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Study design. (A) Schematic of mass spectrometry proteomic serum protein biomarker identification in human and mouse, and statistical analysis framework. (B) Progression of pathological and symptomatic neuropathy of mouse models, and time points of serum collection.
Source publication
Molecular markers, scalable for clinical use are critical for the development of effective treatments, and for design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We co...
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... and human serum samples were analysed by the University of Victoria-Genome BC Proteomics Centre by LC/MRM-MS (liquid chromatography-multiple reaction monitoring mass spectrometry) using SIS (stable isotope-labelled standard) peptides. Mouse and human peptide panels were of 270 and 375 peptides ( Supplementary Tables 1 and 2), respectively, selected as peptides previously validated in LC-MRM experiments according to the Office of Cancer Clinical Proteomics Research guidelines of assay development. 36 For details of mass spectrometry and analysis, see the Supplementary material. ...
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... was collected from Gars C201R and Gars P278KY , Gjb1-null, Hspb8 K141N and C61 het (CMT1A, PMP22d model) mice alongside wild-type littermate controls. Serial serum samples were taken at different time points, corresponding to different phases of neuropathy (Fig. 1B). Fifty-one proteins were significantly altered in at least two time points of each gene (or both gene variants for Gars mut ) in one or more of the four mouse models, but only seven were shared across more than one model. This identified complements C1q-B and C1q-C, neural cell adhesion molecule 1 (Ncam1), proteasome subunit beta type ...
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... was also collected from 41 genetically confirmed CMT patients, and 12 healthy age-matched controls (Fig. 1A). The CMT1 patient group was of mixed genotypes including PMP22d (CMT1A; n = 21), GJB1 (CMT1X, n = 1) and MPZ (CMT1B, n = 1). CMT2 patients carried pathogenic variants in GARS (CMT2D; n = 4), AARS (CMT2N; n = 6), DHTKD1 (CMT2Q, n = 3), DNM2 (CMT2M, n = 1), DYNC1H1 (CMT2O, n = 1), MFN2 (CMT2A, n = 1), MME (CMT2T, n = 1), RAB7 (CMT2B, n = ...
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... the other proteins identified in the patient screen: APOE, KNG1, ITIH2 and VTN were not elevated in two time points for any mouse model. proteomic data, no significant increase was identified in C61 het mouse sera (Supplementary Fig. 1). Together, these data indicate that NCAM1 is increased across different CMT mouse models compared to littermate controls, with the difference becoming more noticeable with increasing age and neuropathy severity. ...
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... post-symptomatic 6-and 10-month time points (Fig. 4E), but was statistically significant only at 6 months (Fig. 4F). In Hspb8 K141N , increased C3 expression compared to control was detectable at 2 and 10 months of age (Fig. 4G), corresponding to both pre-symptomatic and symptomatic phases. In contrast, C3 was stable in Gars C201R , Gars 278KY (Fig. 4E) or C61 (Fig. 4H) het mouse models compared with ...
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... significance indicated by Student's t-test, where *P < 0.05, **P < 0.01. 14); all CMT (n = 49), CMT1 (n = 24), PMP22d (n = 19), CMT2 (n = 17) and GARS/AARS (n = 8) neuropathy patients; GNE myopathy (n = 39), BMD (n = 30) and DMD (n = 4) patients. GDF15 ROC curves for: (B) CMT (all) versus control, AUC = 0.972 (95% CI; 0.936-1); (C) GARS/AARS versus control, AUC = 0.960 (95% CI; 0.886-1); (D) PMP22d versus control, AUC 974 (95% CI; 0.912-1). ...
Citations
... Similarly, in the case of CMT2E, the presence of neurofilaments in the culture media supernatant of patient motor neurons has been identified as a potential in vitro biomarker for axonal degeneration 73 . More complex in vitro models incorporating other cell types, such as muscle cells, may be used to validate recently identified CMT biomarkers, such as GDF15 or NCAM1 113 . Elevated NCAM1 has been observed in various mouse models and patients and may serve as an indicator of disease severity by correlating positively with the CMTES neuropathy severity score. ...
