Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a major cellular energy sensor that is activated by an increase in the AMP/adenosine triphosphate (ATP) ratio. This causes the initiation of adaptive cellular programs, leading to the inhibition of anabolic pathways and increasing ATP synthesis. AMPK indirectly inhibits mammalian target of rapamycin (mTOR) complex 1 (mTORc1), a serine/threonine kinase and central regulator of cell growth and metabolism, which integrates various growth inhibitory signals, such as the depletion of glucose, amino acids, ATP and oxygen. While neuroprotective approaches by definition focus on neurons, that are more sensitive under cell stress conditions, astrocytes play an important role in the cerebral energy homeostasis during ischemia. Therefore, the protection of astrocytic cells or other glial cells may contribute to the preservation of neuronal integrity and function. In the present study, it was thus hypothesized that a preventive induction of energy deprivation-activated signaling pathways via AMPK may protect astrocytes from hypoxia and glucose deprivation. Hypoxia-induced cell death was measured in a paradigm of hypoxia and partial glucose deprivation in vitro in the immortalized human astrocytic cell line SVG. Both the glycolysis inhibitor 2-deoxy-d-glucose (2dG) and the AMPK activator A-769662 induced the phosphorylation of AMPK, resulting in mTORc1 inhibition, as evidenced by a decrease in the phosphorylation of the target ribosomal protein S6 (RPS6). Treatment with both 2dG and A-769662 also decreased glucose consumption and lactate production. Furthermore, A-769662, but not 2dG induced an increase in oxygen consumption, possibly indicating a more efficient glucose utilization through oxidative phosphorylation. Hypoxia-induced cell death was profoundly reduced by treatment with 2dG or A-769662. On the whole, the findings of the present study demonstrate, that AMPK activation via 2dG or A-769662 protects astrocytes under hypoxic and glucose-depleted conditions.

Abstracts iii23NEURO-ONCOLOGY • MAY 2017 ing defects in the IDH1 gene by detecting circulating cell free (ccf) DNA in the CSF derived from the tumor tissue of glioma patients. Moreover, MGMT promoter methylation was evaluated for the same samples in a subset of patients. METHODS: Lumbar puncture was performed to obtain CSF from 7 patients with glioma. ccfDNA was extracted from 1 ml of CSF using the Maxwell rapid sample concentrator system. Subsequently, the presence of point mutation of the IDH1 gene at codon 132 was screened by real-time PCR/high-resolution melting (HRM) curve analysis, and the mutation was confirmed on the basis of DNA sequencing results. Status in the IDH1 gene was also analyzed using the same assay technique for the DNA extracted from the excised fresh tumor tissue, to compare the results with that of CSFderived ccfDNA. In addition, ccfDNA was also extracted from the plasma in 3 patients to additionally examine the presence of the IDH1 gene mutation. For MGMT promoter methylation, a quantitative analysis was performed using the methylation-specific HRM method. RESULTS: CSF-derived ccfDNA was successfully extracted from all patients and analyzed using real-time PCR/ high-resolution melting curve analysis. IDH1 gene mutation was detected in 3 of the 7 glioma patients. The results of the IDH1 gene analysis of CSF-derived ccfDNA and that of the DNA extracted from the surgically excised tumor tissue were consisted in all patients. However, IDH1 gene mutation was not detected all in plasma-derived ccfDNA. MGMT methylation was evaluated in two cases. CONCLUSION: Gene analysis of ccfDNA from the CSF, not from the plasma, enabled the evaluation of IDH1 gene defect in glioma patients less invasively, without directly obtaining any tumor tissue. Moreover, this technique can be applied to analyze MGMT gene promotor methylation.