Alessandra Bolino

Kidney stones (nephrolithiasis), which affect 12% of males and 5% of females in the western world, are familial in 45% of patients and are most commonly associated with hypercalciuria. Three disorders of hypercalciuric nephrolithiasis (Dent's disease, X-linked recessive nephrolithiasis (XRN), and X-linked recessive hypophosphataemic rickets (XLRH)) have been mapped to Xp11.22 (refs 5-7). A microdeletion in one Dent's disease kindred allowed the identification of a candidate gene, CLCN5 (refs 8,9) which encodes a putative renal chloride channel. Here we report the investigation of 11 kindreds with these renal tubular disorders for CLCN5 abnormalities; this identified three nonsense, four missense and two donor splice site mutations, together with one intragenic deletion and one microdeletion encompassing the entire gene. Heterologous expression of wild-type CLCN5 in Xenopus oocytes yielded outwardly rectifying chloride currents, which were either abolished or markedly reduced by the mutations. The common aetiology for Dent's disease, XRN and XLRH indicates that CLCN5 may be involved in other renal tubular disorders associated with kidney stones.

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.

Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2-null mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell–autonomous loss of Mtmr2–Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

The myotubularin family of phosphoinositide phosphatases includes several members mutated in neuromuscular diseases or associated with metabolic syndrome, obesity, and cancer. Catalytically dead phosphatases regulate their active homologs by heterodimerization and potentially represent key players in the phosphatase-kinase balance. Although the enzymatic specificity for phosphoinositides indicates a role for myotubularins in endocytosis and membrane trafficking, recent findings in cellular and animal models suggest that myotubularins regulate additional processes including cell proliferation and differentiation, autophagy, cytokinesis, and cytoskeletal and cell junction dynamics. In this review, we discuss how myotubularins regulate such diverse processes, emphasizing newly identified functions in a physiological and pathological context. A better understanding of myotubularin pathophysiology will pave the way towards therapeutic strategies.

Charcot-Marie-Tooth (CMT) neuropathies are one of the most common neuromuscular disorders. However, despite the identification of more than 100 causative genes, therapeutic options are still missing. The generation of authentic animal models and the increasing insights into the understanding of disease mechanisms, in addition to extraordinary developments in gene and molecular therapies, are quickly changing this scenario, and several strategies are currently being translated, or are getting close to, clinical trials. Here, we provide an overview of the most recent advances for the therapy of CMT at both the preclinical and clinical levels. For clarity, we have grouped the approaches in three different categories: gene therapy based on viral-mediated delivery, molecular therapies based on alternative delivery systems, and pharmacological therapies.

The myotubularin family is a large eukaryotic group within the tyrosine/dual-specificity phosphatase super-family (PTP/DSP). Among the 14 human members, three are mutated in genetic diseases: myotubular myopathy and two forms of Charcot-Marie-Tooth neuropathy. We present an analysis of the myotubularin family in sequenced genomes. The myotubularin family encompasses catalytically active and inactive phosphatases, and both classes are well conserved from nematode to man. Catalytically active myotubularins dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns3,5P2, leading to the production of PtdIns and PtdIns5P. This activity may be modulated by direct interaction with catalytically inactive myotubularins. These phosphoinositides are signaling molecules that are notably involved in vacuolar transport and membrane trafficking. Myotubularins are thus proposed to be implicated in these cellular mechanisms, and recent observations on myotubularins homologues in the nematode Caenorhabditis elegans indicate a role in endocytosis.

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto-oncogene, repeatedly identified in the heterozygous state in both long- and short-segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single-strand conformational polymorphism analysis established for all the 20 exons of the RET proto-oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement). Hum Mutat 9:243–249, 1997. © 1997 Wiley-Liss, Inc.

How membrane biosynthesis and homeostasis is achieved in myelinating glia is mostly unknown.We previously reported that loss of myotubularin-related protein 2 (MTMR2) provokes autosomal recessive demyelinating Charcot-Marie-Tooth type 4B1 neuropathy, characterized by excessive redundant myelin, also known as myelin outfoldings.We generated a Mtmr2-null mouse that models the human neuropathy.We also found that, in Schwann cells, Mtmr2 interacts with Discs large 1 (Dlg1), a scaffold involved in polarized trafficking and membrane addition, whose localization in Mtmr2-null nerves is altered.We here report that, in Schwann cells, Dlg1 also interacts with kinesin 13B (kif13B) and Sec8, which are involved in vesicle transport and membrane tethering in polarized cells, respectively.Taking advantage of the Mtmr2-null mouse as a model of impaired membrane formation, we provide here the first evidence for a machinery that titrates membrane formation during myelination.We established Schwann cell/DRG neuron cocultures from Mtmr2-null mice, in which myelin outfoldings were reproduced and almost completely rescued by Mtmr2 replacement.By exploiting this in vitro model, we propose a mechanism whereby kif13B kinesin transports Dlg1 to sites of membrane remodeling where it coordinates a homeostatic control of myelination.The interaction of Dlg1 with the Sec8 exocyst component promotes membrane addition, whereas with Mtmr2, negatively regulates membrane formation.Myelin outfoldings thus arise as a consequence of the loss of negative control on the amount of membrane, which is produced during myelination.

We previously reported that autosomal recessive demyelinating Charcot-Marie-Tooth (CMT) type 4B1 neuropathy with myelin outfoldings is caused by loss of MTMR2 (Myotubularin-related 2) in humans, and we created a faithful mouse model of the disease. MTMR2 dephosphorylates both PtdIns3P and PtdIns(3,5)P(2), thereby regulating membrane trafficking. However, the function of MTMR2 and the role of the MTMR2 phospholipid phosphatase activity in vivo in the nerve still remain to be assessed. Mutations in FIG4 are associated with CMT4J neuropathy characterized by both axonal and myelin damage in peripheral nerve. Loss of Fig4 function in the plt (pale tremor) mouse produces spongiform degeneration of the brain and peripheral neuropathy. Since FIG4 has a role in generation of PtdIns(3,5)P(2) and MTMR2 catalyzes its dephosphorylation, these two phosphatases might be expected to have opposite effects in the control of PtdIns(3,5)P(2) homeostasis and their mutations might have compensatory effects in vivo. To explore the role of the MTMR2 phospholipid phosphatase activity in vivo, we generated and characterized the Mtmr2/Fig4 double null mutant mice. Here we provide strong evidence that Mtmr2 and Fig4 functionally interact in both Schwann cells and neurons, and we reveal for the first time a role of Mtmr2 in neurons in vivo. Our results also suggest that imbalance of PtdIns(3,5)P(2) is at the basis of altered longitudinal myelin growth and of myelin outfolding formation. Reduction of Fig4 by null heterozygosity and downregulation of PIKfyve both rescue Mtmr2-null myelin outfoldings in vivo and in vitro.

Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by deficient activity of β-galactocerebrosidase (GALC). The infantile forms manifest with rapid and progressive central and peripheral demyelination, which represent a major hurdle for any treatment approach. We demonstrate here that neonatal lentiviral vector-mediated intracerebral gene therapy (IC GT) or transplantation of GALC-overexpressing neural stem cells (NSC) synergize with bone marrow transplant (BMT) providing dramatic extension of lifespan and global clinical–pathological rescue in a relevant GLD murine model. We show that timely and long-lasting delivery of functional GALC in affected tissues ensured by the exclusive complementary mode of action of the treatments underlies the outstanding benefit. In particular, the contribution of neural stem cell transplantation and IC GT during the early asymptomatic stage of the disease is instrumental to enhance long-term advantage upon BMT. We clarify the input of central nervous system, peripheral nervous system and periphery to the disease, and the relative contribution of treatments to the final therapeutic outcome, with important implications for treatment strategies to be tried in human patients. This study gives proof-of-concept of efficacy, tolerability and clinical relevance of the combined gene/cell therapies proposed here, which may constitute a feasible and effective therapeutic opportunity for children affected by GLD.

We describe 10 patients from a large family with early onset motor and sensory neuropathy. Six were still living at the time of the study. In all cases, early motor milestones had been achieved. Mean age at onset of symptoms was 34 months; these included progressive distal and proximal muscle weakness of lower limbs. Pes equinovarus developed in all patients during childhood. Slight facial weakness was present in four patients, and one of them also had bilateral facial synkinesia. Intellectual function was normal in all cases. There was no evidence of thickened peripheral nerves. All three adult patients (mean age, 27 years) were seriously handicapped and wheelchair-bound. Death occurred in the fourth to fifth decade of life and the duration of the illness varied from 27 to 39 years. Motor nerve conduction velocities ranged from 15 to 17 dsec in the upper limbs of the youngest patients, and were undetectable in the adult patients. Sensitive action potentials were almost always absent. In all patients, auditory evoked potentials showed abnormally delayed interpeak I-III latencies. The most prominent pathologic finding was a highly unusual myelin abnormality consisting of irregular redundant loops and folding of the myelin sheath. The genealogic study gave strong evidence of autosomal-recessive inheritance. The molecular analysis failed to demonstrate either duplication in the chromosome 17p11.2-12, point mutations in the four exons of the <i>PMP-22</i> (17p11.2) and the six exons of the <i>PO</i> (1q21–q25) genes, or linkage to chromosome 8q13–21.1.

Hereditary motor and sensory neuropathy (HMSN) with focally folded myelin sheaths, or Charcot-Marie-Tooth type 4B (CMT4B), is a distinct clinical entity belonging to the heterogeneous group of autosomal recessive demyelinating neuropathies. We first described a large pedigree with CMT4B, which showed a high consanguinity level and an autosomal recessive pattern of inheritance. Through conventional linkage analysis, we excluded linkage of the locus segregating in this pedigree to any of the known genes responsible for other HMSNs. Using homozygosity mapping and haplotype sharing analysis, we were able to localize the disease gene in a 4 cM interval on chromosome 11q23, between the D11S1332 and D11S917 loci. On the basis of the clinical characteristics of the disease, we propose that this locus corresponds to the CMT4B gene.

Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth type 4B1 (CMT4B1). This disorder is characterized by childhood onset of weakness and sensory loss, severely decreased nerve conduction velocity, demyelination in the nerve with myelin outfoldings, and severe functional impairment of affected patients, mainly resulting from loss of myelinated fibers in the nerve. We recently generated Mtmr2-null(neo) mice, which show a dysmyelinating neuropathy with myelin outfoldings, thus reproducing human CMT4B1. Mtmr2 is detected in both Schwann cells and neurons, in which it interacts with discs large 1/synapse-associated protein 97 and neurofilament light chain, respectively. Here, we specifically ablated Mtmr2 in either Schwann cells or motor neurons. Disruption of Mtmr2 in Schwann cells produced a dysmyelinating phenotype very similar to that of the Mtmr2-null(neo) mouse. Disruption of Mtmr2 in motor neurons does not provoke myelin outfoldings nor axonal defects. We propose that loss of Mtmr2 in Schwann cells, but not in motor neurons, is both sufficient and necessary to cause CMT4B1 neuropathy. Thus, therapeutical approaches might be designed in the future to specifically deliver the Mtmr2 phospholipid phosphatase to Schwann cells in affected nerves.

Myelination is a complex process that requires coordinated Schwann cell-axon interactions during development and regeneration. Positive and negative regulators of myelination have been recently described, and can belong either to Schwann cells or neurons. Vimentin is a fibrous component present in both Schwann cell and neuron cytoskeleton, the expression of which is timely and spatially regulated during development and regeneration. We now report that vimentin negatively regulates myelination, as loss of vimentin results in peripheral nerve hypermyelination, owing to increased myelin thickness in vivo, in transgenic mice and in vitro in a myelinating co-culture system. We also show that this is due to a neuron-autonomous increase in the levels of axonal neuregulin 1 (NRG1) type III. Accordingly, genetic reduction of NRG1 type III in vimentin-null mice rescues hypermyelination. Finally, we demonstrate that vimentin acts synergistically with TACE, a negative regulator of NRG1 type III activity, as shown by hypermyelination of double Vim/Tace heterozygous mice. Our results reveal a novel role for the intermediate filament vimentin in myelination, and indicate vimentin as a regulator of NRG1 type III function.

