Publications by authors named "Nathalie Lamarche-Vane"

43 Publications

Microexon alternative splicing of small GTPase regulators: Implication in central nervous system diseases.

Wiley Interdiscip Rev RNA 2021 Jun 21:e1678. Epub 2021 Jun 21.

Segal Cancer Center, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.

Microexons are small sized (≤51 bp) exons which undergo extensive alternative splicing in neurons, microglia, embryonic stem cells, and cancer cells, giving rise to cell type specific protein isoforms. Due to their small sizes, microexons provide a unique challenge for the splicing machinery. They frequently lack exon splicer enhancers/repressors and require specialized neighboring trans-regulatory and cis-regulatory elements bound by RNA binding proteins (RBPs) for their inclusion. The functional consequences of including microexons within mRNAs have been extensively documented in the central nervous system (CNS) and aberrations in their inclusion have been observed to lead to abnormal processes. Despite the increasing evidence for microexons impacting cellular physiology within CNS, mechanistic details illustrating their functional importance in diseases of the CNS is still limited. In this review, we discuss the unique characteristics of microexons, and how RBPs participate in regulating their inclusion and exclusion during splicing. We consider recent findings of microexon alternative splicing and their implication for regulating the function of small GTPases in the context of the microglia, and we extrapolate these findings to what is known in neurons. We further discuss the emerging evidence for dysregulation of the Rho GTPase pathway in CNS diseases and the consequences contributed by the mis-splicing of microexons. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease.
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http://dx.doi.org/10.1002/wrna.1678DOI Listing
June 2021

QUAKING Regulates Microexon Alternative Splicing of the Rho GTPase Pathway and Controls Microglia Homeostasis.

Cell Rep 2020 12;33(13):108560

Segal Cancer Center, Lady Davis Institute for Medical Research and Gerald Bronfman Department of Oncology and Departments of Biochemistry, Human Genetics, and Medicine, McGill University, Montréal, QC H3T1E2, Canada. Electronic address:

The role of RNA binding proteins in regulating the phagocytic and cytokine-releasing functions of microglia is unknown. Here, we show that microglia deficient for the QUAKING (QKI) RNA binding protein have increased proinflammatory cytokine release and defects in processing phagocytosed cargo. Splicing analysis reveals a role for QKI in regulating microexon networks of the Rho GTPase pathway. We show an increase in RhoA activation and proinflammatory cytokines in QKI-deficient microglia that are repressed by treating with a Rock kinase inhibitor. During the cuprizone diet, mice with QKI-deficient microglia are inefficient at supporting central nervous system (CNS) remyelination and cause the recruited oligodendrocyte precursor cells to undergo apoptosis. Furthermore, the expression of QKI in microglia is downregulated in preactive, chronic active, and remyelinating white matter lesions of multiple sclerosis (MS) patients. Overall, our findings identify QKI as an alternative splicing regulator governing a network of Rho GTPase microexons with implications for CNS remyelination and MS patients.
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http://dx.doi.org/10.1016/j.celrep.2020.108560DOI Listing
December 2020

The chemokine CCL7 regulates invadopodia maturation and MMP-9 mediated collagen degradation in liver-metastatic carcinoma cells.

Cancer Lett 2020 07 23;483:98-113. Epub 2020 Mar 23.

Department of Surgery, Research Institute of the McGill University Health Centre, 1001 Décarie Blvd, Glen Site, Room E.02.6230, Montréal, QC, H4A 3J1, Canada; Department of Medicine, Research Institute of the McGill University Health Centre, 1001 Décarie Blvd, Glen Site, Room E.02.6230, Montréal, QC, H4A 3J1, Canada; Department of Oncology, Research Institute of the McGill University Health Centre 1001 Décarie Blvd, Glen Site, Room E.02.6230, Montréal, QC, H4A 3J1, Canada; Department of McGill University, Research Institute of the McGill University Health Centre 1001 Décarie Blvd, Glen Site, Room E.02.6230, Montréal, QC, H4A 3J1, Canada; Department of the Cancer Research Program, Research Institute of the McGill University Health Centre 1001 Décarie Blvd, Glen Site, Room E.02.6230, Montréal, QC, H4A 3J1, Canada. Electronic address:

Liver metastases remain a major cause of death from gastrointestinal tract cancers and other malignancies, such as breast and lung carcinomas. Understanding the underlying biology is essential for the design of effective therapies. We previously identified the chemokine CCL7 and its receptor CCR3 as critical mediators of invasion and metastasis in lung and colon carcinoma cells. Here we show that the CCL7/CCR3 axis regulates a late stage in invadopodia genesis namely, the targeting of MMP-9 to the invadopodia complex, thereby promoting invadopodia maturation and collagen degradation. We show that this process could be blocked by overexpression of a dominant negative RhoA in highly invasive cells, while a constitutively active RhoA upregulated invadopodia maturation in CCL7-silenced and poorly invasive and metastatic cells and also enhanced their metastatic potential in vivo, collectively, implicating RhoA activation in signaling downstream of CCL7. Blockade of the ERK or PI3K pathways by chemical inhibitors also inhibited invadopodia formation, but affected the initiation stage of invadopodia genesis. Our data implicate CCL7/CCR3 signaling in invadopodia maturation and suggest that chemokine signaling acts in concert with extracellular matrix-initiated signals to promote invasion and liver metastasis.
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http://dx.doi.org/10.1016/j.canlet.2020.03.018DOI Listing
July 2020

The calcium-activated protease calpain regulates netrin-1 receptor deleted in colorectal cancer-induced axon outgrowth in cortical neurons.

