Peripheral Neuropathy In Humans Research Articles

Functional ultrasound and brain connectivity reveal central nervous system compromise in Trembler-J mice model of Charcot-Marie-Tooth disease

The Charcot-Marie-Tooth-1E (CMT1E) disease is typically described as a peripheral neuropathy in humans, causing decreased nerve conduction, spastic paralysis, and tremor. The Trembler-J (TrJ) mice serve as a high fidelity model of this disease. Here, we use functional ultrasound (fUS) and functional connectivity (FC) to analyze TrJ mice’s brain activity during sensory stimulation and resting state experiments against wild type (WT) mice - the healthy counterpart. fUS is an imaging technique that measures cerebral blood volume (CBV) temporal changes. We study these changes in the primary somatosensory cortex barrel field (S1BF) of both mice populations during periodic vibrissae stimulation, measuring the number of pixels that correlate to the stimulation (i.e., the size of the activation area), the average correlation of these pixels (i.e., the response strength), and the CBV’s rate of change for each stimulation (i.e., the hemodynamic response). Then, we construct a FC matrix for each genotype and experiment by correlating the CBV signals from the eight cortical regions defined by the Paxinos and Franklin atlas. Our results show that TrJ mice have significantly diminished neurovascular responses and altered brain connectivity with respect to WT mice, pointing to central nervous system effects that could shift our understanding of the CMT1E disease.

Hidden hearing loss in a Charcot-Marie-Tooth type 1A mouse model.

Hidden hearing loss (HHL), a recently described auditory neuropathy characterized by normal audiometric thresholds but reduced sound-evoked cochlear compound action potentials, has been proposed to contribute to hearing difficulty in noisy environments in people with normal hearing thresholds and has become a widespread complaint. While most studies on HHL pathogenesis have focused on inner hair cell (IHC) synaptopathy, we recently showed that transient auditory nerve (AN) demyelination also causes HHL in mice. To test the effect of myelinopathy on hearing in a clinically relevant model, we studied a mouse model of Charcot-Marie-Tooth type 1A (CMT1A), the most prevalent hereditary peripheral neuropathy in humans. CMT1A mice exhibited the functional hallmarks of HHL together with disorganization of AN heminodes near the IHCs with minor loss of AN fibers. These results support the hypothesis that mild disruptions of AN myelination can cause HHL and that heminodal defects contribute to the alterations in the sound-evoked cochlear compound action potentials seen in this mouse model. Furthermore, these findings suggest that patients with CMT1A or other mild peripheral neuropathies are likely to suffer from HHL. Furthermore, these results suggest that studies of hearing in patients with CMT1A might help develop robust clinical tests for HHL, which are currently lacking.

Hidden hearing loss in a Charcot-Marie-Tooth type 1A mouse model.

Hidden hearing loss (HHL), a recently described auditory neuropathy characterized by normal audiometric thresholds but reduced sound-evoked cochlear compound action potentials, has been proposed to contribute to hearing difficulty in noisy environments in people with normal hearing thresholds, a widespread complaint. While most studies on HHL pathogenesis have focused on inner hair cell (IHC) synaptopathy, we recently showed that transient auditory nerve (AN) demyelination also causes HHL in mice. To test the impact of myelinopathy on hearing in a clinically relevant model, we studied a mouse model of Charcot-Marie-Tooth type 1A (CMT1A), the most prevalent hereditary peripheral neuropathy in humans. CMT1A mice exhibited the functional hallmarks of HHL together with disorganization of AN heminodes near the IHCs with minor loss of AN fibers. These results support the hypothesis that mild disruptions of AN myelination can cause HHL, and that heminodal defects contribute to the alterations in the sound-evoked cochlear compound action potentials seen in this mouse model. Also, these findings suggest that patients with CMT1A or other mild peripheral neuropathies are likely to suffer from HHL. Furthermore, these results suggest that studies of hearing in CMT1A patients might help develop robust clinical tests for HHL, which are currently lacking.

