Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.     

Unesterified docosahexaenoic acid is protective in neuroinflammation

Docosahexaenoic acid (22:6n‐3) is the major brain n‐3 polyunsaturated fatty acid and it is possible that docosahexaenoic acid is anti‐inflammatory in the brain as it is known to be in other tissues. Using a combination of models including the fat‐1 transgenic mouse, chronic dietary n‐3 polyunsaturated fatty acid modulation in transgenic and wild‐type mice, and acute direct brain infusion, we demonstrated that unesterified docosahexaenoic acid attenuates neuroinflammation initiated by intracerebroventricular lipopolysaccharide. Hippocampal neuroinflammation was assessed by gene expression and immunohistochemistry. Furthermore, docosahexaenoic acid protected against lipopolysaccharide‐induced neuronal loss. Acute intracerebroventricular infusion of unesterified docosahexaenoic acid or its 12/15‐lipoxygenase product and precursor to protectins and resolvins, 17S‐hydroperoxy‐docosahexaenoic acid, mimics anti‐neuroinflammatory aspects of chronically increased unesterified docosahexaenoic acid. LC‐MS/MS revealed that neuroprotectin D1 and several other docosahexaenoic acid‐derived specialized pro‐resolving mediators are present in the hippocampus. Acute intracerebroventricular infusion of 17S‐hydroperoxy‐docosahexaenoic acid increases hippocampal neuroprotectin D1 levels concomitant to attenuating neuroinflammation. These results show that unesterified docosahexaenoic acid is protective in a lipopolysaccharide‐initiated mouse model of acute neuroinflammation, at least in part, via its conversion to specialized pro‐resolving mediators; these docosahexaenoic acid stores may provide novel targets for the prevention and treatment(s) of neurological disorders with a neuroinflammatory component.

     

Peptidylarginine deiminase: a candidate factor in demyelinating disease

In earlier studies we demonstrated that an increase in the relative amounts of citrullinated myelin basic protein (MBP) was found in multiple sclerosis (Moscarello et al. 1994). To determine the temporal relationship between the citrullinated MBP and peptidylarginine deiminase (PAD), the enzyme responsible for deiminating arginyl residues in proteins, we studied enzyme activity, enzyme protein, PAD mRNA in a spontaneously demyelinating transgenic mouse model and we correlated the amount of PAD with citrullinated MBP. Both PAD protein as measured in an immunoslot blot method and PAD RNA were elevated. In fractionation studies we showed that the increase in PAD enzyme was due to an increase in the PAD found in membrane fractions and not the soluble PAD (PADII). From our data we concluded that up-regulation of myelin-associated PAD was responsible for the increase in citrullinated MBP in our transgenic mice prior to onset of clinical or pathological signs of demyelination. We postulate that a similar mechanism may be responsible for the increase in citrullinated MBP in multiple sclerosis.     

CNTF mediates neurotrophic factor secretion and fluid absorption in human retinal pigment epithelium

Ciliary neurotrophic factor (CNTF) protects photoreceptors and regulates their phototransduction machinery, but little is known about CNTF's effects on retinal pigment epithelial (RPE) physiology. Therefore, we determined the expression and localization of CNTF receptors and the physiological consequence of their activation in primary cultures of human fetal RPE (hfRPE). Cultured hfRPE express CNTF, CT1, and OsM and their receptors, including CNTFRα, LIFRβ, gp130, and OsMRβ, all localized mainly at the apical membrane. Exogenous CNTF, CT1, or OsM induces STAT3 phosphorylation, and OsM also induces the phosphorylation of ERK1/2 (p44/42 MAP kinase). CNTF increases RPE survivability, but not rates of phagocytosis. CNTF increases secretion of NT3 to the apical bath and decreases that of VEGF, IL8, and TGFβ2. It also significantly increases fluid absorption (J(V)) across intact monolayers of hfRPE by activating CFTR chloride channels at the basolateral membrane. CNTF induces profound changes in RPE cell biology, biochemistry, and physiology, including the increase in cell survival, polarized secretion of cytokines/neurotrophic factors, and the increase in steady-state fluid absorption mediated by JAK/STAT3 signaling. In vivo, these changes, taken together, could serve to regulate the microenvironment around the distal retinal/RPE/Bruch's membrane complex and provide protection against neurodegenerative disease.     

