Rho proteins, mental retardation and the cellular basis of cognition

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Rho proteins, mental retardation and the cellular basis of cognition Ger J.A. Ramakers For several decades, it has been known that mental retardation (MR) is associated with abnormalities in dendrites and dendritic spines. The recent cloning of seven genes that cause nonspecific MR when mutated provides important insights in the cellular mechanisms that result in the dendritic abnormalities associated with MR. Three of the encoded proteins, oligophrenin1, PAK3 and αPIX, interact directly with Rho GTPases. Rho GTPases are key signaling proteins that integrate extracellular and intracellular signals to orchestrate coordinated changes in the actin cytoskeleton essential for directed neurite outgrowth and the regulation of synaptic connectivity. Although many details of the cell biology of Rho signaling in the CNS are still unclear, a picture is unfolding showing how mutations that alter Rho signaling result in abnormal neuronal connectivity and deficient cognitive functioning in humans. Conversely, these findings illuminate the cellular mechanisms underlying normal cognitive function.

Ger J.A. Ramakers Neurons and Networks, Netherlands Institute for Brain Research, Graduate School Neurosciences Amsterdam, Meibergdreef 33, 1105 AZ Amsterdam ZO, The Netherlands.

Mental retardation (MR) is a developmental disability, characterized by a global deficiency in cognitive abilities, an inability to cope with every day life and an onset during childhood. In practical terms, MR is defined by an intelligence quotient (IQ) below 70, occurring in 2–3% of the population [1]. MR can be part of a clinical syndrome (syndromic MR) together with various congenital abnormalities, such as body and brain malformations, neurological, neuroendocrine or psychiatric symptoms or metabolic defects. More often, in nonsyndromic MR, no other functional or anatomical abnormalities are observed (including abnormalities in brain anatomy), and a low intelligence is thus the only detectable deficit. MR is highly heterogeneous with regard to the severity with which different cognitive abilities are affected, as well as to the known causes, which include both environmental and genetic factors [1]. Among the environmental factors are maternal malnutrition, fetal alcohol exposure and infections, premature birth, birth trauma, anoxia and hypothyroidy. Genetic causes of MR range from chromosomal (cytogenetically detectable) abnormalities, which are often associated with more severe forms of MR, to single-gene mutations and polygenic predisposition, which underlie milder forms of MR. The most common genetic cause of MR is trisomy of chromosome 21, which causes Down’s syndrome, whereas the most frequent single gene deficit is fragile X (fraX) syndrome. No therapy is available for MR, but directed educational efforts can help to improve cognitive performance. Investigating the causes of MR and underlying cellular mechanisms is essential to understand the http://tins.trends.com

(a) TH

(b)

(c)

(d)

MS

ST

10 µm TRENDS in Neurosciences

Fig. 1. Dendritic spine abnormalities in Golgi-stained cerebral cortex of (a) a normal infant at six months, (b) a non-syndromic mentally retarded infant at ten months, (c) a normal infant at seven years, and (d) a profoundly retarded infant at 12 years. The initial overabundance of thin spines results in lowered spine densities later. Abbreviations: MS, mushroom-shaped spines; ST, stubby spines; TH, thin spines. Reproduced, with permission, from Ref. [4].

structural and functional basis of MR, and could eventually result in therapies to ameliorate the mental deficit. At the same time, these studies reveal mechanisms required to attain at least normal intelligence, and thereby contribute to one of the great challenges in neuroscience: establishing the neurobiological basis of human cognition. MR as a deficit in neuronal network connectivity MR is associated with abnormalities in dendritic branching and spines

Severe forms of MR are often associated with brain malformations, microcephaly, lissencephaly (smooth brain) and/or neuronal migration deficits. Here, and in cases where many neurons are lost as a result of toxicity or trauma, the size and connectivity of the neuronal network is impaired to such a degree that its capacity to process information is severely limited. Although few studies have been conducted, milder forms of MR (and nonsyndromic MR by definition) show little, if any, consistent change in brain macroanatomy, including relevant areas like cerebral cortex and hippocampus. About three decades ago, Golgi studies indicated for the first time that MR is associated with

