ORIGINAL ARTICLES AND REVIEWS
Luigi Aloe; Maria Luisa Rocco
Istituto di Biologia Cellulare e Neurobiologia, Consiglio Nazionale delle Ricerche, Rome, Italy
It has been shown that topical nerve growth factor (NGF) administration induces healing action on human cutaneous, corneal and pressure ulcers, glaucoma, maculopathy and retinitis pigmentosa suggesting a therapeutic potential of NGF in human ophthalmology and cutaneous ulcers. A similar therapeutic suggestion has emerged for the NGF gene therapy of Alzheimer's disease and ischemic heart injury. Moreover, over the last few years, the role and biological properties of NGF have also been investigated with transgenic mice over-expressing and down-expressing NGF. However, the results obtained with these transgenic mice seem suitable to confirm and/or support the evidence obtained with exogenous administration of NGF regarding the suggested clinical potentiality of NGF. The aim of the present brief review is to report and comment on these two different findings of NGF's healing properties.
Key words: NGF receptor, transgenic mice, knockout mice, NGF and therapy
NGF: DISCOVERY AND ONGOING FINDINGS
Since its discovery, nerve growth factor (NGF) has long occupied a critical role in developmental neurobiology because of the many important neuronal functions it has been shown to have . NGF is the first discovered and best-characterised member of a family of neurotrophic factors, collectively indicated as neurotrophins, which include brain-derived neurotrophic factors and neurotrophin-3 (NT-3). These neurotrophic factors share significant structural homologies and overlapping actions  exerting its action on growth and survival of peripheral sensory and sympathetic neurons  (see Figure 1A, B) and on a number of brain neurons, particularly forebrain cholinergic neurons (FBCN) that are the major NGF-target cells within the central nervous system [4-6]. The molecule, initially described as a neurotrophic factor acting only or mainly on growth and differentiation of peripheral sympathetic and sensory neurons (Figure 1A, B), resulted to possess a number of other target cells within the nervous system as well as extra-neuronal targets including cells in cutaneous, immune, endocrine [3, 6, 7] and adipose tissue . The biological activity of NGF is mediated by two distinct receptors: TrkA (a tyrosine kinase receptor) and p75 (a member of the tumour necrosis factor receptor superfamily) [2, 9]. NGF's functional roles are supported by findings demonstrating that administration of anti-NGF antibodies in developing rodents down-regulates the circulating level of NGF, induces damages of peripheral and sympathetic NGF-target cells and ultimately leads to immunosympathectomy . Because NGF is a rather high molecular-weight protein, it is unable to cross the blood-brain-barrier, and intracerebral administration produces undesired side effects. The role of NGF on brain target neurons has been studied using of NGF conjugated with small molecules  or gene therapy and transgenic animal models. These findings paved the way for further investigations on the role of NGF in learning and memory that undergo degeneration during age-related disorders including the role of NGF in learning, memory, brain neuronal degenerative diseases and Alzheimer's disease (AD) [4, 5, 12].
We have recently demonstrated that topical NGF administration promotes, in human cutaneous ulcers induced by pressure, diabetes, rheumatoid arthritis and corneal ulcers [13-16], and safety protects damaged retinal cell's degeneration in patients affected by glaucoma , maculopathy  and retinitis pigmentosa . More recently, findings published by others indicated that NGF administered through gene therapy protected FBCN that degenerate in patients with AD, and reduced cell damages in myocardial infarction  and spinal cord injury . These findings are summarised in Table 1.
During the last two decades, the biological properties of NGF have also been investigated using NGF transgenic mice models, over-expressing NGF (Figure 2A) or lacking NGF, knockout (KO) mice (Figure 2B). These transgenic animal models display a number of neuronal and non-neuronal deficits similar to those observed after exogenous administration of purified NGF or anti-NGF-antibody (ANA), but also revealed some contradictory effects, not only among different strains of NGF transgenic mice models, but also between NGF transgenic mice and mice treated with exogenous NGF administration. The aim of this brief review is, therefore, to compare and critically assess these differences and to discuss the NGF transgenic mouse model in order to support the hypothesis.
