Biphasic Myopathic Phenotype of Mouse DUX, an ORF within Conserved FSHD-Related Repeats

Facioscapulohumeral muscular dystrophy (FSHD) is caused by contractions of D4Z4 repeats at 4q35.2 thought to induce misregulation of nearby genes, one of which, DUX4, is actually localized within each repeat. A conserved ORF (mDUX), embedded within D4Z4-like repeats, encoding a double-homeodomain protein, was recently identified on mouse chromosome 10. We show here that high level mDUX expression induces myoblast death, while low non-toxic levels block myogenic differentiation by down-regulating MyoD and Myf5. Toxicity and MyoD/Myf5 expression changes were competitively reversed by overexpression of Pax3 or Pax7, implying mechanistic similarities with the anti-myogenic activity of human DUX4. We tested the effect of mDUX expression on Xenopus development, and found that global overexpression led to abnormalities in gastrulation. When targeted unilaterally into blastomeres fated to become tail muscle in 16-cell embryos, mDUX caused markedly reduced tail myogenesis on the injected side. These novel cell and animal models highlight the myopathic nature of sequences within the FSHD-related repeat array

Contact Hypersensitivity to Oxazolone Provokes Vulvar Mechanical Hyperalgesia in Mice

The interplay among pain, allergy and dysregulated inflammation promises to yield significant conceptual advances in immunology and chronic pain. Hapten-mediated contact hypersensitivity reactions are used to model skin allergies in rodents but have not been utilized to study associated changes in pain perception in the affected skin. Here we characterized changes in mechanical hyperalgesia in oxazolone-sensitized female mice challenged with single and repeated labiar skin exposure to oxazolone. Female mice were sensitized with topical oxazolone on their flanks and challenged 1-3 times on the labia. We then measured mechanical sensitivity of the vulvar region with an electronic pressure meter and evaluated expression of inflammatory genes, leukocyte influx and levels of innervation in the labiar tissue. Oxazolone-sensitized mice developed vulvar mechanical hyperalgesia after a single labiar oxazolone challenge. Hyperalgesia lasted up to 24 hours along with local influx of neutrophils, upregulation of inflammatory cytokine gene expression, and increased density of cutaneous labiar nerve fibers. Three daily oxazolone challenges produced vulvar mechanical hyperalgesic responses and increases in nerve density that were detectable up to 5 days post-challenge even after overt inflammation resolved. This persistent vulvar hyperalgesia is resonant with vulvodynia, an understudied chronic pain condition that is remarkably prevalent in 18-60 year-old women. An elevated risk for vulvodynia has been associated with a history of environmental allergies. Our pre-clinical model can be readily adapted to regimens of chronic exposures and long-term assessment of vulvar pain with and without concurrent inflammation to improve our understanding of mechanisms underlying subsets of vulvodynia and to develop new therapeutics for this condition

RNA Gain-of-Function in Spinocerebellar Ataxia Type 8

Microsatellite expansions cause a number of dominantly-inherited neurological diseases. Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins. Bi-directional expression of the spinocerebellar ataxia type 8 (SCA8) CTG CAG expansion produces CUG expansion RNAs (CUGexp) from the ATXN8OS gene and a nearly pure polyglutamine expansion protein encoded by ATXN8 CAGexp transcripts expressed in the opposite direction. Here, we present three lines of evidence that RNA gain-of-function plays a significant role in SCA8: 1) CUGexp transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons in the brain; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUGexp transcripts trigger splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4). In vivo optical imaging studies in SCA8 mice confirm that Gabt4 upregulation is associated with the predicted loss of GABAergic inhibition within the granular cell layer. These data demonstrate that CUGexp transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUGexp disorders. Moreover, our demonstration that relatively short CUGexp transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders

Toxicity of mDUX.

<p>(A) Morphology of iC2C12-mDUX cells induced for 24 hours (Dox) with 500 ng/ml doxycycline. The majority of induced cells were detached and floating after 24 hours. (B) ATP assay for analysis of viability in iC2C12-mDUX cells induced with various concentrations of doxycyline for 24 and 48 hours. Decreased cell viability was significant in the cells induced with as little as 32 ng/ml doxycycline in the first 24 hours. Results are presented as fold difference compare to untreated cells at 24 hours. (C) FACS analysis of annexin V/7-AAD stained cells for determination of apoptosis and cell death. Single annexin V positive cells (x-axis, bottom right corner) represent cells undergoing apoptosis, and double positive cells (annexin V⁺ and 7-AAD⁺, right top population) represent dead cells. A slight increase of apoptotic and dead cells was detected at 12 hours which progressed to significant after 24 hours of induction. (D) ATP assay on the cells induced for 24 hours demonstrated that antioxidants (AsAc: ascorbic acid (21.25 mM), B-MET: β-mercaptoethanol (0.5 mM), MTG: monothioglycerol (2.25 mM)) did not have any beneficial effect on cell viability even in cells treated with the low dose of doxycycline (32 ng/ml). (E) Morphology of cells, either uninduced (Control), mDUX-induced (Dox, 125 mg/ml) or induced and treated with antioxidants. (F) Morphology of mDUX inducible fibroblasts (i3T3-mDUX) and inducible mDUX embryonic stem cells (iES-mDUX) (G) after 24 hours of induction with 500 ng/ml doxycyline. mDUX expressed at high levels induces cell death in fibroblasts and embryonic stem cells. (H) ATP assay for effects of doxycycline on viability of control C2C12 and iC2C12 cells after 48 hours of treatment. Results are presented as fold difference compare to untreated C2C12 cells.</p

mDUX and myogenic regulators.

