Peripheral Nerve Myelin Proteins Research Articles

Myelin protein zero mutation-related hereditary neuropathies: Neuropathological insight from a new nerve biopsy cohort.

Myelin protein zero (MPZ/P0) is a major structural protein of peripheral nerve myelin. Disease-associated variants in the MPZ gene cause a wide phenotypic spectrum of inherited peripheral neuropathies. Previous nerve biopsy studies showed evidence for subtype-specific morphological features. Here, we aimed at enhancing the understanding of these subtype-specific features and pathophysiological aspects of MPZ neuropathies. We examined archival material from two Central European centers and systematically determined genetic, clinical, and neuropathological features of 21 patients with MPZ mutations compared to 16 controls. Cases were grouped based on nerve conduction data into congenital hypomyelinating neuropathy (CHN; n = 2), demyelinating Charcot-Marie-Tooth (CMT type 1; n = 11), intermediate (CMTi; n = 3), and axonal CMT (type 2; n = 5). Six cases had combined muscle and nerve biopsies and one underwent autopsy. We detected four MPZ gene variants not previously described in patients with neuropathy. Light and electron microscopy of nerve biopsies confirmed fewer myelinated fibers, more onion bulbs and reduced regeneration in demyelinating CMT1 compared to CMT2/CMTi. In addition, we observed significantly more denervated Schwann cells, more collagen pockets, fewer unmyelinated axons per Schwann cell unit and a higher density of Schwann cell nuclei in CMT1 compared to CMT2/CMTi. CHN was characterized by basal lamina onion bulb formation, a further increase in Schwann cell density and hypomyelination. Most late onset axonal neuropathy patients showed microangiopathy. In the autopsy case, we observed prominent neuromatous hyperinnervation of the spinal meninges. In four of the six muscle biopsies, we found marked structural mitochondrial abnormalities. These results show that MPZ alterations not only affect myelinated nerve fibers, leading to either primarily demyelinating or axonal changes, but also affect non-myelinated nerve fibers. The autopsy case offers insight into spinal nerve root pathology in MPZ neuropathy. Finally, our data suggest a peculiar association of MPZ mutations with mitochondrial alterations in muscle.

Hereditary motor sensory neuropathy disease caused by de novo gene mutation of peripheral nerve myelin protein 22

Hereditary motor sensory neuropathy disease caused by de novo gene mutation of peripheral nerve myelin protein 22

Proteolipid protein-deficient myelin promotes axonal mitochondrial dysfunction via altered metabolic coupling.

Hereditary spastic paraplegia (HSP) is a neurological syndrome characterized by degeneration of central nervous system (CNS) axons. Mutated HSP proteins include myelin proteolipid protein (PLP) and axon-enriched proteins involved in mitochondrial function, smooth endoplasmic reticulum (SER) structure, and microtubule (MT) stability/function. We characterized axonal mitochondria, SER, and MTs in rodent optic nerves where PLP is replaced by the peripheral nerve myelin protein, P0 (P0-CNS mice). Mitochondrial pathology and degeneration were prominent in juxtaparanodal axoplasm at 1 mo of age. In wild-type (WT) optic nerve axons, 25% of mitochondria-SER associations occurred on extensions of the mitochondrial outer membrane. Mitochondria-SER associations were reduced by 86% in 1-mo-old P0-CNS juxtaparanodal axoplasm. 1-mo-old P0-CNS optic nerves were more sensitive to oxygen-glucose deprivation and contained less adenosine triphosphate (ATP) than WT nerves. MT pathology and paranodal axonal ovoids were prominent at 6 mo. These data support juxtaparanodal mitochondrial degeneration, reduced mitochondria-SER associations, and reduced ATP production as causes of axonal ovoid formation and axonal degeneration.

Nerve regenerative effects of GABA-B ligands in a model of neuropathic pain.

