ABSTRACT
STUDY QUESTION: Does dynamin regulate human sperm acrosomal exocytosis? SUMMARY ANSWER: Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. WHAT IS KNOWN ALREADY: Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. STUDY DESIGN, SIZE, DURATION: Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic individuals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen samples were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. MAIN RESULTS AND THE ROLE OF CHANCE: Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This was an in vitro study performed mainly on ejaculated human spermatozoa. This experimental paradigm necessarily eliminates the physiological contributions of the female reproductive tract that would normally support capacitation and acrosomal responsiveness. WIDER IMPLICATIONS OF THE FINDINGS: This study identifies a novel causative link between dynamin activity and the ability of human spermatozoa to complete a progesterone-induced acrosome reaction. Such findings encourage a more detailed analysis of the contribution of dynamin dysregulation as an underlying aetiology in infertile males whose spermatozoa are unable to penetrate the zona pellucida. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by a National Health and Medical Research Council of Australia Project Grant (APP1103176) awarded to B.N. and E.A.M. The authors report no conflict of interest.
Subject(s)
Acrosome Reaction/drug effects , CDC2 Protein Kinase/genetics , Dynamin I/genetics , Dynamins/genetics , Progesterone/pharmacology , Spermatozoa/drug effects , Acrosome Reaction/physiology , Animals , Brain/metabolism , CDC2 Protein Kinase/metabolism , Dynamin I/antagonists & inhibitors , Dynamin I/metabolism , Dynamin II , Dynamins/antagonists & inhibitors , Dynamins/metabolism , Epididymis/cytology , Epididymis/metabolism , Exocytosis/drug effects , Gene Expression Regulation , Humans , Hydrazones/pharmacology , Male , Mechanotransduction, Cellular , Mice , Naphthols/pharmacology , Phosphorylation/drug effects , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Spermatozoa/metabolism , Spermatozoa/ultrastructure , Testis/cytology , Testis/metabolismABSTRACT
The large GTPase dynamin mediates membrane fission during clathrin-mediated endocytosis (CME). The aminopyrimidine compounds were reported to disrupt dynamin localization to the plasma membrane via the PH domain and implicate this mechanism in the inhibition of CME. We have used a computational approach of binding site identification, docking, and interaction energy calculations to design and synthesize a new library of aminopyrimidine analogues targeting site-2 of the pleckstrin homology (PH) domain. The optimized analogues showed low micromolar inhibition against both dynamin I (IC50 = 10.6 ± 1.3 to 1.6 ± 0.3 µM) and CME (IC50(CME) = 65.9 ± 7.7 to 3.7 ± 1.1 mM), which makes this series among the more potent inhibitors of dynamin and CME yet reported. In CME and growth inhibition cell-based assays, the data obtained was consistent with dynamin inhibition. CEREP ExpresS profiling identified off-target effects at the cholecystokinin, dopamine D2, histamine H1 and H2, melanocortin, melatonin, muscarinic M1 and M3, neurokinin, opioid KOP and serotonin receptors.
Subject(s)
Dynamin I/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pleckstrin Homology Domains/drug effects , Pyrimidines/pharmacology , Binding, Competitive , Carbon-13 Magnetic Resonance Spectroscopy , Cell Line, Tumor , Drug Screening Assays, Antitumor , Enzyme Inhibitors/metabolism , Humans , Mass Spectrometry , Proton Magnetic Resonance Spectroscopy , Pyrimidines/metabolismABSTRACT
Fragment-based in silico screening against dynamin I (dynI) GTPase activity identified the 1,8-naphthalimide framework as a potential scaffold for the design of new inhibitors targeting the GTP binding pocket of dynI. Structure-based design, synthesis and subsequent optimization resulted in the development of a library of 1,8-naphthalimide derivatives, called the Naphthaladyn™ series, with compounds 23 and 29 being the most active (IC50 of 19.1 ± 0.3 and 18.5 ± 1.7 µM respectively). Compound 29 showed effective inhibition of clathrin-mediated endocytosis (IC50(CME) 66 µM). The results introduce 29 as an optimised GTP-competitive lead Naphthaladyn™ compound for the further development of naphthalimide-based dynI GTPase inhibitors.
