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1.
Sci Transl Med ; 12(531)2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075941

RESUMO

Epilepsy treatments for patients with mechanistic target of rapamycin (mTOR) disorders, such as tuberous sclerosis complex (TSC) or focal cortical dysplasia type II (FCDII), are urgently needed. In these patients, the presence of focal cortical malformations is associated with the occurrence of lifelong epilepsy, leading to severe neurological comorbidities. Here, we show that the expression of the actin cross-linking protein filamin A (FLNA) is increased in resected cortical tissue that is responsible for seizures in patients with FCDII and in mice modeling TSC and FCDII with mutations in phosphoinositide 3-kinase (PI3K)-ras homolog enriched in brain (Rheb) pathway genes. Normalizing FLNA expression in these mice through genetic knockdown limited cell misplacement and neuronal dysmorphogenesis, two hallmarks of focal cortical malformations. In addition, Flna knockdown reduced seizure frequency independently of mTOR signaling. Treating mice with a small molecule targeting FLNA, PTI-125, before the onset of seizures alleviated neuronal abnormalities and reduced seizure frequency compared to vehicle-treated mice. In addition, the treatment was also effective when injected after seizure onset in juvenile and adult mice. These data suggest that targeting FLNA with either short hairpin RNAs or the small molecule PTI-125 might be effective in reducing seizures in patients with TSC and FCDII bearing mutations in PI3K-Rheb pathway genes.

2.
Dalton Trans ; 48(46): 17258-17265, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31710322

RESUMO

The development of effective bifunctional catalysts for both oxygen reduction reactions (ORRs) and oxygen evolution reactions (OERs) is crucial for improving the performance of charge and discharge processes in rechargeable metal-air batteries. Here, we report a sulfonated cobalt phthalocyanine/carbon nanotube hybrid (CoPc-SO3H/CNT) prepared by a facile anchoring method along with sonication and magnetic stirring. The resulting CoPc-SO3H/CNT hybrid exhibits better catalytic activity for ORRs and OERs than the cobalt phthalocyanine/carbon nanotube hybrid (CoPc/CNT), sulfonated cobalt phthalocyanine (CoPc-SO3H), cobalt phthalocyanine (CoPc) and the carbon nanotube (CNT). The onset potential of CoPc-SO3H/CNT for the ORR in 0.1 M KOH is 0.88 V (vs. RHE), which is higher than that of CoPc/CNT (0.85 V), the CNT (0.80 V), CoPc-SO3H (0.77 V) and CoPc (0.66 V). Meanwhile, the CoPc-SO3H/CNT hybrid shows a much lower OER potential (1.62 V) at a current density of 10 mA cm-2 compared to CoPc-SO3H (1.64 V), CoPc/CNT (1.74 V), the CNT (1.96 V) and CoPc (>2.00 V) in 1 M KOH. Similar patterns are also found in 0.1 M KOH solution. Both the conductive CNT and the electron-withdrawing sulfonic groups are confirmed to benefit the electrochemical oxygen reactions (ORRs/OERs).

3.
Sci Rep ; 9(1): 16058, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690770

RESUMO

Tremendous efforts have been made to explore biomarkers for classification and grading on gliomas. The goal of this study was to identify more molecular features that are associated with clinical outcomes by comparing the genomic profiles of primary and recurrent gliomas and determine potential recurrence leading factors that are significantly enriched in relapse tumors. Hybrid capture based next generation sequencing (NGS) analysis was performed on 64 primary and 17 recurrent glioma biopsies. Copy number variation (CNV) was more frequent in recurrent tumors and CDKN2A/B loss was significantly enriched. In addition, overall mutations in cell cycle pathway are more common in relapse tumors. The patterns of gene sets, including IDH1/TERT and IDH1/TP53 exhibited significant difference between the groups. Survival analysis uncovered the worse disease-free survival (DFS) and overall survival (OS) associated with altered copy number and excessive activation of CELL CYCLE pathway. High Tumor Mutation Burden (TMB) was also a biomarker with great potential for poor prognosis. The assessment of genomic characteristics in primary versus recurrent gliomas aids the discovery of potential predictive biomarkers. The prognostic value of TMB in gliomas was raised for the first time.

