Megan C. Wright

Schwann cell phenotype is regulated by axon modality and central–peripheral location, and persists in vitro ☆

Myelinating Schwann cells express distinct sensory and motor phenotypes as defined by their differing patterns of growth factor production (Hoke et al., 2006). The heterogeneous growth factor requirements of sensory and motor neurons, however, suggest that Schwann cell phenotype might vary across a broad spectrum. To explore this possibility, we selectively denervated six discrete Schwann cell populations: dorsal root, cutaneous nerve, cutaneous unmyelinated axons, muscle nerve afferents, muscle nerve efferents, and ventral root. Real-time RT-PCR for 11 growth factors was performed on the 6 target Schwann cell populations 5, 15, and 30 days after their denervation, and on normal cutaneous nerve, muscle nerve, ventral root, and dorsal root to establish baseline expression levels. Within the denervated axon populations, IGF-1 and VEGF were expressed most prominently in cutaneous nerve, HGF, NGF, and BDNF in cutaneous nerve and dorsal root, GDNF in dorsal root and ventral root, PTN in the ventral root and muscle nerve efferents, and IGF-2 in both afferents and efferents within muscle nerve; expression of CNTF, FGF-2 and NT-3 was not modality or location specific. ELISA for NGF, BDNF, and GDNF confirmed that gene expression correlated with protein concentration. These findings demonstrate that growth factor expression by denervated Schwann cells is not only subject to further regulation within the previously-defined sensory and motor groups, but also varies along a central-peripheral axis. The traditional view of myelinating Schwann cells as a homogenous population is modified by the realization that complex regulation produces a wide variety of Schwann cell phenotypes. Additionally, we found that Schwann cell phenotype is maintained for 2 weeks in vitro, demonstrating that it may survive several cell divisions without instructive cues from either axons or basal lamina.

Phrenic motor neuron degeneration compromises phrenic axonal circuitry and diaphragm activity in a unilateral cervical contusion model of spinal cord injury

Respiratory dysfunction is the leading cause of morbidity and mortality following traumatic spinal cord injury (SCI). Injuries targeting mid-cervical spinal cord regions affect the phrenic motor neuron pool that innervates the diaphragm, the primary respiratory muscle of inspiration. Contusion-type injury in the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron-diaphragm circuitry. In an attempt to target the phrenic motor neuron pool, two unilateral contusion injury paradigms were tested, a single injury at level C4 and a double injury both at levels C3 and C4, and animals were followed for up to 6 weeks post-injury. Both unilateral cervical injury paradigms are reproducible with no mortality or need for breathing assistance, and are accompanied by phrenic motor neuron loss, phrenic nerve axon degeneration, diaphragm atrophy, denervation and subsequent partial reinnervation at the diaphragm neuromuscular junction, changes in spontaneous diaphragm EMG recordings, and reduction in phrenic nerve compound muscle action potential amplitude. These findings demonstrate significant and chronically persistent respiratory compromise following mid-cervical SCI due to phrenic motor neuron degeneration. These injury paradigms and accompanying analyses provide important tools both for understanding mechanisms of phrenic motor neuron and diaphragm pathology following SCI and for evaluating therapeutic strategies in clinically relevant cervical SCI models.

Early Phrenic Motor Neuron Loss and Transient Respiratory Abnormalities after Unilateral Cervical Spinal Cord Contusion

Contusion-type cervical spinal cord injury (SCI) is one of the most common forms of SCI observed in patients. In particular, injuries targeting the C3-C5 region affect the pool of phrenic motor neurons (PhMNs) that innervates the diaphragm, resulting in significant and often chronic respiratory dysfunction. Using a previously described rat model of unilateral midcervical C4 contusion with the Infinite Horizon Impactor, we have characterized the early time course of PhMN degeneration and consequent respiratory deficits following injury, as this knowledge is important for designing relevant treatment strategies targeting protection and plasticity of PhMN circuitry. PhMN loss (48% of the ipsilateral pool) occurred almost entirely during the first 24 h post-injury, resulting in persistent phrenic nerve axonal degeneration and denervation at the diaphragm neuromuscular junction (NMJ). Reduced diaphragm compound muscle action potential amplitudes following phrenic nerve stimulation were observed as early as the first day post-injury (30% of pre-injury maximum amplitude), with slow functional improvement over time that was associated with partial reinnervation at the diaphragm NMJ. Consistent with ipsilateral diaphragmatic compromise, the injury resulted in rapid, yet only transient, changes in overall ventilatory parameters measured via whole-body plethysmography, including increased respiratory rate, decreased tidal volume, and decreased peak inspiratory flow. Despite significant ipsilateral PhMN loss, the respiratory system has the capacity to quickly compensate for partially impaired hemidiaphragm function, suggesting that C4 hemicontusion in rats is a model of SCI that manifests subacute respiratory abnormalities. Collectively, these findings demonstrate significant and persistent diaphragm compromise in a clinically relevant model of midcervical contusion SCI; however, the therapeutic window for PhMN protection is restricted to early time points post-injury. On the contrary, preventing loss of innervation by PhMNs and/or inducing plasticity in spared PhMN axons at the diaphragm NMJ are relevant long-term targets.

