Hypermyelination and demyelinating peripheral neuropathy in Pmp22-deficient mice.

Peripheral myelin protein PMP22 has been suggested to have a role in peripheral nerve myelination and cell proliferation. Defects at the PMP22 locus are associated with peripheral neuropathies such as Charcot-Marie-Tooth disease type 1A. We now demonstrate that mice devoid of Pmp22 are retarded in the onset of myelination and develop abundant sausage-like hypermyelination structures (tomacula) at a young age followed by severe demyelination, axonal loss and functional impairment. Mice carrying one functional copy of Pmp22 are less affected but they also exhibit focal tomacula comparable to the morphological features in hereditary neuropathy with liability to pressure palsies (HNPP). We conclude that Pmp22 is required for the correct development of peripheral nerves, the maintenance of axons and the determination of myelin thickness and stability.

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.

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.

A transgenic rat model of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy in humans and has been associated with a partial duplication of chromosome 17 (CMT type 1A). We have generated a transgenic rat model of this disease and provide experimental evidence that CMT1A is caused by increased expression of the gene for peripheral myelin protein-22 (PMP22, gas-3). PMP22-transgenic rats develop gait abnormalities caused by a peripheral hypomyelination, Schwann cell hypertrophy (onion bulb formation), and muscle weakness. Reduced nerve conduction velocities closely resemble recordings in human patients with CMT1A. When bred to homozygosity, transgenic animals completely fail to elaborate myelin. We anticipate that the CMT rat model will facilitate the identification of a cellular disease mechanism and serve in the evaluation of potential treatment strategies.

Myelin: keeping nerves well wrapped up.

The recently determined structure of protein zero, P0, the most abundant peripheral nerve myelin protein, provides new insights into the molecular structure of myelin and potential disease mechanisms in hereditary peripheral neuropathies.

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

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.

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.

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system.

Without medical progress, dementing diseases such as Alzheimer's disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimer's disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimer's patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimer's disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimer's patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

The peripheral myelin protein 22 and epithelial membrane protein family.

The peripheral myelin protein 22 (PMP22) and the epithelial membrane proteins (EMP-1, -2, and -3) comprise a subfamily of small hydrophobic membrane proteins. The putative four-transmembrane domain structure as well as the genomic structure are highly conserved among family members. PMP22 and EMPs are expressed in many tissues, and functions in cell growth, differentiation, and apoptosis have been reported. EMP-1 is highly up-regulated during squamous differentiation and in certain tumors, and a role in tumorigenesis has been proposed. PMP22 is most highly expressed in peripheral nerves, where it is localized in the compact portion of myelin. It plays a crucial role in normal physiological and pathological processes in the peripheral nervous system. Progress in molecular genetics has revealed that genetic alterations in the PMP22 gene, including duplications, deletions, and point mutations, are responsible for several forms of hereditary peripheral neuropathies, including Charcot-Marie-Tooth disease type 1A (CMT1A), Dejerine-Sottas syndrome (DDS), and hereditary neuropathy with liability to pressure palsies (HNPP). The natural mouse mutants Trembler and Trembler-J contain a missense mutation in different hydrophobic domains of PMP22, resulting in demyelination and Schwann cell proliferation. Transgenic mice carrying many copies of the PMP22 gene and PMP22-null mice display a variety of defects in the initial steps of myelination and/or maintenance of myelination, whereas no pathological alterations are detected in other tissues normally expressing PMP22. Further characterization of the interactions of PMP22 and EMPs with other proteins as well as their regulation will provide additional insight into their normal physiological function and their roles in disease and possibly will result in the development of therapeutic tools.

Uncoupling of myelin assembly and schwann cell differentiation by transgenic overexpression of peripheral myelin protein 22.

