Jun Li

Objective: Charcot–Marie–Tooth type 4J (CMT4J) is a rare autosomal recessive neuropathy caused by mutations in FIG4 that result in loss of FIG4 protein. This study investigates the natural history and mechanisms of segmental demyelination in CMT4J.Methods: Over the past 9 years, we have enrolled and studied a cohort of 12 CMT4J patients, including 6 novel FIG4 mutations. We evaluated these patients and related mouse models using morphological, electrophysiological, and biochemical approaches.Results: We found sensory motor demyelinating polyneuropathy consistently in all patients. This underlying myelin pathology was associated with nonuniform slowing of conduction velocities, conduction block, and temporal dispersion on nerve conduction studies, which resemble those features in acquired demyelinating peripheral nerve diseases. Segmental demyelination was also confirmed in mice without Fig4 (Fig4^−/−). The demyelination was associated with an increase of Schwann cell dedifferentiation and macrophages in spinal roots where nerve–blood barriers are weak. Schwann cell dedifferentiation was induced by the increasing intracellular Ca²⁺. Suppression of Ca²⁺ level by a chelator reduced dedifferentiation and demyelination of Schwann cells in vitro and in vivo. Interestingly, cell‐specific knockout of Fig4 in mouse Schwann cells or neurons failed to cause segmental demyelination.Interpretation: Myelin change in CMT4J recapitulates the features of acquired demyelinating neuropathies. This pathology is not Schwann cell autonomous. Instead, it relates to systemic processes involving interactions of multiple cell types and abnormally elevated intracellular Ca²⁺. Injection of a Ca²⁺ chelator into Fig4^−/− mice improved segmental demyelination, thereby providing a therapeutic strategy against demyelination. Ann Neurol 2018;83:756–770

Background and purpose: Apoptosis‐inducing factor mitochondrion‐associated‐1 (AIFM1) in mitochondria has captured a great deal of attention due to its well‐described function in apoptosis. Mutations in AIFM1 have resulted in multiple clinical phenotypes, including X‐linked Charcot–Marie–Tooth disease type 4. These syndromes usually involve multiple locations within the nervous system and/or multiple organs. This study describes a novel missense mutation in AIFM1 and its associated peripheral nerve disease.Methods: Patients with AIFM1 mutation were characterized clinically, electrophysiologically, genetically and by magnetic resonance imaging. The fibroblasts were isolated from the patients to study mitochondrial OXPHOS complexes.Results: We identified a family with a novel missense mutation (Phe210Leu) in AIFM1 who developed an isolated late‐onset axonal polyneuropathy in which the central nervous system and other organs were spared. Interestingly, this Phe210Leu mutation resulted in abnormal assembly of mitochondrial complex I and III, and failed to disrupt AIFM1 binding with mitochondrial intermembrane space import and assembly protein 40 (MIA40) in the patients’ cells. Deficiency of either AIFM1 or MIA40 is known to impair the assembly of mitochondrial complex I and IV. However, levels of both AIFM1 and MIA40 were unchanged.Conclusions: Phe210Leu mutation in AIFM1 induces an axonal polyneuropathy that might be contributed by the misassembly of mitochondrial complex I and III. This misassembly appears to be independent of the traditional mechanism via AIFM1/MIA40 deficiency.

Charcot-Marie-Tooth disease type-1A (CMT1A) is one of the most common types of inherited peripheral nerve diseases. It is caused by the trisomy of chromosome 17p12 (c17p12), a large DNA segment of 1.4 Mb containing PMP22 plus eight other genes. The size of c17p12 is formidable for any cloning technique to manipulate, and thus precludes production of models in vitro and in vivo that can precisely recapitulate the genetic alterations in humans with CMT1A. This limitation and other factors have led to several assumptions, which have yet been carefully scrutinized, serving as key principles in our understanding of the disease. For instance, one extra copy of c17p12 in patients with CMT1A results in a higher gene dosage of PMP22, thereby expected to produce a higher level of PMP22 mRNA/proteins that cause the disease. However, there has been increasing evidence that PMP22 levels are highly variable among patients with CMT1A and may fall into the normal range at a given time point. This raises an alternative mechanism causing the disease by dysregulation of PMP22 expression or excessive fluctuation of PMP22 levels, not the absolute increase of PMP22. This has become a pressing issue since recent clinical trials using ascorbic acid failed to alter the clinical outcome of CMT1A patients, leaving no effective therapy for the disease. In this article, we will discuss how this fundamental issue might be investigated. In addition, several other key issues in CMT1A will be discussed, including potential mechanisms responsible for the uniform slowing of conduction velocities. A clear understanding of these issues could radically change how therapies should be developed against CMT1A.

