Cdc42 and Rac1 Signaling Are Both Required for and Act Synergistically in the Correct Formation of Myelin Sheaths in the CNS

“…known for their roles in regulating signaling pathways linking extracellular stimuli to the assembly and organization of the actin cytoskeleton (Hall, 1998), also control microtubule dynamics, cell polarity, membrane trafficking, and gene transcription (Etienne-Manneville and Hall, 2002; Jaffe…”

“To follow the fate of the recombined cells by detection of -galactosidase expression, we bred the conditional LacZ allele from the ROSA26 reporter mouse strain (Soriano, 1999) into control and mutant mice.”

“Cdc42 is not required for OPC proliferation, directed migration, or morphological differentiation Our results do not support a requirement for cdc42 in OPC proliferation, directed migration, or in vitro morphological differentiation; however, there is a significant body of evidence showing a key role for cdc42 in the…”

“Oligodendrocyte progenitor cells (OPCs) were purified from mixed glial cultures (McCarthy and de Vellis, 1980) and differentiated on extracellular matrix substrates (Relvas et al., 2001).”

“…roles in regulating signaling pathways linking extracellular stimuli to the assembly and organization of the actin cytoskeleton (Hall, 1998), also control microtubule dynamics, cell polarity, membrane trafficking, and gene transcription (Etienne-Manneville and Hall, 2002; Jaffe and Hall, 2005).”

“Consequently, in wells to which 25 or 100 ng/ml of netrin-1 was added to the bottom chamber, control OPC migration toward the lower surface of the filter was inhibited significantly ( p 0.01 and 0.009, respectively), as compared with wells to which no netrin-1 was added; however, control and mutant OPCs migrated equal…”

“This observation raises important questions about the specificity of Rho GTPase dominant-negative approaches and is in line with previous reports showing that dominant-negative mutant expression induced more severe phenotypes than those produced by genetic loss-of-function approaches on identical cell types or organis…”

“Cdc42 is not required for OPC proliferation, directed migration, or morphological differentiation Our results do not support a requirement for cdc42 in OPC proliferation, directed migration, or in vitro morphological differentiation; however, there is a significant body of evidence showing a key role for cdc42 in the…”

“…Chrostek,3 Sven M. Krause,1 Klaus-Armin Nave,4 Robin J. M. Franklin, 5 Cord Brakebusch,2 Ueli Suter,1 and João B. Relvas1 1Institute for Cell Biology, Department of Biology, Federal Institute of Technology (ETH) Zurich, CH-8093 Zurich, Switzerland, 2Department of Molecular Pathology, University…”

“Rac1 activity was measured as described previously (Sander et al., 1998).”

“In the context of CNS myelination, pathways known to influence oligodendrocyte development such as those initiated by the activation of integrin receptors (Liang et al., 2004) or the transmembrane protein Lingo-1 (Mi et al., 2005) can modulate the activities of cdc42, rac1, and rhoA.”

“Many key cellular functions ascribed to Rho GTPases rely on experiments during which their function is blocked by expression of dominant-negative mutants (Feig, 1999).”

“During oligodendrocyte development, the coordinated interpretation of extracellular cues, such as integrin, growth factor, or chemokine receptor activation, allows oligodendrocytes to sense their environment and regulate their intrinsic developmental program (Miller, 2002; Baron et al., 2005).”

“Although rhoA is expressed and active during early progenitor stages, the expression and activity of cdc42 and rac1 increase as differentiation proceeds (Liang et al., 2004).”

“, 2004), control and mutant OPCs were allowed to differentiate in culture (Buttery and ffrench-Constant, 1999; Relvas et al., 2001).”

“the CNS Tina Thurnherr,1* Yves Benninger,1* Xunwei Wu,2 Anna Chrostek,3 Sven M. Krause,1 Klaus-Armin Nave,4 Robin J. M. Franklin, 5 Cord Brakebusch,2 Ueli Suter,1 and João B. Relvas1 1Institute for Cell Biology, Department of Biology, Federal Institute of Technology (ETH) Zurich, CH-8093 Zurich, Switzerland, 2Departm…”

“Recent work (Czuchra et al., 2005) has shown that the transfection of Cdc42-null fibroblastoid cells with a Cdc42 dominant-negative mutant (N17 cdc42) elicits additional phenotypes that are not present in untransfected Cdc42null fibroblasts.”

