Junko Taniguchi

Cytokine and chemokine profiles in neuromyelitis optica: significance of interleukin-6

Background: Neuromyelitis optica (NMO) is assumed to be immunologically distinct from multiple sclerosis (MS). Adequate studies about cytokines and chemokines in NMO have been lacking. Objective: To investigate the contribution of cytokines/chemokines in the pathogenesis of NMO. Methods: We measured 27 cytokines/chemokines and Th17 cell-associated cytokines in the cerebrospinal fluid (CSF) of 31 NMO, 29 MS and 18 other non-inflammatory neurological disorders patients. The serum levels of some cytokines/ chemokines were also measured. The correlations between clinical characteristics/laboratory findings and levels of cytokines/chemokines in NMO were examined. Results: The CSF levels of interleukin (IL)-1 receptor antagonist, IL-6, IL-8, IL-13 and granulocyte colony-stimulating factor were significantly increased in NMO, while IL-9, fibroblast growth factor-basic, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein-1-beta and tumor necrosis factor-alpha were increased in MS. IL-10 and interferon-gamma-inducible protein-10 were elevated in NMO and MS. In serum analyses, only the IL-6 level showed significant elevation in NMO. The CSF IL-6 level had a significant correlation with the CSF glial fibrillary acidic protein level and CSF cells, and a weak correlation with anti-aquaporin-4 antibody titers. Conclusions: Different immunological status and pathophysiologies exist between NMO and MS, and IL-6 may play important roles in the pathogenesis of NMO.

BMP inhibition enhances axonal growth and functional recovery after spinal cord injury

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor‐β superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. The reemergence of BMPs in the injured adult CNS suggests their involvement in the pathogenesis of the lesion. Here, we demonstrate that BMPs are potent inhibitors of axonal regeneration in the adult spinal cord. The expression of BMP‐2/4 is elevated in oligodendrocytes and astrocytes around the injury site following spinal cord contusion. Intrathecal administration of noggin – a soluble BMP antagonist—leads to enhanced locomotor activity and reveals significant regrowth of the corticospinal tract after spinal cord contusion. Thus, BMPs play a role in inhibiting axonal regeneration and limiting functional recovery following injury to the CNS.

RGMa modulates T cell responses and is involved in autoimmune encephalomyelitis

In multiple sclerosis, activated CD4+ T cells initiate an immune response in the brain and spinal cord, resulting in demyelination, degeneration and progressive paralysis. Repulsive guidance molecule-a (RGMa) is an axon guidance molecule that has a role in the visual system and in neural tube closure. Our study shows that RGMa is expressed in bone marrow–derived dendritic cells (BMDCs) and that CD4+ T cells express neogenin, a receptor for RGMa. Binding of RGMa to CD4+ T cells led to activation of the small GTPase Rap1 and increased adhesion of T cells to intracellular adhesion molecule-1 (ICAM-1). Neutralizing antibodies to RGMa attenuated clinical symptoms of mouse myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) and reduced invasion of inflammatory cells into the CNS. Silencing of RGMa in MOG-pulsed BMDCs reduced their capacity to induce EAE following adoptive transfer to naive C57BL/6 mice. CD4+ T cells isolated from mice treated with an RGMa-specific antibody showed diminished proliferative responses and reduced interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-17 secretion. Incubation of PBMCs from patients with multiple sclerosis with an RGMa-specific antibody reduced proliferative responses and pro-inflammatory cytokine expression. These results demonstrate that an RGMa-specific antibody suppresses T cell responses, and suggest that RGMa could be a promising molecular target for the treatment of multiple sclerosis.

Rho‐kinase inhibition enhances axonal regeneration after peripheral nerve injury

Abstract  In injured adult neurons, the process of axonal regrowth and reestablishment of the neuronal function have to be activated. We assessed in this study whether RhoA, a key regulator of neurite elongation, is activated after injury to the peripheral nervous system. RhoA is activated in motoneurons but not in Schwann cells after mouse sciatic nerve injury. To examine whether the activation of RhoA and its effector, Rho‐kinase, retards axon regeneration of injured motoneurons, we employed a Rho‐kinase inhibitor, fasudil. Amplitudes of distally evoked compound muscle action potentials are increased significantly faster after axonal injury in mice treated with fasudil compared with controls. Histological analysis shows that fasudil treatment increases the number of regenerating axons with large diameter, suggesting that axon maturation is facilitated by Rho‐kinase inhibition. In addition, fasudil does not suppress the myelination of regenerating axons. These findings suggest that RhoA/Rho‐kinase may be a practical molecular target to enhance axonal regeneration in human peripheral neuropathies.

Myosin IIA is required for neurite outgrowth inhibition produced by repulsive guidance molecule

Although myelin‐associated neurite outgrowth inhibitors express their effects through RhoA/Rho‐kinase, the downstream targets of Rho‐kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho‐kinase, by using blebbistatin – a specific myosin II inhibitor – and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho‐kinase‐dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up‐regulated, and this effect depends on Rho‐kinase activity. Further, RGMa‐induced F‐actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho‐kinase‐dependent activation of MIIA via MLC phosphorylation induces F‐actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.

