Treatment with IFB-088 Improves Neuropathy in CMT1A and CMT1B Mice.

Charcot-Marie-Tooth disease type 1A (CMT1A), caused by duplication of the peripheral myelin protein 22 (PMP22) gene, and CMT1B, caused by mutations in myelin protein zero (MPZ) gene, are the two most common forms of demyelinating CMT (CMT1), and no treatments are available for either. Prior studies of the MpzSer63del mouse model of CMT1B have demonstrated that protein misfolding, endoplasmic reticulum (ER) retention and activation of the unfolded protein response (UPR) contributed to the neuropathy. Heterozygous patients with an arginine to cysteine mutation in MPZ (MPZR98C) develop a severe infantile form of CMT1B which is modelled by MpzR98C/ + mice that also show ER stress and an activated UPR. C3-PMP22 mice are considered to effectively model CMT1A. Altered proteostasis, ER stress and activation of the UPR have been demonstrated in mice carrying Pmp22 mutations. To determine whether enabling the ER stress/UPR and readjusting protein homeostasis would effectively treat these models of CMT1B and CMT1A, we administered Sephin1/IFB-088/icerguestat, a UPR modulator which showed efficacy in the MpzS63del model of CMT1B, to heterozygous MpzR98C and C3-PMP22 mice. Mice were analysed by behavioural, neurophysiological, morphological and biochemical measures. Both MpzR98C/ + and C3-PMP22 mice improved in motor function and neurophysiology. Myelination, as demonstrated by g-ratios and myelin thickness, improved in CMT1B and CMT1A mice and markers of UPR activation returned towards wild-type values. Taken together, our results demonstrate the capability of IFB-088 to treat a second mouse model of CMT1B and a mouse model of CMT1A, the most common form of CMT. Given the recent benefits of IFB-088 treatment in amyotrophic lateral sclerosis and multiple sclerosis animal models, these data demonstrate its potential in managing UPR and ER stress for multiple mutations in CMT1 as well as in other neurodegenerative diseases. (Left panel) the accumulation of overexpressed PMP22 or misfolded mutant P0 in the Schwann cell endoplasmic reticulum (ER) leads to overwhelming of the degradative capacity, activation of ER-stress mechanisms, and myelination impairment. (Right panel) by prolonging eIF2α phosphorylation, IFB-088 reduces the amount of newly synthesized proteins entering the ER, allowing the protein quality control systems to better cope with the unfolded/misfolded protein and allowing myelination to progress.

Hereditary neuropathies: A pathological perspective.

Hereditary neuropathies may result from mutations in genes expressed by Schwann cells or neurons that affect selectively the peripheral nervous system (PNS) or may represent a minor or major component of complex inherited diseases that involve also the central nervous system and/or other organs and tissues. The chapter is constantly expanding and reworking, thanks to advances of molecular genetics; next-generation sequencing is identifying a plethora of new genes and is revolutionizing the diagnostic approach. In the past, diagnostic sural nerve biopsies paved the way to the discovery and elucidation of major genes and molecular pathways associated to most frequent hereditary motor-sensory neuropathies. Nowadays, a sural nerve biopsy may prove useful in selected cases for the differential diagnosis of an acquired neuropathy when clinical examination, nerve conduction studies, and molecular tests are not sufficiently informative. Skin biopsy has emerged as a minimally invasive window on the PNS, which may provide biomarkers of progression and clues to the physiopathology and molecular pathology of inherited neuropathies. The aim of our review is to illustrate the pathological features of more frequent and paradigmatic hereditary neuropathies and to highlight their correlations with the roles of the involved genes and functional consequences of related molecular defects.

MicroRNAs as Biomarkers of Charcot-Marie-Tooth Disease Type 1A.

