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.

Evidence for a recessive PMP22 point mutation in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant neuropathy that can be caused by dominant point mutations in PMP22 which encodes a peripheral nerve myelin protein. Usually, CMT1A is caused by the duplication of a 1.5-megabase (Mb) region on chromosome 17p11.2-p12 containing PMP22. Deletion of a similar 1.5-Mb region is associated with hereditary neuropathy with liability to pressure palsies (HNPP), a clinically distinct neuropathy. We have identified a severely affected CMT1 patient who is a compound heterozygote for a recessive PMP22 point mutation, and a 1.5 Mb deletion in 17p11.2-p12. A son heterozygous for the PMP22 point mutation had no signs of neuropathy, while two others heterozygous for the deletion had HNPP, suggesting that point mutations in PMP22 can result in dominant and recessive alleles contributing to CMT1A.

Properties of the beta-nerve growth factor receptor in development.

The cell surface receptor for beta-nerve growth factor was used as a probe to study the development of embryonic chick sensory ganglia. The ganglia were shown to lose their responsiveness to nerve growth factor in vitro between 14 and 16 days of embryonic age. This loss occurred by a decrease in the magnitude of the maximum biological response, not by a shifting of the response to higher concentrations. Binding assays for the beta-nerve growth factor receptor, using 125I-radiolabelled beta-nerve growth factor, were performed with cells from sensory ganglia 8, 12, 14, 16, 18, and 21 days of age. The assays revealed a twofold increase in the number of receptor sites per ganglion between 8 and 14 days and a sixfold drop between 14 and 16 days of embryonic life. Neither increase nor decrease was accompanied by a large change in the affinity of the receptor for the protein. Together with the results of the bioassay, the data show that the loss of biological responsiveness is correlated with and may be due to a loss of the cells' ability to bind beta-nerve growth factor. Correlation of the results of the binding assays with the known ontogeny of the chick embryo provides a hint at the role of nerve growth factor in normal development.

Neurons regulate Schwann cell genes by diffusible molecules.

Successful peripheral nerve regeneration and functional recovery require the reestablishment of the neuron-Schwann cell relationship in the regenerating rat sciatic nerve, neurons differentially regulate Schwann cell genes. The message for the low-affinity NGF receptor, p75NGFR, is induced in Schwann cells distal to the injury and is repressed as regenerating axons make contact with these cells. The inverse is true for mRNA of the myelin gene P0; expression decreases distal to injury and increases as new axons contact Schwann cells and a program of myelination is initiated. Using an in vitro co-culture paradigm in which primary neurons and adult Schwann cells are separated by a microporous membrane, we show that axon contact is not an absolute requirement for neuronal regulation of Schwann cell genes. In this system neurons but not other cell types, repress the expression of Schwann cell p75NGFR while inducing the expression of the POU domain transcription factor, suppressed cAMP inducible POU, and myelin P0. These results demonstrate that regenerating axons can direct the Schwann cell genetic program from a distance through diffusible molecules.

Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin.

Incubation of PC12 cells preloaded with 125I-nerve growth factor (NGF) reveals rapidly and slowly dissociating binding components indicative of a heterogeneous population of receptors. If the cells are previously exposed to wheat germ agglutinin (WGA) for 30 min, NGF now binds to an apparently homogeneous receptor population which exhibit slow dissociation kinetics. Total binding is also reduced by 50%. If WGA is added subsequent to 125I-NGF, total binding is not diminished, but rapidly dissociating receptors occupied with NGF are all converted to the slowly dissociating form. This conversion of receptors occurs rapidly, reaching completion within 2 min at 37 degrees or 4 degrees C, and is unaffected by metabolic energy poisons, suggesting that WGA-induced slowly dissociating receptors are not the product of internalization. The effects of the lectin are blocked by the sugar N-acetyl-D-glucosamine, and the lectin-induced slowly dissociating receptors are converted back to rapidly dissociating receptors by addition of this same sugar. WGA also affects the association of the NGF receptor with the Triton X-100 cytoskeleton. Greater than 90% of bound 125I-NGF becomes associated with Triton X-100 insoluble cytoskeletons in the presence of the lectin, compared with less than 20% before lectin addition. Cytoskeleton association of the NGF receptor by WGA shows similar kinetics as the conversion of rapidly to slowly dissociating receptors. This interaction may be involved in the alteration of NGF-receptor binding properties produced by this lectin.

Early changes in the synthesis of nuclear and cytoplasmic proteins are induced by nerve growth factor in differentiating rat PC12 cells.