4.
Epilepsia ; 60(6): 1255-1265, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31125447

RESUMO

OBJECTIVES: Patients with mammalian target of rapamycin (mTOR)-dependent malformations of cortical development (MCDs) associated with seizures display hyperperfusion and increased vessel density of the dysmorphic cortical tissue. Some studies have suggested that the vascular defect occurred independently of seizures. Here, we further examined whether hypervascularization occurs in animal models of global and focal MCD with and without seizures, and whether it is sensitive to the mTOR blocker, rapamycin, that is approved for epilepsy treatment in tuberous sclerosis complex. METHODS: We used two experimental models of mTOR-dependent MCD consisting of conditional transgenic mice containing Tsc1null cells in the forebrain generating a global malformation associated with seizures and of wild-type mice containing a focal malformation in the somatosensory cortex generated by in utero electroporation (IUE) that does not lead to seizures. Alterations in blood vessels and the effects of a 2-week-long rapamycin treatment on these phenotypes were assessed in juvenile mice. RESULTS: Blood vessels in both the focal and global MCDs of postnatal day 14 mice displayed significant increase in vessel density, branching index, total vessel length, and decreased tissue lacunarity. In addition, rapamycin treatment (0.5 mg/kg, every 2 days) partially rescued vessel abnormalities in the focal MCD model, but it did not ameliorate the vessel abnormalities in the global MCD model that required higher rapamycin dosage for a partial rescue. SIGNIFICANCE: Here, we identified hypervascularization in mTOR-dependent MCD in the absence of seizures in young mice, suggesting that increased angiogenesis occurs during development in parallel to alterations in corticogenesis. In addition, a predictive functional outcome is that dysplastic neurons forming MCD will have better access to oxygen and metabolic supplies via their closer proximity to blood vessels. Finally, the difference in rapamycin sensitivity between a focal and global MCD suggest that rapamycin treatment will need to be titrated to match the type of MCD.

5.
Neurosci Res ; 143: 44-52, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29857015

RESUMO

We combined translating ribosome affinity purification (TRAP) with in utero electroporation (IUE), called iTRAP to identify the molecular profile of specific neuronal populations during neonatal development without the need for viral approaches and FACS sorting. We electroporated a plasmid encoding EGFP-tagged ribosomal protein L10a at embryonic day (E) 14-15 to target layer 2-4 cortical neurons of the somatosensory cortex. At three postnatal (P) ages-P0, P7, and P14-when morphogenesis occurs and synapses are forming, TRAP and molecular profiling was performed from electroporated regions. We found that ribosome bound (Ribo)-mRNAs from ∼7300 genes were significantly altered over time and included classical neuronal genes known to decrease (e.g., Tbr1, Dcx) or increase (e.g., Eno2, Camk2a, Syn1) as neurons mature. This approach led to the identification of specific developmental patterns for Ribo-mRNAs not previously reported to be developmentally regulated in neurons, providing rationale for future examination of their role in selective biological processes. These include upregulation of Lynx1, Nrn1, Cntnap1 over time; downregulation of St8sia2 and Draxin; and bidirectional changes to Fkbp1b. iTRAP is a versatile approach that allows researchers to easily assess the molecular profile of specific neuronal populations in selective brain regions under various conditions, including overexpression and knockdown of target genes, and in disease settings.


Assuntos
Eletroporação/métodos , Biossíntese de Proteínas , Células Piramidais/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Desenvolvimento Embrionário , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células Piramidais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Sinapsinas/metabolismo
6.
Neurosci Lett ; 629: 15-18, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27345385

RESUMO

Most individuals with tuberous sclerosis complex (TSC) are born with a mutant allele of either TSC1 or TSC2 and a mosaic of psychological and cognitive defects. Tsc1 loss of heterozygosity contributes to severe dendritic abnormalities that are rescued by normalizing the levels of the actin-cross linking protein, Filamin A (FLNA). However, it is unclear whether dendrites and FLNA levels are abnormal in an heterozygote Tsc1 condition. Here, we examined dendritic morphology and FLNA levels in the olfactory bulb of Tsc1 wild type and heterozygote mice. Using in vivo neonatal electroporation to label newborn neurons followed by sholl analysis, we found that Tsc1 haploinsufficiency is associated with increased dendritic complexity and total dendritic length as well as increased FLNA levels. Since reducing FLNA levels has been shown to decrease Tsc1(+/-) dendritic complexity, these data suggest that increased FLNA levels in Tsc1(+/-) mice contribute to abnormal dendritic patterning in the Tsc1 heterozygote condition of individuals with TSC.


