The role of neurotrophins in axonal growth, guidance, and regeneration

Neurotrophins are proteins that regulate neuronal survival, axonal growth, synaptic plasticity and neurotransmission. They are members of the neurotrophic factors family and include factors such as the nerve growth factor (NGF), the brain derived neurotrophic factor (BDNF), the neurotrophin-3 (NT-3), and the neurotrophin-4/5 (NT-4/5). These molecules bind to two types of receptors: i) tyrosine kinase receptors (TrkA, TrkB, TrkC) and ii) a common neurotrophin receptor (p75NTR). The two receptor types can either suppress or enhance each other's actions. Neurotrophins have a multifunctional role both in the central and peripheral nervous system. They have been suggested as axonal guidance molecules during the growth and regeneration of nerves. It has also been proven that they stimulate axonal growth by mediating the polymerization and accumulation of F-actin in growth cones and axon shafts. Neurotrophins, as other neurotrophic factors, have been shown that they reduce neuronal injury by exposure to excitotoxins, glucose deprivation, or ischemia. Furthermore, the nerve regeneration promoting effect of these growth factors is well documented for many different models of central or peripheral nervous system injury. Several studies have shown that exogenous administration of these factors has protective properties for injured neurons and stimulates axonal regeneration. Based on these properties, these molecules may be used as therapeutic agents for treating degenerative diseases and traumatic injuries of both the central and peripheral nervous system.     

Neurotrophic factors in Alzheimer's and Parkinson's diseases: implications for pathogenesis and therapy

Neurotrophic factors comprise essential secreted proteins that have several functions in neural and non-neural tissues, mediating the development, survival and maintenance of peripheral and central nervous system. Therefore, neurotrophic factor issue has been extensively investigated into the context of neurodegenerative diseases. Alzheimer's disease and Parkinson's disease show changes in the regulation of specific neurotrophic factors and their receptors, which appear to be critical for neuronal degeneration. Indeed, neurotrophic factors prevent cell death in degenerative processes and can enhance the growth and function of affected neurons in these disorders. Based on recent reports, this review discusses the main findings related to the neurotrophic factor support – mainly brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor – in the survival, proliferation and maturation of affected neurons in Alzheimer's disease and Parkinson's disease as well as their putative application as new therapeutic approach for these diseases management.     

Neurotrophic factors and neuromuscular disease: I. General comments, the neurotrophin family, and neuropoietic cytokines.

Neurotrophic factors are growth factors or cytokines that are inducible polypeptides and permit intercellular communication. An explosion of information about neurotrophic factors is setting the stage for significant advances in neural disease therapy in the next century. The effects of these trophic factors are overlapping and pleiotropic, acting on many cell types and tissues to control proliferation and differentiation of developing neurons and to exert a variety of functions on mature neurons. Studies of receptors unique to several neurotrophic factor families have revealed exquisite mechanisms of signal transduction. Preclinical trials in neuromuscular disease were promising, but results from initial clinical trials have been disappointing; new and better designed clinical trials are under way. Laboratory investigators also are exploring techniques to deliver factors directly to the central nervous system by means of viral vectors or to exert neurotrophic signals on the nervous system using novel small molecules that stimulate neurotrophic factor or neuroimmunophilin receptors. Combination therapies, refined delivery techniques, and treatment timing may be the key for successful treatment with neurotrophic factors. In this two-part review, we discuss the neurobiology of neurotrophic factors, the characteristics of the major neurotrophic factors, and their therapeutic potential in neuromuscular disease.     

The role of neuronal growth factors in neurodegenerative disorders of the human brain

Recent evidence suggests that neurotrophic factors that promote the survival or differentiation of developing neurons may also protect mature neurons from neuronal atrophy in the degenerating human brain. Furthermore, it has been proposed that the pathogenesis of human neurodegenerative disorders may be due to an alteration in neurotrophic factor and/or trk receptor levels. The use of neurotrophic factors as therapeutic agents is a novel approach aimed at restoring and maintaining neuronal function in the central nervous system (CNS). Research is currently being undertaken to determine potential mechanisms to deliver neurotrophic factors to selectively vulnerable regions of the CNS. However, while there is widespread interest in the use of neurotrophic factors to prevent and/or reduce the neuronal cell loss and atrophy observed in neurodegenerative disorders, little research has been performed examining the expression and functional role of these factors in the normal and diseased human brain. This review will discuss recent studies and examine the role members of the nerve growth factor family (NGF, BDNF and NT-3) and trk receptors as well as additional growth factors (GDNF, TGF-α and IGF-I) may play in neurodegenerative disorders of the human brain.     

