Rho-Linked Mental Retardation Genes 225 which assumes a configuration that prevents the activation of the C-terminal kinase domain. Upon binding to Rac-GTP or Cdc42-GTP, the autoinhibition is alleviated, resulting in activation of the PAK proteins and their autophosphorylation (Jaffer and Chernoff, 2002; Bokoch, 2003). Noteworthy is a recent study that reported PAK3 binds significantly more Cdc42 than Rac1, and is selectively activated by endoge- nous Cdc42, suggesting that PAK3 is a selective effector of Cdc42 (Kreis et al., 2007). Among the PAK proteins, PAK1 and PAK3 are highly expressed in the brain. Both proteins are present in the hippocampus and cortex, with PAK3 being particu- larly highly expressed in postmitotic neurons of the dentate gyrus and cortical layers II/III and V (Kreis and Barnier, 2009). In neurons, PAK3 shows a diffuse distribu- tion throughout the soma and proximal dendrites and is present in dendritic spines (Boda et al., 2004). As discussed below, both PAK1 and PAK3 proteins have been implicated in spine morphogenesis, however, as of this writing, only mutations in PAK3 have been identified that are associated with nonsyndromic MR. In particular, five dif- ferent mutations in the PAK3 gene have been identified in several X-linked MR pedigrees. The first PAK3 mutation, R419X, found in family MRX30, introduced a premature stop codon that abolishes the kinase activity of the truncated product (Allen et al., 1998). Since then four additional mutations have been identified in MR patients. These include the R67C and the A365E mutations located in the p21- binding domain and in the kinase domain, respectively; the W446S mutation located in the catalytic domain; and, finally, a splice mutation located at the 5 end of intron 6 leading to a disruption of the reading frame with a premature stop codon at posi- tion 128 (Bienvenu et al., 2000; Gedeon et al., 2003; Peippo et al., 2007; Rejeb et al., 2008). Biochemical analysis demonstrated that PAK3 proteins harboring the R419X and A365E mutations, and presumably also the W446S mutation, are devoid of kinase activity, whereas the PAK3 protein with the R67C mutation has a func- tional kinase domain but displays a decrease in binding to Cdc42 and a decrease in its activation by this GTPase (Kreis et al., 2007). Several lines of evidence have demonstrated a role for PAK3 (as well as PAK1) in the regulation of dendritic spine morphogenesis, s ynapse formation, and/or synap- tic plasticity. First, a study using transgenic mice in which the catalytic activity of the PAK family members, PAK1 and PAK3, is inhibited by expression of the PAK- autoinhibitory domain (AID-PAK) revealed that cortical neurons of these mice have fewer spines than control animals and show a shift in the overall spine population towards shorter spines with larger heads and postsynaptic densities. Interestingly, these mice also show enhanced LTP and reduced LTD in the cortex, as well as specific deficits in the consolidation phase of hippocampus-dependent memory, suggesting a role for PAK in memory retention (Hayashi et al., 2004). Secondly, Boda et al. observed that RNAi-mediated suppression of PAK3, or expression of a dominant negative, kinase-dead, PAK3 mutant (R419X), in rat hip- pocampal organotypic slice cultures results in the formation of abnormal elongated dendritic spines and filopodia-like protrusions, as well as a decrease in mature spine synapses. They observed that these defects were associated with reduced expression of AMPARs at the synapse and defective LTP (Boda et al., 2004). Interestingly, a 226 N.N. Kasri and L. Van Aelst more recent study compared the effects of three different PAK3 mutants (R67C, A365E and R419X) on spine morphogenesis and observed that these mutant pro- teins affect spinogenesis differentially (Kreis et al., 2007). Specifically, they found that expression of the PAK3 kinase-dead mutants, A365E and R419X, in CA1 neurons of hippocampal brain slices profoundly altered spine morphology without affecting spine density, whereas expression of the PAK3 R67C mutant drastically decreased spine density. Based on these data, a model was proposed in which PAK3 may act at two different steps during spine formation, namely at (1) the initiation of spines and (2) at spine