Signals that promote myelination must be tightly modulated to adjust myelin thickness to the axonal diameter. In the peripheral nervous system, axonal neuregulin 1 type III promotes myelination by activating erbB2/B3 receptors and the PI3K/AKT/mTOR pathway in Schwann cells. Conversely, PTEN (phosphatase and tensin homolog on chromosome 10) dephosphorylates PtdIns(3,4,5)P3 and negatively regulates the AKT pathway and myelination. Recently, the DLG1/SAP97 scaffolding protein was described to interact with PTEN to enhance PIP3 dephosphorylation. Here we now report that nerves from mice with conditional inactivation of Dlg1 in Schwann cells display only a transient increase in myelin thickness during development, suggesting that DLG1 is a transient negative regulator of myelination. Instead, we identified DDIT4/RTP801/REDD1 as a sustained negative modulator of myelination. We show that DDIT4 is expressed in Schwann cells and its maximum expression level precedes the peak of AKT activation and of DLG1 activity in peripheral nerves. Moreover, loss of DDIT4 expression both in vitro and in vivo in Ddit4-null mice provokes sustained hypermyelination and enhanced mTORC1 activation, thus suggesting that this molecule is a novel negative regulator of PNS myelination.

Abstract Charcot–Marie–Tooth ( CMT ) neuropathies are highly heterogeneous disorders caused by mutations in more than 70 genes, with no available treatment. Thus, it is difficult to envisage a single suitable treatment for all pathogenetic mechanisms. Axonal Neuregulin 1 (Nrg1) type III drives Schwann cell myelination and determines myelin thickness by ErbB2/B3‐ PI 3K–Akt signaling pathway activation. Nrg1 type III is inhibited by the α‐secretase Tace, which negatively regulates PNS myelination. We hypothesized that modulation of Nrg1 levels and/or secretase activity may constitute a unifying treatment strategy for CMT neuropathies with focal hypermyelination as it could restore normal levels of myelination. Here we show that in vivo delivery of Niaspan, a FDA ‐approved drug known to enhance TACE activity, efficiently rescues myelination in the Mtmr2 −/− mouse, a model of CMT 4B1 with myelin outfoldings, and in the Pmp22 +/− mouse, which reproduces HNPP (hereditary neuropathy with liability to pressure palsies) with tomacula. Importantly, we also found that Niaspan reduces hypermyelination of Vim (vimentin) −/− mice, characterized by increased Nrg1 type III and Akt activation, thus corroborating the hypothesis that Niaspan treatment downregulates Nrg1 type III signaling.

Charcot-Marie-Tooth (CMT) neuropathies are very heterogeneous disorders from both a clinical and genetic point of view. The CMT genes identified so far encode different proteins that are variably involved in regulating Schwann cells and/or axonal functions. However, the function of most of these proteins still remains to be elucidated.To characterize a large cohort of patients with demyelinating, axonal, and intermediate forms of CMT neuropathy.A cohort of 131 unrelated patients were screened for mutations in 12 genes responsible for CMT neuropathies. Demyelinating, axonal, and intermediate forms of CMT neuropathy were initially distinguished as usual on the basis of electrophysiological criteria and clinical evaluation. A sural nerve biopsy was also performed for selected cases. Accordingly, patients underwent first-level analysis of the genes most frequently mutated in each clinical form of CMT neuropathy.Although our cohort had a particularly high percentage of cases of rare axonal and intermediate CMT neuropathies, we found mutations in 40% of patients. Among identified changes, 7 represented new mutations occurring in the MPZ, GJB1, EGR2, MFN2, NEFL, and HSBP1/HSP27 genes. Histopathological analysis performed in selected cases revealed morphological features, which correlated with the molecular diagnosis and provided evidence of the underlying pathogenetic mechanism.Clinical and pathological analysis of patients with CMT neuropathies contributes to our understanding of the molecular mechanisms of CMT neuropathies.

Mutations of FIG4 are responsible for Yunis-Varón syndrome, familial epilepsy with polymicrogyria, and Charcot-Marie-Tooth type 4J neuropathy (CMT4J). Although loss of the FIG4 phospholipid phosphatase consistently causes decreased PtdIns(3,5)P2 levels, cell-specific sensitivity to partial loss of FIG4 function may differentiate FIG4-associated disorders. CMT4J is an autosomal recessive neuropathy characterized by severe demyelination and axonal loss in human, with both motor and sensory involvement. However, it is unclear whether FIG4 has cell autonomous roles in both motor neurons and Schwann cells, and how loss of FIG4/PtdIns(3,5)P2-mediated functions contribute to the pathogenesis of CMT4J. Here, we report that mice with conditional inactivation of Fig4 in motor neurons display neuronal and axonal degeneration. In contrast, conditional inactivation of Fig4 in Schwann cells causes demyelination and defects in autophagy-mediated degradation. Moreover, Fig4-regulated endolysosomal trafficking in Schwann cells is essential for myelin biogenesis during development and for proper regeneration/remyelination after injury. Our data suggest that impaired endolysosomal trafficking in both motor neurons and Schwann cells contributes to CMT4J neuropathy.

Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS) and central nervous system (CNS) myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK)-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1), a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K)/v-AKT murine thymoma viral oncogene homolog (AKT) pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS.

Inherited peripheral neuropathies (IPNs) represent a broad group of genetically and clinically heterogeneous disorders, including axonal Charcot-Marie-Tooth type 2 (CMT2) and hereditary motor neuropathy (HMN). Approximately 60%-70% of cases with HMN/CMT2 still remain without a genetic diagnosis. Interestingly, mutations in HMN/CMT2 genes may also be responsible for motor neuron disorders or other neuromuscular diseases, suggesting a broad phenotypic spectrum of clinically and genetically related conditions. Thus, it is of paramount importance to identify novel causative variants in HMN/CMT2 patients to better predict clinical outcome and progression.We designed a collaborative study for the identification of variants responsible for HMN/CMT2. We collected 15 HMN/CMT2 families with evidence for autosomal recessive inheritance, who had tested negative for mutations in 94 known IPN genes, who underwent whole-exome sequencing (WES) analyses. Candidate genes identified by WES were sequenced in an additional cohort of 167 familial or sporadic HMN/CMT2 patients using next-generation sequencing (NGS) panel analysis.Bioinformatic analyses led to the identification of novel or very rare variants in genes, which have not been previously associated with HMN/CMT2 (ARHGEF28, KBTBD13, AGRN and GNE); in genes previously associated with HMN/CMT2 but in combination with different clinical phenotypes (VRK1 and PNKP), and in the SIGMAR1 gene, which has been linked to HMN/CMT2 in only a few cases. These findings were further validated by Sanger sequencing, segregation analyses and functional studies.These results demonstrate the broad spectrum of clinical phenotypes that can be associated with a specific disease gene, as well as the complexity of the pathogenesis of neuromuscular disorders.

Charcot–Marie–Tooth disease type 4B1, CMT4B1, is a severe, autosomal-recessive, demyelinating peripheral neuropathy, due to mutations in the Myotubularin-related 2 gene, MTMR2. MTMR2 is widely expressed and encodes a phosphatase whose substrates include phosphoinositides. However, this does not explain how MTMR2 mutants specifically produce demyelination in the peripheral nerve. Therefore, we analysed the cellular and subcellular distribution of Mtmr2 in nerve. Mtmr2 was detected in all cytoplasmic compartments of myelin-forming Schwann cells, as well as in the cytoplasm of non-myelin-forming Schwann cells and both sensory and motorneurons. In contrast, Mtmr2 was detected in the nucleus of Schwann cells and motorneurons, but not in the nucleus of sensory neurons. As Mtmr2 is diffusely present also within the nerve, a specific function could derive instead from nerve-specific interacting proteins. Therefore, we performed two yeast two-hybrid screenings, using either fetal brain or peripheral nerve cDNA libraries. The neurofilament light chain protein, NF-L, was identified repeatedly in both screenings, and found to interact with MTMR2 in both Schwann cells and neurons. Interestingly, NF-L, encoding NF-L, is mutated in CMT2E. These data may provide a basis for the nerve-specific pathogenesis of CMT4B1, and further support for the notion that hereditary demyelinating and axonal neuropathies may represent different clinical manifestations of a common pathological mechanism.

Charcot-Marie-Tooth type 4B (CMT4B) is a severe autosomal recessive neuropathy with demyelination and myelin outfoldings of the nerve. This disorder is genetically heterogeneous, but thus far, mutations in myotubularin-related 2 (MTMR2) and MTMR13 genes have been shown to underlie CMT4B1 and CMT4B2, respectively. MTMR2 and MTMR13 belong to a family of ubiquitously expressed proteins sharing homology with protein tyrosine phosphatases (PTPs). The MTMR family, which has 14 members in humans, comprises catalytically active proteins, such as MTMR2, and catalytically inactive proteins, such as MTMR13. Despite their homology with PTPs, catalytically active MTMR phosphatases dephosphorylate both PtdIns3P and PtdIns(3,5)P2 phosphoinositides. Thus, MTMR2 and MTMR13 may regulate vesicular trafficking in Schwann cells. Loss of these proteins could lead to uncontrolled folding of myelin and, ultimately, to CMT4B. In this review, we discuss recent findings on this interesting protein family with the main focus on MTMR2 and MTMR13 and their involvement in the biology of Schwann cell and CMT4B neuropathies.

Objective Charcot‐Marie‐Tooth (CMT) disease 4B1 and 4B2 (CMT4B1/B2) are characterized by recessive inheritance, early onset, severe course, slowed nerve conduction, and myelin outfoldings. CMT4B3 shows a more heterogeneous phenotype. All are associated with myotubularin‐related protein (MTMR) mutations. We conducted a multicenter, retrospective study to better characterize CMT4B. Methods We collected clinical and genetic data from CMT4B subjects in 18 centers using a predefined minimal data set including Medical Research Council (MRC) scores of nine muscle pairs and CMT Neuropathy Score. Results There were 50 patients, 21 of whom never reported before, carrying 44 mutations, of which 21 were novel and six representing novel disease associations of known rare variants. CMT4B1 patients had significantly more‐severe disease than CMT4B2, with earlier onset, more‐frequent motor milestones delay, wheelchair use, and respiratory involvement as well as worse MRC scores and motor CMT Examination Score components despite younger age at examination. Vocal cord involvement was common in both subtypes, whereas glaucoma occurred in CMT4B2 only. Nerve conduction velocities were similarly slowed in both subtypes. Regression analyses showed that disease severity is significantly associated with age in CMT4B1. Slopes are steeper for CMT4B1, indicating faster disease progression. Almost none of the mutations in the MTMR2 and MTMR13 genes, responsible for CMT4B1 and B2, respectively, influence the correlation between disease severity and age, in agreement with the hypothesis of a complete loss of function of MTMR2/13 proteins for such mutations. Interpretation This is the largest CMT4B series ever reported, demonstrating that CMT4B1 is significantly more severe than CMT4B2, and allowing an estimate of prognosis. ANN NEUROL 2019

Significance Charcot-Marie-Tooth type 4B1 (CMT4B1) is a very severe demyelinating neuropathy with childhood onset, characterized by myelin outfoldings, a pathological aberrant form of myelination. This morphology may be the consequence of an excessive longitudinal growth of the myelinated internode during postnatal nerve development. We first demonstrated that loss of MTMR2 (Myotubularin-related 2) phosphatase causes CMT4B1. MTMR2 dephosphorylates phospholipids, important regulators of membrane trafficking, which is a key process in Schwann cells, the myelinating cells in the PNS. However, why loss of MTMR2 provokes CMT4B1 remains to be assessed. Here we propose a mechanism by which MTMR2, by regulating PtdIns(3,5) P 2 phosphoinositide levels, coordinates myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics necessary to expand the membrane and promote myelin growth.