OBJECTIVE: To determine whether microRNAs (miRs) are elevated in the plasma of individuals with the inherited peripheral neuropathy Charcot-Marie-Tooth disease type 1A (CMT1A), miR profiling was employed to compare control and CMT1A plasma. METHODS: We performed a screen of CMT1A and control plasma samples to identify miRs that are elevated in CMT1A using next-generation sequencing, followed by validation of selected miRs by quantitative PCR, and correlation with protein biomarkers and clinical data: Rasch-modified CMT Examination and Neuropathy Scores, ulnar compound muscle action potentials, and motor nerve conduction velocities. RESULTS: After an initial pilot screen, a broader screen confirmed elevated levels of several muscle-associated miRNAs (miR1, -133a, -133b, and -206, known as myomiRs) along with a set of miRs that are highly expressed in Schwann cells of peripheral nerve. Comparison to other candidate biomarkers for CMT1A (e.g., neurofilament light) measured on the same sample set shows a comparable elevation of several miRs (e.g., miR133a, -206, -223) and ability to discriminate cases from controls. Neurofilament light levels were most highly correlated with miR133a. In addition, the putative Schwann cell miRs (e.g., miR223, -199a, -328, -409, -431) correlate with the recently described transmembrane protease serine 5 (TMPRSS5) protein biomarker that is most highly expressed in Schwann cells and also elevated in CMT1A plasma. CONCLUSIONS: These studies identify a set of miRs that are candidate biomarkers for clinical trials in CMT1A. Some of the miRs may reflect Schwann cell processes that underlie the pathogenesis of the disease. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that a set of plasma miRs are elevated in patients with CMT1A.

A CADM3 variant causes Charcot-Marie-Tooth disease with marked upper limb involvement.

The CADM family of proteins consists of four neuronal specific adhesion molecules (CADM1, CADM2, CADM3 and CADM4) that mediate the direct contact and interaction between axons and glia. In the peripheral nerve, axon-Schwann cell interaction is essential for the structural organization of myelinated fibres and is primarily mediated by the binding of CADM3, expressed in axons, to CADM4, expressed by myelinating Schwann cells. We have identified-by whole exome sequencing-three unrelated families, including one de novo patient, with axonal Charcot-Marie-Tooth disease (CMT2) sharing the same private variant in CADM3, Tyr172Cys. This variant is absent in 230 000 control chromosomes from gnomAD and predicted to be pathogenic. Most CADM3 patients share a similar phenotype consisting of autosomal dominant CMT2 with marked upper limb involvement. High resolution mass spectrometry analysis detected a newly created disulphide bond in the mutant CADM3 potentially modifying the native protein conformation. Our data support a retention of the mutant protein in the endoplasmic reticulum and reduced cell surface expression in vitro. Stochastic optical reconstruction microscopy imaging revealed decreased co-localization of the mutant with CADM4 at intercellular contact sites. Mice carrying the corresponding human mutation (Cadm3Y170C) showed reduced expression of the mutant protein in axons. Cadm3Y170C mice showed normal nerve conduction and myelin morphology, but exhibited abnormal axonal organization, including abnormal distribution of Kv1.2 channels and Caspr along myelinated axons. Our findings indicate the involvement of abnormal axon-glia interaction as a disease-causing mechanism in CMT patients with CADM3 mutations.

Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes.

Here we report biallelic mutations in the sorbitol dehydrogenase gene (SORD) as the most frequent recessive form of hereditary neuropathy. We identified 45 individuals from 38 families across multiple ancestries carrying the nonsense c.757delG (p.Ala253GlnfsTer27) variant in SORD, in either a homozygous or compound heterozygous state. SORD is an enzyme that converts sorbitol into fructose in the two-step polyol pathway previously implicated in diabetic neuropathy. In patient-derived fibroblasts, we found a complete loss of SORD protein and increased intracellular sorbitol. Furthermore, the serum fasting sorbitol levels in patients were dramatically increased. In Drosophila, loss of SORD orthologs caused synaptic degeneration and progressive motor impairment. Reducing the polyol influx by treatment with aldose reductase inhibitors normalized intracellular sorbitol levels in patient-derived fibroblasts and in Drosophila, and also dramatically ameliorated motor and eye phenotypes. Together, these findings establish a novel and potentially treatable cause of neuropathy and may contribute to a better understanding of the pathophysiology of diabetes.

Author Correction: Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transmembrane protease serine 5: a novel Schwann cell plasma marker for CMT1A.