Abstract 1891: Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window

Abstract Nectin-4 is a well validated tumor target, which is highly expressed in several solid cancers such as urothelial, head-and-neck, breast, lung, pancreatic, ovarian, and cervical cancer, while expression in normal tissues is limited. A first-generation antibody-drug conjugate targeting Nectin-4, called enfortumab vedotin-ejfv (EV), was granted accelerated FDA approval in 2019 for urothelial cancers, and is currently approved for frontline therapy in cisplatin-ineligible patients with locally advanced or metastatic urothelial carcinoma, providing groundbreaking improvement in patient survival. However, clinical use of EV has also revealed safety-driven limitations, which result in reduced and/or interrupted drug exposures impacting efficacy. To overcome these limitations in therapeutic window and to maximize on the potential of Nectin-4 as a tumor target beyond urothelial cancers, Adcentrx has developed ADRX-0706, a Nectin-4-targeting next-generation ADC with a drug-antibody ratio of 8. ADRX-0706 is comprised of a novel fully human IgG1k monoclonal antibody linked to an optimized tubulin inhibitor payload AP052 through a proprietary cleavable linker. ADRX-0706 has strong binding affinity and selectively induces cytotoxicity in Nectin-4-expressing tumor cells. Importantly, ADRX-0706 is designed to have improved bystander effect, which allows tumor regression even in cases of heterogeneous Nectin-4 expression. This translates to superior in vivo efficacy of ADRX-0706 over EV in multiple mouse xenograft models of other indications beyond bladder cancer. In a mouse clinical trial of patient-derived cervical cancers with a range of Nectin-4 expression levels, ADRX-0706 reaches a 73% ORR. Since peripheral neuropathy is a main platform toxicity for this payload class, ADRX-0706 is assessed in an in vitro co-culture model to evaluate its neurotoxic potential. While EV displays a 75% decrease in neurite outgrowth, ADRX-0706 strikingly has no impact at concentrations up to 1mg/ml, suggesting a lower risk of peripheral neuropathy in humans for ADRX-0706. In preclinical repeat dose toxicology study, ADRX-0706 reaches an HNSTD of 18mg/kg in NHPs well above therapeutic exposures in mouse pharmacology studies, indicating a tremendous increase in the therapeutic index compared to EV. In summary, the highly selective and potent anti-tumor activity in multiple tumor types and wide preclinical therapeutic index of ADRX-0706 support clinical evaluation of this next generation anti-Nectin-4 ADC. ADRX-0706 is currently in a phase 1a/b study (NCT06036121). Citation Format: Andrew M. Hau, Maria Shahmoradgoli, Dong Jun Lee, Wes Sisson, Anna Wang, Peter P. Challita, Erin Nye, Mario M. Kuo, Alexis Mahloch, Monica Leung, Oscar Betancourt, Alexander Chu-Kung, Maojun Guo, Hui Li, Sabine Rottmann, Pia M. Challita-Eid. Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1891.

Investigation of the effects of berberine on bortezomib-induced sciatic nerve and spinal cord damage in rats through pathways involved in oxidative stress and neuro-inflammation

Bortezomib (BTZ), a proteasome inhibitor, causes dose-limiting peripheral neuropathy in humans. Berberine (BBR), which has various biological and pharmacological properties, is known to have neuroprotective properties. The possible protective effects of BBR on peripheral neuropathy caused by BTZ were investigated in this study. For this purpose, BTZ was intraperitoneally given to Sprague dawley rats on the 1 st, 3rd, 5th, and 7th days with a cumulative dose of 0.8 mg/kg. Moreover, animals were orally administered 50 or 100 mg/kg BBR daily from day 1 to day 10. As a result of the analyzes performed on the sciatic nerve and spinal cord, it was observed that MDA levels and NRF-2, HO-1, NQO1, GCLC and GCLM mRNA transcript levels increased due to oxidative stress caused by BTZ, and the levels of these markers decreased after BBR administration. Also, it was determined that SOD, CAT, GPx and GSH levels increased after BBR treatment. It was observed that BTZ caused inflammation by triggering NF-κB, TNF-α, IL-1β and IL-6 cytokines, on the other hand, with BBR treatment, these cytokines were suppressed and inflammation was alleviated. In addition, it was determined that the expressions of RAGE, STAT3, NLRP3 and TLR4, which have important roles in inflammation, increased with BTZ administration, but BBR suppressed the expressions of these genes. It was determined that the expressions of SIRT1, which plays an important role in neuropathic pain, and CREB-LI neurons, which has an active role in neurite outgrowth and survival, decreased with BTZ administration. It was observed that GFAP levels increased with BTZ administration and decreased with BBR administration. Given all the findings, it was concluded that BBR exhibits protective qualities in the sciatic nerve and spinal cord induced by BTZ.