Erythropoietin – a novel concept for neuroprotection

Neuroprotection as a means to prevent or oppose pathological neuronal loss in central nervous system disease of various pathophysiological origins represents a novel therapeutic approach. This approach is supported by extensive experimental evidence on cell culture and animal studies demonstrating beneficial effects of growth factors on neuronal survival and functional recovery. The clinical use of neuroprotective agents has been hampered by the toxicity of many of the compounds that showed promising therapeutic potential in animal studies. The focus of this review is on a novel neuroprotective approach with erythropoietin, a hematopoietic growth factor that: 1) is expressed in the human central nervous system, 2) is hypoxia-inducible, 3) has demonstrated remarkable neuroprotective potential in cell culture and animal models of disease, 4) has multiple protective effects (antiapoptotic, neurotrophic, antioxidant, angiogenic), and 5) is a clinically extremely well tolerated compound. Accepted: 25 June 2001     

Neurotrophins and neurodegeneration

There is growing evidence that reduced neurotrophic support is a significant factor in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). In this review we discuss the structure and functions of neurotrophins such as nerve growth factor, and the role of these proteins and their tyrosine kinase (Trk) receptors in the aetiology and therapy of such diseases. Neurotrophins regulate development and the maintenance of the vertebrate nervous system. In the mature nervous system they affect neuronal survival and also influence synaptic function and plasticity. The neurotrophins are able to bind to two different receptors: all bind to a common receptor p75NTR, and each also binds to one of a family of Trk receptors. By dimerization of the Trk receptors, and subsequent transphosphorylation of the intracellular kinase domain, signalling pathways are activated. We discuss here the structure and function of the neurotrophins and how they have been, or may be, used therapeutically in AD, PD, Huntington's diseases, ALS and peripheral neuropathy. Neurotrophins are central to many aspects of nervous system function. However they have not truly fulfilled their therapeutic potential in clinical trials because of the difficulties of protein delivery and pharmacokinetics in the nervous system. With the recent elucidation of the structure of the neurotrophins bound to their receptors it will now be possible, using a combination of in silico technology and novel screening techniques, to develop small molecule mimetics with much improved pharmacotherapeutic profiles.     

Apoptosis and neurotrophic factors

Neurotrophic factors are endogenous soluble proteins regulating development, differentiation, and survival of neurons. They are secreted from target cells or surrounding glial cells and act on the neurons via their receptors on the cell membranes. Several factors are reported to promote survival of motor neurons in vitro and to rescue developing motor neurons from naturally occurring cell death. Administration of the factors has also been shown to rescue motor neurons from degeneration after axotomy in adult as well as neonatal rodents. On the basis of these lines of evidence, neurotrophic factors have been considered to be potential candidates for drugs alleviating human motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Although some factors such as ciliary neurotrophic factor and brain-derived neurotrophic factor slowed down the disease progression in animal models of motor neuron disease, phase III clinical trials showed no therapeutic effects for ALS patients treated with these factors. There may be some reasons for this lack of success in humans. Several important issues remain to be resolved such as the drug delivery systems for neurotrophic factors and combination of neurotrophic factors with complementary effects.     

Translating the therapeutic potential of neurotrophic factors to clinical 'proof of concept': A personal saga achieving a career-long quest

