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Mary Reid		Posted: Thursday, February 02, 2012 5:17:04 AM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		The study directly above seems very important. The full text link here, but I'll post a specific part regarding ALS. Mary http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104322/?tool=pubmed Axonal degeneration caused by Dctn1 dysfunction can be inhibited by Nmnat or caspase 6 inhibition The APP/DR6/caspase 6 signaling pathway plays an important role in NGF-deprivation mediated axonal degeneration. Mutations that disrupt retrograde axonal transport are often associated with neurological disorders in which axonal degeneration is an important component. Dctn1 is a component of the dynactin complex that associates with dynein to drive retrograde axonal transport of multiple cargoes along the microtubule lattice (LaMonte et al., 2002, Schroer, 2004). Mutations in DCTN1 are associated with motor neuron degenerative disorders, ALS associated with frontotemporal dementia (FTD), and Perry syndrome, which has symptoms of Parkinsonism (Puls et al., 2003, 2005, Munch et al., 2004, 2005; Wider and Wszolek, 2008; Farrer et al., 2009; Vilarino-Guell et al., 2009). These mutations impair the transport of trophic factor back to the neuronal cell body (Laird et al., 2008), leading to speculation that neurodegeneration in these patients is due to lack of trophic factors in the soma (Puls et al., 2005). Disruption of dynein function leads to decreased NGF transport and increased death in cultured DRG neurons (Wu et al., 2007). We therefore tested whether depletion of Dctn1 induces axonal degeneration via activation of the APP/DR6/caspase 6 pathway. DRG neurons were infected with lentiviruses expressing Dctn1 siRNA constructs. We constructed many and extensively used three different Dctn1 siRNAs in these studies. The efficiency of knockdown for these three siRNAs was quantified using real-time RT-PCR analysis. We found that Dctn1-1 and Dctn1-2 siRNAs reduced Dctn1 mRNA levels by 96% and 90%, respectively; whereas Dctn1-3 siRNA resulted in only a 15% reduction in Dctn1 mRNA (Fig. S6). DRG neurons were infected with lentiviruses expressing each of these siRNAs constructs a time when they are critically dependent on NGF (DIV1). DRG neurons infected with Dctn1-1 or Dctn-2 siRNA showed extensive axon and soma degeneration, whereas those infected with Dctn1-3 siRNA were indistinguishable from neurons expressing a Luciferase siRNA (Fig. 6A-D). Bcl-xl overexpression blocked both axonal and somal degeneration (Fig. 6A-D), whereas cytNmnat1 blocked only axonal degeneration in Dctn1-deficient neurons. These results were similar to those obtained with neurons after NGF withdrawal, suggesting that the mechanism of neuronal damage caused by Dctn1 knockdown and trophic factor deprivation is similar. To assess whether degeneration induced by Dctn1 knockdown is due to NGF transport deficits, we measured NGF internalization (see Materials and Methods) and found that depletion of Dctn1 decreases the amount of NGF that is internalized (Fig. 7A, B). Indeed, the magnitude of the NGF transport defect with each Dctn siRNA was directly correlated with the amount of Dctn1 knockdown and the extent of axon and soma damage. The axonal degeneration, along with the NGF trafficking defects, observed in neurons deficient in Dctn1 prompted us to examine the APP/DR6/caspase 6 pathway in these neurons. We found that, like NGF withdrawal, Dctn1 knockdown initiated loss of surface APP that could be blocked by BACE inhibition (Fig. 8A, B). However, in contrast to axon degeneration after NGF withdrawal, BACE inhibition or APP blocking antibody did not block Dctn1 induced axonal degeneration (Fig. 8C). This is particularly intriguing as caspase 6 inhibition was effective in blocking axonal degeneration in Dctn1-deficient neurons. Moreover, Bcl-xl overexpression blocked both axon degeneration and cell death in this paradigm (Fig. 6). The surprising dichotomy between blockade of APP surface loss and caspase 6 inhibition prompted us to examine caspase 6 activation in neurons expressing Dctn1 siRNA. We found that caspase 6 was activated in neurons lacking Dctn1 even in the presence of BACE inhibitors (Fig. 8D), indicating that caspase 6 can be activated independently of APP cleavage. Together, these results suggest that deficits in axonal transport cause axonal degeneration via mechanisms that are similar to those invoked by trophic factor deprivation as they both involve caspase 6 activation. However, since caspase 6 is activated independent of loss of surface APP in Dctn-deficient neurons, it appears that multiple pathways are capable of activating this enzyme and inducing axonal degeneration.
