The role of dietary non-heme iron load and peripheral nerve inflammation in the development of peripheral neuropathy (PN) in obese non-diabetic leptin-deficient ob/ob mice.

INTRODUCTION: Here, we investigated inflammatory signs of peripheral nerves in leptin-deficient obese ob/ob mice and the modulating effects of the exogenous iron load. METHODS: Ob/ob and ob/+ control mice were fed with high, standard, or low iron diet for four months. RESULTS: We found intraepidermal nerve fiber degeneration in foot skin and low-grade neuropathic abnormalities including mildly slowed motor and compound sensory nerve conduction velocities and low-grade macrophage and T-cell infiltration without overt neuropathology in sciatic nerves of all ob/ob mice. Low dietary iron load caused more pronounced abnormalities than high iron load in ob/ob mice. DISCUSSION: Our data suggest that dietary non-heme iron deficiency may be a modulating factor in the pathogenesis of peripheral neuropathy in obese ob/ob mice with metabolic syndrome. Once the mechanisms can be further elucidated, how low dietary iron augments peripheral nerve degeneration and dysfunction via pro-inflammatory pathways and new therapeutic strategies could be developed. ABBREVIATIONS: CMAP: compound muscle action potential; cSNCV: compound sensory nerve conduction velocity; IENFD: intraepidermal nerve fiber density; LDL: low-density lipoprotein; MetS: metabolic syndrome; MNCV: motor conduction velocity; NCV: nerve conduction velocity; PN: peripheral neuropathy; PNS: peripheral nervous system; STZ: streptozotocin; T2D: type 2 diabetes mellitus; TNF alpha: tumor necrosis factor alpha; WHO: World Health Organization.

Myelin abnormality in Charcot-Marie-Tooth type 4J recapitulates features of acquired demyelination.

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.

Congenital myasthenic syndrome with episodic apnoea: clinical, neurophysiological and genetic features in the long-term follow-up of 19 patients.

BACKGROUND: Congenital myasthenic syndrome with episodic apnoea (CMS-EA) is a rare but potentially treatable cause of apparent life-threatening events in infancy. The underlying mechanisms for sudden and recurrent episodes of respiratory arrest in these patients are unclear. Whilst CMS-EA is most commonly caused by mutations in CHAT, the list of associated genotypes is expanding. METHODS: We reviewed clinical information from 19 patients with CMS-EA, including patients with mutations in CHAT, SLC5A7 and RAPSN, and patients lacking a genetic diagnosis. RESULTS: Lack of genetic diagnosis was more common in CMS-EA than in CMS without EA (56% n = 18, compared to 7% n = 97). Most patients manifested intermittent apnoea in the first 4 months of life (74%, n = 14). A degree of clinical improvement with medication was observed in most patients (74%, n = 14), but the majority of cases also showed a tendency towards complete remission of apnoeic events with age (mean age of resolution 2 years 5 months). Signs of impaired neuromuscular transmission were detected on neurophysiology studies in 79% (n = 15) of cases, but in six cases, this was only apparent following specific neurophysiological testing protocols (prolonged high-frequency stimulation). CONCLUSIONS: A relatively large proportion of CMS-EA remains genetically undiagnosed, which suggests the existence of novel causative CMS genes which remain uncharacterised. In light of the potential for recurrent life-threatening apnoeas in early life and the positive response to therapy, early diagnostic consideration of CMS-EA is critical, but without specific neurophysiology tests, it may go overlooked.

A novel curcumin derivative for the treatment of diabetic neuropathy.

Neuropathy is a common complication of long-term diabetes. Proposed mechanisms of neuronal damage caused by diabetes that are downstream of hyperglycemia and/or loss of insulin signaling include ischemic hypoxia, inflammation and loss of neurotrophic support. The curcumin derivative J147 is a potent neurogenic and neuroprotective drug candidate initially developed for the treatment of neurodegenerative conditions associated with aging that impacts many pathways implicated in the pathogenesis of diabetic neuropathy. Here, we demonstrate efficacy of J147 in ameliorating multiple indices of neuropathy in the streptozotocin-induced mouse model of type 1 diabetes. Diabetes was determined by blood glucose, HbA1c, and insulin levels and efficacy of J147 by behavioral, physiologic, biochemical, proteomic, and transcriptomic assays. Biological efficacy of systemic J147 treatment was confirmed by its capacity to decrease TNFα pathway activation and several other markers of neuroinflammation in the CNS. Chronic oral treatment with J147 protected the sciatic nerve from progressive diabetes-induced slowing of large myelinated fiber conduction velocity while single doses of J147 rapidly and transiently reversed established touch-evoked allodynia. Conduction slowing and allodynia are clinically relevant markers of early diabetic neuropathy and neuropathic pain, respectively. RNA expression profiling suggests that one of the pathways by which J147 imparts its protection against diabetic induced neuropathy may be through activation of the AMP kinase pathway. The diverse biological and therapeutic effects of J147 suggest it as an alternative to the polypharmaceutical approaches required to treat the multiple pathogenic mechanisms that contribute to diabetic neuropathy.