... Additionally, iPSC-derived Gdap1-null motor neurons were found to induce a fragile phenotype in MNs, characterized by mitochondrial dysfunction and activation of the innate immune response 127 . Another recent study demonstrated that in both demyelinating and axonal CMT patient sera, a significant increase in serum complement system protein levels, including C1q and C3, was observed compared to levels in control sera 113 . Increased C1q and C3 deposition was also detected on the sarcolemma, in part colocalized with the NMJs, of two genetically distinct CMT patients (FIG4 and GDAP1), suggesting that the upregulation of complement proteins could at least partially arise from NMJ degeneration. ...
... In CMT1A, the pathological SC is an abnormally differentiated SCs that constitutes onion bulb cells and demyelinating SCs. These abnormal cells express unique markers, such as neural cell adhesion molecule 1 and growth/differentiation factor-15, and it was recently reported that such marker proteins were elevated in the blood of CMT1A patients 5,7 . It was well established that autophagy dysfunction was implicated in the pathogenesis of CMT1A [16][17][18] and that autophagy receptors could be secreted when autophagic flux is dysfunctional 14,15 . ...
Autophagy is a self-degradation system for recycling to maintain homeostasis. p62/sequestosome-1 (p62) is an autophagy receptor that accumulates in neuroglia in neurodegenerative diseases. The objective of this study was to determine the elevation of plasma p62 protein levels in patients with Charcot–Marie–Tooth disease 1A (CMT1A) for its clinical usefulness to assess disease severity. We collected blood samples from 69 CMT1A patients and 59 healthy controls. Plasma concentrations of p62 were analyzed by ELISA, and we compared them with Charcot-Marie-Tooth neuropathy score version 2 (CMTNSv2). A mouse CMT1A model (C22) was employed to determine the source and mechanism of plasma p62 elevation. Plasma p62 was detected in healthy controls with median value of 1978 pg/ml, and the levels were significantly higher in CMT1A (2465 pg/ml, p < 0.001). The elevated plasma p62 levels were correlated with CMTNSv2 (r = 0.621, p < 0.0001), motor nerve conduction velocity (r = − 0.490, p < 0.0001) and disease duration (r = 0.364, p < 0.01). In C22 model, increased p62 expression was observed not only in pathologic Schwann cells but also in plasma. Our findings indicate that plasma p62 measurement could be a valuable tool for evaluating CMT1A severity and Schwann cell pathology.
... For the quantification of human GDF15 (R&D, DGD150), we followed the manufacturer's instructions. Although GDF15 has not been previously associated with nuclear-encoded complex I respiratory chain complex defects, it has been implicated as a biomarker in peripheral neuropathies [13]. ...
... To further prove the pathogenicity of the homozygous NDUFS6 variant identified in our index patient, we studied small nerve fibers and performed functional studies including determination of mitochondrial copy number in addition to proteomic studies on white blood cells and ELISA based quantification of GDF15 has been undertaken as described previously [13]. ...
Background: The NADH dehydrogenase [ubiquinone] iron-sulfur protein 6 (NDUFS6) gene encodes for an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). Bi-allelic NDUFS6 variants have been linked with a severe disorder mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS). Objective: Here, we identified a homozygous variant (c.309 + 5 G > A) in NDUFS6 in one male patient with axonal neuropathy accompanied by loss of small fibers in skin biopsy and further complicated by optic atrophy and borderline intellectual disability. Methods: To address the pathogenicity of the variant, biochemical studies (mtDNA copy number quantification, ELISA, Proteomic profiling) of patient-derived leukocytes were performed. Results: The analyses revealed loss of NDUFS6 protein associated with a decrease of three further mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4 and NDUFV1). Mitochondrial copy number is not altered in leukocytes and the mitochondrial biomarker GDF15 is not significantly changed in serum. Conclusions: Hence, our combined clinical and biochemical data strengthen the concept of NDUFS6 being causative for a very rare form of axonal neuropathy associated with optic atrophy and borderline intellectual disability, and thus expand (i) the molecular genetic landscape of neuropathies and (ii) the clinical spectrum of NDUFS6-associated phenotypes.
... Важливо, що тваринні моделі хвороби з ІA підтипом, які отримували лікування, показали зниження рівня NF-L, що вказує на потенційний біомаркер, який реагує на лікування [17]. Трансмембранна протеаза, серин 5 (TMPRSS5) також є інформативним біомаркером крові, оскільки підвищується під час прогресування хвороби [18]. Протеомний аналіз нещодавно виявив нові біомаркери крові, як-от нейрональна молекула клітинної адгезії 1 (NCAM1) і фактор диференційованого росту 15 (Gdf-15) [19]. ...
