Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2

Gene-based therapeutic strategies to lower ataxin-2 levels are emerging for neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). To identify additional ways of reducing ataxin-2 levels, we performed a genome-wide screen in human cells for regulators of ataxin-2 and identified RTN4R, the gene encoding the RTN4/NoGo-Receptor, as a top hit. RTN4R knockdown, or treatment with a peptide inhibitor, was sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro and Rtn4r knockout mice have reduced ataxin-2 levels in vivo. Remarkably, we observed that ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy. These data define the RTN4/NoGo-Receptor as a novel therapeutic target for ALS and SCA2 and implicate the targeting of ataxin-2 as a potential treatment following nerve injury.

Bio-Scaffolds as Cell or Exosome Carriers for Nerve Injury Repair

Central and peripheral nerve injuries can lead to permanent paralysis and organ dysfunction. In recent years, many cell and exosome implantation techniques have been developed in an attempt to restore function after nerve injury with promising but generally unsatisfactory clinical results. Clinical outcome may be enhanced by bio-scaffolds specifically fabricated to provide the appropriate three-dimensional (3D) conduit, growth-permissive substrate, and trophic factor support required for cell survival and regeneration. In rodents, these scaffolds have been shown to promote axonal regrowth and restore limb motor function following experimental spinal cord or sciatic nerve injury. Combining the appropriate cell/exosome and scaffold type may thus achieve tissue repair and regeneration with safety and efficacy sufficient for routine clinical application. In this review, we describe the efficacies of bio-scaffolds composed of various natural polysaccharides (alginate, chitin, chitosan, and hyaluronic acid), protein polymers (gelatin, collagen, silk fibroin, fibrin, and keratin), and self-assembling peptides for repair of nerve injury. In addition, we review the capacities of these constructs for supporting in vitro cell-adhesion, mechano-transduction, proliferation, and differentiation as well as the in vivo properties critical for a successful clinical outcome, including controlled degradation and re-absorption. Finally, we describe recent advances in 3D bio-printing for nerve regeneration.

An injury-induced serotonergic neuron subpopulation contributes to axon regrowth and function restoration after spinal cord injury in zebrafish

Spinal cord injury (SCI) interrupts long-projecting descending spinal neurons and disrupts the spinal central pattern generator (CPG) that controls locomotion. The intrinsic mechanisms underlying re-wiring of spinal neural circuits and recovery of locomotion after SCI are unclear. Zebrafish shows axonal regeneration and functional recovery after SCI making it a robust model to study mechanisms of regeneration. Here, we use a two-cut SCI model to investigate whether recovery of locomotion can occur independently of supraspinal connections. Using this injury model, we show that injury induces the localization of a specialized group of intraspinal serotonergic neurons (ISNs), with distinctive molecular and cellular properties, at the injury site. This subpopulation of ISNs have hyperactive terminal varicosities constantly releasing serotonin activating 5-HT1B receptors, resulting in axonal regrowth of spinal interneurons. Axon regrowth of excitatory interneurons is more pronounced compared to inhibitory interneurons. Knock-out of htr1b prevents axon regrowth of spinal excitatory interneurons, negatively affecting coordination of rostral-caudal body movements and restoration of locomotor function. On the other hand, treatment with 5-HT1B receptor agonizts promotes functional recovery following SCI. In summary, our data show an intraspinal mechanism where a subpopulation of ISNs stimulates axonal regrowth resulting in improved recovery of locomotor functions following SCI in zebrafish.

Survival and Axonal Outgrowth of the Mauthner Cell Following Spinal Cord Crush Does Not Drive Post-injury Startle Responses

A pair of Mauthner cells (M-cells) can be found in the hindbrain of most teleost fish, as well as amphibians and lamprey. The axons of these reticulospinal neurons cross the midline and synapse on interneurons and motoneurons as they descend the length of the spinal cord. The M-cell initiates fast C-type startle responses (fast C-starts) in goldfish and zebrafish triggered by abrupt acoustic/vibratory stimuli. Starting about 70 days after whole spinal cord crush, less robust startle responses with longer latencies manifest in adult goldfish, Carassius auratus. The morphological and electrophysiological identifiability of the M-cell provides a unique opportunity to study cellular responses to spinal cord injury and the relation of axonal regrowth to a defined behavior. After spinal cord crush at the spinomedullary junction about one-third of the damaged M-axons of adult goldfish send at least one sprout past the wound site between 56 and 85 days postoperatively. These caudally projecting sprouts follow a more lateral trajectory relative to their position in the fasciculus longitudinalis medialis of control fish. Other sprouts, some from the same axon, follow aberrant pathways that include rostral projections, reversal of direction, midline crossings, neuromas, and projection out the first ventral root. Stimulating M-axons in goldfish that had post-injury startle behavior between 198 and 468 days postoperatively resulted in no or minimal EMG activity in trunk and tail musculature as compared to control fish. Although M-cells can survive for at least 468 day (∼1.3 years) after spinal cord crush, maintain regrowth, and elicit putative trunk EMG responses, the cell does not appear to play a substantive role in the emergence of acoustic/vibratory-triggered responses. We speculate that aberrant pathway choice of this neuron may limit its role in the recovery of behavior and discuss structural and functional properties of alternative candidate neurons that may render them more supportive of post-injury startle behavior.

