Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury

Over a half a century of research has confirmed that neurotrophic factors promote the survival and process outgrowth of isolated neurons in vitro. The mechanisms by which neurotrophic factors mediate these survival-promoting effects have also been well characterized. In vivo, peripheral neurons are critically dependent on limited amounts of neurotrophic factors during development. After peripheral nerve injury, the adult mammalian peripheral nervous system responds by making neurotrophic factors once again available, either by autocrine or paracrine sources. Three families of neurotrophic factors were compared, the neurotrophins, the GDNF family of neurotrophic factors, and the neuropoetic cytokines. Following a general overview of the mechanisms by which these neurotrophic factors mediate their effects, we reviewed the temporal pattern of expression of the neurotrophic factors and their receptors by axotomized motoneurons as well as in the distal nerve stump after peripheral nerve injury. We discussed recent experiments from our lab and others which have examined the role of neurotrophic factors in peripheral nerve injury. Although our understanding of the mechanisms by which neurotrophic factors mediate their effects in vivo are poorly understood, evidence is beginning to emerge that similar phenomena observed in vitro also apply to nerve regeneration in vivo.     

Cellular localization of androgen and estrogen receptors in mouse-derived motoneuron hybrid cells and mouse facial motoneurons

The ability of gonadal steroid hormones to augment axonal regeneration after peripheral nerve injury has been well established in rat and hamster motoneuron systems, and provides a foundation for the use of these agents as neurotherapeutics. With the advent of mouse genetics and the availability of transgenic and knockout mice, the use of mice in studies of neuroprotection is growing. It has recently been demonstrated that both androgens and estrogens rescue motoneurons (MN) from injury in mouse-derived motoneuron hybrid cells in vitro and mouse facial motoneurons (FMN) in vivo (Tetzlaff et al. [2006] J Mol Neurosci 28:53-64). To elucidate the molecular mechanisms of these effects, the present study examined the cellular localization of androgen and estrogen receptors in mouse MN in vitro and in vivo. Immunoblotting and immunocytochemistry studies established the presence of androgen receptor (AR) and estrogen receptor alpha/beta in immortalized mouse motoneuron hybrid cells and AR and estrogen receptor alpha in mouse FMN.     

Interactions between retinoic acid, nerve growth factor and sonic hedgehog signalling pathways in neurite outgrowth

Many studies have shown a role of retinoid signalling in neurite outgrowth in vitro, and that the retinoic acid receptor (RAR) beta2 is critical for this process. We show here that RARbeta2 is expressed predominantly in dorsal root ganglia (DRG) neuronal subtypes that express neurofilament (NF) 200 and calcitonin gene-related peptide (CGRP), and that these neurons extend neurites in response to RA. We demonstrate that retinoid signalling has a role in neurite outgrowth in vivo, by showing that in a peripheral nerve crush model there is less neurite outgrowth from RARbeta null DRG compared to wild-type. We identify sonic hedgehog (Shh) as a downstream target of the RARbeta2 signalling pathway as it is expressed in the injured DRG of wild-type but not RARbeta null mice. This regulation is direct as when RARbeta2 is overexpressed in adult motoneurons Shh is induced in them. Finally we show that Shh alone cannot induce neurite outgrowth but potentiates RARbeta2 signalling in this process.     

Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury.

Interactions between ingrowing nerve fibers and their target tissues form the basis for functional connectivity with the central nervous system. Studies of the developing dental pulp innervation by nerve fibers from the trigeminal ganglion is an excellent example of nerve-target tissue interactions and will allow specific questions regarding development of the dental pulp nerve system to be addressed. Dental pulp cells (DPC) produce an array of neurotrophic factors during development, suggesting that these proteins might be involved in supporting trigeminal nerve fibers that innervate the dental pulp. We have established an in vitro culture system to study the interactions between the dental pulp cells and trigeminal neurons. We show that dental pulp cells produce several neurotrophic factors in culture. When DPC are cocultured with trigeminal neurons, they promote survival and a specific and elaborate neurite outgrowth pattern from trigeminal neurons, whereas skin fibroblasts do not provide a similar support. In addition, we show that dental pulp tissue becomes innervated when transplanted ectopically into the anterior chamber of the eye in rats, and upregulates the catecholaminergic nerve fiber density of the irises. Interestingly, grafting the dental pulp tissue into hemisected spinal cord increases the number of surviving motoneurons, indicating a functional bioactivity of the dental pulp-derived neurotrophic factors in vivo by rescuing motoneurons. Based on these findings, we propose that dental pulp-derived neurotrophic factors play an important role in orchestrating the dental pulp innervation.     

