Charcot-Marie-Tooth disease misdiagnosed as chronic inflammatory demyelinating polyradiculoneuropathy: An international multicentric retrospective study.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth (CMT) disease, an untreatable hereditary polyneuropathy, may mimic chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), a treatable neuropathy. METHODS: In this retrospective study, we analyzed the characteristics of CMT patients misdiagnosed as CIDP at 16 university hospitals in three countries, compared these patients with a reference group of CIDP patients, and estimated the cost of misdiagnosis. RESULTS: Among 1104 CIDP cases, we identified 35 CMT patients misdiagnosed as CIDP (3.2%). All were initially diagnosed with definite or probable CIDP (European Federation of Neurological Societies/Peripheral Nerve Society criteria), and mutations in PMP22, MPZ, and 10 other CMT genes were found in 34%, 31%, and 35% of cases, respectively. In comparison with a reference group of 35 CIDP patients, CMT patients were younger (median age at disease onset = 39 vs. 56 years) and more frequently had motor weakness at disease onset (80% vs. 29%), hearing loss (14% vs. 0%), normal brachial plexus imaging (70% vs. 40%), lower cerebrospinal fluid protein content (median = 0.5 vs. 0.8 g/L), and lower treatment response (20% vs. 69%). Treatment cost in these 35 misdiagnosed patients was estimated at 4.6 million euros (M€), whereas the cost of CMT genetic analysis in 1104 patients was estimated at 2.7 M€. CONCLUSIONS: In this study, 35 of 1104 (3.2%) patients initially diagnosed with CIDP had CMT. Importantly, the cost of treating these 35 misdiagnosed patients was significantly higher than the cost of performing CMT genetic analysis in 1104 patients (4.6 M€ vs. 2.7 M€), suggesting that CMT genetic investigations should be more widely used before diagnosing CIDP.

CLIC3 controls recycling of late endosomal MT1-MMP and dictates invasion and metastasis in breast cancer.

Chloride intracellular channel 3 (CLIC3) drives invasiveness of pancreatic and ovarian cancer by acting in concert with Rab25 to regulate the recycling of α5ß1 integrin from late endosomes to the plasma membrane. Here, we show that in two estrogen receptor (ER)-negative breast cancer cell lines, CLIC3 has little influence on integrin recycling, but controls trafficking of the pro-invasive matrix metalloproteinase MT1-MMP (also known as MMP14). In MDA-MB-231 cells, MT1-MMP and CLIC3 are localized primarily to late endosomal/lysosomal compartments located above the plane of adhesion and near the nucleus. MT1-MMP is transferred from these late endosomes to sites of cell-matrix adhesion in a CLIC3-dependent fashion. Correspondingly, CLIC3-knockdown opposes MT1-MMP-dependent invasive processes. These include the disruption of the basement membrane as acini formed from MCF10DCIS.com cells acquire invasive characteristics in 3D culture, and the invasion of MDA-MB-231 cells into Matrigel or organotypic plugs of type I collagen. Consistent with this, expression of CLIC3 predicts poor prognosis in ER-negative breast cancer. The identification of MT1-MMP as a cargo of a CLIC3-regulated pathway that drives invasion highlights the importance of late endosomal sorting and trafficking in breast cancer.

Diverse Cutaneous Presentations of Langerhans Cell Histiocytosis in Children: A Retrospective Cohort Study.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare disease, frequently affecting young children. PROCEDURE: We performed a retrospective study in patients younger than 16 years old manifesting with skin symptoms, and documented their different cutaneous lesions and systemic symptoms. We compared subgroups of children with single-system, skin-only, and multisystem disease and sought signs predictive for multisystem disease. In a small sample of patients, BRAF mutations were analyzed in archived biopsies. RESULTS: A wide spectrum of cutaneous presentations varying from crusted nodules and papules, blisters, vascular tumor-like lesions, scaling orange to red macules (frequently in seborrheic regions) to purpuric macules, and papules was documented in our cohort of 32 children. Otitis externa was a common manifestation and mucosal lesions were seen in three patients. A novel manifestation was a red-blue nodule that appeared in a patient after a vaccination. None of the cutaneous lesions was predictive for the classification or final outcome as a single-system or multisystem disease. However, later onset and a more protracted course of skin lesions were more frequent findings in multisystem LCH. Mucosal lesions and otitis externa were almost exclusively seen in patients with multisystem disease, a finding that warrants further investigation. Both wild-type (WT) and mutated BRAF were found not only in multisystem LCH, but also in skin-only LCH. Two cases with rapidly resolving congenital lesions had WT BRAF. CONCLUSIONS: Late onset and a protracted course of skin lesions are associated with MS-LCH, whereas WT BRAF is found in rapidly resolving skin lesions.

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression.

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P(1B)-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.

Folding defects in P-type ATP 8B1 associated with hereditary cholestasis are ameliorated by 4-phenylbutyrate.