Neuropathic pain arises as a direct consequence of a lesion or disease affecting the peripheral somatosensory system. It may be associated with allodynia and increased pain sensitivity. Few studies correlated neuropathic pain with nerve morphology and myelin proteins expression. Our aim was to test if neuropathic pain is related to nerve degeneration, speculating whether the modulation of peripheral GABA-B receptors may promote nerve regeneration and decrease neuropathic pain. We used the partial sciatic ligation- (PSL-) induced neuropathic model. The biochemical, morphological, and behavioural outcomes of sciatic nerve were analysed following GABA-B ligands treatments. Simultaneous 7-days coadministration of baclofen (10 mg/kg) and CGP56433 (3 mg/kg) alters tactile hypersensitivity. Concomitantly, specific changes of peripheral nerve morphology, nerve structure, and myelin proteins (P0 and PMP22) expression were observed. Nerve macrophage recruitment decreased and step coordination was improved. The PSL-induced changes in nociception correlate with altered nerve morphology and myelin protein expression. Peripheral synergic effects, via GABA-B receptor activation, promote nerve regeneration and likely ameliorate neuropathic pain.

The dorsal root ganglion in Friedreich’s ataxia

Atrophy of dorsal root ganglia (DRG) and thinning of dorsal roots (DR) are hallmarks of Friedreich's ataxia (FRDA). Many previous authors also emphasized the selective vulnerability of larger neurons in DRG and thicker myelinated DR axons. This report is based on a systematic reexamination of DRG, DR and ventral roots (VR) in 19 genetically confirmed cases of FRDA by immunocytochemistry and single- and double-label immunofluorescence with antibodies to specific proteins of myelin, neurons and axons; S-100alpha as a marker of satellite and Schwann cells; laminin; and the iron-responsive proteins ferritin, mitochondrial ferritin, and ferroportin. Confocal images of axons and myelin allowed the quantitative analysis of fiber density and size, and the extent of DR and VR myelination. A novel technology, high-definition X-ray fluorescence (HDXRF) of polyethylene glycol-embedded fixed tissue, was used to "map" iron in DRG. Unfixed frozen tissue of DRG in three cases was available for the chemical assay of total iron. Proliferation of S-100alpha-positive satellite cells accompanied neuronal destruction in DRG of all FRDA cases. Double-label visualization of peripheral nerve myelin protein 22 and phosphorylated neurofilament protein confirmed the known loss of large myelinated DR fibers, but quantitative fiber counts per unit area did not change. The ratio of myelinated to neurofilament-positive fibers in DR rose significantly from 0.55 to 0.66. In VR of FRDA patients, fiber counts and degree of myelination did not differ from normal. Pooled histograms of axonal perimeters disclosed a shift to thinner fibers in DR, but also a modest excess of smaller axons in VR. Schwann cell cytoplasm in DR of FRDA was depleted while laminin reaction product remained prominent. Numerous small axons clustered around fewer Schwann cells. Ferritin in normal DRG localized to satellite cells, and proliferation of these cells in FRDA caused wide rims of reaction product about degenerating nerve cells. Mitochondrial ferritin was not detectable. Ferroportin was present in the cytoplasm of normal satellite cells and neurons, and in large axons of DR and VR. In FRDA, some DRG neurons lost their cytoplasmic ferroportin immunoreactivity, whereas the cytoplasm of satellite cells remained ferroportin positive. Ferroportin in DR axons disappeared in parallel with atrophy of large fibers. HDXRF of DRG detected regional and diffuse increases in iron fluorescence that matched ferritin expression in satellite cells. The observations support the conclusions that satellite cells and DRG neurons are affected by iron dysmetabolism; and that regeneration and inappropriate myelination of small axons in DR are characteristic of the disease.

