We have developed a simple non-radioactive in situ hybridization procedure for tissue sections and cultured cells using digoxigenin-labelled cRNA probes. This protocol can be applied for the detection of various transcripts present at a wide range of expression levels in the central nervous system. Cerebellar hybridization signals for transcripts estimated to be expressed at high (MBP, myelin basic protein), moderate (GluR1, subunit of AMPA/kainate sensitive glutamate receptors) and low (inositol polyphosphate-5-phosphatase) levels of abundance are demonstrated as examples. The sensitivity and cellular resolution were significantly improved by avoiding any ethanol treatment commonly used in other procedures. The localization of a labelled cell with respect to its environment is shown to be more easily assessed by counterstaining of the tissue with the nuclear dye Hoechst 33258. The present protocol can be combined with immunocytochemistry as demonstrated for glial fibrillary acidic protein (GFAP). All steps of the procedure, including preparation and labelling of the cRNA probes, pretreatment of tissue, hybridization and visualization of the labelled transcripts, are described in detail.

Multiple sclerosis is a chronic inflammatory disease of the CNS. Although progressive axonal injury and diffuse inflammatory damage has been shown in the chronic phase of the disease, little is known about the molecular mechanisms underlying these pathological processes. In order to identify these mechanisms, we have studied the gene expression profile in non-lesion containing tissue, the so-called normal-appearing white matter (NAWM). We performed differential gene expression analysis and quantitative RT-PCR on subcortical white matter from 11 multiple sclerosis and 8 control cases. Differentially expressed genes were further analysed in detail by in situ hybridization and immunofluorescence studies. We show that genes known to be involved in anti-inflammatory and protective mechanisms such as STAT6, JAK1, IL-4R, IL-10, Chromogranin C and Hif-1alpha are consistently upregulated in the multiple sclerosis NAWM. On the other hand, genes involved in pro-inflammatory mechanisms, such as STAT4, IL-1beta and MCSF, were also upregulated but less regularly. Immunofluorescence colocalization analysis revealed expression of STAT6, JAK1, IL-4R and IL-13R mainly in oligodendrocytes, whereas STAT4 expression was detected predominantly in microglia. In line with these data, in situ hybridization analysis showed an increased expression in multiple sclerosis NAWM of HIF-1alpha in oligodendrocytes and HLA-DRalpha in microglia cells. The consistency of the expression levels of STAT6, JAK1, JAK3 and IL-4R between the multiple sclerosis cases suggests an overall activation of the STAT6-signalling pathway in oligodendrocytes, whereas the expression of STAT4 and HLA-DRalpha indicates the activation of pro-inflammatory pathways in microglia. The upregulation of genes involved in anti-inflammatory mechanisms driven by oligodendrocytes may protect the CNS environment and thus limit lesion formation, whereas the activation of pro-inflammatory mechanisms in microglia may favour disease progression. Altogether, our data suggests an endogenous inflammatory reaction throughout the whole white matter of multiple sclerosis brain, in which oligodendrocytes actively participate. This reaction might further influence and to some extent facilitate lesion formation.

RORbeta is an orphan nuclear receptor related to retinoid and thyroid hormone receptors and is exclusively expressed in the central nervous system (CNS). Here we present an in situ hybridization analysis of the distribution of RORbeta mRNA in the developing and adult rat CNS. The receptor localizes to areas involved in the processing of sensory information. In the cerebral cortex, RORbeta mRNA was exclusively detected in non-pyramidal neurons of layer IV and, less so, layer V. The highest expression was found in primary sensory cortices. In the thalamus highest RORbeta expression was found in the sensory relay nuclei projecting to the respective cortical areas. In contrast, sensory projection neurons in the periphery, for example retinal ganglion cells and neurons of the sensory ganglia showed only little RORbeta expression. RORbeta is also expressed in areas involved in the generation and maintenance of circadian rhythms - the suprachiasmatic nucleus, the pineal gland and the retina. In the latter two tissues, RORbeta mRNA abundance oscillates with circadian rhythmicity peaking during the hours of darkness. RORbeta mRNA could not be detected in striatum, hippocampus, cerebellum, the motor nuclei of the cranial nerves or the ventral part of the spinal cord. During development, RORbeta is expressed in many areas as early as embryonic day (E) 15, anticipating the distribution pattern in the adult. Our data suggest that RORbeta regulates genes whose products play essential roles in the context of sensory input integration as well as in the context of circadian timing system.

