P0 Glycoprotein Research Articles

Protein kinase C epsilon activation regulates proliferation, migration, and epithelial to mesenchymal-like transition in rat Schwann cells.

Protein kinase type C-ε (PKCε) plays an important role in the sensitization of primary afferent nociceptors, promoting mechanical hyperalgesia. In accordance, we showed that PKCε is present in sensory neurons of the peripheral nervous system (PNS), participating in the control of pain onset and chronification. Recently, it was found that PKCε is also implicated in the control of cell proliferation, promoting mitogenesis and metastatic invasion in some types of cancer. However, its role in the main glial cell of the PNS, the Schwann cells (SCs), was still not investigated. Rat primary SCs culture were treated with different pharmacologic approaches, including the PKCε agonist dicyclopropyl-linoleic acid (DCP-LA) 500 nM, the human recombinant brain derived neurotrophic factor (BDNF) 1 nM and the TrkB receptor antagonist cyclotraxin B 10 nM. The proliferation (by cell count), the migration (by scratch test and Boyden assay) as well as some markers of SCs differentiation and epithelial-mesenchymal transition (EMT) process (by qRT-PCR and western blot) were analyzed. Overall, we found that PKCε is constitutively expressed in SCs, where it is likely involved in the switch from the proliferative toward the differentiated state. Indeed, we demonstrated that PKCε activation regulates SCs proliferation, increases their migration, and the expression of some markers (e.g., glycoprotein P0 and the transcription factor Krox20) of SCs differentiation. Through an autocrine mechanism, BDNF activates TrkB receptor, and controls SCs proliferation via PKCε. Importantly, PKCε activation likely promoted a partial EMT process in SCs. PKCε mediates relevant actions in the neuronal and glial compartment of the PNS. In particular, we posit a novel function for PKCε in the transformation of SCs, assuming a role in the mechanisms controlling SCs' fate and plasticity.

Serum Myelin Oligodendrocyte Glycoprotein and Myelin Protein Zero as Diagnostic Biomarkers in Diabetic Neuropathy

Background: Diabetic neuropathy can affect any peripheral nerve, including sensory neurons, motor neurons, and the autonomic nervous system. Therefore, diabetic neuropathy has the potential to affect essentially any organ and can affect parts of the nervous system like the optic nerve, spinal cord, and brain. In addition, chronic hyperglycemia affects Schwann cells, and more severe patterns of diabetic neuropathy in humans involve demyelization. Schwann cell destruction might cause a number of changes in the axon. study aims to evaluate serum myelin protein level as a predicting marker in the diagnosis of diabetic neuropathy and to prevent early neuropathy complications of type 2 diabetes. Subjects and methods: To achieve the purpose of the objective, this study involved 120 individuals divided into three groups. The first group included 40 healthy individuals; the second group included 40 type 2 diabetic patients with a diabetes duration of more than 5 years; and the last group included 40 type 2 diabetic patients with a diabetes duration of less than or equal to 5 years. The enzyme-linked immunesorbent assay (ELISA) system is used to detect serum MOG and MPZ. Results: both groups of type 2 diabetes patients had significant (p≤ 0.05) increases in serum myelin protein zero P0 (MPZ) and myelin oligodendrocyte glycoprotein (MOG). Conclusion: According to the results, myelin protein can be used to diagnose patients with diabetic neuropathy at an early stage. But it did not rise to the level of a biomarker due to a lack of sensitivity

Experimental Autoimmune Encephalomyelitis Animal Models Induced by Different Myelin Antigens Exhibit Differential Pharmacologic Responses to Anti-Inflammatory Drugs