Assuntos
Dendritos/genética , Dendritos/patologia , Filaminas/metabolismo , Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/genética , Animais , Haploinsuficiência , Camundongos , Camundongos Transgênicos , Bulbo Olfatório/metabolismo , Bulbo Olfatório/patologia , Proteína 1 do Complexo Esclerose Tuberosa
7.
Neurosci Lett ; 612: 43-47, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26655465

RESUMO

Expression of hypoxia-inducible factor 1a (HIF1a) is increased under several pathological conditions such as hyperactive mechanistic target of rapamycin complex 1 (mTORC1) in tuberous sclerosis complex (TSC). Hyperactive mTORC1 and the resulting increased dendritic complexity of neurons are shared molecular and cellular alterations in several neurological disorders associated with cognitive disabilities. Despite some evidence that HIF1a contributes to dendritic overgrowth in vitro, it remains unknown whether increased HIF1a in TSC neurons could contribute to their increased dendritic complexity. To address this use in vivo, we generated TSC neurons by deleting Tsc1 in newborn olfactory bulb (OB) neurons of conditional Tsc1 transgenic mice using neonatal electroporation. In addition to their increased dendritic complexity, Tsc1(null) neurons have been reported to display increased Hif1a mRNA level and HIF1a transcriptional activity. We found that Tsc1(null)-dependent dendritic overgrowth was prevented by knocking down HIF1a or expressing a dominant negative HIF1a. In addition, overexpressing HIF1a in wild-type developing neurons resulted in increased dendritic complexity in vivo. These data highlight that an increase in HIF1a levels contributes to abnormal dendritic patterning in developing neurons under normal conditions and hyperactive mTORC1 conditions as in TSC.


Assuntos
Dendritos/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Esclerose Tuberosa/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Técnicas de Silenciamento de Genes , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos Transgênicos , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética
8.
Mol Med Rep ; 12(6): 8141-7, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26500043

RESUMO

The present study examined the association between microRNA (miR)­296 and angiogenesis following cerebral ischemic injury, and the underlying mechanisms. A cerebral ischemic model was established in rats via right middle cerebral artery occlusion. The animals were randomly divided into four groups (baseline, 1 day, 3 day and 7 day). Quantitative polymerase chain reaction and western blot analyses were performed to examine the expression levels of miR­296 and hepatocyte growth factor­regulated tyrosine kinase substrate (HGS), respectively. Angiogenesis was assessed by examining microvessel density. The results demonstrated that miR­296 and angiogenesis were significantly upregulated, while HGS was significantly downregulated following ischemic injury. Adenovirus­mediated overexpression of miR­296 markedly enhanced the formation of capillary­like structures in human umbilical vein endothelial cells, parallel with significantly increased expression levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2, and reduced expression levels of DLL4 and Notch1. The results of the present study provided in vivo and in vitro evidence suggesting that miR­296 promotes angiogenesis in the ischemic brain through upregulating VEGF and downregulating Notch1 following cerebral ischemic injury.


