PTEN: a crucial mediator of mitochondria-dependent apoptosis.

The highly frequent mutation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in various cancers has attracted much attention to study its role in tumorigenesis. As an important tumor suppressor, the pro-apoptotic function of PTEN has been linked to its capacity antagonizing the PI3K/Akt signaling pathway. However, less data are available concerning its role in neurodegeneration in which apoptotic processes are also involved. In the present study, we attempted to study the role and the underlying mechanism of PTEN in neuronal apoptosis. Using primary rat hippocampal cultures, staurosporine (STS, 100 nM) induced a time-dependent apoptosis, accompanied by a marked production of reactive oxygen species (ROS), release of cytochrome c and activation of caspase 9 and 3. However, the expression of PTEN, and the levels of phospho-PTEN and phospho-Akt were not changed at all time points tested (0.5-24 h) after STS stimulation, suggesting that the protein level as well as the phosphorylation status of PTEN were not related to the procession of apoptosis. Interestingly, immunostaining revealed a punctate intracellular distribution of PTEN from 2 to 8 h after adding STS. Double labeling and Western blotting of mitochondrial fraction demonstrated a mitochondrial location and accumulation of PTEN, respectively, after challenging with STS. Furthermore, we provide evidence for the first time that PTEN was associated with Bax in the absence and the presence of STS. Of note, the STS-induced marked increase in the cellular ROS level, release of cytochrome c and activation of caspase 3 were inhibited in cultured hippocampal cells when PTEN was knocked down by a specific antisense. Moreover, knockdown of PTEN significantly protected hippocampal cells from apoptotic damage. These findings demonstrated that PTEN is a crucial mediator of mitochondria-dependent apoptosis, and thus could become a molecular target for interfering with neurodegenerative diseases.

Neuroprotective effects of Ginkgo biloba extract.

Ginkgo biloba extract has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of dementia. The extract contains multiple compounds such as flavonoids and terpenoids that are thought to contribute to its neuroprotective and vasotropic effects. In this review, we summarize the experimental results on the mechanism of neuroprotection induced by standardized extract of Ginkgo biloba leaves (EGb 761) and its constituents. The effects described mostly in animals include those on cerebral blood flow, neurotransmitter systems, cellular redox state and nitric oxide level. Furthermore, we discuss the current status of clinical trials as well as undesired side effects of EGb 761.

Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer's disease.

The standardized Ginkgo biloba extract EGb 761(definition see editorial) has been shown to produce neuroprotective effects in different in vivo and in vitro models. Since EGb 761 is a complex mixture containing flavonoid glycosides, terpene lactones (non-flavone fraction) and various other constituents, the question arises as to which of these compounds mediates the protective activity of EGb 761. Previous studies have demonstrated that the non-flavone fraction was responsible for the antihypoxic activity of EGb 761. Thus, we examined the neuroprotective and anti-apoptotic ability of the main constituents of the non-flavone fraction, the ginkgolides A, B, C, J and bilobalide. In focal cerebral ischemia models, the administration of bilobalide (5-20 mg/kg, s. c.) 60 min before ischemia dose-dependently reduced the infarct area in mouse brain and the infarct volume in rat brain 2 days after the onset of the injury. 30 minutes of pretreatment with ginkgolide A (50 mg/kg, s. c.) and ginkgolide B (100 mg/kg, s. c.) reduced the infarct area in the mouse model of focal ischemia. In primary cultures of hippocampal neurons and astrocytes from neonatal rats, ginkgolide B (1 microM) and bilobalide (10 microM) protected the neurons against damage caused by glutamate (1 mM, 1 h) as evaluated by trypan blue staining. In addition, bilobalide (0.1 microM) was able to increase the viability of cultured neurons from chick embryo telencepalon when exposed to cyanide (1 mM, 1h). Furthermore, we attempted to find out whether ginkgolides A, B, and J and bilobalide were also able to inhibit neuronal apoptosis (determined by nuclear staining with Hoechst 33 258 and TUNEL-staining). Ginkgolide B (10 microM), ginkgolide J (100 microM) and bilobalide (1 microM) reduced the apoptotic damage induced by serum deprivation (24h) or treatment with staurosporine (200 nM, 24h) in cultured chick embryonic neurons. Bilobalide (100 microM) rescued cultured rat hippocampal neurons from apoptosis caused by serum deprivation (24h), whereas ginkgolide B (100 microM) and bilobalide (100 microM) reduced apoptotic damage induced by staurosporine (300 nM, 24h). Ginkgolide A failed to affect apoptotic damage neither in serum-deprived nor in staurosporine-treated neurons. The results suggest that some of the constituents of the non-flavone fraction of EGb 761 possess neuroprotective and anti-apoptotic capacity, and that bilobalide is the most potent one. In contrast, ginkgolic acids (100-500 microM) induced neuronal death, which showed features of apoptosis as well as of necrosis, but these constituents were removed from EGb 761 below an amount of 0.0005 %. Taking together, there is experimental evidence for a neuroprotective effect of EGb 761 that agrees with clinical studies showing the efficacy of an oral treatment in patients with mild and moderate dementia.

