JOURNAL OF NEUROCHEMISTRY | 2015 | 133 | 767779 doi: 10.1111/jnc.

13100

, , ,

*Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston,

Texas, USA
Department of Biology, University of Houston, Houston, Texas, USA
Department of Psychology, University of Houston, Houston, Texas, USA
Biology of Behavior Institute (BoBI), University of Houston, Houston, Texas, USA

Abstract retardation, atypical synaptic plasticity and aberrant spine

The small GTPase Rac1 is well known for regulating actin
cytoskeleton reorganization in cells. Formation of extensions
at the surface of the cell is required for migration and even for
cell invasion and metastases. Because an elevated level and
hyperactivation of this protein has been associated with
metastasis in cancer, direct regulators of Rac1 are currently
envisioned as a potential strategy to treat certain cancers.
Less research, however, has been done regarding the role of
this small GTP-binding protein in brain development, where it
has an important role in dendritic spine morphogenesis
through the regulation of actin. Alteration of dendritic devel-
opment and spinogenesis has been often associated with
mental disorders. Rac1 is associated with and required for
learning and the formation of memories in the brain. Rac1
appears to be dysregulated in certain neurodevelopmental
disorders that present all these three alterations: mental
morphology. Thus, to develop novel therapies for rescuing
cognitive impairment, a reasonable approach might be to
target this protein, Rac1, which plays a pivotal role in directing
signals that regulate actin dynamics, which in turn might have
an effect in spine cytoarchitecture and synaptic function. It is
possible that novel drugs that regulate Rac1 activation and
function could modulate actin cytoskeleton and spine dynam-
ics, representing potential candidates to repair intellectual
disability in disorders associated with spine abnormalities.
Herein, we present a list of the current Rac1 inhibitors that
might fulll this role together with a summary of the latest
ndings concerning their function as they relate to neuronal
studies.
Keywords: autism, cognitive disorders, fragile X syndrome,
small GTPase inhibitors.
J. Neurochem. (2015) 133, 767779.

Several neurodevelopmental disorders classied within the
autism spectrum disorders (ASDs) present, among other
characteristics, with mild to severe intellectual disability.
Mounting evidence suggests that this learning impairment
correlates with parallel phenotypes observed in these condi-
tions, such as abnormal morphological rearrangements of
dendritic spines, which ultimately affect the formation of a
functional synapse and its plasticity. Dendritic spine devel-
opment is crucial for the establishment of synaptic connec-
tions, and relies heavily on cytoskeletal reorganization and
actin dynamics upon other factors (Blanpied and Ehlers
2004; Nithianantharajah and Hannan 2013). Thus, an
attractive hypothesis is to suggest a link between cognitive
function and the regulation of proteins involved in actin
dynamics (Huttenlocher 1970, 1991). We have reported that
ablation of one of those proteins, Rac1, alters synaptic
plasticity, learning capability as well as spine morphology in
the mouse (Bongmba et al. 2011; Tejada-Simon and Bon-
gmba 2012). Moreover, Rac1 has been found to be up-
regulated in certain neurodevelopmental disorders (such as
fragile X syndrome, FXS) that present mental retardation,
atypical plasticity and high density of immature dendritic
Received November 5, 2014; revised manuscript received March 11,
2015; accepted March 14, 2015.
Address correspondence and reprint requests to Maria V. Tejada-
Simon, Associate Professor of Pharmacology, 551 Science and Research
building 2, mail code 5037, Department of Pharmacological and
Pharmaceutical Sciences, College of Pharmacy, University of Houston,
TX 77204, USA. E-mail: mvtejada-simon@uh.edu
Abbreviations used: ASDs, autism spectrum disorders; FMRP, fragile
X mental retardation protein; GAPs, GTPase-activating proteins; GEFs,
guanine exchange factors.

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768 M. V. Tejada-Simon

spines (Bongmba et al. 2011). Interestingly, by modulating

expression and activity levels of Rac1 through newly
available inhibitors, several of those aberrant phenotypes
can be rescued. Thus, it seems reasonable to consider that by
modulating the actin cytoskeleton, these new inhibitors could
become potential candidates to reestablish cognitive function
in certain ASDs. The present discussion aims to highlight
recent uses for these particular Rac1 regulators, and indicate
whether they might offer new opportunities for therapeutic
intervention in mental retardation.

