DOI] [PubMed] [Google Scholar]
  41. Lin CH, Chen PS, Gean PW. Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons. Eur J Pharmacol. 2008;589:85–93. doi: 10.1016/j.ejphar.2008.05.047. [DOI] [PubMed] [Google Scholar]
  42. Liu C, Chen S, Kamme F, Hu B. Ischemic preconditioning prevents protein aggregation after transient cerebral ischemia. Neuroscience. 2005;134:69–80. doi: 10.1016/j.neuroscience.2005.03.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mabuchi T, Kitagawa K, Kuwabara K, Takasawa K, Ohtsuki T, Xia Z, Storm D, Yanagihara T, Hori M, Matsumoto M. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo. J Neurosc. 2001;21:9204–9213. doi: 10.1523/JNEUROSCI.21-23-09204.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Malinak C, Silverstein FS. Hypoxic-ischemic injury acutely disrupts microtubule-associated protein 2 immunostaining in neonatal rat brain. Biol Neonate. 1996;69:257–267. doi: 10.1159/000244319. [DOI] [PubMed] [Google Scholar]
  45. Marsden KM, Doll T, Ferralli J, Botteri F, Matus A. Transgenic expression of embryonic MAP2 in adult mouse brain: implications for neuronal polarization. J Neurosci. 1996;16:3265–3273. doi: 10.1523/JNEUROSCI.16-10-03265.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Meller R. The role of the ubiquitin proteasome system in ischemia and ischemic tolerance. Neuroscientist. 2009;15:243–260. doi: 10.1177/1073858408327809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Meller R, Thompson SJ, Lusardi TA, Ordonez AN, Ashley MD, Jessick V, Wang W, Torrey DJ, Henshall DC, Gafken PR. Ubiquitin–Proteasome-Mediated Synaptic Reorganization: A Novel Mechanism Underlying Rapid Ischemic Tolerance. J Neurosci. 2008;28:50–59. doi: 10.1523/JNEUROSCI.3474-07.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Michaelis EK. Selective neuronal vulnerability in the hippocampus: Relationship to neurological disease mechanisms for differential sensitivity of neurons to stress. In: Bartsch T, editor. The hippocampus in neurological disease: An integrative view. Oxford: Oxford University Press; 2012. pp. 54–76. [Google Scholar]
  49. Michaelis EK, Wang X, Pal R, Bao X, Hascup KN, Wang Y, Wang WT, Hui D, Agbas A, Choi IY, Belousov A, Gerhardt GA. Neuronal Glud1 (glutamate dehydrogenase 1) over-expressing mice: increased glutamate formation and synaptic release, loss of synaptic activity, and adaptive changes in genomic expression. Neurochem Int. 2011;59:473–481. doi: 10.1016/j.neuint.2011.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Minnerup J, Sutherland BA, Buchan AM, Kleinschnitz C. Neuroprotection for stroke: current status and future perspectives. Intl J Mol Sci. 2012;13:11753–11772. doi: 10.3390/ijms130911753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. O’Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, Howells DW. 1,026 experimental treatments in acute stroke. Ann Neurol. 2006;59:467–477. doi: 10.1002/ana.20741. [DOI] [PubMed] [Google Scholar]
  52. Okabe S, Hirokawa N. Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons. Proc Natl Acad Sci. 1989;86:4127–4131. doi: 10.1073/pnas.86.11.4127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Orlowski RZ. The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Diff. 1999;6:303. doi: 10.1038/sj.cdd.4400505. [DOI] [PubMed] [Google Scholar]
  54. Parfenova H, Basuroy S, Bhattacharya S, Tcheranova D, Qu Y, Regan RF, Leffler CW. Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection. Am J Physiol-Cell Physiol. 2006;290:C1399–C1410. doi: 10.1152/ajpcell.00386.2005. [DOI] [PubMed] [Google Scholar]