P311 accelerates nerve regeneration of the axotomized facial nerve

In axotomized adult neurons, a process of axonal regrowth and re-establishment of the neuronal function has to be activated. Developmentally regulated factors may be reactivated during neuronal regeneration. Here we identify a gene, previously designated P311, that is up-regulated in the axotomized facial motoneurons. Ectopically expressed P311 localizes in the cytoplasm and the nucleus. Over-expression of P311 induces p21(WAF1/Cip1) expression, leading PC12 cells to differentiate and to have neuron-like morphologies. Adenovirus-mediated P311 gene transfer promotes neurite outgrowth of postnatal dorsal root ganglion neurons and embryonic hippocampal neurons in vitro. This effect is abolished by the activation of Rho kinase. P311 also facilitates nerve regeneration following facial nerve injury in vivo. Our data provide evidence that genes involved in the differentiation process contribute to the regeneration of injured mature neurons, and may provide a practical molecular target.     

Different subsets of axonal guidance cues are essential for sensory neurite outgrowth to cutaneous and muscle targets in the dorsal ramus of the embryonic chick

The dorsal ramus nerve diverges dorsally from each spinal nerve to innervate the epaxial muscle and dermis that are derived in situ from each dermamyotome. The outgrowth of both the sensory and motor components of this nerve are sensitive to the proximity of the dermamyotome. Motoneurons display a direct target response that is not dependent upon the concurrent outgrowth of sensory neurites (Tosney: Dev. Biol. 122:540-588, 1987). Likewise, the outgrowth of sensory neurites could be directly dependent on the dermamyotome. Alternatively, sensory neurites could be dependent on motor axons that in turn require the dermamyotome for outgrowth. To distinguish between these possibilities, motor outgrowth was abolished by unilateral ventral neural tube deletion and the patterns of subsequent sensory neurite outgrowth were assessed. The cutaneous nerve branch formed in all cases. In contrast, neither of the epaxial muscle nerves formed in the absence of epaxial motoneuron outgrowth. Furthermore, sensory neurites could not be detected diverging into muscle from the cutaneous nerve or entering muscle via other novel routes. We conclude that motoneurons are essential for sensory outgrowth to epaxial muscle but not to cutaneous targets. It is clear that different subsets of navigational cues guide sensory afferents to muscle and to cutaneous destinations.     

Shared and unique roles of CAP23 and GAP43 in actin regulation, neurite outgrowth, and anatomical plasticity

CAP23 is a major cortical cytoskeleton-associated and calmodulin binding protein that is widely and abundantly expressed during development, maintained in selected brain structures in the adult, and reinduced during nerve regeneration. Overexpression of CAP23 in adult neurons of transgenic mice promotes nerve sprouting, but the role of this protein in process outgrowth was not clear. Here, we show that CAP23 is functionally related to GAP43, and plays a critical role to regulate nerve sprouting and the actin cytoskeleton. Knockout mice lacking CAP23 exhibited a pronounced and complex phenotype, including a defect to produce stimulus-induced nerve sprouting at the adult neuromuscular junction. This sprouting deficit was rescued by transgenic overexpression of either CAP23 or GAP43 in adult motoneurons. Knockin mice expressing GAP43 instead of CAP23 were essentially normal, indicating that, although these proteins do not share homologous sequences, GAP43 can functionally substitute for CAP23 in vivo. Cultured sensory neurons lacking CAP23 exhibited striking alterations in neurite outgrowth that were phenocopied by low doses of cytochalasin D. A detailed analysis of such cultures revealed common and unique functions of CAP23 and GAP43 on the actin cytoskeleton and neurite outgrowth. The results provide compelling experimental evidence for the notion that CAP23 and GAP43 are functionally related intrinsic determinants of anatomical plasticity, and suggest that these proteins function by locally promoting subplasmalemmal actin cytoskeleton accumulation.     