Actin dynamics in neurons: link to intellectual

disability
It is well accepted that members of the Rho family of small
GTPases control the assembly and disassembly of the actin
cytoskeleton as well as many other cellular activities (Hall
1998, 2005). But, over the past decade, a role for the actin
cytoskeleton in brain development, maturation, and disease
has been strongly established. Studies addressing the function
of actin signaling and cytoskeleton dynamics in synaptic
development, brain plasticity, and neuronal regeneration have
placed the regulatory proteins involved in this pathway in the
spotlight, suggesting that the actin pathway is required for
proper synapse activity, conferring stability and allowing for
normal brain information processing (Smith et al. 2014).
Small GTPases from the Rho family are now considered to be
essential players in linking synaptic plasticity, neuronal spine
formation and cognitive capabilities (Newey et al. 2005).
Over the years, our research interest has focused on one of its
members, Rac1. This protein has a critical role in the brain
and is involved in learning and memory formation (Haditsch
et al. 2009, 2013; Tejada-Simon and Bongmba 2012).
Rac1 belongs to the Rho family of small GTPases and it
has a role in regulating actin polymerization. Rac1 is formed
by one central b-sheet with six strands, ve of which run
parallel to each other and the sixth one is positioned anti-
parallel to the others. It also consists of two short 310 helices,
and six a-helices (Fig. 1; Hirshberg et al. 1997). Structur-
ally, Rac1 holds an insertion domain (mostly exposed and
with a highly charged surface), and a nucleotide-binding
pocket with several residues forming the effector loop
(involved in hydrolysis of GTP, as well as binding of GAPs
and other downstream signal molecules).
As seen with all other small GTPases, there are two phases
on the activation of Rac1. In phase one, Rac1 changes
between inactive (GDP-bound) and active (GTP-bound)
forms. This is done through the catalytic action of guanine
exchange factors (also known as GEFs) and GTPase-
activating proteins (also known as GAPs). In phase two,
Rac1 also cycles from cytosol to membrane-associated forms
through a posttranslational modication that adds a preny-
lated group to the protein. This modication appears to be
necessary for translocation to the membrane (Fig. 2). The
Fig. 1 Tertiary structure of Ras-related C3 botulinum toxin substrate 1
(Rac1) protein structure complexed with guanosine-50 -(beta,gamma-
imido) triphosphate or GTP (structure obtained from RCSD Protein
Data Base at http://www.rcsb.org. DOI: 10.2210/pdb1mh1/pdb, Hirsh-
berg et al. 1997). Rac1 is composed of a central beta sheet with six
strands. It also contains six alpha helices and two short 310 helices.
Shaded area represents Rac1 fold, an area likely related to interaction
with downstream molecules and regulatory compounds.
relationship between these two phases of activation appears
to be very important in the proper interaction of Rac1 with
other targets downstream of the signaling pathway (Boguski
and McCormick 1993; Ridley 1995, 1996; Vojtek and
Cooper 1995; Reif and Cantrell 1998; Harwood and Braga
2003). Recent studies in neurons have suggested that Rac1,
as well as associated GEFs and GAPs, are not only found but
enriched in the postsynaptic density. Thus, GEFs and GAPs
are perfectly placed to respond to the regulation by glutamate
receptors and CaMKII (Calcium/calmodulin-dependent
protein kinase II) activity (Chen et al. 1998; Penzes et al.
2001; Tejada-Simon et al. 2006), both with an important role
in brain function.
Rac1 is expressed at synapses in the adult mouse
hippocampus, where activation of NMDARs induces mem-
brane translocation and activation of Rac1 (Tejada-Simon
et al. 2005, 2006). NMDA receptor-dependent events are
responsible for changes in morphology (Hayashi-Takagi
et al. 2010), which occur during synaptic plasticity as well as
learning and memory formation. Interestingly, Rac1 plays
also an important dynamic role in long-term plasticity
(Martinez and Tejada-Simon 2011; Tejada-Simon and Bon-
gmba 2012), learning (Martinez et al. 2007; Haditsch et al.
2009, 2013; Tejada-Simon and Bongmba 2012), and spine
formation (Bongmba et al. 2011; Tejada-Simon and Bon-
gmba 2012). Therefore, regulation of Rac1 likely provides a