<p>qRT-PCR for mDUX and myogenic genes in iC2C12-mDUX cells evaluated at different times (A, using 500 ng/mL doxycycline) or doses (C, at 12 hours). Results are presented as fold difference compared to uninduced cells (0 ng/ml) except for the expression of mDUX in which 12 hours of induction was taken as the group for comparison. Error bars represent the STDEV. Induction with 8 ng/mL of doxycycline was sufficient for significant down-regulation of MyoD. (B) Immunofluorescence for detection of MyoD (red) in iC2C12-mDUX cells induced during the time course of 12 hours. Nuclei were stained with DAPI (blue). A notable decrease in the number of the positive-staining nuclei and the intensity of the staining was detected as early as 4 hours after induction. (C) Expression of mDUX, MyoD, and Pax7 when mDUX is induced with various concentrations of doxycycline.</p

Pax3 and Pax7 compete with mDUX.

<p>(A) FACS analysis of iC2C12-mDUX cells transduced with MSCV retroviral constructs caring GFP, Pax3-ires-GFP or Pax7-ires-GFP. Almost all of the cells at the time of the experiment stably express GFP (x-axis). (B) Immunofluorescence for Pax3 or Pax7 (red) and GFP (green) reveals that Pax3 and Pax7 are expressed in GFP⁺ cells. Cell number is decreased in induced samples due to toxicity of mDUX. (C) ATP assay for determination of cell viability in iC2C12-mDUX cells transduced with MSCV-ires-GFP, MSCV-Pax3-ires-GFP or MSCV-Pax7-ires-GFP. Cells were induced with various concentrations of doxycycline for 24 and 48 hours. Pax3- and Pax7-expressing cells are largely resistant to the toxicity of mDUX induced by 32 ng/mL dox even after 48 hours of induction. (D) qRT-PCR analyses for MyoD and Myf5 in the cells shown in (C), induced for 18 hours. Expression of MyoD and Myf5 is strongly repressed at 32 ng/mL induction in the control cells, but not the Pax3 or Pax7 expressing cells.</p

mDUX expression in Xenopus.

<p>(A) Neurula stage embryos following injection of <i>GFP</i> mRNA alone (control) or <i>GFP+mDUX</i> mRNA. The green color indicates the domain filled by the RNA injection. The control has a normal neural plate while the mDUX case is very abnormal due to deranged gastrulation movements. (B) Tail muscle pattern in stage 45 tadpoles. The green color shows immunostaining with 12/101 antibody. Control shows the normal pattern of myotomes. Two examples (mDUX) demonstrate how injection into blastomeres V2.1 and 2.2 results in an inhibition of muscle differentiation on the injected (left) side.</p

mDUX and myogenic differentiation.

<p>(A) Phase-contrast microscopy of iC2C12-mDUX cells induced with doxycyline through 6 days of differentiation. (B) Immunofluorescence for detection of MyHC (red, upper panels) and MyoD (red, lower panels) in cells induced with low levels of doxycycline. Nuclei were counterstained with DAPI (blue). iC2C12-mDUX cells were in differentiation medium for 6 days when myotube fusion index was calculated (C). Significantly diminished myogenic differentiation was observed in the cells induced with 10 ng/mL doxycyline. (D) Inhibition of differentiation was confirmed by qRT-PCR. Results are presented as fold difference compared to uninduced cells (0 ng/mL) and the error bars represent the STDEV. (E) Immunofluorescence for detection of MyHC (red) in C2C12 and iC2C12 control cells after 6 days of differentiation and treatment with different concentrations of doxycycline. (F) Calculated fusion index and (G) gene expression analyses of differentiated C2C12 and iC2C12 control cells. Doxycycline by itself did not have any significant effect on myoblast (C2C12 and iC2C12) differentiation.</p

A Newton Algorithm for the Nearest Correlation Matrix

Firstly, we describe and investigate the algorithm of Qi and Sun which solves the problem of finding the nearest correlation matrix to a symmetric matrix. This algorithm claims a quadratic convergence. We discuss improving this algorithm's efficiency and reliability and detect a problem when we are aiming at a nearest correlation matrix with a high accuracy, using small error tolerences. As a consequence, we suggest a modified version, based on the algorithm of Qi and Sun, which is also a quadratically convergent algorithm, has improved efficiency and is modified so that the algorithm can return the nearest correlation matrix to high accuracy showing a robust and reliable behaviour. Secondly, we investigate the general alternating projections method and also Higham's alternating projections method for the nearest correlation matrix. We discuss variations of the latter and include a further projection which allows more constraints to be added to the problem. We introduce a new algorithm and compare its convergence behaviour with Higham's alternating projections method