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Mary Reid		Posted: Saturday, February 04, 2012 5:22:59 AM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Mary Reid wrote: TDP-43 interacts with ARF6. ARF6 involved in cytokinesis. Mutations in CHMP2B cause ALS. CHMP2B also implicated in cytokinesis. Mutations in p150glued cause ALS p150glued implicated in cytokinesis. The authors of one study below suggest disrupted cytokinesis in SOD1 deficiency. Mutations in FIG4 may also cause ALS. Reduced PIP2 levels have been reported in FIG4 mutant mice. PIP2 activates WASP which is part of the cytokinesis machinery. By the way, an interesting last study here concerning PIP2 and the neuromuscular junction. NEDD5 is essential for cytokinesis, Autoantibodies to NEDD5 are reported in systemic sclerosis. Mary PMCID: PMC2951409 http://www.jbc.org/content/279/43/44756.full In addition to RhoA, Rac1, and Cdc42 have also been implicated in the cytokinesis of mammalian cells, based on the appearance of multinucleated cells among cells expressing constitutively active Rac1 or Cdc42 (14, 15). "TDP-43 loss of function in the pathogenesis of neurodegeneration. Here we show that the knockdown of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knockdown cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated, and membrane localization of these molecules was reduced." J Biol Chem. 2009 Aug 14;284(33):22059-66. Epub 2009 Jun 17. TDP-43 depletion induces neuronal cell damage through dysregulation of Rho family GTPases. Iguchi Y, Katsuno M, Niwa J, Yamada S, Sone J, Waza M, Adachi H, Tanaka F, Nagata K, Arimura N, Watanabe T, Kaibuchi K, Sobue G. Source Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan. Abstract The 43-kDa TAR DNA-binding protein (TDP-43) is known to be a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss of function in the pathogenesis of neurodegeneration. Here we show that the knockdown of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knockdown cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated, and membrane localization of these molecules was reduced. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation, a key regulating factor of Rho family activity and intracellular localization. In contrast, overexpression of TDP-43 mitigated the cellular damage caused by pharmacological inhibition of geranylgeranylation. Furthermore administration of geranylgeranyl pyrophosphate partially restored cell viability and neurite outgrowth in TDP-43 knockdown cells. In summary, our data suggest that TDP-43 plays a key role in the maintenance of neuronal cell morphology and survival possibly through protein geranylgeranylation of Rho family GTPases. PMID: 19535326 [PubMed - indexed for MEDLINE] PMCID: PMC2755930
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Mary Reid		Posted: Saturday, February 04, 2012 5:53:56 AM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Mary Reid wrote: NEDD5 is essential for cytokinesis, Autoantibodies to NEDD5 are reported in systemic sclerosis. Mutations in SEPT9 cause hereditary neuralgic amyotrophy. J Cell Biol. 2010 Nov 15;191(4):741-9. Epub 2010 Nov 8. Distinct roles of septins in cytokinesis: SEPT9 mediates midbody abscission. Estey MP, Di Ciano-Oliveira C, Froese CD, Bejide MT, Trimble WS. Source Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. Abstract Septins are a family of GTP-binding proteins implicated in mammalian cell division. Most studies examining the role of septins in this process have treated the family as a whole, thus neglecting the possibility that individual members may have diverse functions. To address this, we individually depleted each septin family member expressed in HeLa cells by siRNA and assayed for defects in cell division by immunofluorescence and time-lapse microscopy. Depletion of SEPT2, SEPT7, and SEPT11 causes defects in the early stages of cytokinesis, ultimately resulting in binucleation. In sharp contrast, SEPT9 is dispensable for the early stages of cell division, but is critical for the final separation of daughter cells. Rescue experiments indicate that SEPT9 isoforms containing the N-terminal region are sufficient to drive cytokinesis. We demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody, providing mechanistic insight into the role of SEPT9 during abscission. PMID: 21059847 [PubMed - indexed for MEDLINE] PMCID: PMC2983063