“The generation of conditional Cdc42 mutant mice (Wu et al., 2006) and Rac1 mutant mice (Chrostek et al., 2006) have been described previously.”

“…that the formation of the multilamelar myelin sheath requires the continuous progression of the inner tongue over the axonal surface (Speidel, 1964; Bunge et al., 1989), it is not immediately evident to us how enlarged inner tongues, sometimes more than several axon diameters in length, are still…”

“Therefore, dominant-negative mutants of cdc42 may inhibit, especially if expressed at high levels (Braga et al., 2000), the activation of other Rho GTPases, making it nearly impossible to discriminate between the specific contributions from different Rho GTPases.”

“This conclusion was also supported by standard in vitro migration assays (Boyden chamber; see Material and Methods) with which we tested the capacity of freshly isolated control and mutant OPC to migrate in response to PDGF, an OPC chemoattractant secreted along OPC migratory pathways (McKinnon et al., 1993), or netri…”

“The generation of conditional Cdc42 mutant mice (Wu et al., 2006) and Rac1 mutant mice (Chrostek et al., 2006) have been described previously.”

“The sporadic presence of such structures during normal development has been reported previously in amphibian cerebellum (Rosenbluth, 1966).”

“, 1993), or netrin-1, an OPC chemorepellent expressed at the ventricular zone during OPC emigration (Jarjour et al., 2003).”

“This observation raises important questions about the specificity of Rho GTPase dominant-negative approaches and is in line with previous reports showing that dominant-negative mutant expression induced more severe phenotypes than those produced by genetic loss-of-function approaches on identical cell types or organis…”

“During oligodendrocyte development, the coordinated interpretation of extracellular cues, such as integrin, growth factor, or chemokine receptor activation, allows oligodendrocytes to sense their environment and regulate their intrinsic developmental program (Miller, 2002; Baron et al., 2005).”

“…Krause,1 Klaus-Armin Nave,4 Robin J. M. Franklin, 5 Cord Brakebusch,2 Ueli Suter,1 and João B. Relvas1 1Institute for Cell Biology, Department of Biology, Federal Institute of Technology (ETH) Zurich, CH-8093 Zurich, Switzerland, 2Department of Molecular Pathology, University of Copenhagen, 2100…”

“…the myelin abnormalities observed in the Cdc42 mutant CNS could be directly attributed to impairment of oligodendrocyte process extension and branching (Liang et al., 2004), control and mutant OPCs were allowed to differentiate in culture (Buttery and ffrench-Constant, 1999; Relvas et al., 2001).”

“…it is thought that the formation of the multilamelar myelin sheath requires the continuous progression of the inner tongue over the axonal surface (Speidel, 1964; Bunge et al., 1989), it is not immediately evident to us how enlarged inner tongues, sometimes more than several axon diameters in…”

“In oligodendrocytes, the organization of the microfilament and microtubule array is likely to provide the driving force for changes in plasma membrane conformation associated with process extension and branching (Simpson and Armstrong, 1999; Richter-Landsberg, 2001; Song et al., 2001).”

“These GTPases are expressed by oligodendrocytes (Erschbamer et al., 2005), and, in primary oligodendrocyte cultures, their expression and activity are developmentally regulated (Liang et al., 2004).”

“6C), which also acts as an OPC mitogen (Besnard et al., 1987).”

“Rac1 is required for proper axon ensheathment and myelination Cdc42 and rac1 cooperate in pathways regulating important cellular functions (Cotteret and Chernoff, 2002).”

“The excluded cytoplasm presumably flows to regions of uncompacted myelin, which in the early developing CNS alternate irregularly with compacted myelin sheaths along the individual axon (Storts and Koestner, 1969; Remahl and Hildebrand, 1990).”

“In oligodendrocytes, the organization of the microfilament and microtubule array is likely to provide the driving force for changes in plasma membrane conformation associated with process extension and branching (Simpson and Armstrong, 1999; Richter-Landsberg, 2001; Song et al., 2001).”

