Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice

Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein and an important constituent of the lysosomal membrane. Here we show that LAMP-2 deficiency in mice increases mortality between 20 and 40 days of age. The surviving mice are fertile and have an almost normal life span. Ultrastructurally, there is extensive accumulation of autophagic vacuoles in many tissues including liver, pancreas, spleen, kidney and skeletal and heart muscle. In hepatocytes, the autophagic degradation of long-lived proteins is severely impaired. Cardiac myocytes are ultrastructurally abnormal and heart contractility is severely reduced. These findings indicate that LAMP-2 is critical for autophagy. This theory is further substantiated by the finding that human LAMP-2 deficiency causing Danon's disease is associated with the accumulation of autophagic material in striated myocytes.     

The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel

TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe(3+)-bound transferrin receptors, or after lysosomal degradation of ferritin-iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe(2+) transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe(2+) transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe(2+) permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe(2+) at varying degrees, which correlate well with the disease severity. A comparison of TRPML1(-/- )ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe(2+) levels, an increase in intralysosomal Fe(2+) levels and an accumulation of lipofuscin-like molecules in TRPML1(-/-) cells. We propose that TRPML1 mediates a mechanism by which Fe(2+) is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.     

Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr?231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr?231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.     

Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J

Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells. Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a phospholipid present in small quantities that regulates membrane trafficking in the endosome-lysosome axis in yeast. Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the 'pale tremor' mouse. Positional cloning identified insertion of ETn2beta (early transposon 2beta) into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC (suppressor of actin) domain PtdIns(3,5)P2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns(3,5)P2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosome-lysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated patients with hereditary motor and sensory neuropathy. This novel form of autosomal recessive Charcot-Marie-Tooth disorder is designated CMT4J.     

The lipid phosphatase SHIP2 controls insulin sensitivity

Insulin is the primary hormone involved in glucose homeostasis, and impairment of insulin action and/or secretion has a critical role in the pathogenesis of diabetes mellitus. Type-II SH2-domain-containing inositol 5-phosphatase, or 'SHIP2', is a member of the inositol polyphosphate 5-phosphatase family. In vitro studies have shown that SHIP2, in response to stimulation by numerous growth factors and insulin, is closely linked to signalling events mediated by both phosphoinositide-3-OH kinase and Ras/mitogen-activated protein kinase. Here we report the generation of mice lacking the SHIP2 gene. Loss of SHIP2 leads to increased sensitivity to insulin, which is characterized by severe neonatal hypoglycaemia, deregulated expression of the genes involved in gluconeogenesis, and perinatal death. Adult mice that are heterozygous for the SHIP2 mutation have increased glucose tolerance and insulin sensitivity associated with an increased recruitment of the GLUT4 glucose transporter and increased glycogen synthesis in skeletal muscles. Our results show that SHIP2 is a potent negative regulator of insulin signalling and insulin sensitivity in vivo.     

A mutation in the tRNA(Leu)(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies

Mitochondrial encephalomyopathies are usually divided into three distinct clinical subgroups: (1) mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS); (2) myoclonus epilepsy associated with ragged-red fibres (MERRF); and (3) chronic progressive external ophthalmoplegia (CPEO) including Kearns-Sayre syndrome. Large deletions of human mitochondrial DNA and a transition mutation at the mitochondrial transfer RNALys gene give rise to CPEO including Kearns-Sayre syndrome and MERRF, respectively. Here we report an A-to-G transition mutation at nucleotide pair 3,243 in the dihydrouridine loop of mitochondrial tRNA(Leu)(UUR) that is specific to patients with MELAS. Because this mutation creates an ApaI restriction site, we could perform a simple molecular diagnostic test for the disease. The mutation was present in 26 out of 31 independent MELAS patients and 1 out of 29 CPEO patients, but absent in the 5 MERRF and 50 controls tested. Southern blot analysis confirmed that the mutant DNA always coexists with the wild-type DNA (heteroplasmy).     

