Nerve Growth Factor Modulation of Retinal Ganglion Cell Physiology
GLORIA ROBERTI,1* FLAVIO MANTELLI,1 ILARIA MACCHI,2 MINA MASSARO-GIORDANO,3 AND MARCO CENTOFANTI1,4 1
IRCCS Fondazione GB Bietti, Rome, Italy
Department of Ophthalmology, Campus Bio-Medico University of Rome, Rome, Italy
Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
DSCMT University of Rome Tor Vergata, Rome, Italy
Nerve growth factor (NGF) is an endogenous neurotrophin involved in the development, maintenance and regeneration of mammalian sympathetic and sensory neurons. Additionally, NGF is known to have trophic and differentiating activity on several populations of cholinergic neurons of the central nervous system (CNS), and to act as a differentiation factor in the development of the visual cortex. The paramount functions of NGF in the visual system are also highlighted by the presence of this neurotrophin and both its receptors TrkA and p75 in most intra-ocular tissues, including lens, vitreous, choroid, iris, and trabecular meshwork. In the retina, NGF is produced and utilized speciﬁcally by retinal ganglion cells (RGC), bipolar neurons and glial cells, and is thought to have crucial protective effects in several disease states. Studies on the role of NGF on RGCs survival following optic nerve transection, ischemic injury, ocular hypertension and glaucoma are discussed in this review. J. Cell. Physiol. 229: 1130–1133, 2014. © 2014 Wiley Periodicals, Inc.
Nerve growth factor (NGF) is the ﬁrst and most studied member of the neurotrophin family. It was discovered in 1953 by Rita Levi-Montalcini in the Victor Hamburger laboratory at Washington University (St. Louis, MA), by grafting a piece of mouse sarcoma tissue onto chick embryos whose wing buds had been extirpated. She discovered that the tumor tissue produced a soluble factor that promoted the growth of nearby sensory and sympathetic ganglia. Collaborating with the biochemist Stanley Cohen, they isolated the substance responsible for this ganglia overgrowth and named it NGF (Levi-Montalcini, 1987). NGF is a pleiotropic factor that extends its biological activity from the central and peripheral nervous systems to the immune, endocrine, and visual systems (Aloe et al., 1997). Signals mediated by NGF are propagated by two distinct receptors: the high-afﬁnity receptor TrkA (tropomyosinrelated kinase receptor A, known as tyrosine kinase receptor A), and the low afﬁnity receptor p75 (pan) neurotrophin receptor (Friedman and Greene, 1999). The duration and magnitude of NGF signaling depends on the ratio of TrkA and p75 co-distributed on cell surface. As anticipated, NGF plays a pivotal role in the development, maintenance, and regeneration of mammalian sympathetic and sensory neurons of the peripheral nervous system (PNS). It has also been suggested to play a speciﬁc role for several populations of cholinergic neurons of the central nervous system (CNS), both during development and in adulthood. These NGF-sensitive PNS and CNS neurons express both the high- and low-afﬁnity NGF receptors. Functionally, signaling by TrkA and p75 may be synergistic, independent, or antagonistic. Interestingly, these opposing NGF effects, ranging from cell growth, differentiation, survival and cell death, are apparently not shared by other neurotrophins. In most cases, TrkA expression exerts protective actions on diseases involving degeneration of NGF target cells, while the NGF precursor proNGF induces apoptosis through p75 (Friedman and Greene, 1999; Klesse and Parada, 1999; Huang and Reichardt, 2003; Fahnestock et al., 2004; Chao et al., 2006; Reichardt, 2006). © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .
The action of NGF seems to be dependent on a receptorcoupling event, followed by the internalization of the NGF– NGF receptor complex and its retrograde axonal transport (Johnson et al., 1987). NGF may affect a variety of additional CNS neurons since NGF binding sites are present early in development in many other neuronal systems, including the visual system (Vantini et al., 1989; Yan and Johnson, 1988, 1989). Under normal conditions, NGF and TrkA are expressed in the anterior segment of the eye (iris, ciliary body, lens, cornea, and conjunctiva), and NGF is released into the aqueous humor (Lambiase et al., 2002). In the retina NGF is produced and utilized by retinal ganglion cells (RGCs), bipolar neurons and glial cells, in a local paracrine/autocrine fashion. During visual system development, NGF, TrkA, and p75, as well as other neurotrophins and their related receptors, are highly expressed in numerous visual centers, from the retina to the visual cortex, where NGF inﬂuences neuronal outgrowth, survival, and selective apoptosis. Studies investigating the role of NGF in RGC survival after optic nerve transection, ischemic injury, ocular hypertension and glaucoma (summarized in Table 1) are discussed in this review.
