Anterograde transport of neurotrophic factors

The actions of neurotrophic factors are classically thought to be mediated by their retrograde transport from target tissues to the cell bodies. There is now evidence that specific trophic factors are trafficked anterogradely along peripheral and central axons and released to postsynaptic cells. This review focuses on recent experiments that demonstrate the involvement of the anterograde transfer of neurotrophic factors in various physiological processes, including the regulation of developmental neuronal death, the modulation of synaptic transmission, and the control of axonal and dendritic architecture. The authors also discuss whether anterograde transport of exogenous trophic factors can be exploited to protect damaged postsynaptic neurons and spare their function. This issue has clear implications for possible therapeutic applications of neurotrophic factors.     

Anterograde axonal transport, transcytosis, and recycling of neurotrophic factors

Traditional views of neurotrophic factor biology held that trophic factors are released from target cells, retrogradely transported along their axons, and rapidly degraded upon arrival in cell bodies. Increasing evidence indicates that several trophic factors such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF-2), glial cell-line derived neurotrophic factor (GDNF), insulin-like growth factor (IGF-I), and neurotrophin-3 (NT-3), can move anterogradely along axons. They can escape the degradative pathway upon internalization and are recycled for future uses. Internalized ligands can move through intermediary cells by transcytosis, presumably by endocytosis via endosomes to the Golgi system, by trafficking of the factor to dendrites or by sorting into anterograde axonal transport with subsequent release from axon terminals and uptake by second- or third-order target neurons. Such data suggest the existence of multiple “trophic currencies,” which may be used over several steps in neural networks to enable nurturing relationships between connected neurons or glial cells, not unlike currency exchanges between trading partners in the world economy. Functions of multistep transfer of trophic material through neural networks may include regulation of neuronal survival, differentiation of phenotypes and dendritic morphology, synapse plasticity, as well as excitatory neurotransmission. The molecular mechanisms of sorting, trafficking, and release of trophic factors from distinct neuronal compartments are important for an understanding of neurotrophism, but they present challenging tasks owing to the low levels of the endogeneous factors.     

Axonal transport and neuronal transcytosis of trophic factors, tracers, and pathogens

Neurons can specifically internalize macromolecules, such as trophic factors, lectins, toxins, and other pathogens. Upon internalization in terminals, proteins can move retrogradely along axons, or, upon internalization at somatodendritic domains, they can move into an anterograde axonal transport pathway. Release of internalized proteins from neurons after either retrograde or anterograde axonal transport results in transcytosis and trafficking of proteins across multiple synapses. Recent studies of binding properties of several such proteins suggest that pathogens and lectins may utilize existing transport machineries designed for trafficking of trophic factors. Specific pathways may protect trophic factors, pathogens, and toxins from degradation after internalization and may target the trophic or pathogenic cargo for transcytosis after either retrograde or anterograde transport along axons. Elucidating the molecular mechanisms of sorting steps and transport pathways will further our understanding of trophic signaling and could be relevant for an understanding and possible treatment of neurological diseases such as rabies, Alzheimer's disease, and prion encephalopathies. At present, our knowledge is remarkably sparse about the types of receptors used by pathogens for trafficking, the signals that sort trophins or pathogens into recycling or degradation pathways, and the mechanisms that regulate their release from somatodendritic domains or axon terminals. This review intends to draw attention to potential convergences and parallels in trafficking of trophic and pathogenic proteins. It discusses axonal transport/trafficking mechanisms that may help to understand and eventually treat neurological diseases by targeted drug delivery.     

Trophic factors in neurodegenerative disorders

Neuronal survival is dependent on continuous trophic stimulation by neurotrophic factors. Anterograde and retrograde transport of neurotrophic factors and their receptors within neurites is essential for the communication of these survival signals. Lack of neurotrophic input has been proposed as a pathomechanism leading to neurodegenerative disease. The present short review provides a summary of some of the recent data on neurotrophic factors in neurodegenerative disorders and describes how disturbances of axonal trafficking might deprive neurons from trophic input.     

Brain-derived neurotrophic factor is an anterograde survival factor in the rat visual system

BACKGROUND: The neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5 and NT-6, are a family of proteins that play fundamental roles in the differentiation, survival and maintenance of peripheral and central neurons. Much research has focused on the role of neurotrophins as target-derived, retrogradely transported trophic molecules. Although there is recent evidence that BDNF and NT-3 can be transported in an anterograde direction along peripheral and central axons, there is as yet no conclusive evidence that these anterograde factors have direct post-synaptic actions. RESULTS: We report that BDNF travels in an anterograde direction along the optic nerve. The anterogradely transported BDNF had rapid effects on retinal target neurons in the superior colliculus and lateral geniculate nucleus of the brain. When endogenous BDNF within the developing superior colliculus was neutralised, the rate of programmed neuronal death increased. Conversely, provision of an afferent supply of BDNF prevented the degeneration of geniculate neurons after removal of their cortical target. CONCLUSIONS: BDNF released from retinal ganglion cells acts as a survival factor for post-synaptic neurons in retinal target fields.     