The article describes the prevalence of inherited neuromuscular disease, specifically neural amyotrophy, or Charcot-Marie-Tooth disease, its key clinical and neurological features, promising blood biomarkers for future diagnosis and therapeutic strategy assessment, along with current treatment methods under investigation, taking into account gene therapy. A clinical case is presented describing Charcot-Marie-Tooth disease type IA in a young man, which was confirmed by molecular genetic testing. The study of diagnostic criteria for nosologies of this spectrum is relevant, taking into account the rarity of the disease and certain difficulties in making a diagnosis.
... Nerve CSA may reflect clinical severity of CMT1A [15], but its usefulness as a longitudinal biomarker has been questioned [16]. Of blood biomarkers, growth differentiation factor 15 (GDF15) was recently found elevated in serum of individuals with CMT1A and other subtypes of CMT [17,18]. Other promising serum or plasma biomarkers include transmembrane protease serine 5 [19], certain microRNAs [20], neural adhesion molecule 1 [17], and neurofilament light chain (NFL) [21], although without confirmation in longitudinal studies [22]. ...
... Of blood biomarkers, growth differentiation factor 15 (GDF15) was recently found elevated in serum of individuals with CMT1A and other subtypes of CMT [17,18]. Other promising serum or plasma biomarkers include transmembrane protease serine 5 [19], certain microRNAs [20], neural adhesion molecule 1 [17], and neurofilament light chain (NFL) [21], although without confirmation in longitudinal studies [22]. Expression of GDF15 is responsive to integrated stress response [18], while NFL is a structural component of neurons that is released upon axon degeneration [23]. ...
... GDF15 was recently shown to have strong potential as a biomarker for CMT [17], in addition to cardiometabolic diseases [52]. Plasma NFL in turn is a marker of axonal degeneration, which is elevated in several neurological diseases including CMT. ...
Charcot-Marie-Tooth disease (CMT) is a heterogeneous set of hereditary neuropathies whose genetic causes are not fully understood. Here, we characterize three previously unknown variants in PMP22 and assess their effect on the recently described potential CMT biomarkers’ growth differentiation factor 15 (GDF15) and neurofilament light (NFL): first, a heterozygous PMP22 c.178G > A (p.Glu60Lys) in one mother-son pair with adult-onset mild axonal neuropathy. The variant led to abnormal splicing, confirmed in fibroblasts by reverse transcription PCR. Second, a de novo PMP22 c.35A > C (p.His12Pro), and third, a heterozygous 3.2 kb deletion predicting loss of exon 4. The latter two had severe CMT and ultrasonography showing strong nerve enlargement similar to a previous case of exon 4 loss due to a larger deletion. We further studied patients with PMP22 duplication (CMT1A) finding slightly elevated plasma NFL, as measured by the single molecule array immunoassay (SIMOA). In addition, plasma GDF15, as measured by ELISA, correlated with symptom severity for CMT1A. However, in the severely affected individuals with PMP22 exon 4 deletion or p.His12Pro, these biomarkers were within the range of variability of CMT1A and controls, although they had more pronounced nerve hypertrophy. This study adds p.His12Pro and confirms PMP22 exon 4 deletion as causes of severe CMT, whereas the previously unknown splice variant p.Glu60Lys leads to mild axonal neuropathy. Our results suggest that GDF15 and NFL do not distinguish CMT1A from advanced hypertrophic neuropathy caused by rare PMP22 variants.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10048-023-00729-5.
... In addition, GDF15 was able to rescue human motoneurons derived from induced pluripotent stem cells, IPSCs, from degeneration. Jennings et al. recently reported an elevation of GDF15 in the serum of patients with Charcot-Marie-Tooth disease and a mouse model of this disorder, proposing that the induction of GDF15 is an adaptive response to stress to promote peripheral neuron regeneration [73]. ...