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.     

FGF-2 Low Molecular Weight Selectively Promotes Neuritogenesis of Motor Neurons In Vitro

In this study, we screened in vitro the different capabilities of trophic factors with promising effect for enhancing selective regeneration and thus promoting specific reinnervation of target organs after peripheral nerve regeneration. We found that FGF-2 (18 kDa) was the trophic factor that exerted the most selective effect in promoting neurite outgrowth of spinal motoneurons both in terms of elongation and arborization. The mechanism underlying this effect on neuritogenesis seems related to FGF-2 enhancing the interaction between FGFR-1 and PSA-NCAM. The interaction of these two receptors is important during the early stages of neuritogenesis and pathfinding, while integrin alpha7B subunit seems to play a role during neurite stabilization.     

Guidance of regenerating motor axons in vivo by gradients of diffusible peripheral nerve-derived factors

During development of the central nervous system, neurons rely on target-derived factors to guide their outgrowing processes. Several CNS target-derived chemoattractive and repellent factors have been isolated and characterized, and their mechanism of action determined. For the peripheral nervous system, the results from numerous experiments suggest that during regeneration axons also respond to concentration gradients of target-derived factors leading to an oriented outgrowth up the gradient to the denervated target in vivo. The results from in vitro experiments have shown that diffusible concentration gradients of factors released from a length of denervated peripheral nerve, composed predominantly of Schwann cells, direct the outgrowth of sensory and motor neuron growth cones over distances of several hundred microns. However, a conclusive demonstration of a chemoattractive influence of diffusible concentration gradients on regenerating adult motor axons in vivo has remained elusive. The present experiments show that concentration gradients of denervated peripheral nerve-released factors direct the regeneration of adult motor axons in vivo, and that these gradients are effective over distances of more than 6.5 mm. Nonconditioned medium exerted no influence on the regenerating axons. Thus, results from in vivo experiments parallel those from in vitro experiments and indicate that isolated peripheral nerve-released factors that are effective in vitro will play a similar role on sensory and motor axons in vivo. Finally, the results show that diffusible concentration gradients of target-derived factors direct axon outgrowth both during both development and regeneration, as well as in vivo and in vitro.     

GDNF及BDNF对受损运动神经元的长期修复

为了研究胶质细胞源神经营养因子（ＧＤＮＦ） 及脑源神经营养因子（ＢＤＮＦ） 对切断轴突的新生运动神经元的长期维持存活及促进神经再生的作用, 我们选用出生时单侧切断坐骨神经的雏鸡模型, 用裸ＤＮＡ 转染方法, 在损伤神经附近的肌肉中转染ＧＤＮＦ ｃＤＮＡ 和ＢＤＮＦ ｃＤＮＡ 的真核表达载体,观察在体表达的神经营养因子对损伤的修复作用。结果显示,在体表达的ＧＤＮＦ 在８ 周内能使切断坐骨神经的腰脊髓运动神经元近９０ ％ 维持存活。切断的坐骨神经从断端向远体端再生,最长再生达９ ．５ｍ ｍ 。表达两个因子比单独表达ＧＤＮＦ 对运动神经元的存活无显著性差异。而两个因子协同作用对坐骨神经的再生更为有效,坐骨神经再生最长的可达１５ ．４ｍ ｍ 。     

Androgen-sensitivity of somata and dendrites of spinal nucleus of the bulbocavernosus (SNB) motoneurons in male C57BL6J mice