The effects of age, gender, and body mass index on amplitude of sensory nerve action potentials: Multivariate analyses

OBJECTIVE Previous studies have shown that age, gender, and body mass index (BMI) affect amplitude of sensory nerve action potentials (SNAP), but the total effects of multiple factors or the most prominently affected nerves have not been elucidated. This study systematically investigated effects of these factors. METHODS Amplitude of SNAP of the median, ulnar, superficial radial, superficial peroneal, and sural nerves was measured in 105 healthy subjects. The effects of age, gender, and BMI on each nerve were estimated by multivariate linear regression analysis. RESULTS SNAP amplitude decreased with age in all five nerves. Women had greater SNAP amplitude than men in the upper limb nerves (median, ulnar, and radial), but not in the lower limb nerves (peroneal and sural). Similarly, greater BMI was associated with smaller amplitudes in the upper limb nerves, but not in the lower limb nerves. Multivariate analyses showed the three factors explained 50% of the variation in the median nerve, 46% in the ulnar nerve, and 22-32% in the remaining nerves. CONCLUSIONS The effects of age, gender, and BMI on SNAP amplitudes are not identical in different sensory nerves. Age was strongly correlated with SNAP amplitude in the nerves tested, whereas gender and BMI affect amplitudes only in the upper limb nerves. SIGNIFICANCE Age, gender, and BMI should be taken into account in clinical practice, but the extent of influence depends on the sensory nerves examined.

LIM-only protein 4 interacts directly with the repulsive guidance molecule A receptor Neogenin

Repulsive guidance molecule A (RGM A) was recently described as a potent inhibitor of neuroregeneration in a rat spinal cord injury model. The receptor mediating RGM A’s repulsive activity was shown to be Neogenin, a member of the Deleted in Colorectal Cancer (DCC) family of netrin receptors. Binding of RGM A to Neogenin induces activation of the small GTPase RhoA and of its effector Rho‐kinase by an unknown mechanism. Here we show, that the cytoplasmic tail of Neogenin interacts directly with the transcriptional coactivator LIM domain only 4 (LMO4) in human SH‐SY5Y cells, human Ntera neurons, and in embryonic rat cortical neurons. RGM A binding to Neogenin but not binding of Netrin‐1, induces release of LMO4 from Neogenin. Down‐regulation of LMO4 neutralizes the repulsive activity of RGM A in neuronal cell lines and embryonic rat cortical neurons and prevents RhoA activation. These results show for the first time that an interaction of Neogenin with LMO4 is involved in the RGM A – Neogenin signal transduction pathway for RhoA activation.

Delayed treatment with Rho-kinase inhibitor does not enhance axonal regeneration or functional recovery after spinal cord injury in rats

Axonal regeneration in the central nervous system is blocked by many different growth inhibitory factors. Some of these inhibitors act on neurons by activating RhoA and Rho-kinase, an effector of RhoA. Several studies have shown that Rho-kinase inhibition immediately after spinal cord injury enhances axonal sprouting and functional recovery. In this study, we ask whether delayed treatment with Rho-kinase inhibitor is effective in promoting regeneration and functional recovery. We administered Fasudil, a Rho-kinase inhibitor, locally to the injury site 4 weeks or immediately after contusion of the thoracic spinal cord in rats. Although the immediate treatment significantly stimulated axonal sprouting and recovery of hindlimb function, treatment started 4 weeks after surgery had no effect on fiber sprouting or locomotor recovery. Our findings suggest that RhoA/Rho-kinase alone may not account for the irreversible arrest of axon outgrowth in the chronic stage of injury in the central nervous system.

Multiple angiogenetic factors are upregulated in POEMS syndrome

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome is a multisystem disorder associated with plasma cell dyscrasia. Elevated serum levels of vascular endothelial growth factor (VEGF), which strongly promotes neovascularization and vasopermeability, are considered to be responsible for the characteristic symptoms such as angiomata, pleural effusion/ascites, edema, and organomegaly in the disorder. To study whether other angiogenetic factors are upregulated in POEMS syndrome, we measured serum levels of basic fibroblast growth factor and hepatocyte growth factor (HGF), as well as VEGF, in 17 patients with POEMS syndrome. All these factors were significantly upregulated in the POEMS syndrome patients. After the treatment with anti-VEGF antibody, the levels of HGF did not change, suggesting that elevation of HGF levels is not secondary to VEGF overproduction. These results suggest that different angiogenetic factors might contribute to the pathogenesis of POEMS syndrome, and this fact might contribute to the insufficient clinical effects obtained by suppression of VEGF alone.

Neogenin regulates neuronal survival through DAP kinase

The repulsive guidance molecule (RGM) is a membrane-bound protein that has diverse functions in the developing central nervous system. Identification of neogenin as a receptor for RGM provided evidence of its cell death-inducing activity in the absence of RGM. Here, we show that the serine/threonine kinase death-associated protein kinase (DAPK) is involved in the signal transduction of neogenin. Neogenin interacts with DAPK and reduces DAPK autophosphorylation on Ser308 in vitro. Neogenin-induced cell death is abolished in the presence of RGM or by blocking DAPK. Although neogenin overexpression or RGM downregulation in the chick neural tube in vivo induces apoptosis, coexpression of the dominant-negative mutant or small-interference RNA of DAPK attenuates this proapoptotic activity. Thus, RGM/neogenin regulates cell fate by controlling the DAPK activity.