Amyotrophic lateral sclerosis (ALS) is a fatal and incurable paralytic disorder caused by the progressive death of upper and lower motoneurons. Although numerous strategies have been developed to slow disease progression and improve life quality, to date only a few therapeutic treatments are available with still unsatisfactory therapeutic benefits. The secretome of dental pulp stem cells (DPSCs) contains numerous neurotrophic factors that could promote motoneuron survival. Accordingly, DPSCs confer neuroprotective benefits to the SOD1G93A mouse model of ALS. However, the mode of action of DPSC secretome on motoneurons remains largely unknown. Here, we used conditioned medium of human DPSCs (DPSCs-CM) and assessed its effect on survival, axonal length, and electrical activity of cultured wildtype and SOD1G93A motoneurons. To further understand the role of individual factors secreted by DPSCs and to circumvent the secretome variability bias, we focused on GDF15 and HB-EGF whose neuroprotective properties remain elusive in the ALS pathogenic context. DPSCs-CM rescues motoneurons from trophic factor deprivation-induced death, promotes axon outgrowth of wildtype but not SOD1G93A mutant motoneurons, and has no impact on the spontaneous electrical activity of wildtype or mutant motoneurons. Both GDF15 and HB-EGF protect SOD1G93A motoneurons against nitric oxide-induced death, but not against death induced by trophic factor deprivation. GDF15 and HB-EGF receptors were found to be expressed in the spinal cord, with a two-fold increase in expression for the GDF15 low-affinity receptor in SOD1G93A mice. Therefore, the secretome of DPSCs appears as a new potential therapeutic candidate for ALS.
... A plethora of new therapies targeting a variety of molecular mechanisms are already under development [11], and new druggable pathways are consistently being identified with hope for a more stratified approach in future patient care. Whilst there is a variety of validated biomarkers in patients for neuromuscular disorders such as Duchenne muscular dystrophy (serum miR-499) [13], or Charcot-Marie-Tooth disease (NCAM1 and GDF15) [14], there is a lack of early biomarkers that could predict muscle-wasting diseases such as sarcopenia or cachexia. Some biomarkers associated with physical frailty and sarcopenia have been identified, such as gut bacteria and markers of inflammation [15], as well as C-reactive protein (CRP) [15,16], also a marker of muscle wasting in cancer cachexia [17], but longitudinal patient studies are yet to be undertaken to evaluate the potential of these biomarkers to predict onset, treatment response and the progression of such muscle wasting disease. ...
Skeletal muscle homeostasis is essential for the maintenance of a healthy and active lifestyle. Imbalance in muscle homeostasis has significant consequences such as atrophy, loss of muscle mass, and progressive loss of functions. Aging-related muscle wasting, sarcopenia, and atrophy as a consequence of disease, such as cachexia, reduce the quality of life, increase morbidity and result in an overall poor prognosis. Investigating the muscle proteome related to muscle atrophy diseases has a great potential for diagnostic medicine to identify (i) potential protein biomarkers, and (ii) biological processes and functions common or unique to muscle wasting, cachexia, sarcopenia, and aging alone. We conducted a meta-analysis using gene ontology (GO) analysis of 24 human proteomic studies using tissue samples (skeletal muscle and adipose biopsies) and/or biofluids (serum, plasma, urine). Whilst there were few similarities in protein directionality across studies, biological processes common to conditions were identified. Here we demonstrate that the GO analysis of published human proteomics data can identify processes not revealed by single studies. We recommend the integration of proteomics data from tissue samples and biofluids to yield a comprehensive overview of the human skeletal muscle proteome. This will facilitate the identification of biomarkers and potential pathways of muscle-wasting conditions for use in clinics.
... In a recent study, Jennings et al., assessed biomarkers in the sera of 55 CMT patients, including six AARS1 patients. They found an increase in NCAM1 and GDF15 and a widespread activation of the inflammatory complement system, of which especially C3 was upregulated among the CMT2 patients [55]. In our study, C3 was the top fourth upregulated protein and we saw an activation of proteins involved in inflammation. ...
... In our study, C3 was the top fourth upregulated protein and we saw an activation of proteins involved in inflammation. It was speculated in the study by Jennings et al., that neuromuscular junction degeneration may cause upregulation of the imflammatory system [55]. Unfortunately, neither NCAM1 or GDF15 were among the detected proteins in our proteomic screen based on PBMCs. ...