The reversible effect of colchicine on the taste bud cells of the central circumvallate papilla in the rat

Summary It is believed that differentiation and maintenance of taste buds in vertebrates is dependent on the trophic function of their sensory nerve supply. In the present work colchicine was injected into the circumvallate papilla of the rat. This produced a reversible blockade of neuroplasmic transport and disappearance of taste buds. Colchicine inhibited the further differentiation of bud cells, but apparently did not change the life cycle of the cells present already at the time of injection. It is speculated that the neurotrophic factors in this particular cell system are effective to induce cell differentiation only.This work was supported by CAIT Grant No 1776     

Mycalolide B dissociates dynactin and abolishes retrograde axonal transport of dense-core vesicles

Axonal transport is critical for maintaining synaptic transmission. Of interest, anterograde and retrograde axonal transport appear to be interdependent, as perturbing one directional motor often impairs movement in the opposite direction. Here live imaging of Drosophila and hippocampal neuron dense-core vesicles (DCVs) containing a neuropeptide or brain-derived neurotrophic factor shows that the F-actin depolymerizing macrolide toxin mycalolide B (MB) rapidly and selectively abolishes retrograde, but not anterograde, transport in the axon and the nerve terminal. Latrunculin A does not mimic MB, demonstrating that F-actin depolymerization is not responsible for unidirectional transport inhibition. Given that dynactin initiates retrograde transport and that amino acid sequences implicated in macrolide toxin binding are found in the dynactin component actin-related protein 1, we examined dynactin integrity. Remarkably, cell extract and purified protein experiments show that MB induces disassembly of the dynactin complex. Thus imaging selective retrograde transport inhibition led to the discovery of a small-molecule dynactin disruptor. The rapid unidirectional inhibition by MB suggests that dynactin is absolutely required for retrograde DCV transport but does not directly facilitate ongoing anterograde DCV transport in the axon or nerve terminal. More generally, MB''s effects bolster the conclusion that anterograde and retrograde axonal transport are not necessarily interdependent.     

Neurotrophins, synaptic plasticity and dementia

The growing realization that neurotrophins, such as brain-derived neurotrophic factor (BDNF), are crucial in modulating synaptic plasticity has broadened the spectrum of their trophic actions. At the same time, it has become clear that Abeta peptides derived from amyloid precursor protein (APP) have dramatic effects on synaptic transmission before the onset of the neurodegenerative disease. Because neurotrophins and Abeta are responsible for affecting both synaptic and cognitive function, it is likely that their mechanisms of action will be related and might even intersect. This review highlights several recent findings that suggest trophic factors and APP use similar pathways to control neuronal activity.     

Serum growth factors and neuroprotective surveillance

The adult brain requires a constant trophic input for appropriate function. Although the main source of trophic factors for mature neurons is considered to arise locally from glial cells and synaptic partners, recent evidence suggests that hormonal-like influences from distant sources may also be important. These include not only relatively well-characterized steroid hormones that cross the brain barriers, but also blood-borne protein growth factors able to cross the barriers and exert unexpected, albeit specific, trophic actions in diverse brain areas. Insulin-like growth factor I (IGF-I) is until now the serum neurotrophic factor whose actions on the adult brain are best-characterized. This is because IGF-I has been known for many years to be present in serum, whereas the presence in the circulation of other more classical neurotrophic factors has only recently been recognized. Thus, new evidence strongly suggests that IGF-I, and other blood-borne neurotrophic factors such as Fibroblast Growth Factor (FGF-2) or the neurotrophins, exert a tonic trophic input on brain cells, providing a mechanism for what we may refer to as neuroprotective surveillance. Protective surveillance includes "first-line" defense mechanisms ranging from blockade of neuronal death after a wide variety of cellular insults to upregulation of neurogenesis when defenses against neuronal death are overcome. Most importantly, surveillance should also encompass modulation of homeostatic mechanisms to prevent neuronal derangement. These will include modulation of basic cellular processes such as metabolic demands and maintainance of cell-membrane potential as well as more complex processes such as regulation of neuronal plasticity to keep neurons able to respond to constantly changing functional demands.     