While the therapeutic potential of neurotrophic factors has been well-recognized for over two decades, attempts to translate that potential to the clinic have been disappointing, largely due to significant delivery obstacles. Similarly, gene therapy (or gene transfer) emerged as a potentially powerful, new therapeutic approach nearly two decades ago and despite its promise, also suffered serious setbacks when applied to the human clinic. As advances continue to be made in both fields, ironically, they may now be poised to complement each other to produce a translational breakthrough. The accumulated data argue that gene transfer provides the 'enabling technology' that can solve the age-old delivery problems that have plagued the translation of neurotrophic factors as treatments for chronic central nervous system diseases. A leading translational program applying gene transfer to deliver a neurotrophic factor to rejuvenate and protect degenerating human neurons is CERE-120 (AAV2-NRTN). To date, over two dozen nonclinical studies and three clinical trials have been completed. A fourth (pivotal) clinical trial has completed all dosing and is currently evaluating safety and efficacy. In total, eighty Parkinson's disease (PD) subjects have thus far been dosed with CERE-120 (some 7years ago), representing over 250 cumulative patient-years of exposure, with no serious safety issues identified. In a completed sham-surgery, double-blinded controlled trial, though the primary endpoint (the Unified Parkinson's Disease Rating Scale (UDPRS) motor off score measured at 12months) did not show benefit from CERE-120, several important motor and quality of life measurements did, including the same UPDRS-motor-off score, pre-specified to also be measured at a longer, 18-month post-dosing time point. Importantly, not a single measurement favored the sham control group. This study therefore, provided important, well-controlled evidence establishing 'clinical proof of concept' for gene transfer to the CNS and the first controlled evidence for clinical benefit of a neurotrophic factor in a human neurodegenerative disease. This paper reviews the development of CERE-120, starting historically with the long-standing interest in the therapeutic potential of neurotrophic factors and continuing with selective accounts of past efforts to translate their potential to the clinic, eventually leading to the application of gene transfer and its role as the 'enabling technology'. Because of growing interest in translational R&D, including its practice in industry, the paper is uniquely oriented from the author's personal, quasi-autobiographic perspective and career-long experiences conducting translational research and development, with a focus on various translational neurotrophic factor programs spanning 30+ years in Big Pharma and development-stage biotech companies. It is hoped that by sharing these perspectives, practical insight and information might be provided to others also interested in translational R&D as well as neurotrophic factors and gene therapy, offering readers the opportunity to benefit from some of our successes, while possibly avoiding some of our missteps.     

A culture model for neurite regeneration of human spinal cord neurons

Effective therapeutic interventions for injuries of the central nervous system such as spinal cord injury are still unavailable, having a great impact on the quality of life of victims and their families, as well as high costs in medical care. Animal models of spinal cord injury are costly, time-consuming and labor-intensive, making them unsuitable for screening large numbers of experimental conditions. Thus, culture models that recapitulate key aspects of neuronal changes in central nervous system injuries are needed to gain further understanding of the pathological and regenerative mechanisms involved, as well as to accelerate the screening of potential therapeutic agents. In this study we differentiated adherent cultures of dissociated human fetal spinal cord neural precursors into postmitotic neurons which we could then detach from culture plates and successfully freeze down in a viable state. When replated in neuronal medium without neurodifferentiating factors, these ready-to-use human spinal cord neurons remained viable, postmitotic and regenerated neurites in a cell density-dependent manner. Insulin-like growth factor 1 and growth hormone had no effect on neurite regeneration while brain-derived neurotrophic factor increased both the number of cells with neurites as well as the average neurite length. Our model can be applied to investigate factors involved in neuroregeneration of the human spinal cord and since adherent dissociated cell cultures are used, this system has significant potential as a screening platform for therapeutic agents to treat spinal cord injury.     

Vascular endothelial growth factor: adaptive changes in the neuroglialvascular unit

Brain postnatal development is modulated by adaptation and experience. Experience-mediated changes increase neuronal activity leading to increased metabolic demands that involve adaptive changes including ones at the microvascular network. Therefore, vascular environment plays a key role in central nervous system (CNS) development and function in health and disease. Trophic factors are crucial in CNS development and cell survival in adults. They participate in protection and proliferation of neuronal, glial and endothelial cells. Among the most important molecules are: the proangiogenic vascular endothelial growth factor (VEGF), the neurotrophin brain derived neurotrophic factor (BDNF), insulin growth factor (IGF-I) and the glycoprotein erythropoietin (EPO). We propose the term angioglioneurins to define molecules acting on the three components of the neurogliovascular unit. We have previously reported the effects of environmental modifications on the three components of the neurogliovascular unit during the postnatal development. We have also described the main role played by VEGF in the experience-induced postnatal changes. Angioglioneurin administration, alone or in combination with other neuroprotective strategies such as environmental enrichment, has been proposed as a non-invasive therapeutic strategy against several CNS diseases.     