2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 767--779


Fig. 2 Key players in membrane translo-
cation and activation cycles for the small
GTPase Rac1. Novel molecules acting as
regulators of the activation process of Rac1
are indicated in red.
relationship among aberrant dendrite morphology, abnormal
plasticity and cognitive deciency. Intriguingly, these repre-
sent altered phenotypes on several neurodevelopmental
disorders (FXS, Tuberous Sclerosis syndrome, Rett syn-
drome, among others) (Table 1), as well as other neuropsy-
Table 1 Several neurological disorders associated with cognitive disability presenting aberrant spine formation possibly associated to altered actin
cytoskeleton dynamics
Disease Spine pathology
Alzheimers disease Loss of dendritic spines
faulty actin depolymerization:
defective Rac1-dependent
activation of PAK
Down syndrome Loss dendritic spines
reduction in spine size
distortion of spine shape
giant spines
Schizophrenia Reduction in spine size
decrease spine density
Fragile X syndrome Increased density of long, thin, and
immature
dendritic spines distortion of spine
shape
Epilepsy Distortion of spine shape
Varicosity formation
Decrease spine density
Rett syndrome Decrease in spine density
Tuberous sclerosis Decrease in spine density
giant spines
Niemann-Pick disease Decrease in spine density
Trisomy 13 Distortion of spine shape
Huntingtons disease Altered number and size of dendritic
spines: decrease in cytoskeletal
proteins
2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 767--779
Rac1 and cognitive function 769
chiatric disorders, including depression (Golden et al. 2013)
which has been associated with long-term reduction on the
transcription of Rac1.
Among the mentioned neurodevelopmental disorders, one
of the most studied is FXS. FXS is the most common type of
References
Ferrer et al. (1990), Pozueta et al. (2013)
Suetsugu and Mehraein (1980), Takashima et al. (1981),
Ferrer et al. (1990), Takashima et al. (1994)
Garey et al. (1998), Roberts et al. (1996), Glantz and
Lewis (2000), Rosoklija et al. (2000), Glausier and Lewis
(2013)
Rudelli et al. (1985), Hinton et al. (1991), Comery et al.
(1997), Irwin et al. (2000), Kaufmann and Moser (2000),
Irwin et al. (2001), Galvez and Greenough (2005),
McKinney et al. (2005), Dolen et al. (2007), Hayashi et al.
(2007), Liu et al. (2011), Su et al. (2011)
Scheibel et al. (1974), Isokawa and Levesque 1991;
Belichenko et al. (1994a), Belichenko and Dahlstro m
(1995), von Campe et al. (1997), Blumcke et al. (1999),
Freiman et al. (2011), Wong and Guo (2013)
Belichenko et al. (1994a,b)
Machado-Salas (1984)
Walkely and Baker (1984), Higashi et al. (1993)
Marin-Padilla (1974, 1976)
Nithianantharajah and Hannan (2013)
770 M. V. Tejada-Simon
inherited cognitive disability syndrome. FXS patients present
a wide range of abnormal features, such as facial dimor-
phism, postpubertal aberrant testicular growth, defective
connective tissue (Hagerman and Cronister 1996), language
problems, hyperactivity, lack of attention, autistic behavior
(ODonnell and Warren 2002), problems with sleep, epileptic
seizures (Hagerman 2002), anxiety, auditory, and other
sensory decits (Hagerman and Cronister 1996; Berry-Kravis
2002). It has been established that lack of fragile X mental
retardation protein, or FMRP, is the underlying cause for
FXS (Verkerk et al. 1991). This is a protein normally
expressed in a multitude of tissues in the body, including
neurons (Bakker et al. 2000), where it functions as a
translation suppressor, taking care of regulating translation
and the transport of mRNAs (Brown et al. 2001; Jin and
Warren 2000, 2003; Miyashiro et al. 2003). Basic research
has shown that, in Fmr1 knockout mice, lack of FMRP
induces aberrant expression and an over-activation of Rac1
in the mouse brain (Bongmba et al. 2011). This exaggerated
expression and possibly function of Rac1 might very well be
responsible for the morphological anomalies reported in
dendritic spines, altered plasticity and learning associated to
FXS. Importantly, using a mouse model bearing a condi-
tional inactivation of the rac1 gene in the hippocampus, it
was reported that ablation of Rac1 induces similar abnor-
malities as the ones described with exaggerated Rac1
expression in the FXS model (Tejada-Simon and Bongmba
2012). These results point to the importance of the tight
regulation of the actin cytoskeleton and balance of the
expression or activation of its regulatory proteins for proper
brain development and function.
Emergence of novel molecules that can regulate
actin dynamics
Thus far, there has not been an effective therapeutic treatment
for all symptoms associated with ASDs and neurodevelop-
mental disorders. However, many characteristic dysfunc-
tional behaviors (aggression, hyperactivity, irritability,
inattention, repetitive behavior) can currently be treated
pharmacologically (Benvenuto et al. 2013). Regrettably,
intellectual disability is not one of them. Antipsychotics have
been occasionally used to treat severe behavior, but these
agents only promoted mild cognitive improvement in
children (Aman et al. 2009). However, based on the results
obtained by our studies and the research of others, it is
possible that targeting the neurochemical signaling involving
actin cytoskeleton regulation via the small GTPase Rac1, may
lead to new treatments specically directed to improve the
intellectual disability of these individuals. This improvement
might provide at least a modest relief to the group of people
affected with the disorder, improving their quality of life as
well as their caretakers. To prove this hypothesis pharmaco-
logically has been a challenge, since specic inhibitors for
2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 767--779
Rac1 were not widely available. The recent development of
new synthetic molecules targeting Rac1 activation and
function is lling this needed gap. In the near future, we
believe these chemicals might get us closer to the develop-
ment of new remedies to nally treat cognitive incapacity
associated with altered neuronal morphology and plasticity.
Novel Rac1 inhibitors/regulators
Several compounds have been characterized and described as
Rac1-specic inhibitors. The initial strategy for the identi-
cation of these compounds has been primarily based in the
information on structure-function of Rac1 interacting with its
GEFs. This approach has revealed that residues located in the
b2/b3 sheet region are essential for at least specic GEF
recognition (Tiam 1). Moreover, manipulation of a single
residue in Rac1, Trp(56), eluded interaction with Tiam1 (Gao
et al. 2001).
Even though the use of structural information to develop
these new Rac1-GEFs regulators has been successful, the wide
role of Rac1 in an organism demanded other strategies, such as
targetting Rac1 effectors, offering additional benets down-
stream from Rac1. Thus, in more recent years other molecules
have been developed that interfere with Rac1 signaling, by
inhibiting Rac1 ability to recruit effector molecules.
Currently, several compounds are available as specic
Rac1 inhibitors (Fig. 2): NSC23766, EHop-016, AZA1 and
EHT1864. A summary of studies investigating the effects
and uses of these substances is presented in Table 2.
NSC23766
NSC23766, [N6-[2-[[4-(diethylamino)-1-methylbutyl]
amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinedi-
amine trihydro-chloride], is a small molecule that acts as a
Rac1-specic inhibitor. It represents a rst generation of
Rac1-specic inhibitors, highly soluble and membrane
permeable (Tables 3 and 4). A vast number of studies in
non-neuronal cell cultures are available describing its
functionality as Rac1 inhibitor, showing that NSC23766
interferes with certain Rac1-GEFs (specically Trio and
Tiam-1), thus impairing Rac1-GDP/GTP exchange (Gao
et al. 2004). NSC23766 has been a great tool for studies
determining the role of Rac1 in the regulation of the cell
cycle in human cancer cells (Liu et al. 2014). Inhibition of
Rac1 alters the behavior of metastatic cells (Hernandez et al.
2010), reduces estrogen receptor levels in breast cancer cells
(Rosenblatt et al. 2011), and the migration and growth of
leukemia cells (Wang et al. 2009a). NSC23766 also offers
cardiovascular protection (Wang et al. 2012). In all systems
tested, NSC23766 was able to induce cell cycle arrest,
control cell migration, proliferation and cell growth, pointing
to these inhibitory effects as a future novel therapeutic
strategy for certain types of cancer.
 Rac1 and cognitive function 771