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Mary Reid		Posted: Saturday, February 04, 2012 6:06:31 AM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Mary Reid wrote: Mutations in FIG4 may also cause ALS. Reduced PIP2 levels have been reported in FIG4 mutant mice. PIP2 activates WASP which is part of the cytokinesis machinery. By the way, an interesting last study here concerning PIP2 and the neuromuscular junction. PMCID: PMC2951409 RAB35 is another gene involved in cytokinesis and neuromuscular junction function. Perhaps a further connection with PIP2 and ALS. "Rab35 is involved in the intercellular bridge localization of two molecules essential for the postfurrowing steps of cytokinesis: the phosphatidylinositol 4,5-bis phosphate (PIP2) lipid and the septin SEPT2." " Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity Cell. 2011 Apr 1;145(1):117-32. Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins. Uytterhoeven V, Kuenen S, Kasprowicz J, Miskiewicz K, Verstreken P. Source VIB, Department of Molecular and Developmental Genetics, Program in Molecular and Developmental Genetics, Program in Cognitive and Molecular Neuroscience, Laboratory of Neuronal Communication, Herestraat 49, 3000 Leuven, Belgium. Abstract Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release. Copyright © 2011 Elsevier Inc. All rights reserved. PMID: 21458671 [PubMed - indexed for MEDLINE] Curr Biol. 2006 Sep 5;16(17):1719-25. Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis. Kouranti I, Sachse M, Arouche N, Goud B, Echard A. Source Laboratoire Mécanismes moléculaires du transport intracellulaire, Institut Curie, Centre National de la Recherche Scientifique UMR144, 26 rue d'Ulm, 75248 Paris cedex 05, France. Abstract Cytokinesis is the final step of cell division and leads to the physical separation of the daughter cells. After the ingression of a cleavage membrane furrow that pinches the mother cell, future daughter cells spend much of the cytokinesis phase connected by an intercellular bridge. Rab proteins are major regulators of intracellular transport in eukaryotes, and here, we report an essential role for human Rab35 in both the stability of the bridge and its final abscission. We find that Rab35, whose function in membrane traffic was unknown, is localized to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway. Consistent with a key requirement for Rab35-regulated recycling during cell division, inhibition of Rab35 function leads to the accumulation of endocytic markers on numerous cytoplasmic vacuoles in cells that failed cytokinesis. Moreover, Rab35 is involved in the intercellular bridge localization of two molecules essential for the postfurrowing steps of cytokinesis: the phosphatidylinositol 4,5-bis phosphate (PIP2) lipid and the septin SEPT2. We propose that the Rab35-regulated pathway plays an essential role during the terminal steps of cytokinesis by controlling septin and PIP2 subcellular distribution during cell division. PMID: 16950109 [PubMed - indexed for MEDLINE]
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Mary Reid		Posted: Saturday, February 04, 2012 7:50:26 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		FEBS Lett. 2009 Apr 2;583(7):1096-101. Epub 2009 Mar 14. Rab35 regulates neurite outgrowth and cell shape. Chevallier J, Koop C, Srivastava A, Petrie RJ, Lamarche-Vane N, Presley JF. Source Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada. Abstract Recent studies have identified Rab35 in the endocytic pathway and as a regulator of cytokinesis; however its molecular mechanisms are currently unknown. Here, we find that Rab35 colocalizes with actin filaments and with Cdc42, Rac1 and RhoA, and that Rab35 can activate Cdc42 both in vivo and in vitro. We find activated Rab35 stimulates neurite outgrowth in PC12 and N1E-115 cells via a Cdc42-dependent pathway and that siRNA knockdown of Rab35 activity abolishes neurite outgrowth in these cell lines. We conclude that one function of Rab35 is to regulate Rho-family GTPases and that this role has consequences for neurite outgrowth. PMID: 19289122 [PubMed - indexed for MEDLINE] J Biol Chem. 2005 Oct 14;280(41):34735-40. Epub 2005 Jul 26. ALS2/Alsin regulates Rac-PAK signaling and neurite outgrowth. Tudor EL, Perkinton MS, Schmidt A, Ackerley S, Brownlees J, Jacobsen NJ, Byers HL, Ward M, Hall A, Leigh PN, Shaw CE, McLoughlin DM, Miller CC. Source Departments of Neuroscience and Neurology, Institute of Psychiatry, Kings College, London SE5 8AF, United Kingdom. Abstract Rac and its downstream effectors p21-activated kinase (PAK) family kinases regulate actin dynamics within growth cones to control neurite outgrowth during development. The activity of Rac is stimulated by guanine nucleotide exchange factors (GEFs) that promote GDP release and GTP binding. ALS2/Alsin is a recently described GEF that contains a central domain that is predicted to regulate the activities of Rac and/or Rho and Cdc42 activities. Mutations in ALS2 cause some recessive familial forms of amyotrophic lateral sclerosis (ALS) but the function of ALS2 is poorly understood. Here we demonstrate that ALS2 is present within growth cones of neurons, in which it co-localizes with Rac. Furthermore, ALS2 stimulates Rac but not Rho or Cdc42 activities, and this induces a corresponding increase in PAK1 activity. Finally, we demonstrate that ALS2 promotes neurite outgrowth. Defects in these functions may therefore contribute to motor neuron demise in ALS. PMID: 16049005 [PubMed - indexed for MEDLINE]