ALS-Related Mutant FUS Protein Is Mislocalized to Cytoplasm and Is Recruited into Stress Granules of Fibroblasts from Asymptomatic FUS P525L Mutation Carriers.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) shows a strong genetic basis, with SOD1, FUS, TARDBP, and C9ORF72 being the genes most frequently involved. This has allowed identification of asymptomatic mutation carriers, which may be of help in understanding the molecular changes preceding disease onset. OBJECTIVES: We studied the cellular expression of FUS protein and the effect of heat-shock- and dithiothreitol-induced stress in fibroblasts from FUS P525L mutation carriers, healthy controls, and patients with sporadic ALS. METHODS: Western blots and immunocytochemistry were performed to study the subcellular localization of FUS protein. Control and stressed cells were double stained with FUS and the stress marker TIA-R. RESULTS: Fibroblasts from healthy controls and sporadic ALS cases showed a prominent nuclear FUS expression. In the 2 FUS P525L mutation carriers, instead, most cells showed a protein localization in both nucleus and cytoplasm, or exclusively in the cytoplasm. Stress prompted the formation of cytoplasmic granules in all subjects and in sporadic ALS FUS mislocalization to the cytoplasm. Cytoplasmic FUS was recruited into stress granules, which showed a time-dependent decrease in all subjects. However, in the FUS P525L fibroblasts, the granules persisted longer, and they were more numerous than those detected in the cells from controls and sporadic ALS patients. CONCLUSIONS: We show that in fibroblasts of FUS P525L mutation carriers, FUS mislocalized to the cytoplasm where it redistributed into stress granules with likely a dose effect, i.e. a higher number of cells with granules, which persist longer, than in controls and ALS cases. These data represent an early molecular change occurring before ALS onset, suggesting a transient preaggregative state.

Acetyl-CoA production from pyruvate is not necessary for preservation of myelin.

Oligodendrocytes and Schwann cells not only form myelin in the central and peripheral nervous system, but also provide metabolic and trophic support to the axons they ensheathe. Acetyl-CoA is potentially a key molecule in Schwann cells and oligodendrocytes because it is at the crossroads of cellular lipid biosynthesis and energy generation. The main route for acetyl-CoA production is the oxidation of pyruvate by the pyruvate dehydrogenase complex (PDC). PDC deficiency in humans results in neurodegeneration and developmental impairments in both white and gray matter structures. To address the importance of PDC in myelinating glia, we deleted Pdha1 gene specifically in oligodendrocytes and Schwann cells. Surprisingly, sciatic and optic nerve morphology and the motor performance of Pdha1f/Y; CnpCre/+ mice are undistinguishable from those of controls at 1 month of age. In addition, myelin is stably maintained for at least 10 months. However, Pdha1f/Y; CnpCre/+ mice showed reduced fiber density and signs of axonal degeneration in both sciatic and optic nerves from 6 months of age. In contrast, 10 month-old mice bearing a floxed Pdha1 gene with either P0-Cre (expressed only by Schwann cells) or NG2-CreER (expressed in oligodendrocyte precursor cells) do not show any sign of axonal pathology or alterations in myelin structure or thickness. This indicates that the axonopathy is specific to the Pdha1f/Y; CnpCre/+ mice. Taken together, these results suggest that acetyl-CoA derived from pyruvate is not necessary for myelin maintenance and, thus, myelin-forming cells are not likely to contribute to the pathophysiology of PDC deficiency.

A novel ATTR L32V mutation causes familial amyloid polyneuropathy in a Bolivian family.