Novel Roles for Osteopontin and Clusterin in Peripheral Motor and Sensory Axon Regeneration

Previous studies demonstrated that Schwann cells (SCs) express distinct motor and sensory phenotypes, which impact the ability of these pathways to selectively support regenerating neurons. In the present study, unbiased microarray analysis was used to examine differential gene expression in denervated motor and sensory pathways in rats. Several genes that were significantly upregulated in either denervated sensory or motor pathways were identified and two secreted factors were selected for further analysis: osteopontin (OPN) and clusterin (CLU) which were upregulated in denervated motor and sensory pathways, respectively. Sciatic nerve transection induced upregulation of OPN and CLU and expression of both returned to baseline levels with ensuing regeneration. In vitro analysis using exogenously applied OPN induced outgrowth of motor but not sensory neurons. CLU, however, induced outgrowth of sensory neurons, but not motor neurons. To assess the functional importance of OPN and CLU, peripheral nerve regeneration was examined in OPN and CLU−/− mice. When compared with OPN+/+ mice, motor neuron regeneration was reduced in OPN−/− mice. Impaired regeneration through OPN−/− peripheral nerves grafted into OPN+/+ mice indicated that loss of OPN in SCs was responsible for reduced motor regeneration. Sensory neuron regeneration was impaired in CLU−/− mice following sciatic nerve crush and impaired regeneration nerve fibers through CLU−/− nerve grafts transplanted into CLU+/+ mice indicated that reduced sensory regeneration is likely due to SC-derived CLU. Together, these studies suggest unique roles for SC-derived OPN and CLU in regeneration of peripheral motor and sensory axons.

Overexpression of the astrocyte glutamate transporter GLT1 exacerbates phrenic motor neuron degeneration, diaphragm compromise, and forelimb motor dysfunction following cervical contusion spinal cord injury.

A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP+ astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.

Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury

Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI.

Degeneration of phrenic motor neurons induces long-term diaphragm deficits following mid-cervical spinal contusion in mice.

A primary cause of morbidity and mortality following cervical spinal cord injury (SCI) is respiratory compromise, regardless of the level of trauma. In particular, SCI at mid-cervical regions targets degeneration of both descending bulbospinal respiratory axons and cell bodies of phrenic motor neurons, resulting in deficits in the function of the diaphragm, the primary muscle of inspiration. Contusion-type trauma to the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron-diaphragm circuitry. We have generated a mouse model of cervical contusion SCI that unilaterally targets both C4 and C5 levels, the location of the phrenic motor neuron pool, and have examined histological and functional outcomes for up to 6 weeks post-injury. We report that phrenic motor neuron loss in cervical spinal cord, phrenic nerve axonal degeneration, and denervation at diaphragm neuromuscular junctions (NMJ) resulted in compromised ipsilateral diaphragm function, as demonstrated by persistent reduction in diaphragm compound muscle action potential amplitudes following phrenic nerve stimulation and abnormalities in spontaneous diaphragm electromyography (EMG) recordings. This injury paradigm is reproducible, does not require ventilatory assistance, and provides proof-of-principle that generation of unilateral cervical contusion is a feasible strategy for modeling diaphragmatic/respiratory deficits in mice. This study and its accompanying analyses pave the way for using transgenic mouse technology to explore the function of specific genes in the pathophysiology of phrenic motor neuron degeneration and respiratory dysfunction following cervical SCI.