GDAP1 Involvement in Mitochondrial Function and Oxidative Stress, Investigated in a Charcot-Marie-Tooth Model of hiPSCs-Derived Motor Neurons.

Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) gene have been associated with demyelinating and axonal forms of Charcot-Marie-Tooth (CMT) disease, the most frequent hereditary peripheral neuropathy in humans. Previous studies reported the prevalent GDAP1 expression in neural tissues and cells, from animal models. Here, we described the first GDAP1 functional study on human induced-pluripotent stem cells (hiPSCs)-derived motor neurons, obtained from normal subjects and from a CMT2H patient, carrying the GDAP1 homozygous c.581C>G (p.Ser194*) mutation. At mRNA level, we observed that, in normal subjects, GDAP1 is mainly expressed in motor neurons, while it is drastically reduced in the patient’s cells containing a premature termination codon (PTC), probably degraded by the nonsense-mediated mRNA decay (NMD) system. Morphological and functional investigations revealed in the CMT patient’s motor neurons a decrease of cell viability associated to lipid dysfunction and oxidative stress development. Mitochondrion is a key organelle in oxidative stress generation, but it is also mainly involved in energetic metabolism. Thus, in the CMT patient’s motor neurons, mitochondrial cristae defects were observed, even if no deficit in ATP production emerged. This cellular model of hiPSCs-derived motor neurons underlines the role of mitochondrion and oxidative stress in CMT disease and paves the way for new treatment evaluation.

Prolonged use of statins and peripheral neuropathy: A systematic review

Peripheral neuropathy is one of the most common neurological disorder. Traditionally, diabetes mellitus is the most common aetiology of peripheral neuropathy. However, there are many other aetiologies of peripheral neuropathy, such as drug-induced, spinal trauma, alcoholism, vitamin B12 deficiency, and many more. In previous studies, long-term statin use was also associated with peripheral neuropathy. We conducted a systematic search through PubMed and Embase using the protocol following the PRISMA statement in making a systematic review. W e included article that published in the last 10 years (from 2011 until July 2020). Two reviewers extracted data from the articles that have been selected. We extracted the study population, study design, type of intervention, control variable, p-value, and the outcome of the study We found 250 journals in our search strategy, and at the end, 4 journals included. Some studies revealed that peripheral neuropathy was observed during the long-term treatment of statin. However, some studies also revealed that statin does not increase the risk of peripheral neuropathy. The possible mechanism of statin-induced peripheral neuropathy in humans are disturbing the function and integrity of cell membrane, the role of alfa-tocopherol or Vitamin E, and the role of ubiquinone (Coenzyme Q10). In contrast, it is believed that statins lowering the risk of peripheral neuropathy through the anti-inflammatory effects of statins. Different result from each study can be caused by various factors. Besides the peripheral neuropathy side effect that described in the literature, statins are well tolerated. Awareness the side effects would lead to better treatment. Future study about association between statins and peripheral neuropathy is needed.