UNLABELLED: Deficiency in P-type ATP8B1 is a severe and clinically highly variable hereditary disorder that is primarily characterized by intrahepatic cholestasis. It presents either as a progressive (progressive familial intrahepatic cholestasis type 1 [PFIC1]) or intermittent (benign recurrent intrahepatic cholestasis type 1 [BRIC1]) disease. ATP8B1 deficiency is caused by autosomal recessive mutations in the gene encoding ATP8B1, a putative aminophospholipid-translocating P-type adenosine triphosphatase. The exact pathogenesis of the disease is elusive, and no effective pharmacological therapy is currently available. Here, the molecular consequences of six distinct ATP8B1 missense mutations (p.L127P, p.G308V, p.D454G, p.D554N, p.I661T, and p.G1040R) and one nonsense mutation (p.R1164X) associated with PFIC1 and/or BRIC1 were systematically characterized. Except for the p.L127P mutation, all mutations resulted in markedly reduced ATP8B1 protein expression, whereas messenger RNA expression was unaffected. Five of seven mutations resulted in (partial) retention of ATP8B1 in the endoplasmic reticulum. Reduced protein expression was partially restored by culturing the cells at 30 degrees C and by treatment with proteasomal inhibitors, indicating protein misfolding and subsequent proteosomal degradation. Protein misfolding was corroborated by predicting the consequences of most mutations onto a homology model of ATP8B1. Treatment with 4-phenylbutyrate, a clinically approved pharmacological chaperone, partially restored defects in expression and localization of ATP8B1 substitutions G308V, D454G, D554N, and in particular I661T, which is the most frequently identified mutation in BRIC1. CONCLUSION: A surprisingly large proportion of ATP8B1 mutations resulted in aberrant folding and decreased expression at the plasma membrane. These effects were partially restored by treatment with 4-phenylbutyrate. We propose that treatment with pharmacological chaperones may represent an effective therapeutic strategy to ameliorate the recurrent attacks of cholestasis in patients with intermittent (BRIC1) disease.

Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin.

UNLABELLED: Wilson disease (WD) is an autosomal recessive copper overload disorder of the liver and basal ganglia. WD is caused by mutations in the gene encoding ATP7B, a protein localized to the trans-Golgi network that primarily facilitates hepatic copper excretion. Current treatment comprises reduction of circulating copper by zinc supplementation or copper chelation. Despite treatment, a significant number of patients have neurological deterioration. The aim of this study was to investigate the possibility that defects arising from some WD mutations are ameliorated by drug treatment aimed at improvement of protein folding and restoration of protein function. This necessitated systematic characterization of the molecular consequences of distinct ATP7B missense mutations associated with WD. With the exception of p.S1363F, all mutations tested (p.G85V, p.R778L, p.H1069Q, p.C1104F, p.V1262F, p.G1343V, and p.S1363F) resulted in reduced ATP7B protein expression, whereas messenger RNA abundance was unaffected. Retention of mutant ATP7B in the endoplasmic reticulum, increased protein expression, and normalization of localization after culturing cells at 30 degrees C, and homology modeling suggested that these proteins were misfolded. Four distinct mutations exhibited residual copper export capacity, whereas other mutations resulted in complete disruption of copper export by ATP7B. Treatment with pharmacological chaperones 4-phenylbutyrate (4-PBA) and curcumin, a clinically approved compound, partially restored protein expression of most ATP7B mutants. CONCLUSION: These findings might enable novel treatment strategies in WD by directly enhancing the protein expression of mutant ATP7B with residual copper export activity. 1795.).

Posttranslational regulation of copper transporters.

Copper is an essential but potentially harmful trace element involved in many enzymatic processes that require redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by posttranslational regulation of copper import and export through the copper import proteins hCTR1 and hCTR2 and the copper exporters ATP7A and ATP7B. Regulation of copper uptake and export is achieved by modulation of transporter expression, copper-dependent and copper-independent trafficking of the different transporters, posttranslational modifications, and interacting proteins. In this review we systematically discuss the contribution of these different mechanisms to the regulation of copper transport.

Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake.

High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2-vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2-vsvG. Furthermore, hCTR2-vsvG was co-immunoprecipitated with hCTR2-FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2-eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)-luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE-luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 muM CuCl2, consistent with the K(m) of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE-luciferase reporter activation in a copper-dependent manner at 40 and 100 microM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.

Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions.

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity.

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.

Rab25 and CLIC3 collaborate to promote integrin recycling from late endosomes/lysosomes and drive cancer progression.

Here we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5ß1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5ß1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo.

New developments in the regulation of intestinal copper absorption.

The transition metal copper is an essential trace element involved in many enzymatic processes that require redox-chemistry. The redox-activity of copper is potentially harmful. Severe imbalance of copper homeostasis can occur with some hereditary disorders of copper metabolism. Copper is acquired from the diet by intestinal absorption and is subsequently distributed throughout the body. The regulation of intestinal copper absorption to maintain whole-body copper homeostasis is currently poorly understood. This review evaluates novel findings regarding the molecular mechanism of intestinal copper uptake. The role of recently identified transporters in enterocyte copper uptake and excretion into the portal circulation is described, and the regulation of dietary copper uptake during physiological and pathophysiological conditions is discussed.

Filamin A stabilizes Fc gamma RI surface expression and prevents its lysosomal routing.

Filamin A, or actin-binding protein 280, is a ubiquitously expressed cytosolic protein that interacts with intracellular domains of multiple receptors to control their subcellular distribution, and signaling capacity. In this study, we document interaction between FcgammaRI, a high-affinity IgG receptor, and filamin A by yeast two-hybrid techniques and coimmunoprecipitation. Both proteins colocalized at the plasma membrane in monocytes, but dissociated upon FcgammaRI triggering. The filamin-deficient cell line M2 and a filamin-reconstituted M2 subclone (A7), were used to further study FcgammaRI-filamin interactions. FcgammaRI transfection in A7 cells with filamin resulted in high plasma membrane expression levels. In filamin-deficient M2 cells and in filamin RNA-interference studies, FcgammaRI surface expression was consistently reduced. FcgammaRI localized to LAMP-1-positive vesicles in the absence of filamin as shown by confocal microscopy indicative for lysosomal localization. Mouse IgG2a capture experiments suggested a transient membrane expression of FcgammaRI before being transported to the lysosomes. These data support a pivotal role for filamin in FcgammaRI surface expression via retention of FcgammaRI from a default lysosomal pathway.