Differential effects of the trophic factors BDNF, NT-4, GDNF, and IGF-I on the isthmo-optic nucleus in chick embryos

The isthmo-optic nucleus (ION) of chick embryos is a model system for the study of retrograde trophic signaling in developing CNS neurons. The role of brain-derived neurotrophic factor (BDNF) is well established in this system. Recent work has implicated neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), and insulin-like growth factor I (IGF-I) as additional trophic factors for ION neurons. Here it was examined in vitro and in vivo whether these factors are target-derived trophic factors for the ION in 13- to 16-day-old chick embryos. Unlike BDNF, neither GDNF, NT-4, nor IGF-I increased the survival of ION neurons in dissociated cultures identified by retrograde labeling with the fluorescent tracer DiI. BDNF and IGF-I promoted neurite outgrowth from ION explants, whereas GDNF and NT-4 had no effect. Injections of NT-4, but not GDNF, in the retina decreased the survival of ION neurons and accelerated cell death in the ION. NT-4-like immunoreactivity was present in the retina and the ION. Exogenous, radiolabeled NT-4, but not GDNF or IGF-I, was retrogradely transported from the retina to the ION. NT-4 transport was significantly reduced by coinjection of excess cold nerve growth factor (NGF), indicating that the majority of NT-4 bound to p75 neurotrophin receptors during axonal transport. Binding of NT-4 to chick p75 receptors was confirmed in L-cells, which express chick p75 receptors. These data indicate that GDNF has no direct trophic effects on ION neurons. IGF-I may be an afferent trophic factor for the ION, and NT-4 may act as an antagonist to BDNF, either by competing with BDNF for p75 and/or trkB binding or by signaling cell death via p75.     

Pseudorabies virus Us9 directs axonal sorting of viral capsids

Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.     

Differential effects of the trophic factors BDNF,NT‐4, GDNF,and IGF‐I on the isthmo‐optic nucleus in chick embryos

The isthmo‐optic nucleus (ION) of chick embryos is a model system for the study of retrograde trophic signaling in developing CNS neurons. The role of brain‐derived neurotrophic factor (BDNF) is well established in this system. Recent work has implicated neurotrophin‐4 (NT‐4), glial cell line–derived neurotrophic factor (GDNF), and insulin‐like growth factor I (IGF‐I) as additional trophic factors for ION neurons. Here it was examined in vitro and in vivo whether these factors are target‐derived trophic factors for the ION in 13‐ to 16‐day‐old chick embryos. Unlike BDNF, neither GDNF, NT‐4, nor IGF‐I increased the survival of ION neurons in dissociated cultures identified by retrograde labeling with the fluorescent tracer DiI. BDNF and IGF‐I promoted neurite outgrowth from ION explants, whereas GDNF and NT‐4 had no effect. Injections of NT‐4, but not GDNF, in the retina decreased the survival of ION neurons and accelerated cell death in the ION. NT‐4–like immunoreactivity was present in the retina and the ION. Exogenous, radiolabeled NT‐4, but not GDNF or IGF‐I, was retrogradely transported from the retina to the ION. NT‐4 transport was significantly reduced by coinjection of excess cold nerve growth factor (NGF), indicating that the majority of NT‐4 bound to p75 neurotrophin receptors during axonal transport. Binding of NT‐4 to chick p75 receptors was confirmed in L‐cells, which express chick p75 receptors. These data indicate that GDNF has no direct trophic effects on ION neurons. IGF‐I may be an afferent trophic factor for the ION, and NT‐4 may act as an antagonist to BDNF, either by competing with BDNF for p75 and/or trkB binding or by signaling cell death via p75. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 289–303, 2000     

Neurotrophic factor regulation of developing avian oculomotor neurons: differential effects of BDNF and GDNF.

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target-derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) may be target-derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio-iodinated BDNF, NT-3, NT-4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT-3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT-3, and NT-4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75(NTR) and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target.     

Kinesin-2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre- and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule-dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin-2 motor subunit Klp64D and separate siRNA-mediated knock-outs of all the three kinesin-2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin-2 subunits but not with each other. Altogether, these suggest that kinesin-2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors.     

Growth factors in Parkinson's disease

The etiology of Parkinson's disease, one of the most frequent neurodegenerative disorders in human, is unknown. New hopes concerning satisfactory therapies include transplants of autologous adrenal medullary chromaffin tissue, fetal mesencephalic dopaminergic neurons, and local application of growth factors with a neurotrophic capacity. A large body of evidence supports the notion that neurons require trophic support not only during a limited period of ontogenesis, but during their whole lifespan. Relevant molecules promote survival, transmitter synthesis and other differentiated properties, and become crucially important when a neuron is metabolically or toxically impaired. Several molecules, most of which occur in the striatum and the substantia nigra, have been identified that protect lesioned dopaminergic nigrostriatal neurons in culture or in animal models of Parkinson's disease. These include members of the neurotrophin, fibroblast growth factor, and insulin-like growth factor families as well as epidermal growth factor/transforming growth factor alpha, interleukins and ciliary neurotrophic factor. Whether their effects are merely pharmacological, or reflect a physiological role in the nigrostriatal system, is unclear as yet. This article reviews experiments that document the trophic effects of these factors on dopaminergic neurons and discusses their possible physiological and therapeutic relevance.     

