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Traumatic brain injury (TBI) in children

Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality after cervical spinal cord injury (SCI). Upon SCI, inspiratory signals originating in the medullary rostral ventral respiratory group (rVRG) become disrupted from their phrenic motor neuron (PhMN) targets, resulting in diaphragm paralysis. Limited growth of both damaged and spared axon populations occurs after central nervous system trauma attributed, in part, to expression of various growth inhibitory molecules, some that act through direct interaction with the protein tyrosine phosphatase sigma (PTPσ) receptor located on axons. In the rat model of C2 hemisection SCI, we aimed to block PTPσ signaling to investigate potential mechanisms of axon plasticity and respiratory recovery using a small molecule peptide mimetic that inhibits PTPσ. The peptide was soaked into a biocompatible gelfoam and placed directly over the injury site immediately after hemisection and replaced with a freshly soaked piece 1 week post-SCI. At 8 weeks post-hemisection, PTPσ peptide significantly improved ipsilateral hemidiaphragm function, as assessed in vivo with electromyography recordings. PTPσ peptide did not promote regeneration of axotomized rVRG fibers originating in ipsilateral medulla, as assessed by tracing after adeno-associated virus serotype 2/mCherry injection into the rVRG. Conversely, PTPσ peptide stimulated robust sprouting of contralateral-originating rVRG fibers and serotonergic axons within the PhMN pool ipsilateral to hemisection. Further, relesion through the hemisection did not compromise diaphragm recovery, suggesting that PTPσ peptide-induced restoration of function was attributed to plasticity of spared axon pathways descending in contralateral spinal cord. These data demonstrate that inhibition of PTPσ signaling can promote significant recovery of diaphragm function after SCI by stimulating plasticity of critical axon populations spared by the injury and consequently enhancing descending excitatory input to PhMNs.

Myelomeningocele (MMC) is a devastating congenital neural tube defect that results in the exposure of spinal cord to the intrauterine environment, leading to secondary spinal cord injury and severe impairment. Although the mechanisms underlying the secondary pathogenesis are clinically relevant, the exact cause of in utero–acquired spinal cord damage remains unclear. The objective of this study was to determine whether the hyaluronic acid (HA) concentration in amniotic fluid (AF) in the retinoic acid-induced model of MMC is different from that in normal controls and whether these differences could have an impact on the viscosity of AF. Our data shows that the concentration of HA in AF samples from fetuses with MMC (MMC-AF) and normal control samples (Norm-AF) were not significantly different at embryonic day 18 (E18) and E20. Thereafter, the HA concentration significantly increased in Norm-AF but not in MMC-AF. Compared with Norm-AF, the concentration of HA in MMC-AF and the viscosity of MMC-AF were significantly lower at E21. Agarose gel electrophoresis confirmed a significant reduction in the HA level of MMC-AF compared with Norm-AF at E21. No HA-degrading activity was detected in MMC-AF. In summary, we identified a deficiency in the AF level of HA and the viscosity of AF in fetal rats with MMC. These data are discussed in relation to a potential role the reduction in the AF viscosity due to the low level of HA may play in the exacerbating effects of mechanical trauma on spinal cord damage at the MMC lesion site.

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons. Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

Increasing the intrinsic growth potential of neurons after injury has repeatedly been shown to promote some level of axonal regeneration in rodent models. One of the most studied pathways involves the activation of the PI3K/AKT/mTOR pathways, primarily by reducing the levels of PTEN, a negative regulator of PI3K. Likewise, activation of signal transducer and activator of transcription 3 (STAT3) has previously been shown to boost axonal regeneration and sprouting within the injured nervous system. Here, we examined the regeneration of the corticospinal tract (CST) after cortical expression of constitutively active (ca) Akt3 and STAT3, both separately and in combination. Overexpression of caAkt3 induced regeneration of CST axons past the injury site independent of caSTAT3 overexpression. STAT3 demonstrated improved axon sprouting compared to controls and contributed to a synergistic improvement in effects when combined with Akt3 but failed to promote axonal regeneration as an individual therapy. Despite showing impressive axonal regeneration, animals expressing Akt3 failed to show any functional improvement and deteriorated with time. During this period, we observed progressive Akt3 dose-dependent increase in behavioral seizures. Histology revealed increased phosphorylation of ribosomal S6 protein within the unilateral cortex, increased neuronal size, microglia activation and hemispheric enlargement (hemimegalencephaly).