Table 2 Summary of reported effects of chemical compounds acting as Rac1 inhibitors

Chemical
administered Effect References

NSC23766 Decrease in active Rac1, PAK1, P-selectin, ATP in platelets Akbar et al. (2006)
Reduces migration of human trophoblast Nicola et al. (2008)
Suppression of interkinetic nuclear migration of mouse neocortical Minobe et al. (2009)
progenitor cells
Inhibition of cell migration and cell spreading in metastatic Hernandez et al. (2010)
breast cancer cells
Reduced aberrant spine density and dimensions in neuropathic Tan et al. (2011)
pain and injury
Attenuation of neurite outgrowth, production of reactive oxygen Kim et al. (2013)
species, in PC12 cells
Inhibition of M2 muscarinic receptor in neonatal Levay et al. (2013)
rat cardiac myocytes
Attenuated production of proinammatory cytokines, reactive Rochfort et al. (2014)
oxygen species in interendothelial adherens and tight junctions
Abrogated gadolinium-promoted proliferation in mouse embryo Shen et al. (2014)
broblast NIH3T3 cells
Arrest of G1/S phase, inhibits proliferation of human cancer cells Liu et al. (2014)
Suppression of PAK1 and reactive oxygen species in mouse DeSantiago et al. (2014)
ventricular myocytes
Abolished cecal ligation and puncture-induced enhancement of Hwaiz et al. (2014)
platelet ligands and MMP-9 in plasma onseptic mice
Decrease neutrophil recruitment and colon tissue damage Yu et al. (2014)
Impaired replication of inuenza virus Dierkes et al. (2014)
Inhibition of alpha1 receptor responses in vascular tissues, Rahman et al. (2014)
potentiation of PGE2a and thromboxane receptor responses
Attenuation of c-Myc transcription in chronic lymphocytic leukemia Hofbauer et al. (2014)
cells
Ameliorates osteoarthritis development in vivo Zhu et al. (2015)
in neuronal system
Decreased changes in spine morphology in post-spinal Tan et al. (2008)
cord injury rats
Decreased rufe formation and lopodia number in NIH3T3 Menna et al. (2009)
cultured neurons
Prevention of IP3K-A-induced lopodia formation in HeLa cells as Kim et al. (2009)
well as spine formation in cultured hippocampal neurons
Increased neuronal density, less apoptotic cells in rat Zhang et al. (2009)
hippocampus
Decreased Rac1 activity and amyloid precursor protein levels Wang et al. (2009b)
Reduction of oxidative damage caused by ischemia in Raz et al. (2010)
hippocampal area CA1
Elimination of DHPG-induce LTD in hippocampal neurons Zhou et al. (2011)
Impairement of LTP and LTD in mouse hippocampal slices Martinez and
Tejada-Simon 2011;
Inhibition of synaptic enhancement, ERK1/2 phosphorylation in rat Kawakami-Mori et al. (2012)
hippocampal slices
Attenuation of dendritic spine dysgenesis, mechanical allodynia Tan et al. (2013)
and electrophysiological signs of burn-induced neuropathic pain
Inhibition of consolidation of cocaine-induced conditioned place Ding et al. (2013)
preference
Avoid redistribution of CYFIP1 complexes, affecting dendritic De Rubeis et al. (2013)
spine morphology in vivo and in vitro. Unbalance between mature
and immature dendritic spines