OBJECTIVE: Development of biomarkers for Charcot-Marie-Tooth (CMT) disease is critical for implementing effective clinical trials. The most common form of CMT, type 1A, is caused by a genomic duplication surrounding the PMP22 gene. A recent report (Neurology 2018;90:e518-3524) showed elevation of neurofilament light (NfL) in plasma of CMT1A disease patients, which correlated with disease severity. However, no plasma/serum biomarker has been identified that is specific to Schwann cells, the most directly affected cells in CMT1A. METHODS: We used the Olink immuno PCR platform to profile CMT1A patient (n = 47, 2 cohorts) and normal control plasma (n = 41, two cohorts) on five different Olink panels to screen 398 unique proteins. RESULTS: The TMPRSS5 protein (Transmembrane protease serine 5) was elevated 2.07-fold (P = <0.0001) in two independent cohorts of CMT1A samples relative to controls. TMPRSS5 is most highly expressed in Schwann cells of peripheral nerve. Consistent with early myelination deficits in CMT1A, TMPRSS5 was not significantly correlated with disease score (CMTES-R, CMTNS-R), nerve conduction velocities (Ulnar CMAP, Ulnar MNCV), or with age. TMPRSS5 was not significantly elevated in smaller sample sets from patients with CMT2A, CMT2E, CMT1B, or CMT1X. The Olink immuno PCR assays confirmed elevated levels of NfL (average 1.58-fold, P < 0.0001), which correlated with CMT1A patient disease score. INTERPRETATION: These data identify the first Schwann cell-specific protein that is elevated in plasma of CMT1A patients, and may provide a disease marker and a potentially treatment-responsive biomarker with good disease specificity for clinical trials.

Schwann cell transcript biomarkers for hereditary neuropathy skin biopsies.

OBJECTIVE: Charcot-Marie-Tooth (CMT) disease is most commonly caused by duplication of a chromosomal segment surrounding Peripheral Myelin Protein 22, or PMP22 gene, which is classified as CMT1A. Several candidate therapies reduce Pmp22 mRNA levels in CMT1A rodent models, but development of biomarkers for clinical trials in CMT1A is a challenge given its slow progression and difficulty in obtaining nerve samples. Quantitative PCR measurements of PMP22 mRNA in dermal nerves were performed using skin biopsies in human clinical trials for CMT1A, but this approach did not show increased PMP22 mRNA in CMT1A patients compared to controls. One complicating factor is the variable amounts of Schwann cells (SCs) in skin. The objective of the study was to develop a novel method for precise evaluation of PMP22 levels in skin biopsies that can discriminate CMT1A patients from controls. METHODS: We have developed methods to normalize PMP22 transcript levels to SC-specific genes that are not altered by CMT1A status. Several CMT1A-associated genes were assembled into a custom Nanostring panel to enable precise transcript measurements that can be normalized to variable SC content. RESULTS: The digital expression data from Nanostring analysis showed reproducible elevation of PMP22 levels in CMT1A versus control skin biopsies, particularly after normalization to SC-specific genes. INTERPRETATION: This platform should be useful in clinical trials for CMT1A as a biomarker of target engagement that can be used to optimize dosing, and the same normalization framework is applicable to other types of CMT. ANN NEUROL 2019;85:887-898.

Myelin protein zero mutations and the unfolded protein response in Charcot Marie Tooth disease type 1B.

OBJECTIVE: To determine the prevalence of MPZ mutations that cause Charcot Marie Tooth neuropathy type 1B (CMT1B) and activate the unfolded protein Response (UPR). BACKGROUND: CMT1B is caused by >200 heterozygous mutations in MPZ, the major protein in peripheral nerve myelin. Mutations Ser63del MPZ and Arg98Cys MPZ cause the mutant protein to be retained in the ER and activate the generally adaptive UPR. Treatments that modulate UPR activation have improved cellular and rodent models of CMT1B raising the possibility that other MPZ mutations that activate the UPR would also respond favorably to similar treatment. The prevalence of MPZ mutations that activate the UPR is unknown. METHODS: We developed a dual luciferase reporter assay of Xbp1 splicing using stably transfected RT4 Schwann cells to assay the ability of cDNA constructs bearing 46 distinct MPZ mutations to activate the UPR. Constructs also carried an HA tag to permit detection of ER retention of mutant proteins. UPR activation and ER retention were correlated with clinical phenotypes. RESULTS: Eighteen mutations demonstrated ER retention and UPR activation to a similar degree as Ser63del and Arg98Cys MPZ. Thirty-five of the mutations activated the UPR > 1.5 fold compared to that of wild-type MPZ. Correlation was high between firefly and Nano-luciferase reporters and between both reporters and ER localization. UPR activity did not correlate with clinical onset or severity. CONCLUSION: Many CMT1B causing mutations activate the UPR and may be susceptible to therapeutic efforts to facilitate UPR function.

Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2.

