Masaaki Kohta

MicroRNA-183 upregulates HIF-1α by targeting isocitrate dehydrogenase 2 (IDH2) in glioma cells

MicroRNAs (miRs) are small, non-coding RNAs that regulate gene expression and contribute to cell proliferation, differentiation and metabolism. Our previous study revealed the extensive modulation of a set of miRs in malignant glioma. In that study, miR microarray analysis demonstrated the upregulation of microRNA-183 (miR-183) in glioblastomas. Therefore, we examined the expression levels of miR-183 in various types of gliomas and the association of miR-183 with isocitrate dehydrogenase 2 (IDH2), which has complementary sequences to miR-183 in its 3′-untranslated region (3′UTR). In present study, we used real-time PCR analysis to demonstrate that miR-183 is upregulated in the majority of high-grade gliomas and glioma cell lines compared with peripheral, non-tumorous brain tissue. The mRNA and protein expression levels of IDH2 are downregulated via the overexpression of miR-183 mimic RNA in glioma cells. Additionally, IDH2 mRNA expression is upregulated in glioma cells expressing anti-miR-183. We verified that miR-183 directly affects IDH2 mRNA levels in glioma cells using luciferase assays. In malignant glioma specimens, the expression levels of IDH2 were lower in tumors than in the peripheral, non-tumorous brain tissues. HIF-1α levels were upregulated in glioma cells following transfection with miR-183 mimic RNA or IDH2 siRNA. Moreover, vascular endothelial growth factor and glucose transporter 1, which are downstream molecules of HIF-1α, were upregulated in cells transfected with miR-183 mimic RNA. These results suggest that miR-183 upregulation in malignant gliomas induces HIF-1α expression by targeting IDH2 and may play a role in glioma biology.

Combined IDH1 mutation and MGMT methylation status on long-term survival of patients with cerebral low-grade glioma

OBJECTIVE The management of low-grade glioma (LGG) still remains controversial because the effectiveness of early and extensive resection is unclear, and the use of radiation therapy or chemotherapy is not well-defined. In particular, the importance of prognostic factors for survival remains a matter of discussion. The purpose of this study was to validate prognostic factors for survival in patients with LGG. MATERIALS AND METHODS A consecutive series of 55 patients with WHO grade II LGG treated in our institute between 1983 and 2013 were retrospectively reviewed to determine the prognostic factors for survival. All data were retrospectively analyzed from the aspect of baseline characteristics, pathological findings, genetic change, surgical treatments, adjuvant therapies, and survival time. Cox multivariate analysis was performed to determine the prognostic factors for survival. RESULTS There were 28 patients with diffuse astrocytoma (DA), 21 patients with oligodendroglioma (OG), and 6 patients with oligoastrocytoma (OA) diagnosed on initial surgery. The median overall survival was 193 months and fifteen patients (27.3%) died. A mutation in isocitrate dehydrogenase-1 (IDH1) was found in 72.9% of LGG, and this mutation was positively correlated with methylation of O6-methylguanine-DNA methyltransferase (MGMT) (p=0.02). A better prognosis was significantly associated with combined IDH1 mutation and MGMT methylation status (both positive vs both negative, HR 0.079 [95% CI 0.008-0.579], p=0.012), as well as histology (OG vs DA and OA, HR 0.158 [95% CI 0.022-0.674], p=0.011) and tumor size (<6 cm vs ≥6 cm, HR 0.120 [95% CI 0.017-0.595], p=0.008). CONCLUSIONS Tumor histology, size and IDH-mutation status are important predictors for prolonged overall survival in patients with LGG and may provide a reliable tool for standardizing future treatment strategies.

Endoscopic endonasal translacerum approach to the inferior petrous apex.

OBJECTIVE The surgical approach to lesions involving the inferior petrous apex (IPA) is still challenging. The purpose of this study is to demonstrate the anatomical features of the IPA and to assess the applicability of an endoscopic endonasal approach through the foramen lacerum (translacerum approach) to the IPA. METHODS The surgical simulation of the endoscopic endonasal translacerum approach was conducted in 3 cadaver heads. The same technique was applied in 4 patients harboring tumors involving the IPA (3 chordomas and 1 chondro-sarcoma). RESULTS By removing the fibrocartilaginous component of the foramen lacerum, a triangular space was created between the anterior genu of the petrous portion of the carotid artery and the eustachian tube, through which the IPA could be approached. The range of the surgical maneuver reached laterally up to the internal auditory canal, jugular foramen, and posterior vertical segment of the petrous portion of the carotid artery. In clinical application, the translacerum approach provided sufficient space to handle tumors at the IPA. Gross-total and partial removal was achieved in 3 and 1 cases, respectively, without permanent surgery-related morbidity and mortality. CONCLUSIONS The endoscopic endonasal translacerum approach provides reliable access to the IPA. It is indicated alone for lesions confined to the IPA and in combination with other approaches for more extensive lesions.

