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Keywords:

  • apoptosis;
  • cerebral ischemia;
  • glutamate;
  • heat-shock proteins 70 and 90;
  • membrane lipid peroxidation;
  • mitochondrial transmembrane potential.

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Mild metabolic stress may increase resistance of neurons in the brain to subsequent, more severe insults, as demonstrated by the ability of ischemic pre-conditioning and dietary restriction to protect neurons in experimental models of stroke- and age-related neurodegenerative disorders. In the present study we employed iodoacetic acid (IAA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, to test the hypothesis that inhibition of glycolysis can protect neurons. Pre-treatment of cultured hippocampal neurons with IAA can protect them against cell death induced by glutamate, iron and trophic factor withdrawal. Surprisingly, protection occurred with concentrations of IAA (2–200 nm) much lower than those required to inhibit glycolysis. Pre-treatment with IAA results in suppression of oxyradical production and stabilization of mitochondrial function in neurons after exposure to oxidative insults. Levels of the stress heat-shock proteins HSP70 and HSP90, and of the anti-apoptotic protein Bcl-2, were increased in neurons exposed to IAA. Our data demonstrate that IAA can stimulate cytoprotective mechanisms within neurons, and suggest the possible use of IAA and related compounds in the prevention and/or treatment of neurodegenerative conditions.

Abbreviations used
HNE

4-hydroxy-2,3-nonenal

HSP

heat-shock protein

GRP

glucose-regulated protein

IAA

iodoacetic acid.

Exposure of neurons to moderate levels of stress can increase their resistance to more severe and otherwise lethal insults in experimental cell culture and animal models relevant to the pathogenesis of ischemic stroke and other neurodegenerative conditions. For example, exposure of cultured neurons to elevated temperatures (heat-shock) increases their resistance to excitotoxicity (Lowenstein et al. 1991), as does prior exposure to a low subtoxic level of N-methyl-d-aspartate (Marini and Paul 1992). In vivo, a mild ‘pre-conditioning’ ischemia can increase resistance of hippocampal and cortical neurons to a subsequent severe ischemic insult (Liu et al. 1993; Amin et al. 1995; Matsushima and Hakim 1995; Kitagawa et al. 1997), and mild impairment of oxidative phosphorylation in cell culture and in vivo can also elicit a cytoprotective response (Riepe et al. 1997). In addition to aerobic metabolism, neurons can also produce energy anaerobically via the process of glycolysis in which glucose is phosphorylated and then metabolized to 3-carbon sugars that undergo oxidation and reactions of phosphate bonds to generate ATP. Under basal conditions, glycolysis is thought to make only a minor contribution to the total ATP production of neurons (Lutz 1992). Under hypoxic conditions, anaerobic glycolysis provides the adult brain with a limited amount of energy and time to maintain ion homoeostasis and other essential processes before several events occur that lead to brain cell damage and death. It is not known whether inhibition of glycolysis can induce a cytoprotective response in neurons. If this were the case, then chemicals that inhibit glycolysis may have potential clinical applications.

Biochemical cascades that result in cell death, particularly those involved in a form of programmed cell death called apoptosis, occur not only in the cytoplasm, but also in the mitochondria and endoplasmic reticulum (Mattson 2000). For example, neuronal apoptosis induced by excitotoxic and oxidative insults involves changes in mitochondrial membrane permeability (Green and Reed 1998), release of calcium from the endoplasmic reticulum (Guo et al. 1998, 1999), and activation of proteolytic cascades involving one or more caspases (Chan and Mattson 1999). Two major families of proteins can prevent apoptosis, the Bcl-2 family (Sadoul 1998) and the inhibitor of apoptosis protein (IAP) family (Robertson et al. 2000). Bcl-2 may prevent cell death by stabilizing mitochondrial membranes and suppressing oxyradical damage, while IAPs can directly inhibit caspases. The mechanisms whereby moderate stress increases neuronal resistance to injury are beginning to be identified. One class of proteins involved in the stress response are molecular chaperones that include heat-shock proteins and glucose-regulated proteins (Koroshetz and Bonventre 1994; Yu et al. 1999). These proteins act by binding to dysfunctional and misfolded proteins, which often targets them for proteolytic degradation. Chaperone proteins are localized in specific subcellular compartments. For example, heat-shock protein 70 (HSP70) is present in the cytosol, HSP75 is primarily in the mitochondria and glucose-regulated protein 78 (GRP78) is located in the endoplasmic reticulum.

