Leukoencephalopathy and conduction blocks in PLEKHG5-associated intermediate CMT disease.

Biallelic variants in PLEKHG5 have been reported so far associated with different clinical phenotypes including Lower motor neuron disease (LMND) [also known as distal hereditary motor neuropathies (dHMN or HMN) or distal spinal muscular atrophy (DSMA4)] and intermediate Charcot-Marie-Tooth disease (CMT). We report four patients from two families presenting with intermediate CMT and atypical clinical and para-clinical findings. Patients presented with predominant distal weakness with none or mild sensory involvement and remain ambulant at last examination (22-36 years). Nerve conduction studies revealed, in all patients, intermediate motor nerve conduction velocities, reduced sensory amplitudes and multiple conduction blocks in upper limbs, outside of typical nerve compression sites. CK levels were strikingly elevated (1611-3867 U/L). CSF protein content was mildly elevated in two patients. Diffuse bilateral white matter lesions were detected in one patient. Genetic analysis revealed three novel frameshift variants c.1835_1860del and c.2308del (family 1) and c.104del (family 2). PLEKHG5-associated disease ranges from pure motor phenotypes with predominantly proximal involvement to intermediate CMT with predominant distal motor involvement and mild sensory symptoms. Leukoencephalopathy, elevated CK levels and the presence of conduction blocks associated with intermediate velocities in NCS are part of the phenotype and may arise suspicion of the disease, thus avoiding misdiagnosis and unnecessary therapeutics in these patients.

Elovl5 is required for proper action potential conduction along peripheral myelinated fibers.

Elovl5 elongates fatty acids with 18 carbon atoms and in cooperation with other enzymes guarantees the normal levels of very long-chain fatty acids, which are necessary for a proper membrane structure. Action potential conduction along myelinated axons depends on structural integrity of myelin, which is maintained by a correct amount of fatty acids and a proper interaction between fatty acids and myelin proteins. We hypothesized that in Elovl5-/- mice, the lack of elongation of Elovl5 substrates might cause alterations of myelin structure. The analysis of myelin ultrastructure showed an enlarged periodicity with reduced G-ratio across all axonal diameters. We hypothesized that the structural alteration of myelin might affect the conduction of action potentials. The sciatic nerve conduction velocity was significantly reduced without change in the amplitude of the nerve compound potential, suggesting a myelin defect without a concomitant axonal degeneration. Since Elovl5 is important in attaining normal amounts of polyunsaturated fatty acids, which are the principal component of myelin, we performed a lipidomic analysis of peripheral nerves of Elovl5-deficient mice. The results revealed an unbalance, with reduction of fatty acids longer than 18 carbon atoms relative to shorter ones. In addition, the ratio of saturated to unsaturated fatty acids was strongly increased. These findings point out the essential role of Elovl5 in the peripheral nervous system in supporting the normal structure of myelin, which is the key element for a proper conduction of electrical signals along myelinated nerves.

Fragile X Mental Retardation Protein Bidirectionally Controls Dendritic Ih in a Cell Type-Specific Manner between Mouse Hippocampus and Prefrontal Cortex.

Channelopathies are implicated in Fragile X syndrome (FXS), yet the dysfunction of a particular ion channel varies with cell type. We previously showed that HCN channel function is elevated in CA1 dendrites of the fmr1-/y mouse model of FXS, but reduced in L5 PFC dendrites. Using male mice, we tested whether Fragile X Mental Retardation Protein (FMRPO), the protein whose absence causes FXS, differentially modulates HCN channels in CA1 versus L5 PFC dendrites. Using a combination of viral tools, intracellular peptide, and dendritic electrophysiology, we found that FMRP regulates HCN channels via a cell-autonomous protein-protein interaction. Virally expressed FMRP restored WT HCN channel-related dendritic properties in both CA1 and L5 neurons. Rapid intracellular perfusion of the non-mRNA binding N-terminal fragment, FMRP1-298, similarly restored dendritic function. In support of a protein-protein interaction, we found that FMRP associated with HCN-TRIP8b complexes in both hippocampus and PFC. Finally, voltage-clamp recordings showed that FMRP modulated Ih by regulating the number of functional dendritic HCN channels rather than individual channel properties. Together, these represent three novel findings as to the nature of the changes in dendritic function in CA1 and PFC neurons based on the presence or absence of FMRP. Moreover, our findings provide evidence that FMRP can regulate its targets in opposite directions depending upon the cellular milieu.SIGNIFICANCE STATEMENT Changes in dendritic function, and voltage-gated ion channels in particular, are increasingly the focus of neurological disorders. We, and others, previously identified cell type-specific channelopathies in a mouse of model of Fragile X syndrome. The present study shows that replacing Fragile X Mental Retardation Protein, which is absent in Fragile X syndrome, in adult CA1 and L5 PFC neurons regulates the number of functional dendritic HCN channels in a cell type-specific manner. These results suggest that Fragile X Mental Retardation Protein regulates dendritic HCN channels via a cell-autonomous protein--protein mechanism.