Nerve Conduction and Neuromuscular Transmission in C57Bl/6 Mice with Genetically Determined Peripheral Neuropathy

Charcot–Marie–Tooth disease is one of the most widespread forms of hereditary peripheral neuropathy in humans. C57Bl/6 mice are considered the most appropriate animal model for studies of this disease. We measured the conduction velocity in the sciatic nerve (NCV) and amplitude of the M-wave in mice of strains C57Bl/6 and C57Bl. It was found that the mean conduction velocity in the right and left sciatic nerves of C57Bl/6 mice was about 3.5-fold lower than that in C57Bl animals. In the former mice, the mean amplitude of compound nerve action potentials (CNAPs) and that of the M-wave in the m. gastrocnemius-soleus after stimulation of the sciatic nerve were 5- and 4-fold, respectively, lower, than those in the control (C57B1). Thus, the data obtained show that the genetically determined pathology of the peripheral nervous system caused by a mutation of the PMP22 gene results in dramatic negative shifts of the characteristics of conduction via the peripheral nerves and of neuromuscular trans-mission.

Hepatic disorder is a risk factor for aggravation of oxaliplatin-induced peripheral neuropathy in humans and mice: Possible involvement of HMGB1

Hepatic disorder is a risk factor for aggravation of oxaliplatin-induced peripheral neuropathy in humans and mice: Possible involvement of HMGB1

Novel Familial Variant of the Desert Hedgehog Gene: Clinical Findings in Two Sisters with 46,XY Gonadal Dysgenesis or 46,XX Karyotype and Literature Review

Background: In humans, Desert Hedgehog (DHH) gene mutations are a very rare cause of 46,XY gonadal dysgenesis (GD), eventually associated with peripheral neuropathy. Patients and Methods: Clinical records of 12 patients with 46,XY GD and unknown genetic background were reviewed and a 46,XY woman with peripheral neuropathy was individuated. Her 46,XX sister affected by similar neuropathy was also investigated. Genomic DNA was extracted and DHH exons sequenced and analyzed. A comparative genomic hybridization array was also performed. Results: In both the 46,XY and 46,XX sisters, a homozygous c.554C>A mutation in exon 2 of the DHH gene was found, determining a premature termination codon (p.Ser 185*). Heterozygous consanguineous carrier parents showed neither reproductive problems nor peripheral neuropathy. In the proband and her sister, a 499-kb duplication in 9p22.1 was also found. Conclusion: A 46,XY European woman with 46,XY GD and a novel homozygous DHH pathogenic variant is reported, confirming that this gene plays a key role in male gonadal development. Her 46,XX sister, harboring the same mutation, showed normal internal and external female phenotype. Thus, DHH seems not to be involved in the ovarian development pathway or its postpubertal function. Homozygous DHH mutations cause a specific peripheral neuropathy in humans with both 46,XY and 46,XX karyotypes.

A GJA9 frameshift variant is associated with polyneuropathy in Leonberger dogs

BackgroundMany inherited polyneuropathies (PN) observed in dogs have clinical similarities to the genetically heterogeneous group of Charcot-Marie-Tooth (CMT) peripheral neuropathies in humans. The canine disorders collectively show a variable expression of progressive clinical signs and ages of onset, and different breed prevalences. Previously in the Leonberger breed, a variant highly associated with a juvenile-onset PN was identified in the canine orthologue of a CMT-associated gene. As this deletion in ARHGEF10 (termed LPN1) does not explain all cases, PN in this breed may encompass variants in several genes with similar clinical and histopathological features.ResultsA genome-wide comparison of 173 k SNP genotypes of 176 cases, excluding dogs homozygous for the ARHGEF10 variant, and 138 controls, was carried out to detect further PN-associated variants. A single suggestive significant association signal on CFA15 was found. The genome of a PN-affected Leonberger homozygous for the associated haplotype was sequenced and variants in the 7.7 Mb sized critical interval were identified. These variants were filtered against a database of variants observed in 202 genomes of various dog breeds and 3 wolves, and 6 private variants in protein-coding genes, all in complete linkage disequilibrium, plus 92 non-coding variants were revealed. Five of the coding variants were predicted to have low or moderate effect on the encoded protein, whereas a 2 bp deletion in GJA9 results in a frameshift of high impact. GJA9 encodes connexin 59, a connexin gap junction family protein, and belongs to a group of CMT-associated genes that have emerged as important components of peripheral myelinated nerve fibers. The association between the GJA9 variant and PN was confirmed in an independent cohort of 296 cases and 312 controls. Population studies showed a dominant mode of inheritance, an average age of onset of approximately 6 years, and incomplete penetrance.ConclusionsThis GJA9 variant represents a highly probable candidate variant for another form of PN in Leonberger dogs, which we have designated LPN2, and a new candidate gene for CMT disease. To date, approximately every third PN-diagnosed Leonberger dog can be explained by the ARHGEF10 or GJA9 variants, and we assume that additional genetic heterogeneity in this condition exists in the breed.