Direct access to serum macromolecules by intraerythrocytic malaria parasites.

Trafficking pathways in malaria-infected erythrocytes are complex because the internal parasite is separated from the serum by the erythrocyte and parasitophorous vacuolar membranes. Intraerythrocytic Plasmodium falciparum parasites can endocytose dextrans, protein A and an IgG2a antibody. Here we show that these macromolecules do not cross the erythrocyte or parasitophorous vacuolar membranes, but rather gain direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Evidence for this structure includes visualization of membranes that are continuous between the parasitophorous vacuolar and erythrocyte membranes, and surface labelling of the parasite with fluorescent macromolecules under conditions that block endocytosis. The parasite can internalize by fluid-phase endocytosis macromolecules from the aqueous compartment surrounding it. Thus, surface antigens on trophozoites and schizonts should be considered as targets for antibody-directed parasiticidal agents.     

Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR.

Cystic fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability, increased mucous sulphation, increased Na+ absorption and defective acidification of lysosomal vesicles. The CF gene encodes a protein (the cystic fibrosis transmembrane conductance regulator, CFTR) that can function as a low-conductance Cl- channel with a linear current-voltage relationship whose regulation is defective in CF patients. Larger conductance, outwardly rectifying Cl- channels are also defective in CF and fail to activate when exposed either to cyclic AMP-dependent protein kinase A or to protein kinase C. The role of the outwardly rectifying Cl- channel in CF has been questioned. We report here that expression of recombinant CF genes using adeno-associated virus vectors in CF bronchial epithelial cells corrects defective Cl- secretion, that it induces the appearance of small, linear conductance Cl- channels, and restores protein kinase A activation of outwardly rectifying Cl- channels. These results re-establish an involvement of outwardly rectifying Cl- channels in CF and suggest that CFTR regulates more than one conductance pathway in airway tissues.     

5例原发性痛风患者HPRT基因的克隆和序列分析

原发性痛风是先天性嘌呤代谢缺陷症，与嘌呤代谢相关两次黄嘌呤—鸟嘌呤磷酸核糖转移酶(HPRT，EC2．4．2．8)、磷酸核糖焦磷酸合成酶(PRS)等缺陷有关。克隆了正常中国人和4个中国原发性痛风家族5位患的HPRT基因，序列分析表明，正常中国人及Case5患的HPRT编码区序列与报道的序列(GeneBank登录号M26434)完全一致，而在3个中国痛风家族4位患的HPRT基因编码区中共发现11个新突变，包括3个同义突变、1个元义突变和7个错义突变。这是关于中国大陆原发性痛风患HPRT基因突变的首次报道。     

Direct enzyme transfer from lymphocytes is specific

Lymphocytes are known to interact directly with other cells in vivo and in vitro, and have recently been shown to transfer the lysosomal enzyme, beta-glucuronidase, to fibroblasts from patients with an inherited deficiency of the enzyme. This process requires cell-cell contact, is unaffected by inhibitors of 'classical' receptor-mediated endocytosis and is abolished by inhibitors of protein synthesis. Although it is not yet known to what extent the transfer of enzymes by direct cellular interaction is a general phenomenon, a similar mechanism could possibly be involved in the transfer of other lysosomal enzymes in vivo and in the exchange of protein in vitro. We show here that the direct transfer of enzymes from lymphocytes to fibroblasts is restricted to only certain lysosomal enzymes.     

ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function

Mutations in ClC-7, a late endosomal/lysosomal member of the CLC family of chloride channels and transporters, cause osteopetrosis and lysosomal storage disease in humans and mice. Severe osteopetrosis is also observed with mutations in the OSTM1 gene, which encodes a membrane protein of unknown function. Here we show that both ClC-7 and Ostm1 proteins co-localize in late endosomes and lysosomes of various tissues, as well as in the ruffled border of bone-resorbing osteoclasts. Co-immunoprecipitations show that ClC-7 and Ostm1 form a molecular complex and suggest that Ostm1 is a beta-subunit of ClC-7. ClC-7 is required for Ostm1 to reach lysosomes, where the highly glycosylated Ostm1 luminal domain is cleaved. Protein but not RNA levels of ClC-7 are greatly reduced in grey-lethal mice, which lack Ostm1, suggesting that the ClC-7-Ostm1 interaction is important for protein stability. As ClC-7 protein levels in Ostm1-deficient tissues and cells, including osteoclasts, are decreased below 10% of normal levels, Ostm1 mutations probably cause osteopetrosis by impairing the acidification of the osteoclast resorption lacuna, which depends on ClC-7 (ref. 3). The finding that grey-lethal mice, just like ClC-7-deficient mice, show lysosomal storage and neurodegeneration in addition to osteopetrosis implies a more general importance for ClC-7-Ostm1 complexes.     

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.     

T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis.

Multiple sclerosis is thought to be an autoimmune disease of the central nervous system mediated by T cells specific for a myelin antigen. Myelin basic protein has been studied as a potential autoantigen in the disease because of its role as an encephalitogen in experimental autoimmune encephalomyelitis and post-viral encephalomyelitis and because of the presence in the blood of multiple sclerosis patients of in vivo-activated T cells reactive to myelin basic protein. Immune involvement in multiple sclerosis has been further suggested by the association with the major histocompatibility complex class II phenotype DR2, DQw1. To define the T-cell specificity toward myelin basic protein, 15,824 short-term T-cell lines were established from multiple sclerosis subjects, subjects with other neurological diseases, and normal controls. Here we report a higher frequency of T-cell lines reactive with a DR2-associated region of myelin basic protein between residues 84-102 in patients with multiple sclerosis compared with controls. A second region, identified between residues 143-168, was recognized equally in multiple sclerosis patients and controls and was associated with the DRw11 phenotype. These DR2 and DRw11 associations were also observed among T-cell lines generated from family members of a multiple sclerosis patient. The immunodominant 84-102 peptide from myelin basic protein was both DR2- and DQw1-restricted among different T-cell lines. These results raise the possibility that this immunodominant region may be encephalitogenic in some DR2+ individuals.     

Clathrin self-assembly is mediated by a tandemly repeated superhelix.

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.     

Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins

Eukaryotic members of the CLC gene family function as plasma membrane chloride channels, or may provide neutralizing anion currents for V-type H(+)-ATPases that acidify compartments of the endosomal/lysosomal pathway. Loss-of-function mutations in the endosomal protein ClC-5 impair renal endocytosis and lead to kidney stones, whereas loss of function of the endosomal/lysosomal protein ClC-7 entails osteopetrosis and lysosomal storage disease. Vesicular CLCs have been thought to be Cl- channels, in particular because ClC-4 and ClC-5 mediate plasma membrane Cl- currents upon heterologous expression. Here we show that these two mainly endosomal CLC proteins instead function as electrogenic Cl-/H+ exchangers (also called antiporters), resembling the transport activity of the bacterial protein ClC-e1, the crystal structure of which has already been determined. Neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion flux to proton counter-transport. ClC-4 and ClC-5 may still compensate the charge accumulation by endosomal proton pumps, but are expected to couple directly vesicular pH gradients to Cl- gradients.     

Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies

In vitro studies of muscle mitochondrial metabolism in patients with mitochondrial myopathy have identified a variety of functional defects of the mitochondrial respiratory chain, predominantly affecting complex I (NADH-CoQ reductase) or complex III (ubiquinol-cytochrome c reductase) in adult cases. These two enzymes consist of approximately 36 subunits, eight of which are encoded by mitochondrial DNA (mtDNA). The increased incidence of maternal, as opposed to paternal, transmission in familial mitochondrial myopathy suggests that these disorders may be caused by mutations of mtDNA. Multiple restriction endonuclease analysis of leukocyte mtDNA from patients with the disease, and their relatives, showed no differences in cleavage patterns between affected and unaffected individuals in any single maternal line. When muscle mtDNA was studied, nine of 25 patients were found to have two populations of muscle mtDNA, one of which had deletions of up to 7 kilobases in length. These observations demonstrate that mtDNA heteroplasmy can occur in man and that human disease may be associated with defects of the mitochondrial genome.     

GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides

Alzheimer's disease is associated with increased production and aggregation of amyloid-beta (Abeta) peptides. Abeta peptides are derived from the amyloid precursor protein (APP) by sequential proteolysis, catalysed by the aspartyl protease BACE, followed by presenilin-dependent gamma-secretase cleavage. Presenilin interacts with nicastrin, APH-1 and PEN-2 (ref. 6), all of which are required for gamma-secretase function. Presenilins also interact with alpha-catenin, beta-catenin and glycogen synthase kinase-3beta (GSK-3beta), but a functional role for these proteins in gamma-secretase activity has not been established. Here we show that therapeutic concentrations of lithium, a GSK-3 inhibitor, block the production of Abeta peptides by interfering with APP cleavage at the gamma-secretase step, but do not inhibit Notch processing. Importantly, lithium also blocks the accumulation of Abeta peptides in the brains of mice that overproduce APP. The target of lithium in this setting is GSK-3alpha, which is required for maximal processing of APP. Since GSK-3 also phosphorylates tau protein, the principal component of neurofibrillary tangles, inhibition of GSK-3alpha offers a new approach to reduce the formation of both amyloid plaques and neurofibrillary tangles, two pathological hallmarks of Alzheimer's disease.     

Recombinant human lipocortin 1 inhibits thromboxane release from guinea-pig isolated perfused lung

The guinea-pig perfused isolated lung, used in conjunction with the cascade superfusion system to measure the release of thromboxane A2(TXA2), is a simple and convenient model for assessing the inhibition by glucocorticoids of eicosanoid formation. Dexamethasone inhibits the release of TXA2 from the lung when it is stimulated by agents such as RCS-RF2 of leukotrienes, but not when bradykinin or arachidonic acid are used. Using this model we have shown that the glucocorticoids suppress eicosanoid generation by cells through the induction of a family of phospholipase A2-inhibitory proteins now termed the 'lipocortins'. Recently the primary structure of one form of lipocortin has been elucidated and the human gene cloned. Lipocortin 1 is a polar monomeric protein with anti-phospholipase properties in vitro and we now report that when infused into guinea-pig lung preparations this protein has the same inhibitory profile as the glucocorticoids but with a more rapid onset of action. This is the first demonstration that eicosanoid formation can be inhibited by a recombinant phospholipase inhibitory protein applied extracellularly.     

厌氧-好氧周期循环条件下厌氧磷吸收现象

在厌氧-好氧周期循环反应器中，稳定运行阶段出现规律性的与生物除磷理论相悖的厌氧磷酸盐吸收现象．为此，从厌氧吸收磷、有机物的效果及两者之间的关系、厌氧有机物吸收能量来源等角度，对厌氧吸磷机理进行探讨．采用乙酸钠为主要有机基质，当进水P／COD从2／100增加到4／100，厌氧阶段磷酸盐的去除率一直稳定在50％-70％．在排除化学除磷的情况下，实验结果表明厌氧吸磷与厌氧吸收乙酸盐基质直接相关，而糖原又是有机基质吸收与胞内聚合物储存过程重要的甚至唯一的能量来源．通过对厌氧磷酸盐、有机物、糖原以及混合液中pH值变化的测定分析，初步推测厌氧吸收磷酸盐是当胞内无可利用的磷源时，糖原分解对磷酸盐的需求，并通过敏感于pH值变化的磷载体蛋白，将胞外液相中的无机磷酸盐运输到细胞内部。