The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant peripheral neuropathy associated with a large DNA duplication on the short arm of human chromosome 17. The trembler (Tr) mouse serves as a model for CMT1A because of phenotypic similarities and because the Tr locus maps to mouse chromosome 11 in a region of conserved synteny with human chromosome 17. Recently, the peripheral myelin gene Pmp-22 was found to carry a point mutation in Tr mice. We have isolated cDNA and genomic clones for human PMP-22. The gene maps to human chromosome 17p11.2-17p12, is expressed at high levels in peripheral nervous tissue and is duplicated, but not disrupted, in CMT1A patients. Thus, we suggest that a gene dosage effect involving PMP-22 is at least partially responsible for the demyelinating neuropathy seen in CMT1A.

Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13).

We have recently described a novel cDNA, SR13 (Welcher, A. A., U. Suter, M. De Leon, G. J. Snipes, and E. M. Shooter. 1991. Proc. Natl. Acad. Sci. USA. 88:7195-7199), that is repressed after sciatic nerve crush injury and shows homology to both the growth arrest-specific mRNA, gas3 (Manfioletti, G., M. E. Ruaro, G. Del Sal, L. Philipson, and C. Schneider, 1990. Mol. Cell Biol. 10:2924-2930), and to the myelin protein, PASII (Kitamura, K., M. Suzuki, and K. Uyemura. 1976. Biochim. Biophys. Acta. 455:806-816). In this report, we show that the 22-kD SR13 protein is expressed in the compact portion of essentially all myelinated fibers in the peripheral nervous system. Although SR13 mRNA was found in the central nervous system, no corresponding SR13 protein could be detected by either immunoblot analysis or by immunohistochemistry. Northern and immunoblot analysis of SR13 mRNA and protein expression during development of the peripheral nervous system reveal a pattern similar to other myelin proteins. Furthermore, we demonstrate by in situ mRNA hybridization on tissue sections and on individual nerve fibers that SR13 mRNA is produced predominantly by Schwann cells. We conclude that the SR13 protein is apparently exclusively expressed in the peripheral nervous system where it is a major component of myelin. Thus, we propose the name Peripheral Myelin Protein-22 (PMP-22) for the proteins and cDNA previously designated PASII, SR13, and gas3.

Neurotrophins and time: different roles for TrkB signaling in hippocampal long-term potentiation.

We examined the role of TrkB ligands in hippocampal long-term potentiation (LTP) using function-blocking TrkB antiserum (Ab) and Trk-IgG fusion proteins. Incubation of hippocampal slices with TrkB Ab had no effect on basal synaptic transmission, short-term plasticity, or LTP induced by several trains of tetanic stimulation. The TrkB Ab-treated slices, however, showed significant deficits in LTP induced by either theta-burst stimulation (TBS) or "pairing." Slices exposed to the same number of inducing stimuli, delivered either as TBS or as a single 100 Hz epoch, only exhibited TrkB-sensitive LTP when TBS was used, indicating that the temporal pattern of stimulation determines the neurotrophin dependence. The late phase of LTP (2-3 hr) was also significantly impaired in slices pretreated with TrkB Ab or a TrkB-IgG. The application of a TrkB-IgG 30 min after LTP induction caused previously potentiated synaptic transmission to return to baseline levels, indicating that TrkB ligands are required to maintain LTP for up to 1 hr after induction. Taken together, these results indicate that both the temporal patterns of synaptic activity and the different temporal phases of synaptic enhancement are important in determining the neurotrophin dependence of plasticity in the hippocampus.

Cloning and characterization of a specific receptor for mouse oncostatin M.

Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor beta [LIFRbeta], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRbeta and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.

Progress in the molecular understanding of hereditary peripheral neuropathies reveals new insights into the biology of the peripheral nervous system.

