Mechanisms Of Nt-3 Mediated Neuronal Regeneration In The Spinal Cord

Spinal cord injury (SCI) is a devastating condition caused by multiple aetiologies that affects more than 130,000 people per year (Thuret et al., 2006). It usually has overwhelming consequences on the life of patients and results in temporary or permanent impairment of the sensory, autonomic, and motor systems among these the recovery of motor and sexual function is rated as most important. SCI is characterised by initial neuronal and glial cell death, resulting in significant loss of function and a strong neuroinflammatory response. Further damage occurs in the chronic stages of the disease, and is characterised by glial scar formation, and demyelination due to oligodendrocyte degeneration. Glial scars are inhibitory for axonal regeneration and prevent the regeneration of neuronal pathways. Multiple treatments are being researched for SCI, including cellular transplantation therapies, and molecular neuroprotective and neurorestorative therapies, which are extensively reviewed in Thuret et al. (2006). Neurotrophins (NTs) are signalling peptides that provide trophic support to neurons during development and adulthood, and regulate their differentiation, neurite outgrowth, plasticity, and transmission. Current research is focusing on exogenous administration of NTs to promote regeneration, and inhibit the secondary degeneration of peripheral and spinal neural circuits after SCI. Members of the NT family of signalling proteins include the more extensively studied nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and the less well-studied neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) (Reichardt, 2006). NTs mediate their physiological functions through the binding to high-affinity trk receptors (trkA, trkB, and trkC), and to low-affinity p75 receptors (p75NTR). trk receptors are intrinsic tyrosine kinase receptors that undergo trans-autophosphorylation upon ligand binding, and stimulate the phosphatidylinositol 3-kinase/Akt (PI3K/Akt), mitogen activated protein kinase (MAPK), phospholipase C (PLC ) prosurvival and prodifferentiation signalling pathways. They also activate the GTPase Rho to promote cytoskeletal remodelling and guide neurite outgrowth. This study will focus on NT-3, which binds with high affinity to trkC receptors, and also associates with trkA and trkB with a low affinity. Before being post-translationally modified, NTs exist as proneurotrophin precursors that are also secreted (Lu et al., 2005). Interestingly, proneurotrophins induce neuronal apoptosis through high affinity interactions with p75NTRs. Thus, post-translational processing of NTs seems to influence their physiological function. The latter might be very important in the characterisation of neuroprotective effects of NTs, but is beyond the scope of this study and will not be discussed in further detail.

NTs have trophic activities in subsets of peripheral and central neuronal populations. In vitro and in vivo genetic and pharmacological studies of NT-3 function have highlighted its importance in the maintenance of survival of all proprioceptive feedback loop elements i.e. muscle spindles, proprioceptive afferents, spinal interneurons , and motor neurons (Ernfors et al., 1994 Kucera et al., 1995 Woolley et al., 1999 B chade et al., 2002 Woolley et al., 2005). NT-3 seems to be necessary for the survival and development of muscle spindles and proprioceptive afferents, whereas spinal interneurons and motor neurons require it for normal development, but don t seem to exclusively rely on it. Wright et al. (1997) investigated the effects of skeletal muscle specific NT-3 overexpression in NT-3+/+ mice (myo-NT-3 mice). As expected, myo-NT-3 mice show a 50% increase in parvalbumin positive (PV+) DRG neuron numbers compared to NT+/+ controls (PV is an established marker of proprioceptive type I afferents and can distinguish them from other DRG neuronal populations). An increase in the number of axons projecting to the intermediate and ventral spinal cord, and muscle hyperinnervation were also observed. This shows that target tissue derived NT-3 has trophic effects in all subcellular compartments of the neuron. Thus, the signal initiated at the sensory axon terminal of the proprioceptor must somehow be retrogradely transported to the neuronal cell body and central axonal projection. Nerve ligation studies of the sciatic nerve reveal that activated trk receptors accumulate distally but not proximally to the ligation, and are colocalised with clathrin, a component of coated vesicles (Bhattacharyya et al., 1997). These results suggest that retrograde signalling is mediated by endosomes that are retrogradely transported in axons to neuronal cell bodies. The retrograde transport of NTs occurs in numerous central and peripheral populations of neurons via signalling endosomes that originate at the axon terminals, or peripheral sensory terminals (DiStefano et al., 1992). The retrograde transport was also shown to be necessary for the trophic actions of NTs (Harrington et al., 2011). It was shown that the cofilin mediated remodelling of the actin cytoskeleton is necessary for the docking of signalling endosomes on microtubules after their internalisation. Importantly, Rac1 recruitment to trkA, Rac1 mediated recruitment of cofilin, and cofilin mediated F-actin breakdown are necessary steps for NT mediated neuronal survival. Thus, NT trophic retrograde signalling is mediated by retrograde transport of signalling endosomes to neuronal cell bodies.

