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PubMed2004

Aberrant neuronal and mitochondrial proteins in hippocampus of transgenic mice overexpressing human Cu/Zn superoxide dismutase 1.

Mutations of Cu/Zn superoxide dismutase 1 (SOD1), a metalloenzyme catalyzing the conversion of superoxide anion to hydrogen peroxide (H(2)O(2)), are linked to motor neuron degeneration. Transgenic mouse strains overexpressing wild-type human SOD1 (Tg-SOD1) were shown to have mitochondrial swelling, vacuolization, or learning and memory deficits and are widely used for biochemical, genetic, and cognitive studies; this, along with the advent of advanced proteomic methods, made us investigate protein expression in hippocampus. Hippocampal tissues of wild-type, hemizygous, and homozygous Tg-SOD1 mice were isolated and used for two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight identification. We identified several synaptosomal, neuronal, antioxidant, and mitochondrial proteins in hippocampus, and expression levels of syntaxin-binding protein 1, N-ethylmaleimide-sensitive factor, synaptosomal-associated protein 25, dynamin-1, neurofilament triplet L protein, neurofilament triplet M protein, neuronal tropomodulin, and neuronal protein 25 were significantly decreased in Tg-SOD1. None of the antioxidant proteins were altered except mouse SOD1. Mitochondrial ATP synthase alpha/beta chain and elongation factor Tu were aberrant in Tg-SOD1. We conclude that derangement of neuronal and mitochondrial proteins may indicate synaptosomal and neuronal loss in Tg-SOD1 hippocampus, already reported in morphological terms. This observation is of relevance to understanding brain deficits in Down syndrome, as SOD1 is encoded on chromosome 21.

Animals↗
PubMed2004

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene.

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.

Amino Acid Motifs↗
PubMed2005

Cationic TAT peptide transduction domain enters cells by macropinocytosis.

Naturally occurring and synthetic short arginine containing protein transduction domains (PTDs), including HIV1 TAT, poly-Arg and Antp, have been used to deliver a wide variety of macromolecular, biologically active therapeutic cargo into cells, including peptides, proteins, antisense oligonucleotides and liposomes, in vitro and to treat pre-clinical models of cancer and stroke. PTDs enter cells in a rapid, receptor-independent fashion. Recently, large TAT-fusion proteins (in excess of 30,000 Da) were shown to transduce into cells by fluid-phase macropinocytosis, a specialized form of endocytosis that is independent of caveolae, clathrin and dynamin. However, it remains controversial as to whether or not PTD peptides (1000-5000 Da) enter cells via macropinocytosis and/or through an unknown alternative mechanism. Due to strong ionic interactions with the cell surface, previous measurements of PTD peptide internalization were inaccurate. Cationic PTD peptides containing variable numbers of arginine residues and conditions entered cells exclusively through macropinocytosis. In addition, no PTD peptide was found to enter cells at 4 degrees C, a long held assumption of transduction. Taken together, these observations provide a solid scientific basis for the development of novel biologically active transducible anticancer PTD peptide therapeutics.

Animals↗
PubMed2004

Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis.

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.

Amino Acid Sequence↗
PubMed2006

Characterization of the urinary albumin degradation pathway in the isolated perfused rat kidney.

This study examines the existence of the urinary albumin degradation pathway and the proposed role of receptor-mediated endocytosis in this process using the isolated perfused rat kidney (IPK) model. Albumin-derived peptides in IPK urine are analyzed in terms of their relative size distribution using radioactivity and absorbance at 214 nm, and their susceptibility to trypsin digestion. The effects of perfusing kidneys with concanamycin A and myristoyl trimethyl ammonium bromide (MTMAB), inhibitors of the receptor-mediated endocytosis regulators vacuolar-type H(+) ATPase (v-ATPase) and dynamin GTPase, respectively, are examined. Normal IPK urine contains mildly degraded (defined as approximately 10-40 kDa; 43.0 +/- 8.3%) and heavily degraded (defined as <10 kDa; 22.6 +/- 7.7%) albumin peptides as well as intact albumin (34.5 +/- 4.1%). The relative size distribution of the peptides is similar by radioactivity and absorbance at 214 nm, and both profiles are reduced to very small peptides following trypsin digestion. Administration of concanamycin A or MTMAB causes a significant increase in the proportion of intact albumin (concanamycin A: 55.8 +/- 11.6%; MTMAB: 50.0 +/- 11.9%) excreted compared with normal IPK urine. This coincides with a reduction in the proportion of mildly (concanamycin A: 27.6 +/- 9.8%; MTMAB: 39.9 +/- 11.5%) and heavily degraded (concanamycin A: 16.6 +/- 7.4%; MTMAB: 10.0 +/- 2.5%) albumin present and is not associated with changes in glomerular permeability to albumin because no significant change is observed in the fractional clearance of Ficoll (radius range 20-60 A) in the presence of concanamycin A. This study demonstrates the existence of albumin peptides in IPK urine and suggests that receptor-mediated endocytosis plays a role in urinary albumin degradation.

