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ObjectiveTo validate sphingomyelin (SM) dosage in the cerebrospinal fluid (CSF) of patients affected by chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and Guillain-Barré syndrome (GBS) as a reliably assessable biomarker.MethodsWe prospectively enrolled 184 patients from six Italian referral centres, in whom CSF SM levels were quantified by a fluorescence-based assay optimised and patented in our laboratory.ResultsWe confirmed increased levels of SM in the CSF of patients affected by typical CIDP (n=35), atypical CIDP (n=18) and acute inflammatory demyelinating polyradiculoneuropathy, AIDP (n=12) compared with patients affected by non-demyelinating neurological diseases, used as controls (n=85) (p=0.0065 and pConclusionsCSF SM is a diagnostic and staging wet biomarker for acquired demyelinating neuropathies and may effectively improve the management of patients affected by GBS and CIDP.

Neuregulin1 (NRG1) is a growth factor playing a pivotal role in peripheral nerve development through the activation of the transmembrane co-receptors ErbB2–ErbB3. Soluble NRG1 isoforms, mainly secreted by Schwann cells, are strongly and transiently up-regulated after acute peripheral nerve injury, thus suggesting that they play a crucial role also in the response to nerve damage. Here we show that in the rat experimental model of the peripheral demyelinating neuropathy Charcot-Marie-Tooth 1A (CMT1A) the expression of the different NRG1 isoforms (soluble, type α and β, type a and b) is strongly up-regulated, as well as the expression of NRG1 co-receptors ErbB2–ErbB3, thus showing that CMT1A nerves have a gene expression pattern highly reminiscent of injured nerves. Because it has been shown that high concentrations of soluble NRG1 negatively affect myelination, we suggest that soluble NRG1 over-expression might play a negative role in the pathogenesis of CMT1A disease, and that a therapeutic approach, aimed to interfere with NRG1 activity, might be beneficial for CMT1A patients. Further studies will be necessary to test this hypothesis in animal models and to evaluate NRG1 expression in human patients. Impact statement Charcot-Marie-Tooth1A (CMT1A) is one of the most frequent inherited neurological diseases, characterized by chronic demyelination of peripheral nerves, for which effective therapies are not yet available. It has been recently proposed that the treatment with soluble Neuregulin1 (NRG1), a growth factor released by Schwann cells immediately after acute nerve injury, might be effective in CMT1A treatment. However, the expression of the different isoforms of endogenous NRG1 in CMT1A nerves has not been yet investigated. In this preliminary study, we demonstrate that different isoforms of soluble NRG1 are strongly over-expressed in CMT1A nerves, thus suggesting that a therapeutic approach based on NRG1 treatment should be carefully reconsidered. If soluble NRG1 is over-expressed also in human CMT1A nerves, a therapeutic approach aimed to inhibit (instead of stimulate) the signal transduction pathways driven by NRG1 might be fruitfully developed. Further studies will be necessary to test these hypotheses.

Fast, accurate and reliable methods to quantify the amount of myelin still lack, both in humans and experimental models. The overall objective of the present study was to demonstrate that sphingomyelin (SM) in the cerebrospinal fluid (CSF) of patients affected by demyelinating neuropathies is a myelin biomarker. We found that SM levels mirror both peripheral myelination during development and small myelin rearrangements in experimental models. As in acquired demyelinating peripheral neuropathies myelin breakdown occurs, SM amount in the CSF of these patients might detect the myelin loss. Indeed, quantification of SM in 262 neurological patients showed a significant increase in patients with peripheral demyelination (p = 3.81 * 10 − 8) compared to subjects affected by non-demyelinating disorders. Interestingly, SM alone was able to distinguish demyelinating from axonal neuropathies and differs from the principal CSF indexes, confirming the novelty of this potential CSF index. In conclusion, SM is a specific and sensitive biomarker to monitor myelin pathology in the CSF of peripheral neuropathies. Most importantly, SM assay is simple, fast, inexpensive, and promising to be used in clinical practice and drug development.

Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy whose pathogenetic mechanisms are still poorly defined and an etiologic treatment is not yet available. An abnormally high intracellular Ca(2+) concentration ([Ca(2+)]i) occurs in Schwann cells from CMT1A rats (CMT1A SC) and is caused by overexpression of the purinoceptor P2X7. Normalization of the Ca(2+) levels through down-regulation of P2X7 appears to restore the normal phenotype of CMT1A SC in vitro. We recently demonstrated that the diadenosine 5',5'''-P1, P2-diphosphate (Ap2A) isomer P18 behaves as an antagonist of the P2X7 purinergic receptor, effectively blocking channel opening induced by ATP. In addition, P18 behaves as a P2Y11 agonist, inducing cAMP overproduction in P2Y11-overexpressing cells. Here we investigated the in vitro effects of P18 on CMT1A SC. We observed that basal levels of intracellular cAMP ([cAMP]i), a known regulator of SC differentiation and myelination, are significantly lower in CMT1A SC than in wild-type (wt) cells. P18 increased [cAMP]i in both CMT1A and wt SC, and this effects was blunted by NF157, a specific P2Y11 antagonist. Prolonged treatment of organotypic dorsal root ganglia (DRG) cultures with P18 significantly increased expression of myelin protein zero, a marker of myelin production, in both CMT1A and wt cultures. Interestingly, P18 decreased the content of non-phosphorylated neurofilaments, a marker of axonal damage, only in CMT1A DRG cultures. These results suggest that P2X7 antagonists, in combination with [cAMP]i-increasing agents, could represent a therapeutic strategy aimed at correcting the molecular derangements causing the CMT1A phenotype.

CMT1A patients commonly share PMP22 genetic overloading but they show phenotypic heterogeneity and variability in PMP22 mRNA and protein expression. Moreover, PMP22 mRNA levels do not correlate with clinical outcome measures in these patients, suggesting their uselessness as a disease biomarker. Thus, in-depth analysis of PMP22 transcription and translation might help to define its pathogenic role in CMT1A. We focused on the alternative splicing of PMP22 gene to verify whether mRNA processing is altered in CMT1A. We identified three new PMP22 transcripts enriched in human sural nerve biopsies. One of them was an untranslated variant, whereas the other two originated from a PMP22 undescribed exon and encoded for a new putative protein localized in the endoplasmic reticulum. As splicing events in the PMP22 gene are differently regulated in tissues and during development, we analyzed the levels of PMP22 transcripts and their splicing pattern in human and experimental CMT1A. We found an altered PMP22 splicing ratio in the CMT1A rat. In addition, we showed a remarkable derangement in rat QKI expression, which is a critical regulator of splicing during myelination. Overall, our data suggest that an alteration of mRNA processing could be a pathogenic mechanism in CMT1A.

Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot-Marie-Tooth type 1A (CMT1A) rats. The latter used as a model of dys-demyelination. O₂ consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O₂ consumption by IMV from Wt and CMT1A 1-month-old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys-demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys-demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.

Background We investigated the extent, modalities and reversibility of changes at cellular level in the expression of genes and proteins occurring upon Hind limb unloading (HU) in the tibiae of young C57BL/6J male mice. We focused on the effects of HU in chondrogenic, osteogenic, and marrow mesenchymal cells. Methods We analyzed for expression of genes and proteins at two time points after HU (7 and 14 days), and at 14 days after recovery from HU. Levels of mRNAs were tested by in situ hybridization. Protein levels were tested by immunohistochemistry. We studied genes involved in osteogenesis (alkaline phosphatase (AP), osteocalcin (OC), bonesialoprotein (BSP), membrane type1 matrix metalloproteinase (MT1-MMP)), in extracellular matrix (ECM) formation (procollagenases (BMP1), procollagenase enhancer proteins (PCOLCE)) and remodeling (metalloproteinase-9 (MMP9), RECK), and in bone homeostasis (Stro-1, CXCL12, CXCR4, CD146). Results We report the following patterns and timing of changes in gene expression induced by HU: 1) transient or stable down modulations of differentiation-associated genes (AP, OC), genes of matrix formation, maturation and remodelling, (BMP1, PCOLCEs MMP9) in osteogenic, chondrogenic and bone marrow cells; 2) up modulation of MT1-MMP in these same cells, and uncoupling of its expression from that of AP; 3) transient down modulation of the osteoblast specific expression of BSP; 4) for genes involved in bone homeostasis, up modulation in bone marrow cells at distal epiphysis for CXCR4, down modulation of CXCL12, and transient increases in osteoblasts and marrow cells for Stro1. 14 days after limb reloading expression returned to control levels for most genes and proteins in most cell types, except AP in all cells, and CXCL12, only in bone marrow. Conclusions HU induces the coordinated modulation of gene expression in different mesenchymal cell types and microenvironments of tibia. HU also induces specific patterns of expression for homeostasis related genes and modulation of mRNAs and proteins for ECM deposition, maturation and remodeling which may be key factors for bone maintenance.