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Table 1. Properties of MR genes involved in nonsyndromic MR and syndromic MR genes involved in Rho signalinga,b Gene

Locus Protein name(s)

Function(s)

Effects of mutation(s)

MRX familyc

MIM no.d

GEF for Rac/Cdc42; activator of PAKs

Exon skipping, 28 amino acid deletion in CH domain, loss-of-function owing to translocation

MRX46

300267

Nonsyndromic MR (MRX) genes

ARHGEF6

Xq26

FMR2

Xq28

FMR2

Transcriptional activator? (AF-4 like)

Truncation, loss-of-expression

GDI1

Xq28

αGDI, RABGDIA

GDI for Rab3a,b; neurotransmitter release and vesicle trafficking

Decrease and loss-of-function, truncation and loss of expression

MRX41, fam R; MRX48 300104

IL1RAPL

Xp22

ILRAPL

IL signaling?

Deletions, truncation and decreased expression

MRX34

300206

OPHN1

Xq12

Oligophrenin-1

Rho family GAP inhibitor of RhoA?

Frame-shift and decreased expression, loss-of-function

MRX60

300127

PAK3

Xq22

PAK3, β-PAK

Ser/Thr protein kinase Cdc42/Rac1 effector

Truncation, loss-of-kinase activity, mutation in GTPase-binding domain

MRX30; MRX47

300142

TM4SF2

Xp11

Tetraspanin 2

Integrin-mediated signaling?

Truncation, mutation in extracellular loop, translocation and decreased expression

aPIX, Cool-2

309548

300096

Syndromic MR genes involved in Rho signaling

FGDY

Xp11

FGD1

GEF for Cdc42

LIMK1

7q11

LIM kinase-1

Tyr kinase downstream of Rac/Cdc42 Deletion of multiple genes, and PAK; phosphorylates and including elastin gene inactivates cofilin

Truncation, mutations in PH and GEF domain

Faciogenital dysplasia 305400 Aarskog–Scott syndrome Williams syndrome (probably involved)

601329

aAbbreviations:

ARHGEF, Rho guanine nucleotide exchange factor; Cool-2, cloned out of library 2; FGD, faciogenital dysplasia; FMR, fragile site mental retardation; GDI, guanine dissociation inhibitor; GEF, guanine nucleotide exchange factor; IL, interleukin; IL1RAPL, IL-1 receptor accessory protein like; LIMK, LIM domain containing kinase; MR, mental retardation; PAK, p21-associated kinase; PH, pleckstrin homology; PIX, PAK-interacting exchange factor; RABGDIA, Rab GDP-dissociation inhibitor a; TM4SF2, transmembrane-4 superfamily 2. bOnly well-confirmed MRX genes (listed in the XLMR Update website) are included. Genes involved in both non-syndromic and syndromic MR are excluded. All genes are expressed in fetal and adult brain. cAs listed in the XLMR Update website (http://xlmr.interfree.it/home.htm). dEntry numbers for Online Mendelian Inheritance in Man database; for further information and references (http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=OMIM).

abnormalities in dendrites and dendritic spines [2–4]. A study by Purpura [4] on children with nonspecific MR revealed a reduction in dendritic spines and a predominance of very long and thin spines at the expense of stubby and mushroom-like spines (Fig. 1). As the long, thin spine morphology resembled immature spines, the term ‘spine dysgenesis’ was introduced. Huttenlocher [2] first described reductions in dendritic branching in MR. Subsequent Golgi studies focused on pyramidal neurons in the cerebral cortex and hippocampus of patients with Down’s, Rett and fraX syndromes. They confirmed an association between MR and abnormalities in spine shape and reduced spine densities and dendritic branching, suggesting that MR is due to abnormal development of connectivity [5]. Similar abnormalities were found in adults with untreated phenylketonuria [6] and in infants with severe malnutrition [7]. Animal models for MR replicate deficits in dendrites and spines