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Mary Reid		Posted: Sunday, February 05, 2012 5:33:17 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Mutations in C9ORF72 cause amyotrophic lateral sclerosis and frontotemporal dementia. RB1CC1 interacts with C9ORF72 as well as DOCK7 (dedicator of cytokinesis 7) http://www.genecards.org/cgi-bin/carddisp.pl?gene=DOCK7 UniProtKB/Swiss-Prot: DOCK7_HUMAN, Q96N67 Function: Functions as a guanine nucleotide exchange factor (GEF), which activates Rac1 and Rac3 Rho small GTPases by exchanging bound GDP for free GTP. Does not have a GEF activity for CDC42. Required for STMN1 'Ser-15' phosphorylation during axon formation and consequently for neuronal polarization Gene Wiki entry for DOCK7 (Dock7)
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Mary Reid		Posted: Sunday, February 05, 2012 5:38:42 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		DOCK7 is necessary for the phosphorylation of stathmin. (See above) Am J Pathol. 2004 Nov;165(5):1701-18. Dysregulation of stathmin, a microtubule-destabilizing protein, and up-regulation of Hsp25, Hsp27, and the antioxidant peroxiredoxin 6 in a mouse model of familial amyotrophic lateral sclerosis. Strey CW, Spellman D, Stieber A, Gonatas JO, Wang X, Lambris JD, Gonatas NK. SourceUniversity of Pennsylvania Medical Center, 609 Stellar-Chance Labs., 422 Curie Blvd., Philadelphia, PA, 19104-6100, USA. Abstract Gain-of-function mutations of the Cu/Zn superoxide dismutase (SOD1) gene cause dominantly inherited familial amyotrophic lateral sclerosis. The identification of differentially regulated proteins in spinal cords of paralyzed mice expressing SOD1(G93A) may contribute to understanding mechanisms of toxicity by mutant SOD1. Protein profiling showed dysregulation of Stathmin with a marked decrease of its most acidic and phosphorylated isoform, and up-regulation of heat shock proteins 25 and 27, peroxiredoxin 6, phosphatidylinositol transfer protein-alpha, apolipoprotein E, and ferritin heavy chain. Stathmin accumulated in the cytoplasm of 30% of spinal cord motor neurons with fragmented Golgi apparatus. Overexpression of Stathmin in HeLa cells was associated with collapse of microtubule networks and Golgi fragmentation. These results, together with the decrease of one Stathmin isoform, suggest a role of the protein in Golgi fragmentation. Mutant SOD1 co-precipitated and co-localized with Hsp25 in neurons and astrocytes. Mutant SOD1 may thus deprive cells of the anti-apoptotic and other protective activities of Hsp25. Astrocytes contained peroxiredoxin 6, a unique nonredundant antioxidant. The up-regulation of peroxiredoxin 6 probably constitutes a defense to oxidative stress induced by SOD1(G93A). Direct effects of SOD1(G93A) or sequential reactions triggered by the mutant may cause the protein changes. PMID:15509539[PubMed - indexed for MEDLINE] PMCID: PMC1618651
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Mary Reid		Posted: Sunday, February 05, 2012 5:44:06 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		J Cell Biochem. 2004 Oct 1;93(2):242-50. The role of stathmin in the regulation of the cell cycle. Rubin CI, Atweh GF. SourceDivision of Hematology/Oncology, Mount Sinai School of Medicine, New York, New York 10029, USA. Abstract Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take. PMID:15368352[PubMed - indexed for MEDLINE]
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Mary Reid		Posted: Sunday, February 05, 2012 5:49:11 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		DOCK7 would appear to have the same effect as DR6 J Neurosci. 2011 Aug 31;31(35):12579-92. The atypical Guanine-nucleotide exchange factor, dock7, negatively regulates schwann cell differentiation and myelination. Yamauchi J, Miyamoto Y, Hamasaki H, Sanbe A, Kusakawa S, Nakamura A, Tsumura H, Maeda M, Nemoto N, Kawahara K, Torii T, Tanoue A. Source Department of Pharmacology and Laboratory Animal Resource Facility, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan. jyamauchi@nch.go.jp Abstract In development of the peripheral nervous system, Schwann cells proliferate, migrate, and ultimately differentiate to form myelin sheath. In all of the myelination stages, Schwann cells continuously undergo morphological changes; however, little is known about their underlying molecular mechanisms. We previously cloned the dock7 gene encoding the atypical Rho family guanine-nucleotide exchange factor (GEF) and reported the positive role of Dock7, the target Rho GTPases Rac/Cdc42, and the downstream c-Jun N-terminal kinase in Schwann cell migration (Yamauchi et al., 2008). We investigated the role of Dock7 in Schwann cell differentiation and myelination. Knockdown of Dock7 by the specific small interfering (si)RNA in primary Schwann cells promotes dibutyryl cAMP-induced