Differentiation of rat pheochromocytoma PC12 cells into neuron-like cells was induced by nerve growth factor (NGF) and changes in the apparent rate of synthesis of cellular proteins were analyzed. Attention was particularly focused on the first few hours of exposure to NGF before significant neurite outgrowth was detectable. Cultures were pulse-labeled for 1-h periods with [35S]methionine and proteins were extracted from various subcellular fractions and analyzed by one-dimensional gradient and two-dimensional equilibrium and nonequilibrium gel electrophoresis. The results showed that although the general level of protein synthesis remained constant, by 8 h NGF increased the apparent rate of synthesis of approximately 11 cytoplasmic and 5 nuclear proteins. For several of these proteins, the effect was apparently NGF-specific, since no induction was observed in dibutyryl cAMP-treated cells. Of interest was the following observation: of the five nuclear proteins, NGF increased the synthesis of two proteins with MrS of 56,000 [doublet] and 50,000 D that were associated with a biochemically and morphologically defined nuclear matrix fraction. A cytoplasmic protein, with an Mr of 92,000 D (pI 4.8) appeared to be induced de novo by NGF. NGF also decreased the rate of synthesis of several cytoplasmic and nuclear proteins of low molecular mass (less than 40,000 D). Since only 1-h pulses of [35S]methionine were used, and since experiments with actinomycin D showed that most of these NGF-induced early changes in rates of synthesis included a transcription-dependent step, it seems likely that early effects of NGF include activation of specific genes.

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors.

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.

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.

Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells.

When a clonal line of rat pheochromocytoma (PC12) was exposed to beta-nerve growth factor (beta NGF), N6, O2-dibutyryl adenosine 3':5' cyclic monophosphate (Bt2cAMP), or a combination of the two, 10, 26, or 70% of the cell clumps, respectively, displayed neurites after 1.d. Increases in the cellular RNA concentration were also found to be additive or greater when both agents were present. Neurites induced by Bt2cAMP alone were not maintained after replacement with beta NGF. The degree of potentiated neurite outgrowth was a function of the time of simultaneous exposure to both agents. The initiation of neurite outgrowth in the presence of Bt2cAMP was independent of RNA synthesis, in contrast to that induced by beta NGF alone. We conclude that beta NGF-induced initiation of morphological differentiation of these cells is not mediated by a cAMP-dependent mechanism. Consideration of Bt2cAMP effects upon other cell lines suggest that Bt2cAMP causes a rapid, but unstable, reorganization of the PC12 cytoskeleton, resulting in the initiation of neurite outgrowth from these cells. In contrast, beta NGF alone achieves a more stable cytoskeleton reorganization by an RNA synthesis-dependent mechanism.

Expression of specific sheath cell proteins during peripheral nerve growth and regeneration in mammals.

Protein synthesis in the nerve sheath of injured as well as intact mature and developing sciatic nerves from rat and rabbit was investigated by incubating segments of nerve with [35S]methionine in vitro. The composition of labeled proteins under the different conditions of nerve growth was analyzed by two-dimensional gel electrophoresis and fluorography. The expression of six secreted proteins in rat sciatic nerve with the apparent molecular weights of 70,000 (70 kD), 54,000 (54 kD), 51,000 (51 kD), 39,000 (39 kD), 37,000 (37 kD), and 30,000 (30 kD) was of particular interest because of the correlation of their synthesis and secretion with aspects of nerve growth and regeneration. The synthesis of the 37-kD protein was significantly stimulated during both sciatic nerve development as well as regeneration but not in the intact mature nerve. The expression of this protein appears to be regulated by signal(s) from the axon but not the target. The 70-kD protein was exclusively synthesized in response to axotomy, thus confining its role to some aspect(s) of nerve repair. In contrast, the 54- and 51-kD proteins were expressed in the intact mature nerve sheath. Their synthesis and release was rapidly inhibited upon axotomy but returned to normal or higher levels towards the end of sciatic nerve regeneration, suggesting a role in the maintenance of the integrity of the mature (nongrowing) rat nerve. The 39- and 30-kD proteins were only transiently synthesized within the first week after axotomy. Two proteins with the apparent molecular masses of 70 and 37 kD were synthesized in denervated rabbit sciatic nerve. The similar molecular weights, net charges, and time-courses of induction suggest a homology between these proteins in rabbit and rat, indicating common molecular responses of peripheral nerve sheath cells to axon injury in both mammalian species.

Expression of apolipoprotein E by cultured vascular smooth muscle cells is controlled by growth state.

