Neurite Outgrowth Inhibitor (NogoA) Is Upregulated in White Matter Lesions of Complex Cortical Malformations.

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.

Exercise modulates the levels of growth inhibitor genes before and after multiple sclerosis.

Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system. The animal model of MS, experimental autoimmune encephalomyelitis (EAE), is commonly used for studies of human inflammatory demyelinating diseases and has been shown to be suitable for studying the effects of exercise on MS pathophysiology. The present study was conducted to determine the impact of forced swimming and voluntary running wheel exercises before and after the induction of EAE on expression of Nogo-A, NgR, and ROCK genes in the brain tissue. A total of 96 C57BL/6 mice were randomly divided into two groups, namely exercises before (EXb, n = 48) and after (EXa, n = 48) induction of EAE. Each group was divided into four subgroups: Forced Swimming + EAE (n = 12), Voluntary Running Wheel + EAE (n = 12), NoEX-EAE (n = 12), and Control group (n = 12). Animals performed either swimming exercise for 30 min per day or running wheel for one hour per day, five days per week for four weeks. Results of Luxal Fast Blue (LFB) staining demonstrated that the degree of demyelination was significantly less in the experimental exercised compared to NoEX-EAE groups (P < .05). Amazingly, both modes of exercise reduced the severity of MS symptoms in mice exposed to swimming and wheel running, evaluated as body weight, clinical scores, degree of demyelination, and gene expressions, regardless of whether the exercise was performed before or after EAE induction.

Anti­Nogo­A antibody promotes brain function recovery after cardiopulmonary resuscitation in rats by reducing apoptosis.

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.

Nogo-A/Pir-B/TrkB Signaling Pathway Activation Inhibits Neuronal Survival and Axonal Regeneration After Experimental Intracerebral Hemorrhage in Rats.

Intracerebral hemorrhage (ICH) leads to widespread pathological lesions in the brain, especially impacting neuronal survival and axonal regeneration. This study aimed to elucidate whether the Nogo-A (a myelin-related protein)/paired immunoglobulin-like receptor B (Pir-B)/tropomyosin receptor kinase B (TrkB) pathway could exert a regulatory effect in ICH. An ICH model was first established in Sprague Dawley rats, followed by different administrations of vehicle, k252a, or NSC 87877. The Morris water maze test was performed to observe ICH-induced cognitive dysfunction in rats. Rats in the ICH + NSC 87877 group showed better cognitive performance compared with those injected with vehicle or k252a. Neurobehavioral scores were identical. By harvesting brain tissues at different time points after ICH, we detected the expression levels of Nogo-A and PirB with western blot and immunofluorescence and found that they were markedly upregulated at 48 h after ICH. TUNEL and Fluoro-Jade B staining showed that NSC 87877 treatment attenuated ICH-induced apoptosis and neuronal death, whereas k252a treatment aggravated these pathological changes. The expression levels of growth-associated protein 43 (GAP43) and neurofilament 200 (NF200) were higher in the ICH + NSC 87877 group compared with the ICH + vehicle group, but were lower in the ICH + k252a group. Finally, we confirmed the protective role of p-TrkB/TrkB in ICH by western blot. To sum up, our study identified the inhibitory role of the Nogo-A/PirB/TrkB pathway in ICH; however, p-TrkB/TrkB may serve as a potential target for secondary brain injury post-ICH.

RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.

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.

Developmental Wiring of Specific Neurons Is Regulated by RET-1/Nogo-A in Caenorhabditis elegans.

Nogo-A is a membrane-bound protein that functions to inhibit neuronal migration, adhesion, and neurite outgrowth during development. In the mature nervous system, Nogo-A stabilizes neuronal wiring to inhibit neuronal plasticity and regeneration after injury. Here, we show that RET-1, the sole Nogo-A homolog in Caenorhabditis elegans, is required to control developmental wiring of a specific subset of neurons. In ret-1 deletion mutant animals, specific ventral nerve cord axons are misguided where they fail to respect the ventral midline boundary. We found that ret-1 is expressed in multiple neurons during development, and, through mosaic analysis, showed that ret-1 controls axon guidance in a cell-autonomous manner. Finally, as in mammals, ret-1 regulates ephrin expression, and dysregulation of the ephrin ligand VAB-2 is partially responsible for the ret-1 mutant axonal defects. Together, our data present a previously unidentified function for RET-1 in the nervous system of C. elegans.

Cornel Iridoid Glycoside Improves Locomotor Impairment and Decreases Spinal Cord Damage in Rats.

Purpose. This study was to investigate the effects of cornel iridoid glycoside (CIG) on spinal cord injury (SCI) in rats. Methods. The thoracic cord (at T9) of rats was injured by clip compression for 30 sec. Locomotor function was assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale. Neuroanatomic stereological parameters as well as Nogo-A, p75 neurotrophin receptor (p75NTR), and ROCKII expression were measured by histological processing, immunohistochemistry, and stereological analyses. The axons passing through the lesion site were detected by BDA tracing. Results. Intragastric administration of CIG (60 and 180 mg/kg) improved the locomotor impairment at 10, 17, 24, and 31 days post-injury (dpi) compared with untreated SCI model rats. CIG treatment decreased the volume of the lesion epicenter (LEp) and increased the volume of spared tissue and the number of surviving neurons in the injured spinal cord at 31 dpi. CIG promoted the growth of BDA-positive axons and their passage through the lesion site and decreased the expression of Nogo-A, p75NTR, and ROCKII both in and around the LEp. Conclusion. CIG improved the locomotor impairment, decreased tissue damage, and downregulated the myelin-associated inhibition signaling pathway in SCI rats. The results suggest that CIG may be beneficial for SCI therapy.

Knockdown of Nogo gene by short hairpin RNA interference promotes functional recovery of spinal cord injury in a rat model.

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.

Isoform-specific localization of Nogo protein in the optic pathway of mouse embryos.

Expression of Nogo protein was investigated in the optic pathway of embryonic mice by using isoform-specific antibodies Bianca and 11C7, which recognize Nogo-A/B and Nogo-A, respectively. Our previous reports from using antibody N18 have shown that Nogo is localized on the radial glia in the retina and at the midline of the ventral diencephalon in mouse embryos during the ingrowth of retinal ganglion cells (RGCs) axons. This glial-specific localization is markedly different from findings in other studies. This study showed Nogo-A/B primarily on radial glia in the retina at E13 and then later on retinal ganglion cells and axons at E14 and E15, whereas Nogo-A was expressed preferentially by RGCs and their axons. In the ventral diencephalon, Nogo-A/B was expressed strongly on radial glia, particularly in those located in the midline region of the chiasm but also on RGC axons. In Nogo-A knockout embryos, the isoform Nogo-B (revealed by Bianca) was observed on radial glia in the ventral diencephalon and on RGCs and their axons. We concluded that Nogo-A is localized on the ganglion cells and retinal axons, whereas Nogo-B is expressed by the radial glia in the optic pathway. Nogo-B may play an important role in guiding axon growth in decisive regions of the visual pathway, which include the optic disc and the optic chiasm. J. Comp. Neurol. 524:2322-2334, 2016. © 2016 Wiley Periodicals, Inc.