Our model indicates that the ectopic ventral GFP::RAB-3 puncta in cyy-1 resulted from the failure of synapse elimination, while those in cdk-5 resulted from the failure of the transportation PF-02341066 price of the disassembled ventral GFP::RAB-3 to the dorsal side. Therefore, it predicts that the ectopic ventral GFP::RAB-3 in cyy-1, but not in cdk-5, might represent functional presynaptic and postsynaptic specializations. To test this, we first examined whether the ectopic ventral GFP::RAB-3 puncta in cyy-1 or cdk-5 colocalize with an active-zone protein SYD-2. Consistent with our prediction, the ectopic ventral GFP::RAB-3 in cyy-1, but not in cdk-5, mutants shows a high degree of colocalization

with mCHERRY::SYD-2 ( Figures 6A and 6B). These results indicate that the ectopic RAB-3 puncta in cyy-1, but not cdk-5, mutants might represent presynaptic specializations. To further address the functionality of GFP::RAB-3 puncta, we tested if the ventral GFP::RAB-3 labeled synaptic vesicles in cyy-1, but not cdk-5, mutants undergo exocytosis. Mutations in unc-13 genes have been reported to have defects in the exocytosis of synaptic vesicles ( Brose et al., 2000) and lead to excessive accumulations of RAB-3 at functional presynaptic terminals www.selleckchem.com/products/DAPT-GSI-IX.html ( Ch’ng et al., 2008). It is conceivable that such an effect of unc-13 mutants would not occur in nonfunctional presynaptic sites. Consistently,

we found that the unc-13(e450) mutation causes increased intensity of GFP::RAB-3 4-Aminobutyrate aminotransferase puncta in DD neurons compared to wild-type background (data not shown). We next generated unc-13; cyy-1 and unc-13; cdk-5 double mutants. The ventral GFP::RAB-3 puncta in unc-13; cyy-1 double mutants are brighter compared to cyy-1 alone ( Figures S5A and S5B), implying that the ventral GFP::RAB-3 puncta in cyy-1 single

mutants might represent functional presynaptic specializations. However, the ventral GFP::RAB-3 puncta in unc-13; cdk-5 double mutants show similar intensity compared to cdk-5 alone ( Figures S5C and S5D), indicating that the ventral GFP::RAB-3 puncta in cdk-5 single mutants are not likely functional presynapses. As internal controls, dorsal GFP::RAB-3 puncta both in unc-13; cyy-1 and unc-13; cdk-5 double mutants are brighter compared to cyy-1 and cdk-5 alone, respectively ( Figure S5). To further clarify the identities of ectopic RAB-3 puncta in cyy-1 and cdk-5, we asked whether the ectopic puncta are associated with postsynaptic specializations. In wild-type animals, the GABAergic presynaptic SNB-1/synaptobrevin from the DD and VD neurons juxtaposes postsynaptic UNC-49/GABA receptors in the dorsal and ventral cord, respectively ( Gally and Bessereau, 2003). A lin-6 mutation that was shown to eliminate the VD neurons ( Hallam and Jin, 1998) facilitates our analysis of the DD ectopic RAB-3 puncta in the ventral side of the animal.

The incorrect stimulus resulted in the reverse (30:70) ratio. Thus, on 30% of trials subjects received “misleading” feedback. After 40 trials

the reinforcement contingencies reversed, so that the frequently rewarded stimulus now became frequently punished and vice versa. Each subject completed a pseudorandom fixed sequence of 80 trials. Subjects were instructed that the identity of the correct stimulus could change, but received no information as to how often such a change might occur (for details see Supplemental Experimental Procedures). Details of DNA extraction from the saliva samples and genotyping are described in the Supplemental Experimental Procedures. For DAT1, two alleles of interest were analyzed: the common 10R allele and the rarer 9R allele. The insertion/deletion polymorphism in the SERT promoter region (5HTTLPR) was genotyped BIBW2992 cost for the long (S) or short (L) alleles in combination with the single nucleotide polymorphism rs25531 A/G substitution in the same region. For the behavioral analysis,

we used a biallelic model, where the S allele was grouped with the rare LG allele (indicated as S′), given that the G-substitution in the L allele results in reduced expression more similar to the S allele ( Hu et al., 2006 and Praschak-Rieder et al., 2007). LA alleles were indicated as L′. Given the large sample size, all genotypes could be analyzed separately, which enabled testing for dose-dependent gene effects. In all analyses, GSK1349572 sex, age, and education level were included as covariates of no interest. The statistical significance threshold for all tests was p = 0.05, using a Bonferroni correction where appropriate.