maturation (Kreis et al., 2007). Finally, mice lacking the PAK3 gene have been generated, and analysis of these mice showed selective impairment in late-phase hippocampal LTP, a distinct form of long-term synaptic plasticity involving new gene expression (Meng et al., 2005). Surprisingly, in this mouse knock-out model, no obvious deficits in spine morphology or density were observed. The differences seen with regard to spine morphology between the knock-out and RNAi studies could potentially reflect dif- ferences between a homogeneous and a heterogeneous cell population, respectively, or could be attributed to compensatory mechanisms (e.g., PAK1 or PAK2) in the knock-out mice. Indeed, it has recently been shown that expression of active PAK1 can revert the long spine phenotype induced by RNAi-mediated suppression of PAK3 (Boda et al., 2008), although it should be noted that PAK1 and PAK3 also seem to have distinct roles in spine morphogenesis (Boda et al., 2008). The PAK3 knock-out mice did, however, show a dramatic decrease in the levels of the phos- phorylated/active form of cAMP-responsive element-binding protein (CREB) in the hippocampus, whereas no changes in the total CREB protein levels were observed (Meng et al., 2005). Several studies have shown that CREB function is important for synaptic plasticity and memory formation in mice (Kandel, 2001; Lonze and Ginty, 2002). Therefore, the reduced CREB function may be responsible for the impairment in late-phase hippocampal LTP in these mice. Together, these studies indicate that mutations in PAK3, which are associ- ated with nonsyndromic MR, cause aberrant spine structure and/or function as a result of altered actin dynamics and/or transcriptional regulation (see also Fig. 2). Interestingly, defects in PAK signaling not only result in MR, but recently have also been associated with Alzheimer disease (AD) (reviewed in Kreis and Barnier, 2009). This may not be that surprising considering the analogy between AD and MR; that is, both conditions share in common spine loss or spine alterations. AD is defined clinically as a gradual loss of cognitive performance with the onset of a slowly pro- gressive impairment of memory during mid-to-late adult life. The neuropathological hallmarks include amyloid deposits (Aß), neurofibrillary tangles, and reductions in the number of neurons and synapses in many areas of the brain, but especially in the cerebral cortex and the hippocampus (LaFerla and Oddo, 2005). Of particular interest is that the Aß oligomers implicated in AD were shown to reduce PAK1 and PAK3 expression levels and activities in the hippocampus and temporal cortex, resulting in a loss of drebrin from the spines and synaptic dysfunctions (Zhao et al., 2006; Ma et al., 2008). Drebrin is localized at spines in adult brains and is required for active clustering and synaptic targeting of PSD95 Rho-Linked Mental Retardation Genes 227 (Takahashi et al., 2003). Expression of active PAK in hippocampal neurons could prevent the effects induced by Aß oligomers, and significantly, pharmacological PAK inhibition in adult mice was sufficient to cause drebrin loss and memory impairment (Zhao et al., 2006). Thus, these findings indicate that loss of PAK3 and/or PAK1 is involved in both developmental-dependent and age-dependent cognitive deficits, such as observed in AD. 2.2.3 Rho Guanine Nucleotide Exchange Factor 6 (ARHGEF6) The ARHGEF6 gene, also known as αPIX or Cool-2, is another Rho GTPase-linked gene shown to be involved in nonsyndromic X-linked MR (Kutsche et al., 2000). It codes for a Cdc42/Rac1 GEF, which harbors a number of interesting motifs impli- cated in protein–protein interactions (Bagrodia et al., 1998; Manser et al., 1998; Kutsche et al., 2000; Koh et al., 2001; Feng et al., 2002). Besides the Dbl homol- ogy (DH) and plextrin homology (PH) domains, it contains an N-terminally located calponin homology (CH) domain, an SH3 domain, a GIT binding domain, and a C-terminally located leucine zipper that mediates the formation of homo- and het- erodimers. The dimeric form of ARHGEF6/Cool-2/αPIX was found to act as a specific GEF for Rac1, whereas the monomeric form as a GEF for Cdc42 as well as Rac (Feng et al., 2004). Significantly, ARHGEF6 has been shown to directly interact