Subject(s)
Dynamin I/antagonists & inhibitors , Naphthalimides/pharmacology , Amines/chemistry , Binding Sites , Cell Line, Tumor , Clathrin/metabolism , Dynamin I/metabolism , Endocytosis/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Guanosine Triphosphate/metabolism , Humans , Models, Molecular , Naphthalimides/chemistry , Phosphatidylserines/pharmacology , Protein Structure, SecondaryABSTRACT
Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, but the precise physiological function of the protein remains ill-defined. Recently, our group proposed a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses in the Drosophila melanogaster neuromuscular junctions.Flies harbor only one Lrrk gene, which might encompass the functions of both mammalian LRRK1 and LRRK2. We therefore studied the role of LRRK2 in mammalian synaptic function and provide evidence that knockout or pharmacological inhibition of LRRK2 results in defects in synaptic vesicle endocytosis, altered synaptic morphology and impairments in neurotransmission. In addition, our data indicate that mammalian endophilin A1 (EndoA1,also known as SH3GL2) is phosphorylated by LRRK2 in vitro at T73 and S75, two residues in the BAR domain. Hence, our results indicate that LRRK2 kinase activity has an important role in the regulation of clathrin-mediated endocytosis of synaptic vesicles and subsequent neurotransmission at the synapse.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Endocytosis/genetics , Protein Serine-Threonine Kinases/genetics , Synaptic Transmission/genetics , Synaptic Vesicles/genetics , Animals , Cells, Cultured , Clathrin/metabolism , Drosophila melanogaster , Dynamin I/antagonists & inhibitors , Endocytosis/drug effects , Hippocampus/cytology , Hydrazones/pharmacology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/physiology , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Long-Evans , Sucrose/pharmacology , Synaptic Transmission/drug effectsABSTRACT
Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI) GTPase activity identified 2,5-bis-(benzylamino)-1,4-benzoquinone 1 as a 273 ± 106 µM inhibitor. In silico lead optimization and focused library-led synthesis resulted in the development of four discrete benzoquinone/naphthoquinone based compound libraries comprising 54 compounds in total. Sixteen analogues were more potent than lead 1, with 2,5-bis-(4-hydroxyanilino)-1,4-benzoquinone (45) and 2,5-bis(4-carboxyanilino)-1,4-benzoquinone (49) the most active with IC50 values of 11.1 ± 3.6 and 10.6 ± 1.6 µM respectively. Molecular modelling suggested a number of hydrogen bonding and hydrophobic interactions were involved in stabilization of 49 within the dynI GTP binding site. Six of the most active inhibitors were evaluated for potential inhibition of clathrin-mediated endocytosis (CME). Quinone 45 was the most effective CME inhibitor with an IC50(CME) of 36 ± 16 µM.
Subject(s)
Dynamin I/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , GTP Phosphohydrolases/antagonists & inhibitors , Quinones/chemistry , Animals , Cell Line , Clathrin/metabolism , Drug Evaluation, Preclinical , Dynamin I/chemistry , Dynamin I/metabolism , Endocytosis/drug effects , Enzyme Inhibitors/metabolism , Humans , Molecular Docking Simulation , Protein Conformation , Structure-Activity RelationshipABSTRACT
Dynamin 1-3 isoforms are known to be involved in endocytotic processes occurring during synaptic transmission. No data has directly linked dynamins yet with normal animal behavior. Here we show that dynamin pharmacologic inhibition markedly impairs hippocampal-dependent associative memory. Memory loss was associated with changes in synaptic function occurring during repetitive stimulation that is thought to be linked with memory induction. Synaptic fatigue was accentuated by dynamin inhibition. Moreover, dynamin inhibition markedly reduced long-term potentiation, post-tetanic potentiation, and neurotransmitter released during repetitive stimulation. Most importantly, the effect of dynamin inhibition onto memory and synaptic plasticity was due to a specific involvement of the dynamin 1 isoform, as demonstrated through a genetic approach with siRNA against this isoform to temporally block it. Taken together, these findings identify dynamin 1 as a key protein for modulation of memory and release evoked by repetitive activity.
