Rainer Kuhn

SCIP: A glial POU domain gene regulated by cyclic AMP

We have isolated cDNA clones encoding SCIP, a POU domain gene expressed by myelin-forming glial of the central and peripheral nervous systems. In purified Schwann cells cultured in the absence of neurons, expression of SCIP is suppressed. This suppression is relieved by cAMP, and induction of SCIP mRNA by this second messenger precedes cAMP induction of myelin-specific genes. Similarly, SCIP expression in vivo precedes full expression of myelin-specific genes in developing oligodendrocytes and Schwann cells. The sequence of the SCIP POU domain is identical to that of Tst-1, a recently identified member of a family of POU domain genes expressed by restricted subsets of neurons. Our results demonstrate that SCIP is also expressed by myelin-forming glia and suggest that it plays a central role in the progressive determination of these cells and their commitment to myelination.

Repression of the myelin P0 gene by the POU transcription factor SCIP.

SCIP is a POU domain transcription factor expressed by Schwann cells, the myelin-forming glial cells of the peripheral nervous system. In this study, we investigate SCIP regulation of the gene encoding P0, the major structural protein of peripheral myelin. We find that SCIP represses transcription of this gene through the joint action of the SCIP POU domain and an amino terminal domain that acts cell specifically. Maximal repression is DNA-binding-dependent, and analysis of the P0 promoter reveals the presence of multiple SCIP binding sites. Surprisingly, none of these sites in their native positions dramatically affect P0 promoter activity or its repression by SCIP, although they mediate repression when moved closer to the P0 transcription start site. We propose that repression occurs through a quenching mechanism mediated by the SCIP POU and amino terminal domains acting in concert with other nuclear proteins, including a Schwann cell-specific adapter.

The gene encoding the transcription factor SCIP has features of an expressed retroposon.

SCIP is a POU domain transcription factor expressed by glial progenitor cells in the peripheral and central nervous systems (dividing Schwann cells and O-2A cells, respectively), where it appears to act as a repressor of myelin-specific genes. We have isolated genomic clones encoding the rat SCIP gene. Comparison of the structure of these clones with genomic Southern blots and SCIP cDNAs demonstrates that SCIP is encoded in a single-copy, intronless gene that has the general features of an expressed retroposon. This gene contributes to an extended CpG island. It is transcribed to produce a 3.1-kb mRNA that encodes a 451-amino-acid protein with a predicted molecular mass of 45 kDa. Immunopurified SCIP antibodies specifically recognize a nuclear protein of this size in cultured proliferating Schwann cells, and gel shift analyses demonstrate that this protein is the predominant octamer-binding protein in these cells.

Growth factors and transcription factors in oligodendrocyte development

Summary O-2A progenitor cells, the precursors of oligodendrocytes in the central nervous system (CNS), probably originate in the subventricular germinal zones of the developing CNS, and subsequently migrate away from there to populate the rest of the CNS with oligodendrocytes. We are trying to understand how the O-2A progenitor cells interact with their changing environment as they migrate, and how this influences each stage of their development into mature, myelinating oligodendrocytes. In this article we summarize evidence that platelet-derived growth factor (PDGF) is important for stimulating O-2A progenitor cell proliferation in vivo, and describe our efforts to map the distribution of PDGF and its receptors in the developing rat CNS by in situ hybridization and immunohistochemistry. These studies suggest that, in the CNS, PDGF α-receptor subunits may be restricted to O-2A lineage cells that have started to migrate away from the subventricular zones towards their final destinations. Many neurons express the A and/or B chains of PDGF, and astrocytes express the A chain, but it is not yet clear which of these cell types might be the major source of PDGF for O-2A lineage cells in vivo. O-2A progenitor cells can be purified and maintained in a proliferating state in vitro by culturing in the presence of PDGF and bFGF. Under these conditions, the POU transcription factor SCIP/Tst-1 is expressed at a high level; when oligodendrocyte differentiation is initiated by withdrawing the growth factors, SCIP/Tst-1 mRNA is rapidly down-regulated, followed by a decline in SCIP/Tst-1 protein and sequential activation of myelin-specific genes. These observations suggest that SCIP/Tst-1 may be mechanistically involved in the transition from proliferation to differentiation in the O-2A lineage. By in situ hybridization, SCIP/Tst-1 appears also to be expressed in developing neurons, so perhaps it fulfils a similar function in several different cell lineages in the CNS.

A Transgenic Mouse Model for Human Hereditary Neuropathy with Liability to Pressure Palsies

Mutations in the gene encoding peripheral myelin protein 22 (PMP22) account for several inherited peripheral neuropathies in humans. We now show that transgenic mice expressing antisense PMP22 RNA exhibit modestly reduced levels of PMP22 together with a phenotype that is reminiscent of hereditary neuropathy with liability to pressure palsies (HNPP), a human disease caused by a 1.5-Mb deletion of a chromosome 17 region that contains the PMP22 gene. Transgenic antisense homozygotes display a striking movement disorder and a slowing of nerve conduction that worsens with age. Morphological analysis of peripheral nerves demonstrates that a subset of axons have thickened myelin sheaths and tomacula in young adults, with significant myelin degeneration detected in older animals. Together with other recent work, these data suggest that dosage of the PMP22 gene alone underlies the pathophysiology observed in HNPP and related disorders.

Single cell analysis of the expression of a nuclear protein, SCIP, by fluorescent immunohistochemistry visualized with confocal microscopy

SummaryA widely applicable method for the accurate quantification or semiquantification of macromolecules at the level of individual cells is described and validated; this is a method which may considerably facilitate the study of many biological processes. This method relies on measuring fluorescent emission in immunocytochemically labelled cells with a confocal microscope. Emission is related quantitatively to the level of the fluorophore by the combination of an analysis of the polarization of the fluorescent emission and fluorophore ratioing methods. The method was applied to the study of the expression of the suppressed cyclic AMP-induced POU protein (SCIP) transcription factor in glial cells of the central nervous system. In particular, the method allowed the study of transcription factor expression in defined cells present in heterogeneous cultures and in cell types which cannot be isolated in sufficient numbers for biochemical analysis using conventional techniques.