A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation

The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/−), or null (−/−) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56140/1/20534_ftp.pd

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins

<p>Abstract</p> <p>Background</p> <p>In multiple sclerosis, inflammatory cells are found in both active and chronic lesions, and it is increasingly clear that cytokines are involved directly and indirectly in both formation and inhibition of lesions. We propose that cytokine mixtures typical of Th1 or Th2 lymphocytes, or monocyte/macrophages each induce unique molecular changes in glial cells.</p> <p>Methods</p> <p>To examine changes in gene expression that might occur in glial cells exposed to the secreted products of immune cells, we have used gene array analysis to assess the early effects of different cytokine mixtures on mixed CNS glia in culture. We compared the effects of cytokines typical of Th1 and Th2 lymphocytes and monocyte/macrophages (M/M) on CNS glia after 6 hours of treatment.</p> <p>Results</p> <p>In this paper we focus on changes with potential relevance for neuroprotection and axon/glial interactions. Each mixture of cytokines induced a unique pattern of changes in genes for neurotrophins, growth and maturation factors and related receptors; most notably an alternatively spliced form of trkC was markedly downregulated by Th1 and M/M cytokines, while Th2 cytokines upregulated BDNF. Genes for molecules of potential importance in axon/glial interactions, including cell adhesion molecules, connexins, and some molecules traditionally associated with neurons showed significant changes, while no genes for myelin-associated genes were regulated at this early time point. Unexpectedly, changes occurred in several genes for proteins initially associated with retina, cancer or bone development, and not previously reported in glial cells.</p> <p>Conclusion</p> <p>Each of the three cytokine mixtures induced specific changes in gene expression that could be altered by pharmacologic strategies to promote protection of the central nervous system.</p

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for molecules associated with metabolism, signaling and regulation in central nervous system mixed glial cell cultures

<p>Abstract</p> <p>Background</p> <p>Cytokines secreted by immune cells and activated glia play central roles in both the pathogenesis of and protection from damage to the central nervous system (CNS) in multiple sclerosis (MS).</p> <p>Methods</p> <p>We have used gene array analysis to identify the initial direct effects of cytokines on CNS glia by comparing changes in early gene expression in CNS glial cultures treated for 6 hours with cytokines typical of those secreted by Th1 and Th2 lymphocytes and monocyte/macrophages (M/M).</p> <p>Results</p> <p>In two previous papers, we summarized effects of these cytokines on immune-related molecules, and on neural and glial related proteins, including neurotrophins, growth factors and structural proteins. In this paper, we present the effects of the cytokines on molecules involved in metabolism, signaling and regulatory mechanisms in CNS glia. Many of the changes in gene expression were similar to those seen in ischemic preconditioning and in early inflammatory lesions in experimental autoimmune encephalomyelitis (EAE), related to ion homeostasis, mitochondrial function, neurotransmission, vitamin D metabolism and a variety of transcription factors and signaling pathways. Among the most prominent changes, all three cytokine mixtures markedly downregulated the dopamine D3 receptor, while Th1 and Th2 cytokines downregulated neuropeptide Y receptor 5. An unexpected finding was the large number of changes related to lipid metabolism, including several suggesting a switch from diacylglycerol to phosphatidyl inositol mediated signaling pathways. Using QRT-PCR we validated the results for regulation of genes for iNOS, arginase and P glycoprotein/multi-drug resistance protein 1 (MDR1) seen at 6 hours with microarray.</p> <p>Conclusion</p> <p>Each of the three cytokine mixtures differentially regulated gene expression related to metabolism and signaling that may play roles in the pathogenesis of MS, most notably with regard to mitochondrial function and neurotransmitter signaling in glia.</p

Autoantibodies to Agrin in Myasthenia Gravis Patients

To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins-1

<p><b>Copyright information:</b></p><p>Taken from "Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins"</p><p>http://www.jneuroinflammation.com/content/4/1/30</p><p>Journal of Neuroinflammation 2007;4():30-30.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2228280.</p><p></p>e IgM and Cy 3-conjugated donkey anti-mouse IgG. Cultures were examined by indirect immunofluoresence as above. Compared to control, the Th1 treated A2B5+ oligodendrocyte precursors have a much more mature appearance including more extensive process formation although the cells still express A2B5, but do not express MHC class II

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins-0

<p><b>Copyright information:</b></p><p>Taken from "Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins"</p><p>http://www.jneuroinflammation.com/content/4/1/30</p><p>Journal of Neuroinflammation 2007;4():30-30.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2228280.</p><p></p>(IgG) followed by Alexa 488-conjugated goat anti-mouse IgM and Cy 3-conjugated donkey anti-mouse IgG. Cultures were examined for indirect immunofluoresence employing a Leitz Orthoplan 2 fluorescent microscope. The Th1 treated microglia (ED-1+ cells) have a different appearance when compared to those incubated with additional medium (control). Control microglia do not express MHC class II whereas Th1 treated microglia strongly express MHC class II molecules

Recognition of agrin from transfected HEK293 cells by agrin+ serum.

<p>Agrin positive serum 2–17 stained positively with HEK293 cells transfected with Flag-agrin construct, co-staining with anti-Flag antibody. Normal human serum cannot recognize agrin-transfected cells.</p

Detection of agrin autoantibodies in MG patient samples.

<p>Optical density readings of normal human serum were 0.18 ± 0.16 (mean ± SD, n  =  25). The green dotted line was set as mean + 3 SD to indicate the cut-off. The red dots indicate positive for agrin antibodies. NHS, normal human serum; OND, other neurological diseases, n  =  55; MG, myasthenia gravis, n  =  93.</p

Distribution of agrin autoantibodies among MG patients.

<p>Of 93 MG samples previously analyzed for antibody to AChR, MuSK and LRP4, 83 were AChR+/MuSK-/LRP4-; 4 were triple seronegative (AChR-/MuSK-/LRP4-) and 6 were AChR-/MuSK+/LRP4-. The cut-off, indicated by the green line, was set as mean + 3 SD. The red dots indicate positive for agrin antibodies.</p