Animals↗
PubMed2006

Differential effects of Cbl isoforms on Egfr signaling in Drosophila.

The Cbl family of proteins downregulate epidermal growth factor receptor (Egfr) signaling via receptor internalization and destruction. These proteins contain two functional domains, a RING finger domain with E3 ligase activity, and a proline rich domain mediating the formation of protein complexes. The Drosophila cbl gene encodes two isoforms, D-CblS and D-CblL. While both contain a RING finger domain, the proline rich domain is absent from D-CblS. We demonstrate that expression of either isoform is sufficient to rescue both the lethality of a D-cbl null mutant and the adult phenotypes characteristic of Egfr hyperactivation, suggesting that both isoforms downregulate Egfr signaling. Interestingly, targeted overexpression of D-CblL, but not D-CblS, results in phenotypes characteristic of reduced Egfr signaling and suppresses the effect of constitutive Egfr activation. The level of D-CblL was significantly correlated with the phenotypic severity of reduced Egfr signaling, suggesting that D-CblL controls the efficiency of downregulation of Egfr signaling. Furthermore, reduced dynamin function suppresses the effects of D-CblL overexpression in follicle cells, suggesting that D-CblL promotes internalization of activated receptors. D-CblL is detected in a punctate cytoplasmic pattern, whereas D-CblS is mainly localized at the follicle cell cortex. Therefore, D-CblS and D-CblL may downregulate Egfr through distinct mechanisms.

Alternative Splicing↗
PubMed2004

Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis.

By transiently or stably overexpressing the mitochondrial fission factor dynamin-related protein-1 (Drp-1), we evaluated the role of mitochondrial division in organelle Ca2+ homeostasis and apoptotic signaling. Quantitative 3D digital microscopy revealed a split mitochondrial network in Drp-1-overexpressing cells without changes in cell viability. High-speed mitochondrial [Ca2+] ([Ca2+]m) imaging revealed propagating intramitochondrial Ca2+ waves in intact cells, which were blocked in the Drp-1-fragmented network, leaving a fraction of individual mitochondria without substantial [Ca2+]m elevation. Consequently, in Drp-1-expressing cells the apoptotic efficacy of ceramide, which causes a Ca2+-dependent perturbation of mitochondrial structure and function, was drastically reduced. Conversely, the sensitivity to staurosporine-induced apoptosis, previously shown to be directly triggered by Drp-1-dependent recruitment of proapoptotic proteins to mitochondria, was enhanced. These results demonstrate that the regulated process of mitochondrial fusion and fission controls the spatiotemporal properties of mitochondrial Ca2+ responses and, thus, physiological and pathological consequences of cellular Ca2+ signals.

Apoptosis↗
PubMed2006

Mitochondria in DRG neurons undergo hyperglycemic mediated injury through Bim, Bax and the fission protein Drp1.

Dorsal root ganglia (DRG) neurons degenerate in diabetic neuropathy (DN) and exhibit mitochondrial damage. We studied mitochondria of cultured DRG neurons exposed to high glucose as an in vitro model of DN. High glucose sequentially increases the expression, activation and localization of the pro-apoptotic proteins Bim and Bax and the mitochondrial fission protein dynamin-regulated protein 1 (Drp1). High glucose causes association of Drp1/Bax, similar to other apoptotic stimuli. Collectively, these events promote mitochondrial fragmentation and reduce mitochondrial number, suggestive of apoptotic mitochondrial fission. Drp1 is also upregulated in DRG from experimentally diabetic rats, suggesting a role for mitochondrial fission in DN. Insulin-like growth factor-I (IGF-I) protects high glucose-treated DRG neurons by preventing mitochondrial accumulation of Bim and Bax but does not modulate Drp1 expression or localization. We propose that mitochondria are compromised by convergence of Bim/Bax proteins with Drp1, which contributes to high glucose-induced injury in DRG neurons.

Animals↗
PubMed2006

Transcriptional regulation of artemin is related to neurite outgrowth and actin polymerization in mature DRG neurons.