Hepatoma-derived growth factor is a neurotrophic factor harbored in the nucleus

Hepatoma-derived growth factor (HDGF) is a heparin-binding proliferating factor originally isolated from conditioned medium of the hepatoma-derived cell line HuH-7. HDGF has greatest homology in an amino acid sequence with high mobility group 1 (HMG1), which has been characterized as a DNA-binding, inflammatory, and potent neurite outgrowth molecule. HDGF is reported to be widely expressed and act as a growth factor in many kinds of cells. However, it has not been investigated in the nervous system. Here, we show by Western blot analysis that HDGF is present in the mouse brain from the embryonic period until adulthood. In situ hybridization and immunohistochemical analyses revealed that HDGF was expressed mainly in neurons, and HDGF protein was localized to the nucleus. HDGF and high mobility group 1 were secreted under physiological conditions and released extracellularly in necrotic conditions. Furthermore, we showed that exogenously supplied HDGF had a neurotrophic effect and was able to partially prevent the cell death of neurons in which endogenous HDGF was suppressed. Therefore, we propose that HDGF is a novel type of neurotrophic factor, on account of its localization in the nucleus and its potential to function in an autocrine manner under both physiological and pathological conditions throughout life.     

CNS expression of B7-H1 regulates pro-inflammatory cytokine production and alters severity of Theiler's virus-induced demyelinating disease

The CNS is a unique organ due to its limited capacity for immune surveillance. As macrophages of the CNS, microglia represent a population originally known for the ability to assist neuronal stability, are now appreciated for their role in initiating and regulating immune responses in the brain. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a mouse model of multiple sclerosis (MS). In response to TMEV infection in vitro, microglia produce high levels of inflammatory cytokines and chemokines, and are efficient antigen-presenting cells (APCs) for activating CD4(+) T cells. However, the regulatory function of microglia and other CNS-infiltrating APCs in response to TMEV in vivo remains unclear. Here we demonstrate that microglia increase expression of proliferating cell nuclear antigen (PCNA), and phenotypically express high levels of major histocompatibility complex (MHC)-Class I and II in response to acute infection with TMEV in SJL/J mice. Microglia increase expression of the inhibitory co-stimulatory molecule, B7-H1 as early as day 5 post-infection, while CNS-infiltrating CD11b(+)CD11c(-)CD45(HIGH) monocytes/macrophages and CD11b(+)CD11c(+)CD45(HIGH) dendritic cells upregulate expression of B7-H1 by day 3 post-infection. Utilizing a neutralizing antibody, we demonstrate that B7-H1 negatively regulates TMEV-specific ex vivo production of interferon (IFN)-γ, interleukin (IL)-17, IL-10, and IL-2 from CD4(+) and CD8(+) T cells. In vivo blockade of B7-H1 in SJL/J mice significantly exacerbates clinical disease symptoms during the chronic autoimmune stage of TMEV-IDD, but only has minimal effects on viral clearance. Collectively, these results suggest that CNS expression of B7-H1 regulates activation of TMEV-specific T cells, which affects protection against TMEV-IDD.     

Macrophage stimulating protein is a novel neurotrophic factor

Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family of the plasminogen-related growth factors (PRGFs). PRGFs are alpha/beta heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target of the hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration.     

Effects of ciliary neurotrophic factor (CNTF) on protein turnover in cultured muscle cells