(continued)

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772 M. V. Tejada-Simon

Table 2 (continued)

Chemical
administered Effect References

Therapeutic potential against cerebral ischemic reperfusion injury, Kiao and Ye (2014)
decrease neuronal apoptosis in diabetic rats
Disruption of reconsolidation of fear memory Wu et al. (2014)

EHop-016 Reduction of lamellipodia formation in breast cancer cells Montalvo-Ortiz et al. (2012)
Inhibits human and murine leukemic cell growth Martin et al. (2013)
Inhibits metastatic cancer cell growth, viability, extension of actin- Dharmawardhane et al. (2013)
based structures and cell migration

AZA 1 Growth suppression of human prostate cancer Zins et al. (2013)

xenografs in mice increasing longevity

EHT 1864 Lowers levels of estrogen receptor, decrease Rosenblatt et al. (2011)
proliferation in breast cancer cells
Avoids spreading of breast cancer cells and reduces proliferation Katz et al. (2012)
Repression of mineralocorticoid receptor signaling and reduced Kawarazaki et al. (2012)
kidney injury in hyperaldosteronemic mice
Reduced lamellipodia, impaired clot retraction, and Stefanini et al. (2012)
granule release in platelets
Protection from DNA damage in HepG2 cells, liver Wartlick et al. (2013)
carcinoma cell line
Decreased Akt and LIMK phosphorylation in pulmonary alveolar Dipaolo et al. (2013)
epithelial cells
Hypersensitivity in monocytic cells Hinterleitner et al. (2013)
Inhibition of contractile responses induced by high K, Rahman et al. (2014)
Phenylephrine, carbachol, PKC in urinary bladder, ileum and
vascular tissue
Alleviation of renal pathology in a mouse model of obesity-related Yoshida et al. (2014)
type 2 diabetes
Blockage of membrane rufing and exocytosis in mast cells Baier et al. (2014)
in neuronal system Inhibition of Abeta-40 release in SH-SY5Y cultured cells, as well Desire et al. (2005)
as reduced levels of Abeta-40 and Abeta-42 in vivo in
albino guinea pigs
Impairement of LTP and LTD in mouse hippocampal slices Martinez and
Tejada-Simon (2011)
Inhibition of macropinosome formation in a human Nara et al. (2012)
neuroblastoma cell line
Decreased NMDAR current density in rat cortical cultures Duffney et al. (2013)
Decreased density of spines in early stages of development of Raynaud et al. (2014)
hippocampal neurons

While fewer studies investigating Rac1 regulation have
been reported in the neuroscience eld, they are still
promising. In vitro experiments have demonstrated that
treatment of NIH3T3 cells, which can differentiate into
neuronal cells (Wang et al. 2011), with NSC23766 indeed
decreases Rac1-GTP levels below baseline (Menna et al.
2009), impairs interkinetic nuclear migration that requires
cytoskeletal factors, preventing proper cytokinesis (Minobe
et al. 2009). In hippocampal neurons, NSC23766 abolishes
DHPG (Dihydroxyphenylglycine)-induced LTD (Martinez
and Tejada-Simon 2011; Zhou et al. 2011), and prevents
development of PI3K-induced lopodia (Kim et al. 2009).
By treating hippocampal slices with NSC23766, NMDA
receptor-dependent induction of LTP (long-term potentia-
tion) and LTD (long-term depression) has been shown to be
impaired, and mGluR (metabotropic glutamate receptor)-
dependent LTD is also altered (Martinez and Tejada-Simon
2011). Moreover, in an animal model of FXS (Fmr1
knockout), it has been demonstrated that, by antagonizing
Rac1 with this drug, the aberrantly enhanced hippocampal
LTD characteristic of these Fmr1 KO mice is mitigated
(Bongmba et al. 2011).