Because inhibition of glycolysis might be expected to induce a mild stress response in neurons without compromising their viability or function, we sought to determine whether inhibition of glycolysis might protect neurons against death. To this end, we employed iodoacetic acid (IAA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (Sabri and Ochs 1971). Our data demonstrate strong neuroprotective effects of IAA against excitotoxic and oxidative insults, and trophic factor withdrawal. The protective effect occurs with concentrations of IAA lower than those which inhibit ATP production, and involves increased production of HSP70, HSP90 and Bcl-2.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Hippocampal cell cultures, experimental treatments and quantification of neuron survival

Hippocampi were removed from embryonic day 18 Sprague–Dawley rats (Harlan, Inc., Indianapolis, IN, USA), and cells were dissociated by mild trypsination and trituration and seeded onto polyethyleneimine-coated plastic 35- or 60-mm diameter dishes or 22-mm2 glass coverslips at a density of approximately 100 cells/mm2 of culture surface. Cultures were maintained in Neurobasal medium containing B-27 supplements (Gibco-BRL, Gaithersbutg, MD, USA), 2 mm l-glutamine, 1 mm HEPES and 0.001% gentamicin sulfate (Sigma, St Louis, MO, USA). All experiments were performed using 8–10-day-old-cultures. IAA, glutamate and FeSO4 (Sigma) were prepared as 200–500× stocks in sterile water, whereas 4-hydroxy-2,3-nonenal (Cayman Chemicals, Ann Arbor, MI, USA) was prepared as a 500× stock in ethanol. IAA was present in the culture medium during the cytotoxic treatments. Withdrawal of trophic support was accomplished by replacing the culture maintenance medium with Locke's buffer as described previously (Chan et al. 1999; Glazner et al. 2000). Neuron survival was quantified by counting viable neurons in pre-marked fields (10× objective; four fields/culture; and a minimum of four cultures per experiment) before experimental treatment and at specified time points thereafter, as described previously (Mattson et al. 1995). Neurons with intact neurites of uniform diameter and a soma with a smooth round appearance were considered viable, whereas neurons with fragmented neurites and a vacuolated soma were considered non-viable.

Measurement of cellular ATP levels

The concentration of ATP in cell lysates was measured using a luciferin/luciferase-based assay. After experimental treatment the cells were rinsed with phosphate-buffered saline (PBS) and lysed with 0.2 mL of cell lysis reagent (Roche, Mannheim, Germany), and10 µL of the lysate was taken for protein determination. ATP concentrations in lysates were quantified using an ATP Bioluminescence Assay Kit CLS II (Roche) and a luminometer (Optocomp II; MGM Instruments, Hamden, CT, USA) according to the manufacturers' protocols. Solutions of known ATP concentrations were used to generate a standard curve, and cell lysates were diluted so that readings fell within the linear range. ATP levels were calculated as nanomole ATP per microgram protein.

Evaluation of mitochondrial membrane potential and oxidative stress

The dye rhodamine 123 (Molecular Probes, Eugene, OR, USA) was employed as a measure of mitochondrial function using methods described previously (Mattson et al. 1993). Briefly, cells were incubated for 30 min in the presence of 10 µm of the dye, washed three times in fresh culture medium, and confocal images of cellular rhodamine 123 fluorescence were acquired using a Zeiss 510 CLSM (488 nm excitation and 510 nm emission). The average pixel intensity in individual cell bodies was determined using the software supplied by the manufacturer (Zeiss, Germany); all images were coded and analyzed without knowledge of experimental treatment history of the cultures. The dye dihydrorhodamine (DHR) was used to quantify relative levels of cellular oxyradicals using methods similar to those described previously (Mattson et al. 1997). DHR localizes to mitochondria and fluoresces when oxidized by hydroxy radical and peroxynitrite to the positively charged rhodamine 123 derivative. Briefly, cells were incubated for 30 min in the presence of 5 µm DHR, washed three times with fresh medium, and confocal images of cellular fluorescence were acquired and analyzed as described for rhodamine 123 fluorescence.