Role of Aldynoglia Cells in Neuroinflammatory and Neuroimmune Responses after Spinal Cord Injury

Aldynoglia are growth-promoting cells with a morphology similar to radial glia and share properties and markers with astrocytes and Schwann cells. They are distributed in several locations throughout the adult central nervous system, where the cells of the aldynoglia interact and respond to the signals of the immune cells. After spinal cord injury (SCI), the functions of resident aldynoglia, identified as ependymocytes, tanycytes, and ependymal stem cells (EpSCs) of the spinal cord are crucial for the regeneration of spinal neural tissue. These glial cells facilitate axonal regrowth and remyelination of injured axons. Here, we review the influence of M1 or M2 macrophage/microglia subpopulations on the fate of EpSCs during neuroinflammation and immune responses in the acute, subacute, and chronic phases after SCI.

Sodium phenylbutyrate inhibits Schwann cell inflammation via HDAC and NFκB to promote axonal regeneration and remyelination

Background Epigenetic regulation by histone deacetylases (HDACs) in Schwann cells (SCs) after injury facilitates them to undergo de- and redifferentiation processes necessary to support various stages of nerve repair. Although de-differentiation activates the synthesis and secretion of inflammatory cytokines by SCs to initiate an immune response during nerve repair, changes in either the timing or duration of prolonged inflammation mediated by SCs can affect later processes associated with repair and regeneration. Limited studies have investigated the regulatory processes through which HDACs in SCs control inflammatory cytokines to provide a favorable environment for peripheral nerve regeneration. Methods We employed the HDAC inhibitor (HDACi) sodium phenylbutyrate (PBA) to address this question in an in vitro RT4 SC inflammation model and an in vivo sciatic nerve transection injury model to examine the effects of HDAC inhibition on the expression of pro-inflammatory cytokines. Furthermore, we assessed the outcomes of suppression of extended inflammation on the regenerative potential of nerves by assessing axonal regeneration, remyelination, and reinnervation. Results Significant reductions in lipopolysaccharide (LPS)-induced pro-inflammatory cytokine (tumor necrosis factor-α [TNFα]) expression and secretion were observed in vitro following PBA treatment. PBA treatment also affected the transient changes in nuclear factor κB (NFκB)-p65 phosphorylation and translocation in response to LPS induction in RT4 SCs. Similarly, PBA mediated long-term suppressive effects on HDAC3 expression and activity. PBA administration resulted in marked inhibition of pro-inflammatory cytokine secretion at the site of transection injury when compared with that in the hydrogel control group at 6-week post-injury. A conducive microenvironment for axonal regrowth and remyelination was generated by increasing expression levels of protein gene product 9.5 (PGP9.5) and myelin basic protein (MBP) in regenerating nerve tissues. PBA administration increased the relative gastrocnemius muscle weight percentage and maintained the intactness of muscle bundles when compared with those in the hydrogel control group. Conclusions Suppressing the lengthened state of inflammation using PBA treatment favors axonal regrowth and remyelination following nerve transection injury. PBA treatment also regulates pro-inflammatory cytokine expression by inhibiting the transcriptional activation of NFκB-p65 and HDAC3 in SCs in vitro.

Genes and miRNAs as Hurdles and Promoters of Corticospinal Tract Regeneration in Spinal Cord Injury

Spinal cord injury (SCI) is a devastating lesion to the spinal cord, which determines the interruption of ascending/descending axonal tracts, the loss of supraspinal control of sensory-motor functions below the injured site, and severe autonomic dysfunctions, dramatically impacting the quality of life of the patients. After the acute inflammatory phase, the progressive formation of the astrocytic glial scar characterizes the acute-chronic phase: such scar represents one of the main obstacles to the axonal regeneration that, as known, is very limited in the central nervous system (CNS). Unfortunately, a cure for SCI is still lacking: the current clinical approaches are mainly based on early vertebral column stabilization, anti-inflammatory drug administration, and rehabilitation programs. However, new experimental therapeutic strategies are under investigation, one of which is to stimulate axonal regrowth and bypass the glial scar. One major issue in axonal regrowth consists of the different genetic programs, which characterize axonal development and maturation. Here, we will review the main hurdles that in adulthood limit axonal regeneration after SCI, describing the key genes, transcription factors, and miRNAs involved in these processes (seen their reciprocal influencing action), with particular attention to corticospinal motor neurons located in the sensory-motor cortex and subjected to axotomy in case of SCI. We will highlight the functional complexity of the neural regeneration programs. We will also discuss if specific axon growth programs, that undergo a physiological downregulation during CNS development, could be reactivated after a spinal cord trauma to sustain regrowth, representing a new potential therapeutic approach.

