A chemoattractant role for NT-3 in proprioceptive axon guidance

Neurotrophin-3 (NT-3) is required for proprioceptive neuron survival. Deletion of the proapoptotic gene Bax in NT-3 knockout mice rescues these neurons and allows for examination of their axon growth in the absence of NT-3 signaling. TrkC-positive peripheral and central axons from dorsal root ganglia follow proper trajectories and arrive in close proximity to their targets but fail to innervate them. Peripherally, muscle spindles are absent and TrkC-positive axons do not enter their target muscles. Centrally, proprioceptive axons branch in ectopic regions of the spinal cord, even crossing the midline. In vitro assays reveal chemoattractant effects of NT-3 on dorsal root ganglion axons. Our results show that survival factor NT-3 acts as a short-distance axon guidance molecule for muscle sensory afferents as they approach their proper targets.     

Smooth-muscle-specific expression of neurotrophin-3 in mouse embryonic and neonatal gastrointestinal tract

Vagal gastrointestinal (GI) afferents are essential for the regulation of eating, body weight, and digestion. However, their functional organization and the way that this develops are poorly understood. Neurotrophin-3 (NT-3) is crucial for the survival of vagal sensory neurons and is expressed in the developing GI tract, possibly contributing to their survival and to other aspects of vagal afferent development. The identification of the functions of this peripheral NT-3 thus requires a detailed understanding of the localization and timing of its expression in the developing GI tract. We have studied embryos and neonates expressing the lacZ reporter gene from the NT-3 locus and found that NT-3 is expressed predominantly in the smooth muscle of the outer GI wall of the stomach, intestines, and associated blood vessels and in the stomach lamina propria and esophageal epithelium. NT-3 expression has been detected in the mesenchyme of the GI wall by embryonic day 12.5 (E12.5) and becomes restricted to smooth muscle and lamina propria by E15.5, whereas its expression in blood vessels and esophageal epithelium is first observed at E15.5. Expression in most tissues is maintained at least until postnatal day 4. The lack of colocalization of β-galactosidase and markers for myenteric ganglion cell types suggests that NT-3 is not expressed in these ganglia. Therefore, NT-3 expression in the GI tract is largely restricted to smooth muscle at ages when vagal axons grow into the GI tract, and when vagal mechanoreceptors form in smooth muscle, consistent with its role in these processes and in vagal sensory neuron survival.     

Target specificity and size of avian sensory neurons supported in vitro by nerve growth factor,brain-derived neurotrophic factor,and neurotrophin-3

To obtain insight into which subpopulations of sensory neurons in dorsal root ganglia are supported by different neurotrophins, we retrogradely labeled cutaneous and muscle afferents in embryonic day 9 chick embryos and followed their survival in neuron-enriched cultures supplemented with either nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3). We found that NGF is a wide survival factor for subpopulations of both cutaneous and muscle afferents, whereas the survival effects of BDNF and NT-3 are restricted primarily to muscle afferents. We also measured soma size in each neurotrophic factor. These new data show that BDNF- and NT-3–dependent cells appear to be a mixture of two populations of neurons: one small diameter and the other large diameter. In contrast, based on size alone, NGF-dependent cells appear to be a single population of only small-diameter neurons. Thus, BDNF and NT-3 may have some new, previously unreported effects on small-diameter afferent neurons. © 1994 John Wiley & Sons, Inc. 1994 John Wiley & Sons, Inc.     

Rho-associated kinase (ROCK) inhibitor, Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurotrophins support neuronal survival and axonal regeneration after injury. To test whether local expression of Neurotrophin-3 (NT-3) would elicit axonal regeneration we lesioned the corticospinal tract (CST) at the level of the hindbrain and measured the number of axons that would grow from the unlesioned CST to the contralateral side where NT-3 was over expressed at the lumbar level of the spinal cord. An adenoviral vector that carried the rat NT-3 gene and the NGF signal peptide driven by the EF1α promoter (Adv.EF-NT-3) was used. This model enabled us to test the effects of NT-3 on axonal regeneration without confounding injury processes. Biotinylated dextran amine (BDA) was injected into the rat cortex on unlesioned side to mark CST axons 10 days postlesion. Adenoviral vectors (1 × 10⁹ pfu, Adv.EF-NT-3 or Adv.EF-LacZ) were delivered to lumbar spinal cord by retrograde transport from the sciatic nerve 4 days later. Histological examination 3 weeks later revealed that more BDA-labelled axons had grown from the unlesioned CST to the denervated side at the lumbar level. Morphometric measurements showed that a significantly larger number of BDA-labelled CST axons ( p  < 0.001) were present in the animals that were treated with Adv.EF-NT-3 than those treated with Adv.EF-LacZ. These data demonstrate that local expression of NT-3 will support axonal regeneration in the injured spinal cord without adverse effects and suggest that gene delivery of neurotrophins may be an effective strategy for nervous system repair after injury.
Acknowledgements:   Funded by NIH Grant NS35280 and by Mission Connect of the TIRR Foundation.     