The use of sensory neurons and assessment of neurite outgrowth in vitro is an important part of understanding neuronal development and plasticity. Cultures of rat dorsal root ganglion (DRG) neurons provide quantitative results very quickly and, when grown on growth promoting or inhibitory substrates, can be utilized to study axonal growth, neurotrophic dependence, and structure and function of growth cones. Since we are interested in axon regeneration and targeting, we have sought to promote neurite outgrowth by refining the techniques of growing DRG neurons in culture. This chapter describes detailed methods for the dissection and purification of DRG neurons and quantitative assessment of neurite on promoting or inhibitory substrates.

A major barrier to intraspinal regeneration after dorsal root (DR) injury is the DR entry zone (DREZ), the CNS/PNS interface. DR axons stop regenerating at the DREZ, even if regenerative capacity is increased by a nerve conditioning lesion. This potent blockade has long been attributed to myelin-associated inhibitors and CSPGs, but incomplete lesions and conflicting reports have prevented conclusive agreement. Here we evaluated DR regeneration in mice, using novel strategies to facilitate complete lesions and analyses, selective tracing of proprioceptive and mechanoreceptive axons, and the first simultaneous targeting of Nogo/Reticulon-4, MAG, OMgp, CSPGs and GDNF. Co-eliminating myelin inhibitors and CSPGs elicited regeneration of only a few conditioning-lesioned DR axons across the DREZ. Their absence, however, markedly and synergistically enhanced regeneration of GDNF-stimulated axons, highlighting the importance of sufficiently elevating intrinsic growth capacity. We also conclude that myelin inhibitors and CSPGs are not the primary mechanism stopping axons at the DREZ.

SUMMARY After optic nerve crush (ONC), the cell bodies and distal axons of most retinal ganglion cells (RGCs) degenerate. RGC somal and distal axon degenerations were previously thought to be controlled by two parallel pathways, involving activation of the kinase dual leucine-zipper kinase (DLK) and loss of the axon survival factor nicotinamide mononucleotide adenylyltransferase-2 (NMNAT2), respectively. Here, we report that palmitoylation of both DLK and NMNAT2 by the palmitoyl acyltransferase ZDHHC17 couples these signals. ZDHHC17-dependent palmitoylation enables DLK-dependent somal degeneration after ONC and also ensures NMNAT-dependent distal axon integrity in healthy optic nerves. We provide evidence that ZDHHC17 also controls survival-versus-degeneration decisions in dorsal root ganglion (DRG) neurons, and we identify conserved motifs in NMNAT2 and DLK that govern their ZDHHC17-dependent regulation. These findings suggest that the control of somal and distal axon integrity should be considered as a single, holistic process, mediated by the concerted action of two palmitoylation-dependent pathways., Graphical Abstract, In Brief Niu et al. show that ZDHHC17 palmitoylates DLK, a mediator of axon-to-soma pro-degenerative signaling, and also NMNAT2, a survival factor whose rapid loss post-injury triggers distal axon degeneration. Palmitoylation of these two key proteins by ZDHHC17 ensures a coordinated response of distal axons and neuronal somas to axonal injury.

Delivery of neurotrophins to the spinal injury site via cellular transplants or viral vectors administration has been shown to promote recovery of locomotion in the absence of locomotor training in adult spinalized animals. These delivery methods involved risks of secondary injury to the cord and do not allow for precise and controlled dosing making them unsuitable for clinical applications. The present study was aimed at evaluating the locomotor recovery efficacy and safety of the neurotrophin BDNF delivered intrathecally to the lumbar locomotor centers using an implantable and programmable infusion mini-pump. Results showed that BDNF treated spinal cats recovered weight-bearing plantar stepping at all velocities tested (0.3-0.8 m/s). Spinal cats treated with saline did not recover stepping ability, especially at higher velocities, and dragged their hind paws on the treadmill. Histological evaluation showed minimal catheter associated trauma and tissue inflammation, underlining that intrathecal delivery by an implantable/programmable pump is a safe and effective method for delivery of a controlled BDNF dosage; it poses minimal risks to the cord and is clinically translational.

