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Impaired neuronal migration and cell death are commonly observed in patients with peroxisomal biogenesis disorders (PBDs), and in mouse models of this diseases. In Pex11β-deficient mice, we observed that the deletion of a single allele of the Pex11β gene (Pex11β+/− heterozygous mice) caused cell death in primary neuronal cultures prepared from the neocortex and cerebellum, although to a lesser extent as compared with the homozygous-null animals (Pex11β−/− mice). In corresponding brain sections, cell death was rare, but differences between the genotypes were similar to those found in vitro. Because PEX11β has been implicated in peroxisomal proliferation, we searched for alterations in peroxisomal abundance in the brain of heterozygous and homozygous Pex11β-null mice compared with wild-type animals. Deletion of one allele of the Pex11β gene slightly increased the abundance of peroxisomes, whereas the deletion of both alleles caused a 30% reduction in peroxisome number. The size of the peroxisomal compartment did not correlate with neuronal death. Similar to cell death, neuronal development was delayed in Pex11β+/− mice, and to a further extent in Pex11β−/− mice, as measured by a reduced mRNA and protein level of synaptophysin and a reduced protein level of the mature isoform of MAP2. Moreover, a gradual increase in oxidative stress was found in brain sections and primary neuronal cultures from wild-type to heterozygous to homozygous Pex11β-deficient mice. SOD2 was upregulated in neurons from Pex11β+/− mice, but not from Pex11β−/− animals, whereas the level of catalase remained unchanged in neurons from Pex11β+/− mice and was reduced in those from Pex11β−/− mice, suggesting a partial compensation of oxidative stress in the heterozygotes, but a failure thereof in the homozygous Pex11β−/− brain. In conclusion, we report the alterations in the brain caused by the deletion of a single allele of the Pex11β gene. Our data might lead to the reconsideration of the clinical treatment of PBDs and the common way of using knockout mouse models for studying autosomal recessive diseases.

Delayed cerebellar development is a hallmark of Zellweger syndrome (ZS), a severe neonatal neurodegenerative disorder. ZS is caused by mutations in PEX genes, such as PEX13, which encodes a protein required for import of proteins into the peroxisome. The molecular basis of ZS pathogenesis is not known. We have created a conditional mouse mutant with brain-restricted deficiency of PEX13 that exhibits cerebellar morphological defects. PEX13 brain mutants survive into the postnatal period, with the majority dying by 35 days, and with survival inversely related to litter size and weaning body weight. The impact on peroxisomal metabolism in the mutant brain is mixed: plasmalogen content is reduced, but very-long-chain fatty acids are normal. PEX13 brain mutants exhibit defects in reflex and motor development that correlate with impaired cerebellar fissure and cortical layer formation, granule cell migration and Purkinje cell layer development. Astrogliosis and microgliosis are prominent features of the mutant cerebellum. At the molecular level, cultured cerebellar neurons from E19 PEX13-null mice exhibit elevated levels of reactive oxygen species and mitochondrial superoxide dismutase-2 (MnSOD), and show enhanced apoptosis together with mitochondrial dysfunction. PEX13 brain mutants show increased levels of MnSOD in cerebellum. Our findings suggest that PEX13 deficiency leads to mitochondria-mediated oxidative stress, neuronal cell death and impairment of cerebellar development. Thus, PEX13-deficient mice provide a valuable animal model for investigating the molecular basis and treatment of ZS cerebellar pathology.

