Challenges and Efficacy of Astrocyte-to-Neuron Reprogramming in Spinal Cord Injury: In Vitro Insights and In Vivo Outcomes.

Traumatic spinal cord injury (SCI) leads to the disruption of neural pathways, causing loss of neural cells, with subsequent reactive gliosis and tissue scarring that limit endogenous repair. One potential therapeutic strategy to address this is to target reactive scar-forming astrocytes with direct cellular reprogramming to convert them into neurons, by overexpression of neurogenic transcription factors. Here we used lentiviral constructs to overexpress Ascl1 or a combination of microRNAs (miRs) miR124, miR9/9*and NeuroD1 transfected into cultured and in vivo astrocytes. In vitro experiments revealed cortically-derived astrocytes display a higher efficiency (70%) of reprogramming to neurons than spinal cord-derived astrocytes. In a rat cervical SCI model, the same strategy induced only limited reprogramming of astrocytes. Delivery of reprogramming factors did not significantly affect patterns of breathing under baseline and hypoxic conditions, but significant differences in average diaphragm amplitude were seen in the reprogrammed groups during eupneic breathing, hypoxic, and hypercapnic challenges. These results show that while cellular reprogramming can be readily achieved in carefully controlled in vitro conditions, achieving a similar degree of successful reprogramming in vivo is challenging and may require additional steps.

Human spinal interneurons repair the injured spinal cord through synaptic integration.

Advances in cell therapy offer promise for some of the most devastating neural injuries, including spinal cord injury (SCI). Endogenous VSX2-expressing spinal V2a interneurons have been implicated as a key component in plasticity and therapeutically driven recovery post-SCI. While transplantation of generic V2a neurons may have therapeutic value, generation of human spinal V2a neurons with rostro-caudal specificity and assessment of their functional synaptic integration with the injured spinal cord has been elusive. Here, we efficiently differentiated optogenetically engineered cervical V2a spinal interneurons (SpINs) from human induced pluripotent stem cells and tested their capacity to form functional synapses with injured diaphragm motor networks in a clinically-relevant sub-acute model of cervical contusion injury. Neuroanatomical tracing and immunohistochemistry demonstrated transplant integration and synaptic connectivity with injured host tissue. Optogenetic activation of transplanted human V2a SpINs revealed functional synaptic connectivity to injured host circuits, culminating in improved diaphragm activity assessed by electromyography. Furthermore, optogenetic activation of host supraspinal pathways revealed functional innervation of transplanted cells by host neurons, which also led to enhanced diaphragm contraction indicative of a functional neuronal relay. Single cell analyses pre- and post-transplantation suggested the in vivo environment resulted in maturation of cervical SpINs that mediate the formation of neuronal relays, as well as differentiation of glial progenitors involved in repair of the damaged spinal cord. This study rigorously demonstrates feasibility of generating human cervical spinal V2a interneurons that develop functional host-transplant and transplant-host connectivity resulting in improved muscle activity post-SCI.

Diseased, differentiated and difficult: Strategies for improved engineering of in vitro neurological systems.

The rapidly growing field of cellular engineering is enabling scientists to more effectively create in vitro models of disease and develop specific cell types that can be used to repair damaged tissue. In particular, the engineering of neurons and other components of the nervous system is at the forefront of this field. The methods used to engineer neural cells can be largely divided into systems that undergo directed differentiation through exogenous stimulation (i.e., via small molecules, arguably following developmental pathways) and those that undergo induced differentiation via protein overexpression (i.e., genetically induced and activated; arguably bypassing developmental pathways). Here, we highlight the differences between directed differentiation and induced differentiation strategies, how they can complement one another to generate specific cell phenotypes, and impacts of each strategy on downstream applications. Continued research in this nascent field will lead to the development of improved models of neurological circuits and novel treatments for those living with neurological injury and disease.

Harnessing Spinal Interneurons for Spinal Cord Repair.

Interest in spinal interneurons (SpINs), their heterogeneity in the naive spinal cord and their varying responses to central nervous system injury or disease has been steadily increasing. Our recent review on this topic highlights the vast phenotypic heterogeneity of SpINs and the efforts being made to better identify and classify these neurons. As our understanding of SpIN phenotype, connectivity, and neuroplastic capacity continues to expand, new therapeutic targets are being revealed and novel treatment approaches developed to harness their potential. Here, we expand on that initial discussion and highlight how SpINs can be used to develop advanced, targeted cellular therapies and personalized medicines.

Respiratory plasticity following spinal cord injury: perspectives from mouse to man.