“Perturbation of cytoskeletal reorganization and eventually of vesicle transport, also a putative function of cdc42 (Larocca and Rodriguez-Gabin, 2002; RodriguezBoulan et al., 2005), may also explain the presence of large vesicles sometimes found in the cytoplasm of the mutant cells (Figs.”

“The excluded cytoplasm presumably flows to regions of uncompacted myelin, which in the early developing CNS alternate irregularly with compacted myelin sheaths along the individual axon (Storts and Koestner, 1969; Remahl and Hildebrand, 1990).”

“In oligodendrocytes — the glial cells of the central nervous system — myelin sheaths are thinner in the absence of CDC42, which seems to be the result of a defect in removing cytoplasm as the oligodendrocyte plasma membranes wrap around the axo”

“(76) In the case of oligodendrocytes, Cdc42 is important for the correct formation of myelin sheaths.(76) In the case of neuronal precursors, Cdc42 plays crucial roles in the establishment of Par6-dependent apico-basal polarity processes of stem cells.”

“For example, the deletion of Cdc42 or Rac1 in oligodendrocytes results in the formation of myelin outfoldings, but with reduced overall myelin thickness (622).”

“…that govern the cytoskeletal function during developmental myelination in the CNS, namely the β1 integrin, the Rho GTPases Rac1 and Cdc42 and the extracellular matrix (ECM) proteins fibronectin and laminin (Relvas et al., 2001; Benninger et al., 2006; Thurnherr et al., 2006; Lourenco et al., 2016).”

“In vivo, signaling pathways that regulate the actin cytoskeleton in other cell types have roles in myelination in both the CNS and peripheral nervous system (PNS), including phosphoinositide signaling (Goebbels et al., 2010; Snaidero et al., 2014), the Rho family GTPases (Thurnherr et al., 2006), and the Arp2/3 activa…”

“Interestingly, conditional knockouts of Rac1 and cdc42 have distinct and less-severe effects on oligodendrocyte myelination (Thurnherr et al. 2006), further underscoring differences between PNS and CNS myelination.”

“These proteins have been independently implicated in oligodendrocyte and Schwann cell development, process extension, axonal sorting, and myelination (Kim et al., 2006; Thurnherr et al., 2006; Bacon et al., 2007; Benninger et al., 2007; Nodari et al., 2007; Stendel et al., 2007).”

“3 Q9CPU4 Mgst3 E Glutathione S transferase Mu1 P10649 Gstm1 W,E Glutathione S transferase Mu2 P15626 Gstm2 E Glutathione S transferase Mu6 O35660 Gstm6 E Glutathione S transferase P1 P19157 Gstp1 S,V,E Glutathione S transferase P2 P46425 Gstp2 T Growth associated protein 43 P06837 Gap43 T GTPase Ran P62827 Ran E H+/K+…”

“RhoA activation in rodent oligodendrocytes inhibits remyelination,(42) and Rac1 ablation results in abnormal myelination.(43) Continued treatment with micromolar concentrations of FTY720 induced process extension in OPCs.”

“…and Myrf, whereas at the late stage of differentiation, they are recruited to the enhancers of cellular morphogenesis-associated regulators, such as Cdc42 and Rac1, critical for cytoskeleton reorganization and curvature-dependent actin polymerization (Thurnherr et al. 2006; Benninger et al. 2007).”

“Myelin aberrations were classified according to criteria described in Peters et al. (2000) and Thurnherr et al. (2006), for example, an axon surrounded by two myelin sheaths (Fig.”

“Similar assays and rationale were applied before to study the role of ECM–integrin signaling and downstream molecules in the regulation of oligodendrocyte process extension (Buttery and ffrench-Constant, 1999; Relvas et al., 2001; Colognato et al., 2002; Thurnherr et al., 2006).”

“In addition, in mice, myelin formation is impaired when the WASP family verprolin homologous (WAVE) or the Cdc42 and Rac1 GTPase cytoskeletal regulators are deleted (Kim et al., 2006; Thurnherr et al., 2006; Benninger et al., 2007).”

