A trifluoromethyl analogue of celecoxib exerts beneficial effects in neuroinflammation.

Celecoxib is a selective cyclooxygenase-2 (COX2) inhibitor. We have previously shown that celecoxib inhibits experimental autoimmune encephalomyelitis (EAE) in COX-2-deficient mice, suggestive for a mode of action involving COX2-independent pathways. In the present study, we tested the effect of a trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2 inhibitory activity in two models of neuroinflammation, i.e. cerebellar organotypic cultures challenged with LPS and the EAE mouse model for multiple sclerosis. TFM-C inhibited secretion of IL-1ß, IL-12 and IL-17, enhanced that of TNF-α and RANTES, reduced neuronal axonal damage and protected from oxidative stress in the organotypic model. TFM-C blocked TNF-α release in microglial cells through a process involving intracellular retention, but induced TNF-α secretion in primary astrocyte cultures. Finally, we demonstrate that TFM-C and celecoxib ameliorated EAE with equal potency. This coincided with reduced secretion of IL-17 and IFN-γ by MOG-reactive T-cells and of IL-23 and inflammatory cytokines by bone marrow-derived dendritic cells. Our study reveals that non-coxib analogues of celecoxib may have translational value in the treatment of neuro-inflammatory conditions.

Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation.

BACKGROUND: Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. METHODS/PRINCIPAL FINDINGS: To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1ß, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. CONCLUSION: The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.

A 4-trifluoromethyl analogue of celecoxib inhibits arthritis by suppressing innate immune cell activation.

INTRODUCTION: Celecoxib, a highly specific cyclooxygenase-2 (COX-2) inhibitor has been reported to have COX-2-independent immunomodulatory effects. However, celecoxib itself has only mild suppressive effects on arthritis. Recently, we reported that a 4-trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2-inhibitory activity inhibits secretion of IL-12 family cytokines through a COX-2-independent mechanism that involves Ca2+-mediated intracellular retention of the IL-12 polypeptide chains. In this study, we explored the capacity of TFM-C as a new therapeutic agent for arthritis. METHODS: To induce collagen-induced arthritis (CIA), DBA1/J mice were immunized with bovine type II collagen (CII) in Freund's adjuvant. Collagen antibody-induced arthritis (CAIA) was induced in C57BL/6 mice by injecting anti-CII antibodies. Mice received 10 µg/g of TFM-C or celecoxib every other day. The effects of TFM-C on clinical and histopathological severities were assessed. The serum levels of CII-specific antibodies were measured by ELISA. The effects of TFM-C on mast cell activation, cytokine producing capacity by macrophages, and neutrophil recruitment were also evaluated. RESULTS: TFM-C inhibited the severity of CIA and CAIA more strongly than celecoxib. TFM-C treatments had little effect on CII-specific antibody levels in serum. TFM-C suppressed the activation of mast cells in arthritic joints. TFM-C also suppressed the production of inflammatory cytokines by macrophages and leukocyte influx in thioglycollate-induced peritonitis. CONCLUSION: These results indicate that TFM-C may serve as an effective new disease-modifying drug for treatment of arthritis, such as rheumatoid arthritis.

Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control.

Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.

A new function for the fragile X mental retardation protein in regulation of PSD-95 mRNA stability.

Fragile X syndrome (FXS) results from the loss of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates a variety of cytoplasmic mRNAs. FMRP regulates mRNA translation and may be important in mRNA localization to dendrites. We report a third cytoplasmic regulatory function for FMRP: control of mRNA stability. In mice, we found that FMRP binds, in vivo, the mRNA encoding PSD-95, a key molecule that regulates neuronal synaptic signaling and learning. This interaction occurs through the 3' untranslated region of the PSD-95 (also known as Dlg4) mRNA, increasing message stability. Moreover, stabilization is further increased by mGluR activation. Although we also found that the PSD-95 mRNA is synaptically localized in vivo, localization occurs independently of FMRP. Through our functional analysis of this FMRP target we provide evidence that dysregulation of mRNA stability may contribute to the cognitive impairments in individuals with FXS.

Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration.

Mitochondrial damage is linked to many neurodegenerative conditions, such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. These diseases are associated with changes in the splicing pattern of individual mRNAs. Here, we tested the hypothesis that mitochondrial damage modulates alternative splicing, not only of a few mRNAs, but in a general manner. We incubated cultured human neuroblastoma cells with the chemical agent paraquat (a neurotoxin that interferes with mitochondrial function, causing energy deficit and oxidative stress) and analysed the splicing pattern of 13 genes by RT-PCR. For all mRNAs that are alternatively spliced, we observed a dose- and time-dependent increase of the smaller isoforms. In contrast, splicing of all constitutive splicing exons that we monitored did not change. Using other drugs, we show that the modulation of alternative splicing correlates with ATP depletion, not with oxidative stress. Such drastic changes in alternative splicing are not observed in cell lines of non-neuronal origin, suggesting a selective susceptibility of neuronal cells to modulation of splicing. As a significant percentage of all mammalian mRNAs undergo alternative splicing, we predict that mitochondrial failure will unbalance a vast number of isoform equilibriums, which would give an important contribution to neurodegeneration.

A novel C-terminal sequence from barley polyamine oxidase is a vacuolar sorting signal.

Barley contains two different isoforms of flavin-containing polyamine oxidase (BPAO1 and BPAO2). We have previously demonstrated that BPAO2 is a symplastic protein in barley leaves. On the contrary, maize polyamine oxidase (MPAO), the best characterized member of this enzyme class, is apoplastic. Comparison of the derived amino-acid sequences of BPAO2 and MPAO has revealed that both precursor proteins include a cleavable N-terminal signal peptide of 25 amino acid residues, but the barley enzyme shows an extra C-terminal extension of eight amino acids. By means of MPAO engineering with BPAO2 C-terminal tail (MPAO-T) and exploiting transient expression in Nicotiana tabacum protoplasts, we demonstrate that this oligopeptide is a signal for protein sorting to the plant vacuole. The vacuolar sorting of MPAO-T was saturable. Specific mutations of the C-terminal tail were constructed to determine which amino acid residues of this novel propeptide affect proper protein sorting. No consensus sequence or common structural determinant is required for the intracellular retention of the MPAO-T protein, but a gradual lowering of the efficiency was observed as a result of progressive deletion of the C-terminus.

The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses.

The Fragile X syndrome, which results from the absence of functional FMRP protein, is the most common heritable form of mental retardation. Here, we show that FMRP acts as a translational repressor of specific mRNAs at synapses. Interestingly, FMRP associates not only with these target mRNAs, but also with the dendritic, non-translatable RNA BC1. Blocking of BC1 inhibits the interaction of FMRP with its target mRNAs. Furthermore, BC1 binds directly to FMRP and can also associate, in the absence of any protein, with the mRNAs regulated by FMRP. This suggests a mechanism where BC1 could determine the specificity of FMRP function by linking the regulated mRNAs and FMRP. Thus, when FMRP is not present, loss of translational repression of specific mRNAs at synapses could result in synaptic dysfunction phenotype of Fragile X patients.