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Pietro De Camilli - One of the best experts on this subject based on the ideXlab platform.
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absence of sac2 inpp5f enhances the phenotype of a parkinson s disease mutation of Synaptojanin 1
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Mian Cao, Daehun Park, Pietro De CamilliAbstract:Numerous genes whose mutations cause, or increase the risk of, Parkinson's disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol phosphatase domain of Synaptojanin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain-containing protein, is located within a PD risk locus identified by genome-wide association studies. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurologic defects. We report a "synthetic" effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 wk, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double-mutant neurons. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.
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absence of sac2 inpp5f enhances the phenotype of a parkinson s disease mutation of Synaptojanin 1
bioRxiv, 2020Co-Authors: Mian Cao, Daehun Park, Pietro De CamilliAbstract:Abstract Many genes whose mutations cause, or increase the risk of, Parkinson’s disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol phosphatase domain of Synaptojanin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain containing protein, was identified as a PD risk locus by GWAS. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurological defects. We report a “synthetic” effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 weeks, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double mutant. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.
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parkinson sac domain mutation in Synaptojanin 1 impairs clathrin uncoating at synapses and triggers dystrophic changes in dopaminergic axons
Neuron, 2017Co-Authors: Mian Cao, Mark H. Ellisman, Ghazaleh Ashrafi, Amber J Mccartney, Heather Wheeler, Eric A Bushong, Daniela Boassa, Timothy A Ryan, Pietro De CamilliAbstract:Synaptojanin 1 (SJ1) is a major presynaptic phosphatase that couples synaptic vesicle endocytosis to the dephosphorylation of PI(4,5)P2, a reaction needed for the shedding of endocytic factors from their membranes. While the role of SJ1's 5-phosphatase module in this process is well recognized, the contribution of its Sac phosphatase domain, whose preferred substrate is PI4P, remains unclear. Recently a homozygous mutation in its Sac domain was identified in early-onset parkinsonism patients. We show that mice carrying this mutation developed neurological manifestations similar to those of human patients. Synapses of these mice displayed endocytic defects and a striking accumulation of clathrin-coated intermediates, strongly implicating Sac domain's activity in endocytic protein dynamics. Mutant brains had elevated auxilin (PARK19) and parkin (PARK2) levels. Moreover, dystrophic axonal terminal changes were selectively observed in dopaminergic axons in the dorsal striatum. These results strengthen evidence for a link between synaptic endocytic dysfunction and Parkinson's disease.
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the sac1 domain of synj1 identified mutated in a family with early onset progressive parkinsonism with generalized seizures
Human Mutation, 2013Co-Authors: Catharine E. Krebs, Siamak Karkheiran, Hossein Darvish, Gholam Ali Shahidi, Joseph D. Buxbaum, Gilbert Di Paolo, Vladimir Makarov, James C Powell, Ruth H. Walker, Pietro De CamilliAbstract:This study aimed to elucidate the genetic causes underlying early-onset Parkinsonism (EOP) in a consanguineous Iranian family. To attain this, homozygosity mapping and whole-exome sequencing were performed. As a result, a homozygous mutation (c.773G>A; p.Arg258Gln) lying within the NH2-terminal Sac1-like inositol phosphatase domain of polyphosphoinositide phosphatase Synaptojanin 1 (SYNJ1), which has been implicated in the regulation of endocytic traffic at synapses, was identified as the disease-segregating mutation. This mutation impaired the phosphatase activity of SYNJ1 against its Sac1 domain substrates in vitro. We concluded that the SYNJ1 mutation identified here is responsible for the EOP phenotype seen in our patients probably due to deficiencies in its phosphatase activity and consequent impairment of its synaptic functions. Our finding not only opens new avenues of investigation in the synaptic dysfunction mechanisms associated with Parkinsonism, but also suggests phosphoinositide metabolism as a novel therapeutic target for Parkinsonism.
