Misprogramming of glucose metabolism impairs recovery of hippocampal slices from neuronal GLT-1 knockout mice and contributes to excitotoxic injury through mitochondrial superoxide production.
S Li, J Wang, J V Andersen, B I Aldana, B Zhang, E V Prochownik, P A Rosenberg
{"title":"Misprogramming of glucose metabolism impairs recovery of hippocampal slices from neuronal GLT-1 knockout mice and contributes to excitotoxic injury through mitochondrial superoxide production.","authors":"S Li, J Wang, J V Andersen, B I Aldana, B Zhang, E V Prochownik, P A Rosenberg","doi":"10.1111/jnc.16205","DOIUrl":null,"url":null,"abstract":"<p><p>We have previously reported a failure of recovery of synaptic function in the CA1 region of acute hippocampal slices from mice with a conditional neuronal knockout (KO) of GLT-1 (EAAT2, Slc1A2) driven by synapsin-Cre (synGLT-1 KO). The failure of recovery of synaptic function is due to excitotoxic injury. We hypothesized that changes in mitochondrial metabolism contribute to the heightened vulnerability to excitotoxicity in the synGLT-1 KO mice. We found impaired flux of carbon from <sup>13</sup>C-glucose into the tricarboxylic acid cycle in synGLT-1 KO cortical and hippocampal slices compared with wild-type (WT) slices. In addition, we found downregulation of the neuronal glucose transporter GLUT3 in both genotypes. Flux of carbon from [1,2-<sup>13</sup>C]acetate, thought to be astrocyte-specific, was increased in the synGLT-KO hippocampal slices but not cortical slices. Glycogen stores, predominantly localized to astrocytes, are rapidly depleted in slices after cutting, and are replenished during ex vivo incubation. In the synGLT-1 KO, replenishment of glycogen stores during ex vivo incubation was compromised. These results suggest both neuronal and astrocytic metabolic perturbations in the synGLT-1 KO slices. Supplementing incubation medium during recovery with 20 mM D-glucose normalized glycogen replenishment but had no effect on recovery of synaptic function. In contrast, 20 mM non-metabolizable L-glucose substantially improved recovery of synaptic function, suggesting that D-glucose metabolism contributes to the excitotoxic injury in the synGLT-1 KO slices. L-lactate substitution for D-glucose did not promote recovery of synaptic function, implicating mitochondrial metabolism. Consistent with this hypothesis, phosphorylation of pyruvate dehydrogenase, which decreases enzyme activity, was increased in WT slices during the recovery period, but not in synGLT-1 KO slices. Since metabolism of glucose by the mitochondrial electron transport chain is associated with superoxide production, we tested the effect of drugs that scavenge and prevent superoxide production. The superoxide dismutase/catalase mimic EUK-134 conferred complete protection and full recovery of synaptic function. A site-specific inhibitor of complex III superoxide production, S3QEL-2, was also protective, but inhibitors of NADPH oxidase were not. In summary, we find that the failure of recovery of synaptic function in hippocampal slices from the synGLT-1 KO mouse, previously shown to be due to excitotoxic injury, is caused by production of superoxide by mitochondrial metabolism.</p>","PeriodicalId":16527,"journal":{"name":"Journal of Neurochemistry","volume":null,"pages":null},"PeriodicalIF":4.2000,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Neurochemistry","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1111/jnc.16205","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
