Neurochemical Mechanisms in Disease P25 pptx

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. NNK is a postdoctoral fellow from the Fund for Scientific Research Flanders and is
supported by the Human Frontiers Science Program.
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