Background and objective Experimental autoimmune encephalomyelitis (EAE) is the most commonly used model for studying autoimmune-mediated myelin degradation in multiple sclerosis (MS). Here, we evaluated the pharmacologic responses of several anti-inflammatory drugs with varying mechanisms of actions (MOAs) using EAE models induced by different MOG immunogens to reveal differential pharmacologic characteristics of the disease models and provide a general guidance in animal model selection for MS research. Methods The pharmacologic responses of anti-inflammatory drugs with different mechanisms of actions (MOAs) were evaluated using EAE models induced by either myelin oligodendrocyte glycoprotein p35-55 （MOG35-55）or p1-128 (MOG1-128). EAE animal models were developed in mice with C57BL/6 background. The animals were treated with different anti-MS medications, including 3 B cell-mediated agents and 2 T cell-mediated agents, respectively. Clinical symptoms were monitored and scored, and pharmacodynamic markers including cytokine secretion, inflammatory cell infiltration, and demyelination in spinal cord were analyzed. Results In MOG35-55 peptide-induced EAE model, T cell modulating agents Secukinumab and Fingolimod significantly alleviated clinical symptoms, while B cell-depleting agents, BTK inhibitors PRN2246 and Telitacicept, displayed minimal therapeutic effects or even exacerbated disease progression. In contrast, both T cell-modulating agents and B cell-depleting agents ameliorated disease severity in MOG1-128-induced EAE model. T cell and B cell infiltration in spinal cord increased with disease progression in MOG1-128-induced EAE model. Conclusions Our results demonstrated that induction of EAE by different myelin antigens resulted in differential pharmacologic responses to drugs with specific MOAs. The MOG35-55 peptide-induced EAE model only responded to T cell-modulating drugs, whereas the MOG1-128 protein-induced EAE model exhibited therapeutic sensitivity to both T cell- and B cell-modulating agents. These data suggest the MOG35-55 peptide-induced EAE model is suitable for assessing T cell-modulating agents while MOG1-128 protein-induced model can be employed to evaluate both T cell- and B cell-modulating agents.

Perk Ablation Ameliorates Myelination in S63del-Charcot-Marie-Tooth 1B Neuropathy.

In peripheral nerves, P0 glycoprotein accounts for more than 20% of myelin protein content. P0 is synthesized by Schwann cells, processed in the endoplasmic reticulum (ER) and enters the secretory pathway. However, the mutant P0 with S63 deleted (P0S63del) accumulates in the ER lumen and induces a demyelinating neuropathy in Charcot–Marie–Tooth disease type 1B (CMT1B)–S63del mice. Accumulation of P0S63del in the ER triggers a persistent unfolded protein response. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER stress sensor that phosphorylates eukaryotic initiation factor 2 alpha (eIF2alpha) in order to attenuate protein synthesis. We have shown that increasing phosphophorylated-eIF2alpha (P-eIF2alpha) is a potent therapeutic strategy, improving myelination and motor function in S63del mice. Here, we explore the converse experiment: Perk haploinsufficiency reduces P-eIF2alpha in S63del nerves as expected, but surprisingly, ameliorates, rather than worsens S63del neuropathy. Motor performance and myelin abnormalities improved in S63del//Perk+/− compared with S63del mice. These data suggest that mechanisms other than protein translation might be involved in CMT1B/S63del neuropathy. In addition, Perk deficiency in other cells may contribute to demyelination in a non–Schwann-cell autonomous manner.

Myelin organization in the nodal, paranodal, and juxtaparanodal regions revealed by scanning x-ray microdiffraction.

X-ray diffraction has provided extensive information about the arrangement of lipids and proteins in multilamellar myelin. This information has been limited to the abundant inter-nodal regions of the sheath because these regions dominate the scattering when x-ray beams of 100 µm diameter or more are used. Here, we used a 1 µm beam, raster-scanned across a single nerve fiber, to obtain detailed information about the molecular architecture in the nodal, paranodal, and juxtaparanodal regions. Orientation of the lamellar membrane stacks and membrane periodicity varied spatially. In the juxtaparanode-internode, 198–202 Å-period membrane arrays oriented normal to the nerve fiber axis predominated, whereas in the paranode-node, 205–208 Å-period arrays oriented along the fiber direction predominated. In parts of the sheath distal to the node, multiple sets of lamellar reflections were observed at angles to one another, suggesting that the myelin multilayers are deformed at the Schmidt-Lanterman incisures. The calculated electron density of myelin in the different regions exhibited membrane bilayer profiles with varied electron densities at the polar head groups, likely due to different amounts of major myelin proteins (P0 glycoprotein and myelin basic protein). Scattering from the center of the nerve fibers, where the x-rays are incident en face (perpendicular) to the membrane planes, provided information about the lateral distribution of protein. By underscoring the heterogeneity of membrane packing, microdiffraction analysis suggests a powerful new strategy for understanding the underlying molecular foundation of a broad spectrum of myelinopathies dependent on local specializations of myelin structure in both the PNS and CNS.