Animal models in which the effects of environmental causes of MR on brain development were investigated (e.g. prenatal alcohol exposure [8], hypothyroidism [9], fetal hypoxemia [10] and protein deprivation [11]) all displayed reduced dendritic arborization, spine http://tins.trends.com

deficits or both. Thus, also in these cases, dendritic and spine abnormalities might be a contributing factor. Importantly, deletion of the fraX gene in mice revealed a similar overabundance of long, tortuous, thin spines to that in fraX patients, as well as mild learning deficits [12,13]. Deletion of the cell adhesion molecule L1, which causes hydrocephalus and syndromic MR, induced several brain defects in mice, including abnormal apical dendrites in cortical pyramidal neurons [14]. Impaired information processing in MR might result from deficiencies in neuronal connectivity or plasticity

Dendrites play a crucial role in synaptic integration, which is directly related to their branching complexity [15]. Dendritic spines receive the large majority of all excitatory glutamatergic synaptic transmission, and are focal points of synaptic plasticity [16]. Thus, it is likely that abnormalities in these structures will impair information processing at the cellular and network level. Although Golgi studies have prevented investigation of presynaptic changes in MR, the abnormalities in dendrites and spines are likely to reflect a more general defect in neuronal connectivity. In addition, because spine shape and function are intricately linked [17], the observed abnormalities in spine shape are likely to

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Box 1. Regulation of Rho GTPase activity and downstream signaling Fig. I. Regulatory cycle for the activation and inactivation of Rho GTPases. Regulation is depicted here for the family prototype, RhoA, but is similar for the other members. For experimental purposes, constitutively activated (obtained by mutating glycine in position 14 for Rho or 12 for Rac1 and Cdc42 to valine) and dominant negative mutants of Rho GTPases (mutating Thr at position 19 for Rho or 17 for Rac1/Cdc42 to Asp) are commonly used. The mutated nonsyndromic and syndromic MR genes involved in Rho signaling are indicated at the appropriate positions in red and green, respectively. Abbreviations: FGD, faciogenital dysplasia; GAP, GTPase-activating proteins; GDI, guanine dissociation inhibitor proteins; GEF, guanine nucleotide exchange factors; LIMK, lens intrinsic membrane protein domain kinase; PAK, p21-activated kinase.

Guidance cues, neurotrophins and electric/synaptic activity

Rho–GDI Rho–GDP

P

Rho–GDP

GTP

Dominant negative (RhoN19, Rac/Cdc42N17)

Rho–GAP

OPHN1

Rho–GEF Constitutively active (RhoV14, Rac/Cdc42V12) Rho–GTP

PAK3

FGD1

GDP

Rho effectors

PAK3 LIMK1 Actin dynamics Cell adhesion Cytokinesis Trafficking Gene expression TRENDS in Neurosciences

impair synaptic transmission or plasticity. Taken together, the microstructural observations strongly indicate that in many cases MR might be due to deficient neuronal network formation and/or plasticity. During postnatal brain development, experience-dependent synaptic rearrangement is crucial to optimize neuronal network circuitry to meet environmental demands [18]. MR could ensue from interference with this process as it would result in a limited ability of the brain to process information. Recently conducted twin studies have demonstrated a strong correlation between global cognitive performance (expressed as Spearman’s g factor) and both the volume of gray matter of the entire brain and certain cortical areas [19]. As the http://tins.trends.com