J Neurochem 2020 02 22;152(3):315-332. Epub 2019 Aug 22.

Cancer Research Program, Research Institute of the McGill University Health Center (RI-MUHC), Montréal, Québec, Canada.

During development, neurons extend axons toward their appropriate synaptic targets to establish functional neuronal connections. The growth cone, a highly motile structure at the tip of the axon, is capable of recognizing extracellular guidance cues and translating them into directed axon outgrowth through modulation of the actin cytoskeleton. Netrin-1 mediates its attractive function through the receptor deleted in colorectal cancer (DCC) to promote axon outgrowth and guidance. The calcium-activated protease calpain is involved in the cleavage of cytoskeletal proteins, which plays an important role during adhesion turnover and cell migration. However, its function during neuronal development is less understood. Here we demonstrate that netrin-1 activated calpain in embryonic rat cortical neurons in an extracellular-regulated kinase 1/2-dependent manner. In addition, we found that netrin-1 stimulation led to an increase in calpain-1 localization in the axon, whereas its endogenous inhibitor calpastatin was decreased in the growth cones of cortical neurons by indirect immunofluorescence. Interestingly, calpain-1 was able to cleave DCC in vitro. Furthermore, netrin-1 induced the cleavage of the cytoskeletal proteins spectrin and focal adhesion kinase concomitantly with the intracellular domain of DCC in a calpain-dependent manner in embryonic rat cortical neurons. Cortical neurons over-expressing calpastatin or calpain-depleted neurons displayed increased basal axon length and were unresponsive to netrin-1 stimulation. Altogether, we propose a novel model whereby netrin-1/DCC-mediated axon outgrowth is modulated by calpain-mediated proteolysis of DCC and cytoskeletal targets in embryonic cortical neurons. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.
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http://dx.doi.org/10.1111/jnc.14837DOI Listing
February 2020

Regulators of Rho GTPases in the Nervous System: Molecular Implication in Axon Guidance and Neurological Disorders.

Int J Mol Sci 2019 Mar 25;20(6). Epub 2019 Mar 25.

Cancer Research Program, Research Institute of the MUHC, Montreal, QC H4A 3J1, Canada.

One of the fundamental steps during development of the nervous system is the formation of proper connections between neurons and their target cells-a process called neural wiring, failure of which causes neurological disorders ranging from autism to Down's syndrome. Axons navigate through the complex environment of a developing embryo toward their targets, which can be far away from their cell bodies. Successful implementation of neuronal wiring, which is crucial for fulfillment of all behavioral functions, is achieved through an intimate interplay between axon guidance and neural activity. In this review, our focus will be on axon pathfinding and the implication of some of its downstream molecular components in neurological disorders. More precisely, we will talk about axon guidance and the molecules implicated in this process. After, we will briefly review the Rho family of small GTPases, their regulators, and their involvement in downstream signaling pathways of the axon guidance cues/receptor complexes. We will then proceed to the final and main part of this review, where we will thoroughly comment on the implication of the regulators for Rho GTPases-GEFs (Guanine nucleotide Exchange Factors) and GAPs (GTPase-activating Proteins)-in neurological diseases and disorders.
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http://dx.doi.org/10.3390/ijms20061497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471118PMC
March 2019

Collagen IV-conveyed signals can regulate chemokine production and promote liver metastasis.

Oncogene 2018 07 13;37(28):3790-3805. Epub 2018 Apr 13.

Department of Surgery, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada.