Subject(s)
Dynamin I/metabolism , Excitatory Postsynaptic Potentials/physiology , Hippocampus/physiology , Memory Disorders/metabolism , Memory/physiology , Animals , Dynamin I/antagonists & inhibitors , Dynamin I/genetics , Electric Stimulation , Excitatory Postsynaptic Potentials/drug effects , Gene Expression , Hippocampus/drug effects , Hydrazones/pharmacology , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Male , Memory/drug effects , Memory Disorders/chemically induced , Memory Disorders/physiopathology , Mice , Mice, Inbred C57BL , Neuronal Plasticity/drug effects , Neuronal Plasticity/physiology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Refractory Period, Electrophysiological/drug effects , Refractory Period, Electrophysiological/physiology , Synapses/drug effects , Synapses/physiology , Synaptic Transmission/drug effects , Synaptic Transmission/physiologyABSTRACT
Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was â¼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.
Subject(s)
Cell Membrane/metabolism , Clathrin/physiology , Dynamin I/physiology , Exocytosis/physiology , Hair Cells, Auditory, Inner/metabolism , Hydrazones/pharmacology , Naphthols/pharmacology , Animals , Cell Membrane/drug effects , Dynamin I/antagonists & inhibitors , Dynamin I/genetics , Exocytosis/drug effects , Female , Hair Cells, Auditory, Inner/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation, Missense/physiology , Organ of Corti/cytology , Organ of Corti/metabolismABSTRACT
Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram-negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella-containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71-CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71-CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71-CDT also secreted OMVs-like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin-loaded OMVs by bystander cells was dependent on dynamin-1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.
Subject(s)
Bacterial Toxins/metabolism , Salmonella typhi/metabolism , Salmonella typhimurium/metabolism , Secretory Vesicles/metabolism , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Brefeldin A/pharmacology , Caco-2 Cells , Cell Line , DNA Damage , Dynamin I/antagonists & inhibitors , Dynamin I/metabolism , Dynamins/antagonists & inhibitors , Endocytosis , Epithelial Cells/metabolism , Epithelial Cells/microbiology , HeLa Cells , Humans , Hydrazones/pharmacology , Mice , Promoter Regions, Genetic , Salmonella typhi/genetics , Salmonella typhi/pathogenicity , Salmonella typhimurium/genetics , Salmonella typhimurium/pathogenicityABSTRACT
Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)propyl)-2-methyl-1H-indol-3-yl)-N-octylacrylamide (2) confirmed the tertiary dimethylamino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety could be replaced with a thiazole-4(5H)-one isostere (19, IC(50(dyn I)) = 7.7 µM), reduced under flow chemistry conditions (20, IC(50(dyn I)) = 5.2 µM) or replaced by a simple amine. The latter provided a basis for a high yield library of compounds via a reductive amination by flow hydrogenation. Two compounds, 24 (IC(50 (dyn I)) = 0.56 µM) and 25 (IC(50(dyn I)) = 0.76 µM), stood out. Indole 24 is nontoxic and showed increased potency against dynamin I and II in vitro and in cells (IC(50(CME)) = 1.9 µM). It also showed 4.4-fold selectivity for dynamin I. The indole 24 compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated endocytosis reported to date.
Subject(s)
Acrylamides/chemical synthesis , Dynamin II/antagonists & inhibitors , Dynamin I/antagonists & inhibitors , Indoles/chemical synthesis , Acrylamides/chemistry , Acrylamides/pharmacology , Animals , Brain/enzymology , Cell Line, Tumor , Dynamin I/chemistry , Dynamin II/chemistry , Endocytosis , Humans , Indoles/chemistry , Indoles/pharmacology , Sheep , Small Molecule Libraries , Structure-Activity RelationshipABSTRACT
BACKGROUND: Several lines of investigation support the notion that endocytosis is crucial for Alzheimer's disease (AD) pathogenesis. Substantial evidence have already been reported regarding the mechanisms underlying amyloid precursor protein (APP) traffic, but the regulation of beta-site APP-Cleaving Enzyme 1 (BACE-1) distribution among endosomes, TGN and plasma membrane remains unclear. Dynamin, an important adaptor protein that controls sorting of many molecules, has recently been associated with AD but its functions remain controversial. Here we studied possible roles for dynamin 1 (dyn1) in Aß biogenesis. PRINCIPAL FINDINGS: We found that genetic perturbation of dyn1 reduces both secreted and intracellular Aß levels in cell culture. There is a dramatic reduction in BACE-1 cleavage products of APP (sAPPß and ßCTF). Moreover, dyn1 knockdown (KD) leads to BACE-1 redistribution from the Golgi-TGN/endosome to the cell surface. There is an increase in the amount of surface holoAPP upon dyn1 KD, with resultant elevation of α-secretase cleavage products sAPPα and αCTF. But no changes are seen in the amount of nicastrin (NCT) or PS1 N-terminal fragment (NTF) at cell surface with dyn1 KD. Furthermore, treatment with a selective dynamin inhibitor Dynasore leads to similar reduction in ßCTF and Aß levels, comparable to changes with BACE inhibitor treatment. But combined inhibition of BACE-1 and dyn1 does not lead to further reduction in Aß, suggesting that the Aß-lowering effects of dynamin inhibition are mainly mediated through regulation of BACE-1 internalization. Aß levels in dyn1(-/-) primary neurons, as well as in 3-month old dyn1 haploinsufficient animals with AD transgenic background are consistently reduced when compared to their wildtype counterparts. CONCLUSIONS: In summary, these data suggest a previously unknown mechanism by which dyn1 affects amyloid generation through regulation of BACE-1 subcellular localization and therefore its enzymatic activities.