We have generated previously transgenic rats that overexpress peripheral myelin protein 22 (PMP22) in Schwann cells. In the nerves of these animals, Schwann cells have segregated with axons to the normal 1:1 ratio but remain arrested at the promyelinating stage, apparently unable to elaborate myelin sheaths. We have examined gene expression of these dysmyelinating Schwann cells using semiquantitative reverse transcription-PCR and immunofluorescence analysis. Unexpectedly, Schwann cell differentiation appears to proceed normally at the molecular level when monitored by the expression of mRNAs encoding major structural proteins of myelin. Furthermore, an aberrant coexpression of early and late Schwann cell markers was observed. PMP22 itself acquires complex glycosylation, suggesting that trafficking of the myelin protein through the endoplasmic reticulum is not significantly impaired. We suggest that PMP22, when overexpressed, accumulates in a late Golgi-cell membrane compartment and uncouples myelin assembly from the underlying program of Schwann cell differentiation.

Ultrastructural distribution of PMP22 in Charcot-Marie-Tooth disease type 1A.

Peripheral Myelin Protein-22 (PMP22) is a membrane glycoprotein which represents up to 5% of total protein in myelin of peripheral nerves. Mutations affecting the PMP22 gene have been linked to the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1A (CMT1A; duplications and point mutations), Dejerine-Sottas syndrome (DSS; point mutations), and hereditary neuropathy with liability to pressure palsies (HNPP; deletions). In this study, we determined the ultrastructural distribution of PMP22 and other myelin proteins in normal human peripheral nervous system (PNS) nerves and in CMT1 patients with or without the CMT1A duplication on chromosome 17. Our results demonstrate that PMP22, P0 protein, and myelin basic protein are present in compact myelin of all patients examined. PMP22 was also present in the plasma membrane of Schwann cells of unmyelinated fibers and onion bulbs. Although the precise biological role of PMP22 remains to be discovered, our results support the hypothesis that this protein serves multiple functions in Schwann cells.

Molecular basis of common hereditary motor and sensory neuropathies in humans and in mouse models.

The Hereditary Motor and Sensory Neuropathies (HMSNs) are well known to be clinically, morphologically, and genetically heterogeneous. Yet, recent advances in the cellular and molecular biology of the peripheral nervous system coupled with remarkable progress in human and mouse genetics have provided a framework that has profoundly changed our understanding of the pathogenesis of these diseases. It now appears that most of the HMSNs are related to mutations affecting genes encoding Schwann cell proteins, specifically the Peripheral Myelin Protein PMP22, Myelin Protein Zero, and one of the gap junction proteins, connexin-32. Accordingly, these findings are discussed in the context of the clinical and pathologic features of the human HMSNs, but are interpreted in the context of basic research findings on the cellular and molecular biology of the peripheral nervous system derived from in vivo and in vitro studies in spontaneously-occurring and genetically engineered animal models for the HMSNs.

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.

Myelin and lymphocyte protein (MAL/MVP17/VIP17) and plasmolipin are members of an extended gene family.

An increasing number of four-transmembrane proteins has been found to be associated with CNS and PNS myelin. Some of these proteins play crucial roles in the development and maintenance of the nervous system. In the CNS, proteolipid protein (PLP) is mutated in the myelin disorder Pelizaeus-Merzbacher disease and in spastic paraplegia, while in the PNS, peripheral myelin protein 22 (PMP22) and connexin32 (C x 32) are culprit genes in the most frequent forms of hereditary peripheral neuropathies. Myelin and lymphocyte protein (MAL; also called MVP17 or VIP17) and plasmolipin are additional tetraspan proteins that are highly expressed by myelinating glial cells. However, little is known about the role of these proteins in the nervous system. As a prerequisite for functional genetic approaches in the mouse, we have isolated and characterized a mouse MAL cDNA and the corresponding structural MAL gene. Computer-aided analysis and database searches revealed that MAL belongs to a larger gene family which also includes plasmolipin, BENE and the expressed sequence tag (EST) H09290. While the overall amino acid sequence identities between mouse MAL and the related proteins are relatively low (29-37%), the conserved motif -[Q/Y-G-W-V-M-F/Y-V]- which is found at the junction of the first extracellular loop and the second membrane-associated domain serves as a fingerprint for the MAL protein family. Expression analysis of the members of the MAL gene family indicates widespread expression in various tissues, suggesting a common role of these proteins in cell biology.

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.

Mutation of tryptophan-21 in mouse nerve growth factor (NGF) affects binding to the fast NGF receptor but not induction of neurites on PC12 cells.