To increase sensitivity, we did not use a Bonferroni correction for any of the control analyses. Using the χ2 test, we assessed whether there were any differences between genotype groups in the proportion of subjects passing the acquisition learning however criterion of eight consecutive correct responses, which we report in the Supplemental Experimental Procedures, where we also report baseline effects of task engagement/learning for both the pass and fail groups. Effects of reinforcement on subsequent choice were operationalized as the probability of repeating responses after reward (“win-stay”) and shifting responses after punishment (“lose-shift”) (Figure 1A). Errors during the reversal phase were divided into two types. Perseverative errors were defined as two or more consecutive incorrect choices of the previously rewarded stimulus. Thus, perseverative errors required subjects to erroneously stay with the previously correct stimulus, despite punishment. The remaining errors during the reversal phase were defined as “chance errors.

This powerful application of systems biology to proteomics can be readily applied to decipher in vivo protein networks for other normal or disease proteins in tissues as complex as the mammalian brain. See the Supplemental Experimental Procedures for additional details. BACHD mice were bred, maintained in the FvB/NJ background, and genotyped as previously

described (Gray et al., 2008). BACHD mice were maintained under standard conditions consistent with the National Institutes of Health guidelines and approved by the University of California, Los Angeles, Institutional Animal Care and Use Committees. Protein was prepared as previously described (Gu et al., 2009). Briefly, BACHD and WT BMS-754807 mouse brains were dissected in ice-cold 100 mM PBS and homogenized in modified RIPA buffer supplemented with Complete Protease Inhibitor Mixture tablets (Roche, selleck compound Indianapolis,

IN, USA) using ten strokes from a Potter-Elvehjem homogenizer followed by centrifugation at 4°C for 15 min at 16,000 × g. The resultant supernatant is the soluble fraction and protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Brain lysates (2.5 mg) were subjected to immunoprecipitation with anti-huntingtin clone HDB4E10 (MCA2050, AbD Serotec, 1:500) using Protein G Dynabeads (Invitrogen, Carlsbad, CA, USA). Immunoprecipitated proteins (500 μg) were washed, eluted with NuPAGE LDS loading buffer, and subjected to western blot analysis. Immunoprecipitated Linifanib (ABT-869) protein samples were separated on NuPAGE 3%–8% Tris-Acetate gels (Invitrogen), stained using GelCode Blue stain reagent (Thermo Fisher Scientific, Rockford, IL, USA), destained in ddH2O, and then cut into approximately 24–27 gel slices. The gel slices were washed three times in alternating solutions of a 50:50 mix of 100 mM NaHCO3 buffer/CH3CN and 100% CH3CN. Disulfide bonds were reduced

by incubation in 10 mM dithiothreitol (DTT) at 60°C for 1 hr. Free sulfhydryl bonds were blocked by incubating in 50 mM iodoactamide at 45°C for 45 min in the dark, followed by washing three times in alternating solutions of 100 mM NaHCO3 and CH3CN. The slices were dried and then incubated in a 20 ng/μl solution of porcine trypsin (Promega, Madison, WI, USA) for 45 min at 4°C, followed by incubation at 37°C for 4 to 6 hr. Afterwards, the supernatant was transferred into a fresh collection tube. The gels were incubated for 10 min in a solution of 50% CH3CN/1% trifluoroacetic acid (TFA), in which the supernatant was removed and combined with the previously removed supernatants. This step was repeated a total of three times. The supernatant samples containing the peptides were then spun to dryness and prepared for LC-MS/MS analysis by resuspension in 10 μl of 0.1% formic acid.

16; 2, orb2ΔQ, LI = 2.15) ( Figure 3; Table S4), suggesting that the residual memory of the orb2ΔA mutants might RO4929097 cell line be mediated by the Q domain

