Neurotrophic factors in combinatorial approaches for spinal cord regeneration

Axonal regeneration is inhibited by a plethora of different mechanisms in the adult central nervous system (CNS). While neurotrophic factors have been shown to stimulate axonal growth in numerous animal models of nervous system injury, a lack of suitable growth substrates, an insufficient activation of neuron-intrinsic regenerative programs, and extracellular inhibitors of regeneration limit the efficacy of neurotrophic factor delivery for anatomical and functional recovery after spinal cord injury. Thus, growth-stimulating factors will likely have to be combined with other treatment approaches to tap into the full potential of growth factor therapy for axonal regeneration. In addition, the temporal and spatial distribution of growth factors have to be tightly controlled to achieve biologically active concentrations, to allow for the chemotropic guidance of axons, and to prevent adverse effects related to the widespread distribution of neurotrophic factors. Here, we will review the rationale for combinatorial treatments in axonal regeneration and summarize some recent progress in promoting axonal regeneration in the injured CNS using such approaches.     

Insulin-Like Growth Factor-1 Enhances Motoneuron Survival and Inhibits Neuroinflammation After Spinal Cord Transection in Zebrafish

Cellular and Molecular Neurobiology - Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor produced locally in the central nervous system which can promote axonal regeneration, protect...     

Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.     

Role of neurotrophic factors in development

Neurotrophic factors are molecules which promote and regulate neuronal survival in the developing nervous system. They are distinguished from ubiquitous metabolites necessary for cellular maintenance and growth by their specificity: each neurotrophic factor promotes the survival of only certain kinds of neurons during a particular stage of their development. In addition, it has been argued that neurotrophic factors are involved in many other aspects of neuronal development ranging from axonal guidance to regulation of neurotransmitter synthesis. Recent developmental studies and the use of specific molecular probes have greatly clarified the role of these molecules.     

Vascular and neuronal effects of VEGF in the nervous system: implications for neurological disorders

Vascular endothelial growth factor (VEGF) was originally discovered as an endothelial-specific growth factor. While the predominant role of this growth factor in the formation of new blood vessels (angiogenesis) is unquestioned, recent observations indicate that VEGF also has direct effects on neurons and glial cells, and stimulates their growth, survival and axonal outgrowth. Because of these pleiotropic effects, VEGF has now been implicated in several neurological disorders both in the preterm infant (leukomalacia) and the adult (stroke, neurodegeneration, cerebral and spinal trauma, ischemic and diabetic neuropathy, nerve regeneration). A challenge for the future is to unravel to what extent the effect of VEGF in these disorders relates to its angiogenic activity or direct neurotrophic effect.     

RasGRF1 mediates brain-derived neurotrophic factor-induced axonal growth in primary cultured cortical neurons

The appropriate development and regulation of neuronal morphology are important to establish functional neuronal circuits and enable higher brain function of the central nervous system. R-Ras, a member of the Ras family of small GTPases, plays crucial roles in the regulation of axonal morphology, including outgrowth, branching, and guidance. GTP-bound activated R-Ras reorganizes actin filaments and microtubules through interactions with its downstream effectors, leading to the precise control of axonal morphology. However, little is known about the upstream regulatory mechanisms for R-Ras activation in neurons. In this study, we found that brain-derived neurotrophic factor (BDNF) has a positive effect on endogenous R-Ras activation and promotes R-Ras-mediated axonal growth. RNA interference knockdown and overexpression experiments revealed that RasGRF1, a guanine nucleotide exchange factor (GEF) for R-Ras, is involved in BDNF-induced R-Ras activation and the promotion of axonal growth. Phosphorylation of RasGRF1 by protein kinase A at Ser916/898 is needed for the full activation of its GEF activity and to facilitate Ras signaling. We observed that BDNF treatment markedly increased this phosphorylation. Our results suggest that BDNF is one of the critical extrinsic regulators for R-Ras activation, and that RasGRF1 is an intrinsic key mediator for BDNF-induced R-Ras activation and R-Ras-mediated axonal morphological regulation.     

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.     