Liver metastases remain a major cause of death from gastrointestinal tract cancers as well as from other malignancies such as breast and lung carcinomas and melanoma. Understanding the underlying biology is essential for the design of effective targeted therapies. We previously reported that collagen IV α1/α2 overexpression in non-metastatic lung carcinoma (M27) cells increased their metastatic ability, specifically to the liver and documented high collagen IV levels in surgical resections of liver metastases from diverse tumor types. Here, we aimed to elucidate the functional relevance of collagen IV to metastatic outgrowth in the liver. Gene expression profiling revealed in M27cells significant increases in the expression of chemokines CCL5 (5.7-fold) and CCL7 (2.6-fold) relative to wild-type cells, and this was validated by qPCR and western blotting. Similarly, in human colon carcinoma KM12C and KM12SM cells with divergent liver-colonizing potentials, CCL7 and CCL5 production correlated with type IV collagen expression and the metastatic phenotype. CCL7 silencing by short hairpin RNA (shRNA) reduced experimental liver metastasis in both cell types, whereas CCL5 silencing reduced metastasis of M27 cells, implicating these cytokines in metastatic expansion in the liver. Subsequent functional analyses implicated both MEK/ERK and PI3K signaling upstream of CCL7 upregulation and identified CCL7 (but not CCL5) as a critical migration/invasion factor, acting via the chemokine receptor CCR3. Chemokine CCL5 was identified as a regulator of the T-cell immune response in the liver. Loss of CCL7 in KM12SM cells was also associated with altered E-cadherin and reduced vimentin and Snail expression, implicating it in epithelial-to-mesenchymal transition in these cells. Moreover, in clinical specimens of colon cancer liver metastases analyzed by immunohistochemistry, CCL5 and CCL7 levels paralleled those of collagen IV. The results identify the chemokines CCL5 and CCL7 as type IV collagen-regulated genes that promote liver metastasis by distinct and complementary mechanisms.
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http://dx.doi.org/10.1038/s41388-018-0242-zDOI Listing
July 2018

CdGAP/ARHGAP31 is regulated by RSK phosphorylation and binding to 14-3-3β adaptor protein.

Oncotarget 2018 02 10;9(14):11646-11664. Epub 2018 Jan 10.

Cancer Research Program, Research Institute of the MUHC, Montreal, Quebec, H4A 3J1, Canada.

Cdc42 GTPase-activating protein (CdGAP, also named ARHGAP31) is a negative regulator of the GTPases Rac1 and Cdc42. Associated with the rare developmental disorder Adams-Oliver Syndrome (AOS), CdGAP is critical for embryonic vascular development and VEGF-mediated angiogenesis. Moreover, CdGAP is an essential component in the synergistic interaction between TGFβ and ErbB-2 signaling pathways during breast cancer cell migration and invasion, and is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer. CdGAP is highly phosphorylated on serine and threonine residues in response to growth factors and is a substrate of ERK1/2 and GSK-3. Here, we identified Ser1093 and Ser1163 in the C-terminal region of CdGAP, which are phosphorylated by RSK in response to phorbol ester. These phospho-residues create docking sites for binding to 14-3-3 adaptor proteins. The interaction between CdGAP and 14-3-3 proteins inhibits the GAP activity of CdGAP and sequesters CdGAP into the cytoplasm. Consequently, the nucleocytoplasmic shuttling of CdGAP is inhibited and CdGAP-induced cell rounding is abolished. In addition, 14-3-3β inhibits the ability of CdGAP to repress the E-cadherin promoter and to induce cell migration. Finally, we show that 14-3-3β is unable to regulate the activity and subcellular localization of the AOS-related mutant proteins lacking these phospho-residues. Altogether, we provide a novel mechanism of regulation of CdGAP activity and localization, which impacts directly on a better understanding of the role of CdGAP as a promoter of breast cancer and in the molecular causes of AOS.
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http://dx.doi.org/10.18632/oncotarget.24126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837747PMC
February 2018

Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides.

FASEB J 2017 08 25;31(8):3467-3483. Epub 2017 Apr 25.

Sainte-Justine University Hospital Research Center, University of Montreal, Montreal, Quebec, Canada;

Gangliosides (sialylated glycolipids) play an essential role in the CNS by regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in the development of severe neurologic disorders, including gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that 2 mammalian enzymes, neuraminidases 3 and 4, play important roles in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In neuraminidase 3 and 4 double-knockout mice, G ganglioside is stored in microglia, vascular pericytes, and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies, and memory loss, whereas their cortical and hippocampal neurons have lower rate of neuritogenesis Double-knockout mice also have reduced levels of G ganglioside and myelin in neuronal axons. Furthermore, neuraminidase 3 deficiency drastically increased storage of G in the brain tissues of an asymptomatic mouse model of Tay-Sachs disease, a severe human gangliosidosis, indicating that this enzyme is responsible for the metabolic bypass of β-hexosaminidase A deficiency. Together, our results provide the first evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies.-Pan, X., De Britto Pará De Aragão, C., Velasco-Martin, J. P., Priestman, D. A., Wu, H. Y., Takahashi, K., Yamaguchi, K., Sturiale, L., Garozzo, D., Platt, F. M., Lamarche-Vane, N., Morales, C. R., Miyagi, T., Pshezhetsky, A. V. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides.
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http://dx.doi.org/10.1096/fj.201601299RDOI Listing
August 2017

Decrease of SYNGAP1 in GABAergic cells impairs inhibitory synapse connectivity, synaptic inhibition and cognitive function.

Nat Commun 2016 11 9;7:13340. Epub 2016 Nov 9.

Department of Neurosciences, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Quebec, Canada H3C 3J7.