3-Ureidopropionate contributes to the neuropathology of 3-ureidopropionase deficiency and severe propionic aciduria: a hypothesis.

3-Ureidopropionate (3-UPA) is a physiologic metabolite in pyrimidine degradation. Pathological accumulation of 3-UPA in body fluids is found in 3-ureidopropionase deficiency and severe forms of propionic aciduria. Both diseases clinically present with a severe neuropathology involving gray and white matter as well as with a dystonic dyskinetic movement disorder. To date nothing is known about the toxic nature of this metabolite. The aim of the present study was to elucidate whether 3-UPA may act as endogenous neurotoxin. Exposure of cultured chick neurons to 3-UPA induced a concentration- and time-dependent neurodegeneration. Neuronal damage was reduced by the antioxidant alpha-tocopherol and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. In contrast, the non-NMDA receptor antagonist CNQX, the metabotropic glutamate receptor antagonist L-AP3, and succinate showed no protective effect. Furthermore, 3-UPA elicited an increased production of reactive oxygen species followed by a delayed increase in intracellular calcium concentrations. Activity measurement of single respiratory chain complexes I-V revealed an inhibition of complex V activity, but not of the electron-transferring complexes I-IV by 3-UPA. In contrast, 3-UPA did not affect the mitochondrial beta-oxidation of fatty acids. In conclusion, our results provide strong evidence that 3-UPA acts as endogenous neurotoxin via inhibition of mitochondrial energy metabolism, resulting in the initiation of secondary, energy-dependent excitotoxic mechanisms.

Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C.

The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.

Intrastriatal administration of 3-hydroxyglutaric acid induces convulsions and striatal lesions in rats.

Glutaryl-CoA dehydrogenase deficiency is an inherited neurometabolic disease complicated by precipitation of acute encephalopathic crises during a vulnerable period of brain development. These crises result in bilateral striatal damage and subsequently a dystonic dyskinetic movement disorder. In previous in vitro studies neuronal damage in this disease has been linked to an excitotoxic mechanism mediated in particular by one of the accumulating metabolites, 3-hydroxyglutaric acid. However, nothing is known about the in vivo effects of this organic acid. In the present study, we used a stereotaxic intrastriatal injection technique to investigate the behavioral and neurotoxic effects of 3-hydroxyglutaric acid exposure in rats. Here, we report that 3-hydroxyglutaric acid induced an increase in convulsion frequency and duration as determined by open field measurement. Nissl-stained coronal sections from treated rats revealed a pale lesion in the striatum following 3-hydroxyglutaric acid exposure. N-methyl-D-aspartate (NMDA) receptor blockade by MK-801 and stimulation of GABA(A) receptors by muscimol prevented the induction of convulsions and striatal damage by 3-hydroxyglutaric acid, whereas blockade of non-NMDA receptors by 6,7-dinitroquinoxaline-2,3-dione (DNQX) was not protective. We conclude that 3-hydroxyglutaric acid induces convulsions and striatal damage via initiation of an imbalance in the excitatory glutamatergic and the inhibitory GABAergic neurotransmission, resulting in an enhanced excitatory input in striatal neurons. These results support the hypothesis of NMDA receptor-mediated excitotoxic cell damage in glutaryl-CoA dehydrogenase deficiency and represent the basis for the development of new neuroprotective treatment strategies.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-kappaB.

