A glutamatergic biomarker panel enables differentiating Grade 4 gliomas/astrocytomas from brain metastases.

Background: The differentiation of high-grade glioma and brain tumors of an extracranial origin is eminent for the decision on subsequent treatment regimens. While in high-grade glioma, a surgical resection of the tumor mass is a fundamental part of current standard regimens, in brain metastasis, the burden of the primary tumor must be considered. However, without a cancer history, the differentiation remains challenging in the imaging. Hence, biopsies are common that may help to identify the tumor origin. An additional tool to support the differentiation may be of great help. For this purpose, we aimed to identify a biomarker panel based on the expression analysis of a small sample of tissue to support the pathological analysis of surgery resection specimens. Given that an aberrant glutamate signaling was identified to drive glioblastoma progression, we focused on glutamate receptors and key players of glutamate homeostasis. Methods: Based on surgically resected samples from 55 brain tumors, the expression of ionotropic and metabotropic glutamate receptors and key players of glutamate homeostasis were analyzed by RT-PCR. Subsequently, a receiver operating characteristic (ROC) analysis was performed to identify genes whose expression levels may be associated with either glioblastoma or brain metastasis. Results: Out of a total of 29 glutamatergic genes analyzed, nine genes presented a significantly different expression level between high-grade gliomas and brain metastases. Of those, seven were identified as potential biomarker candidates including genes encoding for AMPA receptors GRIA1, GRIA2, kainate receptors GRIK1 and GRIK4, metabotropic receptor GRM3, transaminase BCAT1 and the glutamine synthetase (encoded by GLUL). Overall, the biomarker panel achieved an accuracy of 88% (95% CI: 87.1, 90.8) in predicting the tumor entity. Gene expression data, however, could not discriminate between patients with seizures from those without. Conclusion: We have identified a panel of seven genes whose expression may serve as a biomarker panel to discriminate glioblastomas and brain metastases at the molecular level. After further validation, our biomarker signatures could be of great use in the decision making on subsequent treatment regimens after diagnosis.

Studies on Biological and Molecular Effects of Small-Molecule Kinase Inhibitors on Human Glioblastoma Cells and Organotypic Brain Slices.

Glioblastoma is the most common and aggressive primary brain tumor. Multiple genetic and epigenetic alterations in several major signaling pathways-including the phosphoinositide 3-kinases (PI3K)/AKT/mTOR and the Raf/MEK/ERK pathway-could be found. We therefore aimed to investigate the biological and molecular effects of small-molecule kinase inhibitors that may interfere with those pathways. For this purpose, patient-derived glioblastoma cells were challenged with dactolisib, ipatasertib, MK-2206, regorafenib, or trametinib. To determine the effects of the small-molecule kinase inhibitors, assays of cell proliferation and apoptosis and immunoblot analyses were performed. To further investigate the effects of ipatasertib on organotypic brain slices harboring glioblastoma cells, the tumor growth was estimated. In addition, the network activity in brain slices was assessed by electrophysiological field potential recordings. Multi-kinase inhibitor regorafenib and both MK-2206 and dactolisib were very effective in all preclinical tumor models, while with respect to trametinib, two cell lines were found to be highly resistant. Only in HROG05 cells, ipatasertib showed anti-tumoral effects in vitro and in organotypic brain slices. Additionally, ipatasertib diminished synchronous network activity in organotypic brain slices. Overall, our data suggest that ipatasertib was only effective in selected tumor models, while especially regorafenib and MK-2206 presented a uniform response pattern.

Galvanotactic Migration of Glioblastoma and Brain Metastases Cells.

Galvanotaxis, the migration along direct current electrical fields, may contribute to the invasion of brain cancer cells in the tumor-surrounding tissue. We hypothesized that pharmacological perturbation of the epidermal growth factor (EGF) receptor and downstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway prevent galvanotactic migration. In our study, patient-derived glioblastoma and brain metastases cells were exposed to direct current electrical field conditions. Velocity and direction of migration were estimated. To determine the effects of EGF receptor antagonist afatinib and AKT inhibitor capivasertib, assays of cell proliferation, apoptosis and immunoblot analyses were performed. Both inhibitors attenuated cell proliferation in a dose-dependent manner and induced apoptosis. We found that most of the glioblastoma cells migrated preferentially in an anodal direction, while brain metastases cells were unaffected by direct current stimulations. Afatinib presented only a mild attenuation of galvanotaxis. In contrast, capivasertib abolished the migration of glioblastoma cells without genetic alterations in the PI3K/AKT pathway, but not in cells harboring PTEN mutation. In these cells, an increase in the activation of ERK1/2 may in part substitute the inhibition of the AKT pathway. Overall, our data demonstrate that glioblastoma cells migrate in the electrical field and the PI3K/AKT pathway was found to be highly involved in galvanotaxis.

Perampanel Add-on to Standard Radiochemotherapy in vivo Promotes Neuroprotection in a Rodent F98 Glioma Model.