Role of motoneuron-derived neurotrophin 3 in survival and axonal projection of sensory neurons during neural circuit formation

Sensory neurons possess the central and peripheral branches and they form unique spinal neural circuits with motoneurons during development. Peripheral branches of sensory axons fasciculate with the motor axons that extend toward the peripheral muscles from the central nervous system (CNS), whereas the central branches of proprioceptive sensory neurons directly innervate motoneurons. Although anatomically well documented, the molecular mechanism underlying sensory-motor interaction during neural circuit formation is not fully understood. To investigate the role of motoneuron on sensory neuron development, we analyzed sensory neuron phenotypes in the dorsal root ganglia (DRG) of Olig2 knockout (KO) mouse embryos, which lack motoneurons. We found an increased number of apoptotic cells in the DRG of Olig2 KO embryos at embryonic day (E) 10.5. Furthermore, abnormal axonal projections of sensory neurons were observed in both the peripheral branches at E10.5 and central branches at E15.5. To understand the motoneuron-derived factor that regulates sensory neuron development, we focused on neurotrophin 3 (Ntf3; NT-3), because Ntf3 and its receptors (Trk) are strongly expressed in motoneurons and sensory neurons, respectively. The significance of motoneuron-derived Ntf3 was analyzed using Ntf3 conditional knockout (cKO) embryos, in which we observed increased apoptosis and abnormal projection of the central branch innervating motoneuron, the phenotypes being apparently comparable with that of Olig2 KO embryos. Taken together, we show that the motoneuron is a functional source of Ntf3 and motoneuron-derived Ntf3 is an essential pre-target neurotrophin for survival and axonal projection of sensory neurons.     

PlexinA1 signaling directs the segregation of proprioceptive sensory axons in the developing spinal cord

As different classes of sensory neurons project into the CNS, their axons segregate and establish distinct trajectories and target zones. One striking instance of axonal segregation is the projection of sensory neurons into the spinal cord, where proprioceptive axons avoid the superficial dorsal horn-the target zone of many cutaneous afferent fibers. PlexinA1 is a proprioceptive sensory axon-specific receptor for sema6C and sema6D, which are expressed in a dynamic pattern in the dorsal horn. The loss of plexinA1 signaling causes the shafts of proprioceptive axons to invade the superficial dorsal horn, disrupting the organization of cutaneous afferents. This disruptive influence appears to involve the intermediary action of oligodendrocytes, which accompany displaced proprioceptive axon shafts into the dorsal horn. Our findings reveal a dedicated program of axonal shaft positioning in the mammalian CNS and establish a role for plexinA1-mediated axonal exclusion in organizing the projection pattern of spinal sensory afferents.     

Overexpression of neurotrophin-3 in skeletal muscle alters normal and injury-induced limb control

Transgenic overexpression of neurotrophin-3 (NT-3) in mice increases the number of surviving proprioceptive sensory components, including primary sensory neurons, gamma motoneurons and muscle spindles. The numbers of surviving alpha motoneurons are not affected by NT-3 overexpression (Wright et al., Neuron 19: 503- 517, 1997). We have assessed the consequences NT-3-stimulated increase in the proprioceptive sensory system by measuring locomotive abilities of mice that overexpress NT-3 in all skeletal muscles ( myo/NT-3 mice). In adulthood, one myo/NT-3 transgenic line continues to express NT-3 at high levels in muscle and maintains a hypertrophied proprioceptive system (high-OE myo/NT-3 mice). Compared to wildtypes, high-OE myo/NT-3 mice have nine times the amount of NT-3 protein in the medial gastrocnemius at six weeks of age. Although appearing normal during ordinary activity, high-OE myo/NT-3 mice display a distinct clasping phenotype when lifted by the tail. High-OE myo/NT-3 mice show severe locomotor deficits when performing beam walking and rotorod testing. These mice also demonstrate aberrant foot positioning during normal walking. However, following sciatic nerve crush, overexpression of NT-3 prevents further abnormalities in paw positioning, suggesting NT-3 may attenuate sensorimotor deficits that occur in response to sciatic nerve injury. Our results suggest that increases in proprioceptive sensory neurons, spindles and gamma motoneurons, along with continued postnatal NT-3 overexpression in muscle significantly disrupt normal locomotor control. Importantly, however, NT-3 may lessen initial deficits and thus improve functional recovery after peripheral nerve injury, suggesting these mice may serve as a good model to study NT-3's role in neuroprotection of proprioceptive afferents.     