“…molecules mediate inhibitory signals via activation of RhoA Fyn-1 kinase regulates the activity of the small GTPase RhoA (Colognato et al., 2004), which, along with other Rho GTPase subfamily members is an important regulator of oligodendrocyte morphology (Mi et al., 2005; Thurnherr et al., 2006).”

“…the presence of Fyn in myelinating tracts at the time of myelination has not been described, and later studies did not find any proof of a role for Cdc42 and Rac1 in process outgrowth as null animals for both proteins exhibited normal oligodendrocyte differentiation (Thurnherr et al., 2006).”

“Nonetheless, FTY720 can gain access to the CNS, which contains SphK2 for efficient phosphorylation of FTY720 (Billich et al., 2003; Foster et al., in press).”

“An interesting example is the ablation of Cdc42 or Rac1 in oligodendrocytes, which results in the formation of numerous myelin outfoldings with abnormal accumulation of cytoplasm but with reduced overall myelin thickness (Thurnherr et al., 2006).”

“The morphological changes of the inner tongue in CNP deficient mice have some similarity to those caused following a conditional ablation of the Rho GTPases, Cdc42, and Rac1 in oligodendrocytes (Thurnherr et al., 2006).”

“Cd42 and Rac1 play a critical role during the early stages of myelination, particularly in compaction [86].”

“Moreover, the knowledge about the involvement of Rho GTPase signaling in an inherited neuropathy provides a direct path to a better understanding of the development, degeneration, and regeneration of the PNS. Identification of the underlying mechanisms of Schwann cell damage in inherited neuropathies may also hold pro…”

“Interestingly, transgenic mice specifically lacking Cdc42 and Rac1 in oligodendrocytes exhibit normal oligodendroglial differentiation, but show eventual defects in myelin compaction (Thurnherr et al., 2006).”

“Downstream intracellular mediators could involve the small GTPases of the Rho family such as RhoA, Cdc42, and Rac (Thurnherr et al., 2006; Zhao et al., 2007).”

“pated from the in vitro studies), but their ablation results in abnormal accumulation of cytoplasm within myelin and myelin outfoldings [67].”

“RAC1 signaling is important for myelin sheath formation (19) and growth of astrocytic processes (20).”

“Finally, Akt activation in vitro results in activation of Rac1, previously implicated in myelin sheath wrapping (Thurnherr et al., 2006; Benninger et al., 2007; Nodari et al., 2007).”

“For example, VAMP2, VAMP3, SNAP23 (Feldmann et al., 2009), and cdc42 (Thurnherr et al., 2006) were detected as ‘‘trapped’’ in the myelin compartment.”

“Instead, OL-specific conditional knockouts display abnormal cytoplasmic accumulation in the myelin membrane’s inner tongue (Thurnherr et al., 2006).”

“, 2009) deficiencies do not recapitulate Rac1 or cdc42 loss-offunction phenotypes (Thurnherr et al., 2006), it may be that other ECM (or non-ECM) upstream activators of these small GTPases are instead required.”

“These contrasting effects provide compelling evidence for the existence of different mechanisms controlling myosin II activity within the SC, as has been demonstrated in other cell types ( Totsukawa et al., 2000 , 2004 ).”

“There is evidence that cell polarity proteins such as PAK can function in mitotic exit (Höfken and Schiebel, 2002); however, the combined ablation of cdc42 and Rac1 in the oligodendroglial lineage did not affect either proliferation or differentiation of OPCs, but instead regulated myelin sheath formation (Thurnherr e…”

“, 2007) and oligodendrocyte cell biology (Thurnherr et al., 2006).”

“However, recent studies also revealed that Cdc42 and Rac1 intervene in myelination without affecting proliferation and differentiation of oligodendrocytes in culture (Thurnherr et al., 2006).”

“The abundance of 433 mRNAs encoding myelin-associated proteins, such as integrin beta 1 (Itgb1) [98], cell division cycle 42 (Cdc42), ras-related C3 botulinum toxin substrate 1 (Rac1) [99] as well as glyceraldehyde-3-phosphate dehydrogenase (Gapdh), was unchanged during oligodendrocyte differentiation.”

