Microtubules are fibrous structures in the cytoplasm of eukaryotic cells where they play a vital role in cell morphogenesis organization and motility. In mitotic cells microtubules are centrally involved in the mechanics and control of cell division. Microtubules are key components of the mitotic spindle which is the machinery used by eukaryotic cells to segregate chromosomes during mitosis.
There is strong evidence that abnormalities of elements of the mitotic machinery are major factors favoring genomic instability and tumor progression in human cancers. Microtubules are also important for cytoplasmic organization and organelle trafficking in interphasic cells. These microtubule functions are conspicuous in neurons, whose asymmetry can be extreme with cell extensions at the metric scale.
STOP proteins
 Introduction |
| STOP proteins |
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Neurons contain abundant subpopulations of stable microtubules that resist depolymerising conditions such as exposure to cold temperature and to depolymerising drugs [Baas et al., 1991 ; Guillaud et al., 1998 ; Job and Margolis, 1984]. In neurons, microtubule stabilization is mainly due to microtubule association with a family of proteins known as STOPs (Stable Tubule Only Polypeptides). STOP proteins were initially characterized as microtubule cold-stabilizing factors whose activity was inhibited by interaction with Ca2+-calmodulin [Job et al., 1981] and subsequent work has shown that STOPs contain bi-functional modules comprised of overlapping calmodulin-binding and microtubule-stabilizing sequences [Bosc et al., 2001]. Neurons contain two major variants of STOP, E-STOP (89 kD) and N-STOP (116 kD). E-STOP is present in mice brain from embryonic stage E16 to adulthood, whereas N-STOP appears at birth and is subsequently expressed in the adult brain [Guillaud et al., 1998 ; Job and Margolis, 1984 ; Job et al., 1981 ; Bosc et al., 2001 ; Bosc et al., 1996].
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 STOP deficient mice |
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STOP function has been investigated in whole animals by studying STOP null mice [Andrieux et al., 2002]. In these mice, microtubule cold stability is suppressed, with no dramatic consequences for mouse organogenesis, viability or brain anatomy. However, STOP -/- mice display multiple synaptic deficits that affect both long- and short-term synaptic plasticity in the hippocampus. These synaptic defects are associated with depleted vesicular pools in glutamatergic nerve terminals and with severe behavioral disorders [Andrieux et al., 2002]. The dopaminergic (DA) status of STOP null mice has been investigated by the M. F. Suaud Chagny team (U512, Lyon) , at both the behavioral and the neurochemical levels. STOP null mice consistently showed a pronounced increase in locomotor activity in basal conditions, following a mild stress or after amphetamine injection. We showed that this hyper locomotor reactivity is associated with an increased DA transmission in the limbic system. The DA transmission alterations involved increased DA efflux evoked by electrical stimulations mimicking physiological stimuli, in the absence of basal abnormalities [Brun et al., 2005]. STOP null mice also exhibit an altered Prepulse inhibition (PPI), alteration observed in schizophrenic patients and thought to reflect a dysfunction of conserved sensori-motor gating mechanisms [Fradley et al., 2005]. Remarkably, neuroleptics treatment specifically leads to a rescue of the Post Tetanic Potentiation (PTP) within glutamatergic neurons, to a partial rescue of the synaptic vesicle pool and to an improvement of the maternal behaviour sufficient to allow pups survival [Andrieux et al., 2002 ; Brun et al., 2005 ; Fradley et al., 2005].