We report a new transthyretin (ATTR) gene c.272C>G mutation and variant protein, p.Leu32Val, in a kindred of Bolivian origin with a rapid progressive peripheral neuropathy and cardiomyopathy. Three individuals from a kindred with peripheral nerve and cardiac amyloidosis were examined. Analysis of the TTR gene was performed by Sanger direct sequencing. Neuropathologic examination was obtained on the index patient with mass spectrometry study of the ATTR deposition. Direct DNA sequence analysis of exons 2, 3, and 4 of the TTR gene demonstrated a c.272 C>G mutation in exon 2 (p.L32V). Sural nerve biopsy revealed massive amyloid deposition in the perineurium, endoneurium and vasa nervorum. Mass spectrometric analyses of ATTR immunoprecipitated from nerve biopsy showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data. The ATTR L32V is associated with a severe course. This has implications for treatment of affected individuals and counseling of family members.

Deletion of Sarm1 gene is neuroprotective in two models of peripheral neuropathy.

Distal axon degeneration seen in many peripheral neuropathies is likely to share common molecular mechanisms with Wallerian degeneration. Although several studies in mouse models of peripheral neuropathy showed prevention of axon degeneration in the slow Wallerian degeneration (Wlds) mouse, the role of a recently identified player in Wallerian degeneration, Sarm1, has not been explored extensively. In this study, we show that mice lacking the Sarm1 gene are resistant to distal axonal degeneration in a model of chemotherapy induced peripheral neuropathy caused by paclitaxel and a model of high fat diet induced putative metabolic neuropathy. This study extends the role of Sarm1 to axon degeneration seen in peripheral neuropathies and identifies it as a likely target for therapeutic development.

Waardenburg syndrome: a rare cause of inherited neuropathy due to SOX10 mutation.

Waardenburg syndrome (WS) is a rare disorder comprising sensorineural deafness and pigmentation abnormalities. Four distinct subtypes are defined based on the presence or absence of additional symptoms. Mutations in six genes have been described in WS. SOX10 mutations are usually associated with a more severe phenotype of WS with peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, and Hirschsprung disease. Here we report a 32-year-old man with a novel heterozygous missense variant in SOX10 gene, who presented with congenital deafness, Hirschsprung disease, iris heterochromia, foot deformity, and intermediate conduction velocity length-dependent sensorimotor neuropathy. This case highlights that the presence of other non-neuropathic features in a patient with presumed hereditary neuropathy should alert the clinician to possible atypical rare causes.

Adult-onset demyelinating neuropathy associated with FBLN5 gene mutation.

Rare forms of autosomal-dominant Charcot-Marie-Tooth disease (AD-CMT) may be associated with mutations in Fibulin-5 (FBLN5) as AD-CMT is genetically heterogeneous. Here, we report the first pathological study of an Asian family. The proband was a 46-year-old man with slowly progressive distal numbness and weakness for 12 years. He had a history of diabetes mellitus for 12 years. His mother was 81 years old and had mild polyneuropathy. His 16-year-old daughter was asymptomatic. The nerve conduction velocities (NCVs) and compound muscular action potential (CMAP) amplitudes were moderately to severely reduced in the proband, and moderately reduced in his daughter and mother. A sensory response could not be elicited in the proband and was moderately to severely decreased in the daughter and mother. Nerve ultrasound indicated a general enlargement of the peripheral nerves in the proband, daughter, and mother. A sural nerve biopsy from the proband demonstrated a pronounced depletion of myelinated fibers, thin myelinated fibers, and onion-bulb formations. A reported heterozygous mutation of c.1117C>T in FBLN5 was identified in the proband, mother, and daughter. These findings confirm a novel subtype of AD-CMT 1 due to a mutation in the FBLN5 gene.
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Novel NTRK1 mutations associated with congenital insensitivity to pain with anhidrosis verified by functional studies.

Congenital insensitivity to pain with anhidrosis (CIPA), also known as hereditary sensory and autonomic neuropathy type IV, features loss of pain sensation, decreased or absent sweating (anhidrosis), recurrent episodes of unexplained fever, self-mutilating behavior, and variable mental retardation. Mutations in neurotrophic receptor tyrosine kinase 1 (NTRK1) have been reported to be associated with CIPA. We identified four novel NTRK1 mutations in six Korean patients from four unrelated families. Of the four mutations, we demonstrated using a splicing assay that IVS14+3A>T causes aberrant splicing of NTRK1 mRNA, leading to introduction of a premature termination codon. An NTRK1 autophosphorylation assay showed that c.1786G>A (p.Asp596Asn) abolished autophosphorylation of NTRK1. In addition, Western blotting showed that c.704C>G (p.Ser235*) and c.2350_2363del (p.Leu784Serfs*79) blunted NTRK1 expression to undetectable levels. The four novel NTRK1 mutations we report here will expand the repertoire of NTRK1 mutations in CIPA patients, and further our understanding of CIPA pathogenesis.