morphological differentiation, indicating the negative role of Dock7 in Schwann cell differentiation. It also results in a shorter duration of activation of Rac/Cdc42 and JNK, which is the negative regulator of myelination, and the earlier activation of Rho and Rho-kinase, which is the positive regulator of myelination. To obtain the in vivo evidence, we generated Dock7 short hairpin (sh)RNA transgenic mice. They exhibited a decreased expression of Dock7 in the sciatic nerves and enhanced myelin thickness, consistent with in vitro observation. The effects of the in vivo knockdown on the signals to Rho GTPases are similar to those of the in vitro knockdown. Collectively, the signaling through Dock7 negatively regulates Schwann cell differentiation and the onset of myelination, demonstrating the unexpected role of Dock7 in the interplay between Schwann cell migration and myelination. PMID: 21880919 [PubMed - indexed for MEDLINE]
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Mary Reid		Posted: Sunday, February 05, 2012 7:18:59 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		"GMFG was phosphorylated at N-terminal serine, and its phosphorylation was enhanced by coexpression of dominant active Rac1 and Cdc42." "glia maturation factor gamma was concentrated at the NMJ" Circ Res. 2006 Aug 18;99(4):424-33. Epub 2006 Jul 27. Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization. Ikeda K, Kundu RK, Ikeda S, Kobara M, Matsubara H, Quertermous T. Source Donald W. Reynolds Cardiovascular Clinical Research Center, Division of Cardiovascular Medicine, Stanford University School of Medicine, Calif, USA. ikedak@koto.kpu-m.ac.jp Abstract Actin cytoskeleton reorganization is a fundamental process for actin-based cellular functions such as cytokinesis, phagocytosis, and chemotaxis. Regulating actin cytoskeleton reorganization is therefore an attractive approach to control endothelial and inflammatory cells function and to treat cardiovascular diseases. Here, we identified glia maturation factor-gamma (GMFG) as a novel factor in actin cytoskeleton reorganization and is expressed preferentially in microvascular endothelial and inflammatory cells. During mouse embryogenesis, GMFG was expressed predominantly in blood islands of the yolk sac, where endothelial and hematopoietic cells develop simultaneously. In endothelial cells, GMFG was colocalized with F-actin in membrane ruffles and was associated with F-actin assessed by actin co-sedimentation assay. Interestingly, GMFG was phosphorylated at N-terminal serine, and its phosphorylation was enhanced by coexpression of dominant active Rac1 and Cdc42. Furthermore, a pseudophosphorylated form of GMFG (GMFG-S2E) demonstrated higher association with F-actin. Stable expression of GMFG-S2E remarkably enhanced stimulus-responsive lamellipodia and subsequent membrane ruffle formation in HeLa cells presumably through its interaction with Arp2/3 complex. Expression of GMFG enhanced actin-based cellular functions such as migration and tube-formation in endothelial cells. Moreover, we found that GMFG expression was significantly increased in a cardiac ischemia/reperfusion model where inflammation and angiogenesis take place actively. Taken together, our findings define a novel pathway in the regulation of actin-based cellular functions. Regulating GMFG function may provide a novel approach to modulate the pathophysiology of cardiovascular diseases. PMID: 16873721 [PubMed - indexed for MEDLINE] Mol Cell Neurosci. 2005 Oct;30(2):173-85. Analysis of mRNAs that are enriched in the post-synaptic domain of the neuromuscular junction. McGeachie AB, Koishi K, Andrews ZB, McLennan IS. Source Department of Anatomy and Structural Biology, University of Otago, P.O. Box 913, Dunedin, New Zealand. Abstract The identity of synaptically-enriched genes was investigated by comparing the abundance of various mRNAs in the synaptic and extra-synaptic regions of the same muscle fibers. The mRNAs for several known synaptic proteins were significantly elevated in the synaptic region when measured by real-time PCR. The synaptic mRNAs were then further analyzed using microarrays and real-time PCR to identify putative regulators of the neuromuscular junction (NMJ). MRF4 was the only member of the MyoD family that was concentrated at the mature NMJ, suggesting that it may have a unique role in the maintenance of post-synaptic specialization. Three potential regulators of the NMJ were identified and confirmed by real-time PCR: glia maturation factor gamma was concentrated at the NMJ whereas Unr protein and protein tyrosine phosphatase were repressed synaptically. The identification of synaptically-repressed genes may indicate that synaptic specialization is created by a combination of positive and negative signals. PMID: 16095915 [PubMed - indexed for MEDLINE]