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor-kappaB (NF-kappaB) and how this activation contributes to preconditioning-induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine-induced (200 nM, 24 h) apoptosis. The cellular ROS content increased during preconditioning, but returned to basal levels after removal of xanthine/xanthine oxidase or FeSO4. We detected a transient activation of NF-kappaB 4 h after preconditioning as shown by immunocytochemistry, by a decrease in the protein level of IkappaBalpha as well as by electrophoretic mobility shift assay. Preconditioning-mediated neuroprotection was abolished by antioxidants, inhibitors of NF-kappaB activation and cycloheximide suggesting the involvement of ROS, an activation of NF-kappaB and de novo protein synthesis in preconditioning-mediated rescue pathways. Furthermore, preconditioning increased the protein level of Mn-superoxide dismutase which could be blocked by antioxidants, cycloheximide and kappaB decoy DNA. Our data suggest that inhibition of staurosporine-induced neuronal apoptosis by preconditioning with xanthine/xanthine oxidase or FeSO4 involves an activation of NF-kappaB and an increase in the protein level of Mn-superoxide dismutase.

Potentiation of 3-hydroxyglutarate neurotoxicity following induction of astrocytic iNOS in neonatal rat hippocampal cultures.

Neuronal damage in glutaryl-CoA dehydrogenase deficiency (GDD) has previously been addressed to N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity of the accumulating neurotoxic metabolite 3-hydroxyglutarate. However, acute encephalopathic crises in GDD patients are typically precipitated by febrile illness or even routine vaccinations, suggesting a potentiating role of inflammatory cytokines. In the present study we investigated the effect of interleukin-1beta and interferon-gamma on 3-hydroxyglutarate toxicity in rat cortical astrocyte cultures and neonatal rat hippocampal cultures. A cotreatment of both culture systems with interleukin-1beta and interferon-gamma induced the protein expression of astrocytic inducible nitric oxide synthase (iNOS), resulting in increased nitric oxide (NO) production. Cytokine pretreatment alone had no effect on cell viability but potentiated 3-hydroxyglutarate neurotoxicity. NOS inhibition by aminoguanidine and L-NAME prevented an iNOS-mediated potentiation of 3-hydroxyglutarate neurotoxicity but failed to protect neurons against 3-hydroxyglutarate alone. In contrast, superoxide dismutase/catalase as well as MK-801 prevented toxicity of 3-hydroxyglutarate alone as well as its potentiation by iNOS, supporting a central role of NMDA receptor stimulation with subsequently increased superoxide anion production. It is concluded that the potentiation of 3-hydroxyglutarate neurotoxicity is most probably due to an induction of astrocytic iNOS and concomitantly increased NO production, enabling enhanced peroxynitrite formation. Thus, we provide evidence for a neuroimmunological approach to the precipitation of acute encephalopathic crises in GDD by inflammatory cytokines.

Contribution of reactive oxygen species to 3-hydroxyglutarate neurotoxicity in primary neuronal cultures from chick embryo telencephalons.

Glutaryl-CoA dehydrogenase deficiency is an autosomal recessively inherited neurometabolic disorder with a distinct neuropathology characterized by acute encephalopathic crises during a vulnerable period of brain development. 3-Hydroxyglutarate (3-OH-GA), which accumulates in affected patients, has been identified as an endogenous neurotoxin mediating excitotoxicity via N-methyl-D-aspartate receptors. As increased generation of reactive oxygen species (ROS) and nitric oxide (NO) plays an important role in excitotoxic neuronal damage, we investigated whether ROS and NO contribute to 3-OH-GA neurotoxicity. 3-OH-GA increased mitochondrial ROS generation in primary neuronal cultures from chick embryo telencephalons, which could be prevented by MK-801, confirming the central role of N-methyl-D-aspartate receptor stimulation in 3-OH-GA toxicity. ROS increase was reduced by alpha-tocopherol and--less effectively-by melatonin. alpha-Tocopherol revealed a wider time frame for neuroprotection than melatonin. Creatine also reduced neuronal damage and ROS formation but only if it was administered >or=6 h before 3-OH-GA. NO production revealed only a slight increase after 3-OH-GA incubation. NO synthase inhibitor N(omega)-nitro-L-arginine prevented NO increase but did not protect neurons against 3-OH-GA. The NO donor S-nitroso-N-acetylpenicillamine revealed no effect on 3-OH-GA toxicity at low concentrations (0.5-5 microM), whereas it potentiated neuronal damage at high concentrations (50-500 microM), suggesting that weak endogenous NO production elicited by 3-OH-GA did not affect neuronal viability. We conclude from our results that ROS generation contributes to 3-OH-GA neurotoxicity in vitro and that radical scavenging and stabilization of brain energy metabolism by creatine are hopeful new strategies in glutaryl-CoA dehydrogenase deficiency.