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Table 3 Characteristics of novel Rac1 inhibitors
Compound
Name IUPAC name
NSC23766 N6-[2-[[4-(diethylamino)-l-methylbutyl] aminoJ-6-methyl-4-
pyrimidinyl]-2-methyl-4,6-quinolinediamine; trihydrochloride
EHop-016 4-N-(9-ethylcarbazol-3-yl)-2-N-(3-morpholin-4-ylpropyl)pyrimidine-
2,4-diamine
AZA1 N2,N4-bis(2-methyl-lH-indol-5-yl) pyrimidine-2,4-diamine
EHT 1864 2-(Morpholin-4-ylmethyl)-5-[5-[7-(triuoro-methyl) quinolin-4-yl]
sulfanylpentoxyj pyran-4-one;dihydrochloride
*< 1 slightly soluble or insoluble.
Dendritic spine shape, density and distribution have been
strongly associated to synaptic transmission, plasticity and
cognitive function. Many in vivo studies established a link
between Rac1 and altered spine formation. For example,
treatment with NSC23766 inhibits Rac1-GTP activity in
spinal cord-injured rats, reducing dendritic remodeling, spine
density, and excitability (Tan et al. 2008). Spines are also
altered in peripheral nerve injury (Tan et al. 2011) and burn-
injured animals (Tan et al. 2013), presumably because of
abnormal Rac1-dependent down-regulation of dendritic
spines. Thus, according to these studies, subjects with
peripheral nerve, burn or spinal cord injuries exhibit an
increased sensitivity to pain, altered spine density along
dendritic branches, and signs of neuropathic pain. Tan et al.
(2008) described that inhibition of Rac1 with this inhibitor
ameliorated changes in spine morphology after spinal cord
injury was inicted. Additionally, it was reported that the
administration of NSC23766 to peripheral nerve and burn
injured rats not only attenuated spine defects, but also pain
(Tan et al. 2011, 2013). These studies pointed to the
possibility of using NSC23766 to target Rac1 signaling for
pain management after these specic injuries. Moreover, they
demonstrate that Rac1 is a critical signaling molecule
involved in dendritic spine pathology, providing evidence
for a novel association between learning/memory and
neuropathic pain. To further substantiate this theory, other
studies have shown a change in neuronal spine density also
in the hippocampus of rats upon treatment with NSC23766
(Zhang et al. 2009). Normal brain function requires normal
dendritic spine maturation (Penzes et al. 2011). Addition of
NSC23766, both, in vivo and in vitro, appears to affect
dendritic spine morphology, distorting the number of imma-
ture spines (De Rubeis et al. 2013).
Rac1 appears to have a role in other areas of the brain
associated with memory and emotions. Inhibition of Rac1-
associated signaling using NSC23766 has been reported to be
involved in extinction of memory (Sananbenesi et al. 2007). In
FXS mice, which present with abnormally high levels of Rac1,
characteristically altered LTP was somewhat reversed by
2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 767--779
Rac1 and cognitive function 773
Solubility 25C (mg/ml)
Molecular
weight Water DMSO EtOH Empirical formula
530.96 106 106 5 C24H35N7  3HCI
430.55 < 1* 86 < 1* C25H30N60
368.43 < 1* 100 < 1* C22H20N6
581.47 100 100 < 1* C25H27F3N204S  2HCI
pharmacological manipulation of Rac1 with NSC23766,
protecting these mice from particular altered phenotypes
reported (Bongmba et al. 2011), possibly by bringing levels
and activity of Rac1 down to baseline. The use of NSC23766
has demonstrated that Rac1 is essential for the reconsolidation
of auditory-fear memory in the basolateral amygdala and of
contextual-fear memory in the hippocampal CA1 region (Wu
et al. 2014). Additionally, by functionally reducing Rac1
activity with NSC23766 microinjections in rats, it has been
determined that Rac1 has a role in the consolidation and
reconsolidation of cocaine-associated cue memory in nucleus
accumbens and amygdala (Ding et al. 2013).
NSC23766 has brightened a new future for reducing
oxidative stress via inactivation of Rac1. Treatment with the
inhibitor reduced levels of Abeta-40 and Abeta-42 (Desire