Since the first description of the autosomal dominant inherited peripheral neuropathy Charcot-Marie-Tooth (CMT) disease over a century ago, there has been considerable disagreement, based on morphological abnormalities of both the axons of peripheral nerves and their surrounding Schwann cells, as to whether this disorder is due primarily to an autonomous Schwann cell defect or an autonomous neuronal defect. Recently, the Schwann cell protein peripheral myelin protein 22 (PMP-22) has been implicated in the molecular pathogenesis of hereditary peripheral neuropathies in mice and humans. Reinterpretations of morphological studies of the diseased nerves in light of these findings strongly suggest that Schwann cells have a much more pronounced influence on their ensheathed axons than previously anticipated.

cDNA cloning and tissue distribution of five human EPH-like receptor protein-tyrosine kinases.

We have isolated cDNA clones from a human fetal brain library that encode five members of the EPH sub-family of receptor protein tyrosine kinases (PTKs). Comparison of the DNA sequences of these receptors to the Genbank database reveals that two of our clones correspond to the previously identified HEK and ERK receptors, two are apparently human homologues of the mouse receptors Sek and Bsk and one is novel. With these additions, the number of known human EPH sub-family members is nine and the total in all vertebrate species is 13 making it the largest known sub-family of PTKs. Analysis of the expression pattern of EPH sub-family mRNAs reveals that some are expressed in a wide variety of adult tissues while others are quite restricted. Consistent with the amplification of these sequences from a fetal brain cDNA library, all five members which we have isolated are expressed in the brain. We have named these receptors HEK4, HEK5, HEK7, HEK8 and HEK11, following the nomenclature of Wicks et al. (1992) and the numbering convention set forth by Sajjadi et al. (1991). Analysis of these new EPH sub-family members will increase our understanding of the biology of this receptor family and their isolation will provide reagents for the identification of ligands for this large family of orphan receptors.

Endogenous brain-derived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo.

Neuronal growth factors regulate the survival of neurons by their survival and death-promoting activity on distinct populations of neurons. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) promote neuronal survival via tyrosine kinase (Trk) receptors, whereas NGF and BDNF can also induce apoptosis in developing neurons through p75(NTR) receptors in the absence of their respective Trk receptors. Using mutant mice and inactivation of neurotrophins and their receptors with antibodies in rats, we show that endogenous NT-3 induces death of adult BDNF-dependent, axotomized corticospinal neurons (CSNs). When NT-3 is neutralized, the neurons survive even without BDNF, suggesting complete antagonism. Whereas virtually all unlesioned and axotomized CSNs express both trkB and trkC mRNA, p75 is barely detectable in unlesioned CSNs but strongly upregulated in axotomized CSNs by day 3 after lesion, the time point when cell death occurs. Blocking either cortical TrkC or p75(NTR) receptors alone prevents death, indicating that the opposing actions of NT-3 and BDNF require their respective Trk receptors, but induction of death depends on p75(NTR) cosignaling. The results show that neuronal survival can be regulated antagonistically by neurotrophins and that neurotrophins can induce neuronal death in the adult mammalian CNS. We further present evidence that signaling of tyrosine kinase receptors of the trk family can be crucially involved in the promotion of neuronal death in vivo.

Immunocytochemical localization of TrkB in the central nervous system of the adult rat.

The TrkB family of transmembrane proteins serve as receptors for brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-4/5, and possibly NT-3, three members of the neurotrophin family of neurotrophic factors. In order to understand the potential roles played by these receptors, we have examined the distribution of the TrkB receptor proteins in the adult rat brain by using immunohistochemistry. Several different antisera, directed against either synthetic peptides corresponding to different regions of TrkB or a recombinant fusion protein comprising part of the extracellular domain, were generated. Each of these antisera was directed to epitopes found on all known TrkB isoforms (both the tyrosine kinase-possessing isoform and the truncated kinase-lacking isoforms). In addition, a commercially available antibody to the intracellular domain of TrkB was also used. Widespread and distinct staining was observed on the surface of neuronal cell bodies, axons, and dendrites in many structures, including the cerebral cortex, hippocampus, dentate gyrus, striatum, septal nuclei, substantia nigra, cerebellar Purkinje cells, brainstem and spinal motor neurons, and brainstem sensory nuclei. Staining was also observed in the pia matter, on a subpopulation of ependymal cells lining the cerebral ventricle wall, and other nonneuronal cells. The expression pattern of TrkB receptor protein suggests that TrkB plays a broad role in the central nervous system. In addition, the detection of TrkB immunoreactivity on cell bodies and dendrites is consistent with recent models suggesting that neurotrophins may be derived from presynaptic and/or autocrine sources in addition to the classical postsynaptic target.

Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system.

We have generated and characterized a multi-functional polyclonal anti-brain-derived neurotrophic factor antibody. Western blot analysis, dorsal root ganglion neurite outgrowth and dorsal root ganglion neuron survival assays showed that this antibody specifically recognized brain-derived neurotrophic factor and not the other neurotrophins. Furthermore, it was capable of blocking the functional effects of brain-derived neurotrophic factor. Using this antibody, we examined the expression of brain-derived neurotrophic factor in adult rat brains by immunohistochemistry. We found distinct brain-derived neurotrophic factor immunoreactivity in several structures of the brain. These included the neocortex, piriform cortex, amygdaloid complex, hippocampal formation, claustrum, some thalamic and hypothalamic nuclei, the substantia nigra and some brainstem structures. In contrast to brain-derived neurotrophic factor messenger RNA expression, brain-derived neurotrophic factor immunoreactivity was also found in the lateral septum, bed nucleus of the stria teminalis, medial preoptic nucleus, olivery pretectal nucleus, lateral paragigantocellular nucleus and the dorsal horn of the spinal cord. In normal adult rat brains, there was little or no staining in the CA1 region or the granule cell layer of the dentate gyrus of the hippocampus. However, kainate treatments greatly increased brain-derived neurotrophic factor immunoreactivity in the pyramidal cells of the CA1 region, as well as in the dentate gyrus, CA2 and CA3 hippocampal regions. We present evidence for both the subcellular localization and anterograde transport of endogenous brain-derived neurotrophic factor in the central nervous system. The detection of brain-derived neurotrophic factor protein in several discrete regions of the adult brain, and brain-derived neurotrophic factor's dramatic up-regulation following kainate treatment, strongly supports a role of brain-derived neurotrophic factor in the maintenance of adult neurons and synapses. Since several populations of neurons lost during neurodegenerative diseases synthesize brain-derived neurotrophic factor protein, modulation of brain-derived neurotrophic factor levels may be clinically beneficial. The antibody described in this paper will be helpful in determining more precisely the functional activities of brain-derived neurotrophic factor in the adult.

Comparison of neurotrophin regulation of human and rat neuropeptide Y (NPY) neurons: induction of NPY production in aggregate cultures derived from rat but not from human fetal brains.

Previous studies established that brain-derived neurotrophic factor (BDNF) induces neuropeptide Y (NPY) production and accumulation of NPY-mRNA in cultures of rat fetal brain tissues. In this study, we addressed the question: Are cultured human NPY neurons regulated by BDNF and/or by another member of the neurotrophin (NT) family of growth factors? Using aggregate cultures derived from human fetal cortical hemispheres, we assessed the effect of BDNF on NPY production varying the following experimental conditions: fetal and culture age; medium composition (with and without serum), dose and duration of exposure to BDNF, and neurotrophin species tested (BDNF, NT-4/5, NT-3 or NGF). Under none of these conditions did BDNF, NT-4/5, NT-3 or NGF induce an increase in NPY production. This was in contrast to forskolin + phorbol 12 myristate 13-acetate (PMA) which were highly effective in inducing NPY production, verifying that expression of NPY is a regulated process in these cultures. None of these neurotrophins enhanced the response to forskolin + PMA. By comparison, using aggregate cultures derived from rat fetal cortices, BDNF and NT-4/5 were equipotent in inducing NPY production but NT-3 and NGF were essentially ineffective. Moreover, the effects of BDNF or NT-4/5 and forskolin + PMA on NPY production were additive, indicating the involvement of distinct intracellular signalling pathways. Western blot analyses of human- and rat-derived aggregates indicated the presence of full-length Trk receptors which are tyrosine-phosphorylated in response to either BDNF, NT-4/5 or NT-3. Primary cultures of astrocytes (rat as well as human) were devoid of a functional TrkB receptor, strongly suggesting a neuronal expression of TrkB in the aggregates. Thus, a functional TrkB receptor is expressed by both the human and rat aggregates, but only the rat aggregates responded to BDNF or NT-4/5. These results are consistent with a difference in a post TrkB-receptor event(s) mediating BDNF action in the cultured human and rat fetal NPY neurons.

