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Hannu Laaksovirta konsortion jäsenenä. The Genomics England Research Consortium, The International ALS/FTD Genomics Consortium (iAFGC), The International FTD Genetics Consortium (IFGC), The International LBD Genomics Consortium (iLBDGC), The NYGC ALS Consortium, The PROSPECT Consortium,17 James B. Rowe,17 Luisa Benussi,18 Giuliano Binetti,18,19 Roberta Ghidoni,18 Edwin Jabbari,20,21 Coralie Viollet,22 Jonathan D. Glass,23 Andrew B. Singleton,24 Vincenzo Silani,25,26 Owen A. Ross,27 Mina Ryten,8,28,29 Ali Torkamani,30 Toshiko Tanaka,31 Luigi Ferrucci,31 Susan M. Resnick,32 We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered. We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.

Publisher Summary The current fusion machine hypothesis envisions a core complex formed between plasma membrane syntaxin and SNAP-25 and the synaptic vesicle protein synaptobrevinNAMP (S), with vesicular synaptotagmin identified as a low-affinity calcium sensor that interacts with regulatory syntaxin 1. The site of GTP action in exocytosis has been thought to be closely associated with the site of calcium action at some common site. The affinity of this site for Ca 2+ has been shown to be in the 50- to 200- μ M range, as estimated from electrophysiological studies with caged calcium in chromaffin cells, neurons, and digitonin-permeabilized chromaffin cells. Therefore, a good experimental goal would seem to be to search for a embrane fusion protein that is activated by both calcium and GTP. Chromaffin and many other cell types contain a membrane fusion protein, synexin (annexin VII), associated with secretory vesicles and plasma membranes, which have an appropriate intrinsic K d for Ca 2+ of approximately 200 μ M. In addition to binding of GTP and Ca 2+ , a further identifying feature of the hypothetical “fusion scaffold” protein has been proposed to be self-association. Indeed, it has been shown by light scattering and electron microscopy studies that the Ca 2+ -activated form of synexin has a polymeric structure. Consistently, the protein concentration dependence for many synexindependent reactions has consistently been sigmoidal, with Hill coefficients (n H ). It is true that synexin alone is able to mediate efficiently both membrane contact and fusion reactions in vitro . However, the exocytosis process is likely to be much more complicated.

Publisher Summary Membrane fusion processes in cells underlie the structural integrity of the cytoplasm and are crucial to directing synthesis and export of enzymes, hormones, and transmitters. Export is accomplished by a process of exocytosis in which the membranes of secretory vesicles fuse with the plasma membrane. The fusion mechanism may involve a class of calcium-binding proteins termed “annexins.” This chapter discusses the annexin hypothesis for exocytosis. It focuses on function and molecular biology synexin (annexin VII) in secretory cells. Results from several laboratories have also shown that annexins I, II, and VII aggregate chromaffin granules and liposomes in vitro , and that annexin VII drives fusion of granule ghosts and liposomes. These data lend support to the hypothesis that these annexins may be secretory factors in vivo . The above mutagenesis experiments revealed the importance in annexin I of repeat one for aggregation activity and of repeats 2, 3, and 4 for calcium-binding sites. As all annexins bind to phospholipids in a calcium-dependent manner, membranes continue to be a focus for understanding annexin activity. Annexin II was detected in postpartum mammary epithelial cells, when calcium-dependent casein secretion starts and immunogold labeling of annexin VII in chromaffin cells was shown to decrease following treatment with a secretagogue. In addition, annexin II has been reported to insert into artificial membranes in the presence of calcium and to produce voltage-dependent channels.

Publisher Summary Synexin is a cytosolic protein, which in the presence of increased calcium can undergo a conformational change that renders it highly hydrophobic. This chapter discusses some of the assumptions to model the fusion process in vitro, the ways in which synexin promotes different aspects of the model, and some of the physical and chemical evidence underlying the present understanding of synexin action during membrane fusion. The ability of synexin to fuse target membranes lies in the fact that once activated by calcium, the synexin monomers form oligomers, which are capable of interacting with two membranes simultaneously. While the hydrophobic bridge hypothesis has been developed to explain the process of fusion driven by synexin, it need not be limited in principle to just this fusion event. It may be anticipated that any fusion event in which a hydrophobic environment is introduced to the potential fusion site, may proceed by the hydrophobic bridge mechanism and is exemplified by studies on calcium-dependent, synexin driven membrane contact and fusion of chromaffin granules, chromaffin granule ghosts, and phospholipid bilayer liposomes.

Publisher Summary This chapter describes a new protein called synexin that induces Ca 2+ -dependent formation of pentalaminar complexes among secretory granule membranes. Synexin has proved distinct from other more conventional candidates for Ca 2+ -effect mediators, such as calmodulin, actomyosin, and tubulin. The chapter also describes the process of membrane breakage or fission that finally results in the release of secretory vesicle contents. Studies comparing the granule lysis reaction with predicted behavior of secreting chromaffin and other cells are briefly reviewed in the chapter. The biochemical data supporting the exocytosis hypothesis are that the storage organelles from different cells can be isolated and therefore shown to be discrete objects and when secretion occurs, the entire organelle contents ranging from small molecules to large proteins can be found in the extracellular medium. Calcium plays a critical role in regulating exocytosis in a number of systems.

