Nina Raben

Ancient missense mutations in a new member of the RoRet gene family are likely to cause Familial Mediterranean Fever

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation. This report describes the cloning of the gene likely to cause FMF from a 115-kb candidate interval on chromosome 16p. Three different missense mutations were identified in affected individuals, but not in normals. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses.Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation. This report describes the cloning of the gene likely to cause FMF from a 115-kb candidate interval on chromosome 16p. Three different missense mutations were identified in affected individuals, but not in normals. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses.

Targeted Disruption of Pyrin, the FMF Protein, Causes Heightened Sensitivity to Endotoxin and a Defect in Macrophage Apoptosis

Familial Mediterranean fever (FMF) is an inherited disorder characterized by recurrent episodes of fever and inflammation. Most patients with FMF carry missense mutations in the C-terminal half of the pyrin protein. To study the physiologic role of pyrin, we generated mice expressing a truncated pyrin molecule that, similar to FMF patients, retains the full PYRIN domain. Bacterial lipopolysaccharide (LPS) induces accentuated body temperatures and increased lethality in homozygous mutant mice. When stimulated, macrophages from these mice produce increased amounts of activated caspase-1 and, consequently, elevated levels of mature IL-1beta. Full-length pyrin competes in vitro with caspase-1 for binding to ASC, a known caspase-1 activator. Apoptosis is impaired in macrophages from pyrin-truncation mice through an IL-1-independent pathway. These data support a critical role for pyrin in the innate immune response, possibly by acting on ASC, and suggest a biologic basis for the selection of hypomorphic pyrin variants in man.

Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

Autoantibodies to histidyl–tRNA synthetase (HisRS) or to alanyl–, asparaginyl–, glycyl–, isoleucyl–, or threonyl–tRNA synthetase occur in ∼25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl–tRNA synthetases to induce leukocyte migration. HisRS induced CD4+ and CD8+ lymphocytes, interleukin (IL)-2–activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH2-terminal domain, 1–48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl–tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl–tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl–tRNA and lysyl–tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl–tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.

Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease

The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, the genetic deficiency of the glycogen-degrading lysosomal enzyme acid-alpha glucosidase. Accumulation of lysosomal glycogen, presumably transported from the cytoplasm by the autophagic pathway, occurs in multiple tissues, but pathology is most severe in skeletal and cardiac muscle. Skeletal muscle pathology also involves massive autophagic buildup in the core of myofibers. To determine if glycogen reaches the lysosome via autophagy and to ascertain whether autophagic buildup in Pompe disease is a consequence of induction of autophagy and/or reduced turnover due to defective fusion with lysosomes, we generated muscle-specific autophagy-deficient Pompe mice. We have demonstrated that autophagy is not required for glycogen transport to lysosomes in skeletal muscle. We have also found that Pompe disease involves induction of autophagy but manifests as a functional deficiency of autophagy because of impaired autophagosomal-lysosomal fusion. As a result, autophagic substrates, including potentially toxic aggregate-prone ubiquitinated proteins, accumulate in Pompe myofibers and may cause profound muscle damage.

Dysfunction of endocytic and autophagic pathways in a lysosomal storage disease

To understand the mechanisms of skeletal muscle destruction and resistance to enzyme replacement therapy in Pompe disease, a deficiency of lysosomal acid α‐glucosidase (GAA), in which glycogen accumulates in lysosomes primarily in cardiac and skeletal muscles.

Autophagy in lysosomal storage disorders

Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as “autophagy disorders.”