Therapeutic potential of erythropoietin and its structural or functional variants in the nervous system

The growth factor erythropoietin (EPO) and erythropoietin receptors (EPOR) are expressed in the nervous system. Neuronal expression of EPO and EPOR peaks during brain development and is upregulated in the adult brain after injury. Peripherally administered EPO, and at least some of its variants, cross the blood-brain barrier, stimulate neurogenesis, neuronal differentiation, and activate brain neurotrophic, anti-apoptotic, anti-oxidant and anti-inflammatory signaling. These mechanisms underlie their tissue protective effects in nervous system disorders. As the tissue protective functions of EPO can be separated from its stimulatory action on hematopoiesis, novel EPO derivatives and mimetics, such as asialo-EPO and carbamoylated EPO have been developed. While the therapeutic potential of the novel EPO derivatives continues to be characterized in preclinical studies, the experimental findings in support for the use of recombinant human (rh)EPO in human brain disease have already been translated to clinical studies in acute ischemic stroke, chronic schizophrenia, and chronic progressive multiple sclerosis. In this review article, we assess the studies on EPO and, in particular, on its structural or functional variants in experimental models of nervous system disorders, and we provide a short overview of the completed and ongoing clinical studies testing EPO as neuroprotective/neuroregenerative treatment option in neuropsychiatric disease.     

The changing scene of neurotrophic factors.

The purification of brain-derived neurotrophic factor (BDNF), the elucidation of its primary structure, and the subsequent identification of neurotrophin-3 (NT-3) ended the monopoly of NGF as the only well-characterized, target-derived neurotrophic molecule. NGF, BDNF and NT-3 are members of a gene family called neurotrophins. They have strictly conserved domains that determine their basic structure. However, they also have distinctly variable domains that determine their different neuronal specificity mediated by different high affinity receptors, and that share a common low affinity subunit. These similarities and dissimilarities between the members of the neurotrophin gene family are also reflected by their regional distribution, cellular localization and developmental regulation. In this article the neurotrophins are compared with ciliary neurotrophic factor (CNTF), which is a representative of the category of neurotrophic molecules that, according to their regional distribution, developmental expression and cellular localization, do not fulfil the criteria of a target-derived neurotrophic molecule. The physiological and pathophysiological functions of neurotrophins and CNTF are discussed in the context of their potential use for the treatment of traumatic and degenerative diseases of the peripheral and central nervous systems.     

Targeting MicroRNAs Involved in the BDNF Signaling Impairment in Neurodegenerative Diseases

Neurodegenerative diseases are becoming an ever-increasing problem in aging populations. Low levels of brain-derived neurotrophic factor (BDNF) have previously been associated with the pathogenesis of numerous neurodegenerative diseases. Recently, microRNAs (miRNAs) have been proposed as potential novel therapeutic targets for treating various diseases of the central nervous system (CNS), and interestingly, few studies have reported several miRNAs that downregulate the expression levels of BDNF. However, substantial challenges exist when attempting to translate these findings into practical anti-miRNA therapeutics, especially when the targets remain inside the CNS. Thus, in this review, we summarize the specific molecular mechanisms by which several miRNAs negatively modulate the expressions of BDNF, address the potential clinical difficulties that can be faced during the development of anti-miRNA-based therapeutics and propose strategies to overcome these challenges.     

Topical delivery of nerve growth factor for treatment of ocular and brain disorders

Neurotrophins are a family of proteins that support neuronal proliferation, survival, and differentiation in the central and peripheral nervous systems, and are regulators of neuronal plasticity. Nerve growth factor is one of the best-described neurotrophins and has advanced to clinical trials for treatment of ocular and brain diseases due to its trophic and regenerative properties. Prior trials over the past few decades have produced conflicting results, which have principally been ascribed to adverse effects of systemic nerve growth factor administration, together with poor penetrance of the blood-brain barrier that impairs drug delivery. Contrastingly, recent studies have revealed that topical ocular and intranasal nerve growth factor administration are safe and effective, suggesting that topical nerve growth factor delivery is a potential alternative to both systemic and invasive intracerebral delivery. The therapeutic effects of local nerve growth factor delivery have been extensively investigated for different ophthalmic diseases, including neurotrophic keratitis, glaucoma, retinitis pigmentosa, and dry eye disease. Further, promising pharmacologic effects were reported in an optic glioma model, which indicated that topically administered nerve growth factor diffused far beyond where it was topically applied. These findings support the therapeutic potential of delivering topical nerve growth factor preparations intranasally for acquired and degenerative brain disorders. Preliminary clinical findings in both traumatic and non-traumatic acquired brain injuries are encouraging, especially in pediatric patients, and clinical trials are ongoing. The present review will focus on the therapeutic effects of both ocular and intranasal nerve growth factor delivery for diseases of the brain and eye.     

