A Truncated 14-Amino-Acid Myelin Protein-Zero-Targeting Peptide for Fluorescence-Guided Nerve-Preserving Surgery.

BACKGROUND: The occurrence of accidental nerve damage during surgery and the increasing application of image guidance during head-and-neck surgery have highlighted the need for molecular targeted nerve-sparing interventions. The implementation of such interventions relies on the availability of nerve-specific tracers. In this paper, we describe the development of a truncated peptide that has an optimized affinity for protein zero (P0), the most abundant protein in myelin. METHODS AND MATERIALS: Further C- and N-terminal truncation was performed on the lead peptide Cy5-P0101-125. The resulting nine Cy5-labelled peptides were characterized based on their photophysical properties, P0 affinity, and in vitro staining. These characterizations were combined with evaluation of the crystal structure of P0, which resulted in the selection of the optimized tracer Cy5-P0112-125. A near-infrared Cy7-functionalized derivative (Cy7-P0112-125) was used to perform an initial evaluation of fluorescence-guided surgery in a porcine model. RESULTS: Methodological truncation of the 26-amino-acid lead compound Cy5-P0101-125 resulted in a size reduction of 53.8% for the optimized peptide Cy5-P0112-125. The peptide design and the 1.5-fold affinity gain obtained after truncation could be linked to interactions observed in the crystal structure of the extracellular portion of P0. The near-infrared analogue Cy7-P0112-125 supported nerve illumination during fluorescence-guided surgery in the head-and-neck region in a porcine model. CONCLUSIONS: Methodological truncation yielded a second-generation P0-specific peptide. Initial surgical evaluation suggests that the peptide can support molecular targeted nerve imaging.

Nerve Targeting via Myelin Protein Zero and the Impact of Dimerization on Binding Affinity.

BACKGROUND: Surgically induced nerve damage is a common but debilitating side effect. By developing tracers that specifically target the most abundant protein in peripheral myelin, namely myelin protein zero (P0), we intend to support fluorescence-guided nerve-sparing surgery. To that end, we aimed to develop a dimeric tracer that shows a superior affinity for P0. METHODS: Following truncation of homotypic P0 protein-based peptide sequences and fluorescence labeling, the lead compound Cy5-P0101-125 was selected. Using a bifunctional fluorescent dye, the dimeric Cy5-(P0101-125)2 was created. Assessment of the performance of the mono- and bi-labeled compounds was based on (photo)physical evaluation. This was followed by in vitro assessment in P0 expressing Schwannoma cell cultures by means of fluorescence confocal imaging (specificity, location of binding) and flow cytometry (binding affinity; KD). RESULTS: Dimerization resulted in a 1.5-fold increase in affinity compared to the mono-labeled counterpart (70.3 +/- 10.0 nM vs. 104.9 +/- 16.7 nM; p = 0.003) which resulted in a 4-fold increase in staining efficiency in P0 expressing Schwannoma cells. Presence of two targeting vectors also improves a pharmacokinetics of labeled compounds by lowering serum binding and optical stability by preventing dye stacking. CONCLUSIONS: Dimerization of the nerve-targeting peptide P0101-125 proves a valid strategy to improve P0 targeting.

How Does Protein Zero Assemble Compact Myelin?

Myelin protein zero (P0), a type I transmembrane protein, is the most abundant protein in peripheral nervous system (PNS) myelin-the lipid-rich, periodic structure of membrane pairs that concentrically encloses long axonal segments. Schwann cells, the myelinating glia of the PNS, express P0 throughout their development until the formation of mature myelin. In the intramyelinic compartment, the immunoglobulin-like domain of P0 bridges apposing membranes via homophilic adhesion, forming, as revealed by electron microscopy, the electron-dense, double "intraperiod line" that is split by a narrow, electron-lucent space corresponding to the extracellular space between membrane pairs. The C-terminal tail of P0 adheres apposing membranes together in the narrow cytoplasmic compartment of compact myelin, much like myelin basic protein (MBP). In mouse models, the absence of P0, unlike that of MBP or P2, severely disturbs myelination. Therefore, P0 is the executive molecule of PNS myelin maturation. How and when P0 is trafficked and modified to enable myelin compaction, and how mutations that give rise to incurable peripheral neuropathies alter the function of P0, are currently open questions. The potential mechanisms of P0 function in myelination are discussed, providing a foundation for the understanding of mature myelin development and how it derails in peripheral neuropathies.

Upregulation of large myelin protein zero leads to Charcot-Marie-Tooth disease-like neuropathy in mice.