The Neuroprotective Effect of Conditioned Medium from Human Adipose-Derived Mesenchymal Stem Cells is Impaired by N-acetyl Cysteine Supplementation

Oxidative stress is a common feature in neurodegenerative diseases associated with neuroinflammation, and therefore, has been proposed as a key target for novel therapies for these diseases. Recently, adipose-derived stem cell (ASC)-based cell therapy has emerged as a novel strategy for neuroprotection. In this study, we evaluate the therapeutic role of ASC-conditioned medium (ASC-CM) against H₂O₂-induced neurotoxicity in a new in vitro model of ec23/brain-derived neurotrophic factor (BDNF)-differentiated human SH-SY5Y neuron-like cells (SH-SY5Yd). In the presence of ASC-CM, stressed SH-SY5Yd cells recover normal axonal morphology (with an almost complete absence of H₂O₂-induced axonal beading), electrophysiological features, and cell viability. This beneficial effect of ASC-CM was associated with its antioxidant capacity and the presence of growth factors, namely, BDNF, glial cell line-derived neurotrophic factor, and transforming growth factor β1. Moreover, the neuroprotective effect of ASC-CM was very similar to that obtained from treatment with BDNF, an essential factor for SH-SY5Yd cell survival. Importantly, we also found that the addition of the antioxidant agent N-acetyl cysteine to ASC-CM abolished its restorative effect; this was associated with a strong reduction in reactive oxygen species (ROS), in contrast to the moderate decrease in ROS produced by ASC-CM alone. These results suggest that neuronal restorative effect of ASC-CM is associated with not only the release of essential neurotrophic factors, but also the maintenance of an appropriate redox state to preserve neuronal function.     

Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF)

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.     

New insights into the control of neurotrophic growth factor receptor signaling: Implications for nervous system development and repair

Neurotrophic growth factors control neuronal development by activating specific receptor tyrosine kinase positive signaling pathways, such as Ras‐MAPK and PI3K‐Akt cascades. Once activated, neurotrophic factor receptors also trigger a cascade of molecular events, named negative receptor signaling, that restricts the intensity of the positive signals and modulates cellular behavior. Thus, to avoid signaling errors that ultimately could lead to aberrant neuronal physiology and disease, negative signaling mechanisms have evolved to ensure that suitable thresholds of neuronal stimulation are achieved and maintained during right periods of time. Recent findings have revealed that neurotrophic factor receptor signaling is tightly modulated through the coordinated action of many different protein regulators that limit or potentiate signal propagation in spatially and temporally controlled manners, acting at specific points after receptor engagement. In this review, we discuss progress in this field, highlighting the importance of these modulators in axonal growth, guidance, neural connectivity, and nervous system regeneration.     

Masseteric Nerve Injury Increases Expression of Brain-Derived Neurotrophic Factor in Microglia Within the Rat Mesencephalic Trigeminal Tract Nucleus

The distribution of brain-derived neurotrophic factor was examined in the rat mesencephalic trigeminal tract nucleus after transection and crush of the masseteric nerve. In the intact mesencephalic trigeminal tract nucleus, brain-derived neurotrophic factor was detected in small cells with fine processes. These cells and processes were occasionally located adjacent to tyrosine kinase B receptor-immunoreactive sensory neurons. The transection and crush of the masseteric nerve increased expression of brain-derived neurotrophic factor in the nucleus. The number and size of brain-derived neurotrophic factor-immunoreactive cells and processes were dramatically elevated by the nerve injury. As a result, the density of brain-derived neurotrophic factor-immunoreactive profiles in the mesencephalic trigeminal tract nucleus at 7 days after the injury was significantly higher compared with the intact nucleus. Double immunofluorescence method also revealed that brain-derived neurotrophic factor-immunoreactive cells were mostly immunoreactive for OX-42 but not glial fibrillary acidic protein. In addition, the retrograde tracing method demonstrated that brain-derived neurotrophic factor-immunoreactive cells and processes surrounded retrogradely labeled neurons which showed tyrosine kinase B receptor-immunoreactivity. These findings indicate that the nerve injury increases expression of brain-derived neurotrophic factor in microglia within the mesencephalic trigeminal tract nucleus. The glial neurotrophic factor may be associated with axonal regeneration of the injured primary proprioceptor in the trigeminal nervous system.     