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regulation of postsynaptic ampa responses by Synaptojanin 1
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Liang Wei Gong, Pietro De CamilliAbstract:Endocytosis of postsynaptic AMPA receptors is a mechanism through which efficiency of neurotransmission is regulated. We have genetically tested the hypothesis that Synaptojanin 1, a phosphoinositide phosphatase implicated in the endocytosis of synaptic vesicles presynaptically, may also function in the endocytosis of AMPA receptors postsynaptically. Electrophysiological recordings of cultured hippocampal neurons showed that miniature excitatory postsynaptic current amplitudes were larger in Synaptojanin 1 knockout (KO) neurons because of an increase of surface-exposed AMPA receptors. This change did not represent an adaptive response to decreased presynaptic release in KO cultures and was rescued by the expression of wild type, but not catalytically inactive Synaptojanin 1, in the postsynaptic neuron. NMDA-induced internalization of pHluorin-tagged AMPA receptors (GluR2) was impaired in KO neurons. These results reveal a function of Synaptojanin 1 in constitutive and triggered internalization of AMPA receptors and thus indicate a role for phosphatidylinositol(4,5)-bisphosphate metabolism in the regulation of postsynaptic AMPA responses.
Marc Symons - One of the best experts on this subject based on the ideXlab platform.
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abstract a84 Synaptojanin 2 and microrna 31 control invadopodia and metastasis by regulating vesicular trafficking
Cancer Research, 2013Co-Authors: Nir Benchetrit, Marc Symons, David Chetrit, Cindy Koerner, Silvia Carvalho, Fernando Schmitt, Stefan Wiemann, Marcelo Ehrlich, Yosef YardenAbstract:Growth factors propel cell migration and metastasis, but underlying mechanisms are incompletely understood. Employing EGF-stimulated mammary cells we link the lipid phosphatase Synaptojanin 2 (SYNJ2) to an invasive phenotype, and relate high SYNJ2 and low microRNA-31, a metastasis suppressor that restrains SYNJ2, to short survival rates of cancer patients. Knockdown of SYNJ2 robustly impaired both intravasation and extravasation of mammary tumor cells in an animal model. In vitro, SYNJ2-depleted cells exhibited derailed trafficking of EGFR and integrins, resulting in deformed focal adhesions, arrested lamellipodia and disappearance of invadopodia. We conclude that recycling of active EGFRs focally promotes SYNJ2-mediated dephosphorylation of specific phosphoinositol lipids, thereby instigates formation of both invadopodia and lamellipodia and facilitates tumor progression. Citation Format: Nir Ben-Chetrit, David Chetrit, Cindy Koerner, Silvia Carvalho, Marc Symons, Fernando Schmitt, Stefan Wiemann, Marcelo Ehrlich, Yosef Yarden. Synaptojanin-2 and microRNA-31 control invadopodia and metastasis by regulating vesicular trafficking. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A84.
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regulation of Synaptojanin 2 5 phosphatase activity by src
Cell Adhesion & Migration, 2012Co-Authors: Yayu Chuang, Aneta Kwiatkowska, George Tsapraillis, Hyonson Hwang, Konstantinos Petritis, Dan Flynn, Marc SymonsAbstract:Synaptojanin 2 (SYNJ2) is a phosphatidylinositol (PI) phosphatase that controls two distinct functions, clathrin-mediated endocytosis and tumor cell invadopodia formation and invasion. Here, we identify a number of novel SYNJ2 binding partners, several of which have previously been shown to be necessary for invadopodia formation or clathrin-mediated endocytosis. We focus on Src family kinases. We found that Src phosphorylates SYNJ2 on Tyr490, thereby stimulating SYNJ2 5′-phosphatase activity in vitro. We also provide evidence that Src-mediated phosphorylation of SYNJ2 contributes to invadopodia formation.