New novel mutations in Brazilian families with X-linked Charcot-Marie-Tooth disease.

Mutations in the GJB1 gene are the second most frequent cause of Charcot-Marie-Tooth disease (CMT), accounting for approximately 10% of CMT cases worldwide. We retrospectively analyzed detailed clinical and neurophysiological data on four Brazilian families carrying novel mutations of the GJB1 gene. Mutations were identified by bidirectional Sanger sequence analysis on the GJB1 coding region. We identified a total of 12 subjects from four different kindred. There was no male-to-male transmission, and their clinical pictures were within the expected spectrum for GJB1-related neuropathy. Males were more severely affected than females. Five out of the eight females only had subclinical neuropathy. Nerve conduction velocities were in the intermediate range in the male patients and higher in the females affected. These mutations increase the genotypic variability associated with GJB1.

A PRPH splice-donor variant associates with reduced sural nerve amplitude and risk of peripheral neuropathy.

Nerve conduction (NC) studies generate measures of peripheral nerve function that can reveal underlying pathology due to axonal loss, demyelination or both. We perform a genome-wide association study of sural NC amplitude and velocity in 7045 Icelanders and find a low-frequency splice-donor variant in PRPH (c.996+1G>A; MAF = 1.32%) associating with decreased NC amplitude but not velocity. PRPH encodes peripherin, an intermediate filament (IF) protein involved in cytoskeletal development and maintenance of neurons. Through RNA and protein studies, we show that the variant leads to loss-of-function (LoF), as when over-expressed in a cell line devoid of other IFs, it does not allow formation of the normal filamentous structure of peripherin, yielding instead punctate protein inclusions. Recall of carriers for neurological assessment confirms that from an early age, homozygotes have significantly lower sural NC amplitude than non-carriers and are at risk of a mild, early-onset, sensory-negative, axonal polyneuropathy.

Hereditary sensory and autonomic neuropathy type IC accompanied by upper motor neuron abnormalities and type II juxtafoveal retinal telangiectasias.

Hereditary sensory and autonomic neuropathy type I (HSAN-1) is an autosomal dominant sensory neuropathy occurring secondary to mutations in the SPTLC1 and SPTLC2 genes. We present two generations of a single family with Ser384Phe mutation in the SPTLC2 gene located on chromosome 14q24 characterized by a typical HSAN-1c presentation, with additional findings upper motor neuron signs, early demyelinating features on nerve conduction studies, and type II juxtafoveal retinal telangiectasias also known as macular telangiectasias (MacTel II). Although HSAN1 is characterized as an axonal neuropathy, demyelinating features were identified in two subjects on serial nerve conduction studies comprising motor conduction block, temporal dispersion, and prolongation of F-waves. MacTell II is a rare syndrome characterized by bilateral macular depigmentation and Müller cell loss. It has a presumed genetic basis, and these cases suggest that the accumulation of toxic sphingoplipids may lead to Müller cell degeneration, subsequent neuronal loss, depigmentation, and progressive central macular thinning.

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.

Normal audiogram but poor sensitivity to brief sounds in mice with compromised voltage-gated sodium channels (Scn8amedJ).

The Scn8amedJ mutation of the gene for sodium channels at the nodes of Ranvier slows nerve conduction, resulting in motor abnormalities. This mutation is also associated with loss of spontaneous bursting activity in the dorsal cochlear nucleus. However initial tests of auditory sensitivity in mice homozygous for this mutation, using standard 400-ms tones, demonstrated normal hearing sensitivity. Further testing, reported here, revealed a severely compromised sensitivity to short-duration tones of 10 and 2 ms durations. Such a deficit might be expected to interfere with auditory functions that depend on rapid processing of auditory signals.

Clinical and genetic characterization of an Italian family with slow-channel syndrome.

