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Neuroglobin boosts axon regeneration during ischemic reperfusion via p38 binding and activation depending on oxygen signal.

ABSTRACT: Cerebral ischemia causes severe cell death or injury including axon breakdown or retraction in the brain. Axon regeneration is crucial for the functional recovery of injured neurons or brains after ischemia/reperfusion (I/R); however, this process has been proved extremely difficult in adult brains and there is still no effective therapy for it. Here we reported that neuroglobin (Ngb), a novel oxygen-binding or sensor protein existing predominantly in neurons or brains, functions as a driving factor for axon regeneration during I/R. Ngb was upregulated and accumulated in growth cones of ischemic neurons in primary cultures, rat, and human brains, correlating positively to the elevation of axon-regeneration markers GAP43, neurofilament-200, and Tau-1. Ngb overexpression promoted while Ngb knockdown suppressed axon regeneration as well as GAP43 expression in neurons during oxygen-glucose deprivation/reoxygenation (OGD/Re). By using specific pharmacological inhibitors, we identified p38 MAPK as the major downstream player of Ngb-induced axon regeneration during OGD/Re. Mechanistically, Ngb directly bound to and activated p38 in neurons upon OGD/Re. Serial truncation and point mutation of Ngb revealed that the 7-105 aa fragment of Ngb was required and the oxygen-binding site (His64) of Ngb was the major regulatory site for its p38 interaction/activation. Finally, administration of exogenous TAT-Ngb peptides significantly enhanced axon regeneration in cultured neurons upon OGD/Re. Taken together, Ngb promotes axon regeneration via O2-Ngb-p38-GAP43 signaling during I/R. This novel mechanism suggests potential therapeutic applications of Ngb for ischemic stroke and other related axonopathy.

SUBMITTER: Xiong XX

PROVIDER: S-EPMC5833339 | biostudies-other | 2018 Feb

REPOSITORIES: biostudies-other

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Project description:Houshiheisan, a classic prescription in traditional Chinese medicine, contains Flos Chrysanthemi, Radix Saposhnikoviae, Ramulus Cinnamomi, Rhizoma Chuanxiong, Radix et Rhizoma Asari, Radix Platycodonis, Rhizoma Atractylodis macrocephalae, Poria, Rhizoma Zingiberis, Radix Angelicae sinensis, Radix et Rhizoma Ginseng, Radix Scutellariae and Concha Ostreae. According to traditional Chinese medicine theory, Flos Chrysanthemi, Radix Saposhnikoviae, Ramulus Cinnamomi, Rhizoma Chuanxiong, Radix et Rhizoma Asari and Radix Platycodonis are wind-dispelling drugs; Rhizoma Atractylodis macrocephalae, Poria, Rhizoma Zingiberis, Radix Angelicae sinensis and Radix et Rhizoma Ginseng are deficiency-nourishing drugs. A large number of randomized controlled trials have shown that Houshiheisan is effective in treating stroke, but its mechanism of action is unknown. Axonal remodeling is an important mechanism in neural protection and regeneration. Therefore, this study explored the effect and mechanism of action of Houshiheisan on the repair of axons after cerebral ischemia. Rat models of focal cerebral ischemia were established by ligating the right middle cerebral artery. At 6 hours after model establishment, rats were intragastrically administered 10.5 g/kg Houshiheisan or 7.7 g/kg wind-dispelling drug or 2.59 g/kg deficiency-nourishing drug. These medicines were intragastrically administered as above every 24 hours for 7 consecutive days. Houshiheisan, and its wind-dispelling and deficiency-nourishing components reduced the neurological deficit score and ameliorated axon and neuron lesions after cerebral ischemia. Furthermore, Houshiheisan, and its wind-dispelling and deficiency-nourishing components decreased the expression of proteins that inhibit axonal remodeling: amyloid precursor protein, neurite outgrowth inhibitor protein A (Nogo-A), Rho family small GTPase A (RhoA) and Rho-associated kinase 2 (Rock2), and increased the expression of growth associated protein-43, microtubule-associated protein-2, netrin-1, Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42). The effect of Houshiheisan was stronger than wind-dispelling drugs or deficiency-nourishing drugs alone. In conclusion, Houshiheisan, and wind-dispelling and deficiency-nourishing drugs promote the repair of axons and nerve regeneration after cerebral ischemia through Nogo-A/RhoA/Rock2 and Netrin-1/Rac1/Cdc42 signaling pathways. These effects are strongest with Houshiheisan.

Project description:INTRODUCTION:Neonatal hypoxia-ischemic brain damage (HIBD) can lead to serious neuron damage and dysfunction, causing a significant worldwide health problem. bFGF as a protective reagent promotes neuron repair under hypoxia/ischemia (HI). However, how bFGF and downstream molecules were regulated in HI remains elusive. METHODS:We established an in vitro HI model by culturing primary cortical neurons and treated with oxygen-glucose deprivation (OGD). We suppressed the expression of bFGF by using siRNA (small interfering RNA) interference to detect the neuronal morphological changes by immunofluorescence staining. To determine the potential mechanisms regulated by bFGF, the change of downstream molecular including IL-1? was examined in bFGF knockdown condition. IL-1? knockout (KO) rats were generated using CRISPR/Cas9-mediated technologies. We used an accepted rat model of HI, to assess the effect of IL-1? deletion on disease outcomes and carried out analysis on the behavior, histological, cellular, and molecular level. RESULTS:We identified that OGD can induce endogenous expression of bFGF. Both OGD and knockdown of bFGF resulted in reduction of neuron numbers, enlarged cell body and shortened axon length. We found molecules closely related to bFGF, such as interleukin-1? (IL-1?). IL-1? was up-regulated after bFGF interference under OGD conditions, suggesting complex signaling between bFGF and OGD-mediated pathways. We found HI resulted in up-regulation of IL-1? mRNA in cortex and hippocampus. IL-1? KO rats markedly attenuated the impairment of long-term learning and memory induced by HI. Meanwhile, IL-1?-/- (KO, homozygous) group showed better neurite growth and less apoptosis in OGD model. Furthermore, serine/threonine protein kinase (AKT1) mRNA and protein expression was significantly up-regulated in IL-1? KO rats. CONCLUSIONS:We showed that IL-1?-mediated axon regeneration underlie the mechanism of bFGF for the treatment of HIBD in neonatal rats. Results from this study would provide insights and molecular basis for future therapeutics in treating HIBD.

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Neuroglobin boosts axon regeneration during ischemic reperfusion via p38 binding and activation depending on oxygen signal.

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