Chondroitin sulfate proteoglycans (CSPGs), up-regulated in and around the lesion after traumatic spinal cord injury (SCI), are key extracellular matrix inhibitory molecules that limit axon growth and consequent recovery of function. CSPG-mediated inhibition occurs via interactions with axonal receptors, including leukocyte common antigen- related (LAR) phosphatase. We tested the effects of a novel LAR inhibitory peptide in rats after hemisection at cervical level 2, a SCI model in which bulbospinal inspiratory neural circuitry originating in the medullary rostral ventral respiratory group (rVRG) becomes disconnected from phrenic motor neuron (PhMN) targets in cervical spinal cord, resulting in persistent partial-to-complete diaphragm paralysis. LAR peptide was delivered by a soaked gelfoam, which was placed directly over the injury site immediately after C2 hemisection and replaced at 1 week post-injury. Axotomized rVRG axons originating in ipsilateral medulla or spared rVRG fibers originating in contralateral medulla were separately assessed by anterograde tracing via AAV2-mCherry injection into rVRG. At 8 weeks post-hemisection, LAR peptide significantly improved ipsilateral hemidiaphragm function, as assessed in vivo with electromyography recordings. LAR peptide promoted robust regeneration of ipsilateral-originating rVRG axons into and through the lesion site and into intact caudal spinal cord to reach PhMNs located at C3-C5 levels. Furthermore, regenerating rVRG axons re-established putative monosynaptic connections with their PhMNs targets. In addition, LAR peptide stimulated robust sprouting of both modulatory serotonergic axons and contralateral-originating rVRG fibers within the PhMN pool ipsilateral/caudal to the hemisection. Our study demonstrates that targeting LAR-based axon growth inhibition promotes multiple forms of respiratory neural circuit plasticity and provides a new peptide-based therapeutic strategy to ameliorate the devastating respiratory consequences of SCI.

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

The fission/division and fusion of mitochondria are fundamental aspects of mitochondrial biology. The balance of fission and fusion sets the length of mitochondria in cells to serve their physiological requirements. The fission of mitochondria is markedly induced in many disease states and in response to cellular injury, resulting in the fragmentation of mitochondria into dysfunctional units. The mechanism that drives fission is dependent on the dynamin related protein 1 (Drp1) GTPase. mdivi-1 is a quinazolinone originally described as a selective inhibitor of Drp1, over other dynamin family members, and reported to inhibit mitochondrial fission. A recent study has challenged the activity of mdivi-1 as an inhibitor of Drp1. This study raises serious issues regarding the interpretation of data addressing the effects of mdivi-1 as reflective of the inhibition of Drp1 and thus fission. This commentary considers the evidence for and against mdivi-1 as an inhibitor of Drp1 and presents the following considerations; (1) the activity of mdivi-1 toward Drp1 GTPase activity requires further biochemical investigation, (2) as there is a large body of literature using mdivi-1 in vitro with effects as predicted for inhibition of Drp1 and mitochondrial fission, reviewed herein, the evidence is in favor of mdivi-1's originally described bioactivity, and (3) until the issue is resolved, experimental interpretations for the effects of mdivi-1 on inhibition of fission in cell and tissue experiments warrants stringent positive controls directly addressing the effects of mdivi-1 on fission. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1260-1268, 2017.

Neurotrophins are growth factors that have a multitude of roles in the nervous system. We report that neurotrophins induce the fission of mitochondria along embryonic chick sensory axons driven by combined PI3K and Mek-Erk signaling. Following an initial burst of fission, a new steady state of neurotrophin-dependent mitochondria length is established. Mek-Erk controls the activity of the fission mediator Drp1 GTPase, while PI3K may contribute to the actin-dependent aspect of fission. Drp1-mediated fission is required for nerve growth factor (NGF)-induced collateral branching in vitro and expression of dominant negative Drp1 impairs the branching of axons in the developing spinal cord in vivo. Fission is also required for NGF-induced mitochondria-dependent intra-axonal translation of the actin regulatory protein cortactin, a previously determined component of NGF-induced branching. Collectively, these observations unveil a novel biological function of neurotrophins; the regulation of mitochondrial fission and steady state mitochondrial length and density in axons.