Immunoblots

The immunoblot methods were similar to those described previously (Glazner et al. 2000). Briefly, 50 µg of solubilized proteins were separated by electrophoresis in a polyacrylamide gel, transferred to a nitrocellulose sheet, and immunoreacted with primary antibody overnight at 4°C. The nitrocellulose sheet was further processed using horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit secondary antibody and a chemiluminescence detection method (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The primary antibodies included a mouse monoclonal antibody against the inducible form of HSP70 (Sigma; 1 : 5000 dilution), a mouse monoclonal antibody against HSP90 (Transduction Laboratories, Lexington, KY, USA; 1 : 1000 dilution), a rabbit polyclonal antibody against GRP78 (Stressgen, Collegeville, PA, USA; 1: 2000 dilution), a rabbit polyclonal antibody against GRP94 (Stressgen; 1 : 2500 dilution), a mouse monoclonal antibody against Bcl-2 (Transduction Laboratories, 1 : 500 dilution), and a rabbit polyclonal antibody against Bcl-XL (Pharmingen, San Diego, CA, USA; 1 : 2000 dilution).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Iodoacetic acid protects hippocampal neurons against death induced by trophic factor witdrawal, glutamate and iron

IAA is known to selectively inhibit glycolysis, with essentially complete inhibition occurring with concentrations above 100 µm (Sabri and Ochs 1971; Winkler 1981). In a preliminary experiment we found that concentrations of IAA greater than 2 µm were toxic to cultured hippocampal neurons, and we therefore determined whether lower concentrations might exert a neuroprotective effect. Cultures were pre-treated for 24 h with IAA at concentrations of 2, 20, 200 and 2000 nm. The cultures were then exposed to the excitotoxic neurotransmitter glutamate at a concentration of 10 µm for 24 h and neuronal survival was quantified. In cultures not pre-treated with IAA, glutamate killed approximately 80% of the neurons (Fig. 1a). Significantly more neurons survived exposure to glutamate in cultures that had been pre-treated with IAA concentrations of 2, 20 and 200 nm, with a maximum improvement in cell survival to 50–60% with IAA concentrations of 20 and 200 nm. The survival of neurons pre-treated with IAA was maintained through 48 h of exposure to glutamate (Fig. 1a), suggesting a long-term protective effect of IAA pre-treatment rather than a delay of the cell death process. Neuron survival was not improved in cultures pre-treated with 2 µm IAA.

image

Figure 1. Iodoacetic acid protects hippocampal neurons against death induced by glutamate, trophic factor withdrawal and Fe2+. (a) Cultures were pre-treated for 24 h with IAA at the indicated concentrations, and were then exposed to100 µm glutamate for 24 h. Control cultures were pre-treated with vehicle and were not exposed to glutamate. Neuron survival was quantified and values are the mean and SE of determinations made in between four and six cultures. The values for glutamate-treated cultures pre-treated with 20 or 200 nm IAA were significantly greater than the corresponding value for cultures not pre-treated with IAA at both the 24- and 48-h time points (p < 0.01). The value for glutamate-treated cultures pre-treated with 2 nm IAA was significantly greater than the corresponding value for cultures not pre-treated with IAA at both the 24- and 48-h time points (p < 0.05). anova with Scheffe's post hoc tests. (b) Cultures were pre-treated for 24 h with 200 nm IAA, and were then either subjected to trophic factor deprivation (TFD) or were exposed to 10 µm Fe2+ for 24 or 48 h. Neuron survival was quantified and values are the mean and SE of determinations made in 4–6 cultures. *p < 0.05, **p < 0.01 compared with the corresponding value for cells pre-treated with IAA. anova with Scheffe post hoc tests. (c) Cultures were pre-treated for 24 h with 20 or 200 nm IAA, and were then exposed to 5 µm 4-hydroxynonenal (HNE) for 24 h. Neuron survival was quantified and values are the mean and SE of determinations made in between four and six cultures.