THE GENERATION OF TRANSGENIC MICE
In 1953, Watson and Crick published the structure of the double-strand helix model for DNA . This discovery, and subsequent molecular related studies, provided a powerful tool for understanding biological, molecular and genetic mechanisms for a number of pathologies and human therapeutic applications [22-24]. Thus, the knowledge of the DNA structure was the first step to understand and interfere at the genomic level and to generate modified organism, transgenic and KO animals. For most of these studies, the mouse has been selected due to the striking similarity of genetic properties (over 95%) between the mouse and human genome. Indeed, transgenic (gene enhancing) and KO (gene suppressing) mice provided a novel important strategy for studying development and mechanisms of a number of genetic and non-genetic pre- and post-natal diseases by introducing specific loss-of-function or gain-of-function mutations into genes and generating a great number of transgenic rodents. These experimental approaches allowed for the possibility to investigate the mechanism(s) through which specific signals were involved in human physiological and/or pathological events and eventually modify them. The generated NGF transgenic mice proved to be useful for studying a number of diseases, not only those afflicting laboratory and wild animals, but, most importantly, humans. Based on the available finding on the NGF spectrum of action on neuronal and non-neuronal cells and brain neuronal cells, it was reasonable to hypothesise that developing NGF transgenic mice would provide further understanding about the clinical potentiality of NGF.
NGF TRANSGENIC MICE
In 1994, Snider  and Smeyne et al.  generated the first TrkA transgenic mice characterised by severe sensory and sympathetic nerve cell deficits. During the same year, Crowley et al.  published the results of a new generated transgenic NGF KO mouse displaying severe deficits of peripheral sensory and sympathetic neurons and, surprisingly, no deleterious effects were observed in FBCN that received a critical trophic support from the NGF produced and released by the hippocampus and cortex . Why these NGF transgenic mice show peripheral neurons loss and no effect on brain NGF target cells, and why exogenous administration of NGF is unable to compensate for the deficits induced by the endogenous release of NGF in NGF-targets is not clear. In 2004, Coppola, et al.  generated a TrkA KO mouse, characterised also by B-cell abnormalities but normal post-natal development and survival, compared to other generated transgenic mice that die during the first two post-natal weeks .
In 2000, Cattaneo et al.  generated one more transgenic mouse, indicated as AD11, expressing recombinant neutralising anti-NGF monoclonal antibodies and characterised by severe deficits of the sympathetic nervous system, loss of FBCN, but also muscle dystrophy affecting the spinal cord and hind limb extensor muscle, and diffuse cell death in the spleen of adult mice [30-32]. Notably, the AD11 mice displayed a normal postnatal life compared to other NGF or TrkA KO mice that died during the first two post-natal weeks [26, 27]. In addition, no evident signs of neuropathological deficits before 60 days of age were evident. However, these rodents did develop clear signs of neuronal degeneration of the peripheral and central nervous systems that became progressively more evident . Surprisingly, the exogenous administration of NGF can promote complete reversion and recovery of the deficits induced in FBCN by the neutralising NGF antibodies released by the AD11 mouse .
To summarise, while the findings observed with NGF transgenic models confirmed the functional role of NGF on peripheral and brain neurons observed following exogenous NGF administration, they also revealed effects not previously reported using a different experimental approach. For example, the deficits observed in NGF neutralising AD11 mice in cells of the immune and central nervous systems, as well as the action in brain stem cell response, have not been observed with exogenous anti-NGF-antibody administration either during foetal life or during adult life. Likewise, it is not clear why some NGF transgenic mice will die during the early post-natal life and others will survive normally throughout their post-natal life. On the contrary, short or long-term administration of NGF or anti NGF administrations have no deleterious effect on mouse survival induce . Other differences between the two experimental approves include the mechanism through which exogenous administration of NGF reverses the deficits of FBCN in the brain of AD11 mice, in times of constant presence and/or release of neutralising anti-NGF monoclonal antibody by AD11 KO mice. Why the AD11 mice do not develop cutaneous ulcers, similar to those induced by circulating anti-NGF antibodies in NGF autoimmunisation rodents is unaccounted for . A number of other questions remain unresolved. Thus, while NGF transgenic clearly demonstrated that exogenous NGF induces protective and healing action on a number of human disorders (such as cutaneous ulcers and retinal cell degeneration) and NGF genes protect brain cells and cardiac cells, prospecting as potential therapeutic application of NGF, the available published data with NGF transgenic mice seem, despite the numerous contributions regarding the role of NGF and the molecular mechanisms involved, to not allow for the support of the observations obtained with exogenous NGF administration.