Resetting translational homeostasis restores myelination in Charcot-Marie-Tooth disease type 1B mice

P0 glycoprotein is an abundant product of terminal differentiation in myelinating Schwann cells. The mutant P0S63del causes Charcot-Marie-Tooth 1B neuropathy in humans, and a very similar demyelinating neuropathy in transgenic mice. P0S63del is retained in the endoplasmic reticulum of Schwann cells, where it promotes unfolded protein stress and elicits an unfolded protein response (UPR) associated with translational attenuation. Ablation of Chop, a UPR mediator, from S63del mice completely rescues their motor deficit and reduces active demyelination by half. Here, we show that Gadd34 is a detrimental effector of CHOP that reactivates translation too aggressively in myelinating Schwann cells. Genetic or pharmacological limitation of Gadd34 function moderates translational reactivation, improves myelination in S63del nerves, and reduces accumulation of P0S63del in the ER. Resetting translational homeostasis may provide a therapeutic strategy in tissues impaired by misfolded proteins that are synthesized during terminal differentiation.

Developmental regulation of insulin receptor gene in sciatic nerves and role of insulin on glycoprotein P0 in the Schwann cells

In view of the observations that Schwann cells contain insulin receptors, in the present study, we have investigated the developmental regulation of insulin receptor gene in the sciatic nerves of different postnatal age group rats. We have also investigated the role of insulin in the expression of the major PNS myelin glycoprotein P zero (P0) in normal as well as high glucose conditions in primary rat Schwann cells. The expression of insulin receptor gene in sciatic nerves appeared to be differentially regulated. The steady-state levels of insulin receptor mRNA increased remarkably during development and after postnatal day 10, when the peak of myelin structural gene (P0) expression occur and slowly increased further until at least postnatal day 90 in parallel with the growth of the myelin sheath. By employing immunofluorescence and RT-PCR, we observed significant increase in the P0 protein and mRNA levels in Schwann cells in response to the insulin than in insulin deprived counterparts. The presence of insulin in the high glucose medium ameliorated the altered protein and mRNA of P0 in Schwann cells compared to the insulin deprived counterparts. These studies demonstrate the importance of insulin and its receptor as possible regulatory factors in the PNS and also emphasizes their novel therapeutic applications in demyelinating diseases, especially in diabetic poly-neuropathy.

Age-related changes in myelin morphology, electrophysiological property and myelin-associated protein expression of mouse sciatic nerves

This study investigated the morphological and functional changes in peripheral nerves during the maturation and aging process. In a mouse sciatic nerve model, electron micrographs revealed that the number of myelin sheath lamellae gradually increased from 1 week through 12 months of age, when it reached the peak value, and then remained unchanged until 18 months of age; electrophysiological examinations showed that the amplitude of compound muscle action potentials gradually increased from 1 week through 18 months of age and displayed a positive linear correlation with the number of myelin sheath lamellae. Western blot analysis exhibited the age-related expression patterns of four myelin-associated proteins, i.e., myelin-associated glycoprotein (MAG), myelin basic protein (MBP), glycoprotein P0 (P0) and peripheral myelin protein 22 (PMP22). And MAG, MBP and PMP22 showed linear negative or positive correlations with the number of myelin sheath lamellae. Our results suggest that the morphological and functional changes in peripheral nerves are closely related to each other and the myelin-associated proteins perform distinct actions on the formation, maturation, degeneration and regeneration of myelin sheaths.

