Glutaric Acid Mediates Neurotoxicity In Rat Brain Neurons

PART I Primary serum-free culture of rat cortical neurons and the analysis of influencing factorsObjective To explore the factors influencing the purity and viability of primary cultured rat cortical neurons in vitro and find the best conditions of culturing the primary neurons.Methods Firstly, we analyzed the differences in purity and viability of primary cultured neurons between embryonic rat group and newborn rat group by morphological observation, immunocytochemistry and trypan blue staining. Secondly, we evaluated the change of neurons purity and viability in different trypsin digestion time (0 min,5 min and 15 min) at different environment temperatures by immunocytochemistry and trypan blue staining.Results The difference in neurons purity and viability between fetal group and newborn rat group was not significant. Different digestion time affected the neurons purity and viability. Meanwhile the different environment temperature had slight influence on the cell purity and viability.Conclusion Primary cortical neurons culture of the newborn rat is impacted by some factors and optimizing the separating and cultivating condition will improve the neurons purity and viability. PARTⅡThe neurotoxicity of glutaric acid on primary cultured striatal neuronsObjective To investigate the concentration-and time-dependent toxicity of glutaric acid (GA) on primary cultured neonatal rat striatal neurons and the possible mechanism.Methods The neuronal cells purity was determined by neuron-specific enolase (NSE) immunoreactivity, when cultured for 7 days in vitro. The cells, at least>90% pure, cultured for 10 days were submitted to GA at concentration ranging from 1 to 50 mmol/L for 24-96 hours incubation. To evaluate the severity of neuron injury, the changes of morphology were observed by inverted microscopy, whereas the nuclear morphology was observed after an incubation of DNA-binding fluorescent dye Hoechst 33342 or TUNEL staining, the ultrastructure changes were detected by transmission electron microscope (TEM). The mitochondrial function was measured by MTT assay. The rates of neuronal apoptosis and necrosis were assessed via annexin-V and propidium iodide staining by flow cytometer system. To explore the possible apoptosis pathways involved in GA-induced neuronal damage, the expressions of caspase-3, caspase-8 and caspase-9 were detected by quantitative RT-PCR and Western blot.Results Under the inverted microscopy, neurons showed obvious toxic damage in GA treatment group. Compared with controls, the mitochondrial activity was significantly decreased in a concentration-and time-dependent manner. With comparison to the control group, GA (10-50 mmol/L) significantly induced mitochondrial activity(p<0.05) for 24,48, 72,96 hours, whereas at 1 mmol/L concentration, an incubation of 72 h and 96 h made a significant decrease in the mitochondrial activity. Take the 24 h group as the control in the same concentration, the incubation of 50 mmol/L for 48,72,96 h and 10,20 mmol/1 for 96 h dramatically decreased cell viability. The rates of neuronal apoptosis obviously increased: Having incubated for 48,72 h with 50 mmol/L GA, the rates of apoptosis were (40.90±4.09)% and (87.63±9.17)% respectively, and 20 mmol/L GA incubated for 72 h, it was (16.27±1.70)%(p=0.04). For the same concentration group, in 72 hours group, the apoptotic cells were much more than 48 hours group(p<0.05). In all group, the neurons had no increase in necrosis compared with the control group. The apoptotic cells labled by TUNEL were increased. TEM showed nuclear pyknosis The expressions of caspase-9 and-3 mRNA were significantly up regulated at 1h and 6h after 10,25 and 50 mmol/L GA treatment. As well, caspase-9 and-3 were markedly up regulated in active protein level after a 24h treatment of 25,50 mmol/L GA.Conclusions GA reduced the primary rat striatal neuron viability or mitochondrial function in a concentration-and time-dependent fashion. Apoptosis but not necrosis was detected at various stages after an incubation of 24-72 h with GA. GA initiates the apoptotic cascade in the primary cultured striatal neurons, which may be through caspase-9/-3 pathway. This mechanism may contribute to the striatal degeneration observed in glutaric