Therapeutic Development in Charcot Marie Tooth Type 1 Disease.

Charcot-Marie-Tooth disease (CMT) is the most frequent hereditary peripheral neuropathies. It is subdivided in two main groups, demyelinating (CMT1) and axonal (CMT2). CMT1 forms are the most frequent. The goal of this review is to present published data on 1-cellular and animal models having opened new potential therapeutic approaches. 2-exploration of these tracks, including clinical trials. The first conclusion is the great increase of publications on CMT1 subtypes since 2000. We discussed two points that should be considered in the therapeutic development toward a regulatory-approved therapy to be proposed to patients. The first point concerns long term safety if treatments will be a long-term process. The second point relates to the evaluation of treatment efficiency. Degradation of CMT clinical phenotype is not linear and progressive.

Charcot Marie Tooth Disease. A Single Disorder?

Peripheral neuropathies are subdivided into acquired and hereditary transmitted disorders. [...].

Ascorbic acid drives the differentiation of mesoderm-derived embryonic stem cells. Involvement of p38 MAPK/CREB and SVCT2 transporter.

SCOPE: Here we tested the hypothesis that ascorbic acid (AA) is a signaling molecule acting on stem cells via the differentiation of mesoderm derivatives, including myocytes, osteocytes, and adipocytes. MATERIAL AND METHODS: Investigations used a murine embryonic stem cell line CGR8 able to differentiate into different cell types and treated or not with ascorbic acid. Differentiation was tracked mainly through cellular anatomy (including presence of beating cardiomyocytes) and expression of specific markers. CONCLUSION: The study demonstrated that AA drives mesoderm-derived stem cell differentiation toward myogenesis and osteogenesis and also inhibits adipogenesis. Further experiments found that AA competes with retinoic acid (RA) to drive cell differentiation in a dose-dependent manner: AA inhibited neurogenic differentiation and stimulated myogenesis whereas RA did the reverse. The AA-dependent differentiation of embryonic stem cells was shown to involve a p38 MAPK/CREB pathway, probably stimulated by cAMP via adenylate cyclases. In addition, SVCT2, the intracellular transporter of AA, acted as a receptor. Finally, we showed that activation/repression of specific differentiation markers is associated with epigenetic changes in their associated promoters. We discuss the impact of these findings in terms of obesity and aging.

CMTX1 patients' cells present genomic instability corrected by CamKII inhibitors.

BACKGROUND: We previously described that fibroblasts from animal models of CMTX1 present genomic instability and poor connexon activity. In vivo, these transgenic mice present motor deficits. This phenotype could be significantly reverted by treatment with (CamKII) inhibitors. The objective of this study is to translate our findings to patients. METHODS: We cultured fibroblasts from skin biopsies of CMTX1 patients and analyzed cells for genomic instabilty, connexon activity, and potential correction by CamKII inhibitors. RESULTS: The phenotypic analysis of these cells confirmed strong similarities between the GJB1 transgenic mouse cell lines and CMTX1 patient fibroblast cell lines. Both present mitotic anomalies, centrosome overduplication, and connexon activity deficit. This phenotype is corrected by CamKII inhibitors. CONCLUSIONS: Our data demonstrate that fibroblasts from CMTX1 patients present a phenotype similar to transgenic lines that can be corrected by CamKII inhibitors. This presents a track to develop therapeutic strategies for CMTX1 treatment.

Ascorbic acid is a dose-dependent inhibitor of adipocyte differentiation, probably by reducing cAMP pool.

Ascorbic acid (AA) is the active component of vitamin C and antioxidant activity was long considered to be the primary molecular mechanism underlying the physiological actions of AA. We recently demonstrated that AA is a competitive inhibitor of adenylate cyclase, acting as a global regulator of intracellular cyclic adenosine monophosphate (cAMP) levels. Our study, therefore, aimed to determine new targets of AA that would account for its potential effect on signal transduction, particularly during cell differentiation. We demonstrated that AA is an inhibitor of pre-adipocyte cell line differentiation, with a dose-dependent effect. Additionally, we describe the impact of AA on the expression of genes involved in adipogenesis and/or the adipocyte phenotype. Moreover, our data suggest that treatment with AA partially reverses lipid accumulation in mature adipocytes. These properties likely reflect the function of AA as a global regulator of the cAMP pool, since an analog of AA without any antioxidant properties elicited the same effect. Additionally, we demonstrated that AA inhibits adipogenesis in OP9 mesenchymal cell line and drives the differentiation of this line toward osteogenesis. Finally, our data suggest that the intracellular transporter SVCT2 is involved in these processes and may act as a receptor for AA.