Chronic sensorimotor polyradiculopathy with antibodies to P2: An electrophysiological and immunoproteomic analysis

In this study we report a patient with chronic progressive sensory ataxia, proximal weakness, immunoglobulin M (IgM) monoclonal gammopathy, and elevated protein levels in the cerebrospinal fluid, who showed a good response to prednisone. Electrophysiological study disclosed abnormalities predominantly of late responses (F waves and H reflexes), with no evidence of demyelination in the peripheral nerves, suggesting motor and preganglionic sensory nerve roots as the site of the lesion. An immune-mediated pathogenesis was considered and, to identify possible target antigens, we performed bidimensional electrophoresis and a Western blot study. Based on the suspected lesion site, we used human anterior and posterior root extracts. We identified IgM reactivity against peripheral nerve myelin protein P2. Enzyme-linked immunosorbent assay confirmed IgM reactivity toward one synthetic peptide from P2. To our knowledge, reactivity against P2 has not been reported previously in a paraproteinemic neuropathy. Furthermore, we demonstrated that bidimensional electrophoresis and Western blot of the tissue involved, as determined by clinical and electrophysiological studies, may be useful to establish clinical-immunological correlations in paraproteinemic neuropathies.

Peripheral myelin of Xenopus laevis: Role of electrostatic and hydrophobic interactions in membrane compaction

P0 glycoprotein is the major structural protein of peripheral nerve myelin where it is thought to modulate inter-membrane adhesion at both the extracellular apposition, which is labile upon changes in pH and ionic strength, and the cytoplasmic apposition, which is resistant to such changes. Most studies on P0 have focused on structure-function correlates in higher vertebrates. Here, we focused on its role in the structure and interactions of frog (Xenopus laevis) myelin, where it exists primarily in a dimeric form. As part of our study, we deduced the full sequence of X. laevis P0 (xP0) from its cDNA. The xP0 sequence was found to be similar to P0 sequences of higher vertebrates, suggesting that a common mechanism of PNS myelin compaction via P0 interaction might have emerged through evolution. As previously reported for mouse PNS myelin, a similar change of extracellular apposition in frog PNS myelin as a function of pH and ionic strength was observed, which can be explained by a conformational change of P0 due to protonation-deprotonation of His52 at P0's putative adhesive interface. On the other hand, the cytoplasmic apposition in frog PNS myelin, like that in the mouse, remained unchanged at different pH and ionic strength. The contribution of hydrophobic interactions to stabilizing the cytoplasmic apposition was tested by incubating sciatic nerves with detergents. Dramatic expansion at the cytoplasmic apposition was observed for both frog and mouse, indicating a common hydrophobic nature at this apposition. Urea also expanded the cytoplasmic apposition of frog myelin likely owing to denaturation of P0. Removal of the fatty acids that attached to the single Cys residue in the cytoplasmic domain of P0 did not change PNS myelin structure of either frog or mouse, suggesting that the P0-attached fatty acyl chain does not play a significant role in PNS myelin compaction and stability. These results help clarify the present understanding of P0's adhesion role and the role of its acylation in compact PNS myelin.

Transmembrane Domain of Myelin Protein Zero Can Form Dimers: Possible Implications for Myelin Construction

Myelin protein zero (MPZ) is the major integral membrane protein of peripheral nerve myelin in higher vertebrates, mediating homoadhesion of the multiple, spiraling wraps of the myelin sheath. Previous studies have shown that full-length MPZ can form dimers and tetramers, and biochemical studies on the extracellular domain (ECD) indicate that it can form a tetramer, albeit very weakly. On the basis of cross-linking studies and equilibrium sedimentation of a transmembrane (TM) domain peptide (MPZ-TM), we find that the MPZ-TM can form homodimers. We further characterized the dimer by measuring the effects of alanine and leucine substitutions on the ability of the TM to dimerize in Escherichia coli membranes. Our results indicate that the primary packing interface for the MPZ TM homodimer is a glycine zipper (GxxxGxxxG) motif. We also find that the G134R mutation, which lies within the glycine zipper packing interface and causes Charcot-Marie-Tooth disease type 1B, severely inhibits dimerization, suggesting that dimerization of the TM domain may be important for the normal functioning of MPZ. By combining our new results with prior work, we suggest a new model for an MPZ lattice that may form during the construction of myelin.

