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.
Highlights
Ischemic stroke is the most common disease causing disability in the elderly
Representative micrographs showed that Ngb was accumulated in neurites of cortical neurons in the ipsilateral ischemic penumbra (Ipsi) of mouse brain after I/R but not in its Contra or sham controls. e Fluorescent double-staining of Ngb and NF200 in mouse brain tissues after I/R
Western blots showed that axon growth marker growth associated protein-43 (GAP43) was initially decreased within 24 h of reperfusion after 1 h of transient middle cerebral artery occlusion and upregulated at I/R-2, −3 and −7 days (Fig. 1b), reflecting a continuum of axon injury-regeneration process after I/ R
Summary
Ischemic stroke is the most common disease causing disability in the elderly. Neurite or axon damage includesOfficial journal of the Cell Death Differentiation AssociationXiong et al Cell Death and Disease (2018)9:163(see figure on previous page) Fig. 1 Upregulation and accumulation of Ngb in ischemic neurons are associated with axon regeneration during ischemic reperfusion. a Representative photograph showing the ipsilateral ischemic penumbra (Ipsi) and its contralateral counterpart (Contra) (indicated by squares) in the ischemic mouse brain (TTC staining) after I/R (tMCAo-1 h/reperfusion-24 h). b Western blotting analysis of Ngb and GAP43 in the mouse brain after I/ R. Brain slices from sham and I/R-2 day mouse brains were stained with anti-Ngb antibodies (IHC). Representative micrographs showed that Ngb was accumulated in neurites of cortical neurons in the Ipsi of mouse brain (indicated by arrows, right panel) after I/R but not in its Contra or sham controls. Representative micrographs showed that Ngb was well co-localized with NF200 in neurons in the Ipsi of mouse brains subjected to I/R-2 days (indicated by arrows, lower panels). Human brain slices (cerebral cortices) from three cerebral stroke brains and three age-matched normal brains were subjected to IHC analysis of Ngb. Representative micrographs showed that Ngb was upregulated and accumulated in neurite growth cones in the ischemic human brains (indicated by arrowheads) axonal regeneration, is extremely difficult in the adult brain[4]
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