Although mutations in more than 90 genes are known to cause CMT, the underlying genetic cause of CMT remains unknown in more than 50% of affected individuals. The discovery of additional genes that harbor CMT2-causing mutations increasingly depends on sharing sequence data on a global level. In this way-by combining data from seven countries on four continents-we were able to define mutations in ATP1A1, which encodes the alpha1 subunit of the Na+,K+-ATPase, as a cause of autosomal-dominant CMT2. Seven missense changes were identified that segregated within individual pedigrees: c.143T>G (p.Leu48Arg), c.1775T>C (p.Ile592Thr), c.1789G>A (p.Ala597Thr), c.1801_1802delinsTT (p.Asp601Phe), c.1798C>G (p.Pro600Ala), c.1798C>A (p.Pro600Thr), and c.2432A>C (p.Asp811Ala). Immunostaining peripheral nerve axons localized ATP1A1 to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures of myelin sheaths. Two-electrode voltage clamp measurements on Xenopus oocytes demonstrated significant reduction in Na+ current activity in some, but not all, ouabain-insensitive ATP1A1 mutants, suggesting a loss-of-function defect of the Na+,K+ pump. Five mutants fall into a remarkably narrow motif within the helical linker region that couples the nucleotide-binding and phosphorylation domains. These findings identify a CMT pathway and a potential target for therapy development in degenerative diseases of peripheral nerve axons.

PMP22 exon 4 deletion causes ER retention of PMP22 and a gain-of-function allele in CMT1E.

OBJECTIVE: To determine whether predicted fork stalling and template switching (FoSTeS) during mitosis deletes exon 4 in peripheral myelin protein 22 KD (PMP22) and causes gain-of-function mutation associated with peripheral neuropathy in a family with Charcot-Marie-Tooth disease type 1E. METHODS: Two siblings previously reported to have genomic rearrangements predicted to involve exon 4 of PMP22 were evaluated clinically and by electrophysiology. Skin biopsies from the proband were studied by RT-PCR to determine the effects of the exon 4 rearrangements on exon 4 mRNA expression in myelinating Schwann cells. Transient transfection studies with wild-type and mutant PMP22 were performed in Cos7 and RT4 cells to determine the fate of the resultant mutant protein. RESULTS: Both affected siblings had a sensorimotor dysmyelinating neuropathy with severely slow nerve conduction velocities (<10 m/sec). RT-PCR studies of Schwann cell RNA from one of the siblings demonstrated a complete in-frame deletion of PMP22 exon 4 (PMP22Δ4). Transfection studies demonstrated that PMP22Δ4 protein is retained within the endoplasmic reticulum and not transported to the plasma membrane. CONCLUSIONS: Our results confirm that that FoSTeS-mediated genomic rearrangement produced a deletion of exon 4 of PMP22, resulting in expression of both PMP22 mRNA and protein lacking this sequence. In addition, we provide experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain-of-function mutational mechanism consistent with the observed CMT1E in this family. PMP22Δ4 is another example of a mutated myelin protein that is misfolded and contributes to the pathogenesis of the neuropathy.

Absence of Dystrophin Related Protein-2 disrupts Cajal bands in a patient with Charcot-Marie-Tooth disease.

Using exome sequencing in an individual with Charcot-Marie-Tooth disease (CMT) we have identified a mutation in the X-linked dystrophin-related protein 2 (DRP2) gene. A 60-year-old gentleman presented to our clinic and underwent clinical, electrophysiological and skin biopsy studies. The patient had clinical features of a length dependent sensorimotor neuropathy with an age of onset of 50 years. Neurophysiology revealed prolonged latencies with intermediate conduction velocities but no conduction block or temporal dispersion. A panel of 23 disease causing genes was sequenced and ultimately was uninformative. Whole exome sequencing revealed a stop mutation in DRP2, c.805C>T (Q269*). DRP2 interacts with periaxin and dystroglycan to form the periaxin-DRP2-dystroglycan complex which plays a role in the maintenance of the well-characterized Cajal bands of myelinating Schwann cells. Skin biopsies from our patient revealed a lack of DRP2 in myelinated dermal nerves by immunofluorescence. Furthermore electron microscopy failed to identify Cajal bands in the patient's dermal myelinated axons in keeping with ultrastructural pathology seen in the Drp2 knockout mouse. Both the electrophysiologic and dermal nerve twig pathology support the interpretation that this patient's DRP2 mutation causes characteristic morphological abnormalities recapitulating the Drp2 knockout model and potentially represents a novel genetic cause of CMT.

Reduced neurofilament expression in cutaneous nerve fibers of patients with CMT2E.

