Becker syndrome, a recessive nondystrophic myotonia caused by mutations in the chloride channel 1 gene (CLCN1), is characterized by delayed muscle relaxation after contraction. The ADR (arrested development of righting response) mouse is an animal model for Becker syndrome. Skeletal muscles from ADR myotonic animals show an increased number of oxidative fibers with a lack of glycolytic fibers as well as signs of muscle hypertrophy. Through breeding ADR myotonic mice with mice harboring a MEF2-dependent reporter gene, we found that the transcriptional activity of MEF2 was dramatically enhanced in myotonic muscles. Post-translational induction of MEF2 transcriptional activity correlated with the activation of p38 MAPK and did not affect MEF2 DNA-binding affinity. Expression of class II histone deacetylases (HDACs), which repress MEF2-dependent gene expression, was significantly reduced in skeletal muscles from myotonic mice. These findings suggest that the combined effects of class II HDAC deficiency and p38 MAPK activation lead to potent upregulation of MEF2 transcriptional activity, which contributes to the long-term changes in gene expression and fiber-type transformation observed in myotonic skeletal muscles. These findings provide new molecular targets for potential treatment of congenital myotonia.
Hai Wu, Eric N. Olson
Deficiency of the membrane protein FAT/CD36 causes a marked defect in fatty acid uptake by various tissues and is genetically linked to insulin resistance in rats and humans. Here, we examined insulin responsiveness of CD36–/– mice. When fed a diet high in complex carbohydrates and low (5%) in fat, these animals cleared glucose faster than the wild-type. In vivo, uptake of 2-fluorodeoxyglucose by muscle was increased severalfold, and in vitro, insulin responsiveness of glycogenesis by the soleus was enhanced. Null mice had lower glycogen levels in muscle and liver, lower muscle triglyceride levels, and increased liver triglyceride content—all findings consistent with increased insulin-sensitivity. However, when the chow diet was switched to one high in fructose, CD36–/– mice but not wild-type mice developed marked glucose intolerance, hyperinsulinemia, and decreased muscle glucose uptake. High-fat diets impaired glucose tolerance equally in both groups, although CD36 deficiency helped moderate insulin-responsive muscle glucose oxidation. In conclusion, CD36 deficiency enhances insulin responsiveness on a high-starch, low-fat diet. It predisposes to insulin resistance induced by high fructose and partially protects from that induced by high-fat diets. In humans, CD36 deficiency may be an important factor in the metabolic adaptation to diet and in susceptibility to some forms of diet-induced pathology.
Tahar Hajri, Xiao Xia Han, Arend Bonen, Nada A. Abumrad
Pseudophosphatases display extensive sequence similarities to phosphatases but harbor amino acid alterations in their active-site consensus motifs that render them catalytically inactive. A potential role in substrate trapping or docking has been proposed, but the specific requirements for pseudophosphatases during development and differentiation are unknown. We demonstrate here that Sbf1, a pseudophosphatase of the myotubularin family, is expressed at high levels in seminiferous tubules of the testis, specifically in Sertoli’s cells, spermatogonia, and pachytene spermatocytes, but not in postmeiotic round spermatids. Mice that are nullizygous for Sbf1 exhibit male infertility characterized by azoospermia. The onset of the spermatogenic defect occurs in the first wave of spermatogenesis at 17 days after birth during the synchronized progression of pachytene spermatocytes to haploid spermatids. Vacuolation of the Sertoli’s cells is the earliest observed phenotype and is followed by reduced formation of spermatids and eventual depletion of the germ cell compartment in older mice. The nullizygous phenotype in conjunction with high-level expression of Sbf1 in premeiotic germ cells and Sertoli’s cells is consistent with a crucial role for Sbf1 in transition from diploid to haploid spermatocytes. These studies demonstrate an essential role for a pseudophosphatase and implicate signaling pathways regulated by myotubularin family proteins in spermatogenesis and germ cell differentiation.