Haploinsufficiency of the SYNGAP1 gene, which codes for a Ras GTPase-activating protein, impairs cognition both in humans and in mice. Decrease of Syngap1 in mice has been previously shown to cause cognitive deficits at least in part by inducing alterations in glutamatergic neurotransmission and premature maturation of excitatory connections. Whether Syngap1 plays a role in the development of cortical GABAergic connectivity and function remains unclear. Here, we show that Syngap1 haploinsufficiency significantly reduces the formation of perisomatic innervations by parvalbumin-positive basket cells, a major population of GABAergic neurons, in a cell-autonomous manner. We further show that Syngap1 haploinsufficiency in GABAergic cells derived from the medial ganglionic eminence impairs their connectivity, reduces inhibitory synaptic activity and cortical gamma oscillation power, and causes cognitive deficits. Our results indicate that Syngap1 plays a critical role in GABAergic circuit function and further suggest that Syngap1 haploinsufficiency in GABAergic circuits may contribute to cognitive deficits.
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http://dx.doi.org/10.1038/ncomms13340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5105197PMC
November 2016

CdGAP/ARHGAP31, a Cdc42/Rac1 GTPase regulator, is critical for vascular development and VEGF-mediated angiogenesis.

Sci Rep 2016 06 7;6:27485. Epub 2016 Jun 7.

CRCHUM - Centre de recherche du Centre Hospitalier de l'Université de Montréal and Institut du Cancer de Montréal, Montréal, Québec, Canada.

Mutations in the CdGAP/ARHGAP31 gene, which encodes a GTPase-activating protein for Rac1 and Cdc42, have been reported causative in the Adams-Oliver developmental syndrome often associated with vascular defects. However, despite its abundant expression in endothelial cells, CdGAP function in the vasculature remains unknown. Here, we show that vascular development is impaired in CdGAP-deficient mouse embryos at E15.5. This is associated with superficial vessel defects and subcutaneous edema, resulting in 44% embryonic/perinatal lethality. VEGF-driven angiogenesis is defective in CdGAP(-/-) mice, showing reduced capillary sprouting from aortic ring explants. Similarly, VEGF-dependent endothelial cell migration and capillary formation are inhibited upon CdGAP knockdown. Mechanistically, CdGAP associates with VEGF receptor-2 and controls VEGF-dependent signaling. Consequently, CdGAP depletion results in impaired VEGF-mediated Rac1 activation and reduced phosphorylation of critical intracellular mediators including Gab1, Akt, PLCγ and SHP2. These findings are the first to demonstrate the importance of CdGAP in embryonic vascular development and VEGF-induced signaling, and highlight CdGAP as a potential therapeutic target to treat pathological angiogenesis and vascular dysfunction.
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http://dx.doi.org/10.1038/srep27485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895392PMC
June 2016

A Point Mutation in p190A RhoGAP Affects Ciliogenesis and Leads to Glomerulocystic Kidney Defects.

PLoS Genet 2016 Feb 9;12(2):e1005785. Epub 2016 Feb 9.

Goodman Cancer Research Centre and Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease.
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http://dx.doi.org/10.1371/journal.pgen.1005785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747337PMC
February 2016

Direct measurement of oscillatory RhoA activity in embryonic cortical neurons stimulated with the axon guidance cue netrin-1 using fluorescence resonance energy transfer.

Biol Cell 2016 May 10;108(5):115-26. Epub 2016 Feb 10.

Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.

Background Information: Rho GTPases play an essential role during the development of the nervous system. They induce cytoskeletal rearrangements that are critical for the regulation of axon outgrowth and guidance. It is generally accepted that Rac1 and Cdc42 are positive regulators of axon outgrowth and guidance, whereas RhoA is a negative regulator. However, spatiotemporal control of their activity can modify the function of Rho GTPases during axonal morphogenesis. Signalling downstream of the axon guidance cue netrin-1 and its receptor deleted in colorectal cancer (DCC) triggers the activation of Rac1 and the inhibition of RhoA to promote axon outgrowth. However, our previous work also suggests that netrin-1/DCC signalling can activate RhoA in a time- and region-specific manner.

Results: Here, we visualised RhoA activation in response to netrin-1 in live embryonic cortical neurons using fluorescence resonance energy transfer. RhoA activity oscillated in unstimulated neurons and netrin-1 increased the amplitude of the oscillations in growth cones after 5 min of stimulation. Within this period of time, netrin-1 transiently increased RhoA activity and modulated the pattern of RhoA oscillations. We found that the timing of netrin-1-induced RhoA activation was different in whole neurons, cell bodies and growth cones.

Conclusions: We conclude that netrin-1 modulates the spatiotemporal activation of RhoA in embryonic cortical neurons.

Significance: This study demonstrates for the first time the short-term localised activation of RhoA in neuronal growth cones by the axon guidance cue netrin-1.
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http://dx.doi.org/10.1111/boc.201500077DOI Listing
May 2016

p120RasGAP Protein Mediates Netrin-1 Protein-induced Cortical Axon Outgrowth and Guidance.