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Mary Reid		Posted: Thursday, February 09, 2012 4:37:36 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Mary Reid wrote: Mutations in C9ORF72 cause amyotrophic lateral sclerosis and frontotemporal dementia. RB1CC1 interacts with C9ORF72 as well as DOCK7 (dedicator of cytokinesis 7) Olly has mentioned that ALS and vitiligo appeared at the same time. Thought I'd post this study here though to keep it with other info re: DOCK7. Proc Natl Acad Sci U S A. 2009 Feb 24;106(8):2706-11. Epub 2009 Feb 6. Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function. Blasius AL, Brandl K, Crozat K, Xia Y, Khovananth K, Krebs P, Smart NG, Zampolli A, Ruggeri ZM, Beutler BA. SourceDepartments of Genetics and Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Abstract The classical recessive coat color mutation misty (m) arose spontaneously on the DBA/J background and causes generalized hypopigmentation and localized white-spotting in mice, with a lack of pigment on the belly, tail tip, and paws. Here we describe moonlight (mnlt), a second hypopigmentation and white-spotting mutation identified on the C57BL/6J background, which yields a phenotypic copy of m/m coat color traits. We demonstrate that the 2 mutations are allelic. m/m and mnlt/mnlt phenotypes both result from mutations that truncate the dedicator of cytokinesis 7 protein (DOCK7), a widely expressed Rho family guanine nucleotide exchange factor. Although Dock7 is transcribed at high levels in the developing brain and has been implicated in both axon development and myelination by in vitro studies, we find no requirement for DOCK7 in neurobehavioral function in vivo. However, DOCK7 has non-redundant role(s) related to the distribution and function of dermal and follicular melanocytes. PMID:19202056[PubMed - indexed for MEDLINE] PMCID: PMC2650330
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Mary Reid		Posted: Friday, February 17, 2012 9:36:36 AM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Mary Reid wrote: TDP-43 interacts with ARF6. ARF6 involved in cytokinesis. Mutations in CHMP2B cause ALS. CHMP2B also implicated in cytokinesis. Mutations in p150glued cause ALS p150glued implicated in cytokinesis. The authors of one study below suggest disrupted cytokinesis in SOD1 deficiency. Mutations in FIG4 may also cause ALS. Reduced PIP2 levels have been reported in FIG4 mutant mice. PIP2 activates WASP which is part of the cytokinesis machinery. By the way, an interesting last study here concerning PIP2 and the neuromuscular junction. NEDD5 is essential for cytokinesis, Autoantibodies to NEDD5 are reported in systemic sclerosis. "In human endothelial cells, the activated kinase subunit of AMPK in the cytokinetic apparatus is α2, the minority α subunit, which co-localizes with β2 and γ2." "Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals." Cell Cycle. 2012 Mar 1;11(5). [Epub ahead of print] Subunit composition of AMPK trimers present in the cytokinetic apparatus: Implications for drug target identification. Pinter K, Jefferson A, Czibik G, Watkins H, Redwood C. Source Department of Cardiovascular Medicine; University of Oxford; West Wing Level 6; John Radcliffe Hospital; Oxford, United Kingdom. Abstract AMP-activated protein kinase has been shown to be a key regulator of energy homeostasis; it has also been identified as a tumor suppressor and is required for correct cell division and chromosome segregation during mitosis. The enzyme is a heterotrimer, with each subunit having more than one isoform, each encoded by a separate gene (two α, two β and three γ isoforms). In human endothelial cells, the activated kinase subunit of AMPK in the cytokinetic apparatus is α2, the minority α subunit, which co-localizes with β2 and γ2. This is the first demonstration of a trimeric complex of AMPK containing the γ2 regulatory subunit becoming selectively activated and being linked to mitotic processes. We also show that α1 and γ1, the predominant AMPK subunits, are almost exclusively localized in the cytoskeleton, while α2 and γ2 are present in all subcellular fractions, including the nuclei. These data suggest that pharmacological interventions targeted to specific AMPK subunit isoforms have the potential to modify selective functions of AMPK. PMID: 22333580 [PubMed - as supplied by publisher] Mol Cell. 2011 Dec 23;44(6):878-92. Epub 2011 Dec 1. Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis. Banko MR, Allen JJ, Schaffer BE, Wilker EW, Tsou P, White JL, Villén J, Wang B, Kim SR, Sakamoto K, Gygi SP, Cantley LC, Yaffe MB, Shokat KM, Brunet A. Source Department of Genetics, Stanford University, Stanford, CA 94305, USA. Abstract The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells. Copyright © 2011 Elsevier Inc. All rights reserved. PMID: 22137581 [PubMed - in process] PMCID: PMC3246132 [Available on 2012/12/23] J Neurosci. 