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid/metabolism , Aspartic Acid Endopeptidases/metabolism , Dynamin I/genetics , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Aspartic Acid Endopeptidases/genetics , Cell Line, Tumor , Cell Membrane/metabolism , Dynamin I/antagonists & inhibitors , Dynamin I/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Gene Silencing , Humans , Hydrazones/pharmacology , Mice , Mice, Transgenic , Neurons/metabolism , Protein TransportABSTRACT
Mammalian spermatozoa must complete an acrosome reaction prior to fertilizing an oocyte. The acrosome reaction is a unique exocytotic event involving a series of prolonged membrane fusions that ultimately result in the production of membrane vesicles and release of the acrosomal contents. This event requires the concerted action of a large number of fusion-competent signaling and scaffolding proteins. Here we show that two different members of the dynamin GTPase family localize to the developing acrosome of maturing mouse germ cells. Both dynamin 1 and 2 also remain within the periacrosomal region of mature mouse spermatozoa and are thus well positioned to regulate the acrosome reaction. Two pharmacological inhibitors of dynamin, dynasore and Dyngo-4a, blocked the in vitro induction of acrosomal exocytosis by progesterone, but not by the calcium ionophore A23187, and elicited a concomitant reduction of in vitro fertilization. In vivo treatment with these inhibitors also resulted in spermatozoa displaying reduced acrosome reaction potential. Dynamin 1 and 2 phosphorylation increased on progesterone treatment, and this was also selectively blocked by dynasore. On the basis of our collective data, we propose that dynamin could regulate specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa.
Subject(s)
Acrosome/metabolism , Dynamins/metabolism , Exocytosis , Membrane Fusion , Spermatozoa/metabolism , Acrosome/drug effects , Acrosome Reaction/drug effects , Animals , Blotting, Western , Dynamin I/antagonists & inhibitors , Dynamin I/metabolism , Dynamin II/antagonists & inhibitors , Dynamin II/metabolism , Dynamins/antagonists & inhibitors , Female , Fertilization in Vitro , Hydrazones/pharmacology , Male , Mice , Microscopy, Confocal , Naphthols/pharmacology , Oocytes/metabolism , Phosphorylation/drug effects , Progesterone/pharmacology , Progestins/pharmacology , Testis/cytology , Testis/metabolismABSTRACT
BACKGROUND: The amyloid precursor protein (APP) is cleaved by ß- and γ-secretases to generate toxic amyloid ß (Aß) peptides. Alternatively, α-secretases cleave APP within the Aß domain, precluding Aß formation and releasing the soluble ectodomain, sAPPα. We previously showed that inhibition of the GTPase dynamin reduced APP internalization and increased release of sAPPα, apparently by prolonging the interaction between APP and α-secretases at the plasma membrane. This was accompanied by a reduction in Aß generation. In the present study, we investigated whether surface expression of the α-secretase ADAM (a disintegrin and metalloprotease)10 is also regulated by dynamin-dependent endocytosis. RESULTS: Transfection of human embryonic kidney (HEK) cells stably expressing M3 muscarinic receptors with a dominant negative dynamin I mutant (dyn I K44A), increased surface expression of both immature, and mature, catalytically active forms of co-expressed ADAM10. Surface levels of ADAM10 were unaffected by activation of protein kinase C (PKC) or M3 receptors, indicating that receptor-coupled shedding of the ADAM substrate APP is unlikely to be mediated by inhibition of ADAM10 endocytosis in this cell line. Dyn I K44A strongly increased the formation of a C-terminal fragment of ADAM10, consistent with earlier reports that the ADAM10 ectodomain is itself a target for sheddases. The abundance of this fragment was increased in the presence of a γ-secretase inhibitor, but was not affected by M3 receptor activation. The dynamin mutant did not affect the distribution of ADAM10 and its C-terminal fragment between raft and non-raft membrane compartments. CONCLUSIONS: Surface expression and limited proteolysis of ADAM10 are regulated by dynamin-dependent endocytosis, but are unaffected by activation of signaling pathways that upregulate shedding of ADAM substrates such as APP. Modulation of ADAM10 internalization could affect cellular behavior in two ways: by altering the putative signaling activity of the ADAM10 C-terminal fragment, and by regulating the biological function of ADAM10 substrates such as APP and N-cadherin.