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.     

Mitochondrial Regulation of Neuronal Plasticity

The structure and function of neurons is dynamic during development and in adaptive responses of the adult nervous system to environmental demands. The mechanisms that regulate neuronal plasticity are poorly understood, but are believed to involve neurotransmitter and neurotrophic factor signaling pathways. In the present article, I review emerging evidence that mitochondria play important roles in regulating developmental and adult neuroplasticity. In neurons, mitochondria are located in axons, dendrites, growth cones and pre- and post-synaptic terminals where their movements and functions are regulated by local signals such as neurotrophic factors and calcium influx. Mitochondria play important roles in fundamental developmental processes including the establishment of axonal polarity and the regulation of neurite outgrowth, and are also involved in synaptic plasticity in the mature nervous system. Abnormalities in mitochondria are associated with neurodegenerative and psychiatric disorders, suggesting a therapeutic potential for approaches that target mitochondrial mechanisms. Special issue dedicated to John P. Blass.     

Comparative analysis of brain neurite-stimulating protein and nerve growth factor using a culture of chick embryo sensory neurons

A study was made of brain neurite-stimulating proteins separated from lysosomal fractions of mammalian brain tissue. This protein stimulates axonal growth in sensory neurons in organotypic spinal ganglia culture. The physicochemical properties of neurite-stimulating protein differs from nerve growth factor — the familiar neurotrophic factor. Findings showed that nerve growth factor antiserum does not block the action of this protein. Accordingly, brain neuritestimulating protein separated in highly purified form was found to be a low molecular weight protein with the properties of nerve growth promoting factor. It can also be used in the study of conditions promoting sensory neuron ontogenesis and for stimulating regenerative processes within the nervous system.I. P. Kovlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. O. O. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 15–20, January–February, 1988.     

Differential effects of Rho GTPases on axonal and dendritic development in hippocampal neurones

Formation of neurites and their differentiation into axons and dendrites requires precisely controlled changes in the cytoskeleton. While small GTPases of the Rho family appear to be involved in this regulation, it is still unclear how Rho function affects axonal and dendritic growth during development. Using hippocampal neurones at defined states of differentiation, we have dissected the function of RhoA in axonal and dendritic growth. Expression of a dominant negative RhoA variant inhibited axonal growth, whereas dendritic growth was promoted. The opposite phenotype was observed when a constitutively active RhoA variant was expressed. Inactivation of Rho by C3-catalysed ADP-ribosylation using C3 isoforms (Clostridium limosum, C3(lim) or Staphylococcus aureus, C3(stau2)), diminished axonal branching. By contrast, extracellularly applied nanomolar concentrations of C3 from C. botulinum (C3(bot)) or enzymatically dead C3(bot) significantly increased axon growth and axon branching. Taken together, axonal development requires activation of RhoA, whereas dendritic development benefits from its inactivation. However, extracellular application of enzymatically active or dead C3(bot) exclusively promotes axonal growth and branching suggesting a novel neurotrophic function of C3 that is independent from its enzymatic activity.     

Neurotrophic factors in Alzheimer's disease: role of axonal transport

Neurotrophic factors (NTF) are small, versatile proteins that maintain survival and function to specific neuronal populations. In general, the axonal transport of NTF is important as not all of them are synthesized at the site of its action. Nerve growth factor (NGF), for instance, is produced in the neocortex and the hippocampus and then retrogradely transported to the cholinergic neurons of the basal forebrain. Neurodegenerative dementias like Alzheimer's disease (AD) are linked to deficits in axonal transport. Furthermore, they are also associated with imbalanced distribution and dysregulation of NTF. In particular, brain-derived neurotrophic factor (BDNF) plays a crucial role in cognition, learning and memory formation by modulating synaptic plasticity and is, therefore, a critical molecule in dementia and neurodegenerative diseases. Here, we review the changes of NTF expression and distribution (NGF, BDNF, neurotrophin-3, neurotrophin-4/5 and fibroblast growth factor-2) and their receptors [tropomyosin-related kinase (Trk)A, TrkB, TrkC and p75^NTR] in AD and AD models. In addition, we focus on the interaction with neuropathological hallmarks Tau/neurofibrillary tangle and amyloid-β (Abeta)/amyloid plaque pathology and their influence on axonal transport processes in order to unify AD-specific cholinergic degeneration and Tau and Abeta misfolding through NTF pathophysiology.     