A model for in vivo analysis of sudomotor sympathetic C-fiber activation and human sweat gland output.

Quantitative assessment of small-fiber peripheral neuropathy often involves an evaluation of the interaction between the C-fiber sudomotor nerve and local sweat rate (SR). Typically, some sort of quantitative sudomotor axon reflex test (QSART) is performed to aid in diagnosing small-fiber dysfunction. The currently used QSART demonstrates only moderate test-retest reliability and therefore limits its usefulness in tracking small-fiber dysfunction. A new experimental model to examine small C-fiber function in the skin using intradermal electrical stimulation and simultaneous monitoring of SR is proposed. Using intradermal electrical stimulation (1.5 and 2.5 mA) and varying stimulus frequency from 0.2 to 64 Hz, a quantitative relationship between the area under the SR-time curve and log10 stimulus frequency is modeled using a four-parameter logistic equation, providing the following parameters: baseline, plateau, EC50, and Hill slope. The model has good to excellent repeatability within the same day (ICC = 0.98), on different days at the same skin site (ICC = 0.80), and when comparing two different skin sites (ICC = 0.78) with a small bias estimate and the line of identity always lying within the 95% limits of agreement. Atropine sulfate (0.1 mg/ml) blocked 90 ± 5% of the electrically induced sweating. Overall, the model provides control over sudomotor nerve activity and a quantitative assessment of SR. Finally, the ability to reproduce the quantitative stimulus-response curve on different days allows for a robust assessment of the relationship between the activation of a sympathetic C-fiber and local SR.NEW & NOTEWORTHY A model for quantitative assessment of C-fiber function in human skin using intradermal electrical stimulation and local sweat rate measurements has been developed. This new electrically induced sweating model is nonpainful and allows for a complete stimulus-response curve plotting the area under the local sweat rate-time curve vs. the log10 stimulus frequency. The model has good reproducibility and should provide a means of assessing the progression of small C-fiber peripheral neuropathy in humans.