CamKII inhibitors reduce mitotic instability, connexon anomalies and progression of the in vivo behavioral phenotype in transgenic animals expressing a mutated Gjb1 gene.

Mutation in the Gjb1 gene, coding for a connexin (Cx32), is associated with an inherited peripheral neuropathic disorder (X-linked Charcot-Marie-Tooth, CMTX). Our previous work reported that transgenic animals expressing a human Gjb1 transgene present polyploidy and abnormal over-duplication of the centrosome, suggesting a role for Gjb1 in mitotic stability. In this article, we propose mechanisms by which mutations in Gjb1 induce mitotic instability and discuss its potential relation with the CMTX phenotype. We showed that transgenic cells exhibit CamKII over-stimulation, a phenomenon that has been linked to mitotic instability (polyploidy, nuclear volume and centrosome over-duplication), that is reversed by CamKII inhibitors. We also demonstrate that connexon activity is partially restored in transgenic cells with CamKII inhibitors. Our model supports the role for Pim1, a kinase that has been associated with genomic instability in cancers, in genomic instability in Cx32 mutations. Regarding in vivo phenotype, we showed that degradation on the rotarod test in our transgenic mice is significantly lowered by treatment with a CamKII inhibitor (KN93). This effect was seen in two lines with different point mutations in GJB1, and stopping the treatment led to degradation of the phenotype.

Alternative splicing events is not a key event for gene expression regulation in uremia.

BACKGROUND: The control of gene expression in the course of chronic kidney disease (CKD) is not well addressed. Alternative splicing is a common way to increase complexity of proteins. More than 90% of human transcripts are alternatively spliced. We hypothesised that CKD can induce modification of the alternative splicing machinery. METHODS: During mutation screening in autosomal dominant polycystic kidney disease, we identified in mononuclear cells (PBMC), an alternative splicing event on the exon 30 of PKD1 gene, the gene implicated in this disease. This alternative splice variant was not correlated with the cystic disease but with CKD. To confirm the association between this variant and CKD, a monocentric clinical study was performed with 3 different groups according to their kidney function (CKD5D, CKD3-5 and normal kidney function). An exon microarray approach was used to highlight splicing events in whole human genome in a normal cell model (fibroblasts) incubated with uremic serum. Alternative splicing variants identified were confirmed by RT-PCR. RESULTS: The splicing variant of the exon 30 of PKD1 was more frequent in PBMCs from patients with CKD compared to control. With the microarray approach, despite the analysis of more than 230 000 probes, we identified 36 genes with an abnormal splicing index evocating splicing event in fibroblasts exposed to uremic serum. Only one abnormal splicing event in one gene, ADH1B, was confirmed by RT-PCR. CONCLUSION: We observed two alternative spliced genes in two different cell types associated with CKD. Alternative splicing could play a role in the control of gene expression during CKD but it does not seem to be a major mechanism.

Ascorbic acid derivatives as a new class of antiproliferative molecules.

Ascorbic acid (AA) has long been described as an antiproliferative agent. However, the molecule has to be used at a very high concentrations, which necessitates i.v. injection, and the tight regulation of in-blood and in-cell AA concentrations making it impossible to hold very high concentrations for any substantial length of time. Here we report evidence that AA derivates are antiproliferative and cytotoxic molecules at an IC50 lower than AA itself. Among these new molecules, we selected K873 that has cytotoxic and antiproliferative effects on different human tumor cells at tenth micromolar concentration. In a further step, we demonstrated that K873 selectively to kills only cancer cells without being toxic for normal non-dividing (or poorly dividing) cells. Finally, we tested the effect of treatment with K873 (5-10 mg/kg/d by i.p. route) on tumor progression in xenografted immunodeficient mice (BALB/c Nude). Our data suggest that K873 administration strongly inhibits tumor progression. In a previous study using microarrays, we demonstrated that AA decreases the expression of two genes families involved in cell cycle progression, i.e. initiation factor of translation and tRNA synthetases. Here we show that K873 treatment also decreases the expression of four of these genes in xenografted tumors, in proportions similar to that previously observed with AA. Taken together, our data suggest that AA and K873 share similar action. Our findings suggest that AA derivatives could be a promising new class of anti-cancer drugs, either alone or in combination with other molecules.

Connexin 32 is involved in mitosis.