Publisher Summary This chapter describes purification of synexin and methods for analyzing synexin activity. To measure synexin activity, a recording spectrophotometer such as the Gilson 250 can be used, with which four samples can be measured at one time. In addition, a four-cuvet holder is needed, as are the cuvets. The cuvets can either be quartz or acrylic plastic, and of a size suitable for holding 1 ml total volume. The recorder should be set with a full scale of OD 0.2 at 540 nm. The instrument can be temperature statted with a circulating water pump, but routine assays are usually performed at room temperature. The chapter also describes techniques for purifying and assaying endonexin II and lipocortin I.

Adrenal medullary tissue contains a soluble protein of M.W. 47,000 which specifically binds Ca2+ and, in the presence of Ca2+, induces the fusion of the outer leaflets of chromaffin granule membranes. We have named this protein synexin, from the Greek word “synexis” which means “a meeting”. We suggest synexin may be an intracellular receptor for Ca2+ in the process of exocytosis, acting to promote fusion between secretory vesicles and the plasma membrane, or, in the case of compound exocytosis, between vesicles.

Publisher Summary This chapter discusses hormone secretion by exocytosis with an emphasis on information from the chromaffin cell system. Exocytosis is the general mechanism by which many hormones, transmitters, enzymes, and other special proteins and peptides are secreted from cells. Exocytosis means that the chemical species to be secreted is stored in a secretory vesicle, which, upon the appropriate stimulus, fuses with the cell membrane and deposits the vesicle contents outside the cell. The “handmaiden” of this process is usually calcium. In many cases, the ultimate signal for exocytosis seems to be an increase in the intracellular free calcium ion concentration. For many reasons, the chromaffin cell is the very best vantage point from which to examine the hormone-secretion process. From a biochemical viewpoint, the chromaffin cell has no peer, subcellular fractionation is simple, and the cells are easily cultured. Chromaffin cells in the adrenal medulla are joined and electrically coupled into cords of cells surrounded in the intact gland by an abundant fenestrated capillary bed.

A chemiosmotic mechanism is the basis of ATP, Cl–-induced lysis of chromaffin granules, and we have now shown that the granule model successfully predicts the secretory properties of human platelets, bovine parathyroid cells, and bovine chromaffin cells. Like the granule lysis system, secretion from these cells requires specific anions in the medium, is inhibited by anion transport blocking drugs and proton ionophores, and is suppressed by elevated osmotic strength. We suggest that secretory granules fused to plasma membranes in secreting cells may experience net solute uptake and subsequently undergo local, outwardly directed osmotic lysis, or exocytosis.

Chromaffin granules (CG) were found to synthesize cyclic -AMP (cAMP) from exogenous ATP only under conditions which caused release of granule contents. Basal as well as stimulated conditions for release from CG resulted in a rate of c-AMP synthesis directly proportional to the release rate. Release could be evoked by addition of c-AMP or dibutyryl - cAMP, although at relatively high concentrations (0.1 mM). Phosphodiesterase inhibitors were inactive in promoting release from the CG or in potentiating ATP-stimulated release. Permeant anions, required for release of CG contents, were also found to potentiate adenylate cyclase (AC) activity and cAMP formation in vesicles. These data suggest that the release event itself results in activation of the granule membrane cyclase.

Publisher Summary This chapter discusses the chromaffin granule and possible mechanisms of exocytosis. Chromaffin granules and chromaffin cells have been subjects of biological and chemical investigation for over a quarter of a century, and fundamental, seminal information has been obtained about the general process of secretion from cells. Chromaffin granules play several roles in adrenal medullary cells, some of which may be amenable to biochemical analysis. This view presumes that chromaffin granules are assembled in the Golgi apparatus, by analogy to studies on other cells such as those of the pancreas, though in fact studies on the ultrastructural biochemistry of chromaffin-granule assembly are in a relatively primitive stateThis chapter describes current information on the biochemical basis of exocytosis as viewed from the vantage point of the chromaffin granule. It presents a general view of exocytosis and included granule assembly, the mechanism of calcium action, the release event, and recovery of the granule membrane after secretion.

Adrenal chromaffin granules in an isoosmotic medium can be induced to release their contents, including epinephrine, in an all or none fashion. Such release is based on osmotic lysis of the granules and is regulated by Mg-ATP and permeant anions. In this paper, we examine the mechanism of this release as it relates to membrane sites involving Mg-ATP and anions. We conclude that osmotic lysis of chromaffin granules in an isoosmotic medium is the result of electrogenic pumping of protons into the granule via a membrane Mg-ATPase and subsequent anion uptake via a selective transport site. Studies involving ATP-independent osmotic release mechanisms and anion transport blocking drugs further suggest that these two granule membrane sites may be physically as well as functionally coupled.

Publisher Summary This chapter assesses the chemiosmotic hypothesis of secretion in the light of the properties exhibited by isolated secretory granules and by intact and permeabilized secretory cells. The molecular events associated with membrane fusion are far from understood, as are most aspects of the exocytotic process. One hypothesis proposed to explain the membrane fusion event in exocytosis is the chemiosmotic hypothesis of secretion. The original chemiosmotic hypothesis explains the coupling of mitochondrial electron transport to ATP synthesis. The chemiosmotic hypothesis of secretion was formulated following the discovery of the chemiosmotic properties of isolated chromaffin granules. However, subsequent results obtained with intact and permeabilized cells revealed large differences between the predicted and observed secretory properties of these cells. Although the chemiosmotic hypothesis as originally proposed is no longer valid, several alternative hypotheses have been advanced according to which the osmotic swelling of secretory vesicles constitutes the force that drives the fission step in exocytosis.