The Rho GTPases are a subfamily of the Ras superfamily of small (20–30 kDa) GTP-binding proteins. In mammals, they include RhoA, RhoB, RhoC, Rac1, Rac2, Rac3, Cdc42, Rnd1/Rho6, Rnd2/Rho7, Rnd3/RhoE, RhoD/HP1, RhoG, TC10 and RhoH/TTF; of these, most research has focused on RhoA, Rac1 and Cdc42. They act as molecular switches, cycling between an active, GTP-bound state and an inactive, GDP-bound state (Fig. I). Rho proteins are activated by guanine nucleotide exchange factors (GEFs), which mediate exchange of GTP for GDP [a,b]. Inactivation occurs through hydrolysis of the bound GTP by the intrinsic GTPase activity of the protein, and is stimulated by GTPase activating proteins (GAPs) [a,b]. GDP-bound Rho proteins are sequestered in the cytoplasm by guanine dissociation inhibitor (GDI) proteins, which prolong inactivation [c]. In addition, C-terminal geranylgeranylation is essential for membrane binding and functioning of Rho GTPases [d]. Binding of GTP enables interaction with downstream effectors, which are often activated by release from intramolecular autoinhibition. The 30 Rho effector proteins include protein kinases and scaffold proteins involved in the regulation of actin dynamics, and kinases involved in regulating gene expression, lipid kinases and lipases (Box 2) [b,e]. References a Kjøller, L. and Hall, A. (1999) Signaling to Rho GTPases. Exp. Cell Res. 253, 166–179 b Van Aelst, L. and D’Souza-Schorey, C. (1997) Rho GTPases and signaling networks. Genes Dev. 11, 295–322 c Sasaki, T. and Takai, Y. (1998) The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. Biochem. Biophys. Res. Commun. 245, 641–645 d Zhang, F.L. and Casey, P.J. (1996) Protein prenylation: molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65, 241–269 e Bishop, A.L. and Hall, A. (2000) Rho GTPases and their effector proteins. Biochem. J. 348, 241–255

volume of gray matter is, to a large extent, made up of ‘neuropil’ (dendrites, axonal arborizations and synapses), these findings support the idea that the size of the network is an important determinant of overall cognitive function, even in the normal population. Abnormalities in Rho GTPase signaling are a prominent cause of MR Genetics of nonspecific MR implicate abnormal Rho signaling in MR

Although the abnormalities in dendrites and spines offered a plausible explanation for the impaired cognitive abilities in some forms of MR, how they were generated remained an enigma for decades. The

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Box 2. Regulation of actin dynamics by RhoA, Rac1 and Cdc42

Collapsin-1/ sema3A

Extracellular stimuli

GTPases

Neurotrophins (p75NTR)

Ephrin-A5 (EphA-R)

Glutamate (NMDA-R)

Cdc42–GDP

Rac–GDP

Rho–GDP

Cdc42–GTP

Rac–GTP

Rho–GTP

p35/Cdk5 1st Order effectors

N-WASP

2nd Order effectors

Final effectors

Actin cytoskeleton response

PI4P5K

PIP2

Arp2/3 complex

Actin filament assembly Filopodia formation (Cdc42)

IRSp53

WAVE2

PAK1

ROK/ROCK

LIMK

MLCK

MLCP

Capping proteins

Cofilin

Myosin II heavy chain

Myosin light chain

Actin filament extension

Actin filament depolymerization

Actomyosin disassembly

Actomyosin contraction

Lamellipodia formation (Rac)

Outgrowth inhibition/ retraction

Neuronal responses Stimulate outgrowth and spine formation

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Fig. I. Signaling through Cdc42, Rac1 and RhoA within the context of mammalian neuronal development. Abbreviations: Arp 2/3, actin-related protein 2/3; Cdk5, cyclin-dependent kinase 5; EphA-R, ephrin-A-receptor; IRSp53, insulin receptor substrate protein of 53 kDa; LIMK, lens intrinsic membrane protein domain kinase; MLCK, myosin light-chain kinase; MLCP, myosin light-chain phosphatase; NMDA-R, N-methyl-D-aspartate receptor; NTR,