Charcot-Marie-Tooth (CMT) disease is a hereditary neuropathy mainly caused by gene mutation of peripheral myelin proteins including myelin protein zero (P0, MPZ). Large myelin protein zero (L-MPZ) is an isoform of P0 that contains an extended polypeptide synthesized by translational readthrough at the C-terminus in tetrapods, including humans. The physiological role of L-MPZ and consequences of an altered L-MPZ/P0 ratio in peripheral myelin are not known. To clarify this, we used genome editing to generate a mouse line (L-MPZ mice) that produced L-MPZ instead of P0. Motor tests and electrophysiological, immunohistological, and electron microscopy analyses show that homozygous L-MPZ mice exhibit CMT-like phenotypes including thin and/or loose myelin, increased small-caliber axons, and disorganized axo-glial interactions. Heterozygous mice show a milder phenotype. These results highlight the importance of an appropriate L-MPZ/P0 ratio and show that aberrant readthrough of a myelin protein causes neuropathy.

In silico and in vitro effects of the I30T mutation on myelin protein zero instability in the cell membrane.

Charcot-Marie-Tooth (CMT) diseases are a heterogeneous group of genetic peripheral neuropathies caused by mutations in a variety of genes, which are involved in the development and maintenance of peripheral nerves. Myelin protein zero (MPZ) is expressed by Schwann cells, and MPZ mutations can lead to primarily demyelinating polyneuropathies including CMT type 1B. Different mutations demonstrate various forms of disease pathomechanisms, which may be beneficial in understanding the disease cellular pathology. Our molecular dynamics simulation study on the possible impacts of I30T mutation on the MPZ protein structure suggested a higher hydrophobicity and thus lower stability in the membranous structures. A study was also conducted to predict native/mutant MPZ interactions. To validate the results of the simulation study, the native and mutant forms of the MPZ protein were separately expressed in a cellular model, and the protein trafficking was chased down in a time course pattern. In vitro studies provided more evidence on the instability of the MPZ protein due to the mutation. In this study, qualitative and quantitative approaches were adopted to confirm the instability of mutant MPZ in cellular membranes.

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail.

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane.

Ionic strength and calcium regulate membrane interactions of myelin basic protein and the cytoplasmic domain of myelin protein zero.

The formation of a mature myelin sheath in the vertebrate nervous system requires specific protein-membrane interactions. Several myelin-specific proteins are involved in stacking lipid membranes into multilayered structures around axons, and misregulation of these processes may contribute to chronic demyelinating diseases. Two key proteins in myelin membrane binding and stacking are the myelin basic protein (MBP) and protein zero (P0). Other factors, including Ca2+, are important for the regulation of myelination. We studied the effects of ionic strength and Ca2+ on the membrane interactions of MBP and the cytoplasmic domain of P0 (P0ct). MBP and P0ct bound and aggregated negatively charged lipid vesicles, while simultaneously folding, and both ionic strength and calcium had systematic effects on these interactions. When decreasing membrane net negative charge, the level and kinetics of vesicle aggregation were affected by both salt and Ca2+. The effects on lipid membrane surfaces by ions can directly affect myelin protein-membrane interactions, in addition to signalling effects in myelinating glia.

Molecular structure and function of myelin protein P0 in membrane stacking.

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.

Myelin Protein Zero180-199 Peptide Induced Experimental Autoimmune Neuritis in C57BL/6 Mice.

Mouse models of peripheral demyelinating neuropathy play an important role in enabling the study of disease pathogenesis. Further, induction in transgenic mice allows for the precise interrogation of disease mechanisms, as well as the analysis of the efficacy and mechanisms of potential new therapies. Here we describe a method to successfully induce experimental autoimmune neuritis (EAN) using myelin protein zero (P0)180-199 peptide in combination with Freund's complete adjuvant and pertussis toxin in the C57BL/6 mouse strain. We also outline a sensitive paradigm of accurately assessing the extent of functional deficits occurring in murine EAN.

Genetic variants, structural, and functional changes of Myelin Protein Zero and Mannose-Binding Lectin 2 protein involved in immune response and its allelic transmission in families of patients with leprosy in Colombia.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Genetic factors associated with immune response contribute to infection development and disease. M. leprae has the capacity to invade Schwann cells in the peripheral nervous system and cause neuropathy. However, while the responsible molecular mechanisms remain to be fully unveiled, they have begun being elucidated. We studied genetic variants Myelin Protein Zero (MPZ), a major structural component of the myelin sheath, and Mannose Binding Lectin 2 (MBL2), a protein involved in immune response, in 112 family groups of 114 leprosy patients using PCR-RFLP, aiming to calculate the association and allelic transmission of variants associated in first, second and third-degree relatives. Polymorphisms found in MPZ and MBL2 showed association with leprosy. Different probabilities for allelic transmission were found for first and second-degree relatives, a fact that is important to take into account when evaluating risk in contacts of leprosy patients. Structural analysis allows the study of putative amino acids and their possible effect on protein structure and function, as well as on the assembly of a protein homotetramer. Our results suggest that the identified MPZ and MBL2 gene mutations are associated with leprosy in a Colombian population, which correlates with MPZ and MBL2 protein function, and increase the risk of M. leprae infection in leprosy-patients' family members. Additionally, structural analyses were carried out specifically for MPZ protein using information available in databases, and analyzing the substitutions in wildtype and mutant protein. The results show significant structural changes, which may be associated to infection and pathogenicity.