The X-linked form of Charcot-Marie-Tooth disorder (CMTX) is the second most frequent type (15% of CMT forms). It involves the GJB1 gene coding for connexin 32, a protein involved in gap junction formation and function. There is no curative treatment for CMTX. We present data on transgenic lines that was accomplished by inserting a human BAC carrying the GJB1 gene, in which two different mutations in connexin 32 (Cx32) observed in patients were introduced. Investigation of these models implicated Cx32 in the control of mitotic stability. The model in which Gjb1 has been invalidated had the same phenotype. This new function for Cx32 was recently confirmed by results from the Mitocheck program. Locomotor impediment was seen in the behavior of these animals, the severity of which correlated with transgene copy number and RNA expression.

High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. METHODS: We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. RESULTS: We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. CONCLUSION: HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

Association of PKD2 (polycystin 2) mutations with left-right laterality defects.

Mutations in the PKD1 (polycystin 1) and PKD2 (polycystin 2) genes cause autosomal dominant polycystic kidney disease (ADPKD). Most Pkd2-null mouse embryos present with left-right laterality defects. For the first time, we report the association of ADPKD resulting from a mutation in PKD2 and left-right asymmetry defects. PKD1 and PKD2 were screened for mutations or large genomic rearrangements in 3 unrelated patients with ADPKD presenting with laterality defects: dextrocardia in one and situs inversus totalis in 2 others. A large gene deletion, a single-exon duplication, and an in-frame duplication respectively, were found in the 3 patients. These polymorphisms were found in all tested relatives with ADPKD, but were absent in unaffected related individuals. No left-right anomalies were found in other members of the 3 families. A possible association between heterotaxia and a PKD2 mutation in our 3 patients is suggested by: (1) the existence of laterality defects in Pkd2-null mouse and zebrafish models and (2) detection of a pathogenic PKD2 mutation in the 3 probands, although PKD2 mutations account for only 15% of ADPKD families. The presence of left-right laterality defects should be systematically screened in larger cohorts of patients with ADPKD harboring PKD2 mutations.

Ascorbic Acid and gene expression: another example of regulation of gene expression by small molecules?

Ascorbic acid (vitamin C, AA) has long been considered a food supplement necessary for life and for preventing scurvy. However, it has been reported that other small molecules such as retinoic acid (vitamin A) and different forms of calciferol (vitamin D) are directly involved in regulating the expression of numerous genes. These molecules bind to receptors that are differentially expressed in the embryo and are therefore crucial signalling molecules in vertebrate development. The question is: is ascorbic acid also a signalling molecule that regulates gene expression?We therefore present and discuss recent publications that demonstrate that AA regulates the expression of a battery of genes. We offer a clue to understanding the biochemical mechanism by which AA regulates gene expression. Finally we will discuss the question of a receptor for AA and its potential involvement in embryonic development and cell differentiation.

Effect of ascorbic acid in patients with Charcot-Marie-Tooth disease type 1A: a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy that affects roughly one in 5000 births. No specific therapy currently exists for this degenerative disorder, which is characterised by distal progressive muscle atrophy and sensory loss, although ascorbic acid has been shown to reduce demyelination and improve muscle function in a transgenic mouse model of CMT1A. We tested the safety and efficacy of ascorbic acid in adults with CMT1A. METHODS: This 12-month, randomised, double-blind, placebo-controlled study was undertaken between September, 2005, and October, 2008. Patients diagnosed with CMT1A according to clinical examination and confirmation by genotyping were randomly assigned in a 1:1:1 ratio to receive 1 g ascorbic acid per day, 3 g ascorbic acid per day, or placebo. Treatment allocation was based on a computer-generated list of random numbers in blocks of 12, with stratification according to study site and sex; all investigators and participants were unaware of treatment allocation. The primary outcome was the Charcot-Marie-Tooth disease neuropathy score (CMTNS) at 12 months. Analysis was by intention to treat. This study is registered with the Orphanet Database, number ORPHA60779. FINDINGS: The median change in CMTNS from baseline to 12 months was 0.5 points (95% CI -0.3 to 1.4) for the placebo group (n=62), 0.7 points (0.0 to 1.4) for the 1 g ascorbic acid group (n=56), and -0.4 points (-1.2 to 0.4) for the 3 g ascorbic acid group (n=61). We did not find any significant difference in these changes between the groups (p=0.14). The occurrence of adverse events did not differ between the groups (p=0.74). INTERPRETATION: Ascorbic acid at both doses was safe and well tolerated in adults with CMT1A over 12 months. However, there were no significant differences between the groups and the efficacy of ascorbic acid was not shown.

Antiproliferative effect of ascorbic acid is associated with the inhibition of genes necessary to cell cycle progression.