recent cloning of seven genes that cause nonspecific X-linked MR (MRX) has provided a useful starting point for investigating the cellular mechanisms responsible for MR [20] (Table 1). Three of these genes (OPHN1, PAK3 and ARHGEF6, encoding oligophrenin 1 [21], PAK3 [22] and αPIX [23], respectively) participate directly in cellular signaling through Rho GTPases (Box 1). Oligophrenin 1 acts as a Rho–GAP (GTPase-activating protein) and stimulates the GTPase activity of RhoA, Rac1 and Cdc42 in vitro [21]; PAK3 is a serine/threonine http://tins.trends.com

neurotrophin receptor; N-WASP, neuronal Wiscot–Aldrich syndrome protein; PAK, p21-activated kinase; PI4P5K, phosphatidylinositol 4-phosphate 5-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate, ROCK, Rho-associated kinase; WAVE, WASP family verprolin-homologous protein. Note that most of the interrelationships have been established in non-neuronal cell lines and need confirmation in neurons.

protein kinase that mediates effects downstream of Rac and Cdc42 on the actin cytoskeleton and gene expression [24] (Boxes 1,2); and αPIX is a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc4, and at the same time, interacts with PAKs [24,25]. The fact that the Rho-linked MRX proteins each interact with Rho GTPases at different locations in the regulatory cycle (Box 1) emphasizes the importance of aberrant Rho signaling in MR. Rho proteins are highly conserved regulators of the actin cytoskeleton [26] and are emerging as key signaling

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Additional genetic evidence for Rho GTPase signaling in MR Cdc42 and Rac are primarily involved in protrusive activity by promoting actin polymerization through activation of members of the WASP, WAVE/SCAR and PAK families and PI4P5 kinase, which catalyzes PIP2 formation (Fig. I). Cdc42, in synergy with PIP2, activates neuronal N-WASP, which binds the Arp2/3 complex and actin monomers [a], resulting in actin filament formation [b]. Activated Cdc42 induces long filopodia through N-WASP, whereas dominant-negative N-WASP strongly reduces neurite outgrowth [c]. Lamellipodia formation by Rac1 requires activation of WAVE2 through IRSp53 [a], which binds the Arp2/3 complex to stimulate actin filament formation. Extension of actin filaments is stimulated by inhibition of barbed end capping proteins by generation of PIP2 downstream of Rac1 [d]. Rac1 and Cdc42 also block depolymerization of actin filaments at the pointed side by activation of PAK1, which activates LIMK to phosphorylate and inhibit cofilin [e]. Activation of PAK1 also inhibits MLCK, decreasing myosin light-chain phosphorylation and reducing actomyosin contraction [f], thereby opposing the downstream actions of Rho. In addition, Rac1 activation stimulates myosin II heavy-chain phosphorylation, resulting in actomyosin disassembly [g]. The neuronspecific regulator of Cdk5, p35, is a specific effector of Rac1 that inhibits PAK1 [h]. Cdk5 and p35 are required for cerebral cortex lamination and neurite outgrowth. RhoA appears to be involved primarily in the generation of contractile force by the actomyosin cytoskeleton. RhoA activates ROCK, which phosphorylates myosin light-chain directly [i], or indirectly by inhibiting myosin light-chain phosphatase [j]. This results in increased actomyosin contraction [k]. Activation of Rho inhibits neurite outgrowth through ROCK, whereas inhibition of Rho or ROCK induces neurites [l]. Furthermore, ROCK phosphorylates LIMK (like PAK), resulting in actin filament stabilization [m]. LIMK is necessary for reducing the number of neurites activated by ROCK [l]. References a Takenawa, T. and Miki, H. (2001) WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement. J. Cell Sci. 114, 1801–1809 b Mullins, R.D. (2000) How WASP-family proteins and the Arp2/3 complex convert intracellular signals into cytoskeletal structures. Curr. Opin. Cell Biol. 12, 91–96 c Banzai, Y. et al. (2000) Essential role of neural Wiskott–Aldrich syndrome protein in neurite extension in PC12 cells and rat hippocampal primary culture cells. J. Biol. Chem. 275, 11987–11992 d Carpenter, C.L. et al. (1997) Signal transduction pathways involving the small G proteins Rac and Cdc42 and phosphoinositide kinases. Adv. Enzyme Regul. 37, 377–390 e Edwards, D.C. et al. (1999) Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat. Cell Biol. 1, 253–259 f Sanders, L.C. et al. (1999) Inhibition of myosin light-chain kinase by p21-activated kinase. Science 283, 2083–2085 g Van Leeuwen, F.N. et al. (1999) Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading. Nat. Cell Biol. 1, 242–248 h Nikolic, M. et al. (1998) The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity. Nature 395, 194–198 i Amano, M. et al. (1996) Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 271, 20246–20249 j Kimura, K. et al. (1996) Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273, 245–248 k Burridge, K. and Chrzanowska-Wodnicka, M. (1996) Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12, 463–518 l Bito, H. et al. (2000) A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons. Neuron 26, 431–441 m Maekawa, M. et al. (1999) Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285, 895–898