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role of Synaptojanin 2 in glioma cell migration and invasion
Cancer Research, 2004Co-Authors: Yayu Chuang, Nicole Rusk, Marc Symons, Nhan L Tran, Mitsutoshi Nakada, Michael E BerensAbstract:The small GTPase Rac1 is thought to play an important role in cell migration and invasion. We have previously identified Synaptojanin 2, a phosphoinositide phosphatase, as an effector of Rac1. Here, we show that small interfering RNA-mediated depletion of either Rac1 or Synaptojanin 2 inhibits invasion of SNB19 and U87MG glioblastoma cells through Matrigel and rat brain slices. Depletion of Rac1 or Synaptojanin 2 also inhibits migration of SNB19 and U87MG cells on glioma-derived extracellular matrix. In addition, we found that depletion of Rac1 or Synaptojanin 2 inhibits the formation of lamellipodia and invadopodia, specialized membrane structures that are thought to be involved in extracellular matrix degradation. These results suggest that Synaptojanin 2 contributes to the role of Rac1 in cell invasion and migration by regulating the formation of invadopodia and lamellipodia. This study also identifies Synaptojanin 2 as a novel potential target for therapeutic intervention in malignant tumors.
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Synaptojanin 2 functions at an early step of clathrin mediated endocytosis
Current Biology, 2003Co-Authors: Nicole Rusk, Stefania Mariggio, Ginette Guay, Cristiano Lurisci, Ivan R Nabi, Daniela Corda, Marc SymonsAbstract:Synaptojanin 2 is a ubiquitously expressed polyphosphoinositide phosphatase that displays a high degree of homology in its catalytic domains with Synaptojanin 1 [1,2]. Neurons of Synaptojanin 1-deficient mice display an increase in clathrin-coated vesicles and delayed reentry of recycling vesicles into the fusion-competent vesicle pool, but no defects in early steps of endocytosis [3,4]. Here we show that inhibition of Synaptojanin 2 expression via small interfering (si) RNA causes a strong defect in clathrin-mediated receptor internalization in a lung carcinoma cell line. This inhibitory phenotype is rescued by overexpression of wild-type Synaptojanin 2, but not of wild-type Synaptojanin 1 or mutant Synaptojanin 2 that is deficient in 5'-phosphatase activity. In addition, electron-microscopic analysis shows that Synaptojanin 2 depletion causes a decrease in clathrin-coated pits and vesicles. These results suggest a role for Synaptojanin 2 in clathrin-coated pit formation and imply that lipid hydrolysis is required at an early stage of clathrin-mediated endocytosis. Taken together, our results also indicate that Synaptojanin 2 is functionally distinct from Synaptojanin 1.
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Synaptojanin 2 a novel rac1 effector that regulates clathrin mediated endocytosis
Current Biology, 2000Co-Authors: Nicole Malecz, Yayu Chuang, Peter C Mccabe, Caroline Spaargaren, Rongguo Qiu, Marc SymonsAbstract:Abstract The small GTPase Rac has been implicated in a wide range of cellular processes, including the organization of the actin cytoskeleton, transcriptional control and endocytic vesicle trafficking [1–3]. The signaling components that mediate these functions downstream of Rac largely remain to be identified. In this study, we have identified Synaptojanin 2, a polyphosphoinositide phosphatase as a novel Rac1 effector. Synaptojanin 2 directly and specifically interacts with Rac1 in a GTP-dependent manner. Expression of constitutively active Rac1 caused the translocation of Synaptojanin 2 from the cytoplasm to the plasma membrane. Both activated Rac1 and a membrane-targeted version of Synaptojanin 2 inhibited endocytosis of the epidermal growth factor (EGF) and transferrin receptors, a process that is known to be dependent on polyphosphoinositide lipids. Endocytosis of growth factor receptors is thought to play an important role in the regulation of cell proliferation. Thus, these results suggest that Synaptojanin 2 may mediate the inhibitory effect of Rac1 on endocytosis and could contribute to Rac1-mediated control of cell growth.