Temporal regulation of neuropilin-1 expression and sensitivity to semaphorin 3A in NGF- and NT3-responsive chick sensory neurons

The extracellular molecule semaphorin 3A (Sema3A) is proposed to be a negative guidance cue that participates in patterning DRG sensory axons in the developing chick spinal cord. During development Sema3A is first expressed throughout the spinal cord gray matter, but Sema3A expression later disappears from the dorsal horn, where small-caliber cutaneous afferents terminate. Sema3A expression remains in the ventral horn, where large-muscle proprioceptive afferents terminate. It has been proposed that temporal changes in the sensitivity of different classes of sensory afferents to Sema3A contribute to the different pathfinding of these sensory afferents. This study compared the expression of the semaphorin 3A receptor subunit, neuropilin-1, and the collapse response of growth cones to semaphorin 3A for NGF (cutaneous)- and NT3 (proprioceptive)-dependent sensory axons extended from E6-E10 chick embryos. Growth cones extended from E6 DRGs in NT3-containing medium expressed neuropilin-1 and collapsed in response to Sema3A. From E7 until E10 NT3-responsive growth cones expressed progressively lower levels of neuropilin-1, and were less sensitive to Sema3A. On the other hand, growth cones extended from DRGs in NGF-containing medium expressed progressively higher levels of neuropilin-1 and higher levels of collapse response to Sema3A over the period from E6-E10. Thus, developmental patterning of sensory terminals in the chick spinal cord may arise from changes in both Sema3A expression in the developing spinal cord and accompanying changes in neuronal expression of the Sema3A receptor subunit, neuropilin-1.     

NT-3 Expression in Spared DRG and the Associated Spinal Laminae as well as Its Anterograde Transport in Sensory Neurons Following Removal of Adjacent DRG in Cats

Neurotrophin-3 plays an important role in survival and differentiation of sensory and sympathetic neurons, sprouting of neurites, synaptic reorganization, and axonal growth. The present study evaluated changes in expression of NT-3 in the spinal cord and L6 dorsal root ganglion (DRG), after ganglionectomy of adjacent dorsal roots in cats. NT-3 immunoreactivity increased at 3 days post-operation (dpo), but decreased at 10 dpo in spinal lamina II after ganglionectomy of L1–L5 and L7–S2 (leaving L6 DRG intact). Conversely, NT-3 immunoreactivity decreased on 3 dpo, but increased on 10 dpo in the nucleus dorsalis. Very little NT-3 mRNA signal was detected in the spinal cord, despite the changes in NT-3 expression. The above changes may be related to changes in NT-3 expression in the DRG. Ganglionectomy of L1–L5 and L7–S2 resulted in increase in NT-3 immunoreactivity and mRNA in small and medium-sized neurons, but decreased expression in large neurons of L6 DRG at 3 dpo. It is possible that increased NT-3 in spinal lamina II is derived from anterograde transport from small- and medium-sized neurons of L6 DRG, whereas decreased NT-3 immunoreactivity in the nucleus dorsalis is due to decreased transport of NT-3 from large neurons in the DRG at this time. This notion is supported by observations on NT-3 distribution in the dorsal root of L6 after ligation of the nerve root. The above results indicate that DRG may be a source of neurotrophic factors such as NT-3 to the spinal cord, and may contribute to plasticity in the spinal cord after injury.     

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.     