OBJECTIVE: To investigate the effects of NEFL Glu396Lys mutation on the expression and assembly of neurofilaments (NFs) in cutaneous nerve fibers of patients with Charcot-Marie-Tooth disease type 2E (CMT2E). METHODS: A large family with CMT2E underwent clinical, electrophysiologic, and skin biopsy studies. Biopsies were processed by indirect immunofluorescence (IF), electron microscopy (EM), and Western blot analysis. RESULTS: The clinical features demonstrated intrafamilial phenotypic variability, and the electrophysiologic findings revealed nerve conductions that were either slow or in the intermediate range. All patients had reduced or absent compound muscular action potential amplitudes. Skin biopsies showed axons labeled with the axonal markers protein gene product 9.5 and α-tubulin, but not with NFs. The results of Western blot analysis were consistent with those of IF, showing reduced or absent NFs and normal expression of α-tubulin. EM revealed clusters of regenerated fibers, in absence of myelin sheath abnormalities. Both IF and EM failed to show NF aggregates in dermal axons. The morphometric analysis showed a smaller axonal caliber in patients than in controls. The study of the nodal/paranodal architecture demonstrated that sodium channels and Caspr were correctly localized in patients with CMT2E. CONCLUSIONS: Decrease in NF abundance may be a pathologic marker of CMT2E. The lack of NF aggregates, consistent with prior studies, suggests that they occur proximally leading to subsequent alterations in the axonal cytoskeleton. The small axonal caliber, along with the normal molecular architecture of nodes and paranodes, explain the reduced velocities detected in patients with CMT2E. Our results also demonstrate that skin biopsy can provide evidence of pathologic and pathogenic abnormalities in patients with CMT2E.

Demyelinating CMT--what's known, what's new and what's in store?

Inherited neuropathies known collectively as Charcot-Marie-Tooth disease are one of the most common inherited neurological conditions affecting ∼1 in 2500 people. A heterogenous disorder, CMT is divided into subtypes based on the pattern of inheritance and also by neurophysiological studies. Despite the clinical similarities among patients with demyelinating CMT, it is recognized that this group of disorders is both genetically and phenotypically heterogenous. Understanding the pathogenesis of these disorders requires an intimate knowledge of normal myelin development and homeostasis. Improvements in genetic testing techniques over the last 20 years have contributed majorly to the identification of specific genes, proteins, and molecular pathways that are providing the basis for understanding the disease processes and developing rational approaches to therapy.

Unfolded protein response, treatment and CMT1B.

CMT1B is the second most frequent autosomal dominant inherited neuropathy and is caused by assorted mutations of the myelin protein zero (MPZ) gene. MPZ mutations cause neuropathy gain of function mechanisms that are largely independent MPZs normal role of mediating myelin compaction. Whether there are only a few or multiple pathogenic mechanisms that cause CMT1B is unknown. Arg98Cys and Ser63Del MPZ are two CMT1B causing mutations that have been shown to cause neuropathy in mice at least in part by activating the unfolded protein response (UPR). We have recently treated Arg98Cys mice with derivatives of curcumin that improved the neuropathy and reduced UPR activation.(1) Future studies will address whether manipulating the UPR will be a common or rare strategy for treating CMT1B or other forms of inherited neuropathies.

Curcumin derivatives promote Schwann cell differentiation and improve neuropathy in R98C CMT1B mice.

Charcot-Marie-Tooth disease type 1B is caused by mutations in myelin protein zero. R98C mice, an authentic model of early onset Charcot-Marie-Tooth disease type 1B, develop neuropathy in part because the misfolded mutant myelin protein zero is retained in the endoplasmic reticulum where it activates the unfolded protein response. Because oral curcumin, a component of the spice turmeric, has been shown to relieve endoplasmic reticulum stress and decrease the activation of the unfolded protein response, we treated R98C mutant mice with daily gastric lavage of curcumin or curcumin derivatives starting at 4 days of age and analysed them for clinical disability, electrophysiological parameters and peripheral nerve morphology. Heterozygous R98C mice treated with curcumin dissolved in sesame oil or phosphatidylcholine curcumin performed as well as wild-type littermates on a rotarod test and had increased numbers of large-diameter axons in their sciatic nerves. Treatment with the latter two compounds also increased compound muscle action potential amplitudes and the innervation of neuromuscular junctions in both heterozygous and homozygous R98C animals, but it did not improve nerve conduction velocity, myelin thickness, G-ratios or myelin period. The expression of c-Jun and suppressed cAMP-inducible POU (SCIP)-transcription factors that inhibit myelination when overexpressed-was also decreased by treatment. Consistent with its role in reducing endoplasmic reticulum stress, treatment with curcumin dissolved in sesame oil or phosphatidylcholine curcumin was associated with decreased X-box binding protein (XBP1) splicing. Taken together, these data demonstrate that treatment with curcumin dissolved in sesame oil or phosphatidylcholine curcumin improves the peripheral neuropathy of R98C mice by alleviating endoplasmic reticulum stress, by reducing the activation of unfolded protein response and by promoting Schwann cell differentiation.