J Biol Chem 2016 Feb 28;291(9):4589-602. Epub 2015 Dec 28.

From the Department of Anatomy and Cell Biology and Cancer Research Program, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec, H4A 3J1 Canada and

The receptor deleted in colorectal cancer (DCC) mediates the attraction of growing axons to netrin-1 during brain development. In response to netrin-1 stimulation, DCC becomes a signaling platform to recruit proteins that promote axon outgrowth and guidance. The Ras GTPase-activating protein (GAP) p120RasGAP inhibits Ras activity and mediates neurite retraction and growth cone collapse in response to repulsive guidance cues. Here we show an interaction between p120RasGAP and DCC that positively regulates netrin-1-mediated axon outgrowth and guidance in embryonic cortical neurons. In response to netrin-1, p120RasGAP is recruited to DCC in growth cones and forms a multiprotein complex with focal adhesion kinase and ERK. We found that Ras/ERK activities are elevated aberrantly in p120RasGAP-deficient neurons. Moreover, the expression of p120RasGAP Src homology 2 (SH2)-SH3-SH2 domains, which interact with the C-terminal tail of DCC, is sufficient to restore netrin-1-dependent axon outgrowth in p120RasGAP-deficient neurons. We provide a novel mechanism that exploits the scaffolding properties of the N terminus of p120RasGAP to tightly regulate netrin-1/DCC-dependent axon outgrowth and guidance.
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http://dx.doi.org/10.1074/jbc.M115.674846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813483PMC
February 2016

Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1.

J Cell Biol 2015 Aug;210(5):817-32

Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada The Research Institute of McGill University Health Centre, Montreal, Quebec H4A 3J1, Canada

During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1-induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator. Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase-deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1-induced Rac1 activation. Hsc70 was required for netrin-1-mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance.
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http://dx.doi.org/10.1083/jcb.201505084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555821PMC
August 2015

CUX2 protein functions as an accessory factor in the repair of oxidative DNA damage.

J Biol Chem 2015 Sep 28;290(37):22520-31. Epub 2015 Jul 28.

From the Goodman Cancer Research Centre and Departments of Biochemistry, Medicine, Oncology, and

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.
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http://dx.doi.org/10.1074/jbc.M115.651042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566227PMC
September 2015

Rho GTPases in embryonic development.

Small GTPases 2014 ;5(2)

a McGill University ; Department of Anatomy and Cell Biology ; Montreal , QC Canada.

In the last decade, several mouse models for RhoA, Rac1, and Cdc42 have emerged and have contributed a great deal to understanding the precise functions of Rho GTPases at early stages of development. This review summarizes our current knowledge of various mouse models of tissue-specific ablation of Cdc42, Rac1, and RhoA with emphasis on early embryogenesis, epithelial and skin morphogenesis, tubulogenesis, development of the central nervous system, and limb development.
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http://dx.doi.org/10.4161/sgtp.29716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601498PMC
July 2015

Rho GTPases in neurodegeneration diseases.

Exp Cell Res 2013 Sep 2;319(15):2384-94. Epub 2013 Jul 2.

McGill University, Department of Anatomy and Cell Biology, Montreal, QC, Canada H3A 0C7.

Rho GTPases are molecular switches that modulate multiple intracellular signaling processes by means of various effector proteins. As a result, Rho GTPase activities are tightly spatiotemporally regulated in order to ensure homeostasis within the cell. Though the roles of Rho GTPases during neural development have been well documented, their participation during neurodegeneration has been far less characterized. Herein we discuss our current knowledge of the role and function of Rho GTPases and regulators during neurodegeneration, and highlight their potential as targets for therapeutic intervention in common neurodegenerative disorders.
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http://dx.doi.org/10.1016/j.yexcr.2013.06.016DOI Listing
September 2013

Tyrosine phosphorylation of the Rho guanine nucleotide exchange factor Trio regulates netrin-1/DCC-mediated cortical axon outgrowth.

Mol Cell Biol 2013 Feb 10;33(4):739-51. Epub 2012 Dec 10.

Department of Anatomy & Cell Biology, McGill University, Montreal, Quebec, Canada.