with group I PAK kinases, as well as with the synaptic adaptor protein GIT1 (G- protein coupled receptor kinase-interacting protein1) (Bagrodia et al., 1998; Manser et al., 1998; Daniels et al., 1999; Feng et al., 2002; Zhang et al., 2003). The latter protein has been shown to be crucial for spine formation; its loss of expression sig- nificantly decreases the number of spines (Zhang et al., 2005). Furthermore, GIT1 has been found to be important for the localization of the closely related family member ßPIX to dendritic spines and to activate Rac1 and its downstream effector PAK locally (Zhang et al., 2005). The first mutation in ARHGEF6 associated with nonsyndromic X-linked MR was identified in a male carrying a reciprocal X;21 translocation breakpoint located between exons 10 and 11 of the ARHGEF6 gene (Kutsche et al., 2000). Subsequently, additional mutations have been identified in the first intron of the gene that result in preferential skipping of exon 2 and a predicted protein product lacking the first 28 amino acids in affected males in a large MRX family (MRX46) (Kutsche et al., 2000). A recent study demonstrated that the ARHGEF6 protein is present in CA3 and CA1 neurons of the hippocampus and that expression of epitope-tagged ARHGEF6 in hippocampal slice cultures shows a punctate staining in dendritic spines that colocalizes with PSD-95 and other synaptic proteins (Node-Langlois et al., 2006). The same study also revealed a requirement for ARHGEF6 in spine morphogenesis. Whereas overexpression of ARHGEF6 did not alter spine mor- phology, RNAi-mediated knock-down of ARHGEF6 resulted in abnormalities in spine morphology similar to those reported for knock-down of PAK3: a decrease of large mushroom-type spines and an increase of elongated spines and filopodia-like protrusions (Node-Langlois et al., 2006). 228 N.N. Kasri and L. Van Aelst Consistent with a role for ARHGEF6 in the regulation of spine morphogen- esis, the Drosophila homologue, dPIX, was also shown to play a major role in regulating postsynaptic structures and protein localization at the glutamatergic neu- romuscular junction (Parnas et al., 2001). It is important to note that the defect in spine structure in ARHGEF6 RNAi-treated neurons could be rescued by coex- pression of a constitutively active PAK3 protein, but not with wild-type PAK3 (Node-Langlois et al., 2006). By contrast, the phenotype caused by knock-down of PAK3 could not be rescued by overexpression of ARHGEF6. Together, these results indicate that ARHGEF6 is involved in the same signaling pathway as PAK3, thereby controlling spine morphogenesis and plasticity of synaptic networks. Hence, similar mechanisms are likely to underlie cognitive deficits associated with mutations in ARHGEF6 and PAK3. Interestingly, a recent study focusing on the closely related family member βPIX suggested a potential mechanism by which the PIX proteins are regulated in the synapse. As discussed above, Saneyoshi et al. identified a signaling pathway upstream of βPIX by which NMDAR activation during neuronal development or plasticity can modulate spinogenesis. They f ound that CaMKK/CaMKI interacts with βPIX/GIT1 and mediates phosphorylation of Ser516 in βPIX to enhance Rac activity and promote formation/stabilization of mushroom-shaped spines (Saneyoshi et al., 2008). 2.2.4 CYFIP/Rac/PAK and Fragile X Syndrome Fragile X syndrome (FXS) is the most common inherited cause of MR with approxi- mately 1 in 4000 males affected. In the vast majority of cases, this X-linked disorder is caused by an unstable expansion of the CGG trinucleotide repeat and hypermethy- lation of CpG dinucleotides in the 5 untranslated region of the FMR1 gene, which results in transcriptional silencing of FMR1. The first clinical indication of FXS is often delayed developmental milestones, such as mild motor delays and/or language delays. Autistic-like behaviors such as hand flapping, poor eye contact, and hand bit- ing may be observed. The average IQ in adult men with the completely methylated full mutation is approximately 40. Less affected males, which typically have incom- plete methylation and thus resulting in an incomplete activation of FMR1, may have an IQ in the borderline to low normal range. Physical features may include macro- orchidism