2012 Jan 18;32(3):1123-1141. Reduced Activity of AMP-Activated Protein Kinase Protects against Genetic Models of Motor Neuron Disease. Lim MA, Selak MA, Xiang Z, Krainc D, Neve RL, Kraemer BC, Watts JL, Kalb RG. Source Department of Pediatrics, Division of Neurology, Abramson Research Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, Neuroscience Graduate Group and Department of Neurology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, Department of Neurology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, MassGeneral Institute for Neurodegenerative Disease, Charlestown, Massachusetts 02129, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Geriatrics Research Foundation and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108, and School of Molecular Biosciences, Washington State University, Pullman, Washington 99164. Abstract A growing body of research indicates that amyotrophic lateral sclerosis (ALS) patients and mouse models of ALS exhibit metabolic dysfunction. A subpopulation of ALS patients possesses higher levels of resting energy expenditure and lower fat-free mass compared to healthy controls. Similarly, two mutant copper zinc superoxide dismutase 1 (mSOD1) mouse models of familial ALS possess a hypermetabolic phenotype. The pathophysiological relevance of the bioenergetic defects observed in ALS remains largely elusive. AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and thus might be activated in various models of ALS. Here, we report that AMPK activity is increased in spinal cord cultures expressing mSOD1, as well as in spinal cord lysates from mSOD1 mice. Reducing AMPK activity either pharmacologically or genetically prevents mSOD1-induced motor neuron death in vitro. To investigate the role of AMPK in vivo, we used Caenorhabditis elegans models of motor neuron disease. C. elegans engineered to express human mSOD1 (G85R) in neurons develops locomotor dysfunction and severe fecundity defects when compared to transgenic worms expressing human wild-type SOD1. Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals. Similar observations were made with nematodes engineered to express mutant tat-activating regulatory (TAR) DNA-binding protein of 43 kDa molecular weight. Altogether, these data suggest that bioenergetic abnormalities are likely to be pathophysiologically relevant to motor neuron disease. PMID: 22262909 [PubMed - as supplied by publisher]
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Mary Reid		Posted: Friday, February 17, 2012 9:55:52 AM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Mary Reid wrote: Mol Cell. 2011 Dec 23;44(6):878-92. Epub 2011 Dec 1. Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis. Banko MR, Allen JJ, Schaffer BE, Wilker EW, Tsou P, White JL, Villén J, Wang B, Kim SR, Sakamoto K, Gygi SP, Cantley LC, Yaffe MB, Shokat KM, Brunet A. Source Department of Genetics, Stanford University, Stanford, CA 94305, USA. http://www.cell.com/molecular-cell/retrieve/pii/S1097276511008550 Does anyone have the full text of the above study please? Thanks Mary
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Mary Reid		Posted: Saturday, February 18, 2012 6:34:55 AM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		It would appear that increased AMPKalpha2 is detrimental in ALS as its inhibition has been found to prolong survival. Increased AMPKalpha2 activity would result in increased phosphorylation of myosin regulatory light chain. A study here reports that increased phosphorylation of myosin regulatory light chain results in a slow-to-fast transition in soleus muscle. This is reported in ALS. Might the benefit of AMPKalpha2 antagonism in ALS be due to the fact that it preserves slow twitch soleus muscle, thus conserving oxygen? Perhaps that's why swimming is reported to be beneficial. Mary "AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain" http://ajpcell.physiology.org/content/285/3/C575.full MUSCLE CELL BIOLOGY AND CELL MOTILITY MUSCLE CELL BIOLOGY AND CELL MOTILITY Increased phosphorylation of myosin light chain associated with slow-to-fast transition in rat soleus Cyril Bozzo,1,2 Laurence Stevens,2 Luana Toniolo,1 Yvonne Mounier,2 and Carlo Reggiani1 1Department of Anatomy and Physiology, University of Padova, 35131 Padua, Italy; and 2Laboratory of Neuromuscular Plasticity, Institut Fédératif de Recherche 118, University of Sciences and Technologies, 59655 Villeneuve d'Ascq cedex, France Submitted 25 September 2002 ; accepted in final form 10 May 2003 ABSTRACT In striated muscles myosin light chain (MLC)2 phosphorylation regulates calcium sensitivity and mediates sarcomere organization. Little is known about the changes in MLC2 phosphorylation in relation to skeletal muscle plasticity. We studied changes in MLC2 phosphorylation in rats receiving three treatment conditions causing slow-to-fast transitions: 1) atrophy induced by 14 days of hindlimb suspension (HS), 2) hypertrophy induced by 14 days of clenbuterol administration (CB), and 3) 14 days of combined