The study of respiratory plasticity in animal models spans decades. At the bench, researchers use an array of techniques aimed at harnessing the power of plasticity within the central nervous system to restore respiration following spinal cord injury. This field of research is highly clinically relevant. People living with cervical spinal cord injury at or above the level of the phrenic motoneuron pool at spinal levels C3-C5 typically have significant impairments in breathing which may require assisted ventilation. Those who are ventilator dependent are at an increased risk of ventilator-associated co-morbidities and have a drastically reduced life expectancy. Pre-clinical research examining respiratory plasticity in animal models has laid the groundwork for clinical trials. Despite how widely researched this injury is in animal models, relatively few treatments have broken through the preclinical barrier. The three goals of this present review are to define plasticity as it pertains to respiratory function post-spinal cord injury, discuss plasticity models of spinal cord injury used in research, and explore the shift from preclinical to clinical research. By investigating current targets of respiratory plasticity research, we hope to illuminate preclinical work that can influence future clinical investigations and the advancement of treatments for spinal cord injury.

Effects of Chronic High-Frequency rTMS Protocol on Respiratory Neuroplasticity Following C2 Spinal Cord Hemisection in Rats.

High spinal cord injuries (SCIs) lead to permanent diaphragmatic paralysis. The search for therapeutics to induce functional motor recovery is essential. One promising noninvasive therapeutic tool that could harness plasticity in a spared descending respiratory circuit is repetitive transcranial magnetic stimulation (rTMS). Here, we tested the effect of chronic high-frequency (10 Hz) rTMS above the cortical areas in C2 hemisected rats when applied for 7 days, 1 month, or 2 months. An increase in intact hemidiaphragm electromyogram (EMG) activity and excitability (diaphragm motor evoked potentials) was observed after 1 month of rTMS application. Interestingly, despite no real functional effects of rTMS treatment on the injured hemidiaphragm activity during eupnea, 2 months of rTMS treatment strengthened the existing crossed phrenic pathways, allowing the injured hemidiaphragm to increase its activity during the respiratory challenge (i.e., asphyxia). This effect could be explained by a strengthening of respiratory descending fibers in the ventrolateral funiculi (an increase in GAP-43 positive fibers), sustained by a reduction in inflammation in the C1-C3 spinal cord (reduction in CD68 and Iba1 labeling), and acceleration of intracellular plasticity processes in phrenic motoneurons after chronic rTMS treatment. These results suggest that chronic high-frequency rTMS can ameliorate respiratory dysfunction and elicit neuronal plasticity with a reduction in deleterious post-traumatic inflammatory processes in the cervical spinal cord post-SCI. Thus, this therapeutic tool could be adopted and/or combined with other therapeutic interventions in order to further enhance beneficial outcomes.

Transneuronal tracing to map connectivity in injured and transplanted spinal networks.

It has become widely appreciated that the spinal cord has significant neuroplastic potential, is not hard-wired, and that with traumatic injury and anatomical plasticity, the networks that we once understood now comprise a new anatomy. Harnessing advances in neuroanatomical tracing to map the neuronal networks of the intact and injured spinal cord has been crucial to elucidating this new spinal cord anatomy. Many new techniques have been developed to identify these networks using a variety of retrograde and anterograde tracers. One method of tracing that has become more widely used to map anatomical changes is transneuronal tracing. Viral tracers are being increasingly used to map spinal networks, leading to an advanced understanding of spinal circuitry and host-donor-host interactions between the injured spinal cord and neural transplants. This review will highlight advances in neuronal tracing, specifically using pseudorabies virus (PRV), and its use in the intact, injured, and transplanted spinal cord.

Respiratory Training and Plasticity After Cervical Spinal Cord Injury.

While spinal cord injuries (SCIs) result in a vast array of functional deficits, many of which are life threatening, the majority of SCIs are anatomically incomplete. Spared neural pathways contribute to functional and anatomical neuroplasticity that can occur spontaneously, or can be harnessed using rehabilitative, electrophysiological, or pharmacological strategies. With a focus on respiratory networks that are affected by cervical level SCI, the present review summarizes how non-invasive respiratory treatments can be used to harness this neuroplastic potential and enhance long-term recovery. Specific attention is given to "respiratory training" strategies currently used clinically (e.g., strength training) and those being developed through pre-clinical and early clinical testing [e.g., intermittent chemical stimulation via altering inhaled oxygen (hypoxia) or carbon dioxide stimulation]. Consideration is also given to the effect of training on non-respiratory (e.g., locomotor) networks. This review highlights advances in this area of pre-clinical and translational research, with insight into future directions for enhancing plasticity and improving functional outcomes after SCI.