Axonal filopodial asymmetry induced by synaptic target

During vertebrate neuromuscular junction (NMJ) assembly, motor axons and their muscle targets exchange short-range signals that regulate the subsequent steps of presynaptic and postsynaptic specialization. We report here that this interaction is in part mediated by axonal filopodia extended preferentially by cultured Xenopus spinal neurons toward their muscle targets. Immunoblotting and labeling experiments showed that basic fibroblast growth factor (bFGF) was expressed by muscle and associated with the cell surface, and treatment of cultured spinal neurons with recombinant bFGF nearly doubled the normal density of filopodia in neurites. This effect of bFGF was abolished by SU5402, a selective inhibitor of FGF-receptor 1 (FGFR1), and forced expression of wild-type or dominant-negative FGFR1 in neurons enhanced or suppressed the assembly of filopodia, respectively. Significantly, in nerve-muscle cocultures, knocking down bFGF in muscle decreased both the asymmetric extension of filopodia by axons toward muscle and the assembly of NMJs. In addition, neurons expressing dominant-negative FGFR1 less effectively triggered the aggregation of muscle acetylcholine receptors at innervation sites than did control neurons. These results suggest that bFGF activation of neuronal FGFR1 generates filopodial processes in neurons that promote nerve-muscle interaction and facilitate NMJ establishment.     

Progressive endolysosomal deficits impair autophagic clearance beginning at early asymptomatic stages in fALS mice

Autophagy is an important homeostatic process that functions by eliminating defective organelles and aggregated proteins over a neuron''s lifetime. One pathological hallmark in amyotrophic lateral sclerosis (ALS)-linked motor neurons (MNs) is axonal accumulation of autophagic vacuoles (AVs), thus raising a fundamental question as to whether reduced autophagic clearance due to an impaired lysosomal system contributes to autophagic stress and axonal degeneration. We recently revealed progressive lysosomal deficits in spinal MNs beginning at early asymptomatic stages in fALS-linked mice expressing the human (Hs) SOD1^G93A protein. Such deficits impair the degradation of AVs engulfing damaged mitochondria from distal axons. These early pathological changes are attributable to mutant HsSOD1, which interferes with dynein-driven endolysosomal trafficking. Elucidation of this pathological mechanism is broadly relevant, because autophagy-lysosomal deficits are associated with several major neurodegenerative diseases. Therefore, enhancing autophagic clearance by rescuing endolysosomal trafficking may be a potential therapeutic strategy for ALS and perhaps other neurodegenerative diseases.     

Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons

Trigeminal sensory axons project to several epithelial targets, including those of the maxillary and mandibular processes. Previous studies identified a chemoattractant activity, termed Maxillary Factor, secreted by these processes, which can attract developing trigeminal axons in vitro. We report that Maxillary Factor activity is composed of two neurotrophins, neurotrophin-3 (NT-3) and Brain-Derived Neurotrophic Factor (BDNF), which are produced by both target epithelium and pathway mesenchyme and which are therefore more likely to have a trophic effect on the neurons or their axons than to provide directional information, at least at initial stages of trigeminal axon growth. Consistent with this, the initial trajectories of trigeminal sensory axons are largely or completely normal in mice deficient in both BDNF and NT-3, indicating that other cues must be sufficient for the initial stages of trigeminal axon guidance.     

Central Connectivity of Transient Receptor Potential Melastatin 8-Expressing Axons in the Brain Stem and Spinal Dorsal Horn

Transient receptor potential melastatin 8 (TRPM8) ion channels mediate the detection of noxious and innocuous cold and are expressed by primary sensory neurons, but little is known about the processing of the TRPM8-mediated cold information within the trigeminal sensory nuclei (TSN) and the spinal dorsal horn (DH). To address this issue, we characterized TRPM8-positive (+) neurons in the trigeminal ganglion and investigated the distribution of TRPM8+ axons and terminals, and their synaptic organization in the TSN and in the DH using light and electron microscopic immunohistochemistry in transgenic mice expressing a genetically encoded axonal tracer in TRPM8+ neurons. TRPM8 was expressed in a fraction of small myelinated primary afferent fibers (23.7%) and unmyelinated fibers (76.3%), suggesting that TRPM8-mediated cold is conveyed via C and Aδ afferents. TRPM8+ axons were observed in all TSN, but at different densities in the dorsal and ventral areas of the rostral TSN, which dominantly receive sensory afferents from intra- and peri-oral structures and from the face, respectively. While synaptic boutons arising from Aδ and non-peptidergic C afferents usually receive many axoaxonic contacts and form complex synaptic arrangements, TRPM8+ boutons arising from afferents of the same classes of fibers showed a unique synaptic connectivity; simple synapses with one or two dendrites and sparse axoaxonic contacts. These findings suggest that TRPM8-mediated cold is conveyed via a specific subset of C and Aδ afferent neurons and is processed in a unique manner and differently in the TSN and DH.