Reflections

Reflections

Ablation of the UPR-Mediator CHOP Restores Motor Function and Reduces Demyelination in Charcot-Marie-Tooth 1B Mice

Deletion of serine 63 from P0 glycoprotein (P0S63del) causes Charcot-Marie-Tooth 1B neuropathy in humans, and P0S63del produces a similar demyelinating neuropathy in transgenic mice. P0S63del is retained in the endoplasmic reticulum and fails to be incorporated into myelin. Here we report that P0S63del is misfolded and Schwann cells mount a consequential canonical unfolded protein response (UPR), including expression of the transcription factor CHOP, previously associated with apoptosis in ER-stressed cells. UPR activation and CHOP expression respond dynamically to P0S63del levels and are reversible but are associated with only limited apoptosis of Schwann cells. Nonetheless, Chop ablation in S63del mice completely rescues their motor deficit and reduces active demyelination 2-fold. This indicates that signaling through the CHOP arm of the UPR provokes demyelination in inherited neuropathy. S63del mice also provide an opportunity to explore how cells can dysfunction yet survive in prolonged ER stress-important for neurodegeneration related to misfolded proteins.

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.

Cytoplasmic Domain of Human Myelin Protein Zero Likely Folded as β-Structure in Compact Myelin

Myelin protein zero (P0 or P0 glycoprotein), the major integral membrane protein in peripheral nervous system myelin, plays a key role in myelin membrane compaction and stability. While the structure of P0 extracellular domain was determined by crystallography, the paucity of any structural data on the highly positive-charged P0 cytoplasmic domain (P0-cyt) has greatly limited our understanding of the mechanism of P0 function. Here, using circular dichroism and intrinsic fluorescence spectroscopy, we attempted to elucidate the structure of human P0-cyt (hP0-cyt) in membrane mimetic environments composed of detergents or lipid vesicles. We found that the secondary structure of P0-cyt was polymorphic—at the lipid/protein ratio corresponding to that of mature peripheral myelin (∼50:1), hP0-cyt mainly adopted a β-conformation, whereas when the proportion of lipid increased, the structure underwent a β→ α transition. By contrast, the secondary structure of the major isoform of myelin basic protein, another myelin protein with a very large positive charge, remained unchanged across a wide range of lipid/protein ratios. We propose that when hP0-cyt is bound at sufficient concentration to lamellar lipid bilayers such as myelin, it folds into a β-conformation; before this threshold lipid/protein ratio is reached, the domain is α-helical. We suggest that the cytoplasmic apposition (major dense line) in compact myelin may be stabilized via the hydrogen-bonding of β-strands formed as a result of local P0-P0 aggregation.

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.

Proteomics of Bovine Myelin Sheath: Characterization of a Truncated Form of P0 by MALDI-TOF/TOF Mass Spectrometry

The glycoprotein P0, the major structural protein of the peripheral nerve myelin, plays a critical role in holding myelin lamellae together via interaction of both extracellular and cytoplasmic domains. Mutations in the human P0 gene give rise to severe and progressive forms of dominantly inherited peripheral neuropathies like CMT1B. Here we report on the characterization of a bovine P0-derived protein of nearly 26 kD that corresponds to the P0 protein truncated in its cytoplasmic domain. Matrix assisted laser desorption ionization (MALDI)-time-of-flight/time-of-flight (TOF/TOF) mass spectrometry (MS) analysis on its tryptic digest has provided a peptide mapping, the main difference of which from the normal P0 analog was represented by the absence of the cluster of peaks at m/z 1513.7501, 1530.7701, and 1546.7651. The latter corresponds to the P0 fragment QTPVLYAMLDHSR and to its pyroglutamic and methionine-oxidized derivatives. The species at 1530.7701 covering the sequence 186-198 of P0 is not an artifact and might have a functional role in the myelin architecture.

GABA Receptor-Mediated Effects in the Peripheral Nervous System: A Cross-Interaction With Neuroactive Steroids

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult mammalian central nervous system (CNS), exerts its action via an interaction with specific receptors (e.g., GABAA and GABAB). These receptors are expressed not only in neurons but also on glial cells of the CNS, which might represent a target for the allosteric action of neuroactive steroids. Herein, we have demonstrated first that in the peripheral nervous system (PNS), the sciatic nerve and myelin-producing Schwann cells express both GABAA and GABAB receptors. Specific ligands, muscimol and baclofen, respectively, control Schwann-cell proliferation and expression of some specific myelin proteins (i.e., glycoprotein P0 and peripheral myelin protein 22 [PMP22]). Moreover, the progesterone (P) metabolite allopregnanolone, acting via the GABAA receptor, can influence PMP22 synthesis. In addition, we demonstrate that P, dihydroprogesterone, and allopregnanolone influence the expression of GABAB subunits in Schwann cells. The results suggest, at least in the myelinating cells of the PNS, a cross-interaction within the GABAergic receptor system, via GABAA and GABAB receptors and neuroactive steroids.