More than half of spinal cord injury (SCI) cases occur in the cervical region, leading to respiratory dysfunction due to damaged neural circuitry that controls critically important muscles such as the diaphragm. The C3-C5 spinal cord is the location of phrenic motor neurons (PhMNs) that are responsible for diaphragm activation; PhMNs receive bulbospinal excitatory drive predominately from supraspinal neurons of the rostral ventral respiratory group (rVRG). Cervical SCI results in rVRG axon damage, PhMN denervation, and consequent partial-to-complete paralysis of hemidiaphragm. In a rat model of C2 hemisection SCI, we expressed the axon guidance molecule, brain-derived neurotrophic factor (BDNF), selectively at the location of PhMNs (ipsilateral to lesion) to promote directed growth of rVRG axons toward PhMN targets by performing intraspinal injections of adeno-associated virus serotype 2 (AAV2)-BDNF vector. AAV2-BDNF promoted significant functional diaphragm recovery, as assessed by in vivo electromyography. Within the PhMN pool ipsilateral to injury, AAV2-BDNF robustly increased sprouting of both spared contralateral-originating rVRG axons and serotonergic fibers. Furthermore, AAV2-BDNF significantly increased numbers of putative monosynaptic connections between PhMNs and these sprouting rVRG and serotonergic axons. These findings show that targeting circuit plasticity mechanisms involving the enhancement of synaptic inputs from spared axon populations is a powerful strategy for restoring respiratory function post-SCI.—Charsar, B. A., Brinton, M. A., Locke, K., Chen, A. Y., Ghosh, B., Urban, M. W., Komaravolu, S., Krishnamurthy, K., Smit, R., Pasinelli, P., Wright, M. C., Smith, G. M., Lepore, A. C. AAV2-BDNF promotes respiratory axon plasticity and recovery of diaphragm function following spinal cord injury.

Compromise in inspiratory breathing following cervical spinal cord injury (SCI) is caused by damage to descending bulbospinal axons originating in the rostral ventral respiratory group (rVRG) and consequent denervation and silencing of phrenic motor neurons (PhMNs) that directly control diaphragm activation. In a rat model of high-cervical hemisection SCI, we performed systemic administration of an antagonist peptide directed against phosphatase and tensin homolog (PTEN), a central inhibitor of neuron-intrinsic axon growth potential. PTEN antagonist peptide (PAP4) robustly restored diaphragm function, as determined with electromyography (EMG) recordings in living SCI animals. PAP4 promoted substantial, long-distance regeneration of injured rVRG axons through the lesion and back toward PhMNs located throughout the C3–C5 spinal cord. These regrowing rVRG axons also formed putative excitatory synaptic connections with PhMNs, demonstrating reconnection of rVRG-PhMN-diaphragm circuitry. Lastly, re-lesion through the hemisection site completely ablated functional recovery induced by PAP4. Collectively, our findings demonstrate that axon regeneration in response to systemic PAP4 administration promoted recovery of diaphragmatic respiratory function after cervical SCI.

Introduction: Spinal cord injury (SCI) is a complicated and devastating neurological disorder. Patients with SCI usually have dramatically reduced quality of life. In recent years, numerous studies have reported advances in understanding the pathophysiology of SCI and developing preclinical therapeutic strategies for SCI, including various molecular therapies, and yet there is still no cure. Areas covered: After SCI, tissue damage, responses and repair involve interactions among many cellular components, including neurons, axons, glia, leukocytes, and other cells. Accordingly, numerous cellular genes and molecules have become therapeutic targets for neural tissue repair, circuit reconstruction, and behavioral restoration. Here, we review the major recent advances in biological and molecular strategies to enhance neuroprotection, axon regeneration, remyelination, neuroplasticity and functional recovery in preclinical studies of SCI. Expert opinion: Researchers have made tremendous progress in identifying individual and combined molecular therapies in animal studies. It is very important to identify additional highly effective treatments for early neuroprotective intervention and for functionally meaningful axon regeneration and neuronal reconnections. Because multiple mechanisms contribute to the functional loss after SCI, combining the most promising approaches that target different pathophysiological and molecular mechanisms should exhibit synergistic actions for maximal functional restoration. [Databases searched: PubMed; inclusive dates: 6/27/2019].