Retinoic acid reduces apoptosis and oxidative stress by preservation of SOD protein level.

Retinoic acid (RA) has already been shown to exert antiapoptotic and antioxidative activity in various cells. In this study, we determined the effect of RA on the mRNA and protein levels of the Cu-,Zn-superoxide dismutase (SOD-1) and Mn-superoxide dismutase (SOD-2) during staurosporine-induced apoptosis in primary cultures from neonatal rat hippocampus. Exposure to staurosporine (300 nM, 24 h) increased the percentage of apoptotic neurons to 62% compared with 18% in controls. We determined an increase in the reactive oxygen species (ROS) content from 4 up to 48 h after the induction of the injury. Treatment with staurosporine did not significantly change the mRNA levels of SOD-1 and SOD-2. However, the SOD-1 and SOD-2 protein levels markedly decreased 24 and 48 h after the addition of staurosporine. Compared with staurosporine-exposed controls, RA (10 nM)-treated cultures showed a significant increase in neuronal survival, a reduced neuronal ROS content, and enhanced protein levels of SOD-1 and SOD-2 24 and 48 h after the start of the exposure to staurosporine. The results suggest that RA reduced staurosporine-induced oxidative stress and apoptosis by preventing the decrease in the protein levels of SOD-1 and SOD-2, and thus supported the antioxidant defense system.

Neuroprotective effects of NV-31, a bilobalide-derived compound: evidence for an antioxidative mechanism.

In previous studies we have already shown that the extract of Ginkgo biloba, and some of its constituents, such as ginkgolide B and bilobalide, protected cultured neurons against apoptotic and excitotoxic damage and reduced the infarct volume after focal cerebral ischemia in mice and rats. In this work, we determined the neuroprotective and antioxidative effects of 4-hydroxy-4-tert-butyl-2,3,5,6-tetrahydrothiopyran-1-oxide (NV-31), a stable compound which was synthesized to mimic the pharmacological activity profile of bilobalide. In pure neuronal cultures from chick embryo telencephalon, damage was induced by serum deprivation (24 h) and exposure to staurosporine (200 nM, 24 h) which caused an increase in the percentage of apoptotic neurons from 14 (controls) to 30 and 55%, respectively. NV-31 (1-100 nM) protected dose-dependently chick neurons against both serum deprivation- and staurosporine-induced apoptosis. Similarly, NV-31 (100 nM) reduced staurosporine (300 nM, 24 h)-induced neuronal damage in mixed cultures of neurons and astrocytes from neonatal rat hippocampus. The cellular ROS content increased 6-fold 4 h after serum deprivation as well as 4 h after the exposure to staurosporine and this increase was reduced by 50% in the presence of 10 and 100 nM NV-31, respectively. In mice, a treatment with 10 and 20 mg/kg NV-31 60 min before and immediately after focal cerebral ischemia, respectively, significantly reduced the infarct area compared with vehicle-treated animals. In the present study, we show that NV-31 promotes neuronal survival and we suggest that its antioxidative property contributes to the mechanism of neuroprotection.

TGF-beta1 inhibits caspase-3 activation and neuronal apoptosis in rat hippocampal cultures.