Yasuo Nemoto - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of a Synaptojanin 2 splice isoform predominantly expressed in nerve terminals
Journal of Biological Chemistry, 2001Co-Authors: Yasuo Nemoto, Niels Ringstad, Markus R Wenk, Kohji Takei, Laurie Daniell, Masami Watanabe, Tomoe Murakami, Hiroshi Yamada, Pietro De CamilliAbstract:We have previously identified Synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and Synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of Synaptojanin 2 that shares several properties with Synaptojanin 1. This isoform, Synaptojanin 2B, undergoes further alternative splicing to generate Synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners Synaptojanin 1, bind Synaptojanin 2B2, whereas only amphiphysin binds Synaptojanin 2B1. Sequence similar to the endophilin-binding site in Synaptojanin 1 is present only in Synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of Synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of Synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the Synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that Synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of Synaptojanin 2, but not of Synaptojanin 1. These results suggest that Synaptojanin 2B has a partially overlapping function with Synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.
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fission and uncoating of synaptic clathrin coated vesicles are perturbed by disruption of interactions with the sh3 domain of endophilin
Neuron, 2000Co-Authors: Niels Ringstad, Ole Kjaerulff, Jenny S Gustafsson, Gilbert Di Paolo, Yasuo Nemoto, John Crum, Markus R Wenk, Pietro De Camilli, Mark H. Ellisman, Oleg ShupliakovAbstract:Abstract Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve protein–protein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and Synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of Synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.
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Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling
Cell, 1999Co-Authors: Ottavio Cremona, Gilbert Di Paolo, Yasuo Nemoto, Markus R Wenk, Anita Lüthi, Warren T. Kim, Kohji Takei, Laurie Daniell, Stephen B. Shears, Richard A. FlavellAbstract:Abstract Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, Synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that Synaptojanin 1–deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P 2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.
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recruitment of an alternatively spliced form of Synaptojanin 2 to mitochondria by the interaction with the pdz domain of a mitochondrial outer membrane protein
The EMBO Journal, 1999Co-Authors: Yasuo Nemoto, Pietro De CamilliAbstract:Synaptojanin 1 is an inositol 5'-phosphatase highly enriched in nerve terminals with a putative role in recycling of synaptic vesicles. We have previously described Synaptojanin 2, which is more broadly expressed as multiple alternatively spliced forms. Here we have identified and characterized a novel mitochondrial outer membrane protein, OMP25, with a single PDZ domain that specifically binds to a unique motif in the C-terminus of Synaptojanin 2A. This motif is encoded by the exon sequence specific to Synaptojanin 2A. OMP25 mRNA is widely expressed in rat tissues. OMP25 is localized to the mitochondrial outer membrane via the C-terminal transmembrane region, with the PDZ domain facing the cytoplasm. Overexpression of OMP25 results in perinuclear clustering of mitochondria in transfected cells. This effect is mimicked by enforced expression of Synaptojanin 2A on the mitochondrial outer membrane, but not by the Synaptojanin 2A mutants lacking the inositol 5'-phosphatase domain. Our findings provide evidence that OMP25 mediates recruitment of Synaptojanin 2A to mitochondria and that modulation of inositol phospholipids by Synaptojanin 2A may play a role in maintenance of the intracellular distribution of mitochondria.
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Synaptojanin family members are implicated in endocytic membrane traffic in yeast
Journal of Cell Science, 1998Co-Authors: B Singerkruger, Yasuo Nemoto, Laurie Daniell, Susan Ferronovick, P De CamilliAbstract:The Synaptojanins represent a subfamily of inositol 5′-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched Synaptojanin, Synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three Synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for Synaptojanin family members in actin function. These findings provide a first direct evidence for a role of Synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.
Markus R Wenk - One of the best experts on this subject based on the ideXlab platform.