One method for the evaluation of sensorimotor therapeutic interventions, the horizontal ladder walking task, analyzes locomotor changes that may occur after disease, injury, or by external manipulation. Although this task is well suited for detection of large effects, it may overlook smaller changes. The inability to detect small effect sizes may be due to a neural compensatory mechanism known as “cross limb transfer”, or the contribution of the contralateral limb to estimate an injured or perturbed limb’s position. The robust transfer of compensation from the contralateral limb may obscure subtle locomotor outcomes that are evoked by clinically relevant therapies, in the early onset of disease, or between higher levels of recovery. Here, we propose angled rungs as a novel modification to the horizontal ladder walking task. Easily-adjustable angled rungs force rats to locomote across a different locomotion path for each hindlimb and may therefore make information from the contralateral limb less useful. Using hM3Dq (excitatory) Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) expressed in large diameter peripheral afferents of the hindlimb in the intact animal, we characterized the sensitivity of our design to detect stepping differences by comparing locomotor changes observed on angled rungs to those observed on a standard horizontal ladder. On our novel asymmetrical ladder, activation of DREADDs resulted in significant differences in rung misses (p = 0.000011) and weight-supporting events (p = 0.049). By comparison, on a standard ladder, we did not observe differences in these parameters (p = 0.86 and p = 0.98, respectively). Additionally, no locomotor differences were detected in baseline and inactivated DREADDs trials when we compared ladder types, suggesting that the angled rungs do not change animal gait behavior unless intervention or injury is introduced. Significant changes observed with angled rungs may demonstrate more sensitive probing of locomotor changes due to the decoupling of cross limb transfer.

The overall goal of our research is to establish a preformed molecular guidance pathway to direct the growth of dopaminergic axons from embryonic ventral mesencephalon (VM), tissue placed within the substantia nigra (SN), into the striatum to reconstruct the nigrostriatal pathway in a hemi-Parkinson's disease rat model. Guidance pathways were prepared by injecting lentivirus encoding either GFP or a combination of glial-cell-line-derived neurotrophic factor (GDNF) with either GDNF family receptor α1 (GFRα1) or netrin1. In another cohort of animals, adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF) was injected within the striatum after guidance pathway formation. GDNF combined with either GFRα1 or netrin significantly increased growth of dopaminergic axons out of transplants and along the pathway, resulting in a significant reduction in the number of amphetamine-induced rotations. Retrograde tract tracing showed that the dopaminergic axons innervating the striatum were from A9 neurons within the transplant. Increased dopaminergic innervation of the striatum and improved behavioral recovery were observed with the addition of BDNF. Preformed guidance pathways using a combination of GDNF and netrin1 can be used to reconstruct the nigrostriatal pathway and improve motor recovery.

Stem/progenitor cell transplantation delivery of astrocytes is a potentially powerful strategy for spinal cord injury (SCI). Axon extension into SCI lesions that occur spontaneously or in response to experimental manipulations is often observed along endogenous astrocyte "bridges," suggesting that augmenting this response via astrocyte lineage transplantation can enhance axon regrowth. Given the importance of respiratory dysfunction post-SCI, we transplanted glial-restricted precursors (GRPs)-a class of lineage-restricted astrocyte progenitors-into the C2 hemisection model and evaluated effects on diaphragm function and the growth response of descending rostral ventral respiratory group (rVRG) axons that innervate phrenic motor neurons (PhMNs). GRPs survived long term and efficiently differentiated into astrocytes in injured spinal cord. GRPs promoted significant recovery of diaphragm electromyography amplitudes and stimulated robust regeneration of injured rVRG axons. Although rVRG fibers extended across the lesion, no regrowing axons re-entered caudal spinal cord to reinnervate PhMNs, suggesting that this regeneration response-although impressive-was not responsible for recovery. Within ipsilateral C3-5 ventral horn (PhMN location), GRPs induced substantial sprouting of spared fibers originating in contralateral rVRG and 5-HT axons that are important for regulating PhMN excitability; this sprouting was likely involved in functional effects of GRPs. Finally, GRPs reduced the macrophage response (which plays a key role in inducing axon retraction and limiting regrowth) both within the hemisection and at intact caudal spinal cord surrounding PhMNs. These findings demonstrate that astrocyte progenitor transplantation promotes significant plasticity of rVRG-PhMN circuitry and restoration of diaphragm function and suggest that these effects may be in part through immunomodulation., National Institute of Neurological Disorders and Stroke. Grant Number: 2R01NS079702‐06 (to A.C.L.) Paralyzed Veterans of America Research Foundation. Grant Numbers: Grant 476686 (to A.C.L), 476686 NINDS. Grant Number: 2R01NS079702‐06

