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Role of Neurotrophins in Spinal Plasticity and Locomotion Victor Arvanian* VA Medical Center Northport and SUNY Stony Brook, NY, USA Abstract: Synaptic transmission through descending motor pathways to lumbar motoneurons and then to leg muscles is essential for walking in humans and rats. Spinal cord injury (SCI), even when incomplete, results in diminished transmission to motoneurons and very limited recovery of motor function. Neurotrophins have emerged as essential molecules known to promote cell survival and support anatomical reorganization in damaged spinal cord. This review will summarize the evidence implicating the role of neurotrophins in synaptic plasticity in both undamaged and damaged spinal cord, with special emphasis on the potential for neurotrophins to strengthen synaptic connections to motoneurons in support of the application of neurotrophins for recovery of locomotor function after SCI. An important consideration related to therapeutic use of neurotrophins is the successful delivery of these molecules. Prolonged delivery of neurotrophins to the spinal cord of adult mammals has recently become possible through advances in biotechnology. Fibroblasts engineered to secrete neurotrophins and gene transfer of neurotrophins via recombinant viral vectors are among the most promising therapeutic transgene delivery systems for safe and effective neurotrophin delivery. Administration of neurotrophins to the spinal cord using these delivery systems was found to enhance both anatomical and synaptic plasticity and improve functional recovery after SCI. The findings summarized here indicate that neurotrophins have translational research potential for SCI repair, most likely as an essential component of combination therapy.
Keywords: Motoneuron, synaptic transmission, motor function, neurotrophic factor. INTRODUCTION Neurotrophins are small molecules that belong to a family of growth and trophic factors which includes neurotrophin-3 (NT-3), NT-4/5, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) [1-3]. Neurotrophins interact with low-affinity p75 receptors (all neurotrophins) (reviewed in [4]) and the highaffinity tropomyosin-related kinase (trk) family of tyrosine receptors (trkA for NGF, trkB for BDNF and NT-4/5, trkC for NT-3), mediating neurotrophins function (reviewed in [5-7]). Neurotrophins have emerged recently as potent factors that are essential for neuronal survival and formation of sensory and motor pathways in the developing spinal cord (for review see [8-11]). Neurotrophins and their receptors are also expressed in the adult mammalian spinal cord [7; 12; 13]. This observation has prompted investigation of the possible use of neurotrophins in the treatment of spinal cord injury. In adult mammalian spinal cord the level of neurotrophins and expression of their trk receptors in the vicinity of the injury is generally decreased [14-17]. However, increased levels of trk receptors have been detected in partially injured rat and cat spinal cord after ventral or dorsal funiculus spinal cord lesions [18] and dorsal root injuries [19]. In all three models increased levels of trk receptors were associated with axon regrowth. A solid rationale exists for the use of neurotrophins, particularly NT-3 and BDNF, in encouraging spinal cord repair after injury. The ability of neurotrophins to promote sprouting and growth of axons in the damaged spinal cord ([20-29]; reviewed in [6; 30-43]), provide neuroprotection [44-52] and regulate myelination of the axons [24; 53-60] highlights the importance of these neurotrophic factors for regenerative repairs and recovery of function after SCI. The biological effects of neurotrophins in the central nervous system (CNS) are not, however, limited to anatomical plasticity. Neurotrophins have been implicated as important factors that interact with various signaling pathways to induce regulation of synapse formation, modulation of synaptic plasticity and efficacy of synaptic transmission in the mammalian brain [61-73]. In this review, we *Address correspondence to this author at the VA Medical Center, Research Service Bld. 62, 79 Middleville Rd, Northport, NY 11768, USA; Fax: 631 544-5317; Ph: 631 261-4400 ext 7142; E-mail:
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focus specifically on the physiological effects of neurotrophins on synaptic plasticity and transmission in mammalian spinal cord. In conclusion, we discuss the potential for neurotrophins as an important element of combination treatment to improve locomotor function after SCI. DESCENDING INPUTS AND VOLUNTARY ACTIONS UPON STEREOTYPED MOTOR FUNCTIONS Descending input from the brain and plasticity in the ventrolateral reticulospinal tract and dorsal corticospinal tract play a critical role in voluntary modulation of key rhythmic and stereotyped functions such as locomotion, bladder function, ejaculation, etc. (reviewed in [74-81]; see also companion papers from Merrill et al., Courtois and Guertin, and Steuer et al.). Incomplete SCI, both lateral hemisection [82-84] and contusion [85-87], result in an initial loss followed by partial spontaneous recovery of voluntary locomotor function. The ability to gain partial locomotor recovery spontaneously after incomplete SCI correlates with enhanced anatomical plasticity and development of connections