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands that helps to ensure the survival of sensory neurons. We used an in vitro isolated dorsal root ganglia model to study the effects of artemin on the adult rat neuronal system and investigate differentially regulated genes. We found that 285 genes were differentially transcribed by artemin after 3 h of treatment, including genes related to cell adhesion and actin polymerization. A series of genes involved in the regulation of actin dynamics, including coronin, Myr 5, Wiskott-Aldrich syndrome protein interacting protein, cofilin, drebrin and dynamin were down-regulated by artemin, suggesting that it plays a previously undefined role in the regulation of actin polymerization and synaptic vesicle movement. Artemin also down-regulated the expression of genes related to cell adhesion and matrix assembly, including biglycan, plectin, nestin, neuronatin and the neuron-glia-CAM-related cell adhesion molecule, which is functionally relevant to neurite elongation in DRG neurons. Artemin resulted in increases in total neurite length and branching of the DRG neurons. Also artemin caused an increase of synaptic vesicle clustering. Our results showed that the inhibition of DNA methylation suppressed the artemin-dependent neurite growth, suggesting that the genetic regulation could be relevant to neurite elongation in mature DRG.

Actins↗
PubMed2005

Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions.

In a forward screen for genes affecting neurotransmission in Drosophila, we identified mutations in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, we provide evidence that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. Our data suggest a specific role for mitochondria in regulating synaptic strength.

Animals↗
PubMed2006

Arc/Arg3.1: linking gene expression to synaptic plasticity and memory.

Arc/Arg3.1 is an effector immediate-early gene implicated in the consolidation of memories. Although cloned a decade ago, the physiological role of Arc/Arg3.1 in the brain has remained elusive. Four papers in this issue of Neuron address this function. These studies show that Arc/Arg3.1 regulates endophilin 3 and dynamin 2, two components of the endocytosis machinery. Genetic ablation of Arc/Arg3.1 in mice or overexpression in culture suggest that Arc/Arg3.1 regulates AMPA receptor trafficking and synaptic plasticity. Finally, Arc/Arg3.1 knockout mice show memory retention deficits. These recent developments provide new insights into the function of this popular activity-dependent neuronal marker.

Animals↗
PubMed2004

Mobilization and fusion of a non-recycling pool of synaptic vesicles under conditions of endocytic blockade.

At vertebrate central synapses, it has been demonstrated that a resting pool of synaptic vesicles (SVs) exists that normally does not participate in SV release and recycling. It remains unclear whether SVs within the resting pool are capable of mobilization and fusion. Here, we combine live imaging of SV exo- and endocytosis using pH-sensitive GFP (synapto-pHluorins) with pharmacological and genetic manipulations of the SV cycle at the Drosophila NMJ. We demonstrate that a resting pool of SVs exists at this synapse that encompasses 30-41% of the total SV pool. Under conditions of endocytic blockade, using a temperature-sensitive dynamin mutation, the resting pool of SVs can be mobilized and released. We present a model for the presence of a resting pool of SVs that does not require molecular specification of a subpopulation of SVs.

Animals↗
PubMed2006

mu-Opioid receptor internalization-dependent and -independent mechanisms of the development of tolerance to mu-opioid receptor agonists: Comparison between etorphine and morphine.

A growing body of evidences suggests that receptor desensitization is implicated in the development of tolerance to opioids, which is generally regulated by protein kinases and receptor trafficking proteins. In the present study, we demonstrated that repeated s.c. treatment with etorphine, but not morphine, produced a significant increase in protein levels of G protein-coupled receptor kinase 2, dynamin II, beta-arrestin 2 and phosphorylated-conventional protein kinase C in membranes of the mouse spinal cord, suggesting that the etorphine-induced mu-opioid receptor desensitization may result from G protein-coupled receptor kinase 2/dynaminII/beta-arrestin2-dependent phosphorylation of mu-opioid receptors. Unlike etorphine, morphine failed to change the levels of these trafficking proteins. Furthermore, we found that the level of glial fibrillary acidic protein in the mouse spinal cord was clearly increased by chronic in vivo and in vitro treatment with morphine, whereas no such effect was noted by etorphine. In the behavioral study, intraperitoneal pretreatment with the glial-modulating agent propentofylline suppressed the development of tolerance to morphine-induced antinociception. In addition, intrathecal injection of astrocytes and astrocyte-conditioned medium mixture, which were obtained from cultured astrocytes of the newborn mouse spinal cord, aggravated the development of tolerance to morphine. In contrast, these agents failed to affect the development of tolerance induced by etorphine. These findings provide direct evidence for the distinct mechanisms between etorphine and morphine on the development of tolerance to spinal antinociception. These findings raise the possibility that the increased astroglia response produced by chronic morphine could be associated with the lack of mu-opioid receptor internalization.

Animals↗
PubMed2006

Functional reduction in mu-opioidergic system in the spinal cord under a neuropathic pain-like state following chronic ethanol consumption in the rat.