An abnormal glutamate signaling of glioblastoma may contribute to both tumor progression and the generation of glioma-associated epileptic seizures. We hypothesized that the AMPA receptor antagonist perampanel (PER) could attenuate tumor growth and epileptic events. F98 glioma cells, grown orthotopically in Fischer rats, were employed as a model of glioma to investigate the therapeutic efficiency of PER (15 mg/kg) as adjuvant to standard radiochemotherapy (RCT). The epileptiform phenotype was investigated by video-EEG analysis and field potential recordings. Effects on glioma progression were estimated by tumor size quantification, survival analysis and immunohistological staining. Our data revealed that orthotopically-growing F98 glioma promote an epileptiform phenotype in rats. RCT reduced the tumor size and prolonged the survival of the animals. The adjuvant administration of PER had no effect on tumor progression. The tumor-associated epileptic events were abolished by PER application or RCT respectively, to initial baseline levels. Remarkably, PER preserved the glutamatergic network activity on healthy peritumoral tissue in RCT-treated animals. F98 tumors are not only a robust model to investigate glioma progression, but also a viable model to simulate a glioma-associated epileptiform phenotype. Furthermore, our data indicate that PER acts as a potent anticonvulsant and may protect the tumor-surrounding tissue as adjuvant to RCT, but failed to attenuate tumor growth or promote animal survival.

Mitochondrial complex IV mutation increases reactive oxygen species production and reduces lifespan in aged mice.

AIM: Mitochondrial DNA (mtDNA) mutations can negatively influence lifespan and organ function. More than 250 pathogenic mtDNA mutations are known, often involving neurological symptoms. Major neurodegenerative diseases share key etiopathogenetic components ie mtDNA mutations, mitochondrial dysfunction and oxidative stress. METHODS: Here, we characterized a conplastic mouse strain (C57BL/6 J-mtNOD) carrying an electron transport chain complex IV mutation that leads to an altered cytochrome c oxidase subunit III. Since this mouse also harbours adenine insertions in the mitochondrial tRNA for arginine, we chose the C57BL/6 J-mtMRL as control strain which also carries a heteroplasmic stretch of adenine repetitions in this tRNA isoform. RESULTS: Using MitoSOX fluorescence, we observed an elevated mitochondrial superoxide production and a reduced gene expression of superoxide dismutase 2 in the 24-month-old mtNOD mouse as compared to control. Together with the decreased expression of the fission-relevant gene Fis1, these data confirmed that the ageing mtNOD mouse had a mitochondrial dysfunctional phenotype. On the functional level, we could not detect significant differences in synaptic long-term potentiation, but found a markedly poor physical constitution to perform the Morris water maze task at the age of 24 months. Moreover, the median lifespan of mtNOD mice was significantly shorter than of control animals. CONCLUSION: Our findings demonstrate that a complex IV mutation leads to mitochondrial dysfunction that translates into survival.

Uncoupling protein 2 protects mice from aging.

Uncoupling protein (UCP) 2 is a mitochondrial transporter protein that plays various roles in cellular metabolism, including the glucose and lipid metabolism. Polymorphisms in UCP2 are associated with longevity in humans. In line with this, mice carrying the UCP2 transgene under the control of hypocretin promoter were reported to have an extended lifespan, while, conversely, mice deficient in Ucp2 demonstrated a significantly shorter lifespan. In this study, we examined the phenotype of aging in a large colony of Ucp2-deficient (Ucp2(-/-)) mice on the molecular level. We have found that the significantly shorter lives of Ucp2(-/-) mice is the result of an accelerated aging process throughout their entire lifespan. Thus, Ucp2(-/-) mice not only earlier gained sexual maturity, but also earlier progressed into an aging phenotype, reflected by a decrease in body weight, increased neutrophil numbers, and earlier emergence of spontaneous ulcerative dermatitis. Intriguingly, on the molecular level this acceleration in aging predominantly driven by increased levels of circulating IGF-1 in Ucp2(-/-) mice, hinting at a crosstalk between UCP2 and the classical Insulin/IGF-1 signaling aging pathway.

Reduced adolescent-age spatial learning ability associated with elevated juvenile-age superoxide levels in complex I mouse mutants.

Large-scale, heteroplasmic and generally pathogenic mtDNA defects (as induced by defective mitochondrial DNA polymerase, clonal mutations or DNA deletions) are known to negatively impact on life span and can result in apoptosis and tissue loss in, e.g., skeletal muscle or reduce learning abilities. The functional impact of homoplasmic specific mtDNA point mutations, e.g., in genes coding for the electron transport chain, however, remains a matter of debate. The present study contributes to this discussion and provides evidence that a single point mutation in complex I of the respiratory chain is associated with impairment of spatial navigation in adolescent (6-month-old) mice, i.e., reduced performance in the Morris Water Maze, which goes along with increased production of reactive oxygen species (ROS) in juvenile mice (3 months) but not at the age of phenotype expression. A point mutation in complex III goes along with only a mild and non-significant negative effect on cognitive performance and no significant changes in ROS production. These findings suggest to also consider the ontogenetic development of phenotypes when studying mtDNA mutations and highlights a possible impact of complex I dysfunction on the emergence of neurological deficits.