Stimulating regeneration in the damaged spinal cord.

Great progress has been made in recent years in experimental strategies for spinal cord repair. In this review we describe two of these strategies, namely the use of neurotrophic factors to promote functional regeneration across the dorsal root entry zone (DREZ), and the use of synthetic fibronectin conduits to support directed axonal growth. The junction between the peripheral nervous system (PNS) and central nervous system (CNS) is marked by a specialized region, the DREZ, where sensory axons enter the spinal cord from the dorsal roots. After injury to dorsal roots, axons will regenerate as far as the DREZ but no further. However, recent studies have shown that this barrier can be overcome and function restored. In animals treated with neurotrophic factors, regenerating axons cross the DREZ and establish functional connections with dorsal horn cells. For example, intrathecal delivery of neurotrophin 3 (NT3) supports ingrowth of A fibres into the dorsal horn. This ingrowth is revealed using a transganglionic anatomical tracer (cholera toxin subunit B) and analysis at light and electron microscopic level. In addition to promoting axonal growth, spinal cord repair is likely to require strategies for supporting long-distance regeneration. Synthetic fibronectin conduits may be useful for this purpose. Experimental studies indicate that fibronectin mats implanted into the spinal cord will integrate with the host tissue and support extensive and directional axonal growth. Growth of both PNS and CNS axons is supported by the fibronectin, and axons become myelinated by Schwann cells. Ongoing studies are aimed at developing composite conduits and promoting axonal growth from the fibronectin back into the spinal cord.     

Prevention of thrombin-induced motoneuron degeneration with different neurotrophic factors in highly enriched cultures

Previous reports have shown that neuronal and glial cells express functionally active thrombin receptors. The thrombin receptor (PAR-1), a member of a growing family of protease activated receptors (PARs), requires cleavage of the extracellular amino-terminus domain by thrombin to induce signal transduction. Studies from our laboratory have shown that PAR-1 activation following the addition of thrombin or a synthetic thrombin receptor activating peptide (TRAP) induces motoneuron cell death both in vitro and in vivo. In addition to increasing motoneuron cell death, PAR- 1 activation leads to decreases in the mean neurite length and side branching in highly enriched motoneuron cultures. It has been suggested that motoneuron survival depends on access to sufficient target-derived neurotrophic factors through axonal branching and synaptic contacts. However, whether the thrombininduced effects on motoneurons can be prevented by neurotrophic factors is still unknown. Using highly enriched avian motoneuron cultures, we show here that alone, soluble chick skeletal muscle extracts (CMX), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF) significantly increased motoneuron survival compared to controls, whereas nerve growth factor (NGF) did not have a significant effect on motoneuron survival. Furthermore, cotreatment with muscle-derived agents (i.e., CMX, BDNF, GDNF) significantly prevented the death of motoneurons induced by alpha-thrombin. Yet, non-muscle-derived agents (CNTF and NGF) had little or no significant effect in reversing thrombin-induced motoneuron death. CMX and CNTF significantly increased the mean length of neurites, whereas NGF, BDNF, and GDNF failed to enhance neurite outgrowth compared to controls. Furthermore, CMX and CNTF significantly prevented thrombin-induced inhibition of neurite outgrowth, whereas BDNF and GDNF only partially reversed thrombin-induced inhibition of neurite outgrowth. These findings show differential effects of neurotrophic factors on thrombin-induced motoneuron degeneration and suggest specific overlaps between the trophic and stress pathways activated by some neurotrophic agents and thrombin, respectively.