The goal of the study was to evaluate the mechanism by which ciliary neurotrophic factor (CNTF) regulated protein metabolism in skeletal muscle. L8 myotubes were cultured and effects of various times and doses of CNTF on protein synthesis and degradation were evaluated. Effects of CNTF on turnover of specific pools of proteins (myofibrillar and non-myofibrillar) were also evaluated. Protein synthesis was assayed by incorporation of radioactive tyrosine into muscle proteins. Degradation was assessed by release of labelled tyrosine from pre-labelled myotubes. Effects of CNTF on protein turnover were found to be time- and dose-dependent. CNTF (1 and 10 ng/ml) increased myofibrillar protein synthesis after 12 h of exposure but had no effect on non-myofibrillar protein synthesis. Longer exposures of CNTF (24 h) reduced non-myofibrillar protein synthesis and had no effect on myofibrillar protein synthesis. High concentrations of CNTF (10 and 20 ng/ml) reduced myofibrillar protein degradation but had no effect on degradation of non-myofibrillar proteins. To evaluate the mechanism by which CNTF exerts control of protein turnover, we completed a Northern blot for CNTF receptor alpha-subunit (CNTFRalpha). This was non-detectable via conventional northern analysis. Use of RT-PCR, however, confirmed expression of CNTFRalpha, albeit at a low level compared to rat skeletal muscle. This low expression of the receptor in L8 myotubes may explain the limited effect of CNTF in vitro compared to the larger effects typically detected in vivo. CNTF regulated protein turnover through control of protein synthesis and degradation. Effects were dose and timedependent. These observations may explain ability of CNTF to exert both anabolic and catabolic actions in vivo.     

The relationship between ciliary neurotrophic factor (CNTF) genotype and motor unit physiology: preliminary studies

Background  

Ciliary neurotrophic factor (CNTF) is important for neuronal and muscle development, and genetic variation in the CNTF gene has been associated with muscle strength. The effect of CNTF on nerve development suggests that CNTF genotype may be associated with force production via its influence on motor unit size and firing patterns. The purpose of this study is to examine whether CNTF genotype differentially affects motor unit activation in the vastus medialis with increasing isometric force during knee extension.     

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on the ciliary neurotrophic factor receptor alpha chain

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin are members of the four-helix bundle cytokine family. These proteins signal through a common tripartite receptor composed of leukemia inhibitory factor receptor, gp130, and ciliary neurotrophic factor receptor alpha. Binding to ciliary neurotrophic factor receptor alpha occurs through an interaction site located at the C terminus of the cytokine AB loop and alphaD helix, known as site 1. In the present study, we have generated a model of neuropoietin and identified a conserved binding site for the three cytokines interacting with ciliary neurotrophic factor receptor alpha. To identify the counterpart of this site on ciliary neurotrophic factor receptor alpha, its cytokine binding domain was modeled, and the physicochemical properties of its surface were analyzed. This analysis revealed an area displaying properties complementary to the site 1 of ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin. Based on our computational predictions, residues were selected for their potential involvement in the ciliary neurotrophic factor receptor alpha binding epitope, and site-directed mutagenesis was carried out. Biochemical, cell proliferation, and cell signaling analyses showed that Phe(172) and Glu(286) of ciliary neurotrophic factor receptor alpha are key interaction residues. Our results demonstrated that ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on ciliary neurotrophic factor receptor alpha.     

Leukemia inhibitory factor deficiency modulates the immune response and limits autoimmune demyelination: a new role for neurotrophic cytokines in neuroinflammation

The neurotrophic cytokines ciliary neurotrophic factor and leukemia inhibitory factor (LIF) play a key role in neuronal and oligodendrocyte survival and as protective factors in neuroinflammation. To further elucidate the potential of endogenous LIF in modulating neuroinflammation, we studied myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LIF knockout mice (LIF(-/-) mice). In the late phase of active myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, LIF(-/-) mice exhibited a markedly milder disease course. The inflammatory infiltrate in LIF(-/-) mice was characterized by an increase in neutrophilic granulocytes early and fewer infiltrating macrophages associated with less demyelination later in the disease. In good correlation with an effect of endogenous LIF on the immune response, we found an Ag-specific T cell-priming defect with impaired IFN-gamma production in LIF(-/-) mice. On the molecular level, the altered recruitment of inflammatory cells is associated with distinct patterns of chemokine production in LIF(-/-) mice with an increase of CXCL1 early and a decrease of CCL2, CCL3, and CXCL10 later in the disease. These data reveal that endogenous LIF is an immunologically active molecule in neuroinflammation. This establishes a link between LIF and the immune system which was not observed in the ciliary neurotrophic factor knockout mouse.     

