NEP1-40 promotes myelin regeneration via upregulation of GAP-43 and MAP-2 expression after focal cerebral ischemia in rats

Axon regeneration after lesions to the central nervous system (CNS) is largely limited by the presence of growth inhibitory molecules expressed in myelin. Nogo-A is a principal inhibitor of neurite outgrowth, and blocking the activity of Nogo-A can induce axonal sprouting and functional recovery. However, there are limited data on the expression of Nogo-A after CNS lesions, and the mechanism underlying its influences on myelin growth remains unknown. The aim of the present study was to observe the time course of Nogo-A after cerebral ischemia/reperfusion in rats using immunohistochemistry and western blot techniques, and to test the effect of its inhibitor Nogo extracellular peptide 1–40 (NEP1-40) on neural plasticity proteins, growth-associated binding protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2), as a possible mechanism underlying myelin suppression. A classic model of middle cerebral artery occlusion (MCAO) was established in Sprague-Dawley rats, which were divided into three groups: i) MCAO model group; ii) MCAO + saline group; and iii) MCAO + NEP1-40 group. Rats of each group were divided into five subgroups by time points as follows: days 1, 3, 7, 14 and 28. Animals that only received sham operation were used as controls. The Nogo-A immunoreactivity was located primarily in the cytoplasm of oligodendrocytes. The number of Nogo-A immunoreactive cells significantly increased from day 1 to day 3 after MCAO, nearly returning to the control level at day 7, increased again at day 14 and decreased at day 28. Myelin basic protein (MBP) immunoreactivity in the ipsilateral striatum gradually decreased from day 1 to day 28 after ischemia, indicating myelin loss appeared at early time points and continuously advanced during ischemia. Then, intracerebroventricular infusion of NEP1-40, which is a Nogo-66 receptor antagonist peptide, was administered at days 1, 3 and 14 after MCAO. It was observed that GAP-43 considerably increased from day 1 to day 7 and then decreased to a baseline level at day 28 compared with the control. MAP-2 expression across days 1–28 significantly decreased after MCAO. Administration of NEP1-40 attenuated the reduction of MBP, and upregulated GAP-43 and MAP-2 expression at the corresponding time points after MCAO compared with the MCAO + saline group. The present results indicated that NEP1-40 ameliorated myelin damage and promoted regeneration by upregulating the expression of GAP-43 and MAP-2 related to neuronal and axonal plasticity, which may aid with the identification of a novel molecular mechanism of restriction in CNS regeneration mediated by Nogo-A after ischemia in rats.

[1]  Shu Yang,et al.  NEP1-40 alleviates behavioral phenotypes and promote oligodendrocyte progenitor cell differentiation in the hippocampus of cuprizone-induced demyelination mouse model , 2020, Neuroscience Letters.

[2]  Hong Zhao,et al.  Effects of the transcription factor Olig1 on the differentiation and remyelination of oligodendrocyte precursor cells after focal cerebral ischemia in rats , 2019, Molecular medicine reports.

[3]  Ping Xu,et al.  Changes and Significance of SYP and GAP-43 Expression in the Hippocampus of CIH Rats , 2019, International journal of medical sciences.

[4]  Anthony G. Boghdadi,et al.  The Involvement of the Myelin-Associated Inhibitors and Their Receptors in CNS Plasticity and Injury , 2017, Molecular Neurobiology.

[5]  M. Gutiérrez-Fernández,et al.  NogoA Neutralization Promotes Axonal Restoration After White Matter Injury In Subcortical Stroke , 2017, Scientific Reports.

[6]  Lidian Chen,et al.  Electroacupuncture promotes axonal regeneration in rats with focal cerebral ischemia through the downregulation of Nogo-A/NgR/RhoA/ROCK signaling , 2017, Experimental and therapeutic medicine.

[7]  B. Topçu,et al.  The relationship between changes in the expression of growth associated protein-43 and functional recovery of the injured inferior alveolar nerve following transection without repair in adult rats. , 2015, Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-Maxillo-Facial Surgery.

[8]  J. Kriz,et al.  Increased expression and colocalization of GAP43 and CASP3 after brain ischemic lesion in mouse , 2015, Neuroscience Letters.

[9]  R. Giger,et al.  Insights into the physiological role of CNS regeneration inhibitors , 2015, Front. Mol. Neurosci..

[10]  Y. Huang,et al.  Endogenous Endothelial Progenitor Cells Participate in Neovascularization via CXCR4/SDF‐1 axis and Improve Outcome After Stroke , 2014, CNS neuroscience & therapeutics.

[11]  N. Alkayed,et al.  Delayed administration of a PTEN inhibitor BPV improves functional recovery after experimental stroke , 2013, Neuroscience.

[12]  X. Ma,et al.  Neuroprotective effects of NEP1-40 and fasudil on Nogo-A expression in neonatal rats with hypoxic-ischemic brain damage. , 2011, Genetics and molecular research : GMR.

[13]  F. Llorens,et al.  Emerging functions of myelin‐associated proteins during development, neuronal plasticity, and neurodegeneration , 2011, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[14]  M. Murray,et al.  Nogo-66 Receptor Antagonist Peptide (NEP1-40) Administration Promotes Functional Recovery and Axonal Growth After Lateral Funiculus Injury in the Adult Rat , 2008, Neurorehabilitation and neural repair.

[15]  G. Kartje,et al.  Neuronal Plasticity and Functional Recovery After Ischemic Stroke , 2008, Topics in stroke rehabilitation.