Objective: Charcot–Marie–Tooth type 2P (CMT2P) has been associated with frameshift mutations in the RING domain of LRSAM1 (an E3 ligase). This study describes families with a novel missense mutation of LRSAM1 gene and explores pathogenic mechanisms of CMT2P.Methods: Patients with CMT2P were characterized clinically, electrophysiologically, and genetically. A neuronal model with the LRSAM1 mutation was created using CRISPR/Cas9 technology. The neuronal cell line along with fibroblasts isolated from the patients was used to study RNA‐binding proteins.Results: This American family with dominantly inherited axonal polyneuropathy reveals a phenotype similar to those in previously reported non‐US families. The affected members in our family cosegregated with a novel missense mutation Cys694Arg that alters a highly conserved cysteine in the RING domain. This mutation leads to axonal degeneration in the in vitro neuronal cell line. Moreover, using protein mass spectrometry, we identified a group of RNA‐binding proteins (including FUS, a protein critically involved in motor neuron degeneration) that interacted with LRSAM1. The interactions were disrupted by the Cys694Arg mutation, which resulted in reduction of intranuclear RNA‐binding proteins.Interpretation: Our findings suggest that the mutant LRSAM1 may aberrantly affect the formation of transcription machinery. Given that a similar mechanism has been reported in motor neuron degeneration of amyotrophic lateral sclerosis, abnormalities of RNA/RNA‐binding protein complex may play a role in the neuronal degeneration of CMT2P. Ann Neurol 2016;80:834–845

Schwann cells in the peripheral nervous systems extend their membranes to wrap axons concentrically and form the insulating sheath, called myelin. The spaces between layers of myelin are sealed by myelin junctions. This tight insulation enables rapid conduction of electric impulses (action potentials) through axons. Demyelination (stripping off the insulating sheath) has been widely regarded as one of the most important mechanisms altering the action potential propagation in many neurological diseases. However, the effective nerve conduction is also thought to require a proper myelin seal through myelin junctions such as tight junctions and adherens junctions. In the present study, we have demonstrated the disruption of myelin junctions in a mouse model (Pmp22+/-) of hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of Pmp22 gene. We observed a robust increase of F-actin in Pmp22+/- nerve regions where myelin junctions were disrupted, leading to increased myelin permeability. These abnormalities were present long before segmental demyelination at the late phase of Pmp22+/- mice. Moreover, the increase of F-actin levels correlated with an enhanced activity of p21-activated kinase (PAK1), a molecule known to regulate actin polymerization. Pharmacological inhibition of PAK normalized levels of F-actin, and completely prevented the progression of the myelin junction disruption and nerve conduction failure in Pmp22+/- mice. Our findings explain how abnormal myelin permeability is caused in HNPP, leading to impaired action potential propagation in the absence of demyelination. We call it “functional demyelination”, a novel mechanism upstream to the actual stripping of myelin that is relevant to many demyelinating diseases. This observation also provides a potential therapeutic approach for HNPP. Demyelination has been widely regarded as one of the most important mechanisms impairing the nerve conduction of electrical impulses in many neurological diseases. Failure of the nerve conduction results in sensory and motor deficits. In the present study, we investigated a novel mechanism that also impairs nerve conduction without the removal of myelin, called functional demyelination. Deletion of one of two copies of the PMP22 gene causes hereditary neuropathy with liability to pressure palsies (HNPP). Our recent study reports that deficiency of PMP22 in HNPP disrupts myelin junctions of Schwann cells that seal the spaces between layers of myelin membrane. This disruption leads to “leaky” myelin that impairs the conduction of electrical impulses on the nerves. In the present study, using a HNPP mouse model (Pmp22+/-), we have identified a molecular pathway responsible for the disruption of myelin junctions. Our results showed an increase of actin polymerization in the areas where myelin junctions resided. This increase was associated with an up-regulation of PAK1 activity, a kinase function that is known to regulate actin polymerization. We thus treated the HNPP mice with PAK1 inhibitor. This treatment completely prevented the progression of nerve conduction failure and HNPP pathology. This work offers a promising therapeutic approach for HNPP. Moreover, myelin junction disruption takes place long before the actual stripping of myelin (demyelination) in the late phase of HNPP. Therefore, our finding provides a mechanism upstream to segmental demyelination, a pathological process relevant to many demyelinating diseases.

Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5′-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P₂). However, the loss of FIG4 decreases PI3,5P₂levels likely due to FIG4's dominant effect in scaffolding a PI3,5P₂synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4^−/−) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, inFig4^−/−cells. The fission defect was associated with a robust increase of intralysosomal Ca²⁺inFig4^−/−cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca²⁺efflux of lysosomes because the endogenous ligand of lysosomal Ca²⁺channel TRPML1 is PI3,5P₂that is deficient inFig4^−/−cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca²⁺level and rescued abnormal lysosomal storage inFig4^−/−culture cells andex vivoDRGs. Furthermore, we found that the suppressed Ca²⁺efflux inFig4^−/−culture cells andFig4^−/−mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency.

Genetic factors may be learnt from families with gene mutations that render nerve-injury susceptibility even to ordinary physical activities. A typical example is hereditary neuropathy with liability to pressure palsies (HNPP). HNPP is caused by a heterozygous deletion of PMP22 gene. PMP22 deficiency disrupts myelin junctions (such as tight junction and adherens junctions), leading to abnormally increased myelin permeability that explains the nerve susceptibility to injury. This finding should motivate investigators to identify additional genetic factors contributing to nerve vulnerability of injury.