Heat shock protein 27 (HSP27) mutations have been reported to cause both Charcot-Marie-Tooth disease (CMT) type 2F and distal hereditary motor neuropathy (dHMN) although never previously in a single family.To analyse clinical and electrophysiological findings obtained in a single large Sardinian family bearing the HSP27 R127W mutation.Twenty-one members of a five generation Sardinian family have been studied, including thirteen members affected by peroneal muscular atrophy and proved heterozygous for the known HSP27 R127W mutation. Twelve patients and eight unaffected relatives were subjected to clinical examination. A standardised electrophysiological study was performed in eleven patients and six unaffected relatives.Mean age at onset (+/-SD) was 31.2+/-7.2 years. Mean age at investigation was 45.2+/-12.9 years and mean disease duration at the time of investigation was 14+/-12.9 years. According to current criteria for CMT2 and dHMN, of the 10 patients who had undergone both clinical and neurophysiological examination, five were diagnosed as CMT2, two as dHMN and a further two patients were labelled as an intermediate type. Finally, due to the presence of spastic paraplegia, the index patient did not meet established criteria for the diagnosis of CMT or dHMN.Findings obtained in the present study, broadening the spectrum of clinical manifestations of disorders associated with HSP27 mutations, support the hypothesis of a continuum between CMT2 and dHMN forms and suggest a possible common spectrum between these entities and several forms of CMT plus pyramidal features (HMSN V), providing important implications for molecular genetic testing.

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1+ and Rab7+ vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1+perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis.

Abstract Inherited peripheral neuropathies (IPNs) represent a broad group of disorders including Charcot-Marie-Tooth (CMT) neuropathies characterized by defects primarily arising in myelin, axons, or both. The molecular mechanisms by which mutations in nearly 100 identified IPN/CMT genes lead to neuropathies are poorly understood. Here we show that the Ras-related GTPase Rab35 controls myelin growth via complex formation with the myotubularin-related phosphatidylinositol (PI) 3-phosphatases MTMR13 and MTMR2, encoded by genes responsible for CMT-types 4B2 and B1 in humans, and found that it downregulates lipid-mediated mTORC1 activation, a pathway known to crucially regulate myelin biogenesis. Targeted disruption of Rab35 leads to hyperactivation of mTORC1 signaling caused by elevated levels of PI 3-phosphates and to focal hypermyelination in vivo. Pharmacological inhibition of phosphatidylinositol 3,5-bisphosphate synthesis or mTORC1 signaling ameliorates this phenotype. These findings reveal a crucial role for Rab35-regulated lipid turnover by myotubularins to repress mTORC1 activity and to control myelin growth.

Our understanding of the processes controlling peripheral nervous system myelination have been significantly benefited by the development of an in vitro myelinating culture system in which primary Schwann cells are cocultured together with primary sensory neurons. In this chapter, we describe the protocol currently used in our laboratories to establish Schwann cells neuronal myelinating cocultures. We also include a detailed description of the various substrates that can be used to establish it.

Myelin is a key evolutionary specialization and adaptation of vertebrates formed by the plasma membrane of glial cells, which insulate axons in the nervous system. Myelination not only allows rapid and efficient transmission of electric impulses in the axon by decreasing capacitance and increasing resistance but also influences axonal metabolism and the plasticity of neural circuits. In this review, we will focus on Schwann cells, the glial cells which form myelin in the peripheral nervous system. Here, we will describe the main extrinsic and intrinsic signals inducing Schwann cell differentiation and myelination and how myelin biogenesis is achieved. Finally, we will also discuss how the study of human disorders in which molecules and pathways relevant for myelination are altered has enormously contributed to the current knowledge on myelin biology.

Hereditary motor and sensory neuropathy with focally folded myelin sheaths, or Charcot-Marie-Tooth disease neuropathy type 4B (CMT4B), is a distinct clinical and genetic entity belonging to the heterogeneous group of autosomal recessive demyelinating neuropathies. We previously described a large pedigree with CMT4B and found evidence of linkage to chromosome 11q23. We now describe a second, unrelated family in which two individuals were affected with CMT4B. We exclude the disease locus segregating in this smaller pedigree from the 11q23 region as well as from most of the regions where other CMT loci have been mapped. We thus provide evidence for a second locus causing the CMT4B phenotype.

<b>Objective: </b> Recent evidence in animal models suggests that components of the extracellular matrix (ECM) play a primary role in peripheral nerve degeneration and regeneration. <b>Methods: </b> We investigated the expression of several ECM molecules in human sural nerves by immunohistochemistry, Western blot, and reverse transcriptase PCR analysis. To unravel the possible role of these molecules in nerve regeneration, we compared results obtained from nerves with abundant signs of regeneration with those with complete absence of axonal regeneration. The role of some ECM components on neurite extension was further tested in dorsal root ganglion cultures. <b>Results: </b> We observed that the ECM composition significantly differs in regenerating compared with nonregenerating nerves, independently from their etiologic background. Fibronectin was abundantly expressed in regenerating nerves, whereas vitronectin and fibrin(ogen) prevailed in nonregenerating nerves. Whereas fibronectin is secreted by endoneurial cells, in vivo and vitro studies showed that the source of vitronectin and fibrin(ogen) is the bloodstream. <b>Conclusions: </b> These data indicate that nerve regeneration is impaired in the presence of breaches in the blood–nerve barrier or impaired extracellular matrix (ECM) degradation that leads to accumulation of plasma vitronectin and fibrin(ogen). The transformation into mature, fibronectin-enriched ECM is necessary for efficient nerve regeneration in humans.

Abstract Neuromuscular diseases encompass a heterogeneous array of disorders characterized by varying onset ages, clinical presentations, severity, and progression. While these conditions can stem from acquired or inherited causes, this review specifically focuses on disorders arising from genetic abnormalities, excluding metabolic conditions. The pathogenic defect may primarily affect the anterior horn cells, the axonal or myelin component of peripheral nerves, the neuromuscular junction, or skeletal and/or cardiac muscles. While inherited neuromuscular disorders have been historically deemed not treatable, the advent of gene-based and molecular therapies is reshaping the treatment landscape for this group of condition. With the caveat that many products still fail to translate the positive results obtained in pre-clinical models to humans, both the technological development (e.g., implementation of tissue-specific vectors) as well as advances on the knowledge of pathogenetic mechanisms form a collective foundation for potentially curative approaches to these debilitating conditions. This review delineates the current panorama of therapies targeting the most prevalent forms of inherited neuromuscular diseases, emphasizing approved treatments and those already undergoing human testing, offering insights into the state-of-the-art interventions.

Charcot-Marie-Tooth type 4B1 (CMT4B1) is an autosomal recessive motor and sensory demyelinating neuropathy characterized by the association of early-onset neurological symptoms and typical histological findings. The natural history and the clinical variability of the disease are still poorly known, thus further clarification of the different phenotypes is needed. We report on the case of a Pakistani girl born to consanguineous parents harboring a novel mutation in the MTMR2 gene. When aged 18 months, reduced limb tone, muscle wasting associated with proximal and distal weakness prevalent in lower limbs, absence of tendon reflexes, hoarseness and inspiratory stridor were detected. Vocal cord palsy was diagnosed shortly after. We suggest that laryngeal involvement might be a relevant and initial feature of early-onset CMT4B1 neuropathy. Thus, affected patients should undergo early laryngological evaluation in order to prompt an appropriate management.

Charcot-Marie-Tooth type 4B (CMT4B) is caused by mutations in the myotubularin-related 2 gene, MTMR2, on chromosome 11q22. To date, six loss of function mutations and one missense mutation have been demonstrated in CMT4B patients. It remains to be determined how dysfunction of a ubiquitously expressed phosphatase causes a demyelinating neuropathy. An animal model for CMT4B would provide insights into the pathogenesis of this disorder. We have therefore characterized the mouse homologue of MTMR2 by reconstructing the full-length Mtmr2 cDNA as well as the genomic structure. The 1932 nucleotide open reading frame corresponds to 15 coding exons, spanning a genomic region of approximately 55 kilobases, on mouse chromosome 9 as demonstrated by fluorescence in situ hybridization analysis. A comparison between the mouse and human genes revealed a similar genomic structure, except for the number of alternatively spliced exons in the 5'-untranslated region, two in mouse and three in man. In situ hybridization analysis of mouse embryos showed that Mtmr2 was ubiquitously expressed during organogenesis at E9.5, with some areas of enriched expression. At E14.5, Mtmr2 mRNA was more abundant in the peripheral nervous system, including in dorsal root ganglia and spinal roots.

Abstract Numerous transgenic and knockout mouse models of human hereditary neuropathies have become available over the past decade. We describe a simple, reproducible, and safe biopsy of mouse skin for histopathological evaluation of the peripheral nervous system (PNS) in models of hereditary neuropathies. We compared the diagnostic outcome between sciatic nerve and dermal nerves found in skin biopsy (SB) from the hind foot. A total of five animal models of different Charcot‐Marie‐Tooth neuropathies, and one model of congenital muscular dystrophy associated neuropathy were examined. In wild type mice, dermal nerve fibers were readily identified by immunohistochemistry, light, and electron microscopy and they appeared similar to myelinated fibers in sciatic nerve. In mutant mice, SB manifested myelin abnormalities similar to those observed in sciatic nerves, including hypomyelination, onion bulbs, myelin outfolding, redundant loops, and tomacula. In many strains, however, SB showed additional abnormalities—fiber loss, dense neurofilament packing with lower phosphorylation status, and axonal degeneration—undetected in sciatic nerve, possibly because SB samples distal nerves. SB, a reliable technique to investigate peripheral neuropathies in human beings, is also useful to investigate animal models of hereditary neuropathies. Our data indicate that SB may reveal distal axonal pathology in mouse models and permits sequential follow‐up of the neuropathy in an individual mouse, thereby reducing the number of mice necessary to document pathology of the PNS. © 2010 Wiley‐Liss, Inc.

Remodeling of extracellular matrix (ECM) is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR) is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA) promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.

XLMTM (X-linked myotubular myopathy) is a rare recessive disorder. The MTM1 gene is localized on Xq28 and it encompasses 100 kb at the genomic level, with 15 exons coding for a 603 aa protein named myotubularin. Mutations in myotubularin result in the human disease XLMTM, which is characterized by the persistence of muscle fibres that retain an immature phenotype [1].