Glutaryl-CoA dehydrogenase deficiency (also known as glutaric aciduria type I) is an autosomal, recessively inherited neurometabolic disorder with a distinct neuropathology characterized by acute encephalopathy during a vulnerable period of brain development. Neuronal damage in this disease was demonstrated to involve N-methyl-d-aspartate (NMDA) receptor-mediated neurotoxicity of the endogenously accumulating metabolite 3-hydroxyglutarate (3-OH-GA). However, it remained unclear whether NMDA receptors are directly or indirectly activated and whether 3-OH-GA disturbs the intracellular Ca2+homeostasis. Here we report that 3-OH-GA activated recombinant NMDA receptors (e.g. NR1/NR2A) but not recombinant α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (e.g. GluR-A/GluR-B) in HEK293 cells. Fluorescence microscopy using fura-2 as Ca2+indicator revealed that 3-OH-GA increased intracellular Ca2+concentrations in the presence of extracellular Ca2+in cultured chick neurons. Similar to glutamate-induced cell damage, 3-OH-GA neurotoxicity was modulated by extracellular Na+. The large cation N-methyl-d-glucamine, which does not permeate NMDA receptor channels, enhanced 3-OH-GA-induced Ca2+increase and cell damage. In contrast, 3-OH-GA-induced neurotoxicity was reduced after replacement of Na+by Li+, which permeates NMDA channels but does not affect the Na+/Ca2+exchanger in the plasma membrane. Spectrophotometric analysis of respiratory chain complexes I–V in submitochondrial particles from bovine heart revealed only a weak inhibition of 3-OH-GA on complex V at the highest concentration tested (10 mM). In conclusion, the present study revealed that NMDA receptor activation and subsequent disturbance of Ca2+homeostasis contribute to 3-OH-GA-induced cell damage.

Glutaryl-CoA dehydrogenase deficiency (also known as glutaric aciduria type I) is an autosomal, recessively inherited neurometabolic disorder with a distinct neuropathology characterized by acute encephalopathy during a vulnerable period of brain development. Neuronal damage in this disease was demonstrated to involve N-methyl-d-aspartate (NMDA) receptor-mediated neurotoxicity of the endogenously accumulating metabolite 3-hydroxyglutarate (3-OH-GA). However, it remained unclear whether NMDA receptors are directly or indirectly activated and whether 3-OH-GA disturbs the intracellular Ca2homeostasis. Here we report that 3-OH-GA activated recombinant NMDA receptors (e.g.NR1/NR2A) but not recombinant -amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (e.g.GluR-A/GluR-B) in HEK293 cells. Fluorescence microscopy using fura-2 as Ca2indicator revealed that 3-OH-GA increased intracellular Ca2concentrations in the presence of extracellular Ca2in cultured chick neurons. Similar to glutamate-induced cell damage, 3-OH-GA neurotoxicity was modulated by extracellular Na. The large cation N-methyl-d-glucamine, which does not permeate NMDA receptor channels, enhanced 3-OH-GA-induced Ca2increase and cell damage. In contrast, 3-OH-GA-induced neurotoxicity was reduced after replacement of Naby Li, which permeates NMDA channels but does not affect the Na/Ca2exchanger in the plasma membrane. Spectrophotometric analysis of respiratory chain complexes I–V in submitochondrial particles from bovine heart revealed only a weak inhibition of 3-OH-GA on complex V at the highest concentration tested (10 mM). In conclusion, the present study revealed that NMDA receptor activation and subsequent disturbance of Ca2homeostasis contribute to 3-OH-GA-induced cell damage.

GlutarylCoA 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. 3Hydroxyglutarate 3OHGA, which accumulates in affected patients, has been identified as an endogenous neurotoxin mediating excitotoxicity via NmethylDaspartate 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 3OHGA neurotoxicity. 3OHGA increased mitochondrial ROS generation in primary neuronal cultures from chick embryo telencephalons, which could be prevented by MK801, confirming the central role of NmethylDaspartate receptor stimulation in 3OHGA toxicity. ROS increase was reduced by tocopherol and—less effectively—by melatonin. Tocopherol revealed a wider time frame for neuroprotection than melatonin. Creatine also reduced neuronal damage and ROS formation but only if it was administered =6 h before 3OHGA. NO production revealed only a slight increase after 3OHGA incubation. NO synthase inhibitor NnitroLarginine prevented NO increase but did not protect neurons against 3OHGA. The NO donor SnitrosoNacetylpenicillamine revealed no effect on 3OHGA toxicity at low concentrations 0.55 M, whereas it potentiated neuronal damage at high concentrations 50500 M, suggesting that weak endogenous NO production elicited by 3OHGA did not affect neuronal viability. We conclude from our results that ROS generation contributes to 3OHGA neurotoxicity in vitroand that radical scavenging and stabilization of brain energy metabolism by creatine are hopeful new strategies in glutarylCoA dehydrogenase deficiency.