Altogether, these results demonstrate that primary synaptic defects affecting cytoskeletal proteins can cause a combination of neurotransmission disorders that have been expected to occur in human psychosis and more precisely in schizophrenia. In agreement with this hypothesis, the recently reported gene associated with schizophrenia Disrupted-In-Schizophrenia 1 (DISC1) has been described as a multifunctional protein acting on microtubule or microtubule-related components [Morris et al., 2003]. We have thus proposed that microtubules may represent a novel therapeutic target for anti-psychotic agents. As a test for this hypothesis, we have investigated the ability of one class of microtubule stabilizing drugs, the epothilones, to alleviate null mice behavioral and synaptic defects. Epothilone are anti-mitotic drugs recently develop by pharmaceutical companies for anti cancer therapy [Galsky et al., 2005 ; Kolman 2004 ; Kolman 2004 ; Wang et al., 2005]. We found that epothilone D is able to alleviate STOP KO mice disorders when used 10 to 100 times less than the dosage use in human cancer therapy. Thus, epothilone can act on synaptic plasticity and on animal behavior via its microtubule stabilizing properties. Based on these pharmacological results we patented the use of epothilones for neuronal disorders. |
| STOP proteins and synapses |
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STOP KO mice have indicated a probable and intriguing role of STOP at the synapses. Indeed, it has been very surprising that a protein associated with microtubules along the whole neurite turns out to be important for synaptic function, despite apparent microtubule absence in nerve terminals. There is however, biochemical and proteomic evidence that STOP localizes to synapses and this has raised questions concerning the mechanisms that could promote STOP dissociation from microtubules and re-localization in synaptic structures. Long-term plasticity events, which are severely impaired in STOP null mice, are known to involve the calcium/calmodulin-dependent protein kinase II (CaMKII) and we had previous evidence for the presence of potential CaMKII phosphorylation sites on STOP. Thus, we investigate the potential phosphorylation of STOP by CaMKII. We showed that STOP is phosphorylated by CaMKII on at least three independent sites (S139, S198, S491), both in vitro and in vivo and that phosphorylated STOP do not bind to microtubules in vitro. The phosphorylated forms of STOP co-localize with actin-rich structures in cultured neurons and bind to polymerized F-actin in vitro. Thus, STOP phosphorylation by CaMKII may allow STOP association with synaptic actin and be important for synaptic plasticity (PhD thesis of Julie Baratier, 2002-2004, directed by Annie Andrieux. Baratier et al., 2006).
Julie Baratier.Role and regulation of STOP proteins in synaptic plasticity. [Abstract] As an effort to understand STOP function at the synapse, we searched for STOP partners using the two-hybrid system. A large screen using several fragments of STOP protein and several cDNA libraries lead to the identification of only two potential partners, Tctex-1 and Arc proteins. TcTex-1 is one of three dynein light chains of the dynein motor complex and has been implicated in targeting and binding cargoes to cytoplasmic dynein for retrograde or apical transport [Chuang et al., 2001]. Arc, activity-regulated cytoskeleton-associated gene, is an immediate early gene, involved in LTP and other forms of neuroplasticity [Lyford et al., 1995 ; Steward et al., 2001]. In a search for proteins sharing at least one conserved module with STOP we identify of a mammalian protein containing a microtubule-binding domain. In addition, the protein N-terminus is very similar to that of N-STOP and comprises the calmodulin-binding motif Cam1. We have called this protein SL21, for 21 kDa STOP-like protein and we performed molecular and cellular characterization. Surprisingly, in cultured neurons, SL21 mainly associates with the somatic Golgi and with punctuated material in neurites, despite combined in vitro and in vivo evidence that SL21 