Retrogradely-transported neurotrophin signaling plays an important role in regulating neural circuit specificity. Here we investigated whether targeted delivery of neurotrophin-3 (NT-3) to lumbar motoneurons (MNs) caudal to a thoracic (T10) contusive spinal cord injury (SCI) could modulate dendritic patterning and synapse formation of the lumbar MNs. In vitro, Adeno-associated virus serotype two overexpressing NT-3 (AAV-NT-3) induced NT-3 expression and neurite outgrowth in cultured spinal cord neurons. In vivo, targeted delivery of AAV-NT-3 into transiently demyelinated adult mouse sciatic nerves led to the retrograde transportation of NT-3 to the lumbar MNs, significantly attenuating SCI-induced lumbar MN dendritic atrophy. NT-3 enhanced sprouting and synaptic formation of descending serotonergic, dopaminergic, and propriospinal axons on lumbar MNs, parallel to improved behavioral recovery. Thus, retrogradely transported NT-3 stimulated remodeling of lumbar neural circuitry and synaptic connectivity remote to a thoracic SCI, supporting a role for retrograde transport of NT-3 as a potential therapeutic strategy for SCI.

Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality in individuals suffering from traumatic cervical spinal cord injury (SCI). Repair of CNS axons after SCI remains a therapeutic challenge, despite current efforts. SCI disrupts inspiratory signals originating in the rostral ventral respiratory group (rVRG) of the medulla from their phrenic motor neuron (PhMN) targets, resulting in loss of diaphragm function. Using a rat model of cervical hemisection SCI, we aimed to restore rVRG-PhMN-diaphragm circuitry by stimulating regeneration of injured rVRG axons via targeted induction of Rheb (ras homolog enriched in brain), a signaling molecule that regulates neuronal-intrinsic axon growth potential. Following C2 hemisection, we performed intra-rVRG injection of an adeno-associated virus serotype-2 (AAV2) vector that drives expression of a constitutively-active form of Rheb (cRheb). rVRG neuron-specific cRheb expression robustly increased mTOR pathway activity within the transduced rVRG neuron population ipsilateral to the hemisection, as assessed by levels of phosphorylated ribosomal S6 kinase. By co-injecting our novel AAV2-mCherry/WGA anterograde/trans-synaptic axonal tracer into rVRG, we found that cRheb expression promoted regeneration of injured rVRG axons into the lesion site, while we observed no rVRG axon regrowth with AAV2-GFP control. AAV2-cRheb also significantly reduced rVRG axonal dieback within the intact spinal cord rostral to the lesion. However, cRheb expression did not promote any recovery of ipsilateral hemi-diaphragm function, as assessed by inspiratory electromyography (EMG) burst amplitudes. This lack of functional recovery was likely because regrowing rVRG fibers did not extend back into the caudal spinal cord to synaptically reinnervate PhMNs that we retrogradely-labeled with cholera toxin B from the ipsilateral hemi-diaphragm. Our findings demonstrate that enhancing neuronal-intrinsic axon growth capacity can promote regeneration of injured bulbospinal respiratory axons after SCI, but this strategy may need to be combined with other manipulations to achieve reconnection of damaged neural circuitry and ultimately recovery of diaphragm function.

Neurotrophins represent some of the best candidates to enhance regeneration. In the current study, we investigated the effects of artemin, a member of the glial derived neurotrophic factor (GDNF) family, on sensory axon regeneration following a lumbar dorsal root injury and compared these effects with that observed after either NGF or GDNF expression in the rat spinal cord. Unlike previously published data, artemin failed to induce regeneration of large-diameter myelinated sensory afferents when expressed within either the spinal cord or DRG. However, artemin or NGF induced regeneration of calcitonin gene related peptide positive (CGRP(+)) axons only when expressed within the spinal cord. Accordingly, artemin or NGF enhanced recovery of only nociceptive behavior and showed a cFos distribution similar to the topography of regenerating axons. Artemin and GDNF signaling requires binding to different co-receptors (GFRα3 or GFRα1, respectively) prior to binding to the signaling receptor, cRet. Approximately 70% of DRG neurons express cRet, but only 35% express either co-receptor. To enhance artemin-induced regeneration, we co-expressed artemin with either GFRα3 or GDNF. Co-expression of artemin and GFRα3 only slightly enhanced regeneration of IB4(+) non-peptidergic nociceptive axons, but not myelinated axons. Interestingly, this co-expression also disrupted the ability of artemin to produce topographic targeting and lead to significant increases in cFos immunoreactivity within the deep dorsal laminae. This study failed to demonstrate artemin-induced regeneration of myelinated axons, even with co-expression of GFRα3, which only promoted mistargeted regeneration.