Structure and Stability of Internodal Myelin in Mouse Models of Hereditary Neuropathy

Peripheral neuropathies often result in abnormalities in the structure of internodal myelin, including changes in period and membrane packing, as observed by electron microscopy (EM). Mutations in the gene that encodes the major adhesive structural protein of internodal myelin in the peripheral nervous system of humans and mice--P0 glycoprotein--correlate with these defects. The mechanisms by which P0 mutations interfere with myelin packing and stability are not well understood and cannot be provided by EM studies that give static and qualitative information on fixed material. To gain insights into the pathogenesis of mutant P0, we used x-ray diffraction, which can detect more subtle and dynamic changes in native myelin, to investigate myelin structure in sciatic nerves from murine models of hereditary neuropathies. We used mice with disruption of one or both copies of the P0 gene (models of Charcot-Marie-Tooth-like neuropathy [CMT1B] or Dejerine-Sottas-like neuropathy) and mice with a CMT1B resulting from a transgene encoding P0 with an amino terminal myc-tag. To directly test the structural role of P0, we also examined a mouse that expresses P0 instead of proteolipid protein in central nervous system myelin. To link our findings on unfixed nerves with EM results, we analyzed x-ray patterns from unembedded, aldehyde-fixed nerves and from plastic-embedded nerves. From the x-ray patterns recorded from whole nerves, we assessed the amount of myelin and its quality (i.e. relative thickness and regularity). Among sciatic nerves having different levels of P0, we found that unfixed nerves and, to a lesser extent, fixed but unembedded nerves gave diffraction patterns of sufficient quality to distinguish periods, sometimes differing by a few Angstroms. Certain packing abnormalities were preserved qualitatively by aldehyde fixation, and the relative amount and structural integrity of myelin among nerves could be distinguished. Measurements from the same nerve over time showed that the amount of P0 affected myelin's stability against swelling, thus directly supporting the hypothesis that packing defects underlie instability in "live" or intact myelin. Our findings demonstrate that diffraction can provide a quantitative basis for understanding, at a molecular level, the membrane packing defects that occur in internodal myelin in demyelinating peripheral neuropathies.

Expression of P0 glycoprotein in CNS glia: Effects of overexpression in N20.1 cells

To examine effects of expression of the PNS myelin P0 glycoprotein in glial cells of CNS lineage, we transfected murine N20.1 glial cells with a rat P0 cDNA. A stably transfected cell line expressing high levels of P0 message showed P0 immunostaining, along with changes in morphology. Polymerase chain reaction (PCR) identified the predicted rat P0 sequence in the transfected N20.1 cells and further revealed low levels of mouse P0 message in the nontransfected cells and in primary mouse astrocytes. This is the first evidence of endogenous expression of message for P0 glycoprotein in CNS glia. Quantitative RT-PCR confirmed the expression of rat P0 mRNA in the transfected N20.1 cells, at levels about 400 times greater than murine P0 in nontransfected cells. A 27-kD band was detected in the transfected cells by Western blot with P0 antibody, but not in mock-transfected or nontransfected N20.1 cells. Immunocytochemistry following permeabilization showed intracellular vesicular localization of P0 in the cytoplasm and perinuclear rings in transfected cells, with a similar pattern but much lower levels in nontransfected cells. Faint surface staining for P0 protein without permeabilization was seen only on the transfected cells. A few transfected cells with membrane sheets stained more intensely for surface P0. Quantitative RT-PCR was used to determine if P0 overexpression altered expression of other myelin-related genes compared with glial fibrillary acidic protein (GFAP); the ratios of myelin basic protein (MBP)/GFAP and proteolipid protein (PLP)/GFAP were increased 2- to 3-fold in the P0-transfected cells. We conclude that P0 overexpression alters N20.1 gene expression and cell morphology, and shifts the cells from astroglial to oligodendroglial phenotype.