Background
Aminoacyl tRNA-synthetases are ubiquitously-expressed enzymes that attach amino acids to their cognate tRNA molecules. Mutations in several genes encoding aminoacyl tRNA-synthetases, have been associated with peripheral neuropathy, i.e. AARS1, GARS1, HARS1, YARS1 and WARS1 . The pathogenic mechanism underlying AARS1 -related neuropathy is not known.
Methods
From 2012 onward, all probands presenting at Telemark Hospital (Skien, Norway) with peripheral neuropathy were screened for variants in AARS1 using an “in-house” next-generation sequencing panel. DNA from patient’s family members was examined by Sanger sequencing. Blood from affected family members and healthy controls were used for quantification of AARS1 mRNA and alanine. Proteomic analyses were conducted in peripheral blood mononuclear cells (PBMC) from four affected family members and five healthy controls.
Results
Seventeen individuals in two Norwegian families affected by Charcot-Marie-Tooth disease (CMT) were characterized in this study. The heterozygous NM_001605.2:c.976C > T p.(Arg326Trp) AARS1 mutation was identified in ten affected family members. All living carriers had a mild to severe length-dependent sensorimotor neuropathy. Three deceased obligate carriers aged 74–98 were reported to be unaffected, but were not examined in the clinic. Proteomic studies in PBMC from four affected individuals suggest an effect on the immune system mediated by components of a systemic response to chronic injury and inflammation. Furthermore, altered expression of proteins linked to mitochondrial function/dysfunction was observed. Proteomic data are available via ProteomeXchange using identifier PXD023842.
Conclusion
This study describes clinical and neurophysiological features linked to the p.(Arg326Trp) variant of AARS1 in CMT-affected members of two Norwegian families. Proteomic analyses based on of PBMC from four CMT-affected individuals suggest that involvement of inflammation and mitochondrial dysfunction might contribute to AARS1 variant-associated peripheral neuropathy.
... Interestingly, elevated blood concentrations of the integrated stress response marker GDF15 were also seen in patients with not only CMT2D but also mutations in other genes known to cause CMT. 51,52 In the same study, serum concentrations of neural cell adhesion Molecule 1 were also elevated in patients with CMT versus healthy controls. 52 ...
Traditionally, neurophysiology is the primary diagnostic and prognostic biomarker in peripheral neuropathy clinical practice; however, it may lack responsiveness in the context of slowly progressive neuropathies and where there is significant axonal damage. The development of ultrasensitive platforms for measuring serum proteins at the lower limit of detection of traditional ELISA techniques has transformed the field of blood biomarkers of peripheral neuropathy. A variety of blood biomarkers have been identified from inflammatory cytokines and apokines in diabetic neuropathy through to neuron‐specific proteins such as neurofilament light chain, Schwann cell‐specific proteins such as TMPRSS5 and microRNAs in other acquired and hereditary neuropathies. In this article, we review blood biomarkers of disease activity for the common subtypes of peripheral neuropathy including inflammatory demyelinating neuropathies, vasculitic neuropathy, diabetic neuropathy, chemotherapy‐induced neuropathy and Charcot–Marie–Tooth disease and related disorders including TTR amyloidosis.
Purpose of review
Charcot-Marie-Tooth (CMT) neuropathies are rare, genetically heterogeneous and progressive diseases for which there are no approved treatments and their management remains mostly supportive and symptomatic. This review is intended to provide an update on recent developments in gene therapies for different CMT neuropathies.
Recent findings
Increasing knowledge of disease pathomechanisms underlying several CMT types has facilitated the development of promising viral and nonviral gene therapy approaches. Some of these therapies are currently approaching the crucial step of moving from the bench to the clinic, having passed the proof-of-concept stage in rodent models and some also in larger animals. However, questions of optimal delivery route and dose, off-target effects, and possible payload toxicity remain to be clarified for several of these approaches. Furthermore, limited resources, the rarity of most CMT subtypes, and issues of safety and regulatory requirements, create the need for consensus guidelines and optimal clinical trial design.
Summary
Promising gene therapies have been developed for several CMT neuropathies, with proof-of-principle demonstrated in relevant disease models. Advantages and drawbacks of each approach are discussed and remaining challenges are highlighted. Furthermore, we suggest important parameters that should be considered in order to successfully translate them into the clinic.
