Erythropoietin promotes axonal growth in a model of neuronal polarization

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Abstract

Erythropoietin (EPO) enhances neurogenesis, neuroprotection and regeneration. Here, we examined the effects of EPO on axonal and dendritic growth in a model of neuronal polarization. EPO did not effect survival or the polarized morphology of hippocampal neurons but its effect on neurite outgrowth depended upon the stage of polarization. When added to neurons in the process of establishing polarity (0–2 days in vitro (DIV)), it enhanced axonal and dendritic growth, while EPO added to early polarized cultures at 3–4 DIV promoted the growth of axons but not dendrites. EPO stimulated the phosphorylation of Akt at serine-473 and co-incubation of the Akt/PI-3 kinase pathway inhibitor LY294002 with EPO abolished its effects on Akt phosphorylation and axonal growth. However, while Leukemia Inhibitory Factor (LIF) similarly stimulated phosphorylation of Akt, it had no effect on axonal or dendritic growth, indicating that AKT phosphorylation is necessary but not sufficient for neurite outgrowth in hippocampal neurons.

Introduction

The functional restoration of a compromised nervous system is dependent on the ability to re-establish disrupted neuronal networks. Neurogenesis and cytoprotection can contribute to this endeavor; however, the regeneration of injured neurons is also an important modality in the restitution of a damaged brain. Erythropoietin (EPO) has shown potential in treating brain pathologies through stimulating neurogenesis and providing neuroprotection (Noguchi et al., 2007). Interest in using hematopoietic cytokines to treat neurological disorders has arisen since the discovery of their intrinsic expression in the CNS (Tonges et al., 2007). Furthermore, such hematopoietic cytokines, which include EPO, Leukemia Inhibitory Factor (LIF), and Granulocyte-Colony Stimulating Factor, are attractive therapeutic options in the novel treatment of CNS pathologies as a consequence of their established clinical use (Hasselblatt et al., 2006).

Both EPO and its receptor (EPO-R) are endogenously expressed in the brain, play a role in embryonic brain development and can be induced by metabolic disturbances (Bernaudin et al., 2000, Bernaudin et al., 1999, Digicaylioglu et al., 1995, Lewczuk et al., 2000, Tsai et al., 2006, Yu et al., 2002). Aside from intrinsic expression, exogenously administered EPO affects nervous system function, transiently enhancing adult hippocampal neurogenesis under basal conditions (Ransome and Turnley, 2007) and demonstrating therapeutic efficacy in treating neonatal and adult models of stroke and brain trauma (Brines et al., 2000, Chang et al., 2005, Lu et al., 2005, Tsai et al., 2006, Wang et al., 2004a). These studies showed that the efficacy of EPO in restoring functional deficits after the brain insult was attributed in part to neuroprotection and enhanced neurogenesis. It can also enhance neurite outgrowth (Kretz et al., 2005) and axonal regeneration (King et al., 2007) in retinal ganglion cells following axotomy, although these effects are less well characterized.

LIF also has pleiotropic actions which extend to the CNS (Metcalf, 2003). Similar to EPO, intrinsic LIF signaling may play an important role during embryonic brain development (Li and Grumet, 2007). Furthermore, similar to the neuroprotective properties of EPO, the provision of exogenous LIF has also shown efficacy in attenuating the effects of ischemic brain injury (Suzuki et al., 2005) and promoting neurite outgrowth in some types of neurons, such as sensory and auditory neurons (Cafferty et al., 2001, Gillespie et al., 2001).

Similar to the animal model studies above, the use of cultured hippocampal neurons has shown that EPO treatment affords cytoprotection against toxic, hypoxic and excitotoxic insults (Lewczuk et al., 2000, Morishita et al., 1997, Viviani et al., 2005). Hippocampal neurons express LIF (Lemke et al., 1996), and neuronal expression of LIF can be up-regulated in response to ischemic environments (Suzuki et al., 2000). While neuroprotection might be common to both EPO and LIF, little is known regarding the ability of each cytokine to promote neurite outgrowth in hippocampal neurons. Cultured embryonic hippocampal neurons establish polarity in well characterized stages, allowing the study of axonal and dendritic growth in post-mitotic neurons (Bradke and Dotti, 2000b, Dotti et al., 1988). Over the first 48 h in culture, hippocampal neurons begin to extend several similar neurites before one undergoes rapid elongation in a process of axon selection. During the next 48 h the selected axon undergoes further growth, while the remaining putative dendrites experience a slower rate of development. This process is thought to depend on spatially localized phosphatidylinositol-3 kinase (PI-3 kinase)/Akt signaling (Schwamborn and Puschel, 2004, Yan et al., 2006). Activation of the PI-3 kinase/Akt pathway as well as the STAT3 pathway is reported to contribute to the ability of exogenous EPO to promote axon growth in retinal ganglion cells (Kretz et al., 2005). LIF can also stimulate PI-3 kinase/Akt and STAT3 signaling in neuronal cells (Magni et al., 2007) and combat the deleterious effects of perinatal brain hypoxia–ischemia (Covey and Levison, 2007), suggesting an overlap of function between LIF and EPO signaling. However, we have used the established model of neuronal polarization in the current study to investigate whether exogenous EPO and LIF harbor a similar capacity to promote axonal and dendritic growth in hippocampal neurons and found that only EPO could do so, indicating differential effects of LIF and EPO in a cell context dependent manner. Furthermore the effects of EPO on neurite outgrowth depended upon the stage of polarization, with promotion of both dendrites and axons prior to and during the initial stages of polarization and only effects on axon outgrowth post-polarization.

Section snippets

Characterization of neuronal cultures

Before investigating EPO's morphological and biochemical effects on hippocampal neurons we first sought to analyze the basic characteristics of our cultures. Neurons were treated with either vehicle or rhEPO for 24 and 48 h and stained with βIII tubulin and DAPI (Figs. 1A–D). We also used immunoprecipitation analysis with a polyclonal rabbit antibody that recognizes the EPO-R (Verdier et al., 2006) to ensure that the EPO-R was being expressed in our neurons 24 h after plating. We employed both

Discussion

EPO signaling is an integral component of the brain's intrinsic ability to combat metabolic disturbances associated with hypoxia–ischemia. Treatment with exogenous EPO can provide neuronal protection and improve functional recovery in rodent models of stroke and brain trauma. Additionally, EPO has a capacity to promote axonal regeneration in retinal ganglion cells. However, little is known about EPO's effects on neurite growth in other neuronal types in the CNS. In our current report we provide

Primary hippocampal neuron cultures

Neurons were derived from embryonic day 16 (E16) mouse hippocampi and cultured as previously described (Brewer et al., 1993, Meberg and Miller, 2003) with some modifications. Briefly, time-mated pregnant female C57BL/6 mice were sacrificed by asphyxiation and embryos removed into Hank's Balanced Salt Solution (HBSS, Invitrogen, Mount Waverley, Australia) on ice. Hippocampi were isolated in ice-cold HBSS-media containing 1% (vol/vol) Penicillin/Streptomycin (Invitrogen), 1% (vol/vol) GlutaMax

Acknowledgments

This work was supported by the National Health and Medical Research Council of Australia (project ID: 350227). MR is supported by an Australian Postgraduate Research Scholarship and AT is a NH&MRC Senior Research Fellow.

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