Subject(s)
ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/metabolism , Dynamin I/antagonists & inhibitors , Membrane Proteins/metabolism , ADAM10 Protein , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Protein Precursor/metabolism , Carbachol/pharmacology , Carbamates/pharmacology , Cell Line , Dipeptides/pharmacology , Dynamin I/genetics , Dynamin I/metabolism , Endocytosis , Humans , Protein Kinase C/metabolism , Receptor, Muscarinic M3/metabolismABSTRACT
In the study, the expression of clathrin regulatory proteins dynamin I, AP180, and synaptic vesicle protein synaptophysin in multiple brain regions of the patients with Alzheimer's disease (AD), the transgenic mice carrying the Swedish mutation of amyloid-ß protein precursor (AßPP) 670/671 (AßPPSWE), and the rats injected by bilateral hippocampus with amyloid-ß peptide (Aß)1-42 were examined by immunohistochemistry and Nissl staining, Western blotting, and Real-time PCR, respectively. Spatial learning and memory of the rats were evaluated by Morris Water Maze test, and the ability of endocytosis in the cultured rat hippocampal neurons was detected by FM1-43 fluorescence imaging. Significant decreases in protein levels of dynamin I, AP180, and synaptophysin were observed in both AD patients and mice with AßPPSWE as compared to controls. Obvious declines of dynamin I and synaptophysin at protein and mRNA levels and impaired learning and spatial memory ability were found in the rats injected with Aß1-42 as compared to controls. In addition, deposits of Aß localized in the hippocampus around the sites of Aß1-42 injection and the decreased numbers of Nissl bodies in neurons were found. Moreover, the disrupted synaptic vesicle endocytosis and decreased dynamin I protein were detected in stimulated hippocampal neurons treated with Aß1-42. These findings imply a malfunctioning clathrin-mediated endocytosis during AD pathological processes, which might be relevant to the mechanism underlying the cognitive deficit associated with AD.
Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , Alzheimer Disease/metabolism , Clathrin/physiology , Disease Models, Animal , Dynamin I/physiology , Hippocampus/metabolism , Monomeric Clathrin Assembly Proteins/physiology , Synaptophysin/physiology , Adaptor Proteins, Vesicular Transport/biosynthesis , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/administration & dosage , Amyloid beta-Protein Precursor/toxicity , Animals , Cells, Cultured , Clathrin/antagonists & inhibitors , Dynamin I/antagonists & inhibitors , Endocytosis/genetics , Female , Hippocampus/pathology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Rats , Rats, Sprague-DawleyABSTRACT
We report the development of a homology model for the GTP binding domain of human dynamin I based on the corresponding crystal structure of Dictyostelium discoidum dynamin A. Virtual screening identified 2-[(2-biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)amino]-4-chlorobenzoic acid (1) as a approximately 170 microM potent inhibitor. Homology modeling- and focused library-led synthesis resulted in development of a series of active compounds (the "pthaladyns") with 4-chloro-2-(2-(4-(hydroxymethyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (29), a 4.58 +/- 0.06 microM dynamin I GTPase inhibitor. Pthaladyn-29 displays borderline selectivity for dynamin I relative to dynamin II ( approximately 5-10 fold). Only pthaladyn-23 (dynamin I IC(50) 17.4 +/- 5.8 microM) was an effective inhibitor of dynamin I mediated synaptic vesicle endocytosis in brain synaptosomes with an IC(50) of 12.9 +/- 5.9 microM. This compound was also competitive with respect to Mg(2+).GTP. Thus the pthaladyns are the first GTP competitive inhibitors of dynamin I and II GTPase and may be effective new tools for the study of neuronal endocytosis.