treatment (CB-HS). Three variants of the slow (MLC2s) and two variants of the fast MLC2 (MLC2f) isoform were separated with two-dimensional electrophoresis and identified with monoclonal and polyclonal antibodies specific for MLC2; their relative proportions were densitometrically quantified. In control soleus muscle MLC2s predominated over MLC2f (91.4 ± 3.9% vs. 8.5 ± 3.9%) and was separated into two spots, the less acidic spot being 73.5 ± 4.3% of the total. All treatments caused a decrease of the less acidic unphosphorylated spot of MLC2s (CB: 64.1 ± 5.6%, HS: 62.4 ± 6.8%, CB-HS: 56.4 ± 4.4%), the appearance of a third more acidic variant of MLC2s (representing 3.9–5.9% of total MLC2s), an increase of MLC2f (CB: 30.9 ± 3.1%, HS: 23.9 ± 3.3%, CB-HS: 25.3 ± 3.9%), and the phosphorylation of a large fraction of MLC2f (CB: 30.4 ± 6.7%, HS: 28.7 ± 6.5%, CB-HS: 21.8 ± 2.1%). Treatment with alkaline phosphatase or with protein phosphatase 1 (PP1) removed the most acidic spots of both MLC2f and MLC2s. We conclude that in rat skeletal muscles an increase of MLC2 phosphorylation is associated with the slow-to-fast transition regardless of whether hypertrophy or atrophy develops. http://jp.physoc.org/content/587/14/3561.full.pdf In sedentary ALS fast-twitch muscles, we found a significant fast-to-slow transition from fast-twitch type II fibres to slow-twitch type I fibres and, within the type II fibre population, from type IIb/IIx to IIa fibres, concurring with the findings of Hegedus et al. (2008) (Fig. 3C). In contrast, the typology of the slow-twitch soleus indicated a slow-to-fast transition.
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Mary Reid		Posted: Thursday, March 01, 2012 4:15:03 AM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Dev Cell. 2012 Feb 14;22(2):403-17. Death Receptors DR6 and TROY Regulate Brain Vascular Development. Tam SJ, Richmond DL, Kaminker JS, Modrusan Z, Martin-McNulty B, Cao TC, Weimer RM, Carano RA, van Bruggen N, Watts RJ. Source Neurodegeneration Labs, Department of Neuroscience, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Abstract Signaling events that regulate central nervous system (CNS) angiogenesis and blood-brain barrier (BBB) formation are only beginning to be elucidated. By evaluating the gene expression profile of mouse vasculature, we identified DR6/TNFRSF21 and TROY/TNFRSF19 as regulators of CNS-specific angiogenesis in both zebrafish and mice. Furthermore, these two death receptors interact both genetically and physically and are required for vascular endothelial growth factor (VEGF)-mediated JNK activation and subsequent human brain endothelial sprouting in vitro. Increasing beta-catenin levels in brain endothelium upregulate DR6 and TROY, indicating that these death receptors are downstream target genes of Wnt/beta-catenin signaling, which has been shown to be required for BBB development. These findings define a role for death receptors DR6 and TROY in CNS-specific vascular development. Copyright © 2012 Elsevier Inc. All rights reserved.
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Nemesis		Posted: Thursday, March 01, 2012 2:46:41 PM
Rank: Advanced Member Groups: Member Joined: 2/15/2009 Posts: 2,783		Mary Reid wrote: http://www.cell.com/molecular-cell/retrieve/pii/S1097276511008550 Does anyone have the full text of the above study please? Sorry, Mary I did not see this request until now, I'll see what I can do. Don't just ask what scientists can do to speed up the solution for ALS or when they will do it, instead ask yourself what you can do right now to solve ALS asap.
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Mary Reid		Posted: Thursday, March 01, 2012 5:19:31 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Thanks Nem.
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Olly		Posted: Thursday, March 01, 2012 5:49:36 PM
Rank: Advanced Member Groups: Member Joined: 7/4/2011 Posts: 1,289 Location: United Kingdom		Mary, is this the one your after? www.stanford.edu/group/brunet/Max%20Banko%202011.pdf Full document in pfd... Into the heart, an air that kills, from yon far country blows. What are those blue remembered hills, what sphires what farms are those. That is the land of lost content,I see it shining plain, The happy highways where I went and cannot come again
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Nemesis		Posted: Thursday, March 01, 2012 6:12:04 PM
Rank: Advanced Member Groups: Member Joined: 2/15/2009 Posts: 2,783		Don't just ask what scientists can do to speed up the solution for ALS or when they will do it, instead ask yourself what you can do right now to solve ALS asap.
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Mary Reid		Posted: Thursday, March 01, 2012 6:14:03 PM
Rank: Advanced Member Groups: Member Joined: 9/20/2007 Posts: 3,166 Location: Australia		Thanks heaps Olly.
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