intermediates between extracellular stimuli and dynamic changes in neuronal morphology and connectivity [27–31]. In contrast to most other MR genes, because Rho GTPases have been shown to regulate the development of dendrites and spines [27–31], the function of the Rho-linked genes provides a mechanistic link with the microstructural deficits observed in MR. Given the purely cognitive deficit in MRX, currently, the Rho-linked MRX genes offer the most direct approach to studying the neurobiology of cognition. http://tins.trends.com

The remaining MRX genes are also involved in cellular signaling, and several might be linked to Rho signaling indirectly (Table 1). Tetraspanin 2 [32] participates in membrane-associated complexes [33], which are focal points of Rho signaling [34]. Deletion of the Drosophila neuronal tetraspanin, late bloomer, delays neuromuscular junction formation [33]. αGDI is a GDP-dissociation inhibitor for Rab3a and Rab3c, related to Rho GTPases and involved in neurotransmitter release [35]. Although this suggests a role for defective neurotransmission in MR, αGDI can also be involved in neuronal morphological development [35]. Among the syndromic forms of MR, Aarskog–Scott syndrome results from mutation of FGD1 [36], which encodes a GEF for Cdc42 [37]. Moreover, the cognitive deficits in Williams syndrome might be due to loss of LIMK1 (LIM domain kinase 1), which is activated downstream of Rho GTPases [38] (Box 2). Interestingly, the fraX MR protein (FMRP), which is absent in fraX syndrome, was recently shown to bind specifically to two highly homologous proteins (CYFIP1 and CYFIP2) [39]. CYFIP1 turned out to be identical to p140Sra-1, which binds activated Rac and actin and is probably an effector of Rac [40]. Thus, it seems that even FMRP, which is involved in RNA transport or processing, is linked to Rho signaling. The high proportion of Rho-linked genes in MRX indicates an important role for abnormal Rho signaling in MR, and their number will probably increase with further screening. In fact, cloning of the many remaining MRX genes could be speeded up by screening for specific Rho-associated genes that localize to loci linked to MR. Twin studies have shown that both gray matter volume and global cognition are, to a large extent, determined by – as yet unknown – genetic factors [19]. The MR studies suggest that polymorphisms in Rho-linked genes could be important contributors to the natural variance in IQ. Rho GTPases and the development and/or plasticity of neuronal connectivity Rho GTPases regulate the organization of the actin cytoskeleton

Rho proteins are highly conserved regulators of the actin cytoskeleton [26], cell adhesion and migration [34], cytokinesis [41] and gene expression [42], and therefore could also be involved in MR syndromes associated with neuron loss or migration deficits. Of the 11 Rho proteins expressed in mammalian brain, only RhoA, Rac1 and Cdc42 have been studied in detail, and mainly in fibroblast cell lines [43]. At present, our knowledge of the role of Rho GTPases in the development, plasticity and function of the nervous system is fragmented and sometimes contradictory. However, their crucial involvement in these processes is likely to trigger an avalanche of relevant data in the next few years.

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modulating neurite dynamics and decision making, rather than in regulating long-range neurite extension.