Rat vascular smooth muscle cells (SMC) in culture synthesize and secrete a approximately 38,000-Mr protein doublet or triplet that, as previously described (Majack and Bornstein. 1984. J. Cell Biol. 99:1688-1695), rapidly and reversibly accumulates in the SMC culture medium upon addition of heparin. In the present study, we show that this approximately 38,000-Mr heparin-regulated protein is electrophoretically and immunologically identical to apolipoprotein E (apo-E), a major plasma apolipoprotein involved in cholesterol transport. In addition, we show that expression of apo-E by cultured SMC varies according to growth state: while proliferating SMC produced little apo-E and expressed low levels of apo-E mRNA, quiescent SMC produced significantly more apo-E (relative to other proteins) and expressed markedly increased levels of apo-E mRNA. Northern analysis of RNA extracted from aortic tissue revealed that fully differentiated, quiescent SMC contain significant quantities of apo-E mRNA. These data establish aortic SMC as a vascular source for apo-E and suggest new functional roles for this apolipoprotein, possibly unrelated to traditional concepts of lipid metabolism.

Lipoprotein uptake by neuronal growth cones in vitro.

Macrophages that rapidly enter injured peripheral nerve synthesize and secrete large quantities of apolipoprotein E. This protein may be involved in the redistribution of lipid, including cholesterol released during degeneration, to the regenerating axons. To test this postulate, apolipoprotein E-associated lipid particles released from segments of injured rat sciatic nerve and apolipoprotein E-containing lipoproteins from plasma were used to determine whether sprouting neurites, specifically their growth cones, possessed lipoprotein receptors. Pheochromocytoma (PC12) cells, which can be stimulated to produce neurites in vitro, were used as a model system. Apolipoprotein E-containing lipid particles and lipoproteins, which had been labeled with fluorescent dye, were internalized by the neurites and their growth cones; the unmetabolized dye appeared to be localized to the lysosomes. The rapid rate of accumulation in the growth cones precludes the possibility of orthograde transport of the fluorescent particles from the PC12 cell bodies. Thus, receptor-mediated lipoprotein uptake is performed by the apolipoprotein B,E(LDL) (low density lipoprotein) receptors, and in the regenerating peripheral nerve apolipoprotein E may deliver lipids to the neurites and their growth cones for membrane biosynthesis.

A specific 37,000-dalton protein that accumulates in regenerating but not in nonregenerating mammalian nerves.

A 37-kilodalton protein is synthesized at higher rates in the peripheral and central nervous system of newborn rats than in adult animals. As a specific response to denervation, the synthesis of the 37-kilodalton protein is increased in the mature peripheral and central nervous system; however, this protein accumulates only in the peripheral nervous system. The differences in accumulation of the protein correlate with the apparent differences in the ability of peripheral and central axons to regenerate. The synthesis of the 37-kilodalton protein is inhibited when proper innervation or reinnervation is established.

Molecular investigations on the high-affinity nerve growth factor receptor.

Nerve growth factor (NGF) receptors have been investigated by means of affinity labeling with 125I-NGF and chemical cross-linking. Two distinct NGF-receptor complexes are detected on PC12 cells; these correspond to 100 kd and 158 kd for the low-affinity (LNGFR) and the high-affinity (HNGFR) receptors, respectively. Interestingly, three different antibodies directed against distinct epitopes on the LNGFR immunoprecipitate the low-but not the high-affinity NGF-receptor complex. Although the identities of the signaling molecules in the HNGFR are unknown, antibodies to the src, ras, raf-1, and yes products fail to immunoprecipitate either receptor complex, suggesting that these molecules are not a part of, or tightly coupled to, either receptor type. Phosphotyrosine residues are found exclusively on the HNGFR complex, suggesting that tyrosine phosphorylation may be one of the initiating events in the NGF-induced signal transduction cascade.

Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to TrkA on PC12 cells.

The role of the low affinity neurotrophin receptor, p75LNTR, in NGF-mediated signal transduction has been examined. Our results show that treatment of PC12 cells with MC192, a monoclonal antibody directed against p75LNTR, results in reduced NGF binding to TrkA and attenuated TrkA activation. Use of mutant NGF that binds TrkA but not p75LNTR shows that the MC192 effect requires that NGF bind the p75LNTR receptor. To explore the possibility that MC192 disrupts some normal functional role of p75LNTR, BDNF was used to block binding of NGF to p75LNTR on PC12 cells. By preventing NGF binding to p75LNTR, NGF binding to TrkA and NGF-mediated signal transduction were reduced. We propose that p75LNTR normally acts to increase binding of NGF to TrkA, possibly by increasing the local NGF concentration in the microenvironment surrounding the cell surface TrkA receptor.

