Verdon Taylor

Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell populations in the postnatal mouse brain

Notch signaling plays a pivotal role in the regulation of vertebrate neurogenesis. However, in vitro experiments suggest that Notch1 may also be involved in the regulation of later stages of brain development. We have addressed putative roles in the central nervous system by examining the expression of Notch signaling cascade components in the postnatal mouse brain. In situ mRNA hybridization revealed that Notch1 is associated with cells in the subventricular zone, the dentate gyrus and the rostromigratory stream, all regions of continued neurogenesis in the postnatal brain. In addition, Notch1 is expressed at low levels throughout the cortex and olfactory bulb and shows striking expression in the cerebellar Purkinje cell layer. The Notch ligands, including Delta-like1 and 3 and Jagged1 and Jagged2, show distinct expression patterns in the developing and adult brain overlapping that of Notch1. In addition, the downstream targets of the Notch signaling cascade Hes1, Hes3, Hes5 and the intrinsic Notch regulatory proteins Numb and Numblike also show active signaling in distinct brain regions. Hes5 coincides with the majority of Notch1 expression and can be detected in the cerebral cortex, cerebellum and putative germinal zones. Hes3, on the other hand, shows a restricted expression in cerebellar Purkinje cells. The distribution of Notch1 and its putative ligands suggest distinct roles in specific subsets of cells in the postnatal brain including putative stem cells and differentiated neurons.

Epithelial membrane protein-1, peripheral myelin protein 22, and lens membrane protein 20 define a novel gene family.

Peripheral myelin protein 22 (PMP22) is expressed in many tissues but mainly by Schwann cells as a component of compact myelin of the peripheral nervous system (PNS). Mutations affecting PMP22 are associated with hereditary motor and sensory neuropathies. Although these phenotypes are restricted to the PNS, PMP22 is thought to play a dual role in myelin formation and in cell proliferation. We describe the cloning and characterization of epithelial membrane protein-1 (EMP-1), a putative four-transmembrane protein of 160 amino acids with 40% amino acid identity to PMP22. EMP-1 and PMP22 are co-expressed in most tissues but with differences in relative expression levels. EMP-1 is most prominently found in the gastrointestinal tract, skin, lung, and brain but not in liver. In the corpus gastricum, EMP-1 protein can be detected in epithelial cells of the gastric pit and isthmus of the gastric gland in a pattern consistent with plasma membrane association. EMP-1 and PMP22 mRNA levels are inversely regulated in the degenerating rat sciatic nerve after injury and by growth arrest in NIH 3T3 fibroblasts. The discovery of EMP-1 as the second member of a novel gene family led to the identification of the lens-specific membrane protein 20 (MP20) as a third but distant relative. The proteins of this family are likely to serve similar functions possibly related to cell proliferation and differentiation in a variety of cell types.

EPITHELIAL MEMBRANE PROTEIN-2 AND EPITHELIAL MEMBRANE PROTEIN-3 : TWO NOVEL MEMBERS OF THE PERIPHERAL MYELIN PROTEIN 22 GENE FAMILY

Peripheral myelin protein 22 (PMP22) is expressed by Schwann cells in the peripheral nervous system (PNS), and mutations affecting the PMP22 gene are associated with hereditary motor and sensory neuropathies. We have previously defined the PMP22/EMP/MP20 gene family by characterizing the PMP22-related epithelial membrane protein-1 (EMP-1). We now report the identification of two additional members of the same family, epithelial membrane protein-2 and epithelial membrane protein-3 (EMP-2 and EMP-3). Both cDNA-predicted polypeptides share approx. 40% aa identity with PMP22. In human, EMP-2 and EMP-3 mRNA transcripts are found in most tissues with an expression pattern partially overlapping that of PMP22 and EMP-1. EMP-2 is most prominently expressed in the adult ovary, heart, lung and intestine and in fetal lung. The levels of EMP-3 transcripts are highest in peripheral blood leukocytes, ovary, intestine and various embryonic tissues. In contrast to PMP22 and EMP-1, EMP-2 and EMP-3 expression is detectable in the liver. In vitro transcription-translation generates EMP-2 and EMP-3 polypeptides of 18 kDa which is in agreement with their predicted sizes. Since PMP22 has been implicated in the regulation of cell proliferation and apoptosis, it appears likely that these novel members of the PMP22/EMP/MP20 protein family are also involved in similar regulatory processes in a variety of tissues.