“Signaling through these proteins would be important for myelination, as they are involved in cytoskeletal remodeling, which is essential for oligodendrocyte process extension, axonal ensheathment, and formation of the myelin sheath (Bacon, Lakics, Machesky, & Rumsby, 2007; Kim et al., 2006; Thurnherr et al., 2006).”

“Additional components of this pathway are possibly, the Arp2/3 complex, members of the WASP family and small GTPases such as Cdc42 and Rac1 (Benninger et al. 2006; Jin et al. 2011; Kim et al. 2006; Novak et al. 2011; Thurnherr et al. 2006).”

“[70] who specifically ablated Cdc42 or Rac1 in oligodendrocytes in vivo.”

“Persistent enlargement of inner tongues may thus result either from (i) increased membrane growth rate, or (ii) impaired actin disassembly and myelin membrane compaction [48, 61, 66].”

“, 2010) have been elucidated in forebrain and oligodendrocyte development (Thurnherr et al., 2006).”

“Interestingly, loss of Cdc42 or Rac1 in oligodendroctyes results in disorganization of both compact and noncompact myelin membranes (Thurnherr et al., 2006).”

“Cdc42 mutants (Thurnherr et al., 2006; Wu et al., 2006; Benninger et al., 2007), Hb9-Cre (Arber et al., 1999) and Dhh-Cre (Jaegle et al., 2003; Pereira et al., 2009) mice 3568 | Brain 2012: 135; 3567–3583 M. Horn et al. have been described.”

“In contrast, the activity of other Rho GTPases, Cdc42 and Rac1, seems to increase as differentiation proceeds, and recent evidence suggests that its activity is required to regulate a late stage in myelin formation (Liang et al., 2004; Thurnherr et al., 2006).”

“mutants with multi-folded myelin extensions in the central nervous system (24).”

“This phenomenon is also seen in Cnp KO mice (Edgar et al., 2009) and Cdc42 conditional KO mice (Thurnherr et al., 2006).”

“The fundamental role of Rac1 in both CNS (Thurnherr et al. 2006) and PNS (Benninger et al.”

“…under the control of the 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) promoter (‘knock-in’) (Lappe-Siefke et al., 2003; Saher et al., 2005; Grove et al., 2007) and which were heterozygous for the floxed allele, as previously described (Benninger et al., 2006; Thurnherr et al., 2006).”

“…roles for the Rho GTPases Rac1 and Cdc42 in regulating process outgrowth and branching in differentiating OLGs are questioned by the in vivo observation that their conditional knockout in cells of the OLG lineage does not lead to reduced numbers of myelinated fibers (Thurnherr et al., 2006).”

“Recent findings indicate that the stage-specific and synergistic activation of Rac1 and Cdc42 is crucial for myelination (30), stressing how important the proper activation of Rac1 and Cdc42 is in Schwann cells.”

“In OLs ablation of Cdc42 and Rac1, positive regulators of the cytoskeleton, caused enlargement and retention of cytoplasm in the inner tongue of the OL process in the myelin sheath, indicating a failure of compaction in this region (Thurnherr et al., 2006).”

“While the deletion of cdc42 did not affect NG2-glia migration or proliferation in vitro (Thurnherr et al., 2006), a study could show that the proteoglycan NG2 itself can control migration by regulating planar cell polarity via RhoA/ROCK pathway activation (Biname et al., 2013).”

“, 2015), and Rho GTPases Cdc42 and Rac1 regulate myelin sheath formation (Thurnherr et al., 2006).”

“Additionally, Rho GTPases have also been implicated in the correct formation of myelin sheaths [32].”

“Focal contacts are themselves regulated by RhoGTPases (36) and Rac1 that is activated in Schwannomas (20, 35), and has been implicated in the regulation of focal complexes (21).”

“Rac and Cdc42 GTPases have been reported to have essential but distinct roles during SC development (Feltri et al. 2008) but act synergistically in oligodendrocytes to regulate myelin sheath formation (Thurnherr et al. 2006).”

“Furthermore, similar myelin outfoldings characterized by abnormal accumulation of cytoplasm in the inner tongue of the myelin sheath were detected after oligodendrocyte-specific ablation of Rho GTPases Cdc42 (Thurnherr et al., 2006), which is a known activator of N-Wasp (Rohatgi et al., 2000).”