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Previous studies have shown that cultured hippocampal neurons undergo apoptosis when deprived of trophic support (Chan et al. 1999; Glazner et al. 2000). When cultures were subjected to trophic factor deprivation, 40% of the neurons died within 24 h (Fig. 1b). Pre-treatment with IAA afforded complete protection of neurons against trophic factor withdrawal, which was maintained through 48 h of trophic factor deprivation. Oxidative stress plays important roles in the deaths of neurons induced by overactivation of glutamate receptors (Mattson et al. 1995) and trophic factor deprivation (Greenlund et al. 1995; Chan et al. 1999), and is also implicated in an array of neurodegenerative disorders (Markesbery 1997; Albers and Beal 2000). We therefore determined whether IAA pre-treatment affects the vulnerability of neurons to death induced by Fe2+, an agent that induces membrane lipid peroxidation (Mark et al. 1997). In control cultures, approximately 40% of the neurons survived exposure to FeSO4 (10 µm), whereas in cultures pre-treated with IAA, neuronal survival was significantly improved to approximately 60% (Fig. 1b). The protective effect of IAA was maintained through 48 h of exposure to Fe2+. In contrast to its ability to protect neurons against glutamate, trophic factor deprivation and Fe2+, IAA did not afford significant protection against death induced by 4-hydroxynonenal (Fig. 1c), a cytotoxic aldehyde (Kruman et al. 1997). The protective effect of IAA required pre-treatment because IAA was ineffective in protecting neurons against glutamate, trophic factor deprivation and Fe2+ when added to the cultures immediately after exposure to the insults (data not shown).

Neuroprotective concentrations of iodoacetate do not affect cellular ATP levels

Although IAA is a well-known as an inhibitor of glycolysis, we found that IAA was effective in protecting neurons against death at concentrations far below those required to inhibit glycolysis. We therefore measured cellular ATP levels in neurons exposed to increasing concentrations of IAA. At neuroprotective concentrations of 20 and 200 nm, IAA did not decrease ATP levels (Fig. 2). Higher concentrations (2 µm and above) did cause a significant decrease of the ATP level. These results suggested that the neuroprotective effect of IAA did not result from inhibition of glycolysis.

image

Figure 2. ATP levels are unaffected by neuroprotective concentrations of iodoacetic acid. Hippocampal cultures were exposed for 6 h to the indicated concentrations of IAA and ATP levels were quantified. Values are the mean and SE of determinations made in six cultures.

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Iodoacetic acid stabilizes mitochondrial function

Mitochondrial alterations, including oxyradical production and membrane permeability changes, play pivotal roles in the death of neurons induced by excitotoxic and oxidative insults, as well as the programmed cell death resulting from trophic factor deprivation (Nicholls et al. 1999; Fiskum 2000; Fu et al. 2000). In order to evaluate the effects of IAA pre-treatment on mitochondrial changes known to occur in neurons dying in response to oxidative stress, we employed the fluorescent probes rhodamine 123 (which provides a measure of mitochondrial transmembrane potential; Mattson et al. 1993) and dihydrorhodamine (which provides a measure of mitochondrial reactive oxygen species production; Mattson et al. 1997). Exposure of cultures to Fe2+ resulted in a marked decrease in levels of rhodamine 123 fluorescence in neurons within 12 h (Fig. 3a); quantification of the fluorescence revealed that the magnitude of the decrease was approximately 50% (Fig. 3b). In contrast, essentially no decrease in rhodamine 123 fluorescence occurred after exposure to Fe2+ in neurons pre-treated with either 20 or 200 nm IAA (Figs 3a and b). These findings suggest that the neuroprotective effect of IAA is associated with preservation of mitochondrial membrane potential. Exposure of cultures to Fe2+ resulted in a marked increase in the level of dihydrorhodamine fluorescence in neurons within 12 h, indicating an increase in levels of mitochondrial reactive oxygen species (Fig. 4a); quantification of the fluorescence revealed that the magnitude of the increase was approximately 40–50% (Fig. 4b). In contrast, essentially no increase in dihydrorhodamine fluorescence occurred in neurons pre-treated with either 20 or 200 nm IAA (Fig. 4a,b). Mitochondrial potential was also preserved, and oxyradical production suppressed, in neurons pre-treated with IAA and then exposed to glutamate or trophic factor deprivation (data not shown). Collectively, these findings suggested that IAA elicits changes in neurons that stabilize mitochondrial function and thereby protect the cells against death induced by several different insults. In order to examine further the mechanism of action of IAA, we assessed levels of several proteins known to protect neurons against excitotoxicity and apoptosis.