A number of observations obtained with the AD11 transgenic mice support the hypothesis that NGF can play a critical protective role on degenerating FBCN and possibly in the pathogenesis of human AD [34-36]. It should be taken into consideration, however, that AD is characterised not only by the altered presence of NGF and of NGF receptor expression in NGF-target neurons, but also by deregulations of a number of other different molecular signalling and survival factors. It should, therefore, demonstrate that the NGF molecule is the only or a very critical important factor that can prevent the development and/or protect the diverse deleterious events leading to AD. At present, however, no convincing evidence exists supporting the hypothesis of a direct link between NGF and the potential clinical approach in AD. Thus, the initial enthusiastic hope that the generation of NGF would provide mechanisms supporting or denying the potential therapeutic application of NGF needs at the moment is tempered by the different observations obtained with the two experimental approaches.
We have recently reported that topical NGF administration promotes healing of human cutaneous and corneal ulcers, and protects degenerating retinal cells in patients affected by glaucoma, maculopathy and retinitis pigmentosa [5, 17, 18]. Other studies have shown that the delivery of NGF through NGF gene therapy protects damaged brain neurons [33, 36] and myocardial cells . Though the results obtained with NGF transgenic mice models largely confirms the role of NGF on peripheral sensory and sympathetic neurons and on neurons of the central nervous system, not much has been learned by the published findings with NGF transgenic mice about the therapeutic properties of NGF on cutaneous corneal ulcers and retinal cell protections, as has been demonstrated with exogenous purified and gene therapies. From these two experimental approaches have emerged differences that might generate an erroneous interpretation; including the hypothesis, NGF transgenic models are unable or are insufficient to reproduce the effects obtained by exogenous NGF administration. These differences may temper the original enthusiastic belief that the generation of NGF transgenic mice would provide additional important evidence about the therapeutic properties of NGF. Anyhow, further studies are needed to identify the mechanisms through which NGF acts on damaged cells and to elucidate the role of exogenous NGF and ANA administration versus the endogenous release of NGF and the neutralizing NGF proteins before determine the exact therapeutic properties of NGF within and outside the brain. It is reasonable to hope that the development of other NGF transgenic mouse strains and further basic and clinical experimental approaches with exogenous NGF administration will provide further data, a better understanding and, hopefully, the NGF clinical applications.
This study was supported by National Research Council and NGF Onlus to Luigi Aloe. The Authors thank George N. Chaldakov for discussion and critical comments.
Conflict of interest statement
There are no potential conflicts of interest or any financial or personal relationships with other people or organizations that could inappropriately bias conduct and findings of this study.
1. Meakin SO, Shooter EM. The nerve growth factor family of receptors. Trends Neurosci 1992;15(9):323-31.
2. Barde YA. The nerve growth factor family. Prog Growth Factor Res 1990;2(4):237-48.
3. Levi-Montalcini R, Angeletti PU. Nerve growth factor. Physiol Rev 1968;48(3):534-69.
4. Sofroniew MV, Howe CL, Mobley WC. Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci 2001;24:1217-81.
5. Lambiase A, Aloe L, Centofanti M, Parisi V, Mantelli F, Colafrancesco V, Manni GL, Bucci MG, Bonini S, Levi-Montalcini R. Experimental and clinical evidence of neuroprotection by nerve growth factor eye drops: Implications for glaucoma. Proc Natl Acad Sci USA 2009;106:13469-74.
6. Levi-Montalcini R. The nerve growth factor 35 years later. Science 1987;237(4819):1154-62.
7. Aloe L, Calza L (Eds.). NGF and related molecules in health and disease. Amsterdam: Elsevier; 2004. (Progress in Brain Research, vol. 146. )
8. Chalda kov GN, Tonchev AB, Aloe L. NGF and BDNF: from nerves to adipose tissue, from neurokines to metabokines. Riv Psichiatr 2009;44(2):79-87.
9. Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem 2003;72:609-42.
10. Zaimis E. Nerve Growth Factor and its antiserum. 1971.
11. Friden PM, Walus LR, Watson P, Doctrow SR, Kozarich JW, Backman C, et al. Blood-brain barrier penetration and in vivo activity of an NGF conjugate. Science 1993;259(5093):373-7.
12. Connor B, Dragunow M. The role of neuronal growth factors in neurodegenerative disorders of the human brain. Brain Res Brain Res Rev 1998;27(1):1-39.
13. Lambiase A, Rama P, Bonini S, Caprioglio G, Aloe L. Topical treatment with nerve growth factor for corneal neurotrophic ulcers. N Engl J Med 1998;338(17):1174-80.
14. Bernabei R, Landi F, Bonini S, Onder G, Lambiase A, Pola R, et al. Effect of topical application of nerve-growth factor on pressure ulcers. Lancet 1999;354(9175):307.