INTRODUCTION: The slow-channel congenital myasthenic syndrome (SCCMS) is a postsynaptic form of congenital myasthenic syndromes (CMSs), a clinically heterogeneous group of disorders caused by genetic defects leading to an abnormal signal transmission at the endplate. METHODS: We report clinical and molecular data of a multigenerational family in which the presentation of a progressive proximal-distal weakness with ocular involvement led to a number of different clinical diagnoses. RESULTS: A comprehensive genetic study which included whole-genome linkage analysis and whole-exome sequencing identified a heterozygous missense substitution (c.721C>T, p.L241F) in the ε subunit of the acetylcholine receptor (CHRNE) that was consistent with clinical weakness in all patients. DISCUSSION: SCCMS is characterized by a broad and heterogeneous clinical phenotype in which disease onset, symptoms, severity, and progression can be highly variable even between family members. The identification of a CHRNE mutation allowed to make the definitive diagnosis of CMS in this family and contributed to define the clinical spectrum of this disease.

Protection of insulin­like growth factor 1 on experimental peripheral neuropathy in diabetic mice.

The present study investigated whether insulin­like growth factor­1 (IGF­1) exerts a protective effect against neuropathy in diabetic mice and its potential underlying mechanisms. Mice were divided into four groups: Db/m (control), db/db (diabetes), IGF­1­treated db/db and IGF­1­picropodophyllin (PPP)­treated db/db. Behavioral studies were conducted using the hot plate and von Frey methods at 6 weeks of age prior to treatment. The motor nerve conduction velocity (NCV) of the sciatic nerve was measured using a neurophysiological method at 8 weeks of age. The alterations in the expression levels of IGF­1 receptor (IGF­1R), c­Jun N­terminal kinase (JNK), extracellular signal­regulated kinase (ERK), p38 and effect of IGF­1 on the sciatic nerve morphology were observed by western blotting and electron microscopy. Compared with the control group, the diabetes group developed hypoalgesia after 12 weeks, and neurological lesions improved following an intraperitoneal injection of recombinant (r)IGF­1. The sciatic NCV in the diabetes group was significantly lower compared with the control group. The sciatic NCV improved following rIGF­1 intervention; however, was impaired following administration of the IGF­1 receptor antagonist, PPP. The myelin sheath in the sciatic nerve of the diabetes group was significantly more impaired compared with the control group. The myelin sheath in the sciatic nerves of the rIGF­1­treated group was significantly improved compared with the diabetes group; whereas, they were significantly impaired following administration of the IGF­1R inhibitor. In addition, the expression of IGF­1R, phosphorylated (p)­JNK and p­ERK of sciatic nerves in the db/db mice was significantly increased following treatment with IGF­1. The expression levels of these proteins were significantly lower in the IGF­1­PPP group compared with the IGF­1 group; however, no significant difference was observed in the expression levels of p­p38 following treatment with IGF­1. The results of the present study demonstrated that IGF­1 may improve neuropathy in diabetic mice. This IGF­1­induced neurotrophic effect may be associated with the increased phosphorylation levels of JNK and ERK, not p38; however, it was attenuated by administration of an IGF­1R antagonist.

Identification of a pathogenic PMP2 variant in a multi-generational family with CMT type 1: Clinical gene panels versus genome-wide approaches to molecular diagnosis.

Charcot-Marie-Tooth (CMT) disease type 1 is an inherited peripheral neuropathy characterized by demyelination and reduced nerve conduction velocities. We present a multi-generational family with peripheral neuropathy in whom clinical CMT panel testing failed to conclude a molecular diagnosis. We found a PMP2 pathogenic variant c.155T > C, p.(Ile52Thr) that segregates with disease suggesting that PMP2 variants should be considered in patients with neuropathy and that it may be prudent to include in clinical CMT gene panels.

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.

HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease.

Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

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.

Neurofascin-155 IGG4 Neuropathy: Pathophysiological Insights, Spectrum of Clinical Severity and Response To treatment.

INTRODUCTION: Sensorimotor neuropathy associated with IgG4 antibodies to neurofascin-155 (NF155) was recently described. The clinical phenotype is typically associated with young onset, distal weakness, and in some cases, tremor. METHODS: From a consecutive cohort of 55 patients diagnosed with chronic inflammatory demyelinating polyneuropathy, screening for anti-NF155 antibodies was undertaken. Patients underwent clinical assessment, diagnostic neurophysiology, including peripheral axonal excitability studies and nerve ultrasound. RESULTS: Three of 55 chronic inflammatory demyelinating polyneuropathy patients (5%) tested positive for anti-NF155 IgG4. Patients presenting with more severe disease had higher antibody titers. Ultrasound demonstrated diffuse nerve enlargement. Axonal excitability studies were markedly abnormal, with subsequent mathematical modeling of the results supporting disruption of the paranodal seal. DISCUSSION: A broad spectrum of disease severity and treatment response may be observed in anti-NF155 neuropathy. Excitability studies support the pathogenic role of anti-NF155 IgG4 antibodies targeting the paranodal region. Muscle Nerve 57: 848-851, 2018.

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.