In rats, androgens in adulthood regulate the morphology of motoneurons in the spinal nucleus of the bulbocavernosus (SNB), including the size of their somata and the length of their dendrites. There are conflicting reports about whether androgens exert similar influences on SNB motoneurons in mice. We castrated or sham-operated C57BL6J mice at 90 days of age and, thirty days later, injected cholera toxin conjugated horseradish peroxidase into the bulbocavernosus muscle (to label SNB motoneurons) on one side, and into intrinsic foot muscles contralaterally (to label motoneurons of the retrodorsolateral nucleus (RDLN)). Castrated mice had significantly smaller SNB somas compared to sham-operated mice while there were no differences in soma size of RDLN motoneurons. Dendritic length in C57BL6J mice, estimated in 3-dimensions, also decreased significantly after adult castration. In rats, androgens act directly through androgen receptors (AR) in SNB motoneurons to control soma size and nearly all SNB motoneurons contain AR. Since SNB somata in C57BL6J mice shrank after adult castration, we used immunocytochemistry to characterize AR expression in SNB cells as well as motoneurons in the RDLN and dorsolateral nucleus (DLN). A pattern of labeling matched that seen previously in rats: the highest percentage of AR-immunoreactive motoneurons are in the SNB (98%), the lowest in the RDLN (25%) and an intermediate number in the DLN (78%). This pattern of AR labeling is consistent with the possibility that androgens also act directly on SNB motoneurons in mice to regulate soma size in mice.     

Somatic and axonal LIGHT signaling elicit degenerative and regenerative responses in motoneurons,respectively

A receptor–ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type‐specific post‐receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death‐triggering factor in motoneurons through LT‐βR, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT‐induced axonal elongation and branching require ERK and caspase‐9 pathways. This distinct response involves a compartment‐specific activation of LIGHT signals, with somatic activation‐inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons.     

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.     

Growth and differentiation of PC6 cells: the effects of pulsed electromagnetic fields (PEMF)

Previous studies in our laboratory showed that neurite outgrowth in vitro and nerve regeneration in vivo were stimulated by 2 Hz, 0.3 mT (3 G) pulsed electromagnetic fields (PEMF). To learn more about the effects of PEMF on nerve cells, we exposed PC6 cells, a standard neuronal-like cell model, to the same pulsed electromagnetic fields for 2 h/day for 2 days and asked whether two different cell processes, proliferation and differentiation, were affected. The cells were also treated with a differentiating agent, nerve growth factor (NGF), to further define any interactive effects. We found that proliferation was unaffected by either PEMF or NGF alone or in combination. Differentiation, expressed as neurite outgrowth, was strongly upregulated with NGF, but this NGF response was significantly depressed in cells treated with PEMF.     

Inhibition of Neurite Outgrowth by a Neuropilin-1 Binding Peptide Derived from Semaphorin 3A

Semaphorin 3A (Sema3A), an axon guidance molecule, inhibits neurite outgrowth of sensory neurons. Recombinant Sema3A protein has also inhibited scratching behavior and improved skin inflammation in an atopic dermatitis model. In the present study, we investigated whether Sema3A-derived peptides could bind its receptor, neuropilin-1 (NRP1), to inhibit neurite outgrowth. Here, two candidate NRP1-binding (NPB) peptides, NPB7 and NPB15, were found to inhibit NGF-induced survival and neurite outgrowth of PC12 cells and rat primary neurons in serum-free medium. To investigate the preventive effect of the two NPB peptides in vivo, we assessed whether they could inhibit skin inflammation induced by repeated topical application of oxazolone in mice. NPB15 peptide, but not NPB7, inhibited ear swelling. The NPB15 peptide solution and Vaseline ointment groups showed slightly decreased epidermal nerve densities compared with controls. The combination of NPB15 peptide and Vaseline ointment increased the inhibitory effect of NPB15 peptide on epidermal nerve densities. These results suggest that Sema3A-derived peptides can bind to NRP1 and inhibit neurite outgrowth both in vitro and in vivo. Thus, these peptides may be potent candidates for the treatment of atopic dermatitis.     