[16]  W. Kamphuis,et al.  GAP-43 expression is upregulated in retinal ganglion cells after ischemia/reperfusion-induced damage. , 2007, Experimental eye research.

[17]  E. Irving,et al.  Time-dependent increase in Nogo-A expression after focal cerebral ischemia in marmoset monkeys , 2006, Neuroscience Letters.

[18]  Xiao-Ming Xu,et al.  Temporal and spatial distribution of growth‐associated molecules and astroglial cells in the rat corticospinal tract during development , 2005, Journal of neuroscience research.

[19]  S. Halpain,et al.  The MAP2/Tau family of microtubule-associated proteins , 2004, Genome Biology.

[20]  Jisheng Han,et al.  Hyperpolarization‐activated, cyclic nucleotide‐gated cation channels: Roles in the differential electrophysiological properties of rat primary afferent neurons , 2004, Journal of neuroscience research.

[21]  M. Filbin,et al.  Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS , 2003, Nature Reviews Neuroscience.

[22]  John H. Zhang,et al.  HBO suppresses Nogo-A, Ng-R, or RhoA expression in the cerebral cortex after global ischemia. , 2003, Biochemical and biophysical research communications.

[23]  Ying Wang,et al.  Intraneuronal localization of Nogo‐A in the rat , 2003, The Journal of comparative neurology.

[24]  C. Bandtlow,et al.  Nogo-A and Myelin-Associated Glycoprotein Mediate Neurite Growth Inhibition by Antagonistic Regulation of RhoA and Rac1 , 2002, The Journal of Neuroscience.

[25]  B. Tang,et al.  Nogo-A expression in mouse central nervous system neurons , 2002, Neuroscience Letters.

[26]  M. Schwab,et al.  Patterns of Nogo mRNA and Protein Expression in the Developing and Adult Rat and After CNS Lesions , 2002, The Journal of Neuroscience.

[27]  E. Lehrmann,et al.  Focal cerebral ischemia induces increased myelin basic protein and growth-associated protein-43 gene transcription in peri-infarct areas in the rat brain , 2001, Experimental Brain Research.

[28]  A. Fournier,et al.  Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration , 2001, Nature.

[29]  R. Raghupathi,et al.  Bilateral growth‐related protein expression suggests a transient increase in regenerative potential following brain trauma , 2000, The Journal of comparative neurology.

[30]  P. Caroni,et al.  Shared and Unique Roles of Cap23 and Gap43 in Actin Regulation, Neurite Outgrowth, and Anatomical Plasticity , 2000, The Journal of cell biology.

[31]  Martin E. Schwab,et al.  Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1 , 2000, Nature.

[32]  C. Hulsebosch,et al.  Traumatic brain injury in rats results in increased expression of Gap-43 that correlates with behavioral recovery , 1998, Neuroscience Letters.

[33]  M. Chopp,et al.  Neuronal damage and plasticity identified by microtubule-associated protein 2, growth-associated protein 43, and cyclin D1 immunoreactivity after focal cerebral ischemia in rats. , 1998, Stroke.

[34]  R. Schmidt-Kastner,et al.  Transient expression of GAP-43 within the hippocampus after global brain ischemia in rat , 1997, Cell and Tissue Research.

[35]  Aryeh Routtenberg,et al.  GAP-43: an intrinsic determinant of neuronal development and plasticity , 1997, Trends in Neurosciences.

[36]  J. Hallenbeck,et al.  Acute Focal Ischemia-Induced Alterations in MAP2 Immunostaining: Description of Temporal Changes and Utilization as a Marker for Volumetric Assessment of Acute Brain Injury , 1996, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[37]  J. Kapfhammer,et al.  Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice , 1995, Cell.

[38]  R. Lin,et al.  Microtubule‐Associated Protein 2 as an Early Indicator of Ischemia‐Induced Neurodegeneration in the Gerbil Forebrain , 1994, Journal of neurochemistry.

[39]  R. Jope,et al.  The role of microtubule‐associated protein 2 (MAP‐2) in neuronal growth, plasticity, and degeneration , 1992, Journal of neuroscience research.

[40]  Fred H. Gage,et al.  Reactive synaptogenesis assessed by synaptophysin immunoreactivity is associated with GAP-43 in the dentate gyrus of the adult rat , 1991, Experimental Neurology.

[41]  K. Mikoshiba,et al.  Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage—Immunohistochemical investigation of dendritic damage , 1989, Neuroscience.

[42]  G. Wiche.  High-Mr microtubule-associated proteins: properties and functions. , 1989, The Biochemical journal.

[43]  E. Nedivi,et al.  A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS , 1986, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[44]  M. Zimmermann,et al.  Ethical guidelines for investigations of experimental pain in conscious animals , 1983, Pain.

[45]  B. Kolb,et al.  Cerebral Cortex doi:10.1093/cercor/bhi132 Dendritic Plasticity in the Adult Rat Following Middle Cerebral Artery , 2005 .

[46]  F. Silverstein,et al.  Hypoxic-ischemic injury acutely disrupts microtubule-associated protein 2 immunostaining in neonatal rat brain. , 1996, Biology of the neonate.

[47]  J. Rosenstein.  Diminished expression of microtubule-associated protein (MAP-2) and beta-tubulin as a putative marker for ischemic injury in neocortical transplants. , 1995, Cell transplantation.

[48]  P. Weinstein,et al.  Reversible middle cerebral artery occlusion without craniectomy in rats. , 1989, Stroke.