Peripheral Hyperstimulation Alters Site of Disease Onset and Course in SOD1 Rats

In amyotrophic lateral sclerosis (ALS), the exogenous temporal triggers that result in initial motor neuron death are not understood. Overactivation and consequent accelerated loss of vulnerable motor neurons is one theory of disease initiation. The vulnerability of motor neurons in response to chronic peripheral nerve hyperstimulation was tested in the SOD1(G93A) rat model of ALS. A novel in vivo technique for peripheral phrenic nerve stimulation was developed via intra-diaphragm muscle electrode implantation at the phrenic motor endpoint. Chronic bilateral phrenic nerve hyperstimulation in SOD1(G93A) rats accelerated disease progression, including shortened lifespan, hastened motor neuron loss and increased denervation at diaphragm neuromuscular junctions. Hyperstimulation also resulted in focal decline in adjacent forelimb function. These results show that peripheral phrenic nerve hyperstimulation accelerates cell death of vulnerable spinal motor neurons, modifies both temporal and anatomical onset of disease, and leads to involvement of disease in adjacent anatomical regions in this ALS model.

Distinct Muscarinic Acetylcholine Receptor Subtypes Contribute to Stability and Growth, But Not Compensatory Plasticity, of Neuromuscular Synapses

Muscarinic acetylcholine receptors (mAChRs) modulate synaptic function, but whether they influence synaptic structure remains unknown. At neuromuscular junctions (NMJs), mAChRs have been implicated in compensatory sprouting of axon terminals in paralyzed or denervated muscles. Here we used pharmacological and genetic inhibition and localization studies of mAChR subtypes at mouse NMJs to demonstrate their roles in synaptic stability and growth but not in compensatory sprouting. M2 mAChRs were present solely in motor neurons, whereas M1, M3, and M5 mAChRs were associated with Schwann cells and/or muscle fibers. Blockade of all five mAChR subtypes with atropine evoked pronounced effects, including terminal sprouting, terminal withdrawal, and muscle fiber atrophy. In contrast, methoctramine, an M2/4-preferring antagonist, induced terminal sprouting and terminal withdrawal, but no muscle fiber atrophy. Consistent with this observation, M2−/− but no other mAChR mutant mice exhibited spontaneous sprouting accompanied by extensive loss of parental terminal arbors. Terminal sprouting, however, seemed not to be the causative defect because partial loss of terminal branches was common even in the M2−/− NMJs without sprouting. Moreover, compensatory sprouting after paralysis or partial denervation was normal in mice deficient in M2 or other mAChR subtypes. We also found that many NMJs of M5−/− mice were exceptionally small and reduced in proportion to the size of parental muscle fibers. These findings show that axon terminals are unstable without M2 and that muscle fiber growth is defective without M5. Subtype-specific muscarinic signaling provides a novel means for coordinating activity-dependent development and maintenance of the tripartite synapse.

Ciliary neurotrophic factor is not required for terminal sprouting and compensatory reinnervation of neuromuscular synapses: re-evaluation of CNTF null mice.

Loss of synaptic activity or innervation induces sprouting of intact motor nerve terminals that adds or restores nerve-muscle connectivity. Ciliary neurotrophic factor (CNTF) and terminal Schwann cells (tSCs) have been implicated as molecular and cellular mediators of the compensatory process. We wondered if the previously reported lack of terminal sprouting in CNTF null mice was due to abnormal reactivity of tSCs. To this end, we examined nerve terminal and tSC responses in CNTF null mice using experimental systems that elicited extensive sprouting in wildtype mice. Contrary to the previous report, we found that motor nerve terminals in the null mice sprout extensively in response to major sprouting-stimuli such as exogenously applied CNTF per se, botulinum toxin-elicited paralysis, and partial denervation by L4 spinal root transection. In addition, the number, length and growth patterns of terminal sprouts, and the extent of reinnervation by terminal or nodal sprouts, were similar in wildtype and null mice. tSCs in the null mice were also reactive to the sprouting-stimuli, elaborating cellular processes that accompanied terminal sprouts or guided reinnervation of denervated muscle fibers. Lastly, CNTF was absent in quiescent tSCs in intact, wildtype muscles and little if any was detected in reactive tSCs in denervated muscles. Thus, CNTF is not required for induction of nerve terminal sprouting, for reactivation of tSCs, and for compensatory reinnervation after nerve injury. We interpret these results to support the notion that compensatory sprouting in adult muscles is induced primarily by contact-mediated mechanisms, rather than by diffusible factors.