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Synaptojanin 1 linked phosphoinositide dyshomeostasis and cognitive deficits in mouse models of down s syndrome
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Sergey V Voronov, Silvia Giovedi, Markus R Wenk, Samuel G Frere, Elizabeth A Pollina, Christelle Borel, Hong Zhang, Cecilia Schmidt, Ellen C Akeson, Laurent CimasoniAbstract:Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a signaling phospholipid implicated in a wide variety of cellular functions. At synapses, where normal PtdIns(4,5)P(2) balance is required for proper neurotransmission, the phosphoinositide phosphatase Synaptojanin 1 is a key regulator of its metabolism. The underlying gene, SYNJ1, maps to human chromosome 21 and is thus a candidate for involvement in Down's syndrome (DS), a complex disorder resulting from the overexpression of trisomic genes. Here, we show that PtdIns(4,5)P(2) metabolism is altered in the brain of Ts65Dn mice, the most commonly used model of DS. This defect is rescued by restoring Synj1 to disomy in Ts65Dn mice and is recapitulated in transgenic mice overexpressing Synj1 from BAC constructs. These transgenic mice also exhibit deficits in performance of the Morris water maze task, suggesting that PtdIns(4,5)P(2) dyshomeostasis caused by gene dosage imbalance for Synj1 may contribute to brain dysfunction and cognitive disabilities in DS.
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insp3 mediated intracellular calcium signalling is altered by expression of Synaptojanin 1
Biochemical Journal, 2004Co-Authors: Friedrich W Johenning, Markus R Wenk, Pietro De Camilli, Per Uhlen, Brenda Degray, Eunkyung Lee, Barbara E EhrlichAbstract:Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays an important physiological role as a precursor for the InsP3-mediated intracellular calcium (Ca2+) signalling cascade. It also regulates membrane trafficking, actin function and transmembrane proteins. SJ-1 (Synaptojanin-1), a phosphoinositide phosphatase, regulates the turnover of a PtdIns(4,5)P2 pool involved in clathrin and actin dynamics at the cell surface. We tested the interrelationship of this pool with PtdIns(4,5)P2 pools involved in Ca2+ signalling by expressing in Chinese-hamster ovary cells full-length SJ-1 or its 5-Pase (inositol 5-phosphatase) domain. SJ-1 significantly attenuated the generation of Ca2+ oscillations induced by ATP and the 5-Pase domain mimicked this effect. These changes correlated with increased PtdIns(4,5)P2 phosphatase activity of cellular extracts. Overexpression of the endoplasmic reticulum-anchored PtdIns(4)P phosphatase Sac1 did not affect Ca2+ oscillations, although it increased the Ca2+ efflux rate from intracellular stores. The ability of SJ-1 to alter intracellular Ca2+ signalling indicates a close functional interrelationship between plasma membrane PtdIns(4,5)P2 pools that control actin and endocytosis and those involved in the regulation of specific spatio-temporal Ca2+ signals.
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Regulation of Synaptojanin 1 by cyclin-dependent kinase 5 at synapses.
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Markus R Wenk, Angus C. Nairn, Pietro De CamilliAbstract:Synaptojanin 1 is a polyphosphoinositide phosphatase concentrated in presynaptic nerve terminals, where it dephosphorylates a pool of phosphatidylinositol 4,5-bisphosphate implicated in synaptic vesicle recycling. Like other proteins with a role in endocytosis, Synaptojanin 1 undergoes constitutive phosphorylation in resting synapses and stimulation-dependent dephosphorylation by calcineurin. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates Synaptojanin 1 and regulates its function both in vitro and in intact synaptosomes. Cdk5 phosphorylation inhibited the inositol 5-phosphatase activity of Synaptojanin 1, whereas dephosphorylation by calcineurin stimulated such activity. The activity of Synaptojanin 1 was also stimulated by its interaction with endophilin 1, its major binding partner at the synapse. Notably, Cdk5 phosphorylated serine 1144, which is adjacent to the endophilin binding site. Mutation of serine 1144 to aspartic acid to mimic phosphorylation by Cdk5 inhibited the interaction of Synaptojanin 1 with endophilin 1. These results suggest that Cdk5 and calcineurin may have an antagonistic role in the regulation of Synaptojanin 1 recruitment and activity, and therefore in the regulation of phosphatidylinositol 4,5-bisphosphate turnover at synapses.