A Novel Rac1-GSPT1 Signaling Pathway Controls Astrogliosis Following Central Nervous System Injury

Astrogliosis (i.e. glial scar), which is comprised primarily of proliferated astrocytes at the lesion site and migrated astrocytes from neighboring regions, is one of the key reactions in determining outcomes after CNS injury. In an effort to identify potential molecules/pathways that regulate astrogliosis, we sought to determine whether Rac/Rac-mediated signaling in astrocytes represents a novel candidate for therapeutic intervention following CNS injury. For these studies, we generated mice with Rac1 deletion under the control of the GFAP (glial fibrillary acidic protein) promoter (GFAP-Cre;Rac1flox/flox). GFAP-Cre;Rac1flox/flox (Rac1-KO) mice exhibited better recovery after spinal cord injury and exhibited reduced astrogliosis at the lesion site relative to control. Reduced astrogliosis was also observed in Rac1-KO mice following microbeam irradiation-induced injury. Moreover, knockdown (KD) or KO of Rac1 in astrocytes (LN229 cells, primary astrocytes, or primary astrocytes from Rac1-KO mice) led to delayed cell cycle progression and reduced cell migration. Rac1-KD or Rac1-KO astrocytes additionally had decreased levels of GSPT1 (G1 to S phase transition 1) expression and reduced responses of IL-1β and GSPT1 to LPS treatment, indicating that IL-1β and GSPT1 are downstream molecules of Rac1 associated with inflammatory condition. Furthermore, GSPT1-KD astrocytes had cell cycle delay, with no effect on cell migration. The cell cycle delay induced by Rac1-KD was rescued by overexpression of GSPT1. Based on these results, we propose that Rac1-GSPT1 represents a novel signaling axis in astrocytes that accelerates proliferation in response to inflammation, which is one important factor in the development of astrogliosis/glial scar following CNS injury.

Novel role of Rac-Mid1 signaling in medial cerebellar development

Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3−/−) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3−/− mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3−/− CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3−/− mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development. Summary: Double knockout of Rac1 and Rac3 in cerebellar granule neurons impairs their tangential migration and final differentiation. Rac depleted cells exhibit decreased levels of Mid1 and impaired mTORC1 signaling.

Delayed onset massive oedema and deterioration in traumatic brain injury.

A 52-year-old man fell from standing and a computed tomography (CT) scan revealed traumatic intracerebral haematoma and subarachnoid haemorrhage in the temporal cortex. He was treated without surgery and discharged. On day 30 after the accident, he had no neurological deficit. On day 37 he complained of headache and urinary incontinence, and on day 39 he was hospitalized due to progressive neurological deterioration (reduced conciousness, dilated pupils, and left hemiplegia). A CT scan revealed a diffuse low-density in the right cerebral hemisphere with marked midline shift. Emergency decompressive craniectomy and right temporal lobectomy were performed. Angiography after surgery revealed moderate vasospasm in the right middle and anterior cerebral arteries. The patient remained severely disabled. Delayed onset neurological deterioration can be caused by brain oedema and vasospasm after traumatic brain injury, despite an intervening period of improvement.

Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia

The metabolic pathophysiology underlying ischemic stroke remains poorly understood. To gain insight into these mechanisms, we performed a comparative metabolic and transcriptional analysis of the effects of cerebral ischemia on the metabolism of the cerebral cortex using middle cerebral artery occlusion (MCAO) rat model. Metabolic profiling by gas-chromatography/mass-spectrometry analysis showed clear separation between the ischemia and control group. The decreases of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia (120-min MCAO) without reperfusion. Transcriptional profiling by microarray hybridization indicated that the Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathways were upregulated during cerebral ischemia without reperfusion. In relation to the PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through real-time polymerase chain reaction. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation at serine 85 after 60-min and 120-min MCAO without reperfusion. Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD+ ratio were also observed after 120-min MCAO. Furthermore, intracerebroventricular injection of ataxia telangiectasia mutated (ATM) kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD upregulation after MCAO, but that of protein kinase D inhibitor (CID755673) did not affect HSP27 phosphorylation. Consequently, G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during the earliest stages of ischemia. These findings have important therapeutic implications for the treatment of stroke.