crossing the midline of spinal cord above and below the lesion [88-92]. Combined, these results suggest that plasticity of spared fibers in the reticulospinal and dorsal corticospinal tracts might be involved in recovery of locomotion following incomplete spinal cord injury. Clinical correlations in spinal cord-injured patients have demonstrated the validity of the rodent animal for the study of dysfunction after acute and chronic injuries (reviewed in [93; 94]; for a list of chronic SCIrelated systemic and metabolic problems, see companion papers of Steuer et al.). PHYSIOLOGICAL CHANGES IN SYNAPTIC TRANSMISSION IN DAMAGED SPINAL CORD Despite the reported ability of intrinsic spinal circuits to reorganize anatomically during the chronic stage of incomplete SCI [88-92], many spinal tracts suffer delayed secondary injury after acute mechanical trauma. In fact, the degree of functional recovery from even a partial SCI is very limited in adult rats and humans and synaptic plasticity in pre-existing pathways is thought to be a vital component of this recovery process [95]. Functional loss within surviving axons has been attributed to impaired axonal conduction, demyelination and inflammatory damage subsequent to mechanical © 2013 Bentham Science Publishers
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trauma [96-100]. Electrophysiological evaluations of the damaged spinal cord revealed diminished ability of spared fibers to transmit signals during the chronic stage of contusion [101-106], compression [96; 107] and lateral hemisection (HX) [108] injuries. Intraaxonal recordings from individual axons in anesthetized adult rats revealed conduction deficits in uncut rubrospinal/reticulospinal (RST/RtST) tract axons and increased threshold to trigger action potentials in these axons after chronic HX SCI [109]. These electrophysiological changes were associated with axon demyelination and abnormal localization of sodium channels along axons. As a result of these changes, action potentials triggered by stimuli of physiological strength exhibited a lower probability to propagate through spared axons. Consequently, transmission to lumbar motoneurons from segments rostral to the contusion [106] or HX [108] injury level was substantially reduced. Transmission from segments caudal to complete transection [110] was less impaired, but significantly declined as well. These results are consistent with the view that the maintenance of synaptic inputs to motoneurons is activitydependent, i.e. denervation is known to reduce synaptic inputs to motoneurons while activation of afferent pathways may partially restore plasticity [111; 112]. Enhancing plasticity in damaged spinal cord, strengthening spared connections and the formation of new functional connections is thought to target one of the most difficult challenges in experimental and clinical neurology, which is how to facilitate locomotor recovery after incomplete SCI. This crucial task has prompted investigators to examine the effects of activity on the level of neurotrophins in the damage spinal cord and to study the ability of exogenous neurotrophins to modulate synaptic plasticity at motor pathways. NEUROTROPHINS AND ACTIVITY-DEPENDENT PLASTICITY IN DAMAGED SPINAL CORD Beneficial effects of activity-based therapy in human subjects and animals with SCI are well- documented (reviewed in [113117]). Ample evidence indicates that both active task-specific training (reviewed in [118-122]) and passive electric stimulation exercise (reviewed in [123-126]) may enhance plasticity in damaged spinal cord. Consistent with this activity-dependent concept for maintaining plasticity and function, modulation of spinal plasticity and improvement of functional outcomes have been reported in studies using electric fields applied either epidurally to injured mammalian spinal cord [127-132] or through non-invasive electromagnetic stimulation [133-138]. In order to better understand the mechanisms driving activity-dependent plasticity, the relationship between activity and the level of neurotrophins in the vicinity of the injury has been examined. Several studies suggest the ability of exercise to impact spinal circuitry by increasing the level of neurotrophins in damaged spinal cord and muscles thus providing neurotrophic support. In adult mammalian CNS, the bulk of neurotrophic activity is thought to be carried out by NT-3, NT-4 and BDNF [139]. Expression of these neurotrophins is differentially regulated in spinal cord, muscles and brain in response to SCI [140-142]. In models of hemisection SCI, robust recovery of function has been shown to occur after about two weeks post-injury. Interestingly, levels of BDNF and NT-4 in lumbar motoneurons, though initially decreased, recovered after two survival weeks, coinciding with the recovery of hindlimb mobility following the injury [143]. In models of severe SCI, decreased levels of BDNF and NT-3 mRNA were detected in the vicinity of injury in the lumbar region innervating the impaired hindlimb muscles; however, increased level of BDNF were detected in the cervical region rostral to the injury reflecting an increased compensatory use of the forelimbs [144]. These results indicate that the spinal cord level of supraspinal and muscle afferent inputs plays an important role in modulating the levels of BDNF and NT-3 in the spinal cord [144].