The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

Peripheral neuropathy is dose limiting in paclitaxel cancer chemotherapy and can result in both acute pain during treatment and chronic persistent pain in cancer survivors. The hypothesis tested was that paclitaxel produces these adverse effects at least in part by sensitizing transient receptor potential vanilloid subtype 1 (TRPV1) through Toll-like receptor 4 (TLR4) signaling. The data show that paclitaxel-induced behavioral hypersensitivity is prevented and reversed by spinal administration of a TRPV1 antagonist. The number of TRPV1(+) neurons is increased in the dorsal root ganglia (DRG) in paclitaxel-treated rats and is colocalized with TLR4 in rat and human DRG neurons. Cotreatment of rats with lipopolysaccharide from the photosynthetic bacterium Rhodobacter sphaeroides (LPS-RS), a TLR4 inhibitor, prevents the increase in numbers of TRPV1(+) neurons by paclitaxel treatment. Perfusion of paclitaxel or the archetypal TLR4 agonist LPS activated both rat DRG and spinal neurons directly and produced acute sensitization of TRPV1 in both groups of cells via a TLR4-mediated mechanism. Paclitaxel and LPS sensitize TRPV1 in HEK293 cells stably expressing human TLR4 and transiently expressing human TRPV1. These physiological effects also are prevented by LPS-RS. Finally, paclitaxel activates and sensitizes TRPV1 responses directly in dissociated human DRG neurons. In summary, TLR4 was activated by paclitaxel and led to sensitization of TRPV1. This mechanism could contribute to paclitaxel-induced acute pain and chronic painful neuropathy. Significance statement: In this original work, it is shown for the first time that paclitaxel activates peripheral sensory and spinal neurons directly and sensitizes these cells to transient receptor potential vanilloid subtype 1 (TRPV1)-mediated capsaicin responses via Toll-like receptor 4 (TLR4) in multiple species. A direct functional interaction between TLR4 and TRPV1 is shown in rat and human dorsal root ganglion neurons, TLR4/TRPV1-coexpressing HEK293 cells, and in both rat and mouse spinal cord slices. Moreover, this is the first study to show that this interaction plays an important role in the generation of behavioral hypersensitivity in paclitaxel-related neuropathy. The key translational implications are that TLR4 and TRPV1 antagonists may be useful in the prevention and treatment of chemotherapy-induced peripheral neuropathy in humans.

PGC-1α regulation of mitochondrial degeneration in experimental diabetic neuropathy

Mitochondrial degeneration is considered to play an important role in the development of diabetic peripheral neuropathy in humans. Mitochondrial degeneration and the corresponding protein regulation associated with the degeneration were studied in an animal model of diabetic neuropathy. PGC-1α and its-regulated transcription factors including TFAM and NRF1, which are master regulators of mitochondrial biogenesis, are significantly downregulated in streptozotocin diabetic dorsal root ganglion (DRG) neurons. Diabetic mice develop peripheral neuropathy, loss of mitochondria, decreased mitochondrial DNA content and increased protein oxidation. Importantly, this phenotype is exacerbated in PGC-1α (−/−) diabetic mice, which develop a more severe neuropathy with reduced mitochondrial DNA and a further increase in protein oxidation. PGC-1α (−/−) diabetic mice develop an increase in total cholesterol and triglycerides, and a decrease in TFAM and NRF1 protein levels. Loss of PGC-1α causes severe mitochondrial degeneration with vacuolization in DRG neurons, coupled with reduced state 3 and 4 respiration, reduced expression of oxidative stress response genes and an increase in protein oxidation. In contrast, overexpression of PGC-1α in cultured adult mouse neurons prevents oxidative stress associated with increased glucose levels. The study provides new insights into the role of PGC-1α in mitochondrial regeneration in peripheral neurons and suggests that therapeutic modulation of PGC-1α function may be an attractive approach for treatment of diabetic neuropathy.

Restoration of Cytoskeleton Homeostasis After Gigaxonin Gene Transfer for Giant Axonal Neuropathy

Giant axonal neuropathy (GAN) is caused by loss of function of the gigaxonin protein. On a cellular level GAN is characterized by intermediate filament (IF) aggregation, leading to a progressive and fatal peripheral neuropathy in humans. This study sought to determine if re-introduction of the GAN gene into GAN-deficient cells and mice would restore proper cytoskeleton IF homeostasis. Treatment of primary skin fibroblast cultures from three different GAN patients with an adeno-associated virus type 2 (AAV2) vector containing a normal human GAN transgene significantly reduced the number of cells displaying vimentin IF aggregates. A proteomic analysis of these treated cells was also performed, wherein the abundance of 32 of 780 identified proteins significantly changed in response to gigaxonin gene transfer. While 29 of these responding proteins have not been directly described in association with gigaxonin, three were previously identified as being disregulated in GAN and were now shifted toward normal levels. To assess the potential application of this approach in vivo and eventually in humans, GAN mice received an intracisternal injection of an AAV9/GAN vector to globally deliver the GAN gene to the brainstem and spinal cord. The treated mice showed a nearly complete clearance of peripherin IF accumulations at 3 weeks post-injection. These studies demonstrate that gigaxonin gene transfer can reverse the cellular IF aggregate pathology associated with GAN.