Myelomeningocele (MMC) is a devastating spinal cord birth defect, which results in significant life-long disabilities, impaired quality of life, and difficult medical management. The pathological progression of MMC involves failure in neural tube and vertebral arch closure at early gestational ages, followed by subsequent impairment in spinal cord and vertebral growth during fetal development. MMC is irreversible at term. Thus, prenatal therapeutic strategies that interrupt progressive pathological processes offer an appealing approach for treatment of MMC. However, a thorough understanding of pathological progression of MMC is mandatory for appropriate treatment to be rendered. This article is part of a Special Issue entitled SI: Spinal cord injury.

Intrinsic mechanisms that guide damaged axons to regenerate following spinal cord injury remain poorly understood. Manipulation of posttranslational modifications of key proteins in mature neurons could reinvigorate growth machinery after injury. One such modification is acetylation, a reversible process controlled by two enzyme families, the histone deacetylases (HDACs) and the histone acetyl transferases (HATs), acting in opposition. Whereas acetylated histones in the nucleus are associated with upregulation of growth-promoting genes, deacetylated tubulin in the axoplasm is associated with more labile microtubules, conducive to axon growth. This study investigates the effects of HAT and HDAC inhibitors on cultured adult dorsal root ganglia (DRG) neurons and shows that inhibition of HATs by anacardic acid or CPTH2 improves axon outgrowth, whereas inhibition of HDACs by TSA or tubacin inhibits axon growth. Anacardic acid increased the number of axons able to cross an inhibitory chondroitin sulfate proteoglycan border. Histone acetylation but not tubulin acetylation level was affected by HAT inhibitors, whereas tubulin acetylation levels were increased in the presence of the HDAC inhibitor tubacin. Although the microtubule-stabilizing drug taxol did not have an effect on the lengths of DRG axons, nocodazole decreased axon lengths. Determining the mechanistic basis will require future studies, but this study shows that inhibitors of HAT can augment axon growth in adult DRG neurons, with the potential of aiding axon growth over inhibitory substrates produced by the glial scar.

Following nerve injury, denervated Schwann cells (SCs) convert to repair SCs, which enable regeneration of peripheral axons. However, the repair capacity of SCs and the regenerative capacity of peripheral axons are limited. In the present studies we examined a potential therapeutic strategy to enhance the repair capacity of SCs, and tested its efficacy in enhancing regeneration of dorsal root (DR) axons, whose regenerative capacity is particularly weak. We used male and female mice of a doxycycline-inducible transgenic line to induce expression of constitutively active ErbB2 (caErbB2) selectively in SCs after DR crush or transection. Two weeks after injury, injured DRs of induced animals contained far more SCs and SC processes. These SCs had not redifferentiated and continued to proliferate. Injured DRs of induced animals also contained far more axons that regrew along SC processes past the transection or crush site. Remarkably, SCs and axons in uninjured DRs remained quiescent, indicating that caErbB2 enhanced regeneration of injured DRs, without aberrantly activating SCs and axons in intact nerves. We also found that intraspinally expressed glial cell line-derived neurotrophic factor (GDNF), but not the removal of chondroitin sulfate proteoglycans, greatly enhanced the intraspinal migration of caErbB2-expressing SCs, enabling robust penetration of DR axons into the spinal cord. These findings indicate that SC-selective, post-injury activation of ErbB2 provides a novel strategy to powerfully enhance the repair capacity of SCs and axon regeneration, without substantial off-target damage. They also highlight that promoting directed migration of caErbB2-expressing SCs by GDNF might be useful to enable axon regrowth in a non-permissive environment.SIGNIFICANCE STATEMENTRepair of injured peripheral nerves remains a critical clinical problem. We currently lack a therapy that potently enhances axon regeneration in patients with traumatic nerve injury. It is extremely challenging to substantially increase the regenerative capacity of damaged nerves without deleterious off-target effects. It was therefore of great interest to discover that caErbB2 markedly enhances regeneration of damaged dorsal roots, while evoking little change in intact roots. To our knowledge, these findings are the first demonstration that repair capacity of denervated SCs can be efficaciously enhanced without altering innervated SCs. Our study also demonstrates that oncogenic ErbB2 signaling can be activated in SCs but not impede transdifferentiation of denervated SCs to regeneration-promoting repair SCs.