of Orb2B. Since the orb2ΔQΔB mutation was lethal when homozygous, we tested this allele in combination with the viable orb2ΔA allele. These flies, which lack the Q domain specifically in Orb2A, had a normal short-term memory ( Table S5D) but no long-term memory (5, orb2ΔQΔB/orb2ΔA, LI = 2.86) ( Figure 3; Table S4). This lack of memory shows that the Q domain in Orb2A is essential, and that of Orb2B insufficient, for long-term memory. To test for the sufficiency of the Q domain in Orb2A, we tested the memory of the transheterozygotes in which the Q domain is present only in Orb2A. The learning index of these mutants was indistinguishable from control flies in which both isoforms are intact (6, orb2ΔB/orb2ΔQΔA, LI = 16.97; 7, orb2ΔB/orb2ΔA LI = 20.83) ( Figure 3; Table S4). These results indicate that Orb2A has a specific role in long-term memory that requires the Q domain, which in Orb2B is both dispensable and insufficient. Epacadostat solubility dmso To assess the role of the RBD in long-term memory, as a first step we chose to replace the Orb2 RBD with the RBDs of other CPEBs,

reasoning that such chimeric proteins might retain activity toward conserved and common RNA targets but not Orb2-specific targets involved in long-term memory formation. A swap of the Orb2 RBD with the RBD of Orb1 (3, orb2orb1RBD) did not rescue viability, whereas the swap with the RBD of the mCPEB2 (4, orb2mCPEB2RBD) rendered flies viable and healthy ( Figure 4A; Table S5A). This indicated that RNA binding properties of this domain are required during the development, and moreover

suggested a potential conservation in RNA targets between the CPEB II family members at least in development. The conservation tuclazepam of RNA targets is consistent with the high homology in this region, ∼90% ( Theis et al., 2003). Interestingly, orb2mCPEB2RBD mutants showed strong long-term memory impairment in comparison to the control flies (4, orb2mCPEB2RBD LI = 6.16; 1, orb2+, LI = 32.39) ( Figure 4A; Table S5A). In contrast, short-term memory was normal (4, orb2mCPEB2RBD, LI = 40.0; 1, orb2+, LI = 42.34) ( Figure 4A; Table S5A′), indicating that the long-term memory impairment is unlikely the result of developmental defects caused by the RBD swap. Most importantly, this allele provided us with the unique opportunity to assess the role of the RBD in Orb2B in long-term memory, independently of its role in development. In the orb2mCPEB2RBD background, expression of the wild-type Orb2B, but not Orb2A, fully rescued memory (3, orb2ΔA/orb2mCPEB2RBD, LI = 28.5; 4, orb2ΔB/orb2mCPEB2RBD, LI = 1.68) ( Figure 4B; Table S5B), and this rescue was dependent on its RBD (5, orb2RBD∗ΔA/orb2mCPEB2RBD, LI = 1.04, see the paragraph below on the mutated RBD∗).

, 2011), for subsequent clearance by the proteasome. This process constitutes, as it were, a mitochondrial

version of ER-associated degradation ( Heo et al., 2010). Interestingly, mutations in VCP were recently selleck found to cause familial ALS ( Johnson et al., 2010). Thus, mutations in mitochondrial quality control genes could prevent the efficient elimination of damaged mitochondria and the degradation of superfluous and potentially deleterious polypeptides, hence leading to neuronal dysfunction and perhaps ultimately to cell death. In order for quality control to operate at the level of the mitochondrion, cells must be able to distinguish between “good” and “bad” organelles, and in fact, such discrimination does occur. Mitochondria apparently are deemed to be good if they have a high membrane potential (Δψ), and perhaps low levels of reactive oxygen species (ROS) as well, both presumably indicative of a well-functioning respiratory chain. Conversely, they are deemed bad if they have a low Δψ and elevated ROS, indicative of defective OxPhos; these are the organelles that are eliminated

via selective mitophagy (Twig and Shirihai, 2011). Mitophagy of damaged organelles, however, is a last resort, as cells initially try to prevent the accumulation Ku-0059436 mouse of bad mitochondria via maintenance of a dynamic equilibrium between mitochondrial fission and fusion, which “homogenizes” organellar contents. This mixing of a few bad mitochondria within a larger pool of good ones allows for complementation of genes and gene products to take place after mitochondria

Linifanib (ABT-869) have exchanged contents (Gilkerson et al., 2008), thereby blunting, or even eliminating, the deleterious effects of misfolded proteins and randomly mutated mtDNAs (Twig and Shirihai, 2011). Thus, from a quality control standpoint, one might predict that mutations in genes encoding proteins required for mitochondrial dynamics, and especially organellar fission and fusion, would result in compromised organellar “mixing,” leading to an excess accumulation of bad mitochondria, perhaps causing disease, and this is indeed the case. Gene products in this category include four associated with fusion (although interestingly, none with fission): MFN2 and GDAP1, both causing CMT, and OA proteins OPA1 and OPA3, both causing OA. Even though OPA1 and OPA3 (Huizing et al., 2010 and Ryu et al., 2010) and GDAP1 (Niemann et al., 2005) interact with mitofusins to regulate the mitochondrial network, it is again worth noting that the four genes are associated with two totally different clinical presentations. Mitochondrial dynamics are also altered in HD (Bossy-Wetzel et al., 2008, Kim et al., 2010 and Oliveira, 2010), as the expression of mitochondrial fission-related proteins, such as FIS1 and DRP1 (Costa et al., 2010), which happens to interact with HTT (Song et al.