The chemotropic guidance cue netrin-1 mediates attraction of migrating axons during central nervous system development through the receptor Deleted in Colorectal Cancer (DCC). Downstream of netrin-1, activated Rho GTPases Rac1 and Cdc42 induce cytoskeletal rearrangements within the growth cone. The Rho guanine nucleotide exchange factor (GEF) Trio is essential for Rac1 activation downstream of netrin-1/DCC, but the molecular mechanisms governing Trio activity remain elusive. Here, we demonstrate that Trio is phosphorylated by Src family kinases in the embryonic rat cortex in response to netrin-1. In vitro, Trio was predominantly phosphorylated at Tyr(2622) by the Src kinase Fyn. Though the phospho-null mutant Trio(Y2622F) retained GEF activity toward Rac1, its expression impaired netrin-1-induced Rac1 activation and DCC-mediated neurite outgrowth in N1E-115 neuroblastoma cells. Trio(Y2622F) impaired netrin-1-induced axonal extension in cultured cortical neurons and was unable to colocalize with DCC in growth cones, in contrast to wild-type Trio. Furthermore, depletion of Trio in cortical neurons reduced the level of cell surface DCC in growth cones, which could be restored by expression of wild-type Trio but not Trio(Y2622F). Together, these findings demonstrate that Trio(Y2622) phosphorylation is essential for the regulation of the DCC/Trio signaling complex in cortical neurons during netrin-1-mediated axon outgrowth.
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http://dx.doi.org/10.1128/MCB.01264-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571336PMC
February 2013

A stretch of polybasic residues mediates Cdc42 GTPase-activating protein (CdGAP) binding to phosphatidylinositol 3,4,5-trisphosphate and regulates its GAP activity.

J Biol Chem 2012 Jun 19;287(23):19610-21. Epub 2012 Apr 19.

Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada.

The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.
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http://dx.doi.org/10.1074/jbc.M112.344606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365996PMC
June 2012

The activation of ezrin-radixin-moesin proteins is regulated by netrin-1 through Src kinase and RhoA/Rho kinase activities and mediates netrin-1-induced axon outgrowth.

Mol Biol Cell 2011 Oct 17;22(19):3734-46. Epub 2011 Aug 17.

Department of Anatomy and Cell Biology, McGill University, Montreal H3A 2B2, PQ, Canada.

The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42. The molecular mechanisms that link netrin-1/DCC to the actin machinery remain unclear. In this study we seek to demonstrate that the actin-binding proteins ezrin-radixin-moesin (ERM) are effectors of netrin-1/DCC signaling in embryonic cortical neurons. We show that ezrin associates with DCC in a netrin-1-dependent manner. We demonstrate that netrin-1/DCC induces ERM phosphorylation and activation and that the phosphorylation of DCC is required in that context. Moreover, Src kinases and RhoA/Rho kinase activities mediate netrin-1-induced ERM phosphorylation in neurons. We also observed that phosphorylated ERM proteins accumulate in growth cone filopodia, where they colocalize with DCC upon netrin-1 stimulation. Finally, we show that loss of ezrin expression in cortical neurons significantly decreases axon outgrowth induced by netrin-1. Together, our findings demonstrate that netrin-1 induces the formation of an activated ERM/DCC complex in growth cone filopodia, which is required for netrin-1-dependent cortical axon outgrowth.
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http://dx.doi.org/10.1091/mbc.E10-11-0917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183026PMC
October 2011

Gain-of-function mutations of ARHGAP31, a Cdc42/Rac1 GTPase regulator, cause syndromic cutis aplasia and limb anomalies.

Am J Hum Genet 2011 May;88(5):574-85

Department of Medical and Molecular Genetics, King's College London, School of Medicine, Guy's Hospital, London, London, UK.

Regulation of cell proliferation and motility is essential for normal development. The Rho family of GTPases plays a critical role in the control of cell polarity and migration by effecting the cytoskeleton, membrane trafficking, and cell adhesion. We investigated a recognized developmental disorder, Adams-Oliver syndrome (AOS), characterized by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). Through a genome-wide linkage analysis, we detected a locus for autosomal-dominant ACC-TTLD on 3q generating a maximum LOD score of 4.93 at marker rs1464311. Candidate-gene- and exome-based sequencing led to the identification of independent premature truncating mutations in the terminal exon of the Rho GTPase-activating protein 31 gene, ARHGAP31, which encodes a Cdc42/Rac1 regulatory protein. Mutant transcripts are stable and increase ARHGAP31 activity in vitro through a gain-of-function mechanism. Constitutively active ARHGAP31 mutations result in a loss of available active Cdc42 and consequently disrupt actin cytoskeletal structures. Arhgap31 expression in the mouse is substantially restricted to the terminal limb buds and craniofacial processes during early development; these locations closely mirror the sites of impaired organogenesis that characterize this syndrome. These data identify the requirement for regulated Cdc42 and/or Rac1 signaling processes during early human development.
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http://dx.doi.org/10.1016/j.ajhg.2011.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146732PMC
May 2011

Implication of rho GTPases in neurodegenerative diseases.

Curr Drug Targets 2011 Jul;12(8):1202-15

Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montreal, Quebec, H3A 2B2, Canada.