image

Figure 3. Mitochondrial membrane potential is preserved after oxidative insult in hippocampal neurons pre-treated with IAA. (a) Confocal laser scanning micrographs of rhodamine 123 fluorescence in hippocampal neurons that had been pre-treated with the indicated concentrations of IAA and then exposed to 10 µm Fe2+ for 12 h. Note that Fe2+ caused a marked decrease in the level of rhodamine 123 fluorescence, and that the decrease was prevented by pre-treatment with IAA. (b) Levels of rhodamine 123 fluorescence were quantified in hippocampal neurons that had been pre-treated for 24 h with IAA or vehicle, and then exposed for 12 h to 10 µm Fe2+ or vehicle. Values are the mean and SE of determinations made in at least six cultures (measurements were made in at least 30 neurons/culture). **p < 0.01 compared with each of the other values (anova with Scheffe's post hoc tests).

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image

Figure 4. IAA pre-treatment results in a suppression of mitochondrial oxyradical levels after oxidative insult in hippocampal neurons. (a) Confocal laser scanning micrographs of dihydrorhodamine fluorescence in hippocampal neurons that had been pre-treated with the indicated concentrations of IAA and then exposed to 10 µm FeSO4 for 12 h Note that Fe2+ caused a marked increase in the level of dihydrorhodamine fluorescence, and that the increase was prevented by pre-treatment with IAA. (b) Levels of dihydrorhodamine fluorescence were quantified in hippocampal neurons that had been pre-treated for 24 h with IAA or vehicle, and then exposed for 12 h to 10 µm Fe2+ or vehicle. Values are the mean and SE of determinations made in at least six cultures (measurements were made in at least 30 neurons/culture). **p < 0.01 compared with each of the other values (anova with Scheffe's post hoc tests).

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Iodoacetic acid induces increases in levels of heat-shock proteins and Bcl-2

Previous studies have shown that mild metabolic stress, such as mild ischemia and treatment with 2-deoxy-d-glucose, can induce the production of several different types of cytoprotective proteins including heat-shock proteins and anti-apoptotic Bcl-2 family members (Liu et al. 1993; Duan and Mattson 1999; Yu et al. 1999; Brambrink et al. 2000). We therefore performed immunoblot analyses of control and IAA-treated neurons using antibodies against several stress proteins and Bcl-2 family members. Levels of HSP70 were increased within 6 h of exposure to IAA, continued to increase through 12 h, and remained elevated at 24 h (Fig. 5). Levels of HSP90 were also increased in neurons exposed to IAA; the increase was evident at 6 h and was maintained through 24 h (Fig. 5). Levels of the endoplasmic reticulum stress proteins GRP78 and GRP94 were unchanged during a 24-h treatment with IAA (Fig. 6). Levels of Bcl-2 were increased within 6 h of exposure to IAA, continued to increase through 12 h, and remained elevated at 24 h (Fig. 7). In contrast, levels of Bcl-xL another anti-apoptotic member of the Bcl-2 family, were not changed after exposure to IAA (Fig. 7).

image

Figure 5. Inhibition of glycolysis induces an increase in levels of HSP70 and HSP90 in hippocampal neurons. Cultures were exposed to 20 nm IAA for the indicated time periods, and proteins in cell lysates were subjected to immunoblot analysis (50 µg protein/lane) using antibodies against the indicated proteins. Representative immunoblots (upper) and results of densitometric analyses of multiple experiments (n = 4; mean and SE) are shown.

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image

Figure 6. Inhibition of glycolysis does not affect levels of endoplasmic reticulum glucose-regulated proteins in hippocampal neurons. Cultures were exposed to 20 nm IAA for the indicated time periods, and proteins in cell lysates were subjected to immunoblot analysis (50 µg protein/lane) using antibodies against the indicated proteins. Representative immunoblots (upper) and results of densitometric analyses of multiple experiments (n = 4; mean and SE) are shown.