NT mediated neuronal regeneration after injury occurs both in the peripheral and central nervous systems. Furthermore, NTs induce functional recovery in animal models of SCI. Ramer et al. (2002) conducted a study on the effects of NT-3 treatment on neuronal regeneration after dorsal rhizotomy in adult rats. Intrathecally delivered NT-3 was found to promote axon regeneration in the peripheral nervous system (PNS), cause a significant increase in axon penetration into the CNS through dorsal root entry zones, and lead to an increase in neuronal connectivity within the spinal cord. Rats treated with NT-3 also showed a significantly better improvement in motor behavioural tests assessing proprioceptive function compared to untreated rats. Not only does NT-3 promote the recovery of peripheral proprioceptive afferents, it has also been shown to induce the regeneration of descending spinal cord projection neurons. Bradbury et al. (1998) performed lateral spinal hemisection on rats and showed that intraspinal delivery of NT-3 reverses axotomy-induced neuronal atrophy and death in the spinal cord. These results are supported by the recent study in which the rat corticospinal tract was unilaterally lesioned at the level of the medulla (Chen et al., 2006) adenovirus mediated NT-3 overexpression in the spinal cord induced axonal sprouting of the intact corticospinal tract (CST) into the ipsilesional side of the spinal cord at the segmental level of the injury to form projections that persisted for more than six months. These results are in accordance with the study conducted by Takeoka et al. (2014), in which spontaneous motor recovery in wild-type mice was attributed to the development of novel, dual-midline crossing neuronal pathways descending from motor centres in the brainstem to the caudal ipsilesional spinal cord. Interestingly, by using Egr-/- mutant mice that show dysfunctional muscle spindle reflex circuits due to proprioceptive afferent degeneration, Takeoka et al. (2014) also show that muscle reflex circuits are essential mediators of locomotor recovery after spinal cord injury. Egr-/- mice showed severely impaired spontaneous locomotor recovery after spinal cord hemisection compared to wild-type mice. The authors suggest two possible reasons for such a central involvement of proprioceptive afferents in the formation of novel descending spinal cord motor projection pathways after SCI: (1) proprioceptor afferents provide unique inputs to spinal motor neurons, and might thus be indispensable in mediating circuit rearrangement and functional recovery and/or (2) proprioceptors might contribute to activity-dependent release of trophic factors into the spinal cord, thus promoting neuronal regeneration after injury. These mechanisms are graphically outlined in the figure below.

The in vivo study conducted on mouse mutant progressive motor neuropathy (pmn) model highlights the effectiveness of NT-3 within target tissues in the prevention of neurodegeneration (Haase et al., 1997). pmn mice show neurogenic atrophy of their hindlimb and pelvic girdle muscles that leads to paralysis and death by 7 weeks of age. Intramuscular adenovirus-mediated NT-3 overexpression (targeted to the right gastrocnemius, right triceps brachii, and long muscles of the thoracic trunk) led to improved neuromuscular function in gastrocnemius and diaphragm muscles, and reduced axonal degeneration in the periphery. Furthermore, the improved function of the diaphragm was attributed to an increase in the size of motor units, which might be attributable to axonal collateral sprouting. In vivo experiments at our laboratory have shown that viral vector mediated NT-3 overexpression in forelimb muscles of rats leads to the functional improvement in behavioural tasks, such as the horizontal ladder test, and the grip strength test (unpublished data). On the other hand, no improvement was seen in the fine motor movement, which is dependent on the function of the CST (ibid). This suggests a mechanism of NT-3 mediated recovery that relies on the modulation of lower spinal motor circuits. Electrophysiological analysis of these circuits revealed an imbalance in spinal motor circuits, with increased excitation and decreased inhibition onto lower motor neurons after injury, thought to underlie the development of post-injury spasticity. In summary, NT-3 treatment was observed to restore the balance of excitatory and inhibitory spinal networks, reduce motor neuron hyperexcitability, and enable behavioural recovery. A separate study identified that the overexpression of NT-3 in muscle is associated with a decline in monosynaptic excitatory postsynaptic potentials in motor neurons, consistent with decreased motor neuron excitability (Petruska et al., 2010). In addition to altering the properties of spinal motor circuits, intramuscularly overexpressed NT-3 has also been found to increase plasticity of the CST, and induce its intraspinal anatomical reorganisation (Fortun et al., 2009). Together, these results demonstrate that the intramuscular overexpression of NT-3 restores function mainly by its effect on the motor circuitry of the spinal cord. NT-3 reverses the debilitating changes in neuronal excitability caused by injury, and leads to increased plasticity of the CST.