Hirotaka James Okano

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell–mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

Notch Inhibition Induces Cochlear Hair Cell Regeneration and Recovery of Hearing after Acoustic Trauma

Hearing loss due to damage to auditory hair cells is normally irreversible because mammalian hair cells do not regenerate. Here, we show that new hair cells can be induced and can cause partial recovery of hearing in ears damaged by noise trauma, when Notch signaling is inhibited by a γ-secretase inhibitor selected for potency in stimulating hair cell differentiation from inner ear stem cells in vitro. Hair cell generation resulted from an increase in the level of bHLH transcription factor Atoh1 in response to inhibition of Notch signaling. In vivo prospective labeling of Sox2-expressing cells with a Cre-lox system unambiguously demonstrated that hair cell generation resulted from transdifferentiation of supporting cells. Manipulating cell fate of cochlear sensory cells in vivo by pharmacological inhibition of Notch signaling is thus a potential therapeutic approach to the treatment of deafness.

Visualization of peripheral nerve degeneration and regeneration: monitoring with diffusion tensor tractography.

We applied diffusion tensor tractography (DTT), a recently developed MRI technique that reveals the microstructures of tissues based on its ability to monitor the random movements of water molecules, to the visualization of peripheral nerves after injury. The rat sciatic nerve was subjected to contusive injury, and the data obtained from diffusion tensor imaging (DTI) were used to determine the tracks of nerve fibers (DTT). The DTT images obtained using the fractional anisotropy (FA) threshold value of 0.4 clearly revealed the recovery process of the contused nerves. Immediately after the injury, fiber tracking from the designated proximal site could not be continued beyond the lesion epicenter, but the intensity improved thereafter, returning to its pre-injury level by 3 weeks later. We compared the FA value, a parameter computed from the DTT data, with the results of histological and functional examinations of the injured nerves, during recovery. The FA values of the peripheral nerves were more strongly correlated with axon-related (axon density and diameter) than with myelin-related (myelin density and thickness) parameters, supporting the theories that axonal membranes play a major role in anisotropic water diffusion and that myelination can modulate the degree of anisotropy. Moreover, restoration of the FA value at the lesion epicenter was strongly correlated with parameters of motor and sensory functional recovery. These correlations of the FA values with both the histological and functional changes demonstrate the potential usefulness of DTT for evaluating clinical events associated with Wallerian degeneration and the regeneration of peripheral nerves.

Proinflammatory cytokines expression in noise‐induced damaged cochlea

Recent studies have showed that inflammatory responses occur in inner ear under various damaging conditions including noise‐overstimulation. We evaluated the time‐dependent expression of proinflammatory cytokines in noise‐exposed rat cochlea. Among several detected cytokines, real‐time RT‐PCR showed that interleukin‐1beta (IL‐1β) and interleukin‐6 (IL‐6) were significantly induced 3 hr after noise exposure, and quickly downregulated to the basal level. Tumor necrosis factor‐alpha (TNF‐α) was also slightly upregulated immediately after noise exposure. Immunohistochemical analysis showed that IL‐6 expression was distinctively induced within the lateral side of the spiral ligament. Sequential expression analysis showed that IL‐6 immunoreactivity was initially found in the cytoplasm of lateral wall cells, including Type IV and III fibrocytes, and expanded broader throughout the lateral wall, finally to the stria vascularis. Because of the negative Iba‐1 staining, IL‐6 expression in the early‐phase was not due to macrophage or microglia activation. IL‐6 was also detected in spiral ganglion neurons at 12 and 24 hr after noise exposure. Our data demonstrates the production of proinflammatory cytokines, including TNF‐α, IL‐1β, and IL‐6, in early phase of noise overstimulated cochlea. IL‐6 expression was observed in the spiral ligament, stria vascularis, and spiral ganglion neurons. These cytokines, produced by the cochlear structure itself in response to noise exposure, may initiate an inflammatory response and have some role in the mechanism of noise‐induced cochlear damage.

