Dysferlin and Myoferlin Regulate Transverse Tubule Formation and Glycerol Sensitivity

Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy

MicroRNA-30c targets cytoskeleton genes involved in breast cancer cell invasion

Metastasis remains a significant challenge in treating cancer. A better understanding of the molecular mechanisms underlying metastasis is needed to develop more effective treatments. Here we show that human breast tumor biomarker miR-30c regulates invasion by targeting the cytoskeleton network genes encoding Twinfilin 1 (TWF1) and Vimentin (VIM). Both VIM and TWF1 have been shown to regulate epithelial-to-mesenchymal transition (EMT). Similar to TWF1, VIM also regulates F-actin formation, a key component of cellular transition to a more invasive mesenchymal phenotype. To further characterize the role of the TWF1 pathway in breast cancer, we found that IL-11 is an important target of TWF1 that regulates breast cancer cell invasion and STAT3 phosphorylation. The miR-30c-VIM/TWF1 signaling cascade is also associated with clinical outcome in breast cancer patients

MicroRNA-30c inhibits human breast tumour chemotherapy resistance by regulating TWF1 and IL-11

Chemotherapy resistance frequently drives tumour progression. However, the underlying molecular mechanisms are poorly characterized. Epithelial-to-mesenchymal transition (EMT) has been shown to correlate with therapy resistance, but the functional link and signalling pathways remain to be elucidated. We report here that miR-30c, a human breast tumour prognostic marker, plays a pivotal role in chemo-resistance by a direct targeting of TWF1, which encodes an actin-binding protein and promotes EMT. An IL-6 family member, IL-11 was identified as a secondary target of TWF1 in the miR-30c signalling pathway. Expression of miR-30c inversely correlated with TWF1 and IL-11 levels in primary breast tumours and low IL-11 correlated with relapse-free survival in breast cancer patients. Our study demonstrates that miR-30c is transcriptionally regulated by GATA3 in breast tumours. Identification of a novel miRNA-mediated pathway that regulates chemo-resistance in breast cancer will facilitate the development of novel therapeutic strategies

Advances in understanding and treating ADHD

Attention deficit hyperactivity disorder (ADHD) is a neurocognitive behavioral developmental disorder most commonly seen in childhood and adolescence, which often extends to the adult years. Relative to a decade ago, there has been extensive research into understanding the factors underlying ADHD, leading to far more treatment options available for both adolescents and adults with this disorder. Novel stimulant formulations have made it possible to tailor treatment to the duration of efficacy required by patients, and to help mitigate the potential for abuse, misuse and diversion. Several new non-stimulant options have also emerged in the past few years. Among these, cognitive behavioral interventions have proven popular in the treatment of adult ADHD, especially within the adult population who cannot or will not use medications, along with the many medication-treated patients who continue to show residual disability

Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from Ehd1-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining Ehd1-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in Ehd1-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, Ehd1-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. Ehd1-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy

Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

<div><p>We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from <i>Ehd1</i>-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining <i>Ehd1</i>-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in <i>Ehd1</i>-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, <i>Ehd1</i>-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. <i>Ehd1</i>-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.</p></div

Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.

Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy and its associated cardiomyopathy, we used quantitative trait locus mapping and whole genome sequencing in a mouse model. This approach uncovered a modifier locus on chromosome 11 associated with sarcolemmal membrane damage and heart mass. Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene. A synonymous variant in exon 11 creates a cryptic splice donor, resulting in a truncated annexin A6 protein called ANXA6N32. Live cell imaging showed that annexin A6 orchestrates a repair zone and cap at the site of membrane disruption. In contrast, ANXA6N32 dramatically disrupted the annexin A6-rich cap and the associated repair zone, permitting membrane leak. Anxa6 is a modifier of muscular dystrophy and membrane repair after injury

Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.

Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy and its associated cardiomyopathy, we used quantitative trait locus mapping and whole genome sequencing in a mouse model. This approach uncovered a modifier locus on chromosome 11 associated with sarcolemmal membrane damage and heart mass. Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene. A synonymous variant in exon 11 creates a cryptic splice donor, resulting in a truncated annexin A6 protein called ANXA6N32. Live cell imaging showed that annexin A6 orchestrates a repair zone and cap at the site of membrane disruption. In contrast, ANXA6N32 dramatically disrupted the annexin A6-rich cap and the associated repair zone, permitting membrane leak. Anxa6 is a modifier of muscular dystrophy and membrane repair after injury

Misexpression of triad proteins and expansion of the T-tubule compartment in <i>Ehd1-</i>heterozygous muscle.

<p>(A) BIN1 and DHPR levels are increased +2.25 and +5.4 fold in <i>Ehd1</i>+/- quadriceps muscle compared to WT correlating with the increase in T-tubule structures. Gel code bands are shown as a loading control (LC). (B) Junctophilin 2 (JP2) protein levels were decreased 13-fold in <i>Ehd1+/-</i> quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (C) Junctophilin 1 (JP1) protein levels were similar in <i>Ehd1+/-</i> quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (D) Ultrastructural analysis reveals ectopic (dotted arrow) and elongated (arrow) T-tubules in 8-week-old <i>Ehd1-</i>heterozygous muscle (<i>Ehd1+/-</i>) stained with potassium ferricyanide to color the T-tubule structures black. (E) <i>Ehd1-</i>heterozygous muscle contains duplicated triads containing 2 T-tubules (black arrows) and 3 sarcoplasmic reticulum (SR) in 1 triad unit. Scale 0.5μm. (F) <i>Ehd1-</i>heterozygous muscle stained with potassium ferricyanide, outlines duplicated T-tubule structures (two black arrows). Scale 0.5μm. (G) Ultrastructural analysis of 2-D images reveals increased tubule abnormalities in <i>Ehd1-</i>heterozygous muscle, 12.5%, compared to 1.7% in control muscle (n>400 structures per genotype, p = 0.04).</p

Disordered T-tubules in <i>Ehd1-</i>heterozygous muscle.

<p>Myofibers were immunostained with anti-BIN1 (red) and anti-DHPR (green) antibodies. Representative myofibers are shown. <i>Ehd1-</i>heterozygous (<i>Ehd1+/-</i>) fibers displayed disorganized (white arrowhead) and aggregated (white arrow) T-tubule structures in 27% of myofibers, marked by DHPR, also evidenced in DIC images compared to 0% in control fibers. <i>Ehd1</i>-heterozygous muscle with extensive BIN1 fluorescence extending beyond DHPR staining (yellow arrowhead). Scale 5μm.</p