Exogenous CNTF stimulates axon regeneration of retinal ganglion cells partially via endogenous CNTF

https://doi.org/10.1016/j.mcn.2009.03.002Get rights and content

Abstract

Intravitreal injections of exogenous CNTF stimulate axon regeneration of RGCs in vivo. Nevertheless, controversy exists over the ability of exogenous CNTF to directly stimulate axon regeneration of mature RGCs. Here we demonstrate that CNTF potently stimulated axon outgrowth of mature RGCs in culture in a JAK/STAT3- and PI3K/AKT-signaling pathway-dependent fashion and stronger than oncomodulin. Additional cAMP elevation or inhibition of MAPK activity increased these effects. In vivo intravitreal injections of exogenous CNTF induced endogenous CNTF expression in astrocytes in a manner that depended on the MAPK/ERK-signaling pathway activation. Reduction of endogenous CNTF expression by MAPK/ERK pathway inhibitors or its absence in CNTF deficient mice markedly reduced the neurite growth-promoting effects of exogenous CNTF. These data demonstrate that CNTF is a potent axon growth-promoting factor for mature RGCs. However, exogenously applied CNTF stimulates RGCs in vivo partially indirectly via a mechanism that depends on astrocyte-derived CNTF.

Introduction

Mature mammals normally show only a transient local sprouting reaction of axotomized retinal ganglion cells (RGCs) and therefore fail to regenerate axons over long distances into the injured optic nerve. This failure is partially attributed to proteins associated with myelin of the CNS, inhibitory factors in the forming gliotic scar, and an insufficient intrinsic capability of mature neurons to regrow axons (Fischer et al., 2004b, Fournier et al., 2003, GrandPre et al., 2002, Rudge and Silver, 1990, Wang et al., 2002, Yiu and He, 2006). More than 90% of axotomized RGCs undergo apoptosis within a few days after axotomy, leaving only a small time window for therapeutic intervention aimed at stimulating axon regeneration (Berkelaar et al., 1994, Fischer et al., 2000). Intravitreal application of the cytokine ciliary neurotrophic factor (CNTF) in vivo has been shown to be neuroprotective to axotomized RGCs, and upon repeated administration, it stimulates axon regeneration through a peripheral nerve graft or even into the optic nerve (Cui and Harvey, 2000, Cui et al., 1999, Cui et al., 2003, Hu et al., 2007, Lingor et al., 2008, Mey and Thanos, 1993, Muller et al., 2007). Coadministering drugs that elevate intracellular cAMP increased the beneficial effects of CNTF in vivo (Cui et al., 2003, Muller et al., 2007). A continuous supply of CNTF to RGCs seems to be an even more efficient approach to protect RGCs from cell death and stimulate axon regeneration. For instance, continuously released CNTF from virally transfected RGCs protected up to 25% of RGCs from cell death 5 weeks after optic nerve injury, and it stimulated axon regeneration over long distances within the optic nerve, even reaching the optic chiasm (Leaver et al., 2006). Similarly, injecting lens-derived β/γ-crystallins into the vitreous body, releasing these proteins from the injured lens, or applying the yeast wall extract zymosan into the vitreous body results in an induction and release of CNTF from retinal astrocytes. These measures result in robust protection of RGCs from cell death and switch these neurons into an active regenerative state (Fischer et al., 2000, Fischer et al., 2004a, Fischer et al., 2008, Yin et al., 2003, Lorber et al., 2005, Lorber et al., 2008, Pernet and Di Polo, 2006). Nevertheless, although clearly shown for postnatal RGCs (Jo et al., 1999, Lingor et al., 2008, Lorber et al., 2002), controversy exists over the ability of endogenous or exogenous CNTF to directly stimulate axon regeneration of mature RGCs. Moreover, recent studies have proposed that CNTF is insufficient to directly stimulate axon growth of mature RGCs and that it exerts only weak effects when coapplied with elevators of cAMP (Cen et al., 2007, Cui et al., 2008, Yin et al., 2003, Yin et al., 2006). Furthermore, it was recently hypothesized that CNTF exerts its effects in vivo not directly but rather indirectly through an activation of macrophages (Cen et al., 2007).

Here, we show that CNTF directly and potently stimulates neurite outgrowth of mature RGCs in culture if applied at sufficiently high concentrations. Moreover, we analyze the contribution of different signaling pathways in this context and provide evidence that the major effects of intravitreally applied, exogenous CNTF in vivo are indirectly mediated by a mechanism that is dependent on an induction of endogenous CNTF expression in retinal glia.

Section snippets

CNTF potently stimulates neurite outgrowth of mature RGCs in culture

CNTF stimulates neurite outgrowth of cultured postnatal RGCs at low concentrations (10 ng/ml) when cAMP levels are elevated (Jo et al., 1999). However, at these concentrations the cytokine reportedly failed to stimulate neurite outgrowth of adult RGCs in dissociated cultures or retinal explants alone or only barely in the presence of cAMP elevators (Cen et al., 2007, Yin et al., 2003, Yin et al., 2006). These results led to the hypothesis that CNTF is not a potent axon growth-promoting factor

Discussion

The principal new findings of this study are: i) at sufficiently high concentrations CNTF potently stimulates neurite growth of adult RGCs in culture via the JAK/STAT3- and PI3K/AKT-signaling pathways, ii) reducing MAPK/ERK activity increases neurite outgrowth of mature RGCs in culture, iii) in vivo, intravitreally administered CNTF directly activates the JAK/STAT3-signaling pathway in RGCs, iv) intravitreal application of CNTF induces endogenous CNTF expression in retinal astrocytes in a

Animals, optic nerve surgery and intravitreal administration

Surgical procedures were approved by the local authorities (Regierungspräsidium Tübingen). Adult female Sprague–Dawley rats (weighing 200–230 g) were anesthetized by intraperitoneal injections of ketamine (60–80 mg/kg) and xylazine (10–15 mg/kg), and a 1–1.5 cm incision was made in the skin above the right orbit. The optic nerve was surgically exposed under an operating microscope, the dural sheath was longitudinally opened, and the nerve was completely cut 1 mm behind the eye, avoiding injury

Acknowledgments

We thank Prof. Rose John, University of Kiel, Germany for providing us with hyper-interleukine-6, Prof. Sendtner, University of Würzburg, Germany for providing us CNTF deficient mice and Anastasia Andreadaki for technical support. We thank Vetrivel Sengottuvel and Mareike Caesar for critical comments on the manuscript. This work was supported by the state of Baden-Württemberg.

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