Chronic ethanol consumption produces a painful peripheral neuropathy. The aim of this study was then to investigate the mechanism underlying the neuropathic pain-like state induced by chronic ethanol treatment in rats. Mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal, and it lasted for, at least, 14 weeks. At 24 days after ethanol withdrawal, antinociception of morphine was significantly suppressed and the increased guanosine-5'-o-(3-thio) triphosphate ([(35)S]GTPgammaS) binding to membranes of the spinal cord induced by the selective mu-opioid receptor (MOR) agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO), was significantly decreased under the ethanol-dependent neuropathic pain-like state, whereas the increased [(35)S]GTPgammaS binding to membranes of the spinal cord induced by either the selective delta-opioid receptor (DOR) agonist or kappa-opioid receptor (KOR) agonist was not changed under the ethanol-dependent neuropathic pain-like state. Furthermore, total-MOR immunoreactivity was not changed in the spinal cord of ethanol-fed rats. Under these conditions, immunoblotting showed a robust increase in phosphorylated-cPKC immunoreactivity (p-cPKC-IR) in the spinal cord from chronic ethanol fed-rats, whereas phosphorylated-protein kinase A (PKA), dynamin II and G protein-coupled receptor kinase 2 (GRK2) were not affected in the spinal cord of ethanol-fed rats. These findings suggest that the dysfunction of MOR, but not DOR and KOR, linked to cPKC activation in the spinal cord may be, at least in part, involved in the reduced sensitivity to antinociception induced by morphine under the ethanol-dependent neuropathic pain-like state.

Alcohol-Induced Disorders, Nervous System↗
PubMed2026

CRISPR/Cpf1-mediated editing of DNM1L in induced pluripotent stem cells.

The dynamin-1-like protein (DNM1L), also termed DRP1, is essential for mitochondrial fission. Mutations in DNM1L are associated with neurological disorders and cardiac dysfunction. To decipher the role of DNM1L in human induced pluripotent stem cells (hiPSCs) and in their differentiated counterparts, we used CRISPR/Cpf1 and generated a human iPSC line with a mutation by targeting exon 18 of the DNM1L gene. The generated compound heterozygous (biallelic) DNM1L mutant cell line showed normal cell morphology, genomic stability, and expression of classical stem cell markers. Furthermore, the cells can be differentiated efficiently into the three germ layers meso-, endo-, and ectoderm.

Journal Article↗
PubMed2005

Mitochondrial fusion and fission in the control of apoptosis.

Mitochondrial outer membrane permeabilization (MOMP) determines the point-of-no-return of most if not all signal-transduction cascades leading to cell death. It has been postulated that the molecular mechanism leading to MOMP could depend on the activation of the mitochondrial fission machinery mediated by proteins from the dynamin superfamily. However, recent work suggests that, depending on the specific apoptosis induction pathway, mitochondrial fission can occur independently or downstream from MOMP. Moreover, fragmentation of the mitochondrial network can inhibit MOMP and apoptosis in response to a particular range of lethal stimuli, namely those relying on Ca(2+) waves. Failure to transmit the Ca(2+) wave through disconnected mitochondria then interrupts the propagation of the pro-apoptotic signal. Thus, mitochondrial fission can either enhance or reduce the probability of MOMP and consequent cell death, depending on the initial lethal stimulus.

Apoptosis↗
PubMed2004

TIMP-2 is released as an intact molecule following binding to MT1-MMP on the cell surface.

Binding of tissue inhibitor of metalloproteinase-2 (TIMP-2) to pro-MMP-2 and mature membrane type-1 MMP (MT1-MMP) on the cell surface is required for activation of MMP-2. It has been reported that following binding to cell surface receptors, TIMP-2 undergoes endocytosis and extensive degradation in lysosomes. The purpose of this study was to reexamine the fate of TIMP-2 following binding to transfected HT1080 cell surface MT1-MMP at 4 degrees C. Following 37 degrees C incubation, 125I-TIMP-2 release, endocytosis, and degradation were characterized under varying conditions. More than 85% of the total 125I-TIMP-2 bound to cells was released as intact functional molecules; <15% was degraded. Transfection of HT1080 cells with dominant negative mutant dynamin cDNA resulted in delayed endocytosis and release of 125I-TIMP-2 from cells. Pharmacologic agents that induce clustering of cell surface receptors (concanavalin A) and interfere with endosomal/lysosomal function (bafilomycin A(1)) resulted in enhanced binding of 125I-TIMP-2 to cell surface receptors. Abrogation of activation of proMT1-MMP with a furin inhibitor prevented binding and endocytosis of 125I-TIMP-2. Biotinylation of cell surface MT1-MMP followed by Western blotting confirmed the presence of mature MT1-MMP on the cell surface and degraded MT1-MMP in the intracellular compartment. In conclusion, these studies demonstrate that TIMP-2 is released from cells primarily as an intact functional molecule following binding to MT1-MMP on the cell surface.

Animals↗
PubMed2004

A dynamic podosome-like structure of epithelial cells.

Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of beta4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and beta4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion.

Actins↗