Assuntos
Isquemia Encefálica/genética , Regulação da Expressão Gênica , MicroRNAs/fisiologia , Receptor Notch1/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/metabolismo , Artérias Cerebrais/metabolismo , Regulação para Baixo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Neovascularização Patológica , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Notch1/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Hum Mol Genet ; 24(20): 5746-58, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26220974

RESUMO

Abnormal axonal connectivity and hyperactive mTOR complex 1 (mTORC1) are shared features of several neurological disorders. Hyperactive mTORC1 alters axon length and polarity of hippocampal neurons in vitro, but the impact of hyperactive mTORC1 on axon growth in vivo and the mechanisms underlying those effects remain unclear. Using in utero electroporation during corticogenesis, we show that increasing mTORC1 activity accelerates axon growth without multiple axon formation. This was prevented by counteracting mTORC1 signaling through p70S6Ks (S6K1/2) or eukaryotic initiation factor 4E-binding protein (4E-BP1/2), which both regulate translation. In addition to regulating translational targets, S6K1 indirectly signals through GSK3ß, a regulator of axogenesis. Although blocking GSK3ß activity did not alter axon growth under physiological conditions in vivo, blocking it using a dominant-negative mutant or lithium chloride prevented mTORC1-induced accelerated axon growth. These data reveal the contribution of translational and non-translational downstream effectors such as GSK3ß to abnormal axon growth in neurodevelopmental mTORopathies and open new therapeutic options for restoring long-range connectivity.


Assuntos
Axônios/fisiologia , Proteínas de Transporte/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Complexos Multiproteicos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Axônios/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Processos de Crescimento Celular , Fatores de Iniciação em Eucariotos , Feminino , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Fosfoproteínas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais
10.
Oncol Lett ; 10(5): 3183-3190, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26722309

RESUMO

MicroRNA-128 (miR-128) serves an important role in regulating growth, invasiveness, stem cell-like traits, differentiation and apoptosis of different types of tumor cells. Vascular endothelial growth factor-C (VEGF-C) has been associated with angiogenesis, lymphangiogenesis and regional lymph node metastasis and has previously been reported to have an anti-apoptotic and proliferative role in bladder cancer (BC). To investigate the regulation of miR-128 on VEGF-C expression and their effects on proliferation and metastasis of bladder cancer, T24 and 5637 BC cells were transfected with pre-miR-128, anti-miR-128 and their respective negative control. miR-128 was downregulated in BC tissues and cell lines, while the expression levels of VEGF-C were upregulated. The present results indicated that miR-128 negatively regulated VEGF-C expression in BC T24 and 5637 BC cells. VEGF-C is a direct target of miR-128 in BC cells. Overexpression of miR-128 inhibited cell proliferation, migration and invasion. Knockdown of miR-128 promoted proliferation, migration and invasion in BC cells. Therefore, downregulation mediated malignant progression of BC may be partly attributed to increased VEGF-C expression. Consequently, the findings of the present study provide a molecular basis for the role of miR-128/VEGF-C in the progression of human BC and indicate a novel target for treatment of BC.

11.
Oncol Lett ; 6(5): 1390-1396, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24179530

RESUMO

The present study examined the expression of podocalyxin (PODX) in surgically-resected astrocytomas, associated the levels of PODX expression with the clinicopathological characteristics and survival outcomes of astrocytoma and assessed how PODX affected the viability of astrocytoma cells following the administration of chemotherapeutic agents. The immunohistochemical analysis of 102 patient samples revealed that a high expression of PODX was significantly associated with high-grade astrocytomas (P<0.001) and a high Ki-67 labeling index (LI; P<0.001). A Kaplan-Meier survival analysis demonstrated that the high PODX expression group had significantly shorter disease-free survival (DFS) and overall survival (OS) rates compared with the low expression group (P<0.001). The multivariate analysis using the Cox's proportional hazards model revealed that a high expression of PODX, a high World Health Organization grade and a high Ki-67 LI were independent factors for shorter DFS and OS times. A subsequent in vitro study using SW1783 and U-87 human astrocytoma cell lines revealed that knocking down PODX decreased astrocytoma cell viability against temozolomide-induced apoptotic stress through the inhibition of the Akt survival signaling pathway. In conclusion, the in vivo findings indicated that a high expression of PODX is predictive of a poor survival outcome and, thus, may be used as a prognostic factor to predict the survival outcomes of astrocytoma patients. The in vitro findings indicated that PODX may promote astrocytoma cell viability against chemotherapeutic agent-induced apoptotic stress through the Akt pathway, indicating that PODX may be a novel target for overcoming chemoresistance in astrocytomas.

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