that is apparent just before puberty and those related to a connective tissue dysplasia, which include a long, narrow face, prominent ears, joint hypermobility, and flat feet (reviewed in Garber et al., 2006; Bassell and Warren, 2008; Garber et al., 2008) FMR1 encodes a selective RNA-binding protein (FMRP) that regulates the local translation of a subset of mRNAs at synapses in response to activation of metabotropic glutamate receptors (mGluRs) and possibly other receptors. In the absence of FMRP, increased and dysregulated mRNA translation is believed to contribute to altered spine morphology, synaptic function, and loss of protein synthesis-dependent plasticity (reviewed in Bear et al., 2004; Bagni and Greenough, 2005; Bassell and Warren, 2008). As mentioned before, the shape and density of dendritic spines are altered in patients and in FMR1-deficient mice brains. A few Rho-Linked Mental Retardation Genes 229 reports suggested that FMRP could affect spine morphogenesis through regulation of “cargo” mRNAs, such as Map1B and profilin mRNAs (Lu et al., 2004;Reeve et al., 2005). More recent studies, mainly performed in Drosophila, have linked FMRP’s effect on spine morphology to the Rac1 GTPase signaling pathway. One group demonstrated that the mRNA encoding Rac1 is present in Fmr1-messenger ribonucleoprotein complexes (Lee et al., 2003). Furthermore, evidence was provided that Fmr1 and Rac1 genetically interact, and that Rac1 mediates at least in part the effects of Fmr1 (Drosophila fragile X-related protein) on dendritic branching (Lee et al., 2003). An independent study demonstrated a biochemical association between the Fmr1-interacting protein dCYFIP and dRac1 (Schenck et al., 2003). Phenotypic analyses and genetic interaction experiments placed dRac, CYFIP, and dFMRP in a common pathway controlling axonogenesis and synaptogenesis. Furthermore, evi- dence was presented that Rac1 negatively regulates CYFIP, which in turn negatively regulates Fmr1, with the net result that dRac1 positively regulates dFMR1 action on neuronal morphogenesis (Schenck et al., 2003). Together with the above findings, these data suggest that there is a feedback loop between Rac1 and Fmr1 functions in vivo. The mammalian homologues of Drosophila CYPIP, CYFIP1, and CYPIP2, have also been shown to interact with FMRP. In mammals, CYFIP1 (also known as p140/Sra-1) was initially identified as a target of Rac1 (Kobayashi et al., 1998), whereas CYFIP2 (also termed PIR121) was found to be part of the WAVE protein complex, which mediates actin nucleation by Rac (Eden et al., 2002). In its inac- tive state, this complex contains WAVE and four other proteins: HSPC300, Nap125, Abi2, and PIR121. When active Rac1 is added, the complex dissociates, freeing WAVE and HSPC300, thereby allowing WAVE to activate the actin-related protein 2/3 (Arp2/3) complex to induce actin polymerization (see Fig. 2). In analogy to the mechanism of WAVE activation, a model was proposed in which CYFIP dis- sociates from FMRP/Fmr1 upon interaction with activated Rac1, allowing released FMRP/Fmr1 to regulate local protein translation. A recent study also reported an interaction between PAK1 and FMRP and demonstrated that inhibition of group I PAK kinases rescued symptoms of knock-out (KO) FMR1 mice (Hayashi et al., 2007). Specifically, the spine abnormalities observed in FMR1 KO mice were partially restored by postnatal expression of a dominant negative PAK transgene (AID-PAK). Furthermore, the reduced cortical long-term potentiation was fully restored and several of the behavioral abnormalities associated with FMR1 KO mice were ameliorated by the PAK-AID transgene. Whereas the precise underpinnings of the PAK1/FMRP interaction remain to be established, it is tempting to specu- late (analogous to the CYFIP/WAVE complex) that FMRP and PAK1 could inhibit each other to form an inactive complex. Activation of PAK by GTPases would then trigger the dissociation of the two proteins allowing FMRP to regulate protein translation. Together, these data suggest a model in which FMRP, Rac1, CYFIP, and/or PAK act together in a dynamic signaling complex(es) to regulate actin dynamics and control local protein translation, processes that are key to neuronal morphogenesis and connectivity. 