Allele-Specific Gene Editing Rescues Pathology in a Human Model of Charcot-Marie-Tooth Disease Type 2E.

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

Bioengineered optogenetic model of human neuromuscular junction.

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.

High frequency repetitive Transcranial Magnetic Stimulation promotes long lasting phrenic motoneuron excitability via GABAergic networks.

Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABAA and GABAB receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.

Preparation of Neural Stem Cells and Progenitors: Neuronal Production and Grafting Applications.

Neural stem cells (NSCs) are a valuable tool for the study of neural development and function as well as an important source of cell transplantation strategies for neural disease. NSCs can be used to study how neurons acquire distinct phenotypes and how the interactions between neurons and glial cells in the developing nervous system shape the structure and function of the CNS. NSCs can also be used for cell replacement therapies following CNS injury targeting astrocytes, oligodendrocytes, and neurons. With the availability of patient-derived induced pluripotent stem cells (iPSCs), neurons prepared from NSCs can be used to elucidate the molecular basis of neurological disorders leading to potential treatments. Although NSCs can be derived from different species and many sources, including embryonic stem cells (ESCs), iPSCs, adult CNS, and direct reprogramming of nonneural cells, isolating primary NSCs directly from fetal tissue is still the most common technique for preparation and study of neurons. Regardless of the source of tissue, similar techniques are used to maintain NSCs in culture and to differentiate NSCs toward mature neural lineages. This chapter will describe specific methods for isolating and characterizing multipotent NSCs and neural precursor cells (NPCs) from embryonic rat CNS tissue (mostly spinal cord) and from human ESCs and iPSCs as well as NPCs prepared by reprogramming. NPCs can be separated into neuronal and glial restricted progenitors (NRP and GRP, respectively) and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS. This chapter will describe in detail the methods required for the isolation, propagation, storage, and differentiation of NSCs and NPCs isolated from rat and mouse spinal cords for subsequent in vitro or in vivo studies as well as new methods associated with ESCs, iPSCs, and reprogramming.

Spinal Interneurons as Gatekeepers to Neuroplasticity after Injury or Disease.

Spinal interneurons are important facilitators and modulators of motor, sensory, and autonomic functions in the intact CNS. This heterogeneous population of neurons is now widely appreciated to be a key component of plasticity and recovery. This review highlights our current understanding of spinal interneuron heterogeneity, their contribution to control and modulation of motor and sensory functions, and how this role might change after traumatic spinal cord injury. We also offer a perspective for how treatments can optimize the contribution of interneurons to functional improvement.

Transplanting Cells for Spinal Cord Repair: Who, What, When, Where and Why?

Cellular transplantation for repair of the injured spinal cord has a rich history with strategies focused on neuroprotection, immunomodulation, and neural reconstruction. The goal of the present review is to provide a concise overview and discussion of five key themes that have become important considerations for rebuilding functional neural networks. The questions raised include: (i) who are the donor cells selected for transplantation, (ii) what is the intended target for repair, (iii) when is the optimal time for transplantation, (iv) where should the cells be delivered, and lastly (v) why does cell transplantation remain an attractive candidate for promoting neural repair after injury? Recent developments in neurobiology and engineering now enable us to start addressing these questions with multidisciplinary expertise and methods.

Hereditary spastic paraplegia: gain-of-function mechanisms revealed by new transgenic mouse.

Mutations of the SPAST gene, which encodes the microtubule-severing protein spastin, are the most common cause of hereditary spastic paraplegia (HSP). Haploinsufficiency is the prevalent opinion as to the mechanism of the disease, but gain-of-function toxicity of the mutant proteins is another possibility. Here, we report a new transgenic mouse (termed SPASTC448Y mouse) that is not haploinsufficient but expresses human spastin bearing the HSP pathogenic C448Y mutation. Expression of the mutant spastin was documented from fetus to adult, but gait defects reminiscent of HSP (not observed in spastin knockout mice) were adult onset, as is typical of human patients. Results of histological and tracer studies on the mouse are consistent with progressive dying back of corticospinal axons, which is characteristic of the disease. The C448Y-mutated spastin alters microtubule stability in a manner that is opposite to the expectations of haploinsufficiency. Neurons cultured from the mouse display deficits in organelle transport typical of axonal degenerative diseases, and these deficits were worsened by depletion of endogenous mouse spastin. These results on the SPASTC448Y mouse are consistent with a gain-of-function mechanism underlying HSP, with spastin haploinsufficiency exacerbating the toxicity of the mutant spastin proteins. These findings reveal the need for a different therapeutic approach than indicated by haploinsufficiency alone.