acidemia typeⅠ. PARTⅢProtection of MK-801 against glutaric acid-mediated neurotoxicity in primary cultured striatal neuronsBackground Glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids were accumulated in glutaric aciduria I (GAⅠ), a neurometabolic disease characterized by acute striatal degeneration and chronic progressive cortical atrophy. In this study, we investigate the neurotoxic effects of glutaric acid on primary cultured neonatal rat striatal neurons and explore the effect of NMD A receptors antagonist on the neuronal damage.Methods The purity of primary striatal neurons was determined by immunocytochemistry and a purity of>90% was taken as the prerequisite for the following experiments. The neurons were exposed to GA at concentration ranging from 0 to 50mM for a 24h incubation, with or without NMDA receptor antagonist pre-and co-treatment, in 10 days in vitro (DIV). The cell viability was measured by metabolism of MTT. The rates of neuronal apoptosis and necrosis were assessed via annexin-V and PI staining by flow cytometer system. The expressions of caspase 3,9,8 were detected by Western bloting. The mRNA expressions of NR 1, NR 2A and NR 2B were assayed by Real-time PCR.Results GA decreased cell viability in a concentration-dependent fashion shown in MTT test. The NMDA channel blocker MK-801 enhanced the survival of neurons treated with 10,20 and 50 mM GA(p<0.01), but not returning back up to the normal level, whereas the non-NMDA receptor antagonist CNQX did not block GA-induced toxicity. Having applied 30 mM and 50 mM GA to striatal neurons for 24 h, apoptotic cells significantly increased (41±2.66,46.97±2.47;p<0.01 for both). The NMDA receptor antagonist MK801 (30.23±1.27,28.37±3.32; p<0.01 for both) rather than the AMPA antagonist CNQX (39.03±2.14,43.4±2.45; p>0.05 for both) protrudingly reduced cell apoptosis. As well, caspase 9 and 3 were markedly upregulated in active protein level after a 24 h treatment of 30 mM GA, which was significantly suppressed by the NMDA receptor antagonist MK-801 rather than AMPA antagonist CNQX. As well, the expressions of NR 1 and NR 2B mRNA were decreased but that of NR 2A was not changed.Conclusions GA reduced the primary striatal neuron cell viability in a concentration-and time-dependent fashion. GA initiates apoptosis in the primary cultured striatal neurons and that may be through caspase 9/3 pathway. The NMDA receptor may invole in GA neurotoxicity and the NMDA receptor antagonist protected against this neurotoxicity partly. These mechanisms may contribute to the striatal degeneration observed in glutaric aciduria typeⅠ. Part IV Induction of striatal lesion by chronic glutaric acid administration to ratsBackground Glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids were accumulated in glutaric aciduria I (GAⅠ), a neurometabolic disease characterized by acute striatal degeneration and chronic progressive cortical atrophy. In this study, we investigate the neurotoxic effects of glutaric acid on neonatal rat corpus striatum and explore the possible mechanism involved in the striatal lesion in vivo.Methods NS or GA (5 or 10μmol/g body weight, pH 7.2-7.4) were subcutaneously administered three daily through postnatal days 3-22 of the newborns. The rats were killed by decapitation 12 h after the last injection and the change by microscopic pathology in corpus striatum was evalued by HE staining. TUNEL staining was performed to identify the apoptotic cells. The mRNA expressions of caspase 3,8,9, Bax, Bcl-2 were detected by Real-time PCR and the protein expression of procaspase-3 and the active fraction were evalued by Western bloting.Results Cortical atrophy by gross pathology (macroscope) and vacuolations in the bilateral striatum by microscope were observed. Apoptotic cells were increased in the groups of LGA and HGA. The mRNA expressions of caspase 3,9, Bax were markedly upregulated and the mRNA of Bcl-2 was decreased in expression. the protein expression of procaspase-3 and the active fraction were upregulated in LGA and HGA.Conclusions Chronic GA administration induced striatal impairment. GA initiates apoptosis in the corpora striata and that may be through mitochondrial apoptotic pathways. These mechanisms may contribute to the striatal degeneration observed in glutaric aciduria type I.