Finally, in the same neurons, treatment with Aβ42 oligomers led to a slight, albeit reproducible and significant, MK8776 increase in Tau phosphorylation on S262 in control AMPKα1+/+ but not in AMPKα1 null hippocampal neurons (Figures 6E and 6F), suggesting that AMPKα1 mediates the phosphorylation of Tau on S262 induced by Aβ42 oligomers in hippocampal neurons. Loss of synapses begins during the early stages of AD and progressively affects neuronal network activity, leading to cognitive dysfunction (Coleman and Yao, 2003; Palop and Mucke, 2010; Terry

et al., 1991). In vitro and in vivo studies have demonstrated that Aβ oligomers are contributing to early synapse loss (Hsia et al., 1999; Hsieh et al., 2006; Lacor et al., 2007; Mucke et al., 2000; Shankar et al., 2007), whereas recent studies support Tau as one of

the mediators of Aβ toxicity in dendrites (Ittner et al., 2010; Roberson et al., 2007, 2011). However, our understanding of the molecular mechanisms linking Aβ oligomers and Tau synaptotoxicity in dendritic spines remains incomplete. Here, we report that (1) AMPK is overactivated in hippocampal neurons upon application of Aβ42 oligomers, and this activation is dependent on CAMKK2; (2) CAMKK2 or AMPK activation is sufficient to induce dendritic spine loss in hippocampal neurons in vitro and in vivo; (3) Aβ-mediated activation of AMPK induces the phosphorylation of Tau on residue S262 in the microtubule-binding domain; check details and (4) inhibition of either CAMKK2 or AMPK catalytic activity, or expression of a nonphosphorylatable form of Tau (S262A), blocks Aβ42 oligomer-induced PDK4 synaptotoxicity in hippocampal neurons in vitro and in vivo. AMPK is an important homeostatic regulator and is activated by various forms of cellular and metabolic stresses (Mihaylova and Shaw, 2011; Shaw et al., 2004). Oxidative stress such as elevation of ROS can activate AMPK through a mechanism that is still

unclear (reviewed in Hardie, 2007). Because part of the neuronal toxicity induced by Aβ is thought to involve increased ROS production (Schon and Przedborski, 2011), future experiments should test if AMPK function during Aβ-mediated neurodegeneration requires the ability of ROS to activate AMPK. In the brain, AMPK activity is increased in response to metabolic stresses such as ischemia, hypoxia, or glucose deprivation (Culmsee et al., 2001; Gadalla et al., 2004; Kuramoto et al., 2007; McCullough et al., 2005) and is abnormally elevated in several human neurodegenerative disorders, including AD and other tauopathies, amyotrophic lateral sclerosis, and Huntington’s disease (Ju et al., 2011; Lim et al., 2012; Vingtdeux et al., 2011b). Whether activation of AMPK in these different pathological contexts has a neuroprotective or deleterious outcome in various neuronal subtypes remains controversial (Salminen et al., 2011).

We then proceeded as for source-level coherence, but without neighborhood filtering. This resulted in clusters that represent significant changes in signal power across space, time, and frequency. We compared conditions using both random effects (across

subjects) Cilengitide and fixed effects (pooled across subjects) statistics. To visualize the identified networks we separately projected them onto different subspaces. To display the spatial extent (Figures 3A and 4A), we computed for each location the integral of the corresponding cluster in the connection space over time, frequency, and target locations. This integral was then displayed on the brain surface. This visualization reveals the spatial extent of the network independent of its intrinsic synchronization structure and location in time and frequency. Complementary to the spatial projection, we visualized the spectro-temporal projection (Figures 3B and 4B) by integration over all spatial locations (3D × 3D). This projection shows when and at which frequencies a cluster was active irrespective of the spatial location of synchronization. To analyze further properties of a network (modulations in power, other coherence contrasts, and single-trial analysis), we proceeded as follows: To account for interindividual differences, for each subject, we identified the connections within the network that were statistically significant (we computed