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image

Figure 7. Inhibition of glycolysis induces an increase in the level of Bcl-2 in hippocampal neurons. Cultures were exposed to 20 nm IAA for the indicated time periods, and proteins in cell lysates were subjected to immunoblot analysis (50 µg protein/lane) using antibodies against the indicated proteins. Representative immunoblots (upper) and results of densitometric analyses of multiple experiments (n = 4; mean and SE) are shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

The results of the present study demonstrate that IAA can exert a strong cytoprotective action in cultured hippocampal neurons under conditions relevant to the pathogenesis of several different neurodegenerative disorders. Overactivation of glutamate receptors and oxidative stress are believed to contribute to the death of neurons in stroke (Dirnagl et al. 1999), traumatic brain injuries (Obrenovitch and Urenjak 1997), Alzheimer's disease (Mattson 1998), Parkinson's disease (Beal 1998) and amyotrophic lateral sclerosis (Rothstein 1996). IAA pre-treatment protected hippocampal neurons against glutamate toxicity and death induced by iron, a metal that induces hydroxyl radical production and membrane lipid peroxidation (Mark et al. 1997). IAA was also effective in protecting hippocampal neurons against death induced by trophic factor deprivation, a model of developmental programmed cell death (Chan et al. 1999). Previous studies using an identical hippocampal cell culture system showed that the predominant mode of neuronal death after trophic factor withdrawal, or exposure to glutamate or iron, is apoptosis (Mattson et al. 1997; Chan et al. 1999; Glazner et al. 2000). The ability of IAA to protect neurons against these different insults therefore suggests that iodoacetate can prevent neuronal apoptosis. Treatment with IAA resulted in a stabilization of mitochondrial function and suppression of oxyradical production in neurons exposed to the oxidative and apoptotic insults. Previous studies have shown that neuronal deaths induced by glutamate, trophic factor deprivation and iron each involve oxyradical production and mitochondrial dysfunction (Mattson et al. 1993, 1995, 1997; Greenlund et al. 1995; Schinder et al. 1996; White and Reynolds 1996; Keller et al. 1998; Chan et al. 1999; Putcha et al. 1999). It therefore seems likely that IAA acts on one or more cellular pathways that stabilize mitochondrial function and suppress oxyradical production. In contrast to its ability to protect neurons against death induced by glutamate and iron, IAA was ineffective in protecting neurons against death induced by 4-hydroxynonenal, a toxic aldehyde produced when membrane lipids are peroxidized (Kruman et al. 1997; Mark et al. 1997). This suggests that the neuroprotective effect of IAA may act at a step(s) prior to membrane lipid peroxidation. Consistent with this possibility, we found that insult-induced increases in dihydrorhodamine fluorescence, a measure of hydroxyl radical and peroxynitrite levels, were suppressed in neurons treated with IAA.

Our data suggest that the mechanism whereby IAA protects neurons involves up-regulation of certain protein chaperones and the anti-apoptotic protein Bcl-2. Levels of HSP70, HSP90 and Bcl-2 were significantly increased within 6–12 h of exposure to IAA, and levels of these proteins remained elevated through at least 24 h of exposure to IAA. HSP70 and HSP90 are protein chaperones that have been shown to prevent apoptotic death in a variety of cell types (Beere and Green 2001). These proteins may bind to proteins either upstream (e.g. Akt kinase and Bcl-2 family members) or downstream (e.g. caspases and Apaf-1) of mitochondrial alterations involved in cell death cascades (Sato et al. 2000). Previous studies have linked production of stress proteins including HSP70 and GRP78 to preconditioning neuroprotective effects of heat-shock and mild ischemia (Lowenstein et al. 1991; Liu et al. 1993; Yu and Mattson 1999). Although the role of HSP90 in neuroprotection has not been established, studies of ischemic preconditioning in the heart suggest a cytoprotective role for HSP90 (Nayeem et al. 1997).