The establishment of neural connectivity implicates tight regulation of the intracellular signaling pathways mediated by axon guidance molecules. The Rho family of small GTPases, in particular Rho, Rac, and Cdc42, are important regulators of the cytoskeleton in neuronal cells acting, downstream of most, if not all, guidance cue receptors. Furthermore, recent studies using in vivo knockout mouse models provide new evidence of the primary role played by Rho GTPase signaling during the development of the nervous system. Here, we review our recent understanding of Rho GTPase signaling in response to classical axon guidance cues in mammalian cells. We also describe how in vivo knockout mouse models have been useful to implicate Rho GTPase signaling during the formation of the nervous system. Finally, we present several lines of evidence showing the involvement of Rho GTPase signaling in the development and progression of neurodegenerative diseases.
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http://dx.doi.org/10.2174/138945011795906543DOI Listing
July 2011

Cdc42 GTPase-activating protein (CdGAP) interacts with the SH3D domain of Intersectin through a novel basic-rich motif.

FEBS Lett 2011 Mar 22;585(6):847-53. Epub 2011 Feb 22.

Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 2B2.

The small GTPases Rac1 and Cdc42 are key regulators of the cytoskeleton. We have previously identified the endocytic protein Intersectin as a binding partner and regulator of Cdc42 GTPase-activating protein (CdGAP) with activity towards Rac1 and Cdc42. This interaction is mediated through the SH3D domain of Intersectin and the central domain of CdGAP, which does not contain any typical proline-rich domain or known SH3-binding motif. Here, we have characterized the Intersectin-SH3D/CdGAP interaction. We show that Intersectin-SH3D interacts directly with a small region of CdGAP highly enriched in basic residues and comprising a novel conserved xKx(K/R)K motif.
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http://dx.doi.org/10.1016/j.febslet.2011.02.022DOI Listing
March 2011

Spatial and temporal activation of the small GTPases RhoA and Rac1 by the netrin-1 receptor UNC5a during neurite outgrowth.

Cell Signal 2009 Dec 13;21(12):1961-73. Epub 2009 Sep 13.

Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 2B2.

Netrin-1 attracts or repels growing axons during development. The UNC5 receptors mediate the repulsive response, either alone or in complex with DCC receptors. The signaling mechanisms activated by UNC5 are poorly understood. Here, we examined the role of Rho GTPases in UNC5a signaling. We found that UNC5a induced neurite outgrowth in N1E-115 neuroblastoma cells in a netrin-1- and Rac1-dependent manner. UNC5a lacking its cytoplasmic tail also mediated this effect. In fibroblasts, UNC5a was able to activate RhoA and to a lower extent Rac1 and Cdc42 in response to netrin-1. Using Fluorescence Resonance Energy Transfer (FRET) intermolecular probes, we visualized the spatial and temporal activation of Rac1, Cdc42 and RhoA in live N1E-115 cells expressing UNC5a during neurite outgrowth. We found that Rac1 but not Cdc42 was transiently activated at the leading edge of the cell during neurite initiation. However, at later times when well-developed neurites were formed, active RhoA was found in the cell body and at the base of the neuronal leading process in UNC5a-expressing cells. Together, these findings demonstrate that the netrin-1 receptor UNC5a is able to induce neurite outgrowth and to differentially activate RhoA and Rac1 during neurite extension in a spatial and temporal manner.
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http://dx.doi.org/10.1016/j.cellsig.2009.09.004DOI Listing
December 2009

Rab35 regulates neurite outgrowth and cell shape.

FEBS Lett 2009 Apr 14;583(7):1096-101. Epub 2009 Mar 14.

Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.

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.
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http://dx.doi.org/10.1016/j.febslet.2009.03.012DOI Listing
April 2009

An adaptor role for cytoplasmic Sam68 in modulating Src activity during cell polarization.

Mol Cell Biol 2009 Apr 12;29(7):1933-43. Epub 2009 Jan 12.

Terry Fox Molecular Oncology Group and Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Québec, Canada H3T 1E2.

The Src-associated substrate during mitosis with a molecular mass of 68 kDa (Sam68) is predominantly nuclear and is known to associate with proteins containing the Src homology 3 (SH3) and SH2 domains. Although Sam68 is a Src substrate, little is known about the signaling pathway that link them. Src is known to be activated transiently after cell spreading, where it modulates the activity of small Rho GTPases. Herein we report that Sam68-deficient cells exhibit loss of cell polarity and cell migration. Interestingly, Sam68-deficient cells exhibited sustained Src activity after cell attachment, resulting in the constitutive tyrosine phosphorylation and activation of p190RhoGAP and its association with p120rasGAP. Consistently, we observed that Sam68-deficient cells exhibited deregulated RhoA and Rac1 activity. By using total internal reflection fluorescence microscopy, we observed Sam68 near the plasma membrane after cell attachment coinciding with phosphorylation of its C-terminal tyrosines and association with Csk. These findings show that Sam68 localizes near the plasma membrane during cell attachment and serves as an adaptor protein to modulate Src activity for proper signaling to small Rho GTPases.
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http://dx.doi.org/10.1128/MCB.01707-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655613PMC
April 2009

Compartmentalized DCC signalling is distinct from DCC localized to lipid rafts.