230 N.N. Kasri and L. Van Aelst 2.2.5 Oculocerebrorenal Syndrome of Lowe Protein 1 (OCRL1) Oculocerebrorenal syndrome of Lowe (OCRL) or Lowe syndrome is a rare X- linked developmental disorder characterized by MR, congenital cataracts, and renal Fanconi syndrome (Attree et al., 1992). The gene responsible for OCRL was ini- tially identified by positional cloning of X chromosome breakpoints and encodes a protein termed OCRL1, an inositol polyphosphate-5-phosphatase (Attree et al., 1992;Lowe,2005). In addition to the central polyphosphate-5-phosphatase domain, which uses PI(4,5)P 2 and PI(3,4,5)P 3 as the preferred substrates (Zhang et al., 1995; Schmid et al., 2004), the protein also contains at its C-terminus an ASH (ASPM, SPD2, Hydin) domain (Ponting, 2006) and Rho-GAP-like domain. OCRL1 was initially localized to the Golgi complex (Olivos-Glander et al., 1995; Dressman et al., 2000), and it is recruited to membrane ruffles in response to growth factor stimulation and Rac activation (Faucherre et al., 2003). The GAP domain of OCRL1 has been shown to interact with Rac1, however, it does not appear to possess appreciable GAP activity towards Rac1 (Faucherre et al., 2003). More recent studies showed that OCRL1 is also present on endosomes and is important at early steps of the endocytic pathway (Erdmann et al., 2007), including clathrin-coated pits, which is consistent with the ability of OCRL1 to bind to clathrin, the endocytic clathrin adaptor AP-2, and the endosomal protein Rab5 (Ungewickell et al., 2004; Choudhury et al., 2005; Hyvola et al., 2006). In addition, OCRL1 was also found to bind the Rab5 effector APPL1 on peripheral endosomes; this interaction is mediated by the ASH–RhoGAP-like domains of OCRL1 (Erdmann et al., 2007). Mutations that cause Lowe syndrome have been mapped exclusively to the OCRL1 gene. The overwhelming majority of missense mutations are localized to the 5-phosphatase domain, underscoring the importance of the 5-phosphatase activity of this protein (McCrea et al., 2008). A small number of missense mutations are also located in the ASH and RhoGAP-like domains (McCrea et al., 2008), raising the question as to whether Rac and/or APPL1 interaction may play a role in the disease. The observation that OCRL1-deficient fibroblasts derived from Lowe-syndrome patients, in addition to increased PI(4,5)P 2 levels, also had alterations in the actin cytoskeleton, an increased sensitivity to actin depolymerizing agents, and mislocal- ization of the actin-binding proteins α-actinin and gelsolin (Suchy and Nussbaum, 2002), led initially to the postulation that abnormal cytoskeleton may contribute to the disease process, thus possibly involving Rho GTPase signaling. However, a more recent study showed that although all six known disease-causing missense mutations in the ASH and Rho-GAP domains abolished binding to APPL1, some of these mutations preserved the ability to bind Rac (McCrea et al., 2008). Thus far, APPL1 is the only protein whose binding is consistently disrupted by patient missense mutations in the C-terminal region of OCRL. The same group also demonstrated that APPL1 helps localize OCRL1 to specific cellular sites, and a model was proposed in which disruption of OCRL1 binding to APPL1 would impair the proper localization of OCRL1 as well as disconnect OCRL1 from a protein network potentially linked to the disease phenotype (McCrea et al., 2008). Future studies will, however, be required to further unravel the signaling networks involved. Rho-Linked Mental Retardation Genes 231 Surprisingly, OCRL1 knock-out mice do not develop Lowe syndrome. A potential explanation for this observation is that the OCRL1 loss of function is compensated by the phosphatase, Inpp5b, which shares high homology with OCRL1, and which is more expressed in mice than in humans (Jefferson and Majerus, 1995; Janne et al., 1998; Hellsten et al., 2001; Astle et al., 2006). 