et al. 2005), and prevented Rac1-dependent activation of
NADPH oxidase-induced synaptic enhancement (Kawakami-
Mori et al. 2012), reducing oxidative damage caused by
ischemia in the CA1 area of the hippocampus (Raz et al.
2010).
While the Rac1 inhibitor NSC23766 has potential, its
prospective use as a therapeutic agent is not yet clear.
Researchers are using the structural relationship information
between Rac1 and its inhibitor to develop additional
molecules. However, these new small molecules are struc-
turally unrelated to NSC23766 and do not target GEFs
interaction. It is believed that these molecules will lack
specicity for determined GEFs and will more likely block
the activation of the GTPase (Ferri et al. 2009). By inhibiting
the activation of Rac1 independently of the GEF used, these
novel agents may prove benecial not only in neuroscience
research, but also in cancer.
EHop-016
As a Rac1 inhibitor, NSC23766 has a relatively high IC50,
limiting its possibilities as a therapeutic agent. Using
NSC23766 as a model, researchers have identied and
characterized another inhibitor that is 100 fold more effective
774 M. V. Tejada-Simon
Table 4 Structure of novel Rac1 inhibitors (Blue = N; Yellow = S; Red = O; Green = F; White = H)
Name Linear structure
NSC23766
EHop-016
AZA 1
EHT 1864
in inhibiting Rac1, EHop-016 [4-N-(9-ethylcarbazol-3-yl)-2-
N-N-(3-morpholin-4-ulpropyl) pyrimidine-2,4-diamine].
EHop-016 (Tables 3 and 4) is a very novel, scarcely used,
Rac1 inhibitor with an enhanced potency as compared to
NSC23766 (Dharmawardhane et al. 2013).
EHop-016 is specic for Rac1 and Rac3 isoforms when
used at lower concentrations. It acts over Tiam 1, Trio,
P-Rex, and also Vav2, and at higher concentrations it also
inactivates Cdc42, a close homolog of Rac1 that is
activated by the Vav2 GEF (Montalvo-Ortiz et al. 2012).
EHop-016 has potential as a therapeutic drug for metastatic
cancer, but also others investigate its use to modify cellular
responses regulated by Rac1. Moreover, oral administration
of this drug did not result in any physical problems or
toxicity in mice at a concentration of 1 mg/kg body
weight, once a week for nine consecutive weeks (Mont-
alvo-Ortiz et al. 2012), suggesting its possible use for
in vivo studies and eventually human clinical trials. To our
knowledge, no signicant studies have been done reporting
effects in brain, its plasticity and spine formation using this
compound.
AZA 1
AZA1 [N,N-bis(2-methyl-1H-indol-5-yl)pyrimidine-2,4-
diamine] is a new cell permeable compound that effects
2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 767--779
3D structure
both Cdc42 and Rac1. AZA1 (Tables 3 and 4) suppresses
up-regulation of Rac1-GTP and Cdc42-GTP. To date, it has
been used in prostate cancer cells studies, where its
application results in inhibition of cell migration and growth
in a dose-dependent manner by disabling the lopodia
(Cdc42 dependent) and lamellipodia (Rac1 dependent)
formation. Moreover, in vivo experiments in mice have
revealed that, after daily injections of AZA1 for a period of
2 weeks, specic types of tumor expansion (22Rv1 tumor; a
new human prostate carcinoma cell line) are suppressed by at
least 30% (Zins et al. 2013). To our knowledge, no other
studies are available regarding the effect of this compound,
especially in neurons.
EHT1864
As NSC23766 acts by impeding the Rac1-GEF interaction,
the development of other molecules that interfere with Rac1
downstream effectors was of interest. EHT1864 [2-(mor-
pholin-4-ylmethyl)-5-[5-[7-(triuoromethyl) quinolin-4-yl]
sulfanyl-pentoxy] pyran-4-one] is another small molecule
that is capable of inhibiting Rac1 by facilitating GTP
unloading (Shutes et al. 2007; Onesto et al. 2008) (Tables 3
and 4). While the use of this molecule has been thus far
limited in research, it has been determined that EHT1864
selectively inhibits Rac1 downstream signaling by affecting

