James A. Richardson

Orexins and Orexin Receptors: A Family of Hypothalamic Neuropeptides and G Protein-Coupled Receptors that Regulate Feeding Behavior

The hypothalamus plays a central role in the integrated control of feeding and energy homeostasis. We have identified two novel neuropeptides, both derived from the same precursor by proteolytic processing, that bind and activate two closely related (previously) orphan G protein-coupled receptors. These peptides, termed orexin-A and -B, have no significant structural similarities to known families of regulatory peptides. prepro-orexin mRNA and immunoreactive orexin-A are localized in neurons within and around the lateral and posterior hypothalamus in the adult rat brain. When administered centrally to rats, these peptides stimulate food consumption. prepro-orexin mRNA level is up-regulated upon fasting, suggesting a physiological role for the peptides as mediators in the central feedback mechanism that regulates feeding behavior.

Narcolepsy in orexin Knockout Mice: Molecular Genetics of Sleep Regulation

Neurons containing the neuropeptide orexin (hypocretin) are located exclusively in the lateral hypothalamus and send axons to numerous regions throughout the central nervous system, including the major nuclei implicated in sleep regulation. Here, we report that, by behavioral and electroencephalographic criteria, orexin knockout mice exhibit a phenotype strikingly similar to human narcolepsy patients, as well as canarc-1 mutant dogs, the only known monogenic model of narcolepsy. Moreover, modafinil, an anti-narcoleptic drug with ill-defined mechanisms of action, activates orexin-containing neurons. We propose that orexin regulates sleep/wakefulness states, and that orexin knockout mice are a model of human narcolepsy, a disorder characterized primarily by rapid eye movement (REM) sleep dysregulation.

A Calcineurin-Dependent Transcriptional Pathway for Cardiac Hypertrophy

In response to numerous pathologic stimuli, the myocardium undergoes a hypertrophic response characterized by increased myocardial cell size and activation of fetal cardiac genes. We show that cardiac hypertrophy is induced by the calcium-dependent phosphatase calcineurin, which dephosphorylates the transcription factor NF-AT3, enabling it to translocate to the nucleus. NF-AT3 interacts with the cardiac zinc finger transcription factor GATA4, resulting in synergistic activation of cardiac transcription. Transgenic mice that express activated forms of calcineurin or NF-AT3 in the heart develop cardiac hypertrophy and heart failure that mimic human heart disease. Pharmacologic inhibition of calcineurin activity blocks hypertrophy in vivo and in vitro. These results define a novel hypertrophic signaling pathway and suggest pharmacologic approaches to prevent cardiac hypertrophy and heart failure.

A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure

Diverse forms of injury and stress evoke a hypertrophic growth response in adult cardiac myocytes, which is characterized by an increase in cell size, enhanced protein synthesis, assembly of sarcomeres, and reactivation of fetal genes, often culminating in heart failure and sudden death. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs that were regulated during cardiac hypertrophy and heart failure. We describe >12 miRNAs that are up- or down-regulated in cardiac tissue from mice in response to transverse aortic constriction or expression of activated calcineurin, stimuli that induce pathological cardiac remodeling. Many of these miRNAs were similarly regulated in failing human hearts. Forced overexpression of stress-inducible miRNAs was sufficient to induce hypertrophy in cultured cardiomyocytes. Similarly, cardiac overexpression of miR-195, which was up-regulated during cardiac hypertrophy, resulted in pathological cardiac growth and heart failure in transgenic mice. These findings reveal an important role for specific miRNAs in the control of hypertrophic growth and chamber remodeling of the heart in response to pathological signaling and point to miRNAs as potential therapeutic targets in heart disease.

The Endothelial-Specific MicroRNA miR-126 Governs Vascular Integrity and Angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.

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.

Transient Regenerative Potential of the Neonatal Mouse Heart

The heart in a newborn mouse can rebuild itself after injury, but this regenerative capacity is lost within a few days. Certain fish and amphibians retain a robust capacity for cardiac regeneration throughout life, but the same is not true of the adult mammalian heart. Whether the capacity for cardiac regeneration is absent in mammals or whether it exists and is switched off early after birth has been unclear. We found that the hearts of 1-day-old neonatal mice can regenerate after partial surgical resection, but this capacity is lost by 7 days of age. This regenerative response in 1-day-old mice was characterized by cardiomyocyte proliferation with minimal hypertrophy or fibrosis, thereby distinguishing it from repair processes. Genetic fate mapping indicated that the majority of cardiomyocytes within the regenerated tissue originated from preexisting cardiomyocytes. Echocardiography performed 2 months after surgery revealed that the regenerated ventricular apex had normal systolic function. Thus, for a brief period after birth, the mammalian heart appears to have the capacity to regenerate.

Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human β2m : an animal model of HLA-B27-associated human disorders

Humans who have inherited the human class I major histocompatibility allele HLA-B27 have a markedly increased risk of developing the multi-organ system diseases termed spondyloarthropathies. To investigate the role of B27 in these disorders, we introduced the B27 and human beta 2-microglobulin genes into rats, a species known to be quite susceptible to experimentally induced inflammatory disease. Rats from one transgenic line spontaneously developed inflammatory disease involving the gastrointestinal tract, peripheral and vertebral joints, male genital tract, skin, nails, and heart. This pattern of organ system involvement showed a striking resemblance to the B27-associated human disorders. These results establish that B27 plays a central role in the pathogenesis of the multi-organ system processes of the spondyloarthropathies. Elucidation of the role of B27 should be facilitated by this transgenic model.

Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice

Endothelins act on two subtypes of G protein-coupled receptors, termed endothelin-A and endothelin-B receptors. We report a targeted disruption of the mouse endothelin-B receptor (EDNRB) gene that results in aganglionic megacolon associated with coat color spotting, resembling a hereditary syndrome of mice, humans, and other mammalian species. Piebald-lethal (sl) mice exhibit a recessive phenotype identical to that of the EDNRB knockout mice. In crossbreeding studies, the two mutations show no complementation. Southern blotting revealed a deletion encompassing the entire EDNRB gene in the sl chromosome. A milder allele, piebald (s), which produces coat color spotting only, expresses low levels of structurally intact EDNRB mRNA and protein. These findings indicate an essential role for EDNRB in the development of two neural crest-derived cell lineages, myenteric ganglion neurons and epidermal melanocytes. We postulate that defects in the human EDNRB gene cause a hereditary form of Hirschsprungs disease that has recently been mapped to human chromosome 13, in which EDNRB is located.

Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.

Defects in the gene encoding the endothelin-B receptor produce aganglionic megacolon and pigmentary disorders in mice and humans. We report that a targeted disruption of the mouse endothelin-3 ligand (EDN3) gene produces a similar recessive phenotype of megacolon and coat color spotting. A natural recessive mutation that results in the same developmental defects in mice, lethal spotting (ls), failed to complement the targeted EDN3 allele. The ls mice carry a point mutation of the EDN3 gene, which replaces the Arg residue at the C-terminus of the inactive intermediate big EDN3 with a Trp residue. This mutation prevents the proteolytic activation of big EDN3 by ECE-1. These findings indicate that interaction of EDN3 with the endothelin-B receptor is essential in the development of neural crest-derived cell lineages. We postulate that defects in the human EDN3 gene may cause Hirschsprungs disease.