“…(Liang et al., 2004), but tissuespecific conditional ablation of cdc42 or Rac1 in OLs does not affect proliferation, migration, or in vitro differentiation; instead it causes enlargement of the inner tongue of OL processes and formation of a different type of myelin sheath (Thurnherr et al., 2006).”

“Further, the WAVE complex regulates ARP2/3 which is necessary for initiation of myelination due to its role in actin filament assembly [86], and Cdc42 and Rac1 signaling, both affected by the WAVE complex, and are also necessary for myelin sheath formation [87].”

“…2008), ventral telencephalic neuron formation (Chen et al., 2007) and migration across the midline commissure during development (Kassai et al., 2008), proper axon outgrowth and guidance (Briancon-Marjollet et al., 2008), and myelin sheath formation in oligodendrocytes (Thurnherr et al., 2006).”

“Earlier studies have documented that the differential activation of RhoA, Rac1, and Cdc42 synergize in the process of OL differentiation and myelination (Czopka, Von Holst, Schmidt, Ffrench-Constant, & Faissner, 2009; Liang, Draghi, & Resh, 2004; Thurnherr et al., 2006).”

“Earlier studies have shown that Cdc42 and Rac1 synergize in the process of myelination without affecting proliferation and differentiation of oligodendrocytes (Thurnherr et al., 2006).”

“It is also responsible for the formation of myelin sheaths in central nervous system (29).”

“The ablation of Cdc42 does not affect OPC proliferation or differentiation but it does lead to the extraordinary enlargement of the OL process and the abnormal accumulation of cytoplasmin [47].”

“Cx43 is widely expressed in the CNS, especially neurons, microglia, astrocytes, and oligodendrocytes [26–28], and has been shown to be involved in various neurodegenerative diseases, including Alzheimer’s disease [29], Parkinson’s disease [30], multiple sclerosis [31], epilepsy [32] and prion diseases [33].”

“Also, the expression and activation of Rac1 are known to increase with oligodendroglial differentiation leading to correct myelination, which is supported by studies showing more myelin outfoldings in Rac1-KO mice than in control mice [41].”

“…has garnered signi fi cant attention due to its role in various neurodevelopmental 48,49 and neurodegenerative diseases 50 as well as its critical role in supporting oligodendrocyte functions including OPC migration, differentiation, and the formation of myelin sheaths around axons 40,41,51,52 .”

“The activation of ROCK may increase Ab42 and the activation of PKC may decrease Ab42.”

“During this shift from pre-myelinating OLs to mature myelinating OLs, myelin structural proteins, including proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin basic protein (MBP), are upregulated, coinciding with increased myelin ensheathment of axons 84,85 .”

“Interestingly, Cdc42 and Rac1 act synergistically to regulate appropriate formation of myelin sheath in the CNS, as conditional depletion of Cdc42 and/or Rac1 results in abnormal myelin outfoldings and enlargement of the inner tongue of the myelin sheath (Thurnherr et al., 2006).”

“Indeed, loss of Cdc42 and Rac1 leads to accumulation of cytoplasm of myelin sheaths and myelin out folding, revealing their role in coordinating myelin sheath wrapping [76, 77].”

“In addition, several intracellular signaling molecules including cdc42 and rac1 have been demonstrated to influence myelin architecture (Thurnherr et al., 2006).”

“The roles of Rho, Rac1, and Cdc42 in OPC proliferation and survival are unknown, although Cdc42 activity is not critical for these processes (55).”

“In particular, we have previously shown a relevant role of Rac1 in OL myelination (Thurnherr et al., 2006).”

“For the classification of myelin anomalies, we utilized criteria as reported before (Peters et al., 2000; Thurnherr et al., 2006).”

“In oligodendrocytes, Rac1 cooperates with another Rho GTPase cdc42 to regulate myelin formation, not OPC proliferation, migration, and differentiation (Thurnherr et al., 2006 ).”

“Notably, RAC1 acts as a regulator of OL differentiation and myelination [119,126], suggesting that RAC1 dysregulation may not only affect synapse maintenance but also contribute to white matter loss in schizophrenia.”