Impact of I30T and I30M substitution in MPZ gene associated with Dejerine-Sottas syndrome type B (DSSB): A molecular modeling and dynamics.

Myelin protein zero (MPZ) gene encodes MPZ protein is a vital component of the myelin sheath. Mutationsassociated with MPZ gene leads to severe de-hypomyelination Dejerine-Sottas syndrome type B (DSSB) also termed as Charcot-Marie-Tooth disease (CMT) type 3. In this work, we employed a set of various in silico prediction methods to screen 97 nsSNPs associated with MPZ gene. Based on this, we identified the nsSNPs to be most deleterious and pathogenic associated with DSSB. To get more insight into the mutational effect at three-dimensional structural level, we modeled the homology structure of native type as well as I30T and I30M mutant of MPZ protein using Modeler 9.13 software. Molecular dynamics simulation was initiated to explain the impact of the mutation on its structure and function. The obtained results depict that the protein with I30T mutation had variable structural conformation and dynamic behavior than native and mutant I30M of MPZ protein. We hope our computational insight might be helpful in rationalizing the deleterious mutations in DSSB and the advancement of novel pharmacological strategy.

Characterization of a new rat model for chronic inflammatory demyelinating polyneuropathies.

Our objective was to develop a chronic model of EAN which could be used as a tool to test treatment strategies for CIDP. Lewis rats injected with S-palmitoylated P0(180-199) peptide developed a chronic, sometimes relapsing-remitting type of disease. Our model fulfills electrophysiological criteria of demyelination with axonal degeneration, confirmed by immunohistopathology. The late phase of the chronic disease was characterized by accumulation of IL-17(+) cells and macrophages in sciatic nerves and by high serum IL-17 levels. In conclusion, we have developed a reliable and reproducible animal model resembling CIDP that can now be used for translational drug studies.

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.

Berberine ameliorates experimental autoimmune neuritis by suppressing both cellular and humoral immunity.

Berberine (BBR), an isoquinoline derivative alkaloid, has been extensively used in traditional Chinese medicine for the treatment of diarrhoea, rheumatic diseases, diabetes, etc. Recent studies have demonstrated new biological properties of BBR and suggested the possibility of BBR to be a therapeutic agent for some autoimmune diseases. To explore the effect of BBR on the development of experimental autoimmune neuritis (EAN), BBR was administered intragastrically daily to Lewis rats immunized with P0 peptide 180-199 in Freund's complete adjuvant. We found BBR treatment resulted in amelioration of EAN, accompanied by suppressed lymphocyte (in particular CD4(+) T cell) proliferation, downregulated Th1 (TNF-α) and Th2 (IL-10) cytokines and reduced anti-P0 peptide 180-199 IgG1 and IgG2a. In brief, BBR played a role in ameliorating EAN by suppressing both cellular and humoral immunity. Thus, our study suggests that BBR may be a potential therapeutic agent for the autoimmune disease in the peripheral nervous system, such as Guillain-Barré syndrome.

Specialization of endoplasmic reticulum chaperones for the folding and function of myelin glycoproteins P0 and PMP22.

Peripheral myelin protein 22 (PMP22) and protein 0 (P0) are major peripheral myelin glycoproteins, and mutations in these two proteins are associated with hereditary demyelinating peripheral neuropathies. Calnexin, calreticulin, and ERp57 are critical components of protein quality control responsible for proper folding of newly synthesized glycoproteins. Here, using confocal microscopy, we show that cell surface targeting of P0 and PMP22 is not affected in the absence of the endoplasmic reticulum chaperones. However, the folding and function (adhesiveness) of PMP22 and P0, measured using the adhesion assay, are affected significantly in the absence of calnexin but not in the absence of calreticulin. Deficiency in oxidoreductase ERp57 results in impaired folding and function of P0, a disulfide bond-containing protein, but does not have any effect on folding or function of PMP22 (a protein that does not contain a disulfide bond). We concluded that calnexin and ERp57, but not calreticulin, play an important role in the biology of peripheral myelin proteins PMP22 and P0, and, consequently, these chaperones may contribute to the pathogenesis of peripheral neuropathies and the diversity of these neurological disorders.