BACKGROUND: Ascorbic acid (AA), or Vitamin C, is most well known as a nutritional supplement with antioxidant properties. Recently, we demonstrated that high concentrations of AA act on PMP22 gene expression and partially correct the Charcot-Marie-Tooth disease phenotype in a mouse model. This is due to the capacity of AA, but not other antioxidants, to down-modulate cAMP intracellular concentration by a competitive inhibition of the adenylate cyclase enzymatic activity. Because of the critical role of cAMP in intracellular signalling, we decided to explore the possibility that ascorbic acid could modulate the expression of other genes. METHODS AND FINDINGS: Using human pangenomic microarrays, we found that AA inhibited the expression of two categories of genes necessary for cell cycle progression, tRNA synthetases and translation initiation factor subunits. In in vitro assays, we demonstrated that AA induced the S-phase arrest of proliferative normal and tumor cells. Highest concentrations of AA leaded to necrotic cell death. However, quiescent cells were not susceptible to AA toxicity, suggesting the blockage of protein synthesis was mainly detrimental in metabolically-active cells. Using animal models, we found that high concentrations of AA inhibited tumor progression in nude mice grafted with HT29 cells (derived from human colon carcinoma). Consistently, expression of tRNA synthetases and ieF2 appeared to be specifically decreased in tumors upon AA treatment. CONCLUSIONS: AA has an antiproliferative activity, at elevated concentration that could be obtained using IV injection. This activity has been observed in vitro as well in vivo and likely results from the inhibition of expression of genes involved in protein synthesis. Implications for a clinical use in anticancer therapies will be discussed.

Centrosome overduplication and mitotic instability in PKD2 transgenic lines.

Polycystin-2 (PC-2), a protein encoded by PKD2 and involved in autosomal dominant polycystic kidney disease (ADPKD), is a non-selective cationic channel recently implicated in the function of primary cilia. We recently constructed a new animal model in the form of a transgenic mouse with a BAC-containing human PKD2 inserted in its genome. Two transgenic mouse lines overexpressing human PKD2 showed mitotic instability. Fibroblasts from these transgenic mouse lines have abnormal chromosomal numbers. These lines also have supernumerary centrosomes. PC-2 overexpression is associated with mitotic instability and centrosome overduplication. PC-2 therefore seems to play a role in centrosome duplication, and this hypothesis is being evaluated in other models.

Analysis of the benefits of vitamin cocktails in treating Charcot-Marie-Tooth disease type 1A.

We recently proposed that the use of high doses of ascorbic acid (AA) could constitute the first potential treatment for Charcot-Marie-Tooth disease type 1A (CMT1A).4 We investigated the potential benefits of using cocktails of vitamins for CMT1A therapy. We used transient transfection of Schwann cells with a construction placing the expression of a reporter gene under the control of the Schwann cell-specific promoter of PMP22. Transfected cells were cultured with or without addition of ascorbic acid, vitamin A, vitamin E, or a cocktail of these vitamins. Adding vitamin A or E counteracts the effect of ascorbic acid in inhibiting PMP22 expression. We thus recommend that vitamins A and E should not be included in combination with AA in clinical trials.

Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD.

BACKGROUND: The evolution of the variant Creutzfeldt-Jakob disease (vCJD) epidemic is hazardous to predict due to uncertainty in ascertaining the prevalence of infection and because the disease might remain asymptomatic or produce an alternate, sporadic-like phenotype. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic mice were produced that overexpress human prion protein with methionine at codon 129, the only allele found so far in vCJD-affected patients. These mice were infected with prions derived from variant and sporadic CJD (sCJD) cases by intracerebral or intraperitoneal route, and transmission efficiency and strain phenotype were analyzed in brain and spleen. We showed that i) the main features of vCJD infection in humans, including a prominent involvement of the lymphoid tissues compared to that in sCJD infection were faithfully reproduced in such mice; ii) transmission of vCJD agent by intracerebral route could lead to the propagation of either vCJD or sCJD-like prion in the brain, whereas vCJD prion was invariably propagated in the spleen, iii) after peripheral exposure, inefficient neuroinvasion was observed, resulting in an asymptomatic infection with life-long persistence of vCJD prion in the spleen at stable and elevated levels. CONCLUSION/SIGNIFICANCE: Our findings emphasize the possibility that human-to-human transmission of vCJD might produce alternative neuropathological phenotypes and that lymphoid tissue examination of CJD cases classified as sporadic might reveal an infection by vCJD-type prions. They also provide evidence for the strong propensity of this agent to establish long-lasting, subclinical vCJD infection of lymphoreticular tissues, thus amplifying the risk for iatrogenic transmission.