2.2.6 Mental-Disorder-Associated GAP (MEGAP) Mutations in Rho-linked genes that give rise to mental retardation are not only found on the X-chromosome, but have also been identified on autosomes. For example, the MEGAP (mental-disorder-associated GAP) gene was identified by positional cloning, as the only gene disrupted with a balanced de novo translocation of chro- mosome t(X;3)(p11.2;p25) (Endris et al., 2002). This patient exhibited severe MR and locomotor impairments that are associated with 3p-syndrome. Whereas other genes have also been implicated (Angeloni et al., 1999; Sotgia et al., 1999), 11 patients with 3p-syndrome MR displayed loss of heterozygosity for MEGAP, sup- porting the notion that reduced levels of this protein are causally linked to this form of MR. Notably, the MEGAP gene product had previously been identified as a WAVE-associated protein (WRP) (Soderling et al., 2002), and as a ROBO interacting protein (srGAP3) (Wong et al., 2001). The mRNA transcript of the MEGAP/WRP/srGAP3 gene is predominantly expressed in fetal and adult brain, and is enriched in the neurons of the hippocampus, cortex, and amygdala (Endris et al., 2002). Biochemical studies showed that MEGAP/WRP/srGAP3 s trongly enhances the intrinsic hydrolytic activity of Rac1 and to a significant lesser extent of Cdc42 (Endris et al., 2002). Together with the observation that MEGAP/WRP/srGAP3 directly binds to WAVE-1, a model was proposed in which MEGAP/WRP/srGAP3 functions in a negative feedback loop that inactivates Rac1 associated with WAVE- 1, thereby controlling actin dynamics and spine morphogenesis. Significantly, Soderling et al. generated and characterized WAVE-1 knock-out mice and reported that WAVE-1 knock-out mice exhibited defects in balance and coordination, reduced anxiety, and deficits in learning and memory (Soderling et al., 2003). Interestingly, these phenotypes are strikingly similar to those observed in 3p – syndrome patients. Morphological analysis of neurons in both the CA1 region of the hippocampus and the outer layer of the cortex of WAVE-1 knock-out mice revealed a reduction in spine density and abnormal spine morphology. Furthermore, electrophysiological recordings from hippocampal slices showed that WAVE-1 knock-out mice exhibit increased LTP and reduced LTD (Soderling et al., 2007). To determine whether the MEGAP/WRP/srGAP3’s interaction with WAVE-1 contribute to WAVE’s effect on spine density, synaptic plasticity, and memory, Soderling et al. generated mice that express WAVE-1 without the MEGAP/WRP/srGAP3 binding site. They observed that these WAVE-1 knock-in mice have reduced spine density and altered s ynaptic plasticity, as well as specific deficits in memory retention (Soderling et al., 2007). Thus, MEGAP/WRP/srGAP3’s interaction with WAVE is important for WAVE’s function in neural plasticity and cognitive behavior. 232 N.N. Kasri and L. Van Aelst Together these findings imply that signaling through MEGAP/WRP/srGAP3 and WAVE-1 to the actin cytoskeleton is important for normal neuronal function and connectivity and that alteration of this pathway (e.g., upon loss or reduced expression of MEGAP/WRP/srGAP3) affects the expression of normal behaviors, including learning and memory. 3 Conclusions Rho GTPase mediated signaling pathways modulate actin cytoskeleton dynamics and gene expression, which are critical for structural and functional plasticity in the developing and mature nervous system. Such synaptic remodeling and plastic- ity are thought to underlie the anatomic basis for learning and memory formation and normal cognitive function. Consistent with this are the findings demonstrat- ing an association between various MR conditions and mutations in Rho-linked genes. The current view of how mutations in Rho-linked genes contribute to MR is by disrupting the normal development, structure, and/or plasticity of neuronal networks via perturbations in the actin cytoskeleton and gene regulation networks. Evidence supporting such a view has come from MR patients, mouse models of MR, and RNAi studies in hippocampal and cortical slices. Further elucidation of the molecular and cellular mechanisms by which Rho signaling contributes to the above disorders will not only shed light on the epidemiology of these diseases, but also on basic mechanisms of neuronal development and function and may provide candidates for therapeutic intervention. Acknowledgments Because of space limitations, we are not able to cite the work of many of our colleagues who have made valuable contributions to this field. LVA is supported by NSF and NIH grants. 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