The role of posttranslational modifications in axonal injury and regeneration has been widely studied but there has been little consensus over the mechanism by which each modification affects adult axonal growth. Acetylation is known to play an important role in a variety of neuronal functions and its homeostasis is controlled by two enzyme families: the Histone Deacetylases (HDACs) and Histone Acetyl Transferases (HATs). Recent studies show that HDAC5 deacetylates microtubules in the axonal cytoplasm as part of an injury-induced regeneration response, but little is known about how acetylation of microtubules plays a role. Alpha-tubulin acetyl transferase (αTAT1) is a microtubule specific acetyl transferase that binds to microtubules and directly affects microtubule stability in cells. We hypothesize that increasing tubulin acetylation may play an important role in increasing the rate of axonal growth. In this study, we infected cultured adult DRG neurons with αTAT1 and αTAT1-D157N, a catalytically inactive mutant, and HDAC5, using lentiviruses. We found that αTAT1 significantly increases tubulin acetylation in 293T cells and DRG neurons but αTAT1-D157N does not. Furthermore, in neurons infected with αTAT1, a significant increase in acetylated tubulin was detected towards the distal portion of the axon but this increase was not detected in neurons infected with αTAT1-D157N. However, we found a significant increase in axon lengths of DRG neurons after αTAT1 and αTAT1-D157N infection, but no effect on axon lengths after infection with HDAC5. Our results suggest that while αTAT1 may play a role in axon growth in vitro, the increase is not directly due to acetylation of axonal microtubules. Our results also show that HDAC5 overexpression in the axonal cytoplasm does not play a crucial role in axonal regeneration of cultured DRG neurons. We expressed these genes in DRG neurons in adult rats and performed a sciatic nerve crush. We found that axons did not regenerate any better when infected with any of the constructs compared with control animals. Thus, while αTAT1 may be important for axonal growth in vitro, neither αTAT1 nor HDAC5 had an effect in vivo on the regeneration of sciatic nerves.

Parkinson's disease (PD) represents a challenging condition where different therapeutic options have evolved over the course of the last 50 years. The potential for therapeutic use of cell transplantation for cell replacement or for gene delivery of neurotrophic factors has received a great deal of attention. Currently, all available treatment options are directed towards the amelioration of symptoms. A greater understanding of the distinctive pathology underlying PD might offer some novel therapeutic approaches. Transplantation of embryonic ventral mesencephalon (VM) dopaminergic neurons has shown promise in animal studies, but similar transplant procedures have shown limited success in clinical trials. One important issue may be the site of transplantation. Previous studies have transplanted VM into the striatum, which is the target of these neurons. With increased understanding of growth and guidance molecule effecting dopaminergic neurons, it may be feasible to place transplants in the damaged substantia nigra and direct the growth of axons into target regions to reconstruction of midbrain dopamine (DA) circuitry. Our established and on-going understanding of the molecular cues which support directed growth of DA neurons form an important basis for the refinement and optimization of VM grafting procedures, and also the development of new procedures based on the use of stem cells. In this review, we discuss transplantation therapy and how selective guidance molecules could be used to reconstruction of nigrostriatal circuit.

Different experimental and clinical strategies have been used to promote survival of transplanted embryonic ventral mesencephalic (VM) neurons. However, few studies have focused on the long-distance growth of dopaminergic axons from VM transplants. The aim of this study is to identify some of the growth and guidance factors that support directed long-distance growth of dopaminergic axons from VM transplants. Lentivirus encoding either glial cell line-derived neurotrophic factor (GDNF) or netrin-1, or a combination of lenti-GDNF with either lenti-GDNF family receptor α1 (GFRα-1) or lenti-netrin-1 was injected to form a gradient along the corpus callosum. Two weeks later, a piece of embryonic day 14 VM tissue was transplanted into the corpus callosum adjacent to the low end of the gradient. Results showed that tyrosine hydroxylase (TH(+)) axons grew a very short distance from the VM transplants in control groups, with few axons reaching the midline. In GDNF or netrin-1 expressing groups, more TH(+) axons grew out of transplants and reached the midline. Pathways co-expressing GDNF with either GFRα-1 or netrin-1 showed significantly increased axonal outgrowth. Interestingly, only the GDNF/netrin-1 combination resulted in the majority of axons reaching the distal target (80%), whereas along the GDNF/GFRα-1 pathway only 20% of the axons leaving the transplant reached the distal target. This technique of long-distance axon guidance may prove to be a useful strategy in reconstructing damaged neuronal circuits, such as the nigrostriatal pathway in Parkinson's disease.