By using in vitro DNA mutagenesis, we replaced the tryptophan residue at position 21 in mouse nerve growth factor (NGF) with either phenylalanine, leucine or serine. Yield, biological activity, immunological reactivity and receptor binding of the recombinant proteins were determined. All three mutants were produced at considerably lower yields than wild-type NGF, with the serine mutant being undetectable. The results of competitive binding assays show that tryptophan-21 is involved in recognition of the fast NGF receptor of PC12 cells. However, specific biological activity of NGF is not altered by the replacement of tryptophan-21. Our results therefore suggest that biological activity of NGF is not directly coupled to binding to the fast NGF receptor.

The in vivo expression of type B CD23 mRNA in B-chronic lymphocytic leukemic cells is associated with an abnormally low CD23 upregulation by IL-4: comparison with their normal cellular counterparts.

We recently reported that the sera of chronic lymphocytic leukemia (CLL) patients contained 3-500 times more soluble CD23 (or IgE-BF) than the sera of patients with other lymphoproliferative diseases or normal individuals and that their B cells (B-CLLs) overexpressed CD23 Ag. In the present report, we extended these studies and showed that CD5+ B cells from all CLL patients (n = 15) co-express CD23 Ag. We next identified two additional major differences between B-CLLs and normal adult B cells. First, in contrast to normal adult B cells which exclusively express type A CD23 mRNA, freshly isolated B-CLLs expressed both type B and type A CD23 mRNA. Second, although IL-4 is a potent inducer of type B CD23 mRNA on normal B cells, an optimal concentration of IL-4 infranormally upregulated CD23 on highly purified B-CLLs both at the protein and at the molecular levels. However, co-stimulation of CLL PBMC with phytohemagglutinin (PHA) and IL-4 strongly upregulated CD23 on B-CLLs, reconstituting the high level of CD23 expression observed in vivo. We next attempted to relate B-CLLs to the CD5+ B cell subpopulations present in peripheral blood mononuclear cells (PBMC, n = 3), cord blood mononuclear cells (CBMC, n = 6) and tonsillar lymphocytes (TONS, n = 3) by analysing their co-expression of CD20, CD5 and CD23 Ag and their phenotypic regulation by IL-4. Our results indicated that B-CLLs presented some features in common with the CD23+ umbilical cord blood B cells in as much as, like in B-CLLs; (i) all CD23+ cord blood cells co-expressed CD5 Ag, (ii) freshly isolated CBMC expressed both type A and type B CD23 mRNA, and finally (iii) these cells weakly re-expressed CD23 Ag upon IL-4 stimulation as compared to adult PBMC.

In vivo performance of a new biodegradable polyester urethane system used as a nerve guidance channel.

Biodegradable nerve guidance channels (NGCs) represent a promising alternative to current clinical nerve repair procedures. To be suitable as a NGC material, the polymer system should possess elastomeric properties and degrade at a defined rate without interfering with the regenerating environment. Polymers made of non-crystallizable blocks of poly[glycolide-co-(epsilon-caprolactone)]-diol and crystallizable blocks of poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid]-diol (PHB) can be modulated so as to respond to those criteria. Tubular structures were fabricated from three different types of materials containing either 41, 17 or 8 wt% PHB. Nerve regeneration through a 10 mm long NGC using a transected sciatic nerve model with an 8 mm gap was studied in rats at 4, 12 and 24 weeks. Out of 26 implanted NGCs, 23 contained regenerated tissue cables centrally located within the channel lumen and composed of numerous myelinated axons and Schwann cells. No significant difference in the degree of regeneration was observed between the various channel types. The inflammatory reaction associated with the polymer degradation had not interfered with the nerve regeneration process. Macrophages and giant cells surrounded polymer material remnants. A weight loss of 33, 74 and 88% for polymers containing 41, 17 and 8 wt% PHB was observed after 24 weeks by nuclear magnetic resonance (NMR) anaylsis, respectively. In all cases, the polymer fragments had a porous appearance with multiple surface cracks as evidenced by scanning electron microscopical analysis. Guidance channels made of 8 wt% PHB containing polymer displayed the highest degree of degradation at 24 weeks with only small polymer fragments remaining. The present study suggests that this new biodegradable elastomeric polymeric material holds promises for its utilization as nerve guidance channels.