Bcl-2 family members are thought to exert their anti- or pro-apoptotic actions at the level of the mitochondrial membrane (Kroemer 1999). Bcl-2 can stabilize mitochondrial membrane potential and suppress oxyradical production in several different paradigms of neuronal apoptosis (Borner et al. 1994; Ellerby et al. 1996; Guo et al. 1997; Kruman et al. 1997). Several different environmental signals have been shown to induce production of Bcl-2 in neurons, including neurotrophic factors (Allsopp et al. 1995), cytokines (Tamatani et al. 1999), cell adhesion (Gary and Mattson 2001) and exposure to insults such as ischemia (Zhu et al. 1999b) and seizures (Henshall et al. 2000). In addition, it was recently reported that subtoxic levels of the succinate dehydrogenase inhibitor 3-nitropropionic acid can induce an increase in the levels of Bcl-2 in neurons in the rat brain (Brambrink et al. 2000). The increased level of Bcl-2 in hippocampal neurons exposed to IAA suggests that an adaptive anti-apoptotic response occurs in neurons. Previous studies have shown that Bcl-2 can stabilize mitochondrial function in neurons subjected to excitotoxic and oxidative insults (Bruce-Keller et al. 1998), suggesting an important role for Bcl-2 in the neuroprotective effects of IAA demonstrated in the present study. In the hippocampal culture system employed in the present study, the mode of cell death induced by glutamate, trophic factor withdrawal and iron is predominantely apoptosis (Kruman et al. 1997; Chan et al. 1999; Glazner et al. 2000). It therefore appears to be the case that IAA pre-treatment protects neurons against apoptosis. The possible effects of IAA in modulating necrotic neuronal death remains to be determined.

Interestingly, we found that IAA increases HSP70 levels, but does not increase GRP78 levels. This contrasts with results obtained in studies in which cultured hippocampal neurons were exposed to the non-metabolizable glucose analog 2-deoxy-d-glucose, which induced an increase in levels of both HSP70 and GRP78 (Lee et al. 1999). Therefore, the cellular response to limiting glucose availability by inhibition of glycolysis with 2-deoxy-d-glucose can be distinguished from that induced by IAA based upon the profile of stress proteins produced. GRP78 is localized in endoplasmic reticulum and is responsive to stress in that organelle. The lack of effect of IAA on GRP78 levels therefore suggests that IAA may not induce an endoplasmic reticulum stress response. Another distinction between the neuroprotective mechanisms of IAA and 2-deoxy-d-glucose is that, whereas IAA induces an increase in Bcl-2 levels (present study), 2-deoxy-d-glucose may not (Lee et al. 1999). It will be of considerable interest to identify the specific signaling cascades and transcription factors involved in neuroprotective responses to different types of metabolic inhibitors.

Finally, the implications of the present findings for physiological regulation of neuronal survival by changes in energy metabolism, and for neuroprotective strategies for neurodegenerative disorders, should be considered. In addition to the neuroprotective effect of preconditioning ischemia, recent studies have demonstrated quite striking neuroprotective effects of dietary restriction in rodents. Dietary restriction induces cytoprotective mechanisms in neurons in several different brain regions in experimental models relevant to the pathogenesis of Alzheimer's disease (Bruce-Keller et al. 1999; Zhu et al. 1999a), Parkinson's disease (Duan and Mattson 1999) and stroke (Yu and Mattson 1999). The latter studies, and more recent findings (Lee et al. 2000; Duan et al. 2001), suggest that dietary restriction protects neurons by inducing the up-regulation of HSP70 and brain-derived neurotrophic factor. By modulating the expression of heat-shock proteins, Bcl-2 family members and neurotrophic factors changes in brain energy metabolism may affect neuronal plasticity and survival. Interestingly, administration of 2-deoxy-d-glucose to adult rats and mice can protect neurons against excitotoxic, oxidative and ischemic injury (Duan and Mattson 1999; Lee et al. 1999; Yu and Mattson 1999). In light of the present findings, future studies should therefore be peformed to determine whether IAA and related compounds can exert neuroprotective effects in vivo. Agents that modify cellular energy metabolism may prove valuable in the prevention and treatment of various neurodegenerative conditions.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
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