Subject(s)
Benzoates/chemical synthesis , Dynamin II/antagonists & inhibitors , Dynamin I/antagonists & inhibitors , Dynamins/chemistry , Guanosine Triphosphate/metabolism , Isoindoles/chemical synthesis , Phthalimides/chemical synthesis , Protozoan Proteins/chemistry , ortho-Aminobenzoates/chemical synthesis , Benzoates/chemistry , Benzoates/pharmacology , Binding Sites , Brain/drug effects , Brain/metabolism , Dictyostelium , Endocytosis/drug effects , Humans , Isoindoles/chemistry , Isoindoles/pharmacology , Kinetics , Ligands , Models, Molecular , Phthalimides/chemistry , Phthalimides/pharmacology , Protein Conformation , Protein Structure, Tertiary , Structure-Activity Relationship , Synaptosomes/drug effects , Synaptosomes/metabolism , ortho-Aminobenzoates/chemistry , ortho-Aminobenzoates/pharmacologyABSTRACT
Herein we report the synthesis of discrete iminochromene ("iminodyn") libraries (14-38) as potential inhibitors of dynamin GTPase. Thirteen iminodyns were active (IC(50) values of 260 nM to 100 microM), with N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (17), N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (22), and N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (23) (IC(50) values of 330 +/- 70, 450 +/- 50, and 260 +/- 80 nM, respectively) being the most potent. Five of the most potent iminodyns all inhibited dynamins I and II approximately equally. Iminodyn-22 displayed uncompetitive inhibition with respect to GTP. Selected iminodyns were evaluated for their ability to block receptor mediated endocytosis (RME, mediated by dynamin II) and synaptic vesicle endocytosis (SVE, mediated by dynamin I), with 17 showing no activity while 22 returned RME and SVE IC(50) values of 10.7 +/- 4.5 and 99.5 +/- 1.7 microM, respectively. The iminodyns reported herein represent a new chemical class of the first nanomolar potent dynamin inhibitors that are also effective endocytosis inhibitors.
Subject(s)
Benzopyrans/chemical synthesis , Dynamin II/antagonists & inhibitors , Dynamin I/antagonists & inhibitors , Endocytosis/drug effects , Animals , Benzopyrans/chemistry , Benzopyrans/pharmacology , Cell Line, Tumor , Humans , In Vitro Techniques , Kinetics , Male , Rats , Rats, Sprague-Dawley , Receptors, Transferrin/metabolism , Sheep , Structure-Activity Relationship , Synaptic Vesicles/drug effects , Synaptic Vesicles/metabolism , Synaptosomes/drug effects , Synaptosomes/metabolismABSTRACT
Dynamin (Dyn) 1 plays a role in recycling of synaptic vesicles, and thus in nervous system function. We previously showed that sertraline, a selective serotonin reuptake inhibitor (SSRI), is a mixed-type inhibitor of Dyn 1 with respect to both GTP and L-alpha-phosphatidyl-L-serine (PS) in vitro, and we suggested that it may regulate the neurotransmitter transport by modulating synaptic vesicle endocytosis via inhibition of Dyn 1 GTPase. Here, we investigated the effect of sertraline on endocytosis of marker proteins in human neuroblastoma SH-Sy5Y cells and HeLa cells. Sertraline inhibited endocytosis in both cell lines. Western blotting showed that SH-Sy5Y expresses Dyn 1 and Dyn 2, while HeLa expresses only Dyn 2. GTPase assay showed that sertraline inhibited Dyn 2 as well as Dyn 1. Therefore, the effect of sertraline on endocytosis was mediated by Dyn 2, at least in HeLa cells, as well as by Dyn 1 in cell lines that express it. Moreover, the inhibition mechanism of transferrin (Tf) uptake by sertraline differed from that in cells expressing Dyn 1 K44A, a GTP binding-defective variant, and sertraline did not interfere with the interaction between Dyn 1 and PS-liposomes.