has calmodulin-binding and microtubule-stabilizing activity, similar to STOPs. We show that the Golgi-targeting sequence of SL21 is located within the N-terminal domain that SL21 shares with STOPs, and that Golgi targeting most probably requires SL21 palmitoylation. We find that deletion mutants of N-STOP, containing the N-terminal domain of N-STOP but lacking microtubule stabilizing modules, are targeted to the Golgi apparatus and that the preferential localization of STOP or of STOP mutants on microtubules depends on the presence of at least two microtubule stabilizing modules in the peptide chain. Recent studies indicate the presence of Golgi material in neurites and in nerve terminals, which may be important for synaptic plasticity. Thus, the STOP protein family, which has capacity to couple Golgi vesicles with the neuronal cytoskeleton, may be important for Golgi trafficking in neurites (Gory-Fauré et al., 2006). |
| References |
| Fournet V, Schweitzer A, Chevarin C, Deloulme JC, Hamon M, Giros B, Andrieux A, Martres MP The deletion of STOP/MAP6 protein in mice triggers highly altered mood and impaired cognitive performances. Journal of Neurochemistry, 2012, 121(1): 99-114 Benardais K, Kasem B, Couegnas A, Samama B, Fernandez S, Schaeffer C, Antal MC, Job D, Schweitzer A, Andrieux A, Giersch A, Nehlig A and Boehm N Loss of STOP protein impairs peripheral olfactory neurogenesis. PLoS One, 2010, 5(9): e12753 Charlet A, Muller AH, Laux A, Kemmel V, Schweitzer A, Deloulme JC, Stuber D, Delalande F, Bianchi E, Van Dorsselaer A, Aunis D, Andrieux A, Poisbeau P and Goumon Y Abnormal nociception and opiate sensitivity of STOP null mice exhibiting elevated levels of the endogenous alkaloid morphine. Molecular Pain, 2010, 6: 96-109 Delotterie D, Ruiz G, Brocard J, Schweitzer A, Roucard C, Roche Y, Suaud-Chagny MF, Bressand K and Andrieux A Chronic administration of atypical antipsychotics improves behavioral and synaptic defects of STOP null mice. Psychopharmacology (Berl), 2010, 208(1): 131-141 Fournet V, Jany M, Fabre V, Chali F, Orsal D, Schweitzer A, Andrieux A, Messanvi F, Giros B, Hamon M, Lanfumey L, Deloulme JC and Martres MP The deletion of the microtubule-associated STOP protein affects the serotonergic mouse brain network. Journal of Neurochemistry, 2010, 115(6): 1579-1594 Merenlender-Wagner A, Pikman R, Giladi E, Andrieux A and Gozes I NAP (davunetide) enhances cognitive behavior in the STOP heterozygous mouse-A microtubule-deficient model of schizophrenia. Peptides, 2010, 31(7): 1368-1373 Richard M, Sacquet J, Schweitzer A, Jourdan F, Andrieux A and Pellier-Monnin V STOP proteins contribute to the maturation of the olfactory system. Molecular and Cellular Neuroscience, 2009, 41(2): 120-134 Bégou M, Volle J, Bertrand JB, Brun P, Job D, Schweitzer A, Saoud M, d'Amato T, Andrieux A and Suaud-Chagny MF The STOP null mice model for schizophrenia display cognitive and social deficits partly alleviated by neuroleptics. Neuroscience, 2008, 157(1): 29-39 Bouvrais-Veret C, Weiss S, Hanoun N, Andrieux A, Schweitzer A, Job D, Hamon M, Giros B and Martres MP Microtubule-associated STOP protein deletion triggers restricted changes in dopaminergic neurotransmission. Journal of Neurochemistry, 2008, 104(3): 745-756 Hanaya R, Koning E, Ferrandon A, Schweitzer A, Andrieux A and Nehlig A Deletion of the STOP gene, a microtubule stabilizing factor, leads only to discrete cerebral metabolic changes in mice. Journal of Neurosciense Research, 2007, 86(4): 813-820 Bouvrais-Veret C, Weiss S, Andrieux A, Schweitzer A, McIntosh JM, Job D, Giros B and Martres MP Sustained increase of alpha7 nicotinic receptors and choline-induced improvement of learning deficit in STOP knock-out mice. Neuropharmacology, 2007, 52(8): 1691-1700 Brenner E, Sonnewald U, Schweitzer A, Andrieux A, Nehlig A Hypoglutamatergic activity in the STOP knockout mouse: A potential model for chronic untreated schizophrenia. Journal of Neuroscience Research, 2007, 85(15):3487-3493 Eastwood SL, Lyon L, George L, Andrieux A, Job D and Harrison PJ Altered expression of synaptic protein mRNAs in STOP (MAP-6) mutant mice. Journal