Characterisation of autoantibodies to peripheral myelin protein 22 in patients with hereditary and acquired neuropathies

To investigate the possibility that an autoimmune mechanism may play a role in the hereditary neuropathy Charcot-Marie-Tooth type 1A (CMT1A), sera were analysed by Western blot for anti-peripheral myelin protein 22 (PMP22) autoantibodies. These sera were compared with sera from patients with CMT type 2 (CMT2), acquired peripheral neuropathies such as chronic inflammatory demyelinating neuropathy (CIDP), anti-MAG IgM neuropathy, Miller-Fisher syndrome (MFS), diabetic neuropathy and with control blood donors. Anti-PMP22 positive sera were detected in 70% of patients with CMT1 and unexpectedly in 60% of patients with CMT2. Interestingly, 44% of the patients with other peripheral neuropathies and 23% of the apparently healthy controls showed also anti-PMP22 antibody reactivity. Immunohistochemical analysis of the human anti-PMP22 antisera on healthy sural nerve sections and on PMP22-expressing COS cells revealed that these sera did not recognise endogenous PMP22. Our results indicate that anti-PMP22 autoantibodies are found in sera of patients with different types of peripheral neuropathies, but their role in the pathogenesis of these diseases remains to be determined.

Neural Membrane Protein 35 (NMP35): A Novel Member of a Gene Family Which Is Highly Expressed in the Adult Nervous System

We have identified and isolated a cDNA that codes for the novel protein NMP35 (neural membrane protein 35) using RNA arbitrarily primed PCR (RAP-PCR) for differential display in the developing rat sciatic nerve. NMP35 is predominantly found in the adult nervous system where both transcripts and protein are strongly upregulated during postnatal development. In situ hybridization studies show that NMP35 mRNA is widely distributed in the brain and spinal cord with a neuronal expression pattern. Database comparisons reveal that NMP35 shares significant homologies with the rat glutamate-binding protein (GBP), the Drosophila NMDARA1, and two orphan C. elegans genes. Comparative analysis of NMP35 and GBP expression indicates that they are similarly regulated during development and display regionally overlapping cellular patterns. We conclude that NMP35 and GBP are members of a gene family which is likely to play a role in the development and the maintenance of the nervous system.

Neural membrane protein 35/Lifeguard is localized at postsynaptic sites and in dendrites

We have previously identified and characterized a cDNA coding for neural membrane protein 35 (NMP35). We showed that NMP35 mRNA is predominantly expressed in the adult CNS with a neuronal expression pattern. Functional analysis indicates that the human homologue of NMP35, Lifeguard, plays a role in Fas-mediated cell death. In this study we used affinity-purified antibodies raised against the putative cytoplasmic N-terminal domain of NMP35 to determine its precise subcellular localization in the adult CNS. NMP35 protein is widely expressed throughout the brain and spinal cord, most prominently in dendrites of several neuronal cell types and in the surrounding neuropil. Immunofluorescence confocal microscopy reveals colocalization of NMP35 with the glutamate receptor GluR2 and adjacent localization to the presynaptic vesicle protein synaptophysin. These data suggest that NMP35 may be localized to the postsynaptic membrane. Immunoelectron microscopy with NMP35 antibodies confirms the expression of the protein in dendritic processes and in a subset of synapses at the postsynaptic membrane and density. These findings suggest a role for NMP35 in synapses of the adult central nervous system.

Membrane topology of peripheral myelin protein 22

Peripheral myelin protein 22 (PMP22) is a structural component of compact peripheral nerve myelin and is likely to play a role in the modulation of cell proliferation and cell spreading. Molecular genetics revealed that mutations affecting the PMP22 gene are responsible for the most common forms of hereditary motor and sensory neuropathies in humans. Computer analysis predicts a tetraspan‐membrane structure for the PMP22 protein. We have assessed the topology of PMP22 experimentally using chimeric proteins consisting of different PMP22 domains fused to reporter genes and internally tagged molecules. Based on in vitro transcription/translation assays and immunohistochemical analysis of transfected cells, we propose that PMP22 can adopt a non‐tetraspan topology that has functional implications in normal and disease processes. J. Neurosci. Res. 62:15–27, 2000.

Extensive splice variation and localization of the EHK-1 receptor tyrosine kinase in adult human brain and glial tumors

EHK-1 is a neuronal ELK-related receptor tyrosine kinase which interacts with multiple, membrane-anchored ligands. Recent experiments have suggested a role for some of these ligands in the formation of neuronal pathways. Here, we report the isolation of human EHK-1 cDNAs and the localization of the human EHK-1 gene to chromosome 4q12. Six EHK-1 mRNA splice variants encoding cell-surface receptors with catalytic domains were identified in adult human brain where a 120-kDa EHK-1 protein predominates. Immunohistochemistry for EHK-1 reveals a dendritic staining pattern in cortical neurons and cerebellar Purkinje cells and a marked accumulation of EHK-1 in the somas of pyramidal neurons within the cortex and hippocampus. Interestingly, we have identified lineage aberrant expression of EHK-1 in a number of human gliomas. In addition to functions during development, EHK-1 may be involved in the maintenance of the adult nervous system and contribute to glioma development.