EPO Promotes Axonal Sprouting Via Upregulating GDF10

Objective:Erythropoietin(EPO)has the exact neuroprotective effect of stroke without knowing whether it is involved in axonal sprouting after neuronal damage.Growth and differentiation factor 10(GDF10)has been shown to be a trigger for sudden sprouting in the posterior axis of stroke.We hypothesized that EPO promotes axial sprouting mainly through GDF10,and EPO stimulates GDF10 production through the JAK2-PI3K-NF-κB pathway.And verify the conjecture through experiments.Methods:Neonatal mice were selected within 48 hours of birth to prepare primary cortical neuron medium.Neurons were treated with different concentrations of EPO,and the expression level of GDF10 was observed to correspond to the length of axons.Antagonize signaling molecules with different signal antagonists to determine EPO-regulated GDF10 signaling pathway.Results:In the present in vitro experiment,we found that EPO could promote axonal sprouting and expression of GDF10 in a dose–dependent manner.Knockdown of GDF10 using siRNA abolished the effect of EPO-mediated axonal sprouting,which indicates GDF10 is the executor of EPO-mediated axonal sprouting.Treatment of neuron with nuclear factor-kappaB(NF-κB)inhibitor JSH-23 could inhibit the accumulation of NF-κB phospho-p65(p-p65)in nucleus,upregualtion of GDF10 and extending of axonal length.Moreover,addition of Janus kinase 2(JAK2)inhibitor CEP-33779 or phosphoinositide 3-kinase(PI3K)inhibitor LY294002 into culture medium also blocked the nuclear translocation of p-p65,expression of GDF10 and axonal sprouting,which suggests that EPO induces axonal sprouting via activating cellular JAK2 and PI3 K signaling.When impeding the JAK2 signaling with CEP-33779 could suppress the phosphorylation of PI3 K,which confirms that the upstream of PI3 K signaling is JAK2.Our results provide a novel insight into the role of the EPO and the molecular mechanism of axonal sprouting,which is beneficial to develop novel approaches for neurological recovery after brain injury including stroke.Conclusions:This study firstly report that EPO promotes axonal sprouting through upregulating the expression of GDF10 in a dose-dependent manner.EPO activates JAK2-PI3K-NF-κB signaling pathway and leads to upregulate the level of GDF10 in neurons,which presents a novel approach for axonal sprouting.