MpzR98C arrests Schwann cell development in a mouse model of early-onset Charcot-Marie-Tooth disease type 1B.

Mutations in myelin protein zero (MPZ) cause Charcot-Marie-Tooth disease type 1B. Many dominant MPZ mutations, including R98C, present as infantile onset dysmyelinating neuropathies. We have generated an R98C 'knock-in' mouse model of Charcot-Marie-Tooth type 1B, where a mutation encoding R98C was targeted to the mouse Mpz gene. Both heterozygous (R98C/+) and homozygous (R98C/R98C) mice develop weakness, abnormal nerve conduction velocities and morphologically abnormal myelin; R98C/R98C mice are more severely affected. MpzR98C is retained in the endoplasmic reticulum of Schwann cells and provokes a transitory, canonical unfolded protein response. Ablation of Chop, a mediator of the protein kinase RNA-like endoplasmic reticulum kinase unfolded protein response pathway restores compound muscle action potential amplitudes of R98C/+ mice but does not alter the reduced conduction velocities, reduced axonal diameters or clinical behaviour of these animals. R98C/R98C Schwann cells are developmentally arrested in the promyelinating stage, whereas development is delayed in R98C/+ mice. The proportion of cells expressing c-Jun, an inhibitor of myelination, is elevated in mutant nerves, whereas the proportion of cells expressing the promyelinating transcription factor Krox-20 is decreased, particularly in R98C/R98C mice. Our results provide a potential link between the accumulation of MpzR98C in the endoplasmic reticulum and a developmental delay in myelination. These mice provide a model by which we can begin to understand the early onset dysmyelination seen in patients with R98C and similar mutations.

Trauma does not accelerate neuronal degeneration in Fig4 insufficient mice.

Fig4 null reduces phosphatidylinositol-3,5-diphosphate concentration and causes severe neuronal degeneration in both pale-tremor (plt) mice and patients with Charcot-Marie-Tooth disease type 4J (CMT4J), an inherited condition with recessive mutations in FIG4. Our previous study shows that minor trauma is associated with an accelerated course of motor neuron degeneration in patients with CMT4J. Heterozygous loss of FIG4 function has been suggested to be a risk factor in developing sporadic amyotrophic lateral sclerosis. We therefore hypothesize that minor trauma may trigger or exacerbate motor neuron degeneration in mice with fig4 haploinsufficiency (plt+/-). We have studied 18 wild-type and 18 plt+/- mice and created nerve injury by compressing the sciatic nerve. Outcomes in the mice were evaluated by nerve conduction study, Rotarod, and nerve morphology. No differences were found between wild-type and plt+/- mice. Taken together, our results demonstrate that haploinsufficiency of fig4 does not impose risks in rodents to develop neuronal degeneration in either naïve or traumatic conditions.

Distinct pathogenic processes between Fig4-deficient motor and sensory neurons.

Loss of function of the FIG4 gene causes Charcot-Marie-Tooth disease (CMT)-4J with many features also found in motor neuron disease (MND). Mechanisms for the degeneration are unknown. We investigated this using Fig4-deficient pale tremor (plt) mice, a mouse model of CMT4J. Ultrastructural studies in sensory neurons of dorsal root ganglion (DRG) confirmed abundant vacuoles with membrane disruption. The vacuoles became detectable as early as postnatal day 4 in the DRG. However, the vacuoles were absent or minimal in the spinal motor neurons or cortical neurons in 2- to 5-week-old plt mice. Instead, a large number of electron-dense organelles, reminiscent of those in lysosomal storage disorders, accumulated in the motor neurons, but not in the sensory neurons of DRG. This accumulation was associated with increased levels of lysosomal proteins, such as LAMP2 and NPC1, but not mannose-6-phosphate receptor, an endosomal protein that is usually excluded from the lysosomes. Our results suggest that Fig4 deficiency affects motor neurons differently from sensory neurons by mechanisms involving excessive retention of molecules in lysosomes or disruption of vacuolated organelles. These two distinct pathological changes may contribute to neuronal degeneration.