Journal of the Peripheral Nervous SystemVolume 20, Issue 4 p. 419-421 LETTER TO THE EDITOR A novel heat shock protein 27 homozygous mutation: widening of the continuum between MND/dHMN/CMT2 Marina Scarlato, Corresponding Author Marina Scarlato Department of Neurology, INSPE and Division of Neuroscience, Milan, ItalyAddress correspondence to: Marina Scarlato, MD, PhD, Division of Neuroscience, Department of Neurology & INSPE, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy. Tel: +39 02 26433837; Fax: +39 02 2643 2974; E-mail: [email protected]Search for more papers by this authorFiammetta Viganò, Fiammetta Viganò Human Inherited Neuropathies Unit, INSPE and Division of Neuroscience, Milan, ItalySearch for more papers by this authorPaola Carrera, Paola Carrera Unit of Genomics for Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, and Laboratory of Clinical Molecular Biology, IRCCS San Raffaele Scientific Institute, Milan, ItalySearch for more papers by this authorStefano Carlo Previtali, Stefano Carlo Previtali Department of Neurology, INSPE and Division of Neuroscience, Milan, ItalySearch for more papers by this authorAlessandra Bolino, Alessandra Bolino Human Inherited Neuropathies Unit, INSPE and Division of Neuroscience, Milan, ItalySearch for more papers by this author Marina Scarlato, Corresponding Author Marina Scarlato Department of Neurology, INSPE and Division of Neuroscience, Milan, ItalyAddress correspondence to: Marina Scarlato, MD, PhD, Division of Neuroscience, Department of Neurology & INSPE, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy. Tel: +39 02 26433837; Fax: +39 02 2643 2974; E-mail: [email protected]Search for more papers by this authorFiammetta Viganò, Fiammetta Viganò Human Inherited Neuropathies Unit, INSPE and Division of Neuroscience, Milan, ItalySearch for more papers by this authorPaola Carrera, Paola Carrera Unit of Genomics for Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, and Laboratory of Clinical Molecular Biology, IRCCS San Raffaele Scientific Institute, Milan, ItalySearch for more papers by this authorStefano Carlo Previtali, Stefano Carlo Previtali Department of Neurology, INSPE and Division of Neuroscience, Milan, ItalySearch for more papers by this authorAlessandra Bolino, Alessandra Bolino Human Inherited Neuropathies Unit, INSPE and Division of Neuroscience, Milan, ItalySearch for more papers by this author First published: 13 December 2015 https://doi.org/10.1111/jns.12139Citations: 11Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References Benndorf R, Martin JL, Kosakovsky Pond SL, Wertheim JO (2014). Neuropathy- and myopathy- associated mutations in human small heat shock proteins: characteristics and evolutionary history of the mutation sites. Mutat Res Rev Mutat Res 761: 15–30. Datskevich PN, Mymrikov EV, Gusev NB (2012). Utilization of fluorescent chimeras for investigation of heterooligomeric complexes formed by human small heat shock proteins. Biochimie 94: 1794–1804. Houlden H, Laura M, Wavrant-De Vrièze F, Blake J, Wood N, Reilly MM (2008). Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2. Neurology 71: 1660–1668. Rossor AM, Kalmar B, Greensmith L, Reilly MM (2012). The distal hereditary motor neuropathies. J Neurol Neurosurg Psychiatry 83: 6–14. Solla P, Vannelli A, Bolino A, Marrosu G, Coviello S, Murru MR, Tranquilli S, Corongiu D, Benedetti S, Marrosu MG (2010). Heat shock protein 27 R127W mutation: evidence of a continuum between axonal Charcot-Marie-Tooth and distal hereditary motor neuropathy. J Neurol Neurosurg Psychiatry 81: 958–962. Wettstein G, Bellaye PS, Micheau O, Bonniaud P (2012). Small heat shock proteins and the cytoskeleton: an essential interplay for cell integrity? Int J Biochem Cell Biol 44: 1680–1686. Citing Literature Volume20, Issue4December 2015Pages 419-421 ReferencesRelatedInformation

Charcot–Marie–Tooth disease type 4B (CMT4B) is a demyelinating autosomal recessive motor and sensory neuropathy characterized by focally folded myelin sheaths in the peripheral nerve. We recently mapped the CMT4B gene to a 5-cM interval on chromosome 11q22, using homozygosity mapping and haplotype sharing analysis on a large inbred pedigree. In the present study, we report the construction of a YAC-based transcript map across the 5-cM critical region, including 26 YACs, 35 STSs, and 52 ESTs. Furthermore, using 15 additional physically ordered microsatellite markers from the 11q22 region on the original inbred family, we were able to narrow the critical interval for the gene to 2 Mb, which is now flanked by markers D11S1757 and CHLC-GATA3B05. Finally, after computer analysis of the 33 ESTs assigned to the 2-Mb interval, we demonstrated that 21 different transcripts as well as 3 known genes might represent potential candidates for the disease.

Neurofibromatosis type 2 is an inherited disorder characterized by the development of benign and malignant tumors on the auditory nerves and central nervous system with symptoms including hearing loss, poor balance, skin lesions, and cataracts. Here, we report a novel protein-protein interaction between NF2 protein (merlin or schwannomin) and erythrocyte p55, also designated as MPP1. The p55 is a conserved scaffolding protein with postulated functions in cell shape, hair cell development, and neural patterning of the retina. The FERM domain of NF2 protein binds directly to p55, and surface plasmon resonance analysis indicates a specific interaction with a kD value of 3.7 nM. We developed a specific monoclonal antibody against human erythrocyte p55, and found that both p55 and NF2 proteins are colocalized in the non-myelin-forming Schwann cells. This finding suggests that the p55-NF2 protein interaction may play a functional role in the regulation of apico-basal polarity and tumor suppression pathways in non-erythroid cells.

Human MutationVolume 9, Issue 2 p. 185-187 Mutations in Brief Startle disease in an Italian family by mutation (K276E): The α-subunit of the inhibiting glycine receptor Marco Seri, Marco Seri Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorAlessandra Bolino, Alessandra Bolino Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorLuis Juan Vicente Galietta, Luis Juan Vicente Galietta Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorMargherita Lerone, Margherita Lerone Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorMargherita Silengo, Margherita Silengo Istituto di Discipline Pediatriche, Università degli Studi di Torino, 10126 Torino, ItalySearch for more papers by this authorGiovanni Romeo, Corresponding Author Giovanni Romeo Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this author Marco Seri, Marco Seri Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorAlessandra Bolino, Alessandra Bolino Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorLuis Juan Vicente Galietta, Luis Juan Vicente Galietta Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorMargherita Lerone, Margherita Lerone Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorMargherita Silengo, Margherita Silengo Istituto di Discipline Pediatriche, Università degli Studi di Torino, 10126 Torino, ItalySearch for more papers by this authorGiovanni Romeo, Corresponding Author Giovanni Romeo Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this author First published: 07 January 1999 https://doi.org/10.1002/(SICI)1098-1004(1997)9:2<185::AID-HUMU14>3.0.CO;2-ZCitations: 18AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References Andermann F, Keene DL, Andermann E, Quesney LF (1980) Startle disease or hyperekplexia: further delineation of the syndrome. Brain 103: 985– 997. Gatzi J-L, Devillers-Thiery A, Hussy N, Bertrand S, Changeux J-P, Bertrand D (1992) Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature 359: 500– 505. Grenningloh G, Schmieden V, Schofield PR, Seeburg PH, Siddique T, Mohandas TK, Becker C-M, Betz H (1990) Alpha subunit variants of the human glycine receptor: Primary structures, functional expression and chromosomal localization of the corresponding genes. EMBO J 9: 771– 776. Kirstein L, Silfverskiold BP (1958) A family with emotionally precipitated “drop seizures”. Acta Psychiatr Neurol Scand 33: 471– 476. Kok O, Bruyn GW (1962) An unidentified hereditary disease. Lancet 1: 1359. Langosch D, Becker C-M, Betz H (1990) The inhibitory glycine receptor: A ligand-gated chloride channel of the central nervous system. Eur J Biochem 194: 1– 8. Langosch D, Hartung K, Grell E, Bamberg E, Betz H (1991) Ion channel formation by synthetic transmembrane segments of the inhibitory glycine receptor: A model study. Biochim Byophys Acta 1063: 36– 44. Nigro MA, Lim H-CN (1992) Hyperekplexia and sudden neonatal death. Pediatr Neurol 8: 221– 225. Rajendra S, Lynch JW, Pierce KD, French CR, Barry PH, Schofield PR (1994) Startle disease mutations reduce the agonist sensitivity of the human inhibitory glycine receptor. J Biol Chem 269: 18739– 18742. Rees MI, Andrew M, Jawad S, Owen MJ (1994) Evidence for recessive as well as dominant forms of startle disease (hyperekplexia) caused by mutations in the α1 subunit of the inhibitory glycine receptor. Hum Mol Genet 3: 2175– 2179. Ryan SG, Sherman SL, Terry JC, Sparkes, R, Torres C, Mackey R (1992) Startle disease or hyperekplexia: Response to Clonazepam and assignment of the gene (STHE) to chromosome 5q by linkage analysis. Ann Neurol 31: 663– 668. Schorderet DF, Pescia G, Bernasconi A, Regli F (1994) An additional family with Startle disease and a G1192A mutation at the α1 subunit of the inhibitory glycine receptor gene. Hum Mol Genet 3: 1201. Shiang R, Ryan SG, Zhu Y-Z, Hahn AF, O'Connell P, Wasmuth J (1993) Mutations in the α1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nature Genet 5: 351– 358. Shiang R, Ryan SG, Zhu Y-Z, Fielder TJ, Allen RJ, Fryer A, Yamashita S, O'Connell P, Wasmuth JJ (1995) Mutational analysis of famailaial and sporadic hyperekplexia. Ann Neurol 38: 85– 91. Suhren O, Bruyn GW, Tuynman JA (1966) Hyperekplexia: A hereditary startle syndrome. J Neurol Sci 3: 577– 605. Citing Literature Volume9, Issue21997Pages 185-187 ReferencesRelatedInformation

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.

Evaluation of: Moreno-Navarrete JM, Catalȥn V, Whyte L et al. The l-a-lysophosphatidylinositol/ GPR55 system and its potential role in human obesity. Diabetes 61(2), 281–291 (2012). GPR55, first identified as an orphan G?protein-coupled receptor, was initially suggested to be the ‘atypical’ cannabinoid receptor but later lysophosphatidylinositol (LPI) was recognized as its principal endogenous agonist. In the evaluated study,the LPI–GPR55 system in adipose tissue was examined. GPR55 is expressed in white adipose tissue and its expression is higher in visceral than in subcutaneous fat. GPR55 expression is higher in obese than in lean subjects, and in the obese group is greater in diabetic than in nondiabetic patients. In addition, plasma LPI concentration is increased in obese individuals. In vitro, LPI increases intracellular Ca2+ concentration and stimulates the expression of PPAR?g and genes involved in fatty acid synthesis. In contrast to human adipocytes, LPI has no effect on triglyceride accumulation in rodent adipocytes and adipose tissue GPR55 expression is downregulated in both leptin-deficient mice and rats made obese by a high-fat diet, indicating that the role of GPR55 in adipose tissue is species specific.

A cytogenetically detectable deletion, del(10) (q11.2-->q21.2), was observed in a patient with total colonic aganglionosis with small bowel involvement (TCSA), a variant of Hirschsprung disease (HSCR). A similar deletion is present in another TCSA patient (S.M. Huson, personal communication). To reveal cytogenetically undetectable deletions of chromosome 10 in further patients, we developed a strategy for mapping chromosome 10 DNA markers with respect to the observed deletions. To this end, the two chromosome 10 homologs (deleted and normal) were segregated in two distinct somatic cell hybrids obtained after fusion of the patient's fibroblasts with a Chinese hamster ovary cell line (YH21). Hybrid cells containing chromosome 10 were selected for the expression of the gene coding for the beta subunit of the fibronectin receptor (FNRB), which maps to 10p11.2, using a monoclonal antibody against FNRB. Hybrid 185.O contains the deleted chromosome, whereas hybrid 179.Q contains the nondeleted one. Southern blot and PCR analysis of DNA from these two hybrids mapped the markers RBP3H4, RET, D10S15, D10S5, D10S22, and D10S88 inside the deletion and D10S170, CDC2, EGR2, and D10S19 outside the deletion. MEN2A and MEN2B have recently been mapped within the centromeric region closely linked to RBP3 and D10S15 (which are located inside the deletion) and cosegregate with HSCR in at least two different pedigrees. Since HSCR, MEN2A, and MEN2B represent defects of neural crest cell development, we hypothesize that they originate from mutations in different genes clustered in the centromeric region of 10q.