Long-Term Outcomes of Carotid Endarterectomy and Carotid Artery Stenting for Carotid Artery Stenosis: Real-World Status in Japan

BACKGROUND AND PURPOSE We investigated long-term outcomes of carotid endarterectomy (CEA) and carotid artery stenting (CAS) in our institute to evaluate the outcomes of real-world practice in Japan. METHODS Between August 2006 and July 2013, 203 consecutive carotid revascularizations with either CEA or CAS were performed in our institute. The initial treatment was regarded as the starting point in the cases of the patients who received treatment by bilateral carotid artery stenosis or retreatment. We assessed the long-term outcomes with survival analyses. RESULTS A total of 182 patients (CEA 111, CAS 71), including 86 symptomatic patients, were included in the current study with a mean follow-up period of 42.9 months. The periprocedural stroke/death/myocardial infarction (MI) rate was 3.6% for CEA and 5.6% for CAS groups (P = .71). Estimates of the 4-year event-free rate from the primary end point (the composite of any stroke, death, or MI within 30 days, and any ipsilateral stroke thereafter) using competing risk analysis were 3.6% for CEA and 7.1% for CAS (P = .156). Kaplan-Meier estimates of the 4-year event-free rate from the secondary end point (the composite of any stroke, death, or MI within 30 days, and any stroke or death thereafter) were 13.8% for CEA and 19.1% for CAS (P = .072). Age was the only significant predictor for the primary end point. Both age and CAS were significant predictors for the secondary end point. CONCLUSIONS The current study on real-world practices demonstrated perioperative and long-term outcomes that were comparable to previous major studies of large numbers of patients.

Endoscopic endonasal surgery for pituitary adenomas extending to the oculomotor cistern

Extension of a pituitary adenoma to the oculomotor cistern harbors the risk of oculomotor nerve impairment and further extension into the interpeduncular cistern. The role of endoscopic endonasal surgery for those lesions was investigated.

Pentose phosphate pathway activation via HSP27 phosphorylation by ATM kinase: A putative endogenous antioxidant defense mechanism during cerebral ischemia-reperfusion

Molecular mechanism underlying ischemic stroke remains poorly understood. We previously reported glucose 6-phosphate dehydrogenase (G6PD) activity in pentose phosphate pathway (PPP) is activated via heat shock protein 27 (HSP27) phosphorylation at serine 85 (S85) by ataxia telangiectasia mutated (ATM) kinase during cerebral ischemia. This mechanism seems to be endogenous antioxidative system. To determine whether this system also works during reperfusion, we performed comparative metabolic analysis of reperfusion effect on metabolism in rat cortex using middle cerebral artery occlusion (MCAO). Metabolic profiling using gas-chromatography/mass-spectrometry analysis showed changes in metabolic state that depended on reperfusion time. Enrichment analysis showed PPP was significantly upregulated during ischemia-reperfusion. Significant increases in fructose 6-phosphate and ribulose 5-phosphate after reperfusion also suggested enhancement of PPP. In relation to PPP, ischemia-reperfusion induced an increase of up to 69-fold in HSP27 transcripts after 24-h reperfusion. Immunoblotting showed gradual increase in HSP27 protein and marked increase in HSP27 phosphorylation (S85) that were time-dependent (4.5-fold after 24-h reperfusion). G6PD activity was significantly elevated after 1-h MCAO (20%), reduced after 1-h reperfusion, increased gradually thereafter and significantly elevated after 24-h reperfusion. The NADPH/NAD+ ratio displayed similar increasing pattern. Intracerebroventricular injection of ATM kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD activity, significantly increased protein carbonyl, and resulted in increase in infarct size (100%) 24-h after reperfusion following 90-min MCAO. Consequently, G6PD activation via HSP27 phosphorylation by ATM kinase may be part of endogenous antioxidant defense neuroprotection mechanism that is activated during ischemia-reperfusion. These findings have important implications for treatment of stroke.