Inverted Human Umbilical Artery as a 3D Scaffold for Sciatic Nerve Regeneration in Rats.

Background: Treatment of peripheral nerve injuries (PNIs) remains a challenge. Interposing a graft delivers better regenerative outcomes. Autografts present major drawbacks which have given rise to the development of alternatives such as artificial scaffolds, some of which are very promising. This study was designed to investigate the potential use of an inverted human umbilical cord artery (iHUA) as a 3D scaffold nerve chamber, for nerve regeneration after transection of the sciatic nerve (SN) in rats.Methods: Rats underwent surgical SN transection in their right hindlimb, followed by suture of the device at the resected stumps. Local tolerance, insert biodegradability and nerve reconstruction over time were thoroughly studied by histopathological and morphometric analysis, completed by functional test assessment of sensitivity and motricity recovery.Results: We have demonstrated that nerve reconstruction in the presence of an iHUA insert is effective. The device is well tolerated and highly biodegraded. Although the regenerated nerve is still immature at the end of our study, signs of sensitivity and partial functional recovery were witnessed, confirming our histological findings. Conclusions: Our results support the potential clinical use of iHUA as a 3D scaffold to bridge nerve discontinuity and guide axonal regrowth in selected cases of PNIs.

Nerve grafts and transfers

Following Cruickshank’s (1795) ingenious (and at first disbelieved) demonstration of the regenerative capacity of mammalian nerves, the eighteenth and nineteenth centuries saw a pan-European enthusiasm to redress the nihilism surrounding nerve injury. The first recorded experimental nerve grafts were performed by Philipeaux and Vulpian who attempted both nerve autografting as well as allografting in dogs. At that time, and for many years, allografts were thought to behave similarly to autografts, a belief that persisted well into the twentieth century in some clinics and laboratories. These early attempts at nerve grafting yielded poor results and most surgeons aimed for primary nerve repair despite nerve gaps. Other techniques to allow direct repair involved alteration of position, transposition of the nerve, and even sometimes bone shortening. Although primary repair was frequently possible, after these measures the repair was under tension and mechanical failure was common. Spurling (1945), Whitcomb (1946), and Woodall (1956) showed failure rates of 4%, 7.5%, and 22.4% respectively. Some recovery of function following nerve grafting was documented by Sanders (1942), Seddon (1954), and Brooks (1955). Millesi subsequently published his results for nerve grafting for injuries to the upper limb in 1984. These papers demonstrated more significant recovery of function and highlighted the detriment of delay in treatment to final outcome. Microsurgical advances were central to Millesi’s results, and he emphasized atraumatic dissection and the deleterious effect of tension at the repair site resulting in fibrosis preventing axonal regrowth. Nerve autograft is now the standard for orthotopic nerve reconstruction when primary repair cannot be achieved.

Plasticity of the Injured Spinal Cord

Complete spinal cord injury (SCI) leads to permanent motor, sensitive and sensory deficits. In humans, there is currently no therapy to promote recovery and the only available treatments include surgical intervention to prevent further damage and symptomatic relief of pain and infections in the acute and chronic phases, respectively. Basically, the spinal cord is classically viewed as a nonregenerative tissue with limited plasticity. Thereby the establishment of the “glial” scar which appears within the SCI is mainly described as a hermetic barrier for axon regeneration. However, recent discoveries have shed new light on the intrinsic functional plasticity and endogenous recovery potential of the spinal cord. In this review, we will address the different aspects that the spinal cord plasticity can take on. Indeed, different experimental paradigms have demonstrated that axonal regrowth can occur even after complete SCI. Moreover, recent articles have demonstrated too that the “glial” scar is in fact composed of several cellular populations and that each of them exerts specific roles after SCI. These recent discoveries underline the underestimation of the plasticity of the spinal cord at cellular and molecular levels. Finally, we will address the modulation of this endogenous spinal cord plasticity and the perspectives of future therapeutic opportunities which can be offered by modulating the injured spinal cord microenvironment.