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identification and characterization of a Synaptojanin 2 splice isoform predominantly expressed in nerve terminals
Journal of Biological Chemistry, 2001Co-Authors: Yasuo Nemoto, Niels Ringstad, Markus R Wenk, Kohji Takei, Laurie Daniell, Masami Watanabe, Tomoe Murakami, Hiroshi Yamada, Pietro De CamilliAbstract:We have previously identified Synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and Synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of Synaptojanin 2 that shares several properties with Synaptojanin 1. This isoform, Synaptojanin 2B, undergoes further alternative splicing to generate Synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners Synaptojanin 1, bind Synaptojanin 2B2, whereas only amphiphysin binds Synaptojanin 2B1. Sequence similar to the endophilin-binding site in Synaptojanin 1 is present only in Synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of Synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of Synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the Synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that Synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of Synaptojanin 2, but not of Synaptojanin 1. These results suggest that Synaptojanin 2B has a partially overlapping function with Synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.
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a Synaptojanin homologous region of salmonella typhimurium sigd is essential for inositol phosphatase activity and akt activation
FEBS Letters, 2001Co-Authors: Sandra L Marcus, Markus R Wenk, Olivia Steelemortimer, Brett B FinlayAbstract:The Ser–Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium. The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. Here, we investigated the inositol phospholipid substrate preferences of SigD. Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5-biphosphate and phosphatidylinositol 3,4,5-triphosphate over other phosphatidylinositol lipids. Phosphatidylinositol 3-phosphate was not a substrate, suggesting the 5′ phosphate moiety is one of the preferred substrates. Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5-phosphatase Synaptojanin. Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively. The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a ΔsigD Salmonella strain. A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt. Thus, we have characterized a region near the carboxyl-terminus of SigD which is important for phosphatase activity. We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.
Gilbert Di Paolo - One of the best experts on this subject based on the ideXlab platform.
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screening assay for small molecule inhibitors of Synaptojanin 1 a synaptic phosphoinositide phosphatase
Journal of Biomolecular Screening, 2014Co-Authors: Laura Beth J Mcintire, Gilbert Di Paolo, Kyuin Lee, Belle Changileto, Taewan KimAbstract:Elevation of amyloid β-peptide (Aβ) is critically associated with Alzheimer disease (AD) pathogenesis. Aβ-induced synaptic abnormalities, including altered receptor trafficking and synapse loss, have been linked to cognitive deficits in AD. Recent work implicates a lipid critical for neuronal function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], in Aβ-induced synaptic and behavioral impairments. Synaptojanin 1 (Synj1), a lipid phosphatase mediating the breakdown of PI(4,5)P 2 , has been shown to play a role in synaptic vesicle recycling and receptor trafficking in neurons. Heterozygous deletion of Synj1 protected neurons from Aβ-induced synaptic loss and restored learning and memory in a mouse model of AD. Thus, inhibition of Synj1 may ameliorate Aβ-associated impairments, suggesting Synj1 as a potential therapeutic target. To this end, we developed a screening assay for Synj1 based on detection of inorganic phosphate liberation from a water-soluble, short-chain PI(4,5)P 2 . The assay displayed saturable kinetics and detected Synj1’s substrate preference for PI(4,5)P 2 over PI(3,4,5)P 3 . The assay will enable identification of novel Synj1 inhibitors that have potential utility as chemical probes to dissect the cellular role of Synj1 as well as potential to prevent or reverse AD-associated synaptic abnormalities.