The effect of TGF-beta1 on apoptosis varies depending on the cell type, the kind of stimulus and the experimental conditions. The present study attempted to identify whether TGF-beta1 can prevent neuronal apoptosis and interrupt caspase-3 activation in rat primary hippocampal cultures after staurosporine treatment. TGF-beta1 at the concentration of 1 and 10 ng/ml significantly reduced neuronal damage as detected by trypan blue exclusion. Nuclear staining with Hoechst 33258 and TUNEL-staining further demonstrated that TGF-beta1 at the same concentration range effectively diminished neuronal apoptosis 24 h after staurosporine treatment, whereas 0.1 ng/ml of TGF-beta1 did not. Furthermore, TGF-beta1 (1 and 10 ng/ml) markedly inhibited the activation of caspase-3 induced by staurosporine as demonstrated by both caspase-3 activity assay and Western blotting. This study provides evidence that TGF-beta1 is able to efficiently inhibit caspase-3 activation, and thereby protects cultured hippocampal neurons against apoptosis.

Staurosporine-induced apoptosis in cultured chick embryonic neurons is reduced by polyethylenimine of low molecular weight used as a coating substrate.

The survival of neurons largely depends on adhesion to extracellular matrix proteins. This study investigated the influence of polycationic macromolecules of different molecular weights used as coating substrates on apoptosis in primary cultures of chick embryonic neurons. Coating of the culture flasks with positively charged polyethylenimine (PEI) of 12, 32 and 1616 kDa led to different susceptibilities of the neurons to apoptosis induced by staurosporine and serum deprivation. In cultures grown as usual on polylysine (PL)-coated flasks, we found, after 24 h of incubation in medium with serum (controls), serum-free medium and staurosporine (200 nM)-containing serum-free medium, 15, 35 and 63% apoptotic neurons, respectively as evaluated by nuclear staining with Hoechst 33258. Using 12-kDa PEI as a coating substrate, only 11, 15 and 47% apoptotic neurons could be determined in controls, serum-deprived and staurosporine-treated cultures, respectively. No change in the percentage of apoptotic neurons was found after 24 h of serum deprivation or treatment with staurosporine in cultures grown on 32-kDa PEI compared with cultures grown on PL. However, in staurosporine-treated cultures grown on 1616-kDa PEI, the percentage of apoptotic neurons was even higher than in cultures grown on PL. Immunostaining using neurofilament (NF) antibodies revealed that the reduction of staurosporine-induced apoptosis using 12-kDa PEI instead of PL as a coating substrate was accompanied by a reduced disruption and aggregation of the neurofilaments. Thus, the usage of the newly synthesized 12-kDa PEI as a coating substrate enhanced neuronal resistance to apoptosis.

Retinoic acid potentiated the protective effect of NGF against staurosporine-induced apoptosis in cultured chick neurons by increasing the trkA protein expression.

Nerve growth factor (NGF) has already been shown to protect neurons and PC12 cells from cell death induced by different stimuli. When chick embryonic neurons were exposed to staurosporine (200 nM, 24 hr), the percentage of apoptotic neurons increased from 15% in controls to 80%, but the treatment with NGF alone did not show any neuroprotection. In the presence of retinoic acid (RA, 5 microM), however, NGF (20 pg/ml) reduced staurosporine-induced damage to 42% apoptotic neurons compared to 58% in the presence of RA (5 icroM) alone. TrkA protein expression in chick neurons was markedly reduced by staurosporine, but was found to be increased in the presence of RA and NGF compared with the treatment with staurosporine alone. The antiapoptotic effect caused by RA and NGF was abolished by the tyrosine kinase inhibitor K-252a, as well as by anti-trkA antibodies and anti-NGF antibodies suggesting that the increase in trkA protein expression contributed to its mechanism of action. In addition, RA-enhanced 2.6-fold the NGF secretion from cultured rat cortical astrocytes and conditioned medium of RA-treated astrocytes reduced the percentage of apoptotic chick neurons after a 24 hr-incubation with staurosporine in the same manner as the external addition of RA and NGF. Increasing the endogenous synthesis of growth factors as well as the expression of their receptors by small, blood-brain barrier-permeable drugs was suggested as a promising concept for neuroprotection.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species.