Clinical features of familial amyloid polyneuropathy carrying transthyretin mutations in four Chinese kindreds.

Transthyretin-related familial amyloid polyneuropathy (TTR-FAP) is a rare hereditary disorder, characterized by a length-dependent polyneuropathy and dysfunction of various organs. Wide phenotypic heterogeneity makes early diagnosis difficult. In this study, we reviewed the clinical and electrophysiological features of four unrelated Chinese families with genetically confirmed TTR-FAP. Sequence analysis of TTR gene revealed the presence of four different mutations: Thr49Ala(p.Thr69Ala), Leu55Arg(p.Leu75Arg), Tyr116Ser(p.Tyr136Ser), and Ala36Pro(p.Ala56Pro) from six affected patients and two asymptomatic individuals. Two mutations, Thr49Ala(p.Thr69Ala) and Tyr116Ser(p.Tyr136 Ser), were detected in Chinese FAP patients for the first time. All affected patients manifested a progressive sensorimotor polyneuropathy starting in the lower limbs. The majority of the examined patients displayed cardiomyopathy and vitreous opacities. To avoid misdiagnosis, clinicians should consider screening for TTR variants in patients presenting with progressive polyneuropathy of undetermined origins.

Histopathological features of a patient with Charcot-Marie-Tooth disease type 2U/AD-CMTax-MARS.

Charcot-Marie-Tooth (CMT) disease is a complex of peripheral nervous system disorders. CMT type 2U (CMT2U) is an autosomal dominant (AD) disease caused by mutations in the MARS gene encoding methionyl-tRNA synthetase; this disease has thus been newly called AD-CMTax-MARS. A few families with mutations in the MARS gene have been reported, without detailed histopathological findings. We describe a 70-year-old woman who had bilateral dysesthesia of the soles since the age of 66 years. Sural nerve biopsy showed a decrease in the density of large myelinated nerve fibers. Increased clusters of regenerating myelinated nerve fibers were noted. Electron microscopic analyses revealed degeneration of unmyelinated nerves. There was no vasculitis or inflammatory cell infiltration. Genetic analysis identified a heterozygous p.P800T mutation, a reported mutation in the MARS gene. We report the detailed histopathological findings in a patient with CMT2U/AD-CMTax-MARS. The findings are similar to those found in CMT2D caused by mutations in the GARS gene, encoding glycyl-tRNA synthetase.

Charcot-Marie-Tooth disease type 2G redefined by a novel mutation in LRSAM1.

OBJECTIVE: To identify the unknown genetic cause in a large pedigree previously classified with a distinct form of axonal Charcot-Marie-Tooth disease type 2G (CMT2G) and to explore its transcriptional consequences. METHODS: Clinical reevaluation of the pedigree was performed, followed by linkage analysis with the redefined disease statuses, and whole genome and exome sequencing. The impact of the mutation was investigated by immunoblotting and transcriptome sequencing. RESULTS: Thirteen affected individuals over 3 generations displayed mild and quiescent lower-limb axonal sensorimotor neuropathy. Magnetic resonance imaging (MRI) of lower-limb musculature systematically showed fatty atrophy in clinical and subclinical mutation carriers. We redefined the disease-linked region to chr9q31.3-q34.2 and subsequently identified a novel missense variant in the E3 ubiquitin-protein ligase LRSAM1 (p.Cys694Tyr). Unlike previous reports, we demonstrated in patients' lymphoblasts that the mutation does not influence overall protein levels of LRSAM1, nor of its ubiquitylation target TSG101. The mutation is associated with several transcriptional changes, including a significant upregulation of another E3 ubiquitin-protein ligase, NEDD4L, and of TNFRSF21, a key regulator of axonal degeneration. INTERPRETATION: Our findings demonstrate that the isolated genetic entity CMT2G is caused by a missense mutation in LRSAM1 and should be reclassified as CMT2P. MRI of lower-limb musculature can be used to detect minimal signs of the disease. Transcriptome analysis of patients' cells highlights novel molecular players associated with LRSAM1 dysfunction, and reveals pathways and therapeutic targets shared with amyotrophic lateral sclerosis and Alzheimer disease. Ann Neurol 2016;80:823-833.