Biol Cell 2009 Feb;101(2):77-90

Department of Anatomy and Cell Biology, McGill University, Quebec, Canada.

Background Information: Netrin-1 is a bi-functional cue that attracts or repels different classes of neurons during development. The netrin-1 receptor DCC (deleted in colorectal cancer) acts as a tyrosine kinase-associated receptor to mediate the attractive response towards netrin-1. The lipid raft-localized Src family kinase Fyn is required for DCC-mediated axon guidance. DCC functions are also dependent on lipid rafts, membrane microdomains corresponding to a low-density, detergent-resistant membrane fraction. However, it remains unclear how the association of DCC with lipid rafts controls netrin-1 signalling.

Results: DCC targeted to lipid rafts represented a minor proportion of total DCC inside the cell, but predominated on the cell surface of both IMR-32 human neuroblastoma cells and embryonic cortical neurons. Netrin-1 accumulated in lipid rafts, but had no effect on the targeting of DCC to that compartment, with DCC remaining on the cell surface in lipid rafts through 60 min post-treatment. However, DCC was able to interact with Fyn, both in the lipid rafts and soluble compartments isolated from embryonic E19 rat brains, whereas early downstream signalling components such as Nck-1, and total and active focal adhesion kinase were mainly localized to the non-lipid raft compartment.

Conclusions: Together, these results suggest that DCC can be found in raft and non-raft portions of the plasma membrane, with early signalling events propagated by non-raft associated DCC.
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http://dx.doi.org/10.1042/BC20070108DOI Listing
February 2009

[A brief overview of the small Rho GTPases].

Med Sci (Paris) 2008 Feb;24(2):157-62

Université McGill, Département d'anatomie et biologie cellulaire, 3640 University, room 1-38, Montréal, Québec, H3A 2B2 Canada.

RhoA, Rac1, and Cdc42, the founding members of the Rho subfamily of small GTPases, have been the focus of many research studies since the first discovery of their primary roles in the reorganisation of the actin cytoskeleton. Since then, it is clear that they are involved in a great deal of cellular functions, including cell migration and adhesion, cell growth control, and membrane trafficking. The complete sequencing of the human genome has now highlighted a total of 20 genes encoding Rho-like proteins. Little is known about their distinct cellular functions, however, numerous studies are now beginning to unravel that each of the Rho GTPase must play a specific role in the cell in a timely and spatially regulated fashion. Here, we are presenting a brief overview of the distinct functional roles and similarities known to date for each of the Rho members.
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http://dx.doi.org/10.1051/medsci/2008242157DOI Listing
February 2008

Trio mediates netrin-1-induced Rac1 activation in axon outgrowth and guidance.

Mol Cell Biol 2008 Apr 22;28(7):2314-23. Epub 2008 Jan 22.

CRBM-CNRS, UMR5237, 1919 Route de Mende, 34293 Montpellier Cédex 05, France.

The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio(-/-) cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio(-/-) spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.
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http://dx.doi.org/10.1128/MCB.00998-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268419PMC
April 2008

Role of Rho-GTPases in complement-mediated glomerular epithelial cell injury.

Am J Physiol Renal Physiol 2007 Jul 20;293(1):F148-56. Epub 2007 Mar 20.

Department of Medicine, McGill University Health Centre, Montreal, Quebec H3A 2B4.

Visceral glomerular epithelial cells (GEC) are essential for maintenance of normal glomerular permselectivity. The actin cytoskeleton is a key determinant of GEC morphology and function. In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces nonlytic GEC injury associated with morphological changes of GEC and proteinuria. The current study addresses the role of Rho family of small GTPases in complement-mediated GEC injury. When cultured rat GEC were stimulated with complement C5b-9 for 18 h, RhoA activity increased, whereas Rac1/Cdc42 activities decreased, compared with control cells. Similar changes in Rho-GTPase activities were observed in glomeruli from rats with PHN. The amount of active p190RhoGAP, a negative upstream regulator of RhoA, was decreased in complement-stimulated GEC, potentially contributing to increased RhoA activity. To address the functional effects of Rho-GTPases, GEC were transfected with constitutively active (CA) or dominant negative (DN) Rho-GTPase mutants. GEC transfected with CA-RhoA showed a smaller and round contour and prominent cortical F-actin. In contrast, GEC transfected with CA-Rac1 demonstrated morphological changes that resembled process formation. In addition, expression of CA-RhoA attenuated complement-mediated cytotoxicity, whereas cytotoxicity was augmented by DN-RhoA. Thus exposure of GEC to complement alters the balance of RhoA, Rac1, and Cdc42 activities. The activity of Rac1 may contribute to process formation, while activation of RhoA (e.g., in the setting of complement attack), with or without blunting of Rac1 activity, may have an opposite effect, i.e., contribute to foot process effacement. Activation of RhoA increases the resistance of GEC to complement-mediated injury.
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http://dx.doi.org/10.1152/ajprenal.00294.2006DOI Listing
July 2007
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