The current study was performed to determine the role of reactive oxygen species (ROS) in preconditioning against different forms of neuronal damage. Primary cultures of chick embryonic neurons were treated with either FeSO(4) (100 microM; 15 min) to generate hydroxyl radicals or xanthine/xanthinoxidase (10 microM/0.5 mU ml(-1); 15 min; =X/XO (pre)) to produce superoxide radicals. Both stimuli moderately enhanced ROS formation as measured by fluorescence microscopy. This preconditioning significantly protected the neurons against subsequent glutamate (1 mM)-induced excitotoxic damage, staurosporine (200 nM)-induced neuronal apoptosis and oxidative damage caused by exposure to xanthine/xanthinoxidase (500 microM/5 mU ml(-1); 1 h; =X/XO (dam)). The antioxidants vitamin E (10 microM) and 2-OH-estradiol (1 microM), present during the 15-min preconditioning period, completely abolished the protective effect of X/XO (pre). Furthermore, glutamate, staurosporine or X/XO (dam) markedly enhanced oxygen radical formation. Preceding preconditioning by mild ROS stimulation with X/XO (pre) or Fe(2+) reduced this oxygen radical burst. Again, the effect of X/XO (pre) could be blocked by coadministration of vitamin E or 2-OH-estradiol. However, the FeSO(4)-mediated preconditioning was not abolished by the radical scavengers. To address this phenomenon, the effect of vitamin E and 2-OH-estradiol on Fe(2+)- and X/XO (pre)-induced ROS formation kinetics within the 15 min of preconditioning was monitored. The moderate rise of intracellular ROS content during preconditioning was only reduced permanently by the antioxidants, when the neurons were treated with X/XO (pre), but not when Fe(2+) was used. Thus, an immediate and constant radical scavenging seems to be indispensable to abolish the ROS-induced neuronal preconditioning. The current results indicate that preconditioning by moderate ROS-stimulation protects cultured neurons against different damaging agents and prevents against the subsequent massive oxygen radical formation.

Cerebral organic acid disorders induce neuronal damage via excitotoxic organic acids in vitro.

Glutaryl-CoA dehydrogenase deficiency (GDD), which is one of the most frequent organic acid disorders, is characterized by a specific age- and regional-dependent neuropathology. We hypothesized that the distinct brain damage in GDD could be caused by the main pathologic metabolites, the organic acids glutaric (GA) and 3-hydroxyglutaric (3-OH-GA) acids, through an excitotoxic sequence. Therefore, we investigated the effects of 3-OH-GA and GA on primary neuronal cultures from chick embryonic telencephalons. Here we report that 3-OH-GA and GA decreased cell viability concentration- and time-dependently, which could be only totally prevented by preincubation with MK-801, ifenprodil and NR2B antibodies. Furthermore, cell viability decreased in parallel with the increasing expression of NR2B subunit on cultured neurons from 2nd to 6th DIV. We conclude that GA and 3-OH-GA act as excitotoxic organic acids (EOA) specifically through NR1/NR2B and that the extent of induced neurotoxicity is dependent on NR1/NR2B expression during maturation.

Maturation-dependent neurotoxicity of 3-hydroxyglutaric and glutaric acids in vitro: a new pathophysiologic approach to glutaryl-CoA dehydrogenase deficiency.

Glutaryl-CoA dehydrogenase deficiency is a neurometabolic disorder with a specific age- and region-dependent neuropathology. Between 6 and 18 mo of age, unspecific illnesses trigger acute encephalopathic crises resulting in acute striatal and cortical necrosis. We hypothesized that acute brain damage in glutaryl-CoA dehydrogenase deficiency is caused by the main pathologic metabolites 3-hydroxyglutaric and glutaric acids through an excitotoxic sequence. Therefore, we investigated the effect of 3-hydroxyglutaric acid and glutaric acid on primary neuronal cultures from chick embryo telencephalons and mixed neuronal and glial cell cultures from neonatal rat hippocampi. Exposure to glutaric acid and 3-hydroxyglutaric acid decreased cell viability in a concentration- and time-dependent fashion. This neurotoxic effect could be totally prevented by preincubation with an N-methyl-D-aspartate receptor subunit 2B (NR2B)-specific antagonist, NR2B antibodies, and an unspecific N-methyl-D-aspartate receptor blocker and was partially blocked with an NR2A-specific antagonist but not with NR2A antibodies or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor and metabotropic glutamate receptor antagonists. Furthermore, metabolite toxicity increased in parallel with the increasing expression of the NR2B subunit on cultured neurons from second to sixth day in vitro. We conclude from these results that 3-hydroxyglutaric acid and glutaric acid act as false neurotransmitters, in particular through NR1/2B, and that the extent of induced neurotoxicity is dependent on the temporal and spatial expression of NR1/2B in the CNS during maturation. Beyond favorable implications for treatment and long-term prognosis, glutaryl-CoA dehydrogenase deficiency is the first neurologic disease in which specific neuropathology could be experimentally linked to ontogenetic expression of a particular neurotransmitter receptor subtype.