Ethylene as a modulator of disease resistance in plants

The role of ethylene in the hormonal regulation of plant development has been well established. In addition, it has been implicated in biotic stress, both as a virulence factor of fungal and bacterial pathogens and as a signaling compound in disease resistance. This apparent discrepancy has stimulated research on the effects of various types of pathogens on mutant and transgenic plants that are impaired in ethylene production or perception. It has become clear that ethylene differentially affects resistance against pathogens with different lifestyles and plays an important role in mediating different types of induced resistance.     

Loss of protein structure stability as a major causative factor in monogenic disease

The most common cause of monogenic disease is a single base DNA variant resulting in an amino acid substitution. In a previous study, we observed that a high fraction of these substitutions appear to result in reduction of stability of the corresponding protein structure. We have now investigated this phenomenon more fully. A set of structural effects, such as reduction in hydrophobic area, overpacking, backbone strain, and loss of electrostatic interactions, is used to represent the impact of single residue mutations on protein stability. A support vector machine (SVM) was trained on a set of mutations causative of disease, and a control set of non-disease causing mutations. In jack-knifed testing, the method identifies 74% of disease mutations, with a false positive rate of 15%. Evaluation of a set of in vitro mutagenesis data with the SVM established that the majority of disease mutations affect protein stability by 1 to 3 kcal/mol. The method's effective distinction between disease and non-disease variants, strongly supports the hypothesis that loss of protein stability is a major factor contributing to monogenic disease.     

Brain-derived neurotrophic factor in patients with Huntington's disease

Reduced Brain-Derived Neurotrophic Factor (BDNF) levels have been described in a number of patho-physiological conditions, most notably, in Huntington's disease (HD), a progressive neurodegenerative disorder. Since BDNF is also produced in blood, we have undertaken the measurement of its peripheral levels in the attempt to identify a possible link with HD prognosis and/or its progression. Here we evaluated BDNF level in 398 blood samples including 138 controls, 56 preHD, and 204 HD subjects. We found that BDNF protein levels were not reliably different between groups, whether measured in plasma (52 controls, 26 preHD, 105 HD) or serum (39 controls, 5 preHD, 29 HD). Our experience, and a re-analysis of the literature highlighted that intra-group variability and methodological aspects affect this measurement, especially in serum. We also assessed BDNF mRNA levels in blood samples from 47 controls, 25 preHD, and 70 HD subjects, and found no differences among the groups. We concluded that levels of BDNF in human blood were not informative (mRNA levels or plasma protein level) nor reliable (serum protein levels) as HD biomarkers. We also wish to warn the scientific community in interpreting the significance of changes measured in BDNF protein levels in serum from patients suffering from different conditions.     

Ubiquitination is a major modulator for the activation of inflammasomes and pyroptosis

Inflammasomes are a central node of the innate immune defense system against the threat of homeostatic perturbance caused by pathogenic organisms or host-derived molecules. Inflammasomes are generally composed of multimeric protein complexes that assemble in the cytosol after sensing danger signals. Activated inflammasomes promote downstream proteolytic activation, which triggers the release of pro-inflammatory cytokines therefore inducing pyroptotic cell death. The inflammasome pathway is finely tuned by various mechanisms. Recent studies found that protein post-translational modifications such as ubiquitination also modulate inflammasome activation. Targeting the ubiquitination modification of the inflammasome pathway might be a promising strategy for related diseases. In this review, we extensively discuss the advances in inflammasome activation and pyroptosis modulated by ubiquitination which help in-depth understanding and controlling the inflammasome and pyroptosis in various diseases.     

Stem cell factor is a chemoattractant and a survival factor for CNS stem cells

Migration of neural cells to their final positions is crucial for the correct formation of the central nervous system. Several extrinsic factors are known to be involved in the regulation of neural migration. We asked if stem cell factor (SCF), well known as a chemoattractant and survival factor in the hematopoietic lineage, could elicit similar responses in neural stem cells. For that purpose, a microchemotaxis assay was used to study the effect of SCF on migration of neural stem cells from the embryonic rat cortex. Our results show that SCF-induced chemotaxis and that specific antibodies to SCF or tyrosine kinase inhibitors abolished the migratory response. The SCF-receptor, Kit, was expressed in neural stem cells and in their differentiated progeny. We also show that SCF is a survival factor, but not a mitogen or a differentiation factor for neural stem cells. These data suggest a role for SCF in cell migration and survival in the developing cortex.