Fig. 3 Hypothetical mechanistic involve-
ment of Rac1 in cognitive function.
the displacement of GTP. In this manner, Rac1 remains in an
inactive state, as Rac1-GDP. It has been reported that the
action of EHT1864 likely mediates changes in cellular
morphology and cellular transformation (Shutes et al. 2007),
making it a good candidate also for cancer therapy.
In vitro experiments in neuronal studies have demonstrated
that treatment with EHT1864 results in decreased NMDA
receptor density in rat cortical cultures, similar to the effects
of transfecting those cultures with a dominant negative Rac1
plasmid (Duffney et al. 2013). Lamellipodia formation, a
Rac1-dependent process, has been reported to be reduced in
PDGF (Platelet-derived growth factor)-stimulated neuronal
cells treated with EHT1864, but not when cells are treated
with an inactive form of this compound, such as EHT8560,
which has a similar structure to EHT1864, but has no effect
on the inactivation of Rac1. Moreover, treatment of cultured
hippocampal pyramidal neurons during early stages of
development with EHT1864 yielded inactive Rac1, resulting
in decreased density of dendritic spines (Raynaud et al.
2014). These observations suggest that EHT1864 is indeed a
potent and specic inhibitor of endogenous Rac1 activation
that can be also used to control spine morphogenesis
regulated by Rac1.
In vivo studies are more limited for this drug. Our laboratory
showed that, by treating hippocampal slices with EHT1864,
NMDA receptor-dependent induction of LTP and LTD
appears impaired in a dose-dependent manner (Martinez and
Tejada-Simon 2011). Also, and as previously reported for
NSC23766, inhibition of Rac1 with EHT1864 both, in vitro
and in vivo, results in the decrease of Abeta production (Desire
et al. 2005), pointing to Rac1 inhibition as a possible therapy
also for Alzheimers disease. This is very interesting
because animal models of Alzheimers disease and other
2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 767--779
Rac1 and cognitive function 775
neurodegenerative disorders have shown increases in Rac1
and its downstream kinase, PAK (p21-activated kinase)
(Barnes et al. 2008; Ma et al. 2008; DeGeer and Lamarche-
Vane 2013).
Conclusion
Regulatory proteins of the actin cytoskeleton in the central
nervous system, including Rac1, appear to be important for
brain development and function, as well as cognitive
function (Fig. 3). Alterations on the expression and func-
tional levels of these proteins materialize in learning and
memory disabilities observed in neurodevelopmental and
neurodegenerative disorders. Thus, we proposed a promising
idea, to develop and use novel drugs that could normalize
the levels of these proteins whenever they are aberrant. In
FXS mice, which is associated with hyperactivation of Rac1,
we have already shown that treatment with Rac1 inhibitors,
most likely lowers activated Rac1 to normal levels, affecting
plasticity, thereby rescuing decits characteristic to this
mouse model of FXS (Bongmba et al. 2011). Moreover,
these Rac1 inhibitors are interesting because they function
via different inhibitory mechanisms: for example,
NSC23766 inhibits the interaction with GEFs while
EHT1864 interferes with Rac1 downstream effectors. Thus,
they could offer a synergistic activity when a greater Rac1
regulation is desired.
While it is undeniable that, in the brain, the actin
cytoskeleton has a very important role in regulating synaptic
plasticity, dendritic spine development and cognitive capa-
bilities, the cytoskeleton also coordinates a number of
different processes in an organism, such as transcriptional
activation, cell proliferation and motility, morphogenesis,
776 M. V. Tejada-Simon
cytokinesis, and membrane trafcking. All these are neces-
sary and normal cellular processes. Moreover, GEFs and
GAPs associated with proteins that modulate actin dynamics
may be very specic to a particular small GTPase, expressed
in a particular tissue, or totally promiscuous. Thus, the
question remains as to how best exploit this proposed
chemical biology approach to nd new therapies by teasing
out other important cellular functions attributed to Rac1.
How do we target pathway-selective inhibition of Rac1 in the
brain to rescue altered cognitive function, avoiding global
inhibition of its activity? Without this specicity, there is a
high risk of unplanned lateral outcomes.
Other questions need to be addressed regarding these new
drugs and their successful use in neuroscience, such as their
capacity for brain penetration, toxicity, and function revers-
ibility. Most of the compounds described here appear to be
selective, reversible, and dose-dependent in the systems tested.
While more research needs to be done with respect to the
action of these inhibitors and the safe pharmacology for their
use in humans, some of the preliminary results in animal
models are very promising and suggest that these drugs are
suitable for neuronal tissue, as well as in vivo testing. Thus,
the design of these novel inhibitors and their function can
represent a step forward to the discovery of other small
molecules that might be useful for modulating pathological
conditions associated with alteration of actin dynamics and
spine morphogenesis, and perhaps the rst real approach to
treat cognitive function.
Acknowledgments and conflict of interest
disclosure
This article has been possible with funding from the FRAXA
Research Foundation, the Jerome LeJeune Foundation (France),
GEAR-UH grant program, and SGP-UH grant program (to
M.V.T.S). Special thanks to Dr L. Schwarz, for critical review of
this manuscript. The author has no conicts of interest to declare.
All experiments were conducted in compliance with the ARRIVE
guidelines.
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