Expression of the NGF receptor gene in sensory neurons and their cutaneous targets prior to and during innervation.

We have studied the expression of NGF receptor (NGFR) mRNA in the mouse trigeminal system at closely staged intervals throughout development. The level of NGFR mRNA per neuron is at a constant low level before the earliest axons reach the target field and increases 5-fold to plateau shortly after the arrival of the last axons. NGFR mRNA expression in developing target skin is restricted to mesenchyme, precedes the arrival of the earliest axons, and increases throughout the phase of target field innervation. These findings suggest that NGFR expression on sensory neurons occurs at a low level prior to target field innervation and is up-regulated with this event, and they raise the possibility that NGFRs on mesenchymal cells restrict the distribution of NGF in the target field.

Structure and developmental expression of the nerve growth factor receptor in the chicken central nervous system.

Chicken nerve growth factor (NGF) receptor cDNAs have been isolated and sequenced in an effort to identify functionally important receptor domains and as an initial step in determining the functions of the NGF receptor in early embryogenesis. Comparisons of the primary amino acid sequences of the avian and mammalian NGF receptors have identified several discrete domains that differ in their degree of conservation. The highly conserved regions include an extracellular domain, likely to be involved in ligand binding, in which the positions of 24 cysteine residues and virtually all negatively charged residues are conserved; a transmembrane region, including flanking stretches of extracellular and cytoplasmic amino acids, which has properties suggesting it interacts with other proteins; and a cytoplasmic PEST sequence, which may regulate receptor turnover. Transient expression of NGF receptor mRNA has been seen in many regions of the developing CNS. Experiments suggest that both NGF and its receptor help regulate development of the retina.

Developmental and regional expression of beta-nerve growth factor receptor mRNA in the chick and rat.

Hybridization probes from the transmembrane region of the chick NGF receptor (NGF-R) that show high homology with the rat NGF-R were used to demonstrate an abundant 4.5 kb NGF-R mRNA in the chick embryo at E3.5. The level remained high until E12 but decreased to adult levels by E18. The highest levels at E8 were in spinal cord, bursa of Fabricius, gizzard, femoralis muscle, and skin. In situ hybridization to E7 embryos showed high expression of the NGF-R gene in spinal cord, particularly the lateral motor column, and in dorsal root, sympathetic, and nodose ganglia. NGF-R mRNA expression was observed throughout brain development and in all regions of the adult brain, with high levels in cerebellum and septum. Lymphoid tissues of chick and rat also expressed the receptor. The complex and widespread expression of NGF-R mRNA in areas not known to be NGF targets suggests broader functions for NGF.

The nerve growth factor family of receptors.

The neurotrophins, of which nerve growth factor (NGF) is the best known example, support the survival and differentiation of chick embryo sensory neurons at extremely low concentrations, 10(-12) M or less. These same neurons display two different classes of neurotrophin receptors with dissociation constants of 10(-11) M and 10(-9) M, respectively, implying that only low occupancy of the higher affinity receptor is required to mediate the biological actions of the neurotrophins. Two structurally unrelated receptors have now been characterized for NGF, and one of them, p75NGFR, serves as a receptor for all the known neurotrophins. This is the receptor with a dissociation constant of 10(-9) M. The second NGF receptor is a member of the trk family of tyrosine kinase receptors, p140trkA. Other members, p145trkB and p145trkC, are receptors for brain-derived neurtrophic factor (BDNF) and neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3), respectively, when assayed in fibroblasts. The specificity of neurotrophin binding to these receptors appears to be much higher in neurons than in the non-neuronal cells. The receptor p140trkA has many of the properties of the higher affinity class of NGF receptors, and is able to mediate survival and differentiation of the PC12 cell line, and cell growth and transformation in fibroblast cells. On the other hand, expression of p75NGFR in several types of cells displaying p140trkA induces a component of higher affinity NGF binding not seen in its absence. Since it is unlikely that p75NGFR and p140trkA interact at the level of the receptors, the crosstalk between receptors probably occurs through their signal transduction mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

The genetics of myelin.

Myelin formation and maintenance requires complex interactions between neurons and glia, and between the integral protein and lipid components of the myelin sheath. Many of the underlying mechanisms may be examined by studying the perturbations caused by spontaneous and targeted mutations in myelin protein genes. This review summarizes the progress in our understanding of these mutations with an emphasis on integrating the recent advances in the genetics of myelin into a more generalized view of myelin organization and function.