Respecification of larval proleg motoneurons during metamorphosis of the tobacco hornworm, Manduca sexta: segmental dependence and hormonal regulation

The principal locomotory appendages of the Manduca sexta caterpillar, the prolegs, are present on the third through sixth abdominal segments (anal prolegs located on the terminal segment were not included in this study). Previous studies have characterized some of the proleg retractor muscles and their motoneurons. In the present study we identified additional proleg motoneurons and their putative homologs in the non-proleg-bearing segments. One of the motoneurons present in the proleg-bearing segments is absent in the non-proleg-bearing segments. At pupation the prolegs are lost, their muscles degenerate, and some of their motoneurons regress structurally. Subsequently, subsets of the proleg motoneurons and their homologs in other segments die in a segment-specific pattern. This is the first report of segment-specific motoneurons, and of segment-specific death of identified motoneurons, in Manduca. During adult development the surviving proleg motoneurons innervate the tergosternal muscle (TSM) and grow bilateral dendritic arbors. Dendritic growth is completed by about the 12th of the 18 days of adult development. Following adult emergence all but one of the respecified proleg motoneurons dies. The hormonal dependence of dendritic outgrowth was tested by isolating abdomens to eliminate the ecdysteroid-secreting glands in the thorax. Between the second and fifth days after pupation the motoneurons became progressively more competent to undergo dendritic outgrowth following abdomen isolation. The extent of dendritic outgrowth paralleled the degree of morphological development attained by isolated abdomens. It is concluded that ecdysteroids are required for motoneuron outgrowth, but our findings suggest that, unless an abdominal source of ecdysteroids exists in pupae, a relatively small exposure may be sufficient.     

Fas engagement induces neurite growth through ERK activation and p35 upregulation

Fas (also known as CD95), a member of the tumour-necrosis receptor factor family of 'death receptors', can induce apoptosis or, conversely, can deliver growth stimulatory signals. Here we report that crosslinking Fas on primary sensory neurons induces neurite growth through sustained activation of the extracellular-signal regulated kinase (ERK) pathway and the consequent upregulation of p35, a mediator of neurite outgrowth. In addition, functional recovery after sciatic nerve injury is delayed in Fas-deficient lpr mice and accelerated by local administration of antibodies against Fas, which indicates that Fas engagement may contribute to nerve regeneration in vivo. Our findings define a role for Fas as an inducer of both neurite growth in vitro and accelerated recovery after nerve injury in vivo.     

Prostaglandin J2 and its metabolites promote neurite outgrowth induced by nerve growth factor in PC12 cells

Although A- and J-type prostaglandins (PG's) arrest the cell cycle at the G1 phase in vitro and suppress tumor growth in vivo, their effects on neuronal cells have not so far been clarified. Here, we found promotion of neurite outgrowth as a novel biological function of PGJ's. In PC12h cells, PGJ's (PGJ2, Delta12-PGJ2 and 15-deoxy-Delta12,14-PGJ2) promoted neurite outgrowth in the presence of nerve growth factor (NGF), whereas they themselves did not show such a promotion. The potency of promoting neurite outgrowth was PGJ2 < Delta12-PGJ2 < 15-deoxy-Delta12,14-PGJ2. However, troglitazone, an activator of peroxisome proliferator-activated receptorgamma (PPARgamma), and other PG's including PGA1, PGA2 and PGD2 did not promote neurite outgrowth. These results suggest that PGJ's promote neurite outgrowth independently of PPARgamma activation.     

Electromagnetic field treatment of nerve crush injury in a rat model: effect of signal configuration on functional recovery

Electromagnetic fields (EMFs) have been demonstrated to enhance mammalian peripheral nerve regeneration in vitro and in vivo. Using an EMF signal shown to enhance neurite outgrowth in vitro, we tested this field in vivo using three different amplitudes. The rat sciatic nerve was crushed. Whole body exposure was performed for 4 h/day for 5 days in a 96-turn solenoid coil controlled by a signal generator and power amplifier. The induced electric field at the target tissue consisted of a bipolar rectangular pulse, having 1 and 0.3 ms durations in each polarity, respectively. Pulse repetition rate was 2 per second. By varying the current, the coils produced fields consisting of sham (no current) and peak magnetic fields of 0.03 mT, 0.3 mT, and 3 mT, corresponding to peak induced electric fields of 1, 10, and 100 microV/cm, respectively, at the tissue target. Walking function was assessed over 43 days using video recording and measurement of the 1-5 toe-spread, using an imaging program. Comparing injured to uninjured hind limbs, mean responses were evaluated using a linear mixed statistical model. There was no difference found in recovery of the toe-spread function between any EMF treatments compared to sham.