Objective: To 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.Methods: We 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.Results: We 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<0.0001, p=0.0065 and p<0.0001, respectively). In patients with CIDP classified for disease stage, SM was higher in active CIDP compared with both controls and stable CIDP (p<0.0001), applying for a selective tool to treatment tailoring or withdrawal. SM was also increased in AIDP compared with axonal GBS, discerning the demyelinating from axonal variant of the disease. SM did not correlate with CSF protein levels, stratifying patients independently from commonly used CSF indexes, and displaying high specificity to avoid potential misdiagnosis. Finally, SM correlated with the main clinical scores and some neurophysiological parameters in patients with CIDP and AIDP.Conclusions: CSF 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.

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.

Findings accumulated over time show that neurophysiological, neuropathological, and molecular alterations are present in CMT1A and support the dysmyelinating rather than demyelinating nature of this neuropathy. Moreover, uniform slowing of nerve conduction velocity is already manifest in CMT1A children and does not improve throughout their life. This evidence and our previous studies displaying aberrant myelin composition and structure in adult CMT1A rats prompt us to hypothesize a myelin and axon developmental defect in the CMT1A peripheral nervous system. Peripheral myelination begins during the early stages of development in mammals and, during this process, chemical and structural features of myelinated fibers (MFs) evolve towards a mature phenotype; deficiencies within this self-modulating circuit can cause its blockage. Therefore, to shed light on pathophysiological mechanisms that occur during development, and to investigate the relationship among axonal, myelin, and lipidome deficiencies in CMT1A, we extensively analyzed the evolution of both myelin lipid profile and MF structure in WT and CMT1A rats. Lipidomic analysis revealed a delayed maturation of CMT1A myelin already detectable at P10 characterized by a deprivation of sphingolipid species such as hexosylceramides and long-chain sphingomyelins, whose concentration physiologically increases in WT, and an increase in lipids typical of unspecialized plasma membranes, including phosphatidylcholines and phosphatidylethanolamines. Consistently, advanced morphometric analysis on more than 130,000 MFs revealed a delay in the evolution of CMT1A axon and myelin geometric parameters, appearing concomitantly with lipid impairment. We here demonstrate that, during normal development, MFs undergo a continuous maturation process in both chemical composition and physical structure, but these processes are delayed in CMT1A.

In Charcot–Marie–Tooth type 1A (CMT1A), Schwann cells exhibit a preponderant transcriptional deficiency of genes involved in lipid biosynthesis. This perturbed lipid metabolism affects the peripheral nerve physiology and the structure of peripheral myelin. Nevertheless, the identification and functional characterization of the lipid species mainly responsible for CMT1A myelin impairment currently lack. This is critical in the pathogenesis of the neuropathy since lipids are many and complex molecules which play essential roles in the cell, including the structural components of cellular membranes, cell signaling, and membrane trafficking. Moreover, lipids themselves are able to modify gene transcription, thereby affecting the genotype–phenotype correlation of well-defined inherited diseases, including CMT1A. Here we report for the first time a comprehensive lipid profiling in experimental and human CMT1A, demonstrating a previously unknown specific alteration of sphingolipid (SP) and glycerophospholipid (GP) metabolism. Notably, SP, and GP changes even emerge in biological fluids of CMT1A rat and human patients, implying a systemic metabolic dysfunction for these specific lipid classes. Actually, SP and GP are not merely reduced; their expression is instead aberrant, contributing to the ultrastructural abnormalities that we detailed by X-ray diffraction in rat and human internode myelin. The modulation of SP and GP pathways in myelinating dorsal root ganglia cultures clearly sustains this issue. In fact, just selected molecules interacting with these pathways are able to modify the altered geometric parameters of CMT1A myelinated fibers. Overall, we propose to exploit the present SP and GP metabolism impairment to select effective drugs and validate a set of reliable biomarkers, which remain a challenge in CMT1A neuropathy.

Pathological changes and the cellular and molecular mechanisms underlying axonopathy and myelinopathy are key to understanding a wide range of inherited and acquired peripheral nerve disorders. While the clinical indications for nerve biopsy have diminished over time, its diagnostic value remains significant in select conditions, offering a unique window into the pathophysiological processes of peripheral neuropathies. Evidence highlights the symbiotic relationship between axons and myelinating Schwann cells, wherein disruptions in axo-glial interactions contribute to neuropathogenesis. This review synthesizes recent insights into the pathological and molecular underpinnings of axonopathy and myelinopathy. Axonopathy encompasses Wallerian degeneration, axonal atrophy, and dystrophy. Although extensively studied in traumatic nerve injury, the mechanisms of axonal degeneration and Schwann cell-mediated repair are increasingly recognized as pivotal in non-traumatic disorders, including dying-back neuropathies. We briefly outline key transcription factors, signaling pathways, and epigenetic changes driving axonal regeneration. For myelinopathy, we discuss primary segmental demyelination and dysmyelination, characterized by defective myelin development. We describe paranodal demyelination in light of recent findings in nodopathies, emphasizing that it is not an exclusive indicator of demyelinating disorders. This comprehensive review provides a framework to enhance our understanding of peripheral nerve pathology and its implications for developing targeted therapies.

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.