Study of antibodies to PMP22, IL-6 and TNF-α concentrations in serum in a CMTX1 family

To further understand X-linked dominant Charcot-Marie-Tooth disease (CMTX1), we followed a family of 22 members in China, including 8 patients, 2 asymptomatic carriers and 12 normal family members. Twenty-two family members as well as 60 normal controls unrelated to this family were screened for point mutation by denaturing high performance liquid chromatography (DHPLC). All patients and asymptomatic carriers from this family, but none of the normal population controls, showed a T-C transition at position 266 in codon 89 of exon 2 of connexin 32, resulting in a leucine to proline (L89P) exchange. To study whether the immune system is involved in the pathogenesis of CMTX1 patients and asymptomatic carriers, we measured serum concentrations of antibodies to peripheral nerve myelin protein 22 (PMP22), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) by ELISA. Serological results were also compared with those from GBS patients (n=11) and with normal subjects (n=20). Our analysis showed anti-PMP22 sera reactivity in 50.0% of CMTX1 patients, 63.6% of GBS patients and 10% of normal controls. Our results also indicated that anti-PMP22 antibodies in the CMTX1 family varied with sex. Anti-PMP22 antibodies were found in all male patients but not in all females, which may be one of the reasons that male patients usually have more severe clinical symptoms than that of female patients. There was no statistical difference in serum concentrations of IL-6 and TNF-alpha between CMTX1 patients and normal subjects. In conclusion, we identified a L89P mutation for the first time in a CMTX1 family in China and an associated response to PMP22 in males.

Antibodies to peripheral nerve myelin proteins in chronic inflammatory demyelinating polyradiculoneuropathy and Guillain–Barré syndrome

Antibodies to peripheral nerve myelin proteins in chronic inflammatory demyelinating polyradiculoneuropathy and Guillain–Barré syndrome

Antibodies to peripheral nerve myelin proteins in chronic inflammatory demyelinating polyradiculoneuropathy

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an acquired disorder of the peripheral nervous system with a probable auto-immune pathogenesis. The nature of the responsible autoantigens is unclear in most patients. We used the Western immunoblot technique to seek antibodies to peripheral nerve protein antigens. Sera from eight of 32 (25%) CIDP patients, 12 of 37 (32%) Guillain-Barré syndrome (GBS) patients, zero of 30 (0%) chronic idiopathic axonal polyneuropathy patients and two of 39 (5%) healthy control subjects contained anti-peripheral nerve protein antibodies. The frequency of such antibodies was significantly greater in both CIDP (p = 0.04) and GBS (p = 0.003) patients than in normal control subjects. For CIDP patients, there were non-significant trends for antibodies to be more common in females and in those who responded to treatment with either intravenous immunoglobulin or plasma exchange. The commonest antibodies were directed against a band at 28 kDa, resembling that labelled by a monoclonal antibody against myelin protein zero (P0). Six CIDP and seven GBS patients' sera reacted with this band. These results support the view that antibodies to myelin proteins, and especially P0, are present in the serum of some patients with CIDP and GBS.

Insertion of mutant proteolipid protein results in missorting of myelin proteins.

Two brothers with a leukodystrophy, progressive spastic diplegia, and peripheral neuropathy were found to have proteinaceous aggregates in the peripheral nerve myelin sheath. The patients' mother had only subclinical peripheral neuropathy, but the maternal grandmother had adult-onset leukodystrophy. Sequencing of the proteolipid protein (PLP) gene showed a point mutation IVS4 + 1 G-->A within the donor splice site of intron 4. We identified one transcript with a deletion of exon 4 (Deltaex4, 169bp) encoding for PLP and DM20 proteins and lacking two transmembrane domains, and a second transcript with exon 4 + 10bp encoding three transmembrane domains. Immunohistochemistry showed abnormal aggregation in the myelin sheath of MBP and P0. Myelin-associated glycoprotein was present in the Schmidt-Lanterman clefts but significantly reduced in the periaxonal region. Using immunogold electron microscopy, we demonstrated the presence of mutated PLP/DM20 and the absence of the intact protein in the patient peripheral myelin sheath. We conclude that insertion of mutant PLP/DM20 with resulting aberrant distribution of other myelin proteins in peripheral nerve may constitute an important mechanism of dysmyelination in disorders associated with PLP mutations.

