RESUMO
Complex cortical malformations (CCMs), such as hemimegalencephaly and polymicrogyria, are associated with drug-resistant epilepsy and developmental impairment. They share certain neuropathological characteristics including mammalian target of rapamycin (mTOR) activation and an atypical number of white matter neurons. To get a better understanding of the pathobiology of the lesion architecture, we investigated the role of neurite outgrowth inhibitor A (NogoA), a known regulator of neuronal migration. Epilepsy surgery specimens from 16 CCM patients were analyzed and compared with sections of focal cortical dysplasia IIB (FCD IIB, n = 22), tuberous sclerosis complex (TSC, n = 8) as well as healthy controls (n = 15). Immunohistochemistry was used to characterize NogoA, myelination, and mTOR signaling. Digital slides were evaluated automatically with ImageJ. NogoA staining showed a significantly higher expression within the white matter of CCM and FCD IIB, whereas cortical tubers presented levels similar to controls. Further analysis of possible associations of NogoA with other factors revealed a positive correlation with mTOR and seizure frequency. To identify the main expressing NogoA cell type, double staining revealed dysmorphic neuronal white matter cells. Increased NogoA expression is associated with profound inhibition of neuritic sprouting and therefore contributes to a decrease in neuronal network complexity in CCM patients.
Assuntos
Malformações do Desenvolvimento Cortical/metabolismo , Malformações do Desenvolvimento Cortical/patologia , Proteínas Nogo/biossíntese , Regulação para Cima/fisiologia , Substância Branca/metabolismo , Substância Branca/patologia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , MasculinoRESUMO
Brain injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) is the main cause of neurological dysfunction and death in cardiac arrest. To assess the effect of NogoA antibody on brain function in rats following CPR and to explore the underlying mechanisms, CA/CPR (ventricular fibrillation) rats were divided into the CPR+NogoA, CPR+saline and sham groups. Hippocampal caspase3 levels were detected by RTPCR and immunoblotting. Next, NogoA, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cysteinyl aspartate specific proteinase12 (casapse12), Bcl2 and Bax protein levels in the hippocampus were detected by immunoblotting. Coronal brain sections were analyzed by TUNEL assay to detect apoptosis at 72 h, while Nissl staining and electron microscopy were performed to detect Nissl bodies and microstructure at 24 h, respectively. Finally, rats were assessed for neurologic deficits at various times. Nissl staining revealed morphological improvement after NogoA antibody treatment. Suborganelle structure was preserved as assessed by electron microscopy in model animals postantibody treatment; neurological function was improved as well (P<0.05), while the apoptosis index was decreased (26.2±9.85 vs. 46.6±12.95%; P<0.05). Hippocampal caspase3 mRNA and protein, NogoA protein levels were significantly decreased after antibody treatment (P<0.05). Hippocampal NogoA expression was positively correlated with caspase3 (Pearson's correlation; r=0.790, P=0.000). Hippocampal GRP78 and Bcl2 protein levels were higher after antibody treatment than these levels noted in the model animals (P<0.05), while CHOP, caspase12 and Bax levels were reduced (P<0.05). NogoA antibody ameliorates neurological function after restoration of spontaneous circulation (ROSC), possibly by suppressing apoptosis induced by endoplasmic reticulum stress.
Assuntos
Anticorpos/farmacologia , Apoptose , Reanimação Cardiopulmonar , Parada Cardíaca/metabolismo , Hipocampo/metabolismo , Proteínas Nogo/antagonistas & inibidores , Recuperação de Função Fisiológica , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Parada Cardíaca/patologia , Parada Cardíaca/terapia , Hipocampo/patologia , Masculino , Proteínas Nogo/biossíntese , Ratos , Ratos WistarRESUMO
Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.
Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nogo/biossíntese , Osteonectina/biossíntese , Biossíntese de Proteínas , Substância Branca/patologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Ligação Competitiva , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Proteínas Nogo/genética , Osteonectina/genética , Domínios Proteicos , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Receptores de Esfingosina-1-Fosfato/fisiologia , Células Tumorais Cultivadas , Substância Branca/metabolismoRESUMO
The specific myelin component Nogo protein is one of the major inhibitory molecules of spinal cord axonal outgrowth following spinal cord injury. The present study aimed to investigate the effects of silencing Nogo protein with shRNA interference on the promotion of functional recovery in a rat model with spinal cord hemisection. Nogo-A short hairpin RNAs (Nogo shRNAs) were constructed and transfected into rats with spinal cord hemisection by adenovirus-mediated transfection. Reverse transcriptionpolymerase chain reaction and western blotting were performed to analyze the expression of Nogo-A and Growth Associated Protein 43 (GAP-43). In addition, Basso Beattie Bresnahan (BBB) scores were used to assess the functional recovery of rats following spinal cord injury. The results demonstrated that expression of the NogoA gene was observed to be downregulated following transfection and GAP43 expression was observed to increase. The BBB scores were increased following treatment with Nogo shRNAs, indicating functional recovery of the injured nerves. Thus, Nogo-A shRNA interference can knockdown Nogo gene expression and upregulate GAP-43 to promote the functional recovery of spinal cord injury in rats. This finding may advance progress toward assisting the regeneration of injured neurons through the use of Nogo-A shRNA.