Elevated glial brain-derived neurotrophic factor in Parkinson's diseased nigra.

We show the cellular distribution of immunoreactivity (IR) for brain-derived-neurotrophic-factor (BDNF), neurotrophin-3 (NT-3) and tyrosine kinase receptors TRKB and TRKC in idiopathic Parkinson's disease (IPD) and controls at post-mortem. In both groups, nigral neurons, astrocytes, ramified and amoeboid microglia expressed all antigens. Caudate-putamen neurons expressed all antigens except BDNF with similar distribution between groups. In IPD nigra, increased numbers of BDNF-IR and, less frequently, NT-3-IR ramified glia surrounded fragmented neurons, accompanied by BDNF-IR in surrounding neuropil. Amoeboid microglia were abundant only in IPD nigral scars. In IPD, glia might up-regulate neurotrophins in response to signals released from failing nigral neurons.

Selective enrichment of specific DNA, cDNA and RNA sequences using biotinylated probes, avidin and copper-chelate agarose.

We have developed a general procedure for the rapid and efficient enrichment of specific DNA, RNA or cDNA sequences. Biotinylated DNA or RNA is used as a hybridization probe in solution, avidin is then added to label both the probe and hybrid molecules, and the hybridization mixture chromatographed over cupric-iminodiacetic acid agarose beads. Avidin-probe and avidin-hybrid molecules are selectively retained on the column; non-hybridized sequences are contained in the flow-through fraction. Sequences retained on the column are recovered in high yield by the addition of ethylenediamine tetracetic acid in the buffer. The method can be used in both subtractive enrichment and positive selection protocols. Here we report its application to the isolation of Neisseria gonorrhoeae specific genomic DNA clones and the purification of a cDNA subpopulation representing mRNA sequences that are over-expressed in murine Friend cells after dimethylsulfoxide induction.

BDNF/NT4-5 receptor TrkB and cadherin participate in cell-cell adhesion.

The neurotrophin receptor TrkB plays a key role in promoting cell survival and differentiation in the nervous system. Two adhesive motifs in the extracellular domain of TrkB have been proposed based on its predicted secondary structure. To investigate the potential adhesive function of trkB, a full length trkB cDNA was stably transfected into NIH 3T3 cells and TrkB-expressing clones isolated. Transfectant clones producing different levels of TrkB protein were subjected to a homotypic aggregation assay. Results showed that parental cells were non-adhesive during the assay while TrkB-expressing cells displayed varying degrees of aggregation depending on the amount of TrkB protein expressed. The observed adhesion was Ca(2+)-, Mg(2+)-, and temperature-dependent, characteristics shared by the cadherin family of adhesion molecules. The transfected cell lines also expressed cadherin in proportion to TrkB expression and both molecules were required for cell adhesion. Double immunofluorescence staining studies showed that TrkB was colocalized with cadherin and catenin at cell-cell contact sites. Whether TrkB and cadherin mediate adhesion separately or synergistically remains to be determined.

A myelin protein is encoded by the homologue of a growth arrest-specific gene.