The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode–axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

Wrapping and compaction of myelin sheaths around axons require specific membrane and membrane-associated proteins. Transmembrane proteins like proteolipid protein (PLP), the peripheral myelin protein 22 (PMP-22) and P0 as well as myelin basic protein (MBP) are crucial for this process. We have isolated a rat cDNA, initially denominated NS 3, that is mainly expressed in the myelinating cells of the nervous system, the oligodendrocytes and Schwann cells. The cDNA encodes a highly hydrophobic protein of 16.8 kDa with four putative transmembrane domains. The putative NS 3 protein lacks a N-terminal hydrophobic leader sequence and has no consensus sequence for N-linked glycosylation. In contrast to PLP and PMP-22, the first and third putative transmembrane domain of the NS 3 protein contain charged amino acids, a feature which resembles the structure of gap junction proteins. Sequence analysis showed that NS 3 is the rat homolog of a human gene called MAL that was cloned from, and is expressed in various T-cell lines. Therefore, we call this gene rMAL (rat MAL). In the nervous system, the expression of rMAL, mRNA begins after birth and is highest during myelination. In situ hybridization shows that rMAL mRNA is exclusively expressed in white and gray matter oligodendrocytes in the CNS and in myelinating Schwann cells in peripheral nerves. Immunohistochemistry using a peptide-specific antibody localized the rMAL protein in the myelinated areas of the CNS and PNS. Furthermore, we demonstrate by immunoblot analysis that rMAL is a component of myelin. Its structure and distribution suggest that the rMAL protein might play an important role in compact myelin. We propose that the name rMAL protein refers to rat Myelin And Lymphocyte protein.

Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system that develops in genetically susceptible individuals and likely requires environmental triggers. The autoantigens and molecular mimics triggering the autoimmune response in multiple sclerosis remain incompletely understood. By using a brain-infiltrating CD4+ T cell clone that is clonally expanded in multiple sclerosis brain lesions and a systematic approach for the identification of its target antigens, positional scanning peptide libraries in combination with biometrical analysis, we have identified guanosine diphosphate (GDP)–l-fucose synthase as an autoantigen that is recognized by cerebrospinal fluid–infiltrating CD4+ T cells from HLA-DRB3*–positive patients. Significant associations were found between reactivity to GDP-l-fucose synthase peptides and DRB3*02:02 expression, along with reactivity against an immunodominant myelin basic protein peptide. These results, coupled with the cross-recognition of homologous peptides from gut microbiota, suggest a possible role of this antigen as an inducer or driver of pathogenic autoimmune responses in multiple sclerosis.

Oligodendrocytes, the myelinating glial cells of the central nervous system (CNS), are due to their high specialization and metabolic needs highly vulnerable to various insults. This led to a general view that oligodendrocytes are defenseless victims during brain damage such as occurs in acute and chronic CNS inflammation. However, this view is challenged by increasing evidence that oligodendrocytes are capable of expressing a wide range of immunomodulatory molecules. They express various cytokines and chemokines (e.g. Il-1β, Il17A, CCL2, CXCL10), antigen presenting molecules (MHC class I and II) and co-stimulatory molecules (e.g. CD9, CD81), complement and complement receptor molecules (e.g. C1s, C2 and C3, C1R), complement regulatory molecules (e.g. CD46, CD55, CD59), tetraspanins (e.g. TSPAN2), neuroimmune regulatory proteins (e.g. CD200, CD47) as well as extracellular matrix proteins (e.g. VCAN) and many others. Their potential immunomodulatory properties can, at specific times and locations, influence ongoing immune processes as shown by numerous publications. Therefore, oligodendrocytes are well capable of immunomodulation, especially during the initiation or resolution of immune processes in which subtle signaling might tip the scale. A better understanding of the immunomodulatory oligodendrocyte can help to invent new, innovative therapeutic interventions in various diseases such as Multiple Sclerosis. This article is part of a Special Issue entitled SI: Myelin Evolution.

We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte‐free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide‐synthesizing enzyme UDP‐galactose:ceramide galactosyl‐transferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T‐lymphocyte‐specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl‐CoA‐desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long‐chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the β‐tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into this complex mechanism.