Ron Firestein, Peter L. Nagy, Megan Daly, Phil Huie, Marco Conti, Michael L. Cleary
Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from loss of acid sphingomyelinase (ASM) activity. We have used a knockout mouse model of NPD (ASMKO mice) to evaluate the effects of direct intracerebral transplantation of bone marrow–derived mesenchymal stem cells (MSCs) on the progression of neurological disease in this disorder. MSCs were transduced with a retroviral vector to overexpress ASM and were injected into the hippocampus and cerebellum of 3-week-old ASMKO pups. Transplanted cells migrated away from the injection sites and survived at least 6 months after transplantation. Seven of 8 treated mice, but none of the untreated controls, survived for ≥ 7 months after transplant. Survival times were greater in sex-matched than in sex-mismatched transplants. Transplantation significantly delayed the Purkinje cell loss that is characteristic of NPD, although the protective effect declined with distance from the injection site. Overall ASM activity in brain homogenates was low, but surviving Purkinje cells contained the retrovirally expressed human enzyme, and transplanted animals showed a reduction in cerebral sphingomyelin. These results reveal the potential of treating neurodegenerative lysosomal storage disorders by intracerebral transplantation of bone marrow–derived MSCs.
Hee Kyung Jin, Janet E. Carter, George W. Huntley, Edward H. Schuchman
Gaucher disease, the most common lysosomal storage disease, is caused by a deficiency of glucocerebrosidase resulting in the impairment of glucosylceramide degradation. The hallmark of the disease is the presence of the Gaucher cell, a macrophage containing much of the stored glucosylceramide found in tissues, which is believed to cause many of the clinical manifestations of the disease. We have developed adult mice carrying the Gaucher disease L444P point mutation in the glucocerebrosidase (Gba) gene and exhibiting a partial enzyme deficiency. The mutant mice demonstrate multisystem inflammation, including evidence of B cell hyperproliferation, an aspect of the disease found in some patients. However, the mutant mice do not accumulate large amounts of glucosylceramide or exhibit classic Gaucher cells in tissues.
Hiroki Mizukami, Yide Mi, Ryuichi Wada, Mari Kono, Tadashi Yamashita, Yujing Liu, Norbert Werth, Roger Sandhoff, Konrad Sandhoff, Richard L. Proia
The treatment of chronic inflammatory diseases is complicated by their unpredictable, relapsing clinical course. Here, we describe a new strategy in which an inflammation-regulated therapeutic transgene is introduced into the joints to prevent recurrence of arthritis. To this end, we designed a recombinant adenoviral vector containing a two-component, inflammation-inducible promoter controlling the expression of human IL-10 (hIL-10) cDNA. When tested in vitro, this system had a low-level basal activity and was activated four to five orders of magnitude by various inflammatory stimuli, including TNF-α, IL-1β, IL-6, and LPS. When introduced in joints of rats with recurrent streptococcal cell wall–induced arthritis, the IL-10 transgene was induced in parallel with disease recurrence and effectively prevented the influx of inflammatory cells and the associated swelling of the joints. Levels of inflammation-inducible hIL-10 protein within the joints correlated closely with the severity of recurrence. An endogenously regulated therapeutic transgene can thus establish negative feedback and restore homeostasis in vivo while minimizing host exposure to the recombinant drug.
A.V. Miagkov, A.W. Varley, R.S. Munford, S.S. Makarov
Dominant mutations in sarcomere protein genes cause hypertrophic cardiomyopathy, an inherited human disorder with increased ventricular wall thickness, myocyte hypertrophy, and disarray. To understand the early consequences of mutant sarcomere proteins, we have studied mice (designated αMHC403/+) bearing an Arg403Gln missense mutation in the α cardiac myosin heavy chain. We demonstrate that Ca2+ is reduced in the sarcoplasmic reticulum of αMHC403/+ mice, and levels of the sarcoplasmic reticulum Ca2+-binding protein calsequestrin are diminished in advance of changes in cardiac histology or morphology. Further evidence for dysregulation of sarcoplasmic reticulum Ca2+ in these animals is seen in their decreased expression of the ryanodine receptor Ca2+-release channel and its associated membrane proteins and in an increase in ryanodine receptor phosphorylation. Early administration of the L-type Ca2+ channel inhibitor diltiazem restores normal levels of these sarcoplasmic reticular proteins and prevents the development of pathology in αMHC403/+ mice. We conclude that disruption of sarcoplasmic reticulum Ca2+ homeostasis is an important early event in the pathogenesis of this disorder and suggest that the use of Ca2+ channel blockers in advance of established clinical disease could prevent hypertrophic cardiomyopathy caused by sarcomere protein gene mutations.
Christopher Semsarian, Imran Ahmad, Michael Giewat, Dimitrios Georgakopoulos, Joachim P. Schmitt, Bradley K. McConnell, Steven Reiken, Ulrike Mende, Andrew R. Marks, David A. Kass, Christine E. Seidman, J.G. Seidman
LMX1B encodes a LIM-homeodomain transcription factor. Mutations in LMX1B cause nail-patella syndrome (NPS), an autosomal dominant disease with skeletal abnormalities, nail hypoplasia, and nephropathy. Expression of glomerular basement membrane (GBM) collagens is reduced in Lmx1b–/– mice, suggesting one basis for NPS nephropathy. Here, we show that Lmx1b–/– podocytes have reduced numbers of foot processes, are dysplastic, and lack typical slit diaphragms, indicating an arrest in development. Using antibodies to podocyte proteins important for podocyte function, we found that Lmx1b–/– podocytes express near-normal levels of nephrin, synaptopodin, ZO-1, α3 integrin, and GBM laminins. However, mRNA and protein levels for CD2AP and podocin were greatly reduced, suggesting a cooperative role for these molecules in foot process and slit diaphragm formation. We identified several LMX1B binding sites in the putative regulatory regions of both CD2AP and NPHS2 (podocin) and demonstrated that LMX1B binds to these sequences in vitro and can activate transcription through them in cotransfection assays. Thus, LMX1B regulates the expression of multiple podocyte genes critical for podocyte differentiation and function. Our results indicate that reduced levels of proteins associated with foot processes and the glomerular slit diaphragm likely contribute, along with reduced levels of GBM collagens, to the nephropathy associated with NPS.
Jeffrey H. Miner, Roy Morello, Kaya L. Andrews, Cong Li, Corinne Antignac, Andrey S. Shaw, Brendan Lee
Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the α4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV α4).
Claudia Rohr, Jürgen Prestel, Laurence Heidet, Hiltraud Hosser, Wilhelm Kriz, Randy L. Johnson, Corinne Antignac, Ralph Witzgall
Regulatory subunit KCNE3 (E3) interacts with KCNQ1 (Q1) in epithelia, regulating its activation kinetics and augmenting current density. Since E3 is expressed weakly in the heart, we hypothesized that ectopic expression of E3 in cardiac myocytes might abbreviate action potential duration (APD) by interacting with Q1 and augmenting the delayed rectifier current (IK). Thus, we transiently coexpressed E3 with Q1 and KCNE1 (E1) in Chinese hamster ovary cells and found that E3 coexpression increased outward current at potentials by ≥ –80 mV and accelerated activation. We then examined the changes in cardiac electrophysiology following injection of adenovirus-expressed E3 into the left ventricular cavity of guinea pigs. After 72 hours, the corrected QT interval of the electrocardiogram was reduced by ∼10%. APD was reduced by >3-fold in E3-transduced cells relative to controls, while E-4031–insensitive IK and activation kinetics were significantly augmented. Based on quantitative modeling of a transmural cardiac segment, we demonstrate that the degree of QT interval abbreviation observed results from electrotonic interactions in the face of limited transduction efficiency and that heterogeneous transduction of E3 may actually potentiate arrhythmias. Provided that fairly homogeneous ectopic ventricular expression of regulatory subunits can be achieved, this approach may be useful in enhancing repolarization and in treating long QT syndrome.
Reza Mazhari, H. Bradley Nuss, Antonis A. Armoundas, Raimond L. Winslow, Eduardo Marbán