Targeting neurotrophin receptors in the central nervous system

Neurotrophic factors, and in particular the neurotrophins, restore the function of damaged neurons and prevent apoptosis in adults. The potential therapeutic property of the neurotrophins is however, complicated by the peptidergic structure of these trophic factors, which impairs their penetration into the brain parenchyma, and therefore makes their pharmaco-therapeutic properties difficult to evaluate. In this article we will focus on the neurotrophin Brain-derived neurotrophic factor (BDNF) and its receptors to address various therapeutic strategies that may overcome this problem. We will call this strategy "small molecule approach" because it relies on increasing the function of endogenous neurotrophins by pharmacological compounds that induce synthesis and release of neurotrophins in relevant brain areas or by small synthetic molecules that bind and activate specific neurotrophin receptors. The ability of small molecules to mimic BDNF has a potential therapeutic importance in preventing neuronal damage in several chronic neurodegenerative diseases including Parkinson's Disease, Alzheimer's Disease, and AIDS dementia.     

The neurotrophin family of NGF-related neurotrophic factors

The recent molecular cloning of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) has established the existence of an NGF-related family of neurotrophic factors - the neurotrophins. Purification and recombinant production of BDNF and NT-3 has allowed the initiation or extension of in vitro studies of the neuronal specificity of each of these factors. We have found that NT-3, like NGF and BDNF, promotes survival and neurite outgrowth from certain populations of sensory neurons. There appear to be both distinct and overlapping specificities of the 3 neurotrophins towards peripheral neurons - sympathetic neurons and subpopulations of neural crest and neural placode-derived sensory neurons. Using cultures of central nervous system neurons, we have recently established that BDNF: (i) promotes the survival and phenotypic differentiation of rat septal cholinergic neurons, a property consistent with the discovery of high levels of BDNF mRNA expression within the hippocampus; (ii) promotes the survival of rat nigral dopaminergic neurons and furthermore protects these neurons from two dopaminergic neurotoxins, 6-hydroxydopamine (6-OHDA) and MPTP. Thus the neurotrophic effects of these factors towards peripheral neurons and neuronal populations known to degenerate in two of the major human neurodegenerative diseases - Alzheimer's and Parkinson's disease - provokes the question of whether neurotrophic factors may have therapeutic potential in halting the progression and ameliorating the symptoms of devastating neurological disorders of the CNS or PNS, or improving regeneration of neurons of CNS or PNS after traumatic injury.     

The role of neurotrophic factors in psychostimulant-induced behavioral and neuronal plasticity

Several neurotrophic factors influence the development, maintenance and survival of dopaminergic neurons in the mammalian central nervous system (CNS), including neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and glial derived neurotrophic factor (GDNF). This review focuses on the role of these neurotrophic factors in psychostimulant-induced behavioral sensitization, a form of dopamine-mediated neuronal plasticity that models aspects of paranoid schizophrenia as well as drug craving among psychostimulant addicts. Whereas NT-3, CNTF and bFGF appear to play a positive role in psychostimulant-induced behavioral sensitization, GDNF inhibits this form of behavioral plasticity. The role of BDNF in behavioral sensitization, however, remains elusive. While it has been shown that neurotrophic factors can influence the behavioral, structural and biochemical phenomena related to psychostimulant-induced neuronal plasticity, it is unclear which neurotrophic factors are important physiologically and which have purely pharmacological effects. In either case, examining the role of neurotrophic factors in behavioral sensitization may enhance our understanding of the mechanisms underlying the development of paranoid psychosis and drug craving and lead to the development of novel pharmacological treatments for these disorders.     

The pharmacological potential of neurotrophins: a perspective.

Until recently nerve growth factor (NGF) was the only widely characterized neurotrophic factor which had been shown both in vitro and in vivo to be essential for the survival of selected populations of neurons during development and to be important for maintenance of the differentiated phenotype of mature neurons. The recent cloning of new members of the NGF family, namely brain-derived neurotrophic factor neurotrophin-3 (NT-3), NT-4 and NT-5, has greatly expanded our knowledge of the structural properties and neurotrophic activities of these proteins. Elucidation of their developmental and topographical expression and associated receptors in both the central nervous system and peripheral nervous system is proceeding at a brisk pace, leading to proposals for a potential pharmacological use of these proteins. This possibility will ultimately rely upon a more complete understanding of the roles of these trophic factors in human nervous system physiology and pathology.     

Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons

In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.