Acid a-Glucosidase Deficiency (Glycogenosis Type II, Pompe Disease)

Glycogenosis type II (GSDII, Pompe disease) is an autosomal recessive lysosomal storage disease caused by a deficiency of acid alpha-glucosidase (acid maltase, GAA). The enzyme degrades alpha -1,4 and alpha -1,6 linkages in glycogen, maltose, and isomaltose. Deficiency of the enzyme results in accumulation of glycogen within lysosomes and in cytoplasm eventually leading to tissue destruction. The discovery of the acid a-glucosidase gene has led to rapid progress in understanding the molecular basis of glycogenosis type II and the biological properties of the GAA protein. The last decade has seen several developments: 1) extensive mutational analysis in patients with different forms of the disease, 2) characterization of the enzyme biosynthesis, processing, and lysosomal targeting, 3) generation of knockout mouse models, 4) development of viral vectors for gene replacement therapy, 5) the production of recombinant human enzyme, and 6) a shift in the enzyme replacement therapy approach from theory to practice. It is anticipated that the enzyme replacement therapy will be widely available for human use in the near future. Several recent reviews (including the most comprehensive one by R. Hirschhorn and A. Reuser [1]), address clinical, biochemical and genetic aspects of the disease, as well as development of new therapies for GSDII [2, 3, 4]. In this article we will review recent findings in the area including rapidly accumulating molecular genetic data (more than 20 mutations need to be added to the list), transcriptional control of gene expression, studies in mouse models, and new approaches to gene therapy. We will also highlight some emerging questions following the introduction of enzyme replacement therapy.

A variety of cytokines and immunologically relevant surface molecules are expressed by normal human skeletal muscle cells under proinflammatory stimuli

Muscle is an attractive target for gene therapy and for immunization with DNA vaccines and is also the target of immunological injury in myositis. It is important therefore to understand the immunologic capabilities of muscle cells themselves. In this study, we show that proinflammatory stimuli induce the expression of other cytokines such as IL‐6, transforming growth factor‐beta (TGF‐β), and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) by muscle cells themselves, as well as the up‐regulation of human leucocyte antigen (HLA) class I, class II and intercellular adhesion molecule‐1 (ICAM‐1). Thus, muscle cells have an inherent ability to express and respond to a variety of cytokines and chemokines. The levels of HLA class I, class II and ICAM‐1 in inflamed muscle may be affected by the secreted products of the stimulation.

The Nutrient-Responsive Transcription Factor TFE3 Promotes Autophagy, Lysosomal Biogenesis, and Clearance of Cellular Debris

Enhancing TFE3 activity to promote expression of lysosome-associated genes may be beneficial in treating lysosomal storage disorders. Regulating Lysosomes and Autophagy When deprived of nutrients, cells inhibit anabolic processes, such as protein production, and promote catabolic processes, such as those mediated by lysosomes and autophagosomes. Disruption in lysosomal function causes lysosomal storage disorders. Martina et al. discovered that TFE3, like TFEB, another member of the MiTF/TFE (microphthalmia-associated transcription factor and transcription factor E) family, was inhibited at the lysosome under nutrient-replete conditions and translocated to the nucleus to stimulate genes involved in lysosome biogenesis and function and autophagy in response to nutrient deprivation. Data from various tissues and cell lines indicated that TFE3 and TFEB may be cell-specific mediators of lysosomal homeostasis. Overexpression of TFE3 stimulated lysosomal exocytosis and release of debris in a cellular model of a lysosomal storage disorder, thereby providing a potential therapeutic target. The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator transcription factor EB (TFEB) revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. We report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag guanosine triphosphatases (GTPases) and exhibited mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1)–dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. After starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders.

Myositis: Immunologic Contributions to Understanding Cause, Pathogenesis, and Therapy