Striking features of the cellular response to sciatic nerve injury are the proliferation of Schwann cells in the distal nerve stump and the downregulation of myelin-specific gene expression. Once the axons regrow, the Schwann cells differentiate again to reform the myelin sheaths. We have isolated a rat cDNA, SR13, which is strongly downregulated in the initial phase after sciatic nerve injury. This cDNA encodes a glycoprotein that shares striking amino acid similarity with a purified myelin protein and is specifically precipitated by a myelin-specific antiserum. Immunohistochemistry experiments using peptide-specific polyclonal antibodies localize the SR13 protein to the myelin sheath of the sciatic nerve. Computer-aided sequence analysis identified a pronounced homology of SR13 to a growth arrest-specific mRNA (Gas-3) that is expressed in resting but not in proliferating 3T3 mouse fibroblasts. SR13 is similarly downregulated during Schwann cell proliferation in the rat sciatic nerve. The association of the SR13 as well as the Gas-3 mRNA with nonproliferating cells in two different experimental systems suggests a common role for these molecules in maintaining the quiescent cell state.

Rapid plasmid library screening using RecA-coated biotinylated probes.

A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

Nerve growth factor binding domain of the nerve growth factor receptor.

A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptor to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cysteine-rich sequence of the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.

Molecular approaches to nerve regeneration.

Current research into regeneration of the nervous system has focused on defining the molecular events that occur during regeneration. One well-characterized system for studying nerve regeneration is the sciatic nerve of rat. Numerous studies have characterized the sequence of events that occur after a crush injury to the sciatic nerve (Cajal 1928; Hall 1989). These events include axon and myelin breakdown, changes in the permeability of the blood vessels, proliferation of Schwann cells, invasion of macrophages, and the phagocytosis of myelin fragments by Schwann cells and macrophages. The distal segment of the injured sciatic nerve provides a supportive environment for the regeneration of the nerve fibres (Cajal 1928; David & Aguayo 1981). Within a period of weeks, the injured sciatic nerve is able to regrow and successfully reinnervate the appropriate targets. Some of the molecules that provide trophic support for the regrowing nerve fibres have been identified, including nerve growth factor (NGF) (Heumann et al. 1987) and glial maturation factor beta (Bosch et al. 1989). Another class of molecules show changes in their rates of synthesis during regeneration, including both proteins (Skene & Shooter 1983; Muller et al. 1986) and mRNA species (Trapp et al. 1988; Meier et al. 1989). To better understand nerve regeneration, we have taken two, parallel molecular approaches to study the events associated with regeneration. The first of these is to study in detail the mechanism of action of a molecule that has been implicated in the regeneration process, nerve growth factor. The second approach is to identify novel gene sequences which are regulated during regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of transcriptionally regulated genes after sciatic nerve injury.

Mammalian peripheral nerve fibres can regenerate after injury. In an attempt toward a better understanding of the underlying molecular events, we have isolated novel and known rat cDNA sequences, the expression of which are regulated during sciatic nerve regeneration. For this purpose, cDNA libraries were constructed from either the nerve segment distal to the crush site or the corresponding contralateral uninjured nerve of the same animals. These libraries were screened by differential hybridization and several transcriptionally repressed and induced sequences were isolated. Out of 2,000 cDNA clones screened from the distal library, 11 sequences were found to be induced in the distal nerve segment. This set of induced cDNAs included the rat homolog of vimentin, 28 S and 18 S ribosomal RNA species, and two novel sequences. Of 5,000 screened colonies of the contralateral library, 30 colonies contained sequences that were repressed in the distal segment after nerve crush. They were identified as myelin basic protein, myelin P0, alpha-globin, cytochrome oxidase subunit 1, creatine kinase (muscle type, M) and collagen type I. In addition, five novel sequences were found that were dramatically repressed after sciatic nerve crush. Representative clones were tested by northern blot analysis to study their time course of transcriptional regulation during nerve regeneration. The observed patterns suggest that the regeneration phenomenon shows complex gene regulation in which the nonneuronal cells of the distal segment play an important role. Further characterization of the isolated regulated known and unknown sequences will increase our understanding of the molecular events associated with neuronal regeneration.