Chondroitinase ABC combined with neural stem/progenitor cell transplantation enhances graft cell migration and outgrowth of growth-associated protein-43-positive fibers after rat spinal cord injury

We previously reported that the transplantation of neural stem/progenitor cells (NSPCs) can contribute to the repair of injured spinal cord in adult rats and monkeys. In some cases, however, most of the transplanted cells adhered to the cavity wall and failed to migrate and integrate into the host spinal cord. In this study we focused on chondroitin sulfate proteoglycan (CSPG), a known constituent of glial scars that is strongly expressed after spinal cord injury (SCI), as a putative inhibitor of NSPC migration in vivo. We hypothesized that the digestion of CSPG by chondroitinase ABC (C‐ABC) might promote the migration of transplanted cells and neurite outgrowth after SCI. An in vitro study revealed that the migration of NSPC‐derived cells was inhibited by CSPG and that this inhibitory effect was attenuated by C‐ABC pre‐treatment. Consistently, an in vivo study of C‐ABC treatment combined with NSPC transplantation into injured spinal cord revealed that C‐ABC pre‐treatment promoted the migration of the transplanted cells, whereas CSPG‐immunopositive scar tissue around the lesion cavity prevented their migration into the host spinal cord in the absence of C‐ABC pre‐treatment. Furthermore, this combined treatment significantly induced the outgrowth of a greater number of growth‐associated protein‐43‐positive fibers at the lesion epicentre, compared with NSPC transplantation alone. These findings suggested that the application of C‐ABC enhanced the benefits of NSPC transplantation for SCI by reducing the inhibitory effects of the glial scar, indicating that this combined treatment may be a promising strategy for the regeneration of injured spinal cord.

Noninvasive and real-time assessment of reconstructed functional human endometrium in NOD/SCID/γcnull immunodeficient mice

Human uterine endometrium exhibits unique properties of cyclical regeneration and remodeling throughout reproductive life and also is subject to endometriosis through ectopic implantation of retrogradely shed endometrial fragments during menstruation. Here we show that functional endometrium can be regenerated from singly dispersed human endometrial cells transplanted beneath the kidney capsule of NOD/SCID/γcnull immunodeficient mice. In addition to the endometrium-like structure, hormone-dependent changes, including proliferation, differentiation, and tissue breakdown and shedding (menstruation), can be reproduced in the reconstructed endometrium, the blood to which is supplied predominantly by human vessels invading into the mouse kidney parenchyma. Furthermore, the hormone-dependent behavior of the endometrium regenerated from lentivirally engineered endometrial cells expressing a variant luciferase can be assessed noninvasively and quantitatively by in vivo bioluminescence imaging. These results indicate that singly dispersed endometrial cells have potential applications for tissue reconstitution, angiogenesis, and human–mouse chimeric vessel formation, providing implications for mechanisms underlying the physiological endometrial regeneration during the menstrual cycle and the establishment of endometriotic lesions. This animal system can be applied as the unique model of endometriosis or for other various types of neoplastic diseases with the capacity of noninvasive and real-time evaluation of the effect of therapeutic agents and gene targeting when the relevant cells are transplanted beneath the kidney capsule.

Significance of Remyelination by Neural Stem/Progenitor Cells Transplanted into the Injured Spinal Cord†‡§

Previous reports of functional recovery from spinal cord injury (SCI) in rodents and monkeys after the delayed transplantation of neural stem/progenitor cells (NS/PCs) have raised hopes that stem cell therapy could be used to treat SCI in humans. More research is needed, however, to understand the mechanism of functional recovery. Oligodendrocytes derived from grafted NS/PCs remyelinate spared axons in the injured spinal cord. Here, we studied the extent of this remyelinations contribution to functional recovery following contusive SCI in mice. To isolate the effect of remyelination from other possible regenerative benefits of the grafted cells, NS/PCs obtained from myelin‐deficient shiverer mutant mice (shi‐NS/PCs) were used in this work alongside wild‐type NS/PCs (wt‐NS/PCs). shi‐NS/PCs behaved like wt‐NS/PCs in vitro and in vivo, with the exception of their myelinating potential. shi‐NS/PC‐derived oligodendrocytes did not express myelin basic protein in vitro and formed much thinner myelin sheaths in vivo compared with wt‐NS/PC‐derived oligodendrocytes. The transplantation of shi‐NS/PCs promoted some locomotor and electrophysiological functional recovery but significantly less than that afforded by wt‐NS/PCs. These findings establish the biological importance of remyelination by graft‐derived cells for functional recovery after the transplantation of NS/PCs into the injured spinal cord. STEM CELLS 2011;29:1983–1994.