Subject(s)
Dynamin II/antagonists & inhibitors , Dynamin I/antagonists & inhibitors , Endocytosis/drug effects , Neurons/drug effects , Selective Serotonin Reuptake Inhibitors/pharmacology , Sertraline/pharmacology , Blood Proteins/chemistry , Cell Line, Tumor , Dynamin I/genetics , Dynamin I/metabolism , Dynamin II/genetics , Dynamin II/metabolism , HeLa Cells , Humans , Neurons/metabolism , Phosphatidylserines/metabolism , Phosphoproteins/chemistry , Protein Structure, Tertiary/genetics , Transferrin/metabolismABSTRACT
Screening identified two bisindolylmaleimides as 100 microM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 +/- 0.3 microM dynamin I inhibitor. Dynole 34-2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC(50) approximately 15 microM; RME IC(50) approximately 80 microM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells.
Subject(s)
Acrylamides/chemical synthesis , Acrylamides/pharmacology , Dynamin I/antagonists & inhibitors , Endocytosis/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Indoles/chemical synthesis , Indoles/pharmacology , Acrylamides/chemistry , Animals , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Dynamin I/metabolism , Enzyme Inhibitors/chemistry , Humans , Indoles/chemistry , Mice , Molecular Structure , NIH 3T3 Cells , Small Molecule Libraries , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Probing the dynamin binding site: Bis-tyrphostin (1, Bis-T), is a potent inhibitor of the phospholipid-stimulated GTPase activity of dynamin I. Analogues of Bis-T have significant potential as a biological probes for the dissection of endocytic pathways. Bis-T-derived compounds were synthesised and evaluated for their ability to inhibit the GTPase activity of dynamin I. Two analogues (23 and 24) represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibition.Two azidobenzyl amide (4 and 23) and one 3-trifluoromethyl-3H-diazirin-3-ylphenyl (24) analogues of bis-tyrphostin (1, Bis-T) were synthesised as potential photoaffinity labels for the elucidation of the binding site of compound 1 in dynamin I. Of the two azidobenzyl amide analogues (4 and 23), the terminally substituted 23 retained dynamin I GTPase inhibition (IC(50)=6.4+/-2.8 microM) whilst 4, which was substituted on the central carbon of the amide linker, displayed no activity. Analogue 24 also retained inhibitory activity (IC(50)=36+/-9 microM). Photoaffinity labelling experiments did not unequivocally elucidate the binding pocket of compound 1. However, compounds 23 and 24 represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibitory activity, providing a new direction for analogue synthesis.
Subject(s)
Dynamin I/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Tyrphostins/chemistry , Animals , Dynamin I/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Sheep , Tyrphostins/chemical synthesis , Tyrphostins/pharmacologyABSTRACT
Neuronal dynamin I plays a critical role in the recycling of synaptic vesicles, and thus in nervous system function. We expressed and purified dynamin I to explore potentially clinically useful endocytosis inhibitors and to examine the mechanism of their action. We estimated the IC(50) of nineteen psychotropic drugs for dynamin I. The IC(50) values of two selective serotonin reuptake inhibitors (sertraline and fluvoxamine) were 7.3+/-1.0 and 14.7+/-1.6 microM, respectively. Kinetic analyses revealed that fluvoxamine is a noncompetitive inhibitor of dynamin I guanosine triphosphatase (GTPase) with respect to guanosine 5'-triphosphate (GTP) and a competitive inhibitor with respect to L-phosphatidylserine (PS). Fluvoxamine may compete with PS for binding to the pleckstrin homology domain of dynamin I. On the other hand, sertraline was a mixed type inhibitor with respect to both GTP and PS. Our results indicate that sertraline and fluvoxamine may regulate the transportation of neurotransmitters by modulating synaptic vesicle endocytosis via the inhibition of dynamin I GTPase.
Subject(s)
Dynamin I/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Selective Serotonin Reuptake Inhibitors , Animals , Dynamin I/genetics , Endocytosis/drug effects , Escherichia coli/genetics , Fluvoxamine/pharmacology , Genetic Vectors , Kinetics , Mice , Plasmids/genetics , Psychotropic Drugs/pharmacology , Sertraline/pharmacologyABSTRACT
Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiTMAB inhibited dynamin GTPase activity, with a Ki of 940 +/- 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophosphorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells.