John M. Shelton

Reeler/Disabled-like Disruption of Neuronal Migration in Knockout Mice Lacking the VLDL Receptor and ApoE Receptor 2

Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. Here, we report the requirement for two other proteins, cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1.

Histone Deacetylase 4 Controls Chondrocyte Hypertrophy during Skeletogenesis

Histone deacetylases (HDACs) modulate cell growth and differentiation by governing chromatin structure and repressing the activity of specific transcription factors. We showed previously that HDAC9 acts as a negative regulator of cardiomyocyte hypertrophy and skeletal muscle differentiation. Here we report that HDAC4, which is expressed in prehypertrophic chondrocytes, regulates chondrocyte hypertrophy and endochondral bone formation by interacting with and inhibiting the activity of Runx2, a transcription factor necessary for chondrocyte hypertrophy. HDAC4-null mice display premature ossification of developing bones due to ectopic and early onset chondrocyte hypertrophy, mimicking the phenotype that results from constitutive Runx2 expression in chondrocytes. Conversely, overexpression of HDAC4 in proliferating chondrocytes in vivo inhibits chondrocyte hypertrophy and differentiation, mimicking a Runx2 loss-of-function phenotype. These results establish HDAC4 as a central regulator of chondrocyte hypertrophy and skeletogenesis and suggest general roles for class II HDACs in the control of cellular hypertrophy.

Cardiac Failure in Transgenic Mice With Myocardial Expression of Tumor Necrosis Factor-α

BACKGROUND Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has been detected in several human cardiac-related conditions, including congestive heart failure and septic cardiomyopathy. In these conditions, the origin of TNF-alpha secretion is, at least in part, cardiac myocytes. METHODS AND RESULTS To determine the consequences of TNF-alpha production by cardiac myocytes in vivo, we developed transgenic mice in which expression of a murine TNF-alpha coding sequence was driven by the murine alpha-myosin heavy chain promoter. Four transgenic founders developed an identical illness consisting of tachypnea, decreased activity, and hunched posture. In vivo, ECG-gated MRI of symptomatic transgenic mice documented a severe impairment of cardiac function evidenced by biventricular dilatation and depressed ejection fractions. All transgenic mice died prematurely. Pathological examination of affected animals revealed a globular dilated heart, bilateral pleural effusions, myocyte apoptosis, and transmural myocarditis in both the right and left ventricular free walls, septum, and atrial chambers. In all terminally ill animals, there was significant biventricular fibrosis and atrial thrombosis. CONCLUSIONS This is the first report detailing the effects of tissue-specific production of TNF-alpha by cardiac myocytes in vivo. These findings indicate that production of TNF-alpha by cardiac myocytes is sufficient to cause severe cardiac disease and support a causal role for this cytokine in the pathogenesis of human cardiac disease.

Cytochrome c Deficiency Causes Embryonic Lethality and Attenuates Stress-Induced Apoptosis

Cytochrome c released from mitochondria has been proposed to be an essential component of an apoptotic pathway responsive to DNA damage and other forms of cell stress. Murine embryos devoid of cytochrome c die in utero by midgestation, but cell lines established from early cytochrome c null embryos are viable under conditions that compensate for defective oxidative phosphorylation. As compared to cell lines established from wild-type embryos, cells lacking cytochrome c show reduced caspase-3 activation and are resistant to the proapoptotic effects of UV irradiation, serum withdrawal, or staurosporine. In contrast, cells lacking cytochrome c demonstrate increased sensitivity to cell death signals triggered by TNFalpha. These results define the role of cytochrome c in different apoptotic signaling cascades.

Activated glycogen synthase-3β suppresses cardiac hypertrophy in vivo

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3β under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3β diminished hypertrophy in response to chronic β-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3β as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3β activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

Interactions of the Low Density Lipoprotein Receptor Gene Family with Cytosolic Adaptor and Scaffold Proteins Suggest Diverse Biological Functions in Cellular Communication and Signal Transduction

The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.

MEF2 responds to multiple calcium‐regulated signals in the control of skeletal muscle fiber type

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Recently, we proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin‐dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium‐regulated signaling inputs, specifically calmodulin‐dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber‐specific enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up‐regulates a MEF2‐dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained.

Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice

Hypoxia-inducible factor (HIF) transcription factors respond to multiple environmental stressors, including hypoxia and hypoglycemia. We report that mice lacking the HIF family member HIF-2α (encoded by Epas1) have a syndrome of multiple-organ pathology, biochemical abnormalities and altered gene expression patterns. Histological and ultrastructural analyses showed retinopathy, hepatic steatosis, cardiac hypertrophy, skeletal myopathy, hypocellular bone marrow, azoospermia and mitochondrial abnormalities in these mice. Serum and urine metabolite studies showed hypoglycemia, lactic acidosis, altered Krebs cycle function and dysregulated fatty acid oxidation. Biochemical assays showed enhanced generation of reactive oxygen species (ROS), whereas molecular analyses indicated reduced expression of genes encoding the primary antioxidant enzymes (AOEs). Transfection analyses showed that HIF-2α could efficiently transactivate the promoters of the primary AOEs. Prenatal or postnatal treatment of Epas1−/− mice with a superoxide dismutase (SOD) mimetic reversed several aspects of the null phenotype. We propose a rheostat role for HIF-2α that allows for the maintenance of ROS as well as mitochondrial homeostasis.

Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy

Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science, this issue p. 400, p. 403, p. 407 Gene editing via CRISPR-Cas9 restores dystrophin protein and improves muscle function in mouse models of muscular dystrophy. CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus–9 (AAV9) to deliver gene-editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intraperitoneal at postnatal day 1 (P1), intramuscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after injection. Postnatal gene editing also enhanced skeletal muscle function, as measured by grip strength tests 4 weeks after injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth.

Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA

Genome editing corrects a muscle disease Patients with Duchenne muscular dystrophy find their muscles growing progressively weaker. Studies identified dystrophin as the culprit gene, which galvanized research into gene-targeted therapies. Long et al. apply genome editing to “correct” the disease-causing mutation in mice genetically destined to develop the disease. This germline editing strategy kept muscles from degenerating, even in mice harboring only a small percentage of corrected cells. Although not feasible for humans, this proof of concept sets the stage for applying genome editing to specific cell types involved in the disease. Science, this issue p. 1184 A mutation that causes muscular dystrophy in mice can be corrected by genome editing, which prevents the disease from developing. Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD is characterized by progressive muscle weakness and a shortened life span, and there is no effective treatment. We used clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)–mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germ line of mdx mice, a model for DMD, and then monitored muscle structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene. The degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected cells and their contribution to regenerating muscle. With the anticipated technological advances that will facilitate genome editing of postnatal somatic cells, this strategy may one day allow correction of disease-causing mutations in the muscle tissue of patients with DMD.