Because the spectrum of anorectal malformations is wide, genetic investigations of these anomalies should include the study of multigenic models presenting variable penetrance and expressivity. Current knowledge in clinical genetics, cytogenetics, and molecular genetics of anorectal anomalies are reviewed. The analysis of associated anomalies (that are found in more than 60% of anorectal malformations) is an important aspect of the molecular study, because the association of anomalies with mendelian transmission or with a recognized causative gene can be an essential starting point for further investigations. In the present study, the authors focus on associated sacral anomalies, urethral malformations, and intestinal dysganglionoses. In particular, associated sacral anomalies could be a partial expression of the Currarino syndrome, which represents the only association for which genetic evidence has been demonstrated by linkage analysis. The authors studied a four-generation pedigree with recurrence of the Currarino syndrome, and the haplotype reconstruction confirmed that the gene segregating in this family is located in the 7q36 region. The collection and study of families with multiple cases of anorectal malformations could show whether different phenotypes are caused by single genes.

Journal of the Peripheral Nervous SystemVolume 18, Issue 2 p. 192-194 LETTER TO THE EDITOR A novel homozygous mutation in the MTMR2 gene in two siblings with ‘hypermyelinating neuropathy’ Marco Luigetti, Corresponding Author Marco Luigetti Institute of Neurology, Catholic University of Sacred Heart, Rome, Italy These authors contributed equally to this work.Address correspondence to: Dr. Marco Luigetti, Institute of Neurology, Largo F. Vito 1, 00168 Rome, Italy. Tel: +(39)06-30154435; Fax: +(39)06-35501909; E-mail: [email protected].Search for more papers by this authorAlessandra Bolino, Alessandra Bolino Dulbecco Telethon Institute and INSPE, San Raffaele Scientific Institute, Milan, Italy These authors contributed equally to this work.Search for more papers by this authorStefania Scarlino, Stefania Scarlino Dulbecco Telethon Institute and INSPE, San Raffaele Scientific Institute, Milan, ItalySearch for more papers by this authorMario Sabatelli, Mario Sabatelli Institute of Neurology, Catholic University of Sacred Heart, Rome, ItalySearch for more papers by this author Marco Luigetti, Corresponding Author Marco Luigetti Institute of Neurology, Catholic University of Sacred Heart, Rome, Italy These authors contributed equally to this work.Address correspondence to: Dr. Marco Luigetti, Institute of Neurology, Largo F. Vito 1, 00168 Rome, Italy. Tel: +(39)06-30154435; Fax: +(39)06-35501909; E-mail: [email protected].Search for more papers by this authorAlessandra Bolino, Alessandra Bolino Dulbecco Telethon Institute and INSPE, San Raffaele Scientific Institute, Milan, Italy These authors contributed equally to this work.Search for more papers by this authorStefania Scarlino, Stefania Scarlino Dulbecco Telethon Institute and INSPE, San Raffaele Scientific Institute, Milan, ItalySearch for more papers by this authorMario Sabatelli, Mario Sabatelli Institute of Neurology, Catholic University of Sacred Heart, Rome, ItalySearch for more papers by this author First published: 15 May 2013 https://doi.org/10.1111/jns5.12019Citations: 6 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume18, Issue2June 2013Pages 192-194 RelatedInformation

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders caused by mutations in at least 100 genes. However, approximately 60% of cases with axonal neuropathies (CMT2) still remain without a genetic diagnosis. We aimed at identifying novel disease genes responsible for CMT2.We performed whole exome sequencing and targeted next generation sequencing panel analyses on a cohort of CMT2 families with evidence for autosomal recessive inheritance. We also performed functional studies to explore the pathogenetic role of selected variants.We identified rare, recessive variants in the MYO9B (myosin IX) gene in two families with CMT2. MYO9B has not yet been associated with a human disease. MYO9B is an unconventional single-headed processive myosin motor protein with signaling properties, and, consistent with this, our results indicate that a variant occurring in the MYO9B motor domain impairs protein expression level and motor activity. Interestingly, a Myo9b-null mouse has degenerating axons in sciatic nerves and optic nerves, indicating that MYO9B plays an essential role in both peripheral nervous system and central nervous system axons, respectively. The degeneration observed in the optic nerve prompted us to screen for MYO9B mutations in a cohort of patients with optic atrophy (OA). Consistent with this, we found compound heterozygous variants in one case with isolated OA.Novel or very rare variants in MYO9B are associated with CMT2 and isolated OA.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterised by selective susceptibility to Epstein-Barr virus and frequent association with malignant lymphomas chiefly located in the ileocecal region, liver, kidney and CNS. Taking advantage of a large bacterial clone contig, we obtained a genomic sequence of 197620 bp encompassing a deletion (XLP-D) of 116 kb in an XLP family, whose breakpoints were identified. The study of potential exons from this region in 40 unrelated XLP patients did not reveal any mutation. To define the critical region for XLP and investigate the role of the XLP-D deletion, detailed haplotypes in a region of approximately 20 cM were reconstructed in a total of 87 individuals from 7 families with recurrence of XLP. Two recombination events in a North American family and a new microdeletion (XLP-G) in an Italian family indicate that the XLP gene maps in the interval between DXS1001 and DXS8057, approximately 800 kb centromeric to the previously reported familial microdeletion XLP-D.

Genetic deficiency of β-N-acetylhexosaminidase (Hex) functionality leads to accumulation of GM2 ganglioside in Tay-Sachs disease and Sandhoff disease (SD), which presently lack approved therapies. Current experimental gene therapy (GT) approaches with adeno-associated viral vectors (AAVs) still pose safety and efficacy issues, supporting the search for alternative therapeutic strategies. Here we leveraged the lentiviral vector (LV)-mediated intracerebral (IC) GT platform to deliver Hex genes to the CNS and combined this strategy with bone marrow transplantation (BMT) to provide a timely, pervasive, and long-lasting source of the Hex enzyme in the CNS and periphery of SD mice. Combined therapy outperformed individual treatments in terms of lifespan extension and normalization of the neuroinflammatory/neurodegenerative phenotypes of SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in brain tissues that single treatments failed to achieve. Our results highlight the synergic mode of action of LV-mediated IC GT and BMT, clarify the contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of corrective enzymatic activity. These results have important implications for interpretation of ongoing experimental therapies and for design of more effective treatment strategies for GM2 gangliosidosis.

Journal of the Peripheral Nervous SystemVolume 18, Issue 3 p. 197-198 EDITORIAL Meeting Report: 2013 Peripheral Nerve Society Biennial Meeting, Saint-Malo, France, June 29–July 3, 2013 Eduardo Nobile-Orazio, Eduardo Nobile-Orazio Past PNS PresidentSearch for more papers by this authorJean-Marc Léger, Jean-Marc Léger Chair Local Organizing CommitteeSearch for more papers by this authorRichard A. Lewis, Richard A. Lewis Co-Chairs Scientific Program CommitteeSearch for more papers by this authorAlessandra Bolino, Alessandra Bolino Co-Chairs Scientific Program CommitteeSearch for more papers by this authorMichael E. Shy, Michael E. Shy PNS PresidentSearch for more papers by this authorDavid R. Cornblath, David R. Cornblath PNS Secretary-TreasurerSearch for more papers by this author Eduardo Nobile-Orazio, Eduardo Nobile-Orazio Past PNS PresidentSearch for more papers by this authorJean-Marc Léger, Jean-Marc Léger Chair Local Organizing CommitteeSearch for more papers by this authorRichard A. Lewis, Richard A. Lewis Co-Chairs Scientific Program CommitteeSearch for more papers by this authorAlessandra Bolino, Alessandra Bolino Co-Chairs Scientific Program CommitteeSearch for more papers by this authorMichael E. Shy, Michael E. Shy PNS PresidentSearch for more papers by this authorDavid R. Cornblath, David R. Cornblath PNS Secretary-TreasurerSearch for more papers by this author First published: 12 September 2013 https://doi.org/10.1111/jns5.12041Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume18, Issue3September 2013Pages 197-198 RelatedInformation

Charcot-Marie-Tooth disease type 4B (CMT4B) is a demyelinating autosomal recessive motor and sensory neuropathy characterised by focally folded myelin sheaths in the peripheral nerve. The CMT4B gene has been localised by homozygosity mapping and haplotype sharing in the 11q23 region. A cDNA encoding for the β2 subunit of the human brain sodium channel, SCN2B, has been recently assigned to the same chromosomal interval by FISH. The SCN2B gene has been considered a good candidate for CMT4B on the basis of protein homology, chromosomal localisation, and putative biological function of the coded product. In this paper, we report the genomic structure of the SCN2B gene consisting of 4 exons and 3 introns spanning a region of approximately 12 Kb. In addition, a search for mutations in patients affected with CMT4B as well as a refined physical localisation excludes SCN2B as the CMT4B gene.

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto-oncogene, repeatedly identified in the heterozygous state in both long- and short-segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single-strand conformational polymorphism analysis established for all the 20 exons of the RET proto-oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement). Hum Mutat 9:243–249, 1997. © 1997 Wiley-Liss, Inc.