The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

On the role of glucocorticoid receptors in brain plasticity

Summary 1. The mapping of glucocorticoid receptors (GR) in the rat central nervous system (CNS) has demonstrated their widespread presence in large numbers of nerve and glial cell populations also outside the classical stress regions.2. The present paper summarizes the evidence that glucocorticoids via GR in the CNS can act as lifelong organizing signals from development to aging. The following examples are given. (a) In the prepubertal and adult offspring, prenatal corticosterone treatment can produce long-lasting changes in striatal dopaminergic communication. (b) In adulthood, the evidence suggests complex regulation by adrenocortical hormones of neurotrophic factors and their receptors in the hippocampal formation. (c) In aging, the strongly GR-immunoreactive pyramidal cell layer of the CA1 hippocampal area appears to be preferentially vulnerable to neurotoxic actions of glucocorticoids, especially in some rat strains.3. Strong evidence suggests that each nerve cell in the CNS is supported by a trophic unit, consisting of other nerve cells and glial cells, blood vessels, and extracellular matrix molecules. Due to multiple actions on nerve and glial cell populations of the different trophic units, the glucocorticoids may exert either an overall trophic or a neurotoxic action. It seems likely that with increasing age, the endangering actions of glucocorticoids on nerve cells prevail over the neurotrophic ones, leading to reduced nerve cell survival in some trophic units.     

Neurotrophic regulation of the development and function of the neuromuscular synapses

Recent studies have established that one of the major functions of neurotrophic factors is to regulate synaptic development and plasticity. This owes a great deal to the studies using the neuromuscular junction (NMJ) as a model system. In this review, we summarize the effects of various neurotrophic factors on the development and function of the neuromuscular synapses. We describe experiments addressing the role of neurotrophins, as well as that of other factors (GFLs, TGF-βs, and Wnts). The synaptic effects of neurotrophic factors are divided into two categories: acute effects on synaptic transmission and plasticity occurring within seconds or minutes after cells are exposed to a particular factor, and long-term regulation of synaptic structure and function that takes days to accomplish. We consider the presynaptic effects on the release of the neurotransmitter ACh, as well as the postsynaptic effects on the clustering of ACh receptors. Further studies of the mechanisms underlying these regulatory effects will help us better understand how neurotrophic factors can achieve diverse and synapse-specific modulation in the brain.     

Simultaneous GDNF and BDNF application leads to increased motoneuron survival and improved functional outcome in an experimental model for obstetric brachial plexus lesions

Motoneurons of the neonate rat respond to proximal axonal injury with morphologic and functional changes and ultimately with neuronal death. Recent studies showed that both glial cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) reduce induced degeneration of motoneurons after axotomy and avulsion. Whether rescued motoneurons are functionally intact has been argued. In the present investigation, the authors have used a proximal crush lesion of the brachial plexus in neonatal rats as the experimental model of neuronal injury. This allowed the authors to study the effects of trophic factor administration on injured motoneurons and the relationship between motoneuron survival and extremity function. Trophic factors were locally released by small polymer implants in a low-dose slow-release mode. Six groups of 10 animals were prepared: BDNF, GDNF, GDNF/BDNF, control, sham, and normals. The number of surviving motoneurons was determined by retrograde tracer techniques using Fluorogold and Fastblue. Extremity function was quantitatively evaluated with functional muscle testing at day 56. The results of this study demonstrate that trophic factors applied separately had no effect, whereas combined trophic factor application (GDNF/BDNF group) had a dramatic rescue effect on motoneuron survival as compared with the control groups, which also effected significantly greater strength. The authors conclude that a combination of trophic factors leads to enhanced motoneuron survival, with improved voluntary function as the animal enters adulthood so that exogenous trophic support of motoneurons might have a role in the treatment of all types of severe neonatal plexopathies, maintaining the viability of motoneurons until reconstructive surgery provides them with a pathway for regeneration and endogenous trophic support.     

Neurotrophic factor regulation of developing avian oculomotor neurons: Differential effects of BDNF and GDNF

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target‐derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain‐derived neurotrophic factor (BDNF), neurotrophin 3 (NT‐3), neurotrophin 4 (NT‐4), and glial cell line–derived neurotrophic factor (GDNF) may be target‐derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio‐iodinated BDNF, NT‐3, NT‐4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT‐3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT‐3, and NT‐4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75^NTR and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 295–315, 1999