Th1-like responses to peptides from peripheral nerve myelin proteins in patients with polyneuropathy associated with monoclonal gammopathy.

Polyneuropathy associated with monoclonal gammopathy is usually regarded as an immune-mediated disorder with autoantibody activity against myelin glycoproteins. The pathogenic mechanisms are, however, not fully understood. Several reports have indicated an additional involvement of T cells. We investigated the occurrence of myelin-specific T cells as well as their functional characteristics. Cytokine responses generated after stimulation of peripheral blood mononuclear cells with selected peptides of myelin proteins P0 and P2 were investigated with an ELISPOT method. Three P0 peptides caused significantly elevated levels of interferon-gamma (IFN-gamma)-secreting cells in patients with polyneuropathy associated with monoclonal gammopathy (n = 8) compared with controls (n = 8), whereas none of the peptides caused elevated levels of IL-4-secreting cells. Patients with non-immunological types of neuropathy (n = 4) did not reveal any cytokine responses to myelin peptides. Our results indicate that a Th1-like response against myelin proteins occurs in polyneuropathy associated with monoclonal gammopathy.

Immunological Study Of Hereditary Motor And Sensory Neuropathy Type 1 A (HMSN1 A)

Objectives: Fifty three patients were studied to investigate whether autoimmune or inflammatory mechanisms could explain the phenotypic heterogeneity of patients with hereditary motor and sensory neuropathy type 1a (HMSN1a). Methods: Serum samples were examined for antibodies to peripheral nerve myelin protein 22 (PMP22), ganglioside GM1 and cauda equina homogenate, and interleukin‐6 (IL‐6) and soluble tumour necrosis factor receptor 1 (sTNF R1) concentrations. Serological results were compared with those from patients with other neuropathies (ONPs, n = 30) and with normal subjects (n = 51). Results: In the group as a whole, no relation emerged between clinical severity and any immune parameters. Immunohistochemical examination of four sural nerve biopsies did not show significant inflammatory infiltration. In a subset of 12 patients who experienced stepwise progression of disease, there was a trend towards a higher proportion having anti‐PMP22 antibodies (33% v 15% of those with gradual disease progression, 3% ONPs, and no normal controls) and complement fixing antibodies to human cauda equina (25% v 5% with gradual progression, 8.6% ONPs, 3.9% normal controls, p = 0.07). Conclusions: Patients with HMSN1a and a stepwise disease progression may have an inflammatory, autoimmune component superimposed on the genetic condition.

Immunological study of hereditary motor and sensory neuropathy type 1a (HMSN1a)

Objectives: Fifty three patients were studied to investigate whether autoimmune or inflammatory mechanisms could explain the phenotypic heterogeneity of patients with hereditary motor and sensory neuropathy type 1a (HMSN1a). Methods:...

Morphological And Electrophysiological Signs Of Dysmyelination In Transgenic Mice Expressing CMT1B (MPZ DELSer34) or DSS (MPZ Ser34Cys) Mutations