  55. Popa-Wagner A, Schroder E, Schmoll H, Walker LC, Kessler C. Upregulation of MAP1B and MAP2 in the rat brain after middle cerebral artery occlusion: effect of age. J Cereb Blood Flow Metab. 1999;19:425–434. doi: 10.1097/00004647-199904000-00008. [DOI] [PubMed] [Google Scholar]
  56. Rehbein M, Kindler S, Horke S, Richter D. Two trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs. Brain Res Mol Brain Res. 2000;79:192–201. doi: 10.1016/s0169-328x(00)00114-5. [DOI] [PubMed] [Google Scholar]
  57. Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron. 1996;16:675. doi: 10.1016/s0896-6273(00)80086-0. [DOI] [PubMed] [Google Scholar]
  58. Sims NR, Muyderman H. Mitochondria, oxidative metabolism and cell death in stroke. Biochim Biophys Acta. 2010;1:80–91. doi: 10.1016/j.bbadis.2009.09.003. [DOI] [PubMed] [Google Scholar]
  59. Sutherland GR, Dix GA, Auer RN. Effect of age in rodent models of focal and forebrain ischemia. Stroke. 1996;27:1663–1667. doi: 10.1161/01.str.27.9.1663. [DOI] [PubMed] [Google Scholar]
  60. Tanahashi N, Murakami Y, Minami Y, Shimbara N, Hendil KB, Tanaka K. Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis. J Biol Chem. 2000;275:14336–14345. doi: 10.1074/jbc.275.19.14336. [DOI] [PubMed] [Google Scholar]
  61. Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, Iwama H, Nishikawa T, Ichihara N, Kikuchi T. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science. 1997;276:1699–1702. doi: 10.1126/science.276.5319.1699. [DOI] [PubMed] [Google Scholar]
  62. Ullrich O, Reinheckel T, Sitte N, Hass R, Grune T, Davies KJA. Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones. Proc Natl Acad Sci. 1999;96:6223. doi: 10.1073/pnas.96.11.6223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wang X, Bao X, Pal R, Agbas A, Michaelis EK. Transcriptomic responses in mouse brain exposed to chronic excess of the neurotransmitter glutamate. BMC genomics. 2010;11:360. doi: 10.1186/1471-2164-11-360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wang X, Pal R, Chen XW, Limpeanchob N, Kumar KN, Michaelis EK. High intrinsic oxidative stress may underlie selective vulnerability of the hippocampal CA1 region. Brain Res Mol Brain Res. 2005;140:120–126. doi: 10.1016/j.molbrainres.2005.07.018. [DOI] [PubMed] [Google Scholar]
  65. Wang X, Patel ND, Hui D, Pal R, Hafez MM, Sayed-Ahmed MM, Al-Yahya AA, Michaelis EK. Gene expression patterns in the hippocampus during the development and aging of Glud1 (Glutamate Dehydrogenase 1) transgenic and wild type mice. BMC Neurosci. 2014;15:37. doi: 10.1186/1471-2202-15-37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yan J, Sun XB, Wang HQ, Zhao H, Zhao XY, Xu YX, Guo JC, Zhu CQ. Chronic restraint stress alters the expression and distribution of phosphorylated tau and MAP2 in cortex and hippocampus of rat brain. Brain Res. 2010;6:132–141. doi: 10.1016/j.brainres.2010.05.074. [DOI] [PubMed] [Google Scholar]
  67. Yan J, Zhou B, Taheri S, Shi H. Differential effects of HIF-1 Inhibition by YC-1 on the overall outcome and blood-brain barrier damage in a rat model of ischemic stroke. PloS one. 2011;6:e27798. doi: 10.1371/journal.pone.0027798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yoshimi K, Takeda M, Nishimura T, Kudo T, Nakamura Y, Tada K, Iwata N. An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia. Brain Res. 1991;560:149–158. doi: 10.1016/0006-8993(91)91225-p. [DOI] [PubMed] [Google Scholar]
  69. Zeng L-H, Ouyang Y, Gazit V, Cirrito JR, Jansen LA, Ess KC, Yamada KA, Wozniak DF, Holtzman DM, Gutmann DH, Wong M. Abnormal glutamate homeostasis and impaired synaptic plasticity and learning in a mouse model of tuberous sclerosis complex. Neurobiol Dis. 2007;28:184–196. doi: 10.1016/j.nbd.2007.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]

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