S-100beta protects cultured neurons against glutamate- and staurosporine-induced damage and is involved in the antiapoptotic action of the 5 HT(1A)-receptor agonist, Bay x 3702.

The serotonin (5-HT)(1A) receptor agonists have already been shown to protect cultured neurons from excitotoxic as well as from apoptotic damage [B. Ahlemeyer, J. Krieglstein, Stimulation of 5-HT(1A) receptors inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo, Brain Res. 777 (1997) 179-186. ; B. Ahlemeyer, A. Glaser, C. Schaper, I. Semkova, J. Krieglstein, The 5-HT(1A) receptor agonist, Bay x 3702, inhibited apoptosis induced by serum deprivation in cultured neurons, Eur. J. Pharmacol. 370 (1999) 211-216.; J.H.M. Prehn, M. Welsch, C. Backhauss, J. Nuglisch, F. Ausmeier, C. Karkoutly, J. Krieglstein, Effects of serotonergic drugs in experimental brain ischemia: evidence for a protective role of serotonin in cerebral ischemia, Brain Res. 630 (1993) 110-120.; I. Semkova, P. Wolz, J. Krieglstein, Neuroprotective effect of 5-HT(1A) receptor agonist, Bay x 3702, demonstrated in vitro and in vivo, Eur. J. Pharmacol. 359 (1998) 251-260.; B. Suchanek, H. Struppeck, T. Fahrig, The 5-HT(1A) receptor agonist, Bay x 3702, prevents staurosporine-induced apoptosis, Eur. J. Pharmacol. 355 (1998) 95-101.] and to increase the release of the neurotrophic protein, S-100beta [P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, Stimulation of astroglial 5-HT(1A) receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology, Brain Res. 497 (1989) 80-86. ; P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, S-100 protein is released from astroglial cells by stimulation of 5-HT(1A) receptors, Brain Res. 528 (1990) 155-158.]. In this study, we tried to find out whether S-100beta can protect cultured neurons from glutamate- and staurosporine-induced damage and whether the neuroprotective activity of the highly selective 5-HT(1A) receptor agonist, Bay x 3702, is mediated by an induction of S-100beta. Extracellularly added S-100beta (1-10 ng/ml) reduced staurosporine-induced damage in pure neuronal cultures from chick embryo telencephalon as well as in mixed neuronal/glial cultures from neonatal rat hippocampus. In addition, S-100beta (1 ng/ml) reduced neuronal death induced by exposure to glutamate (0.25 mM, 30 min) in mixed neuronal/glial cultures from neonatal rat hippocampus. In cultured rat cortical astrocytes, a 24 h-treatment with Bay x 3702 (1 nM) increased the S-100beta content in the culture medium from 2.2+/-0.3 (controls) to 6.2+/-0.7 ng/ml. In the adult rat, a 4 h-infusion of 4 microg/kg Bay x 3702 (i.v.) was found to increase the S-100beta content in the striatum 6 h after the beginning of the infusion to 153+/-37 microg/g compared with 60+/-20 microg/g in vehicle-treated rats. Bay x 3702 had no effect on the S-100beta content in the rat hippocampus. Finally, we tried to block the protective effect of Bay x 3702 against staurosporine-induced damage in mixed neuronal/glial cultures from rat neonatal hippocampus by anti-S-100beta antibodies. We found only a partial blockade, although the antibodies fully blocked the antiapoptotic effect of S-100beta itself demonstrating that the antibody was effective in blocking neuroprotection by S-100beta. Thus, we conclude that S-100beta was able to protect cultured neurons against glutamate- and staurosporine-induced damage. Furthermore, S-100beta mediated partially the protective effect of the 5-HT(1A) receptor agonist, Bay x 3702, against staurosporine-induced apoptosis in mixed neuronal/glial cultures from neonatal rat hippocampus.