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1+ and Rab7+ vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1+perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis. https://doi.org/10.7554/eLife.13023.001 eLife digest Neurons communicate with each other through long cable-like extensions called axons. An insulating sheath called myelin (or white matter) surrounds each axon, and allows electrical impulses to travel more quickly. Cells in the brain called oligodendrocytes produce myelin. If the myelin sheath is not properly formed during development, or is damaged by injury or disease, the consequences can include paralysis, impaired thought, and loss of vision. Oligodendrocytes have complex shapes, and each can generate myelin for as many as 50 axons. Oligodendrocytes produce the building blocks of myelin inside their cell bodies, by following instructions encoded by genes within the nucleus. However, the signals that regulate the trafficking of these components to the myelin sheath are poorly understood. Mironova et al. set out to determine whether signaling molecules called phosphoinositides help oligodendrocytes to mature and move myelin building blocks from the cell bodies to remote contact points with axons. Genetic techniques were used to manipulate an enzyme complex in mice that controls the production and turnover of a phosphoinositide called PI(3,5)P2. Mironova et al. found that reducing the levels of PI(3,5)P2 in oligodendrocytes caused the trafficking of certain myelin building blocks to stall. Key myelin components instead accumulated inside bubble-like structures near the oligodendrocyte’s cell body. This showed that PI(3,5)P2 in oligodendrocytes is essential for generating myelin. Further experiments then revealed that reducing PI(3,5)P2 in the neurons themselves indirectly prevented the oligodendrocytes from maturing. This suggests that PI(3,5)P2 also takes part in communication between axons and oligodendrocytes during development of the myelin sheath. A key next step will be to identify the regulatory mechanisms that control the production of PI(3,5)P2 in oligodendrocytes and neurons. Future studies could also explore what PI(3,5)P2 acts upon inside the axons, and which signaling molecules support the maturation of oligodendrocytes. Finally, it remains unclear whether PI(3,5)P2signaling is also required for stabilizing mature myelin, and for repairing myelin after injury in the adult brain. Further work could therefore address these questions as well. https://doi.org/10.7554/eLife.13023.002 Introduction In the vertebrate CNS, the majority of long axons are myelinated. Myelin greatly increases the conduction velocity of action potentials and provides metabolic support for axons. Bidirectional axo-glial signaling is critical for nervous system myelination and fiber stability (Nave and Trapp, 2008; Simons and Lyons, 2013). Myelin development is regulated by oligodendrocyte (OL) intrinsic mechanisms (Zuchero and Barres, 2013), astrocyte secreted factors (Ishibashi et al., 2006), neuronal electrical activity (Barres and Raff, 1993; Ishibashi et al., 2006) and axon derived chemical signals (Coman et al., 2005; Ohno et al., 2009; Winters et al., 2011, Yao et al., 2014). Disorders associated with defective CNS white matter range from multiple sclerosis and inherited leukodystrophies to psychiatric disorders (Fields, 2008; Makinodan et al., 2012; Perlman and Mar, 2012). FIG4 is an evolutionarily conserved lipid phosphatase that removes the 5’ phosphate group from phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2] to produce PI(3)P. Together with its antagonistic kinase PIKFYVE and the scaffold protein VAC14, FIG4 forms an enzyme complex that regulates the interconversion of PI(3)P and PI(3,5)P2 on membranes of the late endosomal/ lysosomal (LE/Lys) compartment (Jin et al., 2008; McCartney et al., 2014). In addition to its 5’-phosphatase activity, Fig4 is required to stabilize the enzyme complex. PI(3,5)P2 directly regulates the lysosomal cation channels TRPML1, TPC1 and TPC2 (Dong et al., 2010; Wang et al., 2012; 2014). Reduced activity of these lysosomal channels and the resulting osmotic enlargement of the LE/Lys may underlie vacuolization in Fig4 null cells (Lenk and Meisler, 2014). Consistent with this model, overexpression of TRPML1 in Vac14 and Fig4 mutant cells appears to rescue vacuolization (Dong et al., 2010; Zou et al., 2015). In Drosophila, loss of TRPML1 generates a muscle vacuolization phenotype reminiscent of FIG4 deficiency (Bharadwaj et al., 2016). FIG4 deficiency is particularly harmful for neural cells with elaborate morphologies, including projection neurons and myelinating glia. Mutations of human FIG4 result in neurological disorders including Charcot-Marie-Tooth type 4J, a severe form of peripheral neuropathy (Chow et al., 2007; Nicholson et al., 2011), polymicrogyria with epilepsy (Baulac et al., 2014), and Yunis-Varon syndrome (Campeau et al., 2013). Mice null for Fig4 exhibit severe tremor, brain region-specific spongiform degeneration, hypomyelination, and juvenile lethality (Chow et al., 2007; Ferguson et al., 2009; Winters et al., 2011). We previously demonstrated that a Fig4 transgene driven by the neuron-specific enolase (NSE) promoter rescued juvenile lethality and neurodegeneration in global Fig4 null mice, and that these phenotypes were not rescued by an astrocyte-specific Fig4 transgene (Ferguson et al., 2012). The neuron-specific transgene also rescued conduction in peripheral nerves (Ferguson et al., 2012) and structural defects in CNS myelination (Winters et al., 2011). Conversely, inactivation of Fig4 specifically in neurons resulted in region-specific neurodegeneration (Ferguson et al., 2012). The cellular and molecular mechanisms relating loss of Fig4 to hypomyelination are poorly understood. To further characterize the requirement of PI(3,5)P2 for CNS myelination, we manipulated individual components of the PI(3,5)P2 biosynthetic complex. Pikfyve and Vac14 global null mice die prematurely, before the onset of CNS myelination (Zhang et al., 2007; Ikonomov et al., 2011). To circumvent this limitation, we employed a combination of conditional null alleles and hypomorphic alleles in the mouse. Our study shows that multiple strategies to perturb the FIG4/PIKFYVE/VAC14 enzyme complex, and by extension the lipid product PI(3,5)P2, result in the common endpoints of arrested OL differentiation, impaired myelin protein trafficking through the LE/Lys compartment, and severe CNS hypomyelination. We demonstrate that these defects in myelin biogenesis are functionally relevant and result in faulty conduction of electrical impulses. Results Conditional ablation of Fig4 in neurons or the OL lineage results in CNS hypomyelination In the early postnatal brain, Fig4 is broadly expressed and enriched in oligodendrocyte progenitor cells (OPCs) and newly formed OLs (NFOs) (Zhang et al., 2014). Mice in which exon 4 of the Fig4 gene is flanked by loxP sites (Ferguson et al., 2012) were used to generate Fig4-/flox,SynCre and Fig4-/flox,Olig2Cre mice deficient for Fig4 in neurons or OLs, respectively. Myelin development in these conditional mutants, as well as the Fig4 global mutant (Fig4-/-) and control mice (Fig4+/+ and Fig4flox/+), was analyzed by Fluoromyelin Green labeling (Figure 1). In control brains, the corpus callosum and internal capsule were prominently labeled (Figure 1A and A’). Staining of these structures was weaker in Fig4-/flox,SynCre brains and further reduced in Fig4-/flox,Olig2Cre and Fig4-/- brains (Figure 1B-D’). For a quantitative comparison of the myelination defects, whole brain membranes were prepared from P21 pups and analyzed by immunoblotting with antibodies specific for the myelin markers myelin-associated glycoprotein (MAG), 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNPase), proteolipid protein (PLP), and myelin basic protein (MBP) (Figure 1E). Compared to Fig4+/+ membranes, a significant reduction in myelin proteins was evident in Fig4-/- mice, Fig4-/flox,SynCre mice and Fig4-/flox,Olig2Cre mice (Figure 1F -I). The finding that the neuronal marker classIII β-tubulin is not significantly decreased in any of these mice indicates that the decrease in CNS myelin is not secondary to neuronal loss. While the Olig2 promoter is highly active in the OL lineage, activity has also been reported in astrocytes and a subset of neurons (Dessaud et al., 2007; Zhang et al., 2014). To independently assess the role of Fig4 in the OL lineage, we generated Fig4-/flox,PdgfraCreER mice that permit tamoxifen inducible gene ablation. At postnatal-days (P)5 and 6, before the onset of CNS myelination, Fig4-/flox,PdgfraCreER pups were injected with 4-hydroxytamoxifen and brains were analyzed at P20-P21. Inducible ablation of Fig4 in the OL-linage resulted in reduced expression of the myelin proteins CNPase, MAG, and MBP, as assessed by Western blot analysis (Figure 1—figure supplement 1A–B’) as well as myelin loss in forebrain structures and cerebellar white matter (Figure 1—figure supplement 1C–D’). Fewer Plp1+ OLs were present in optic nerve sections of Fig4-/flox,PdgfraCreER mice (Figure 1—figure supplement 1E and E’). Together, these studies indicate that proper CNS myelination is dependent upon OL cell-autonomous (intrinsic) functions of Fig4, in addition to non-OL-autonomous (extrinsic) functions of Fig4 provided by neurons. Figure 1 with 2 supplements see all Download asset Open asset Conditional ablation of Fig4 in neurons or OLs leads to CNS hypomyelination. (A-D) Coronal sections of juvenile (P21-30) mouse forebrain stained with FluoroMyelin Green. (A) Fig4 control mice (harboring at least one Fig4 WT allele), (B) Fig4 germline null mice (Fig4-/-), (C) Fig4-/flox,SynCre mice and (D) Fig4-flox,Olig2Cre mice. Thinning of the corpus callosum and internal capsule (white arrowheads) is observed in Fig4-/-, Fig4-/flox,SynCre, and Fig4-flox,Olig2Cre mice. (A’-D’) Higher magnification images of the corpus callosum. Scale bar (A-D), 1 mm and (A’-D’), 400 µm. (E) Representative Western blots of P21 brain membranes prepared from Fig4+/+ (WT), Fig4-/-, Fig4-/flox,SynCre and Fig4-/flox,Olig2Cre mice probed with antibodies specific for the myelin proteins MAG, CNPase, PLP, and MBP. To control for protein loading, membranes were probed for the neuronal marker class III β-tubulin (βIII Tub). (F-I) Quantification of Western blot signals for MAG, MBP, CNPase, and PLP in Fig4+/+ (black bars), Fig4-/- (purple bars), Fig4-/flox,SynCre (light blue bars), and Fig4-flox,Olig2Cre (red bars) brain membranes. Quantification of myelin protein signals is normalized to βIII Tub. Relative protein intensities compared to WT brain are shown as mean value ± SEM. For each of the four genotypes, three independent membrane preparations were carried out. One-way ANOVA with multiple comparisons, Dunnett posthoc test; **p<0.01, ***p<0.001 and ****p<0.0001. An independent strategy for OL-specific Fig4 deletion results in a similar phenotype as shown in Figure 1—figure supplement 1. Histochemical staining of brain, spinal cord and dorsal root ganglion tissue sections of Fig4 conditional knock-out mice, as well as Kaplan-Meier plots for Fig4-/flox,SynCre and Fig4-flox,Olig2Cre mice are shown in Figure 1—figure supplement 2. https://doi.org/10.7554/eLife.13023.003 As previously described, Fig4-/flox,SynCre mice exhibit impaired movement and region-specific vacuolization and neurodegeneration (Figure 1—figure supplement 2A”,B”,C”,D”) (Ferguson et al., 2012). In contrast, Fig4-/flox,Olig2Cre mice exhibit very mild vacuolization in brain (Figure 1—figure supplement 2A’’’,B’’’,C’’’,D’’’). Consistent with the known expression of the Olig2 promoter in motor neurons (Mizuguchi et al., 2001) ventral spinal cord of Fig4-/flox,Olig2Cre mice shows extensive vacuolization (Figure 1—figure supplement 2D’’’), similar to Fig4-/flox, Mnx1Cre (otherwise referred to as Fig4-/flox,Hb9Cre) mice (Figure 1—figure supplement 2E) (Vaccari et al., 2015). Analysis of Fig4-/flox,Hb9Cre spinal cord identified enlarged vacuoles within motoneuron axons, greatly extending their diameter and pushing the axoplasm into a thin peripheral rim near the plasma membrane (Figure 1—figure supplement 2F). In contrast to the movement disability and reduced survival of Fig4-/flox,SynCre mice,(Ferguson et al., 2012) the movement of Fig4-/flox,Olig2Cre mice is normal and no premature death was observed, with the oldest now surviving beyond 14 months of age (Figure 1—figure supplement 2G). There are no obvious defects in mobility of littermate controls and Fig4-/flox,Olig2Cre conditional mutant mice at P23, as demonstrated in the Videos 1 and 2. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Normal locomotion of juvenile Fig4+/flox,Olig2Cre mice. A representative video of a control Fig4+/flox,Olig2Cre mouse at P23. N = 10 https://doi.org/10.7554/eLife.13023.006 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Normal locomotion of juvenile Fig4-/flox,Olig2Cre mice. A representative video of a Fig4-/flox,Olig2Cre conditional mutant mouse at P23 shows no obvious pathology in locomotion. N = 10 https://doi.org/10.7554/eLife.13023.007 Fig4 deficiency in neurons or OLs leads to developmental dysmyelination of the optic nerve Analysis of P21 retina revealed the presence of numerous vacuoles in the inner retina of Fig4-/flox,SynCre mice but no defects in overall morphology or stratification (Figure 2A’). No vacuoles were detected in the Fig4-/flox,Olig2Cre retina (Figure 2A’’). For ultrastructural analysis, optic nerves of Fig4 conditional knock-out mice were processed for transmission electron microscopy (TEM). In P21 Fig4 control mice (retaining at least one intact allele of Fig4), the fraction of myelinated axons in the optic nerve is 79± 2%. In optic nerves of Fig4-/flox,SynCre mice, only 9± 3% of axons are myelinated and in Fig4-/flox,Olig2Cre mice only 12± 1% of axons are myelinated (Figure 2B–B’’ and D). To assess myelin health, we determined the g-ratio (the ratio of the inner axonal diameter to the total fiber diameter) of myelinated axons in the optic nerve of Fig4 control and conditional mutants. Compared to control mice, a small but significant increase in g-ratio was observed in Fig4-/flox,SynCre and Fig4-/flox,Olig2Cre mice, an indication of myelin thinning (Figure 2E). To determine whether the optic nerve hypomyelination at P21 reflects a transient delay in myelin development, rather than a lasting defect, we repeated the analysis with adult mice. Similar to P21 optic nerves, ultrastructural analysis of both types of adult optic nerves revealed profound hypomyelination (Figure 2C–C’’). At P60-75, 92± 2% of axons are myelinated in Fig4 control nerves. This is reduced to 16± 4% in Fig4-/flox,SynCre mice and 12± 2% in Fig4-/flox,Olig2Cre mice (Figure 2F). It is noteworthy that conditional ablation of Fig4 either in neurons or OLs leads to preferential absence of myelin sheaths on small and intermediate caliber axons, while many large caliber axons undergo myelination (Figure 2B’,B”,C’ and C”). Figure 2 Download asset Open asset Conditional ablation of Fig4 in neurons or in OLs leads to severe dysmyelination of the optic nerve. (A-A’’) Sagittal sections of juvenile (P21) mouse retina embedded in epoxy resin and stained with toluidine blue. (A) Fig4 control mice, harboring at least one Fig4 WT allele, (A’) Fig4-/flox,SynCre mice and (A’’) Fig4-/flox,Olig2Cre mice. Scale bar, 100 µm. (B-B’’) Representative TEM images of optic nerve cross sections of P21 (B) Fig4 control, (B’) Fig4-/flox,SynCre and (B’’) Fig4-/flox,Olig2Cre mice. (C-C’’) Representative TEM images of optic nerve cross sections of adult (P60-75) mice. (C) Fig4 control, (C’) Fig4-/flox,SynCre and (C’’) Fig4-/flox,Olig2Cre mice. Black arrows in C’ indicate the presence of dystrophic axons. Scale bar (B-C’’) = 1 μm. (D) Quantification of percentage of myelinated fibers in the optic nerve at P21 and P60-75. At P21, Fig4 controls (n = 3 mice, 3 nerves); Fig4-/flox,SynCre (n = 2 mice, 3 nerves) and Fig4-/flox,Olig2Cre (n = 3 mice, 3 nerves). (E) Quantification of myelinated fiber g-ratios in the optic nerve at P21, n = 3 animals, 3 nerves for all groups. (F) Quantification of myelinated fibers in the optic nerve at P60-P75. Fig4 control (n = 4 mice, 4 nerves), Fig4-/flox,SynCre (n = 4 mice, 4 nerves); Fig4-/flox,Olig2Cre (n = 3 mice, 4 nerves). Results are shown as mean value ± SEM, one-way ANOVA with multiple comparisons, Tukey posthoc test; n.s. p>0.05, *p=0.0211, **p=0.0055, ****p<0.0001. https://doi.org/10.7554/eLife.13023.008 Few axons in the optic nerve of adult Fig4-/flox,SynCre mice showed signs of degeneration (Figure 2C’). No evidence for axonal degeneration was observed in Fig4-/flox,Olig2Cre optic nerves. CNS hypomyelination in Fig4-/flox,Olig2Cre mice was still present at P150, the oldest time point examined by TEM (data not shown). Thus, the optic nerve hypomyelination observed at P21 is not transient in nature but persists into adulthood. We conclude that selective ablation of Fig4 either in neurons or in the OL lineage leads to profound CNS dysmyelination. Conditional ablation of Fig4 in neurons or the OL lineage impairs nerve conduction To determine whether the morphological defects in CNS myelin of Fig4 conditional mutants result in functional deficits, we performed electrophysiological recordings. We measured the conduction velocity and amplitude of compound action potentials (CAPs) in optic nerves acutely isolated from P21 mice. Global deletion of Fig4 (Fig4-/-) results in a dramatic reduction in a population of fast conducting fibers and a corresponding increase in the proportion of slowly conducting fibers (Figure 3A,B,E) (Winters et al., 2011). The average velocity of the largest peak in Fig4 control nerves carrying at least one intact allele of Fig4 is 1.9 ± 0.1 m/s but in Fig4-/- nerves this is reduced to 0.7 ± 0.2 m/s. A similar CAP redistribution was observed in optic nerves prepared from Fig4-/flox,SynCre mice (0.7 ± 0.1 m/s) and Fig4-/flox,Olig2Cre mice (0.6 ± 0.03 m/s) (Figure 3C,D,E). Thus, consistent with biochemical and morphological analyses (Figures 1 and 2), loss of Fig4 in neurons or in the OL-lineage results in slowed nerve conduction. Figure 3 Download asset Open asset Conditional ablation of Fig4 in neurons or OLs leads to impaired conduction of electrical impulses in the optic nerve. Compound action potential (CAP) recordings from acutely isolated optic nerves of P21 mice. (A) Representative CAP traces recorded from Fig4 control mice, harboring at least one Fig4 WT allele (n = 14 nerves), (B) Fig4-/- mice (n = 5 nerves), (C) Fig4-/flox,SynCre mice (n = 11 nerves) and (D) Fig4-/flox,Olig2Cre mice (n = 9 nerves). For each graph, the arrow indicates the largest amplitude peak, as identified by Gaussian fit. (E) Quantification of average conduction velocity of largest amplitude peaks identified in A-D. Results are shown as mean value ± SEM, one-way ANOVA with multiple comparisons, Dunnett posthoc, ****p<0.0001. https://doi.org/10.7554/eLife.13023.009 Reduced number of mature OLs in Fig4-/flox,Olig2Cre and Fig4-/flox,SynCre optic nerves To assess the cellular basis of the CNS hypomyelination phenotype, we stained optic nerve cross sections from Fig4 conditional mutants for markers in the OL lineage. Compared to Fig4 control optic nerves, the diameter of nerves from P21 Fig4-/flox,SynCre and Fig4-/flox,Olig2Cre mice were each reduced by 20%. The density of NG2+ progenitor cells in optic nerve tissue sections is comparable among the three genotypes (Figure 4A–A’’ and D). The density of Olig2+ cells, a marker that labels immature and mature OLs, is reduced, as is labeling of Plp1, a mature OL marker (Figure 4B-B’’,C–C’’,E and F). These studies indicate that OPCs are present at normal density and tissue distribution in the Fig4 conditional null optic nerves, but they fail to generate the normal population of mature myelin-forming OLs. Figure 4 Download asset Open asset Conditional ablation of Fig4 in neurons or OLs results in a decrease of mature OLs. (A, B, C) Optic nerve cross sections from P21 Fig4 control mice, harboring at least one Fig4 WT allele, (A’, B’, C’) Fig4-/flox,SynCre mice and (A’’, B’’, C’’) Fig4-/flox,Olig2Cre mice were stained with anti-NG2, anti-Olig2 or probed for Plp1 mRNA expression. Scale bar = 100 µm. (D-F) Quantification of labeled cells in optic nerve cross sections normalized to area in arbitrary units (A.U.). (D) The density of NG2+ cells in Fig4 control (n = 4 mice), Fig4-/flox,SynCre (n = 3 mice) and Fig4-/flox,Olig2Cre (n = 3 mice) optic nerves is not significantly (n.s.) different. (E) Quantification of the density of Olig2+ cells in Fig4 control (n = 6 mice), Fig4-/flox,SynCre (n = 3 mice) and Fig4-/flox,Olig2Cre (n = 4 mice) optic nerves. (F) Quantification of the density of Plp1+ cells in Fig4 control (n = 8 mice), Fig4-/flox,SynCre (n = 4 mice) and Fig4-/flox,Olig2Cre (n = 4 mice) optic nerves. Results are shown as mean value ± SEM, one-way ANOVA with multiple comparisons, Dunnett’s posthoc test. **p=0.001, ***p=0.0002 and ****p<0.0001. https://doi.org/10.7554/eLife.13023.010 Loss of Fig4 attenuates OL differentiation in vitro For a more detailed analysis of the OL lineage, we isolated primary OPCs from P6-P14 Fig4 pups by anti-PDGFRα immunopanning (Emery and Dugas, 2013). Yields of OPCs per brain did not differ between control and Fig4-deficient mice (data not shown). OPCs were cultured for two days in vitro (DIV2) under proliferating conditions, fixed and analyzed by double-immunofluorescence staining of Ki67 and PDGFRα. The density of Ki67+/PDGFRα+ cells in Fig4+/+ and Fig4-/- cultures is very similar (Figure 5—figure supplement 1A–B). After culture under standard differentiation conditions for 4 days, absence of PDGF and presence of triiodothyronine (T3), OPCs isolated from Fig4+/+ (control) or Fig4-/- pups both acquire a highly arborized morphology and positive staining for OL markers. The density of NG2+ cells and CNPase+ cells, normalized to Hoechst 33,342 dye+ nuclei, is comparable among wildtype and Fig4-deficient cultures (Figure 5A–B’, and C). However, the fraction of cells expressing the more mature OL markers MAG and MBP was significantly reduced in Fig4-/- cultures (Figure 5A–B’, and C). A more detailed categorization of post-mitotic OLs, based on actin and MBP double-labeling, revealed a significantly decreased number of Fig4-deficient OLs that matured to a stage with lamellar MBP+ membrane sheets (Zuchero et al., 2015) (Figure 5D–E). The reduced number of mature OLs in Fig4-/- cultures was not caused by increased cell death (Figure 5—figure supplement 1C–E). For a quantitative assessment of protein expression in primary OLs from Fig4+/+ and Fig4-/- brains, DIV 3 cultures were lysed and analyzed by capillary Western blotting (Figure 5—figure supplement 2A–C). FIG4 is clearly detected in Fig4+/+ OL lysates but not in Fig4-/- OL lysates. In Fig4-/- lysates MAG is significantly reduced. Collectively, these data suggest that the initial programs of OL maturation progress normally in the absence of Fig4 while later stages of OL-differentiation, including lamellar membrane expansion, are Fig4-dependent. Figure 5 with 2 supplements see all Download asset Open asset Fig4-deficient OLs show impaired differentiation and membrane expansion in vitro. Representative images of Fig4 control (Fig4+/+ or Fig4+/-) and Fig4-/- primary OLs after 4 days in differentiation medium, fixed and stained for the OL-lineage markers (A and A’) NG2 and MAG; (B and B’) CNPase and MBP. Scale bar in A-B’, 200 µm. (C) Quantification of NG2, CNPase, MAG, and MBP/CNPase labeled cells in Fig4 control (n = 3) and Fig4-/-(n = 3) cultures normalized to Hoechst 33342 dye labeled cells. The ratio of immunolabeled cells over Hoechst+ cells in Fig4 control cultures was set at 1. Results are shown as mean value ± SEM, multiple t-test analysis with Holm-Sidak method. **p=0.0075 (MAG), *p=0.012 (MBP). (D and D’) Confocal images of MBP+ and Actin Red 555+ OLs in Fig4 control and Fig4-/- cultures. Nuclei were labeled with TO-PRO-3, scale bar = 20 µm. (E) Quantification of the fraction of “arborized” (actin rich, no MBP), “partial” (partial actin disassembly, onset of MBP expansion), and “ring + lamellar” (full MBP expansion, actin disassembly) in Fig4 control cultures (n = 4) and Fig4-/- (n = 4) cultures. Results are shown as mean value ± SEM, multiple t-test analysis with Holm-Sidak method. *p=0.0008 (“partial”), *p=0.009 (“ring + lamellar”). The effects of Fig4 deletion on OPC proliferation and OL survival are shown in Figure 5—figure supplement 1. Quantitative Western blot analysis of myelin proteins in primary OL lysates is shown in Figure 5—figure supplement 2. https://doi.org/10.7554/eLife.13023.011 Independent perturbation of three components of the PI(3,5)P2 biosynthetic complex all result in severe CNS hypomyelination Together with the kinase PIKFYVE and the scaffolding protein VAC14, FIG4 forms a biosynthetic complex necessary for acute interconversion of PI(3) and PI(3,5)P2. The complex is located on the cytosolic surface of vesicles trafficking through the LE/Lys compartment (McCartney et al., 2014). As an independent test of the effect of perturbation of the FIG4/PIKFYVE/VAC14 enzyme complex on CNS myelination, we generated Pikfyveflox/flox,Olig2cre mice predicted to be more severely deficient in PI(3,5)P2 than the FIG4 and VAC14 mutants. Consistent with this expectation, the phenotype of the Pikfyve mutant mice is much more severe, with a significant tremor (Videos 3 and 4) and death at 2 weeks of age (n = 16 pups). FluoroMyelin Green staining of P13 brain tissue revealed profound hypomyelination of the corpus callosum, internal capsule and cerebellar white matter of Pikfyveflox/flox,Olig2cre pups (Figure 6A–A’). In situ hybridization of Plp1 revealed a virtual absence of mature OLs in the Pikfyveflox/flox,Olig2cre brain, including structures in the forebrain and cerebellar white matter (Figure 6B–D’). Toluidine blue staining of P13 optic nerve sections revealed many fibers with clearly visible myelin profiles in Pikfyve positive control mice and a striking absence of myelin profiles in Pikfyveflox/flox;Olig2cre conditional mutants (Figure 6—Supplement 1B–D). Moreover, deficiency of Pikfyve in OLs results in a pronounced accumulation of large perinuclear vesicles in the optic nerve (Figure 6—figure supplement 1B,D). Defects in differentiation of Pikfyve-/- OL cultures are more pronounced than in Fig4-/-OL cultures. Deficiency of Pikfyve reduces OPC proliferation (Figure 6E–E' and G) and results in a 95 ± 1% reduction in cells that progress to the MBP+ stage, compared with wildtype cells (Figure 6F–F’ and H). In addition to Fig4 and Pikfyve mutants, we also examined myelinogenesis in the well-characterized recessive Vac14 mouse mutant L156R (Vac14L156R) (Jin et al., 2008). The L156R missense mutation impairs the interaction of VAC14 with PIKFYVE, but not with FIG4 (Figure 7A). Similar to Fig4-/- mice, Vac14L156R/L156R mice exhibit ~50% reduction in PI(3,5)P2. Immunoblots of brain membranes prepared from Vac14L156R/L156R mice showed significantly reduced levels of the myelin markers MAG, CNPase, and MBP (Figure 7B–E). The electrical properties of optic nerve from Vac14L156R homozygous mice were also impaired, with a significant increase in the population of slowly conducting fibers (Figure 7F–H). Consistent with this observation, toluidine blue staining of optic nerve sections of adult wild-type mice revealed many myelinated fibers but optic nerves of adult Vac14L156R/L156R mice showed few myelinated fibers (Figure 7—figure supplement 1A–D). Thus, independent genetic disruptions of the FIG4/PIKFYV