A novel missense mutation of CMT2P alters transcription machinery.

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.

Fingolimod promotes peripheral nerve regeneration via modulation of lysophospholipid signaling.

BACKGROUND: The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are pleiotropic signaling molecules with a broad range of physiological functions. Targeting the S1P1 receptor on lymphocytes with the immunomodulatory drug fingolimod has proven effective in the treatment of multiple sclerosis. An emerging body of experimental evidence points to additional direct effects on cells of the central and peripheral nervous system. Furthermore, fingolimod has been reported to reduce LPA synthesis via inhibition of the lysophospholipase autotaxin. Here we investigated whether modulation of particular signaling aspects of S1P as well as LPA by fingolimod might propagate peripheral nerve regeneration in vivo and independent of its anti-inflammatory potency. METHODS: Sciatic nerve crush was performed in wildtype C57BL/6, in immunodeficient Rag1 (-/-) and Foxn1 (-/-) mice. Analyses were based on walking track analysis and electrophysiology, histology, and cAMP formation. Quantification of different LPA species was performed by liquid chromatography coupled to tandem mass spectrometry. Furthermore, functional consequences of autotaxin inhibition by the specific inhibitor PF-8380 and the impact of fingolimod on early cytokine release in the injured sciatic nerve were investigated. RESULTS: Clinical and electrophysiological measures indicated an improvement of nerve regeneration under fingolimod treatment that is partly independent of its anti-inflammatory properties. Fingolimod treatment correlated with a significant elevation of axonal cAMP, a crucial factor for axonal outgrowth. Additionally, fingolimod significantly reduced LPA levels in the injured nerve. PF-8380 treatment correlated with improved myelin thickness. Sciatic nerve cytokine levels were not found to be significantly altered by fingolimod treatment. CONCLUSIONS: Our findings provide in vivo evidence for direct effects of fingolimod on cells of the peripheral nervous system that may propagate nerve regeneration via a dual mode of action, differentially affecting axonal outgrowth and myelination by modulating relevant aspects of S1P and LPA signaling.

Inhibition of the neuronal NFκB pathway attenuates bortezomib-induced neuropathy in a mouse model.

Bortezomib is a proteasome inhibitor with a remarkable antitumor activity, used in the clinic as first line treatment for multiple myeloma. One hallmark of bortezomib mechanism of action in neoplastic cells is the inhibition of nuclear factor kappa B (NFκB), a transcription factor involved in cell survival and proliferation. Bortezomib-induced peripheral neuropathy is a dose-limiting toxicity that often requires adjustment of treatment and affects patient's prognosis and quality of life. Since disruption of NFκB pathway can also affect neuronal survival, we assessed the role of NFκB in bortezomib-induced neuropathy by using a transgenic mouse that selectively provides blockage of the NFκB pathway in neurons. Interestingly, we observed that animals with impaired NFκB activation developed significantly less severe neuropathy than wild type animals, with particular preservation of large myelinated fibers, thus suggesting that neuronal NFκB activation plays a positive role in bortezomib induced neuropathy and that bortezomib treatment might induce neuropathy by inhibiting NFκΒ in non-neuronal cell types or by targeting other signaling pathways. Therefore, inhibition of NFκB might be a promising strategy for the cotreatment of cancer and neuropathy.

Congenital hypomyelinating neuropathy due to the association of a truncating mutation in PMP22 with the classical HNPP deletion.

Congenital hypomyelinating neuropathy appears early in life, resulting in a delay of motor and sensory development. Mutations involve genes such as myelin protein zero (MPZ), peripheral myelin protein 22 (PMP22), and early growth response 2 (EGR2). We present a patient with two compound mutations in PMP22: a point mutation causing a premature STOP codon in exon 3 was inherited from the mother on the first allele, and the "typical" PMP22 deletion in the 17p11.2-p12 region was inherited from the father on the other allele. A sural biopsy was performed at age four. The patient has been followed from 28 months to 21 years of age; he presented significant sensory disturbances, with a slight motor deficit. PMP22 mRNA quantitation showed a severe decrease of PMP22 protein. No myelin sheaths were observed in the biopsy; mesaxons failed to form. The absence of PMP22 provides new insights into the role of this protein.