*Correspondence to: Gloria Roberti, IRCCS-Fondazione GB Bietti, Rome, Italy. E-mail: [email protected]
Manuscript Received: 30 January 2014 Manuscript Accepted: 3 February 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 6 February 2014. DOI: 10.1002/jcp.24573
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TABLE 1. Studies investigating the role of nerve growth factor in retinal ganglion cell survival Topic Optic nerve transection
Ocular hypertension Glaucoma
Study design Animal models: - Severing of the orbital portion of the left optic nerve; - intracranial transection of the optic nerve Animal models: - Block of the choroidal and retinal circulations due to induced ocular hypertension; - bilateral carotid occlusion Animal models: - Ocular hypertension induced by intra-anterior chamber injection of methylcellulose Animal models: - Ocular hypertension induced by hypertonic saline injection into the episcleral vein; - Exploratory study in 3 advanced glaucoma patients
RGCs surviving after axotomy
Turner and Delaney (1979), Carmignoto et al. (1989)
Electroretinogram and visual evoked potentials responses; morphological and molecular changes in the retina and optic nerve
Siliprandi et al. (1993), Savilia et al. (2009)
Protective effect of exogenous and endogenous NGF on RGCs
Lambiase et al. (1997)
Role of NGF in VC, LGN and CSF; prevention of RGCs degeneration and evaluation of visual field, optic nerve function, contrast sensitivity, and visual acuity
Sposato et al. (2009), Lambiase et al. (2009)
RGCs, retinal ganglion cells; NGF, nerve growth factor; VC, visual cortex; LGN, lateral geniculate nucleus; CSF, cerebrospinal ﬂuid.
Optic Nerve Transection
The regenerative potential of NGF in the CNS has been suggested by its stimulatory effects on the regeneration of axons in the rat dorsal catecholamine bundle, in rabbit vestibular neurons, in vitro, and in the dorsal funiculus of young kittens (Bjerre et al., 1973, 1974). In the 1970s, studies from Turner et al. also demonstrated a pronounced effect of NGF and its antiserum on optic nerve regeneration in the neuron (Turner and Glaze, 1977; Turner et al., 1978). Light and electron microscopic analyses indicated that NGF treatment, given as a single 200 BU (i.e., 400 ng) intraocular injection at the time of optic nerve transection, signiﬁcantly accelerates the retinal ganglion cell response to axotomy (Turner and Delaney, 1979). Speciﬁcally, the authors demonstrated that NGF administration can ilicite a number of retinal ganglion cell organelle changes a week earlier than it would normally occur. The earliest changes detected following NGF injection were nuclear chromatin and nucleolar alterations. It is possible that these observations reﬂect the ability of exogenous NGF to stimulate a DNA dependent RNA synthesis in the regenerating retinal ganglion cells. Cellular and nuclear hypertrophy, as well as dramatic increases in Golgi ﬁeld densities and in unbound ribosomes in response to NGF administration have also been reported. In the 1980s, Carmignoto et al. (1989) investigated the effect of repetitive intraocular injections of NGF on the survival of RGC after intracranial section of the optic nerve. To objectively assess the effect of NGF, they counted the optic nerve ﬁbers by electron microscopy. This analysis showed that the number of myelinated ﬁbers was signiﬁcantly greater in NGF-treated animals than in controls, and they conﬁrmed that repetitive intraocular injections of NGF promote the survival of a remarkable number of RGCs, for at least 7 weeks following optic nerve transection. In another study, the same research group demonstrated the anterograde and retrograde transport of NGF receptors by RGC axons, suggesting that RGCs axonal terminals may bind the NGF produced in target regions and that NGF is then internalized and retrogradely transported back to RGC bodies (Carmignoto et al., 1991). Ischemic Injury
The effect of repetitive intraocular administration of NGF after a single episode of complete retinal ischemia has been JOURNAL OF CELLULAR PHYSIOLOGY