Peripheral nerve regeneration by transplantation of bone marrow stromal cell—derived Schwann cells in adult rats

Bone marrow stromal cells (BMSCs) can be induced to form Schwann cells by sequentially treating the cells with beta-mercaptoethanol and retinoic acid, followed by forskolin and neurotrophic factors including heregulin. In this study the authors made artificial grafts filled with BMSC-derived Schwann cells (BMSC-DSCs) and transplanted them into the transected sciatic nerve in adult rats to evaluate the potential of BMSCs as a novel alternative method of peripheral nerve regeneration. The BMSC-DSCs were suspended in Matrigel and transferred into hollow fibers (12 mm in length), which were transplanted into the transected sciatic nerve in adult Wistar rats. Six months after cell transplantation, electrophysiological evaluation and walking track analysis were performed. Results of these studies showed significant improvement in motor nerve conduction velocity and sciatic nerve functional index in the BMSC-DSC-transplanted group compared with the control group (Matrigel graft only). Immunohistochemical study data demonstrated that transplanted BMSCs labeled with retrovirus green fluorescent protein were positive for P0 and myelin-associated glycoprotein and had reconstructed nodes of Ranvier and remyelinated regenerated nerve axons. The number of regenerated axons in the axial section of the central portion of the graft was significantly greater in the transplanted group. Although BMSCs can differentiate into several types of cells, tumor formation did not occur 6 months after engraftment. Results in this study indicate that BMSC-DSCs have great potential to promote regeneration of peripheral nerves. The artificial graft made with BMSC-DSCs represents an alternative method for the difficult reconstruction of a long distance gap in a peripheral nerve.

Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 15

Charcot‐Marie‐Tooth neuropathies and Déjérine‐Sottas Syndrome are associated with mutations in the gene encoding Myelin Protein Zero. Here we report the characterization of two lines of transgenic mice expressing, in addition to the wild type protein, different point mutations in the extracellular domain of P0: the first is a deletion of Ser63 (DSer63), described in Charcot‐Marie‐Tooth 1B patients, whereas the second is a substitution of Ser 63 with a Cys (Ser63Cys) reported in a Déjérine‐Sottas patient. By crossing the transgenic mice with P0 null mice, we produced animals that expressed only the mutated P0. We then investigated in vivo the expression and the effect of the two mutated proteins by morphological, biochemical and immunohistochemical analyses. Sciatic nerves of mice expressing P0DSer63 show severe hypomyelination, with the presence of onion bulbs and some uncompacted myelin sheaths. P0DSer63 does not reach the myelin sheaths and it accumulates in the cytoplasm, where it is probably degraded. In contrast, sciatic nerves of mice expressing P0Ser63Cys present a milder hypomyelination with rare onion bulbs and, surprisingly, normal myelin sheaths. The mutated protein is a full‐length protein that, presumably, is integrated into the myelin sheaths. This study demonstrates that different mutations in P0 glycoprotein, leading to disorders of different severity in human, have different fates in the Schwann cells.

Factorially designed crystallization trials of the full-length P0 myelin membrane glycoprotein. I. Precipitation diagram

P0 glycoprotein is the abundant membrane protein of myelin of the peripheral nervous system. We report now the statistical design of the crystallization experiments; based on our belief that important information regarding supersaturation of protein or its solubility nature, as well as metastable state, nucleation or precipitation, are hidden in the trials in which no crystals grow. It is possible to work out this information when the whole set of experiments is designed in such a way as to allow statistical analyses. We selected seven factors, which we believe to be important for crystallization: protein concentration, pH of buffer, nature of precipitant, concentration of precipitant, nature of detergent, additives and temperature. The experimental matrix and detailed work sheet to make 148 solutions having random but balanced combination of these levels were calculated using the program DESIGN. A visual evaluation of crystallization drops was performed using light microscopy. We were able to plot the precipitation boundary diagram. Based on this diagram we have eliminated factors (and levels) that were driving the protein into precipitation. It is known that the precipitation boundary is related to the solubility curves for protein crystals, in the neighborhood of which nucleation and further crystallization is most likely to occur. These conditions are currently being refined to identify important factors (or its levels) that can be crucial in obtaining large and well diffracting crystals. Full-length P0 protein has never been crystallized for structural determination.