High-resolution structural model of porcine P2 myelin membrane protein with associated fatty acid ligand: fact or artifact?

Myelin membrane is a biological complex of glial cells origin; it is composed of 25% (w/w) proteins and 75% lipids, and more than 300 proteins are associated with central nervous system myelin (for peripheral nervous system myelin, such data are lacking). Myelin plays an important role in maintaining propagation of nerve signals. To uncover the nature of propagation phenomena, it is essential to study biochemistry of myelin proteins and lipids, myelin composition, and myelin structure. Nearly all myelin proteins are like antigens, causing clinically well-defined devastating diseases; multiple sclerosis and Guillain-Barré syndrome are two of them. In this article, a high-resolution study (1.8 Å) of porcine myelin P2 protein is presented. Myelin was purified from porcine intradural spinal roots, which were stored at -80°C for 10 years before myelin and P2 protein were purified (spinal roots were a gift of Prof. Kunio Kitamura, Saitama Medical School). The three-dimensional structural analysis uncovered embedded 18-carbons-long fatty acid. Some speculative interpretation is presented, to uncover how this ligand of fatty acid may form cholesterol ester and stabilize the myelin structure or form simple raft microdomain. Protein crystallography indicates that the ligand may be 18-carbons-long fatty acid. This is unlike previous work with mass spectrometry, in which three ligands were determined. In other protein crystallography-based studies of P2 (bovine), an oleic fatty acid was suggested, but, for recombinant (human) protein, palmitic acid was found. There is no fatty acid ligand in equine P2 protein.

[Clinical-genetic correlations in the hereditary motor-sensor neuropathy caused by mutations in the MPZ (P0) gene].

Hereditary motor-sensor neuropathy (HMSN) caused by mutations in the MPZ (P0) gene is a rare variant of hereditary demyelinating polyneuropathies that makes up 5-10% of all cases in different populations. Based on the complex examination of patients of the Russian Federation with different MPZ (P0) mutations, we obtained clinical-genetic, electromyographic and molecular-genetic characteristics of HMSN caused by mutations in the MPZ (P0) gene. Peculiarities of clinical presentations in patients with HMSN, types 1B and 2I, are presented. Diagnostic criteria of these genetic variants have been formed. The new allelic variants of HMSN caused by mutations in the MPZ (P0) gene are described. The distribution of mutations by protein domains has been analyzed.

Thiopalmitoylated peptides from the peripheral nervous system myelin p0 protein: synthesis, characterization, and neuritogenic properties.

Thiopalmitoylation (i.e., the covalent attachment of palmitic acid via a thioester linkage to cysteine residues in the polypeptide backbone) is a common post-translational modification of proteins. Several proteins that have been identified as putative autoantigens in a variety of T-cell mediated autoimmune diseases are thiopalmitoylated, and thus, we have hypothesized that endogenous thiopalmitoylated peptides released during tissue breakdown may play a role in the development and chronicity of autoimmune diseases. To investigate this, we have studied the effect of thiopalmitoylation on the immunogenic and neuritogenic properties of P0, the major peripheral nervous system (PNS) myelin protein, which is thiopalmitoylated at cysteine 153, and described as a candidate autoantigen in Guillain-Barre syndrome (GBS), a human inflammatory demyelinating disease of the PNS. This paper describes the synthesis of palmitoylated peptide P0(180-199) and P0(152-171) by on-resin acylation using specific cysteine side-chain protecting groups: Mmt (labile in diluted acid) and StBu (labile in the presence of tributylphosphine). Our results show that the thiol protecting group had to be adjusted to the peptide sequence: Mmt was efficiently used for P0(180-199) thioacylation, but it was not suitable for thiopalmitoylation of P0(152-171) because of a premature deprotection of the Boc protecting group on the epsilon-NH(2) Lys in the presence of 2% TFA, leading to dipalmitoylation. Palmitoylated P0(152-171) was successfully obtained by using StBu as the thiol protecting group. We could show by circular dichroism that palmitoylation has no influence on the structuration of the peptide in solution but palmitoylation increased the stability of the peptide in the presence of serum. Using EAN (experimental autoimmune neuritis), the rat model of GBS, we have compared the immunological properties of palm and non-palm P0 peptides and showed that thiopalmitoylation has indeed a great influence on their neuritogenic and immunogenic properties. This study provides further support for our hypothesis concerning the role of thiopalmitoylation in the development and chronicity of inflammatory demyelinating diseases and confirms that thiopalmitoylation of peptides may provide a simple means to increase MHC class II restricted responses.