The Neuroplastic and Therapeutic Potential of Spinal Interneurons in the Injured Spinal Cord.

The central nervous system is not a static, hard-wired organ. Examples of neuroplasticity, whether at the level of the synapse, the cell, or within and between circuits, can be found during development, throughout the progression of disease, or after injury. One essential component of the molecular, anatomical, and functional changes associated with neuroplasticity is the spinal interneuron (SpIN). Here, we draw on recent multidisciplinary studies to identify and interrogate subsets of SpINs and their roles in locomotor and respiratory circuits. We highlight some of the recent progress that elucidates the importance of SpINs in circuits affected by spinal cord injury (SCI), especially those within respiratory networks; we also discuss potential ways that spinal neuroplasticity can be therapeutically harnessed for recovery.

Transplantation of Neural Progenitors and V2a Interneurons after Spinal Cord Injury.

There is growing interest in the use of neural precursor cells to treat spinal cord injury (SCI). Despite extensive pre-clinical research, it remains unclear as to which donor neuron phenotypes are available for transplantation, whether the same populations exist across different sources of donor tissue (e.g., developing tissue vs. cultured cells), and whether donor cells retain their phenotype once transplanted into the hostile internal milieu of the injured adult spinal cord. In addition, while functional improvements have been reported after neural precursor transplantation post-SCI, the extent of recovery is limited and variable. The present work begins to address these issues by harnessing ventrally derived excitatory pre-motor V2a spinal interneurons (SpINs) to repair the phrenic motor circuit after cervical SCI. Recent studies have demonstrated that Chx10-positive V2a SpINs contribute to anatomical plasticity within the phrenic circuitry after cervical SCI, thus identifying them as a therapeutic candidate. Building upon this discovery, the present work tests the hypothesis that transplantation of neural progenitor cells (NPCs) enriched with V2a INs can contribute to neural networks that promote repair and enhance respiratory plasticity after cervical SCI. Cultured NPCs (neuronal and glial restricted progenitor cells) isolated from E13.5 Green fluorescent protein rats were aggregated with TdTomato-mouse embryonic stem cell-derived V2a INs in vitro, then transplanted into the injured cervical (C3-4) spinal cord. Donor cells survive, differentiate and integrate with the host spinal cord. Functional diaphragm electromyography indicated recovery 1 month following treatment in transplant recipients. Animals that received donor cells enriched with V2a INs showed significantly greater functional improvement than animals that received NPCs alone. The results from this study offer insight into the neuronal phenotypes that might be effective for (re)establishing neuronal circuits in the injured adult central nervous system.

Integration of Transplanted Neural Precursors with the Injured Cervical Spinal Cord.

Cervical spinal cord injuries (SCI) result in devastating functional consequences, including respiratory dysfunction. This is largely attributed to the disruption of phrenic pathways, which control the diaphragm. Recent work has identified spinal interneurons as possible contributors to respiratory neuroplasticity. The present work investigated whether transplantation of developing spinal cord tissue, inherently rich in interneuronal progenitors, could provide a population of new neurons and growth-permissive substrate to facilitate plasticity and formation of novel relay circuits to restore input to the partially denervated phrenic motor circuit. One week after a lateralized, C3/4 contusion injury, adult Sprague-Dawley rats received allografts of dissociated, developing spinal cord tissue (from rats at gestational days 13-14). Neuroanatomical tracing and terminal electrophysiology was performed on the graft recipients 1 month later. Experiments using pseudorabies virus (a retrograde, transynaptic tracer) revealed connections from donor neurons onto host phrenic circuitry and from host, cervical interneurons onto donor neurons. Anatomical characterization of donor neurons revealed phenotypic heterogeneity, though donor-host connectivity appeared selective. Despite the consistent presence of cholinergic interneurons within donor tissue, transneuronal tracing revealed minimal connectivity with host phrenic circuitry. Phrenic nerve recordings revealed changes in burst amplitude after application of a glutamatergic, but not serotonergic antagonist to the transplant, suggesting a degree of functional connectivity between donor neurons and host phrenic circuitry that is regulated by glutamatergic input. Importantly, however, anatomical and functional results were variable across animals, and future studies will explore ways to refine donor cell populations and entrain consistent connectivity.