Cannabidiol Prevents the Development of Cold and Mechanical Allodynia in Paclitaxel-Treated Female C57Bl6 Mice

The taxane chemotherapeutic paclitaxel frequently produces peripheral neuropathy in humans. Rodent models to investigate mechanisms and treatments are largely restricted to male rats, whereas female mouse studies are lacking. We characterized a range of paclitaxel doses on cold and mechanical allodynia in male and female C57Bl/6 mice. Because the nonpsychoactive phytocannabinoid cannabidiol attenuates other forms of neuropathic pain, we assessed its effect on paclitaxel-induced allodynia. Paclitaxel produced allodynia that was largely dose independent and more robust in female mice, and this effect was prevented by treatment with cannabidiol. Our preliminary findings therefore indicate that cannabidiol may prevent the development of paclitaxel-induced allodynia in mice and therefore be effective at preventing dose-limiting paclitaxel-induced peripheral neuropathy in humans.

Drosophila as a platform to predict the pathogenicity of novel aminoacyl-tRNA synthetase mutations in CMT

Charcot-Marie-Tooth disease (CMT) is the major form of inherited peripheral neuropathy in humans. CMT is clinically and genetically heterogeneous andfour aminoacyl-tRNA synthetases have been implicated in disease etiology. Mutations in the YARS gene encoding a tyrosyl-tRNA synthetase (TyrRS) lead to Dominant Intermediate CMT type C (DI-CMTC). Three dominant YARS mutations were so far associated with DI-CMTC. To further expand the spectrum of CMT causing genetic defects in this tRNA synthetase, we performed DNA sequencing of YARS coding regions in a cohort of 181 patients with various types of peripheral neuropathy. We identified a novel K265N substitution that in contrast to all previously described mutations is located at the anticodon recognition domain of the enzyme. Further genetic analysis revealed that this variant represents a benign substitution. Using our recently developed DI-CMTC Drosophila model, we tested in vivo the pathogenicity of this new YARS variant. We demonstrated that the developmental and behavioral defects induced by all DI-CMTC causing mutations were not present upon ubiquitous or panneuronal TyrRS K265N expression. Thus, in line with our genetic studies, functional analysis confirmed that the K265N substitution does not induce toxicity signs in Drosophila. The consistency observed throughout this work underscores the robustness of our DI-CMTC animal model and identifies Drosophila as a valid read-out platform to ascertain the pathogenicity of novel mutations to be identified in the future.

Attenuation of MCP-1/CCL2 expression ameliorates neuropathy in a mouse model for Charcot–Marie–Tooth 1X

The chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) has been previously shown to be an important mediator of macrophage-related neural damage in models of two distinct inherited neuropathies, Charcot-Marie-Tooth (CMT) 1A and 1B. In mice deficient in the gap junction protein connexin 32 (Cx32def), an established model for the X-chromosome-linked dominant form of CMT (CMT1X), we investigated the role of the chemokine in macrophage immigration and neural damage by crossbreeding the Cx32def mice with MCP-1 knockout mutants. In Cx32def mutants typically expressing increased levels of MCP-1, macrophage numbers were strongly elevated, caused by an MCP-1-mediated influx of haematogenous macrophages. Curiously, the complete genetic deletion of MCP-1 did not cause reduced macrophage numbers in the nerves due to compensatory proliferation of resident macrophages. In contrast, and as already seen in other CMT models, heterozygous deletion of MCP-1 led to reduced numbers of phagocytosing macrophages and an alleviation of demyelination. Whereas alleviated demyelination was transient, axonal damage was persistently improved and even robust axonal sprouting was detectable at 12 months. Other axon-related features were alleviated electrophysiological parameters, reduced muscle denervation and atrophy, and increased muscle strength. Similar to models for CMT1A and CMT1B, we identified MEK-ERK signalling as mediating MCP-1 expression in Cx32-deficient Schwann cells. Blocking this pathway by the inhibitor CI-1040 caused reduced MCP-1 expression, attenuation of macrophage increase and amelioration of myelin- and axon-related alterations. Thus, attenuation of MCP-1 upregulation by inhibiting ERK phosphorylation might be a promising approach to treat CMT1X and other so far untreatable inherited peripheral neuropathies in humans.

Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage

Mutations in the transcription factor SOX10 cause neurocristopathies, including Waardenburg-Hirschsprung syndrome and peripheral neuropathies in humans. This is partly attributed to a requirement for Sox10 in early neural crest for survival, maintenance of pluripotency, and specification to several cell lineages, including peripheral glia. As a consequence, peripheral glia are absent in Sox10-deficient mice. Intriguingly, Sox10 continues to be expressed in these cells after specification. To analyze glial functions after specification, we specifically deleted Sox10 in immature Schwann cells by conditional mutagenesis. Mutant mice died from peripheral neuropathy before the seventh postnatal week. Nerve alterations included a thinned perineurial sheath, increased lipid and collagen deposition, and a dramatically altered cellular composition. Nerve conduction was also grossly aberrant, and neither myelinating nor nonmyelinating Schwann cells formed. Instead, axons of different sizes remained unsorted in large bundles. Schwann cells failed to develop beyond the immature stage and were unable to maintain identity. Thus, our study identifies a novel cause for peripheral neuropathies in patients with SOX10 mutations.

N,N-diethyldithiocarbamate promotes oxidative stress prior to myelin structural changes and increases myelin copper content

Dithiocarbamates are a commercially important class of compounds that can produce peripheral neuropathy in humans and experimental animals. Previous studies have supported a requirement for copper accumulation and enhanced lipid peroxidation in dithiocarbamate-mediated myelinopathy. The study presented here extends previous investigations in two areas. Firstly, although total copper levels have been shown to increase within the nerve it has not been determined whether copper is increased within the myelin compartment, the primary site of lesion development. Therefore, the distribution of copper in sciatic nerve was characterized using synchrotron X-ray fluorescence microscopy to determine whether the neurotoxic dithiocarbamate, N,N-diethyldithiocarbamate, increases copper levels in myelin. Secondly, because lipid peroxidation is an ongoing process in normal nerve and the levels of lipid peroxidation products produced by dithiocarbamate exposure demonstrated an unusual cumulative dose response in previous studies the biological impact of dithiocarbamate-mediated lipid peroxidation was evaluated. Experiments were performed to determine whether dithiocarbamate-mediated lipid peroxidation products elicit an antioxidant response through measuring the protein expression levels of three enzymes, superoxide dismutase 1, heme oxygenase 1, and glutathione transferase α, that are linked to the antioxidant response element promoter. To establish the potential of oxidative injury to contribute to myelin injury the temporal relationship of the antioxidant response to myelin injury was determined. Myelin structure in peripheral nerve was assessed using multi-exponential transverse relaxation measurements (MET 2) as a function of exposure duration, and the temporal relationship of protein expression changes relative to the onset of changes in myelin integrity were determined. Initial assessments were also performed to explore the potential contribution of dithiocarbamate-mediated inhibition of proteasome function and inhibition of cuproenzyme activity to neurotoxicity, and also to assess the potential of dithiocarbamates to promote oxidative stress and injury within the central nervous system. These evaluations were performed using an established model for dithiocarbamate-mediated demyelination in the rat utilizing sciatic nerve, spinal cord and brain samples obtained from rats exposed to N,N-diethyldithiocarbamate (DEDC) by intra-abdominal pumps for periods of 2, 4, and 8 weeks and from non exposed controls. The data supported the ability of DEDC to increase copper within myelin and to enhance oxidative stress prior to structural changes detectable by MET 2. Evidence was also obtained that the excess copper produced by DEDC in the central nervous system is redox active and promotes oxidative injury.