Victor Arvanian
Solid evidence exists that the level of neurotrophins altered by SCI could be partially restored by exercise in an activity-dependent manner. Opposing the effects of SCI, exercise was found to partially restore the levels of neurotrophins and plasticity following spinal cord injury [114; 141; 145; 146]. An interesting recent observation was that the combination of active exercise (step-training) and passive bicycling increased the levels of BDNF, NT-3 and NT4 in the lumbar enlargement of SCI rats, whereas step-training alone increased glial cell-derived neurotrophic factor (GDNF) levels. These increases in neurotrophin levels positively correlated with the recovery of the H-reflex [147]. Together, these experiments support the suggestion that modulation of neurotrophins by exercise may be a potential mechanism underlying the effects of exercise on neuronal plasticity in injured spinal cord [140-148]. This view highlights the necessity for examination of endogenous neurotrophins in mechanisms of synaptic plasticity in intact and injured spinal cord. MODULATION OF SYNAPTIC FUNCTION BY NT-3 AND BDNF IN DEVELOPING SPINAL CORD Many initial electrophysiological studies investigating the effects of neurotrophins on synaptic transmission to spinal motoneurons were conducted in the neonatal rat and involved intracellular recording from single motoneurons over several hours, allowing the examination of the long-lasting effects of neurotrophins and other pharmacological agents. Another convenient feature of this preparation is that it allows the examinination and comparison of the effect of neurotrophins at both segmental and descending inputs on the same motoneuron; both sensory [149] and descending axons [150] terminating on motoneurons express trk receptors. Numerous lines of investigation now indicate a potentially important role for neurotrophins in regulating the strength of synaptic connections to motoneurons during development (for review [151-153]). During the immediate postnatal stage, when connections are strengthening [154], NT-3 is important in regulating the strength of connections to motoneurons [155]. Brief exposure to NT-3 and BDNF induced long-lasting but differential modulation of intracellularly recorded monosynaptic responses of lumbar motoneurons at both segmental and descending inputs. The effect of NT-3 was strictly facilitatory and persisted several hours following the removal of NT-3 [156]. BDNF exerted complex effects and induced an initial facilitation followed by persistent depression of motoneuron monosynaptic responses [157]. In lamina II neurons, BDNF facilitated responses to high threshold primary afferent inputs [158]. These acute modulatory effects of both neurotrophins were restricted to very young animals. The age-dependent action of neurotrophins on AMPAkainate receptor-mediated monosynaptic responses was determined by alteration of functional NMDA receptors, which is the key factor required for the action of both neurotrophins. Because function of NMDA receptors in motoneurons declines during the first postnatal week as a result of enhanced magnesium block [159; 160], the effect of neurotrophins is restricted to this early postnatal period. The age-dependent modulatory action of NT-3 and BDNF is consistent with the decrease in the contribution of NMDA receptors at motoneuron synapses during the perinatal period [160-163]. The loss of NMDA receptor function at synaptic inputs on motoneurons associated with an age-dependent decrease in the expression of neuronal NMDA-2D subunits. Viral vector-mediated delivery of NR2D reversed the age-dependent loss of NMDA receptor function and restored the ability of NT-3 to facilitate synaptic transmission [160]. In addition to modulation of excitatory glutamatergic transmission to lumbar motoneurons, BDNF was found to augment the inhibitory glycinergic and GABAergic transmissions in neonatal mouse spinal neurons [164]. The long-lasting modulations of synaptic transmission by neurotrophins in neonatal intact spinal cord provided a basis for the use of neurotrophins for enhancing the function of surviving fibers in partially or incompletely damaged spinal cord in adults. It remains to be explored if Central Pattern
NT-3 and BDNF in Spinal Cord
Generator-activating approaches such as SpinalonTM (see companion papers of Guertin or Steuer et al.) combined with neurotrophin administration could, in turn, promote regeneration and sprouting even in completely damaged spinal cord models. DELIVERY OF NEUROTROPHINS TO ADULT MAMMALIAN SPINAL CORD In adult cats, application of NT-3 to the cut peripheral muscle nerve via osmotic minipump reversed reductions in both axonal conduction velocity and synaptic responses in target motoneurons [165; 166]. In mutant mice, delivery of NT3 from muscle spindles regulates the synaptic connectivity between muscle sensory and motor neurons [167]. Delivery of neurotrophins directly to the damaged spinal cord of adult mammals, however, is more complicated because neurotrophins do not cross the blood-brain barrier in adult mammals [168]. Prolonged delivery of neurotrophins to the spinal cord of adult mammals has recently become possible through advances in biotechnology and clinically relevant methods for neurotrophin delivery to the spinal cord have been developed. Fibroblasts engineered to secrete neurotrophins and gene transfer of neurotrophins via recombinant viral vectors are among the most promising therapeutic transgene delivery systems for safe and effective neurotrophin delivery (reviewed in [169-174]). In the growing list of available viral vectors, the adenoassociated virus (AAV) has emerged as the most clinically relevant and safe. An important feature of the AAV system is that it is capable of retrograde transgene delivery of neurotrophins to the spinal cord motoneurons via injections into the corresponding innervated muscle [175-178]. This viral vector is derived from a replicationdeficient, non-pathogenic parvovirus with a single-stranded DNA genome. AAV vectors have been found to successfully manipulate various CNS functions and therapeutic expression often lasts for months to years [179; 180]. Because of long-lasting sustained expression, nonpathogenic and nonimmunogenic AAV vectors have recently emerged as a preferred gene transfer system to the spinal cord for preclinical studies in animal models of human disease (reviewed in [181-184]). Recent studies of single-cell electrophysiological parameters confirmed AAV-mediated delivery of neurotrophins to lumbar motoneurons in non-injured [185] and completely transected [186] spinal cord. It should be recognized, however, that an obstacle for the use of AAV as a delivery system might be the difficulty in terminating vector-mediated expression at the desired time point. Other disadvantages of the AAV system include its relatively prolonged establishment of stable transgene expression (up to 2 weeks following the initial injection) and its limited transgene capacity (