t-statistics for each connection in the cluster between conditions using STCP;

p < 0.05, one tailed). We averaged the property www.selleckchem.com/screening/anti-cancer-compound-library.html of interest (e.g., signal power) across each subject’s significant connections and used the resulting values for further analyses and tests. Importantly, the statistical sensitivity of these secondary tests is much higher than for the initial network-identification. The network-identification accounts for a massive multiple-comparison problem, whereas the secondary analyses use only a single test. This explains why the beta network differs between bounce and pass trials, as shown by a secondary analysis, but is not identified in the less sensitive network identification based on the bounce versus Bumetanide pass contrast. To analyze the synchronization pattern of the beta network (Figures 3C and 3E), we defined seven regions of interest (ROIs) in source space (Table S1). We selected sources that constitute a local maximum in the spatial network pattern and summed the connections between any two ROIs in the network. For each connection between two ROIs, the result was normalized by the maximum across all ROI-pairs, thresholded at 0.1, and visualized as the width of lines connecting the ROIs on the brain surface. We used ROC analysis to test whether coherence within a network predicted the subjects’ percept on a single-trial level (Green and Swets, 1966). We computed a predictive index that approximates the probability with which an ideal observer can predict the percept from the coherence on a single trial.

, 2009). In addition, how corridor neurons have acquired their internal guidepost function during evolution remains to be elucidated. Here, we address how TA pathfinding is differentially guided in mammal and reptile/bird embryos along an internal or external path, respectively. We found that species-specific TA trajectories diverge as PI3K Inhibitor Library in vivo they cross the MGE even though essential internal corridor neurons

are conserved in mouse, human, sheep, turtle, snake, and chicken embryos. Combination of grafts in chicken and mouse embryos shows that a cardinal difference between mammals and birds lies in the local positioning of corridor neurons that have otherwise remarkably conserved axonal guidance properties. At the molecular level, the secreted factor Slit2 is differently expressed BAY 73-4506 in the ventral telencephalon of the two species and acts as a short-range repellent on the migration of corridor cells. Using a combination of in vivo and ex vivo experiments in mice, we demonstrate that Slit2 is

required to locally orient the migration of mammalian corridor cells and thereby switches the path of TAs from a default external route into an internal path to the neocortex. Taken together, our results show that the minor differences in the positioning of conserved neurons, which is controlled by Slit2, play an essential role in the species-specific pathfinding of TAs, thereby providing a framework

to understand the shaping and evolution of a major forebrain projection. TAs reach the mammalian neocortex via the internal capsule, whereas they join an external lateral forebrain bundle toward other structures in nonmammalian vertebrates (Butler, 1994, Cordery and Molnar, 1999 and Redies et al., 1997). To understand how this major change in brain connectivity occurred, we first reexamined in detail the positioning of TAs in the ventral telencephalon of different species. We observed that already within the MGE mantle, TAs navigate internally in mammals, whereas they grow Astemizole externally in reptiles/birds (Cordery and Molnar, 1999, Redies et al., 1997 and Verney et al., 2001), as observed in mouse and chick embryos (Figure 1; data not shown). This difference can be further visualized by a comparison with early midbrain dopaminergic projections: whereas TAs and dopaminergic axons both navigate externally to the MGE mantle of reptiles/birds, they grow at distinct internal and external levels, respectively, in mammals (Cordery and Molnar, 1999, Redies et al., 1997 and Verney et al., 2001) (Figures 1C, 1D, 1G, and 1H). Thus, TAs undertake different internal/external trajectories in the MGE, thereby supporting a role for this intermediate target. We previously showed that TA pathfinding in the mouse MGE is controlled by short-range guidepost corridor cells (Lopez-Bendito et al., 2006).