Myelination is a complex process that requires coordinated Schwann cell-axon interactions during development and regeneration. Positive and negative regulators of myelination have been recently described, and can belong either to Schwann cells or neurons. Vimentin is a fibrous component present in both Schwann cell and neuron cytoskeleton, the expression of which is timely and spatially regulated during development and regeneration. We now report that vimentin negatively regulates myelination, as loss of vimentin results in peripheral nerve hypermyelination, owing to increased myelin thickness in vivo, in transgenic mice and in vitro in a myelinating co-culture system. We also show that this is due to a neuron-autonomous increase in the levels of axonal neuregulin 1 (NRG1) type III. Accordingly, genetic reduction of NRG1 type III in vimentin-null mice rescues hypermyelination. Finally, we demonstrate that vimentin acts synergistically with TACE, a negative regulator of NRG1 type III activity, as shown by hypermyelination of double Vim/Tace heterozygous mice. Our results reveal a novel role for the intermediate filament vimentin in myelination, and indicate vimentin as a regulator of NRG1 type III function.

Charcot‐Marie‐Tooth type 4B (CMT4B) disease is a severe autosomal recessive peripheral neuropathy with childhood onset, characterised by progressive muscular atrophy and weakness in the distal extremities, sensory loss, severely decreased nerve conduction velocities, and demyelination with myelin outfoldings in the peripheral nerve. We demonstrated that CMT4B is caused by loss of function mutations in the Myotubularin‐related 2 gene, MTMR2 , on chromosome 11q22 (Bolino et al ., Nat Genet 25:17–19, 2000). MTMR2 belongs to the myotubularin family of protein phosphatases, of which, myotubularin (MTM), mutated in the X‐linked myotubular myopathy (XLMTM) is the founder member. MTMR2 shows specific activity towards phosphatidylinositol 3‐phosphate and 3,5‐biphosphate, PI(3)P and PI(3,5)P2, respectively. However, how abrogation of this lipid phosphatase activity is leading to the specific disease phenotype has not yet been demonstrated. To elucidate the biological role of MTMR2 in the nerve, we performed an extensive expression analysis of this protein in the peripheral nervous system. Since MTMR2 was demonstrated to be ubiquitously expressed also within the nerve, we sought nerve‐specific interactors using the yeast two‐hybrid approach. The neurofilament light chain protein, NF‐L, mutated in various CMTs including axonal type CMT2E, and demyelinating Dejerine‐Sottas syndrome, was found to interact with MTMR2 in Schwann cells as well as in neurons. Since NF‐L is specifically expressed in the nervous system, the interaction between MTMR2 and NF‐L would explain why loss of a ubiquitously expressed phosphatase affects specifically the nerve (Previtali and Bolino, Hum Mol Genet 12:1713–1723, 2003). To model the CMT4B pathology, we generated a general knock‐out mouse arising from inactivation f Mtmr2 in all cells. The characterisation of this animal model is underway. Overall, Mtmr2 null mice display a milder phenotype with respect to the human disorder. The morphological analysis of the peripheral nerve of this mouse line performed at P28 revealed the presence of myelin outfoldings, which are the hallmark of CMT4B pathology.

CMT4B1 is an autosomal recessive peripheral neuropathy characterized by severe progressive muscular atrophy with childhood onset, decreased conduction velocities, demyelination with myelin outfolding, and subsequently axonal loss. The disease gene has been recently identified as myotubularin‐related 2 (MTMR2), encoding a protein phosphatase (Bolino et al., Nature Genetics 25: 17–19; 2000). Although this phosphatase is widely expressed, the disorder is restricted to the peripheral nervous system, just as mutations to the related and broadly expressed phosphatase MTM1 produce only myotubular myopathy. The function of MTMR2 and the pathogenetic mechanism of CMT4B are still unknown. We produced several new polyclonal antibodies to MTMR2 in order to better dissect the expression and potential function of MTMR2 in the peripheral nervous system. Although CMT4B manifests primarily as demyelinating neuropathy, our expression data show that MTMR2 protein is present in both Schwann cells and axons in peripheral nerve. In Schwann cells, MTMR2 is localized in the cytoplasm and, at lower level, in the nucleus, while no major differences were observed between non‐myelin forming and myelin‐forming Schwann cells. DRG sensory neurons and motor neurons, as well as their axons, show MTMR2 expression in their cytoplasm. Thus, it remains unclear whether demyelination in CMT4B is the consequence of a cell‐autonomous or a cell‐extrinsic defect, the latter possibly due to a defective interaction between Schwann cells and axons.

Abstract To understand the pathology of axonal degeneration and demyelination in peripheral neuropathy, histological investigations in different animal models that mimic some aspects of human peripheral neuropathy are needed. Thus, in the following section of this special issue, the main pathological features of experimental autoimmune neuritis, animal models of chemotherapy‐induced peripheral neuropath and of human inherited peripheral neuropathies (IPNs) will be illustrated. When possible, micrographs from animal models and selected human biopsy will be shown side by side.

ABSTRACT The GM2 gangliosidoses Tay-Sachs disease and Sandhoff disease (SD) are respectively caused by mutations in the HEXA and HEXB genes encoding the α and β subunits of β-N-acetylhexosaminidase (Hex). The consequential accumulation of ganglioside in the brain leads to severe and progressive neurological impairment. There are currently no approved therapies to counteract or reverse the effects of GM2 gangliosidosis. Adeno-associated vector (AAV)-based investigational gene therapy (GT) products have raised expectations but come with safety and efficacy issues that need to be addressed. Thus, there is an urgent need to develop novel therapies targeting the CNS and other affected tissues that are appropriately timed to ensure pervasive metabolic correction and counteract disease progression. In this report, we show that the sequential administration of lentiviral vector (LV)-mediated intracerebral (IC) GT and bone marrow transplantation (BMT) in pre-symptomatic SD mice provide a timely and long-lasting source of the Hex enzyme in the central and peripheral nervous systems and peripheral tissues, leading to global rescue of the disease phenotype. Combined therapy showed a clear therapeutic advantage compared to individual treatments in terms of lifespan extension and normalization of the neuroinflammatory and neurodegenerative phenotypes of the SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in the brain tissues that single treatments failed to achieve. Our results highlight the complementary and synergic mode of action of LV-mediated IC GT and BMT, clarify the relative contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of enzymatic activity that is required to achieve a significant therapeutic benefit, with important implications for the monitoring and interpretation of ongoing experimental therapies, and for the design of more effective treatment strategies for GM2 gangliosidosis.

Charcot‐Marie‐Tooth disease type 4B (CMT4B) is a demyelinating autosomal recessive motor and sensory neuropathy characterized by focally folded myelin sheaths in the peripheral nerve. We recently mapped the CMT4B gene to a 5‐cM interval on chromosome 11q22, using homozygosity mapping and haplotype sharing analysis on a large inbred pedigree. In the present study, we report the construction of a YAC‐based transcript map across the 5‐cM critical region, including 26 YACs, 35 STSs, and 52 ESTs. Furthermore, using 15 additional physically ordered microsatellite markers from the 11q22 region on the original inbred family, we were able to narrow the critical interval for the gene to 2 Mb, which is now flanked by markers D11S1757 and CHLC‐GATA3B05. Finally, after computer analysis of the 33 ESTs assigned to the 2‐Mb interval, we demonstrated that 21 different transcripts as well as 3 known genes might represent potential candidates for the disease.

Human MutationVolume 9, Issue 2 p. 185-187 Mutations in Brief Startle disease in an Italian family by mutation (K276E): The α-subunit of the inhibiting glycine receptor Marco Seri, Marco Seri Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorAlessandra Bolino, Alessandra Bolino Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorLuis Juan Vicente Galietta, Luis Juan Vicente Galietta Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorMargherita Lerone, Margherita Lerone Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorMargherita Silengo, Margherita Silengo Istituto di Discipline Pediatriche, Università degli Studi di Torino, 10126 Torino, ItalySearch for more papers by this authorGiovanni Romeo, Corresponding Author Giovanni Romeo Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this author Marco Seri, Marco Seri Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorAlessandra Bolino, Alessandra Bolino Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorLuis Juan Vicente Galietta, Luis Juan Vicente Galietta Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorMargherita Lerone, Margherita Lerone Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this authorMargherita Silengo, Margherita Silengo Istituto di Discipline Pediatriche, Università degli Studi di Torino, 10126 Torino, ItalySearch for more papers by this authorGiovanni Romeo, Corresponding Author Giovanni Romeo Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; Fax 39-10-3779797Search for more papers by this author First published: 07 January 1999 https://doi.org/10.1002/(SICI)1098-1004(1997)9:2<185::AID-HUMU14>3.0.CO;2-ZCitations: 18AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References Andermann F, Keene DL, Andermann E, Quesney LF (1980) Startle disease or hyperekplexia: further delineation of the syndrome. Brain 103: 985– 997. 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