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 viaN-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 -tocopherol and—less effectively—by melatonin. -Tocopherol revealed a wider time frame for neuroprotection than melatonin. Creatine also reduced neuronal damage and ROS formation but only if it was administered ≥6 h before 3-OH-GA. NO production revealed only a slight increase after 3-OH-GA incubation. NO synthase inhibitor N-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 M), whereas it potentiated neuronal damage at high concentrations (50–500 M), 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 vitroand that radical scavenging and stabilization of brain energy metabolism by creatine are hopeful new strategies in glutaryl-CoA dehydrogenase deficiency.

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 a-tocopherol and—less effectively—by melatonin. a-Tocopherol revealed a wider time frame for neuroprotection than melatonin. Creatine also reduced neuronal damage and ROS formation but only if it was administered =6 h before 3-OH-GA. NO production revealed only a slight increase after 3-OH-GA incubation. NO synthase inhibitor N?-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 µM), whereas it potentiated neuronal damage at high concentrations (50–500 µM), 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.

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 -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.

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 a-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.

Estrogens have been suggested for the treatment of neurodegenerative disorders, including stroke, because of their neuroprotective activities against various neurotoxic stimuli such as glutamate, glucose deprivation, iron, or β-amyloid. Here, the authors report that 17β-estradiol (0.3 to 30 mg/kg) and 2-OH-estradiol (0.003 to 30 mg/kg) reduced brain tissue damage after permanent occlusion of the middle cerebral artery in male NMRI mice. In vitro, 17β-estradiol (1 to 10 μmol/L) and 2-OH-estradiol (0.01 to 1 μmol/L) reduced the percentage of damaged chick embryonic neurons treated with FeSO4. In these primary neurons exposed to FeSO4, the authors also found reactive oxygen species to be diminished after treatment with 17β-estradiol (1 to 10 μmol/L) or 2-OH-estradiol (0.01 to 10 μmol/L), suggesting a strong antioxidant activity of the estrogens that were used. Neither the neuroprotective effect nor the free radical scavenging properties of the estrogens were influenced by the estrogen receptor antagonist tamoxifen. The authors conclude that estrogens protect neurons against damage by radical scavenging rather than through estrogen receptor activation.

The present study investigated the effect of cytotoxic hypoxia on cerebral and non-cerebral endothelial cells. Hypoxia was induced by inhibiting the cellular respiratory chain with 1 mM sodium cyanide. Cerebral endothelial cells were damaged after 2 h of hypoxia as assessed by a decrease in cell viability by 25% and by a 2.7-fold higher lactate dehydrogenase release compared to controls. Additional glucose deprivation did not significantly exacerbate hypoxic injury. In addition, we found after 2 h of hypoxia an increase in the release of lactate of 1.02 and 0.42 mg/mg protein compared to 0.27 and 0.07 mg/mg protein in controls in the presence and absence of glucose, respectively. While the activity of ALP of cerebral endothelial cells was maintained at the control level, we found a significant decrease in the γ-GT activity from 3.8 ± 1.3 to 1.09 ± 0.3 U/mg protein after 3 h of hypoxia in the presence as well as in the absence of glucose. The paracellular permeability of the cell monolayer decreased after 1 h and returned to control level after 3 h of hypoxia in the presence of glucose. Non-cerebral endothelial cells remained 98% viable with no change in the release of lactate dehydrogenase and lactate after 2 h of hypoxia in the presence and absence of glucose. The activities of ALP and γ-GT in non-cerebral endothelial cells were 10 and 3 times lower and remained unchanged during hypoxia. We conclude from our experiments that sodium cyanide is useful to study hypoxic injury and that cerebral endothelial cells are more sensitive than non-cerebral endothelial cells to cytotoxic hypoxia. © 1997 Elsevier Science Ltd