Dr. Paul H. Plotz (National Institutes of Health [NIH], Bethesda, Maryland): Myositis, or idiopathic inflammatory myopathy, is one of the rarest inflammatory illnesses in the family of autoimmune diseases. With an incidence of only about 10 new cases per 1 million persons per year in the United States, a busy rheumatologist, neurologist, or dermatologist (the three consultants most likely to make a diagnosis) is unlikely to meet more than one or two new patients with the disorder per year [1, 2]. The rarity of the condition has held back both scholarly and therapeutic study. The notable reports of large series of cases, especially a large experience in Los Angeles reviewed by Bohan and colleagues [3], have provided a framework for all subsequent studies. For the past decade, our group in the Arthritis and Rheumatism Branch of NIH has tried to understand the connection between disease-specific autoantibodies and the diseases in which they are found. We have met and examined approximately 400 patients with myositis or a disease that mimics myositis, and we have done both laboratory and therapeutic studies on many of them. In this report, we concentrate on the clinical and immunologic observations we and others have made in attempting to understand the cause and pathogenesis of an autoimmune disease by understanding the relation of the autoantibodies to the disease. Clinical Features and the Differential Diagnosis of Myositis The study of myositis must begin with a correct diagnosis. Myositis, or, as it is perhaps more usefully called, idiopathic inflammatory myopathy, is a disease of muscle inflammation. Muscle weakness and sometimes pain, often but not always symmetrical and proximal, are limited to the trunk, neck, and limbs. These symptoms are accompanied by signs of muscle damage: the liberation of muscle enzymes, including creatine kinase, aldolase, and other intracellular enzymes (such as aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase); characteristic changes on electromyography; and the presence of degenerating and regenerating myocytes with inflammatory cells, especially lymphocytes, in and around muscle cells and sometimes also around vessels. We have also observed a characteristic series of patterns on magnetic resonance images of inflamed muscles (Figure 1). Figure 1. Magnetic resonance image in a patient with myositis. The differential diagnosis of the weak patient includes a broad spectrum of diseases and toxicities. The borders separating myositis from its close relatives among the rheumatologic diseases are not sharply defined. For example, systemic lupus erythematosus shares both clinical and serologic manifestations with myositis. The late-onset, indolent myositisinclusion body myositisis difficult to distinguish from a bewildering variety of rare cases that in the literature are usually called dystrophies [4]. Some cases treated as myositis are examples of rare or as-yet unrecognized metabolic disorders. The term metabolic myopathy is used for diseases that feature a recognizable genetic deficiency of an enzyme that is vital to the energy production of a muscle cell; the term dystrophy is used for diseases in which no such deficiency is present but in which muscle-cell degeneration has occurred that is presumably caused by genetic defects of one or more structural proteins. This latter assumption is based on the discovery of dystrophin mutations in Duchenne dystrophy. However, because inflammation may be seen in some cases of dystrophy, and because many cases of both metabolic muscle diseases and dystrophies represent either new mutations or the first manifestations of a recessive disease in a family, the real boundaries of all of these illnesses remain unknown. Therefore, it is important to consider them in every case of suspected myositis. For each new case, a review of the major classes of similar diseases catches many of the masqueraders (Table 1). Clinicians should remember that aminotransferase abnormalities do not always indicate liver disease; that fatigue and weakness may be present in the muscles as well as in the head; that drugs and toxins can cause muscle disease; and that a family history of a similar illness, neurologic signs, asymmetry, cranial nerve involvement, and an onset of symptoms related to exercise or eating militate against a diagnosis of myositis. Table 1. Differential Diagnosis of Myositis We studied myositis because some patients with myositis have autoantibodies directed against the ubiquitous intracellular protein, histidyl-transfer RNA (tRNA) synthetase, the enzyme that joins histidine to its cognate tRNA [5]. These antibodies are not seen in any other disease. This strikingly specific association was made even more tantalizing by the subsequent discovery that a small group of patients developed autoantibodies directed against the synthetases responsible for ligating alanine, glycine, isoleucine, or threonine to their respective cognate