of Psychopharmacology, 2007, 21: 635-344 Powell KJ, Hori SE, Leslie R, Andrieux A, Schellinck H, Thorne M and Robertson GS Cognitive impairments in the STOP null mouse model of schizophrenia. Behavioral Neuroscience, 2007, 121(5): 826-835 Baratier J, Peris L, Brocard J, Gory-Faure S, Dufour F, Bosc C, Fourest-Lieuvin A, Blanchoin L, Salin P, Job D and Andrieux A Phosphorylation of microtubule-associated protein STOP by calmodulin kinase II. Journal of Biological Chemistry, 2006, 281(28): 19561-19569 Gory-Faure S, Windscheid V, Bosc C, Peris L, Proietto D, Franck R, Denarier E, Job D and Andrieux A STOP-like protein 21 is a novel member of the STOP family, revealing a Golgi localization of STOP proteins. Journal of Biological Chemistry, 2006, 281(38): 28387-28396 Brun P, Begou M, Andrieux A, Mouly-Badina L, Clerget M, Schweitzer A, Scarna H, Renaud B, Job D and Suaud-Chagny MF Dopaminergic transmission in STOP null mice. Journal of Neurochemistry, 2005, 94(1): 63-73 Fradley RL, O'Meara GF, Newman RJ, Andrieux A, Job D and Reynolds DS STOP knockout and NMDA NR1 hypomorphic mice exhibit deficits in sensorimotor gating. Behavioural Brain Research, 2005, 163(2): 257-264 Galsky MD, Small EJ, Oh WK, Chen I, Smith DC, Colevas AD, Martone L, Curley T, Delacruz A, Scher HI and Kelly WK Multi-institutional randomized phase II trial of the epothilone B analog ixabepilone (BMS-247550) with or without estramustine phosphate in patients with progressive castrate metastatic prostate cancer. Journal of Clinical Oncology, 2005, 23(7): 1439-1446 Wang H et al. Cancer Chemother Pharmacol., 2005 Kolman A Current Opinion in Investigational Drugs, 2004, 5(12): 1292-1297 Kolman A Epothilone D (Kosan/Roche). Current Opinion in Investigational Drugs, 2004, 5(6): 657-667 Morris JA, Kandpal G, Ma L and Austin CP DISC1 (Disrupted-In-Schizophrenia 1) is a centrosome-associated protein that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: Regulation and loss of interaction with mutation. Human Molecular Genetics, 2003, 12(13): 1591-1608 Andrieux A, Salin PA, Vernet M, Kujala P, Baratier J, Gory-Faure S, Bosc C, Pointu H, Proietto D, Schweitzer A, Denarier E, Klumperman J and Job D The suppression of brain cold-stable microtubules in mice induces synaptic defects associated with neuroleptic-sensitive behavioral disorders. Genes & Developement, 2002, 16(18): 2350-2364 Bosc C, Frank R, Denarier E, Ronjat M, Schweitzer A, Wehland J and Job D Identification of novel bifunctional calmodulin-binding and microtubule-stabilizing motifs in STOP proteins. Journal of Biological Chemistry, 2001, 276(33): 30904-30913 Chuang JZ, Milner TA and Sung CH Subunit heterogeneity of cytoplasmic dynein: Differential expression of 14 kDa dynein light chains in rat hippocampus. Journal of Neuroscience, 2001, 21(15): 5501-5512 Steward O and Worley PF Selective targeting of newly synthesized Arc mRNA to active synapses requires NMDA receptor activation. Neuron, 2001, 30(1): 227-240 Lyford GL, Yamagata K, Kaufmann WE, Barnes CA, Sanders LK, Copeland NG, Gilbert DJ, Jenkins NA, Lanahan AA and Worley PF Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites. Neuron. 1995, 14(2): 433-445 Guillaud L, Bosc C, Fourest-Lieuvin A, Denarier E, Pirollet F, Lafanechere L and Job D STOP proteins are responsible for the high degree of microtubule stabilization observed in neuronal cells. Journal of Cellular Biology, 1998, 142(1): 167-179 Bosc C, Cronk JD, Pirollet F, Watterson DM, Haiech J, Job D and Margolis RL Cloning, expression, and properties of the microtubule-stabilizing protein STOP. Proceedings of the National Academy of Science USA, 1996, 93(5): 2125-2130 Baas PW, Slaughter T, Brown A and Black MM Microtubule dynamics in axons and dendrites. Journal of Neuroscience Researches, 1991, 30(1):134-153 Job D and Margolis RL Isolation from bovine brain of a superstable microtubule subpopulation with microtubule seeding activity. Biochemistry, 1984, 23(13): 3025-3031 Job D, Fischer EH and Margolis RL Rapid disassembly of cold-stable microtubules by calmodulin. Proceedings of the National Academy of Sciences USA, 1981, 78(8): 4679-4682 |