investigated (Siliprandi et al., 1993) in cats by pattern electroretinogram (PERG) and pattern visual evoked potentials (PVEP). The recorded PERG and PVEP amplitudes were signiﬁcantly reduced by the experimentally induced retinal ischemia, but were signiﬁcantly worse in the eyes of cats treated with cytochrome c as compared to cats treated with NGF, particularly in the high spatial frequency domain. Additionally, a signiﬁcantly larger number of presumed RGCs survived the insult in NGF-treated retinas as compared to cytochrome c treated retinas. It is conceivable that NGF reduces the entry of calcium ions into retinal neurons after ischemia, thereby protecting them against the ischemic insult. This NGF effect has been later conﬁrmed in a model of bilateral carotid occlusion (two-vessel occlusion, 2VO) by Savilia et al. (2009). The authors investigated morphological and molecular changes occurring in the retina and optic nerve of adult rats at different time points (8, 30, and 75 days) following 2VO. They demonstrated that a single intravitreal injection of NGF (5 mg/3 ml performed 24 h after 2VO ligation) had a longlasting effect in protecting retina and optic nerve from degeneration. This protective effect of NGF is induced by the regulation of Bax/Bcl-2 gene expression and by the expression of the early gene c-jun in the retina. It is interesting to focus on the long lasting effect of a single NGF administration, as indicated by the preservation of optic nerve diameter and myelination, as well as by RGC with nuclear proﬁle counting. The effect was also mediated by an increased synthesis of endogenous NGF due to the mechanical injury caused by the intraocular injection. Ocular Hypertension and Glaucoma
The experimental ﬁndings on the role of NGF in visual system development and RGC physiology, prompted ocular researchers to study the impact of this neurotrophin in retinal damage induced by ocular hypertension and glaucoma, the second most frequent cause of irreversible vision loss in the world (Kingman, 2004). These studies range from animal models of ocular hypertension treated with NGF delivered topically as eye drop formulations or intravitreally, to exploratory cases of advanced glaucoma patients treated with NGF eye drops. Speciﬁcally, Lambiase et al. (1997) investigated the presence of NGF in the aqueous humor and evaluated the protective effect of endogenous and exogenous NGF in retinal damage using an experimental animal model of intraocular
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hypertension in rabbits. They showed that the highest NGF level was observed after 4 days of intraocular hypertension and histological examination revealed that the number of RGC remained unchanged during the ﬁrst 4 days but decreased signiﬁcantly after 10 days. This study also showed that retroocular administration of NGF reduced RGC loss, whereas intraocular injection of NGF antibodies, that inhibit endogenous NGF, exacerbated the retinal insult. These preliminary results suggested that NGF may be helpful in developing a new clinical approach to the treatment of human hypertensive ocular diseases. Subsequently, after reporting that conjunctival application of NGF could reach brain cholinergic neurons and suggesting that high molecular weight proteins could be safely delivered into the brain via the ocular surface to promote the recovery of damaged brain cells, Sposato et al. (2009) used a rat model of glaucoma as an approach to investigate the effects of glaucoma on cells of the lateral geniculate nucleus (LGN) and visual cortex (VC) and for understanding the role of topically applied NGF eye drops on neurodegenerative diseases. They found that glaucoma reduces the concentration of NGF in the cerebrospinal ﬂuid (CSF), LGN, and VC, without causing signiﬁcant changes in NGF serum levels. Topical ocular NGF application for 35 consecutive days enhanced the concentration of NGF in the CSF of glaucomatous rats and normalized its presence in the VC and LGN. Exogenous NGF application induced up-regulation of TrkA in the LGN (but not in the VC), enhanced the expression of p75 in the LGN and reduced its presence in the VC. There has been great scientiﬁc interest and discussion on the mechanism through which NGF ocular application could reach the brain. Several explanations have been proposed, including the anatomical connections between the eye and the brain, via nasal mucosa, nasolacrimal duct, brain ventricles, or CSF diffusion (Ambati et al., 2000; Koevary, 2003; Lambiase et al., 2005, 2007). Recently, Lambiase et al. (2009) demonstrated that topical application of exogenous NGF to the eye prevents RGC degeneration in an experimental rat model of glaucoma and based on these ﬁndings they used the same dosage regimen to treat three glaucoma