Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.
Wnt proteins are a large family of diffusible factors that play important roles in embryonic development, including axis patterning, cell fate specification, proliferation, and axon development. It was recently demonstrated that Ryk (receptor related to tyrosine kinase) is a conserved high-affinity Wnt receptor, and that Ryk-Wnt interactions guide corticospinal axons down the spinal cord during development. Here, we report that the Ryk-Wnt signal mediates the inhibition of corticospinal axon growth in the adult spinal cord. The expression of Wnt-5a is induced in reactive astrocytes around the injury site following a spinal cord injury. In vitro, Wnt-5a inhibits the neurite growth of postnatal cerebellar neurons by activating RhoA/Rho-kinase. In rats with thoracic spinal cord contusion, intrathecal administration of a neutralizing antibody to Ryk resulted in significant axonal growth of the corticospinal tract and enhanced functional recovery. Thus, reexpression of the embryonic repulsive cues in adult tissues contributes to the failure of axon regeneration in the central nervous system.
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.
Inflammatory pain, characterized by a decrease in the nociceptive threshold, arises through the actions of inflammatory mediators, and one of the key molecules is nerve growth factor (NGF). Here we report that the administration of neutralizing antibody to the neurotrophin receptor p75 (p75(NTR)) blocks hyperalgesia, which develops with complete Freund's adjuvant (CFA)-induced inflammation or with an intraplantar injection of NGF. Although CFA injection results in the up-regulation of calcitonin gene-related peptide (CGRP) levels in the primary sensory neurons, blocking p75(NTR) abolishes this effect. We further demonstrate that pro-NGF is the predominant ligand of p75(NTR) in vivo. Plasmin treatment, which is intended to decompose pro-NGF, ameliorates CFA-induced hyperalgesia. In addition, an intraplantar injection of pro-NGF induces hyperalgesia. These data together suggest that pro-NGF, as well as mature NGF, binding to p75(NTR) plays an important role in inflammation-induced hyperalgesia. Interference in the binding may provide a therapeutic approach for the treatment of inflammatory pain.
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