15. Tuveri M, Generini S, Matucci-Cerinic M, Aloe L. NGF, a useful tool in the treatment of chronic vasculitic ulcers in rheumatoid arthritis. Lancet 2000;356(9243):1739-40.
16. Generini S, Tuveri MA, Matucci Cerinic M, Mastinu F, Manni L, Aloe L. Topical application of nerve growth factor in human diabetic foot ulcers. A study of three cases. Exp Clin Endocrinol Diabetes 2004;112(9):542-4.
17. Lambiase A, Coassin M, Tirassa P, Mantelli F, Aloe L. Nerve growth factor eye drops improve visual acuity and electrofunctional activity in age-related macular degeneration: a case report. Ann Ist Super Sanità 2009;45(4):439-42.
18. Lenzi L, Coassin M, Lambiase A, Bonini S, Amendola T, Aloe L. Effect of exogenous administration of nerve growth factor in the retina of rats with inherited retinitis pigmentosa. Vision Res 2005;45(12):1491-500.
19. Meloni M, Caporali A, Graiani G, Lagrasta C, Katare R, Van Linthout S, et al. Nerve growth factor promotes cardiac repair following myocardial infarction. Circ Res 2010;106(7):1275-84.
20. Feng SQ, Kong XH, Liu Y, Ban DX, Ning GZ, Chen JT, et al. Regeneration of spinal cord with cell and gene therapy. Orthop Surg 2009;1(2):153-63.
21. Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 1953;171(4356):737-8.
22. Kiermer V. Protein-protein interactions: better by the dozen. Nat Methods 2007;4(5):389.
23. Melo EO, Canavessi AM, Franco MM, Rumpf R. Animal transgenesis: state of the art and applications. J Appl Genet 2007;48(1):47-61.
24. Blake DJ, Weir A, Newey SE, Davies KE. Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 2002;82(2):291-329.
25. Snider WD. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell 1994;77(5):627-38.
26. Smeyne RJ, Klein R, Schnapp A, Long LK, Bryant S, Lewin A, et al. Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 1994;368(6468):246-9.
27. Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armanini MP, et al. Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 1994;76(6):1001-11.
28. Coppola V, Barrick CA, Southon EA, Celeste A, Wang K, Chen B, et al. Ablation of TrkA function in the immune system causes B cell abnormalities. Development 2004;131(20):5185-95.
29. Ruberti F, Capsoni S, Comparini A, Di Daniel E, Franzot J, Gonfloni S, et al. Phenotypic knockout of nerve growth factor in adult transgenic mice reveals severe deficits in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy. J Neurosci 2000;20(7):2589-601.
30. D'Onofrio M, Arisi I, Brandi R, Di Mambro A, Felsani A, Capsoni S, et al. Early inflammation and immune response mRNAs in the brain of AD11 anti-NGF mice. Neurobiol Aging 2011;32(6):1007-22.
31. De Rosa R, Garcia AA, Braschi C, Capsoni S, Maffei L, Berardi N, et al. Intranasal administration of nerve growth factor (NGF) rescues recognition memory deficits in AD11 anti-NGF transgenic mice. Proc Natl Acad Sci USA 2005;102(10):3811-6.
32. Scardigli R, Capelli P, Vignone D, Brandi R, Ceci M, La Regina F, et al. Neutralization of nerve growth factor impairs proliferation and differentiation of adult neural progenitors in the subventricular zone. Stem Cells 2014;32(9):2516-28.
33. Johnson EM, Jr., Gorin PD, Brandeis LD, Pearson J. Dorsal root ganglion neurons are destroyed by exposure in utero to maternal antibody to nerve growth factor. Science 1980;210(4472):916-8.
34. Tuszynski MH, Thal L, Pay M, Salmon DP, U HS, Bakay R, et al. A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat Med 2005;11(5):551-5.
35. Eriksdotter Jonhagen M, Nordberg A, Amberla K, Backman L, Ebendal T, Meyerson B, et al. Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer's disease. Dement Geriatr Cogn Disord 1998;9(5):246-57.
36. Ferreira D, Westman E, Eyjolfsdottir H, Almqvist P, Lind G, Linderoth B, et al. Brain changes in Alzheimer's disease patients with implanted encapsulated cells releasing nerve growth factor. J Alzheimers Dis 2015;43(3):1059-72.
Address for correspondence:
Istituto di Biologia Cellulare e Neurobiologia, Consiglio Nazionale delle Ricerche,
Via del Fosso di Fiorano 54, 00143
Received on 11 September 2014.
Accepted on 2 December 2014.