patients with rapid and progressive visual ﬁeld loss despite successful control of intraocular pressure. In this study, the beneﬁcial effect of NGF on RGC survival was induced by the inhibition of RGC apoptosis, as shown by the reduction in TUNEL immunostaining and the greater retinal Bcl-2/Bax ratio. According to these experimental ﬁndings, glaucoma patients treated with NGF eye drops demonstrated long lasting improvements in visual ﬁeld, optic nerve function, contrast sensitivity and visual acuity. PERV and VEP amplitude and times-to-peak were the ﬁrst electrofunctional parameters to improve in these patients, suggesting a functional recovery of RGCs and an improvement of neural conduction along postretinal visual pathways. It is important to stress that the preliminary results obtained in these few glaucoma patients, as well as in many other patients with ocular surface diseases such as neurotrophic keratitis (Bonini et al., 2000), were obtained using a murine NGF which was extracted and puriﬁed using the Bocchini–Angeletti method (Bocchini and Angeletti, 1969). These results will need to be conﬁrmed in large randomized clinical trials that have been long expected and are now ﬁnally beginning, following the recent introduction of a new method to obtain a recombinant human NGF produced in Escherichia coli suitable for human use. Conclusions
Since its discovery, NGF has gained substantial interest and many experimental and clinical studies have been developed to understand its role in several diseases like peripheral JOURNAL OF CELLULAR PHYSIOLOGY
neuropathies, diabetes, human immunodeﬁceincy virus, CNS diseases, and skin ulcers. As elucidated in this review, NGF is a crucial neurotrophine for visual system development. It is produced and utilized in the retina by RGC, and it is expressed in the anterior segment of the eye and released into the aqueous humor. These ﬁndings triggered researches to develop different animal models to investigate the role of NGF in RGCs survival. Their studies conﬁrmed that the main mechanism of action of this neurotrophine after binding to its high-afﬁnity receptor TrkA, is the up-regulation of the Bcl-2 protein, which protects cells from apoptosis by preventing caspase activation. Therefore, it soon became clear that NGF has a great pharmacological potential in ophthalmic diseases. The evidence that topically applied NGF eye drops can reach the retina and optic nerve, greatly increased the expectations on the clinical use of this neurotrophin for currently untreatable ocular diseases. Thanks to the recent introduction of a recombinant human NGF as an eye drop formulation, that has already been proven safe in Phase-I clinical trials (Ferrari et al., 2013), wide randomized controlled Phase-II clinical trials have already been planned to investigate the use of NGF in widespread ocular neurodegenerative diseases, like glaucoma. Literature Cited Aloe L, Bracci-Laudiero L, Bonini S, Manni L. 1997. The expanding role of nerve growth factor: From neurotrophic activity to immunologic diseases. Allergy 52:883–894. Ambati J, Canakis CS, Miller JW, Gragoudas ES, Edwards A, Weissgold DJ, Kim I, Delori FC, Adamis AP. 2000. Diffusion of high molecular weight compounds through sclera. Invest Ophthalmol Vis Sci 41:1181–1185. Bjerre B, Bjorklund A, Stenevi V. 1973. Stimulation of growth of new axon sprouts from lesioned monoamine neurons in adult rat brain by nerve growth factor. Brain Res 60: 161–176. Bjerre B, Bjorklund A, Stenevi V. 1974. Inhibition of regenerative growth of central noradrenergic neurons by intracerebrally administered anti NGF-serum. Brain Res 74: 1–18. Bocchini V, Angeletti PU. 1969. The nerve growth factor: Puriﬁcation as a 30,000-molecular weight protein. Proc Natl Acad Sci USA 64:787–794. Bonini S, Lambiase A, Rama P, Caprioglio G, Aloe L. 2000. Topical treatment with nerve growth factor for neurotrophic keratitis. Ophthalmology 107:1347–1351. Carmignoto G, Maffei L, Candeo P, Canella R, Comelli C. 1989. Effect of NGF on the survival of rat retinal ganglion cells following optic nerve section. J Neurosci 9:1263–1272. Carmignoto G, Comelli MC, Candeo P, Cavicchioli L, Yan Q, Meringhi A, Maffei L. 1991. Expression of NGF receptor and NGF receptor mRNA in the developing and adult rat retina. Exp Neurol 111:302–311. Chao MV, Rajagopal R, Lee FS. 2006. Neurotrophin signalling in health and disease. Clin Sci (Lond) 110:167–173. Fahnestock