The effect of the timing regimens on FEV1 was minor. Although some between-group comparisons were of borderline statistical significance, HKI-272 clinical trial the mean differences and their 95% CIs were all well below 150 mL (the a priori smallest worthwhile effect), and equated to ≤ 2% of the predicted normal value. Therefore, although these borderline results favoured inhalation of hypertonic saline before airway clearance techniques, any differences between the effects of the timing regimens on FEV1 are probably too

small to be clinically important. However, in the long term, clinically worthwhile differences in lung function from the use of a particular timing regimen could occur – possibly through differences in clearance effects and differences in adherence. This could be investigated in future research. For FVC, the between-group comparisons were again either of borderline

statistical significance or were non-significant. However, Apoptosis inhibitor unlike the narrow confidence intervals seen in the FEV1 data, some of the between-group comparisons for FVC had 95% CIs that did not exclude the possibility of substantial effects. For example, inhaling hypertonic saline before airway clearance techniques might increase the improvement in FVC by as much as 180 mL more than inhaling it during or after the techniques. Therefore, further data could be obtained to make the estimate of the effect on FVC much more precise and then to determine whether it is large enough to be clinically worthwhile. As with FEV1, the effect of long-term

use of a timing regimen on FVC could also be investigated. Perceived efficacy and satisfaction were significantly lower when hypertonic saline was inhaled after airway clearance techniques than with the other timing regimens. Inhalation of hypertonic saline after the techniques may fail to capitalise on effects of hypertonic saline on mucus clearance if techniques to promote expectoration are not undertaken until 4–6 hours later. Although these results were statistically significant, some may not be clinically worthwhile because the 95% CIs contain effects smaller than the a priori smallest worthwhile effect of 10 mm on the 100 mm visual analogue scale. However, the effect of inhaling hypertonic saline before rather than after the techniques increased satisfaction by 20 mm (95% CI 12 to 29), which clearly exceeds the smallest worthwhile effect. The data did not support our hypothesis that inhaling hypertonic saline after airway clearance techniques would reduce tolerability. We expected that inhaling the hypertonic saline after the techniques may have delivered it to a more exposed airway epithelium because the amount of overlying mucus would be minimised. However, this timing regimen did not reduce subjective or objective tolerability.

Rnd2 can also bind p190RhoGAP ( Wennerberg et al., 2003) and this interaction is similarly disrupted by mutation of residue BMS-754807 research buy T39 in its effector domain into valine ( Figure S6B). However, Rnd2T39V was as effective as wild-type

Rnd2 at rescuing the migration of Rnd2-silenced neurons ( Figure S6F), indicating that Rnd2 activity in the cortex does not require interaction with p190RhoGAP and that Rnd2 and Rnd3 inhibit RhoA signaling via distinct mechanisms. As RhoA has previously been well characterized for its role in regulating the actin cytoskeleton (Govek et al., 2005 and Ridley et al., 2003), we investigated whether Rnd2 and/or Rnd3 knockdown were altering actin dynamics in cortical neurons. We examined filamentous actin (F-actin) levels in electroporated cerebral cortical cells by coelectroporating a fluorescent F-actin probe based

on the see more actin-binding domain of the Utrophin protein (EGFP-UTRCH-ABD). The UTRCH-ABD probe has been shown to faithfully report the presence of F-actin without altering F-actin concentrations in cells expressing the probe ( Burkel et al., 2007). Knockdown of Rnd3 resulted in a marked accumulation of F-actin in the processes of electroporated cells, while F-actin accumulated in both cell body and processes of Rnd2 knockdown cells ( Figure 6A), suggesting that both Rnd2 and Rnd3 regulate actin cytoskeleton organization in migrating neurons. To determine whether F-actin accumulation is responsible for the first migration defects of Rnd3- and Rnd2-silenced neurons, we coelectroporated cofilinS3A, a nonphosphorylatable form of cofilin that constitutively depolymerizes F-actin, together with Rnd2 or Rnd3 shRNA. Overexpression of cofilinS3A fully rescued the migration defect of Rnd3-silenced

neurons, thus indicating that Rnd3 promotes cortical neuron migration by inhibiting RhoA-mediated actin polymerization ( Figure 6B). In contrast, cofilinS3A expression had no effect on the migration of Rnd2-silenced neurons demonstrating that actin remodeling does not contribute to Rnd2 migratory function and that inhibition of RhoA by Rnd2 activates another unidentified process required for neuronal migration. Our results so far have established that both Rnd3 and Rnd2 inhibit RhoA activity (Figure 5), but that they nevertheless promote migration via distinct mechanisms involving p190RhoGAP and F-actin depolymerization in the case of Rnd3 and not Rnd2 ( Figure 6 and Figure S6). An explanation for this apparent paradox could be that Rnd2 and Rnd3 interact with RhoA in different cell compartments, because Rho GTPases have been shown to interact with different effectors and to trigger different cellular responses when located in different cell compartments ( Pertz, 2010).