Implantation of dendritic cells in injured adult spinal cord results in activation of endogenous neural stem/progenitor cells leading to de novo neurogenesis and functional recovery

We report a treatment for spinal cord injury involving implantation of dendritic cells (DCs), which act as antigen‐presenting cells in the immune system. The novel mechanisms underlying this treatment produce functional recovery. Among the immune cells tested, DCs showed the strongest activity inducing proliferation and survival of neural stem/progenitor cells (NSPCs) in vitro. Furthermore, in DC‐implanted adult mice, endogenous NSPCs in the injured spinal cord were activated for mitotic de novo neurogenesis. These DCs produced neurotrophin‐3 and activated endogenous microglia in the injured spinal cord. Behavioral analysis revealed the locomotor functions of DC‐implanted mice to have recovered significantly as compared to those of control mice. Our results suggest that DC‐implantation exerts trophic effects, including activation of endogenous NSPCs, leading to repair of the injured adult spinal cord.

The long non-coding RNA nuclear-enriched abundant transcript 1_2 induces paraspeckle formation in the motor neuron during the early phase of amyotrophic lateral sclerosis

BackgroundA long non-coding RNA (lncRNA), nuclear-enriched abundant transcript 1_2 (NEAT1_2), constitutes nuclear bodies known as “paraspeckles”. Mutations of RNA binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), have been described in amyotrophic lateral sclerosis (ALS). ALS is a devastating motor neuron disease, which progresses rapidly to a total loss of upper and lower motor neurons, with consciousness sustained. The aim of this study was to clarify the interaction of paraspeckles with ALS-associated RNA-binding proteins, and to identify increased occurrence of paraspeckles in the nucleus of ALS spinal motor neurons.ResultsIn situ hybridization (ISH) and ultraviolet cross-linking and immunoprecipitation were carried out to investigate interactions of NEAT1_2 lncRNA with ALS-associated RNA-binding proteins, and to test if paraspeckles form in ALS spinal motor neurons. As the results, TDP-43 and FUS/TLS were enriched in paraspeckles and bound to NEAT1_2 lncRNA directly. The paraspeckles were localized apart from the Cajal bodies, which were also known to be related to RNA metabolism. Analyses of 633 human spinal motor neurons in six ALS cases showed NEAT1_2 lncRNA was upregulated during the early stage of ALS pathogenesis. In addition, localization of NEAT1_2 lncRNA was identified in detail by electron microscopic analysis combined with ISH for NEAT1_2 lncRNA. The observation indicating specific assembly of NEAT1_2 lncRNA around the interchromatin granule-associated zone in the nucleus of ALS spinal motor neurons verified characteristic paraspeckle formation.ConclusionsNEAT1_2 lncRNA may act as a scaffold of RNAs and RNA binding proteins in the nuclei of ALS motor neurons, thereby modulating the functions of ALS-associated RNA-binding proteins during the early phase of ALS. These findings provide the first evidence of a direct association between paraspeckle formation and a neurodegenerative disease, and may shed light on the development of novel therapeutic targets for the treatment of ALS.

Fbxo45 Forms a Novel Ubiquitin Ligase Complex and Is Required for Neuronal Development

ABSTRACT Fbxo45 is an F-box protein that is restricted to the nervous system. Unlike other F-box proteins, Fbxo45 was found not to form an SCF complex as a result of an amino acid substitution in the consensus sequence for Cul1 binding. Proteomics analysis revealed that Fbxo45 specifically associates with PAM (protein associated with Myc), a RING finger-type ubiquitin ligase. Mice deficient in Fbxo45 were generated and found to die soon after birth as a result of respiratory distress. Fbxo45−/− embryos show abnormal innervation of the diaphragm, impaired synapse formation at neuromuscular junctions, and aberrant development of axon fiber tracts in the brain. Similar defects are also observed in mice lacking Phr1 (mouse ortholog of PAM), suggesting that Fbxo45 and Phr1 function in the same pathway. In addition, neuronal migration was impaired in Fbxo45−/− mice. These results suggest that Fbxo45 forms a novel Fbxo45-PAM ubiquitin ligase complex that plays an important role in neural development.