Charcot‐Marie‐Tooth 1B neuropathy (CMT1B), Déjèrine‐Sottas syndrome (DSS) and Congenital Hypomyelination are each associated with mutations in MPZ, encoding P0 glycoprotein, the major structural protein of peripheral nerve myelin. To explore whether new abnormal functions of mutant MPZ can explain this phenotypic diversity, we expressed either of two MPZ mutations: DELSer34 (causing CMT1B) or Ser34Cys (causing DSS) in addition to the two normal endogeneous copies of Mpz in transgenic mice. We have shown previously that overexpression of wild type Mpz causes dose‐dependent dysmyelination. However, multiple lines of mice containing the MPZ mutants showed not only hypomyelination, but also abnormalities in myelin sheaths and onion bulbs that were never observed in Mpz overexpressor mice. To create a mouse model of CMT1B with no Mpz overexpression, one line of DELSer34 that expresses mutant Mpz at levels similar to one wildtype Mpz allele was crossed with heterozygous Mpz knock‐out mice, to obtain the genotype Mpzwt/−/MpzDELSer34. These mice manifest progressive peripheral neuropathy with hypomyelination, and onion bulbs that appear around 6 months of age. As a first step towards longitudinal studies of phenotype, we performed detailed neurophysiological analyses at 12 months of age. We found signs of demyelination, with statistically significant increases of both motor latency after proximal stimulation and F‐wave latencies, and decreases in both motor and mixed afferent nerve conduction velocities of sciatic nerve. The motor response was polyphasic and there was a trend towards reduced CMAP amplitudes. Thus, the clinical onset and progression, pathological features and neurophysiological findings provide a reasonable model of CMT1B. Longitudinal studies to correlate the onset and progression of morphological and electrophysiological abnormalities in these mice are ongoing.

Antigen-specific immunosuppression: nasal tolerance to P0 protein peptides for the prevention and treatment of experimental autoimmune neuritis in Lewis rats

Experimental autoimmune neuritis (EAN) is an autoimmune inflammatory demyelinating disease of the peripheral nervous system (PNS), and represents an animal model of the human Guillain–Barré syndrome (GBS). In this study, we report that nasal administration of the neuritogenic peptide 180–199 and of the cryptic peptide 56–71 of the rat neuritogenic P0 protein of peripheral nerve myelin prevents EAN and attenuates ongoing EAN. Both peptides effectively decreased the severity and shortened clinical EAN. Both a prophylactic and a therapeutic approach proved to be beneficial. These effects were associated with T and B cells hyporesponsiveness to the peptide antigens, reflected by downregulated Th1 cell responses (interferon-γ secretion) and macrophage function, whereas Th2 cell responses (IL-4 secretion) and transforming growth factor-β mRNA expression were upregulated.

The glycoprotein P0 in peripheral gliogenesis.

Recent data show that during development protein zero, P0, the most abundant peripheral nerve myelin protein, is detecable long before myelination. In particular, the expression of P0 in a fraction of migrating neural crest reveals progenitor cell heterogeneity in the developing PNS. Here we review the regulation and potential function of P0 during peripheral gliogenesis.

T cell receptor Vβ gene usage in Guillain-Barré syndrome

We set out to determine whether the T cell receptor (TCR) Vβ gene usage in acute inflammatory demyelinating polyradiculoneuropathy (AIDP) is restricted. We separated activated from non-activated peripheral blood T cells with anti-IL2 receptor (anti-CD25) antibody-labelled magnetic beads from four AIDP patients and four normal control (NC) subjects. The TCR Vβ gene usage of circulating activated and non-activated T cells was heterogeneous in all the patients and controls, but the activated T cells of all four of the AIDP patients showed a more limited usage of Vβ genes and enhanced Vβ 15 usage, as compared to the non-activated T cells. This was not seen in the healthy controls. The activated and non-activated T cells from a patient with acute motor and sensory axonal neuropathy (AMSAN) showed a similar Vβ gene usage to that of the controls. From a further patient with AIDP, we studied the Vβ gene usage of short-term T cell lines reactive to the peripheral nerve myelin proteins P2, P0 and the P0 peptide amino acid sequence 194–208. The Vβ gene usage of the lines was heterogeneous, with enhanced usage of Vβ 15 in the cell line responsive to the P0 peptide. We conclude that T cells activated during the immune response associated with AIDP preferentially used Vβ 15, which may indicate a restricted response to a common antigen, or a role for an as yet undefined superantigen in the pathogenesis of AIDP. © 1997 Elsevier Science B.V. All rights reserved.

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.