We have previously reported a failure of recovery of synaptic function in the CA1 region of acute hippocampal slices from mice with a conditional neuronal knockout (KO) of GLT-1 (EAAT2, Slc1A2) driven by synapsin-Cre (synGLT-1 KO). The failure of recovery of synaptic function is due to excitotoxic injury. We hypothesized that changes in mitochondrial metabolism contribute to the heightened vulnerability to excitotoxicity in the synGLT-1 KO mice. We found impaired flux of carbon from 13C-glucose into the tricarboxylic acid cycle in synGLT-1 KO cortical and hippocampal slices compared with wild-type (WT) slices. In addition, we found downregulation of the neuronal glucose transporter GLUT3 in both genotypes. Flux of carbon from [1,2-13C]acetate, thought to be astrocyte-specific, was increased in the synGLT-KO hippocampal slices but not cortical slices. Glycogen stores, predominantly localized to astrocytes, are rapidly depleted in slices after cutting, and are replenished during ex vivo incubation. In the synGLT-1 KO, replenishment of glycogen stores during ex vivo incubation was compromised. These results suggest both neuronal and astrocytic metabolic perturbations in the synGLT-1 KO slices. Supplementing incubation medium during recovery with 20 mM D-glucose normalized glycogen replenishment but had no effect on recovery of synaptic function. In contrast, 20 mM non-metabolizable L-glucose substantially improved recovery of synaptic function, suggesting that D-glucose metabolism contributes to the excitotoxic injury in the synGLT-1 KO slices. L-lactate substitution for D-glucose did not promote recovery of synaptic function, implicating mitochondrial metabolism. Consistent with this hypothesis, phosphorylation of pyruvate dehydrogenase, which decreases enzyme activity, was increased in WT slices during the recovery period, but not in synGLT-1 KO slices. Since metabolism of glucose by the mitochondrial electron transport chain is associated with superoxide production, we tested the effect of drugs that scavenge and prevent superoxide production. The superoxide dismutase/catalase mimic EUK-134 conferred complete protection and full recovery of synaptic function. A site-specific inhibitor of complex III superoxide production, S3QEL-2, was also protective, but inhibitors of NADPH oxidase were not. In summary, we find that the failure of recovery of synaptic function in hippocampal slices from the synGLT-1 KO mouse, previously shown to be due to excitotoxic injury, is caused by production of superoxide by mitochondrial metabolism.
我们以前曾报道过,在突触素-Cre(synapsin-Cre,synGLT-1 KO)驱动的条件性神经元敲除(KO)GLT-1(EAAT2,Slc1A2)的小鼠急性海马切片的CA1区,突触功能未能恢复。突触功能无法恢复的原因是兴奋毒性损伤。我们推测,线粒体代谢的变化是导致 synGLT-1 KO 小鼠更易受兴奋毒性伤害的原因。与野生型(WT)切片相比,我们发现在 synGLT-1 KO 小鼠的皮质和海马切片中,13C-葡萄糖进入三羧酸循环的碳通量受损。此外,我们还发现两种基因型的神经元葡萄糖转运体 GLUT3 均出现下调。在 synGLT-KO 海马切片中,被认为是星形胶质细胞特异性的 [1,2-13C] 乙酸的碳通量增加了,但在大脑皮层切片中却没有增加。糖原储存主要定位于星形胶质细胞,切片后会迅速耗尽,并在体外培养过程中得到补充。在 synGLT-1 KO 中,体内外培养期间糖原储存的补充受到影响。这些结果表明,在 synGLT-1 KO 切片中,神经元和星形胶质细胞的代谢都受到了干扰。在恢复期间用 20 mM D-葡萄糖补充培养基可使糖原补充正常化,但对突触功能的恢复没有影响。与此相反,20 mM 不可代谢的 L-葡萄糖大大改善了突触功能的恢复,这表明 D-葡萄糖代谢是 synGLT-1 KO 切片兴奋毒性损伤的原因之一。用 L-乳酸替代 D-葡萄糖并不能促进突触功能的恢复,这与线粒体代谢有关。与这一假设相一致的是,丙酮酸脱氢酶的磷酸化会降低酶的活性,而在恢复期间,WT 切片的丙酮酸脱氢酶的磷酸化会增加,但在 synGLT-1 KO 切片中则不会。由于线粒体电子传递链的葡萄糖代谢与超氧化物的产生有关,我们测试了清除和防止超氧化物产生的药物的效果。超氧化物歧化酶/催化酶模拟物 EUK-134 能提供完全保护并完全恢复突触功能。复合体 III 超氧化物产生的位点特异性抑制剂 S3QEL-2 也具有保护作用,但 NADPH 氧化酶抑制剂则没有保护作用。总之,我们发现,之前被证明是兴奋毒性损伤导致的 synGLT-1 KO 小鼠海马切片突触功能恢复失败,是由线粒体代谢产生的超氧化物引起的。
期刊介绍:
Journal of Neurochemistry focuses on molecular, cellular and biochemical aspects of the nervous system, the pathogenesis of neurological disorders and the development of disease specific biomarkers. It is devoted to the prompt publication of original findings of the highest scientific priority and value that provide novel mechanistic insights, represent a clear advance over previous studies and have the potential to generate exciting future research.