M, Yu G, Coughlin MD. 2004. ProNGF: A neurotrophic or an apoptotic molecule? Prog Brain Res 146:101–110. Ferrari MP, Mantelli F, Sacchetti M, Antonangeli MI, Cattani F, D’Anniballe G, Sinigaglia F, Rufﬁni PA, Lambiase A. 2013. Safety and pharmacokinetics of escalating doses of human recombinant nerve growth factor eye drops in a double-masked, randomized clinical trial. BioDrugs. [Epub ahead of print] Friedman WJ, Greene LA. 1999. Neurotrophin signalling via Trks and p75. Exp Cell Res 253:131–142. Huang EJ, Reichardt LF. 2003. TRK receptors: Roles in neuronal signal transduction. Annu Rev Biochem 72:609–642. Johnson EM, Taniuchi M, Brent Clark H, Springer JE, Sookyong Koh, Tayrien MW, Loy R. 1987. Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and central nervous system. J Neurosci 7:923–929. Kingman S. 2004. Glaucoma is second leading cause of blindness globally. Bull World Health Organ 82:887–888. Klesse LJ, Parada LF. 1999. Trks: Signal transduction and intracellular pathways. Microsc Res Tech 45:210–216. Koevary SB. 2003. Pharmacokinetics of topical ocular drug delivery: Potential uses for the treatment of diseases of the posterior segment and beyond. Curr Drug Metab 4:213– 222. Lambiase A, Centofanti M, Micera A, Manni GL, Mattei E, De Gregorio A, de Feo G, Bucci MG, Aloe L. 1997. Nerve growth factor (NGF) reduces and NGF antibody exacerbates retinal damage induced in rabbit by experimental ocular hypertension. Graefes Arch Clin Exp Ophthalmol 235:780–785. Lambiase A, Bonini S, Manni L, Ghinelli E, Tirassa P, Rama P, Aloe L. 2002. Intraocular production and release of nerve growth factor after iridectomy. Invest Ophthalmol Vis Sci. 43:2334–2340. Lambiase A, Tirassa P, Micera A, Aloe L, Bonini S. 2005. Pharmacokinetics of conjunctivally applied nerve growth factor in the retina and optic nerve of adult rats. Invest Ophthalmol Vis Sci 46:3800–3806. Lambiase A, Pagani L, Di Fausto V, Sposato V, Coassin M, Bonini S, Aloe L. 2007. Nerve growth factor eye drop administrated in the ocular surface of rodents affects the nucleus basalis and septum; biochemical and structural evidence. Brain Res 1127:45–51. Lambiase A, Aloe L, Centofanti M, Parisi V, Mantelli F, Colafrancesco V, Manni GL, Bucci MG, Bonini S, Levi-Montalcini R. 2009. Experimental and clinical evidence of neuroprotection by nerve growth factor eye drops: Implications for glaucoma. Proc Natl Acad Sci USA 32:13469–13475. Levi-Montalcini R. 1987. The nerve growth factor: Thirty-ﬁve years later. Science 237:1154– 1162.
NERVE GROWTH FACTOR MODULATION
Reichardt LF. 2006. Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci 361:1545–1564. Savilia S, Giuliani A, Fernandez M, Turba ME, Forni M, Massella A, De Sordi N, Giardino L, Calzà L. 2009. Intravitreal NGF administration counteracts retina degeneration after permanent carotid artery occlusion in rat. BMC Neurosci 10:52. Siliprandi R, Canella R, Carmignoto G. 1993. Nerve growth factor promotes functional recovery of retinal ganglion cells after ischemia. Invest Ophthalmol Vis Sci 34:3232–3245. Sposato V, Parisi V, Manni L, Antonucci MT, Di Fausto V, Sornelli F, Aloe L. 2009. Glaucoma alters the expression of NGF and NGF receptors in visual cortex and geniculate nucleus of rats: Effects of eye NGF application. Vision Res 49:54–63. Turner JE, Delaney RK. 1979. Retinal ganglion cell response to axotomy and nerve growth factor in the regenerating visual system of the newt (notophthalmus viridescens): An ultrastructural morphometric analysis. Brain Res 171:197–212.
JOURNAL OF CELLULAR PHYSIOLOGY
Turner JE, Glaze KA. 1977. Regenerative repair in the severed optic nerve of the newt (triturus viridescens) effect of nerve growth factor. Exp Neurol 57:687–697. Turner JE, Delaney RK, Powell R. 1978. Retinal ganglion cell response to axotomy in the regenerating visual system of the newt (triturus viridescens): An ultrastructural morphometric analysis. Exp Neurol 62:444–462. Vantini G, Schiavo N, Di Martino A, Polato P, Triban C, Callegaro L, Toffano G, Leon A. 1989. Evidence for a physiological role of nerve growth factor in the central nervous system. Neuron 3:267–273. Yan Q, Johnson EM, Jr. 1988. An immunohistochemical study of the nerve growth factor receptor in developing rats. J Neurosci 8:3481–3498. Yan Q, Johnson EM, Jr. 1989. Immunoistochemical localization and biochemical characterization of nerve growth receptor in adult rat brain. J Comp Neurol 290: 585–598.