tRNAs. Furthermore, other myositis-specific autoantibodies have been described in the past decade [6]. In a startling findingthe result of observations by several groups, including Drs. Lori Love, Fred Miller, and colleagues at NIHthe myositis-specific autoantibodies delineated groups of patients who have a remarkably similar clinical illness and share many other features. The common syndromes are summarized in Table 2. Patients with antisynthetase autoantibodies can have either polymyositis or dermatomyositis; those with anti-signal recognition particle (anti-SRP) autoantibodies always have polymyositis; and those with anti-Mi-2 autoantibodies always have dermatomyositis. In the traditional categories, patients with cancer-associated myositis or inclusion body myositis only rarely have myositis-specific autoantibodies, although there are a few exceptions. Table 2. Syndromes Associated with Myositis-Specific Autoantibodies Childhood Myositis: Newly Recognized Diversity Dr. Lisa G. Rider (Food and Drug Administration, Bethesda, Maryland): The idiopathic inflammatory myopathies of childhood are a diverse group of acquired diseases of unknown cause that are all characterized by chronic inflammation in skeletal muscle. Although juvenile dermatomyositis is the most common of these disorders in children, each clinicopathologic entity described in adults has also been reported in children [7], including polymyositis [8], myositis associated with another connective tissue disease (overlap myositis) [9], cancer-associated myositis [10], focal myositis [11], orbital myositis [12], inclusion body myositis [13], eosinophilic myositis [14], and granulomatous myositis [7]. Although the frequency of these clinical conditions in children and adults probably differs, the clinical features, histopathologic findings, and clinical courses appear to be similar in both populations. The most common idiopathic inflammatory myopathy in children is juvenile dermatomyositis, which in children has a peak incidence between ages 5 and 14 years. Juvenile dermatomyositis is similar to adult dermatomyositis and is characterized by the classic Gottron and heliotrope rashes, proximal and symmetrical muscle weakness, and perivascular muscle inflammation. Juvenile and adult dermatomyositis also share immunopathogenetic features, including damage to the endothelial cells of the primary muscle capillaries, perivascular infiltration of B lymphocytes that is associated with deposition of immunoglobulins and the terminal C5b-9 membrane attack complex on the intramuscular microvasculature, and infiltration of T lymphocytes [15]. Juvenile dermatomyositis may differ from the adult disease in the following ways: 1) The clinical presentation in children is more frequently insidious and may be dominated by constitutional symptoms of fatigue, malaise, fever, anorexia, and weight loss [16]; 2) children more often have a multisystem vasculitis that may involve the skin, gastrointestinal mucosa, muscle, heart, and retina [16]; 3) calcinosis develops more frequently in children, particularly in children with longstanding, untreated disease, those with generalized cutaneous vasculitis, or those with a chronic and severe disease course [17]; and 4) once remission is achieved, children appear to return to normal strength and function more frequently than adults with dermatomyositis [18]. The association of malignancy with the development of myositis has been well described in adults but only rarely reported in children. No cases of malignancy have been reported in retrospective studies of large cohorts of children with myositis [19]. However, tumors (most commonly leukemia and lymphoma) have been described in 10 patients with juvenile dermatomyositis and 3 children with polymyositis [7, 10]. In many of these children, the myositis showed atypical features, such as the absence of Gottron papules or the presence of unusual rashes, unilateral muscle weakness, or distal rather than proximal muscle weakness. Some children developed adenopathy, splenomegaly, or an abdominal mass that was seen on examination; the latter prompted a diagnosis of cancer. The myositis followed a paraneoplastic course in that muscle strength, skin rash, and muscle enzyme levels improved with treatment of the underlying cancer. The myositis-specific autoantibodies also develop in children. To date, autoantibodies to threonyl-tRNA synthetase [20], alanyl-tRNA synthetase [21], and histidyl-tRNA synthetase (HRS) (22; unpublished observations) have been found in six children. Anti-signal recognition particle autoantibodies [20] have been detected in one child with polymyositis, and anti-Mi-2 autoantibodies have been found in 10 children with juvenile dermatomyositis [20, 23, 24]. We have found that the clinical features of children with myositis-specific autoantibodies for whom complete clinical data are available are similar to those of adults with the same autoantibodies [20]. Some a