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the sac1 domain of synj1 identified mutated in a family with early onset progressive parkinsonism with generalized seizures
Human Mutation, 2013Co-Authors: Catharine E. Krebs, Siamak Karkheiran, Hossein Darvish, Gholam Ali Shahidi, Joseph D. Buxbaum, Gilbert Di Paolo, Vladimir Makarov, James C Powell, Ruth H. Walker, Pietro De CamilliAbstract:This study aimed to elucidate the genetic causes underlying early-onset Parkinsonism (EOP) in a consanguineous Iranian family. To attain this, homozygosity mapping and whole-exome sequencing were performed. As a result, a homozygous mutation (c.773G>A; p.Arg258Gln) lying within the NH2-terminal Sac1-like inositol phosphatase domain of polyphosphoinositide phosphatase Synaptojanin 1 (SYNJ1), which has been implicated in the regulation of endocytic traffic at synapses, was identified as the disease-segregating mutation. This mutation impaired the phosphatase activity of SYNJ1 against its Sac1 domain substrates in vitro. We concluded that the SYNJ1 mutation identified here is responsible for the EOP phenotype seen in our patients probably due to deficiencies in its phosphatase activity and consequent impairment of its synaptic functions. Our finding not only opens new avenues of investigation in the synaptic dysfunction mechanisms associated with Parkinsonism, but also suggests phosphoinositide metabolism as a novel therapeutic target for Parkinsonism.
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Synaptojanin 1 mediated pi 4 5 p2 hydrolysis is modulated by membrane curvature and facilitates membrane fission
Developmental Cell, 2011Co-Authors: Belle Changileto, Sergey V Voronov, Samuel G Frere, Robin B Chan, Aurelien Roux, Gilbert Di PaoloAbstract:Summary Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] plays a fundamental role in clathrin-mediated endocytosis. However, precisely how PI(4,5)P 2 metabolism is spatially and temporally regulated during membrane internalization and the functional consequences of endocytosis-coupled PI(4,5)P 2 dephosphorylation remain to be explored. Using cell-free assays with liposomes of varying diameters, we show that the major synaptic phosphoinositide phosphatase, Synaptojanin 1 (Synj1), acts with membrane curvature generators/sensors, such as the BAR protein endophilin, to preferentially remove PI(4,5)P 2 from curved membranes as opposed to relatively flat ones. Moreover, in vivo recruitment of Synj1's inositol 5-phosphatase domain to endophilin-induced membrane tubules results in fragmentation and condensation of these structures largely in a dynamin-dependent fashion. Our study raises the possibility that geometry-based mechanisms may contribute to spatially restricting PI(4,5)P 2 elimination during membrane internalization and suggests that the PI(4,5)P 2 -to-PI4P conversion achieved by Synj1 at sites of high curvature may cooperate with dynamin to achieve membrane fission.
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delayed reentry of recycling vesicles into the fusion competent synaptic vesicle pool in Synaptojanin 1 knockout mice
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Warren T. Kim, Ottavio Cremona, Gilbert Di Paolo, Laurie Daniell, Sunghoe Chang, Pietro De CamilliAbstract:Synaptojanin 1 is a polyphosphoinositide phosphatase implicated in synaptic vesicle recycling. We used FM1-43 imaging and electron microscopy in cultured cortical neurons from control and Synaptojanin 1 knockout mice to study how the absence of this protein affects specific steps of the synaptic vesicle cycle. Exo/endocytosis after a moderate stimulus was unchanged. However, during prolonged stimulation, the regeneration of fusion-competent synaptic vesicles was severely impaired. In stimulated nerve terminals, there was a persistent accumulation of clathrin-coated vesicles and a backup of newly reformed vesicles in the cytomatrix-rich area around the synaptic vesicle cluster. These findings demonstrate that Synaptojanin 1 function is needed for the progression of recycling vesicles to the functional synaptic vesicle pool.
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fission and uncoating of synaptic clathrin coated vesicles are perturbed by disruption of interactions with the sh3 domain of endophilin
Neuron, 2000Co-Authors: Niels Ringstad, Ole Kjaerulff, Jenny S Gustafsson, Gilbert Di Paolo, Yasuo Nemoto, John Crum, Markus R Wenk, Pietro De Camilli, Mark H. Ellisman, Oleg ShupliakovAbstract:Abstract Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve protein–protein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and Synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of Synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.