Extracellular and intracellular signals

αPIX

Regulatory proteins (GEFs, GAPs, GDIs)

Rho family GTPases

Rac1/Cdc42

PAK3

Effector proteins

Actin filament assembly

Cytoskeleton organization

Development Growth cone/filopodia responsiveness

OPHN1

RhoA

Actomyosin contraction Adulthood

Synapse/spine responsiveness

Pathfinding errors Neurite branching and outgrowth

Synapse/spine dynamics/rearrangement

(Spine) synapse formation

Decreased/aberrant connectivity

Increased threshold for structural plasticity

Impaired information processing and cognition TRENDS in Neurosciences

Fig. 2. Working hypothesis for the effects of (loss-of-function) mutations in OPHN1, PAK3 and ARHGEF6 (the gene encoding aPIX) on neuronal morphological development and connectivity. The arrows and cross bars in red indicate the presumed effects of the mutations on neuronal signaling and development. Abbreviations: GAP, GTPase-activating proteins; GDI, guanine dissociation inhibitor proteins; GEF, guanine nucleotide exchange factor; OPHN, oligophrenin; PAK, p21-activated kinase.

In neuroblastoma cells, Cdc42 and Rac1 direct the formation of filopodia and lamellipodia, respectively [44], and activation of RhoA induces neurite retraction, whereas inactivation of RhoA induces neurite formation [44]. These morphological changes depend on regulation of the dynamics of the actin cytoskeleton (Box 2), which is concentrated in growth cones, filopodia and lamellipodia in the developing brain [45,46], and in dendritic spines in the adult brain [47]. The actin cytoskeleton is required for growth cone motility, neurite pathfinding and branching [45,46], and for the formation and motility of dendritic spines [47]. Neurite elongation, however, is mainly dependent on tubulin polymerization. Thus, Rho proteins are primarily involved in http://tins.trends.com

Rho GTPases regulate neuronal morphogenesis and connectivity in response to many signals

During development, Rho proteins integrate many extracellular signals to direct the outgrowth of axons and dendrites and the formation and/or dynamics of dendritic spines [27–31]. RhoA, Rac1 and Cdc42 affect the size and shape of growth cones [48], the generation of neurites [49,50], axonal and dendritic elongation, dendritic branching [31,49–53] and axonal pathfinding [27]. The actions of Rho and Rac1 on neurite outgrowth and branching are often antagonistic [44,53–55] because of opposite effects on effectors (e.g. myosin light-chain phosphorylation; Box 2) or more direct interactions at the GTPase level [56]. Generalizing, activation of Rho reduces growth cone motility, neurite elongation and dendritic branching, largely through increased actomyosindependent contraction [50,52,54] (Box 2). Conversely, Rac1 stimulates lamellipodia formation, dendrite initiation, elongation, branching complexity and dynamics [44,49,51,52,54]. Effects of Cdc42 on neuronal morphogenesis appear to be similar to those of Rac1, but more limited: Cdc42 stimulates filopodia formation [44], neurite formation [49] and dendritic branch dynamics [52], whereas dominant-negative Cdc42 induces fewer and shorter dendrites [51]. Rho proteins modulate neurite outgrowth in response to extracellular guidance cues such as myelin, collapsin-1/Sema3A and ephrin-A5 [48,57,58], and neurotrophins [53,59]. Whereas ephrin-A5 and the p75 neurotrophin receptor primarily involve RhoA, myelin and collapsin-1/ Sema3A act through Rac1. Both RhoA and Rac1 appear to act downstream of NGF, BDNF and NT-3 [53], probably through Trk receptors. In addition, Rho proteins could mediate the effects of synaptic activity on neurite outgrowth, because NMDA-dependent dendritic branch elongation in Xenopus tectal neurons requires inhibition of Rho activity [54]. This might turn out to be an important mechanism whereby Rho GTPases are involved in experiencedependent fine-tuning of neuronal connectivity [18]. Rho GTPases modulate dendritic spine formation, plasticity and function

Rho GTPases also regulate dendritic spine formation. In transgenic mice and cultured pyramidal neurons, activated Rac1 produced numerous but small dendritic spine-like structures [29–31], whereas dominant-negative Rac1 decreased spine densities [30]. Activated RhoA reduced the density of spines and the length of spine necks, whereas inactivation of RhoA induced opposite effects [29]. These experiments indicate that the generation and maintenance of dendritic spines requires inhibition of Rho and activation of Rac1,

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whereas activation of Rho induces spine retraction. An activator of Rac1 involved in spine formation could be kalirin-7, a GEF for Rac1, which is targeted to dendritic spines and increases spine formation when overexpressed [60]. Rho proteins regulate cell adhesion through transmembrane proteins linked to the actin cytoskeleton, such as cadherins and integrins [34,61]. Because cadherins are present at synaptic contacts [62] and involved in synaptic plasticity [63], they might be part of the mechanism by which Rho proteins modulate synapse formation or plasticity. Recent studies have shown that spine formation continues into adulthood in response to plastic stimuli, dependent on neurotransmission through NMDA receptors and Ca2+ mobilization [64]. Furthermore, spine morphology is highly dynamic and responsive to glutamatergic activity [65]. Because this dynamic behavior depends on actin polymerization [47], it is probably regulated by Rho GTPases. Rho proteins are also involved in organizing postsynaptic structure and receptor clustering in neuromuscular [66] and inhibitory synapses [67], with potential functional consequences [68]. The Rho signaling network acts as an epigenetic system to regulate neuronal morphology

Rho GTPases have emerged as key regulators of neuronal pathfinding and morphogenesis and (post)synaptic connectivity, setting up the ‘hardware’ for information processing. Moreover, they will probably turn out to be crucially involved in plastic phenomena like activity-dependent synapse rearrangement and the modulation of spine shape and function in response to synaptic activity and other factors. An expanding range of extracellular signals converges onto a network of mutually interacting Rho GTPases through at least 30 GEFs, 13 GAPs and several GDIs (Box 1), mostly expressed in the brain and each specific for one or more Rho proteins. In addition, up- or downregulation of the expression of specific Rho regulators could alter both the basal activity state and the responsiveness of Rho Useful websites • http://xlmr.interfree.it/home.htm XLMR genes update website. An excellent database on loci for syndromic and nonsyndromic mental retardation (MR) and cloned MR genes on the X chromosome, regularly updated by P. Chiurazzi. The site contains information on the associated symptoms in syndromic MR, literature references and links to the Online Mendelian Inheritance in Man database and Medline (PubMed). • http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=OMIM Online Mendelian Inheritance in Man. A database on human genes and genetic disorders authored and edited by V.A. McKusick et al. The database contains textual information, literature references and links to Medline and other resources. The site can be searched for keywords such as mental retardation, MRX, or specific genes or proteins, and yields a wealth of information. • http://www.fmi.ch/members/andrew.matus/video.actin.dynamics.htm Web page of the Matus group at the Friedrich Miescher Institute in Basel, from which movies can be downloaded showing actin dynamics in growth cones and dendritic spines.

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proteins as a function of cell type, maturation or extracellular signals that alter gene expression. In this way, the Rho signaling network acts as an epigenetic system, integrating extracellular and nuclear signals. In concert with other factors (e.g. microtubule-associated proteins), a particular profile of Rho proteins and their regulators could determine the basic morphology of a specific type of neuron (e.g. pyramidal versus multipolar or bipolar [49]), and at the same time, enable modulation by extracellular signals and synaptic activity. All signals would converge on the same pool of local monomeric and filamentous actin, resulting in spatial integration of several cues, directing the cytoskeleton to mediate adaptive changes in cell morphology. The Rho signaling network and MR A nonspecific hypothesis

How could alterations in Rho signaling result in the structural and/or functional network changes that give rise to MR? As yet, studies on the effects of the Rho-linked MR genes on neuronal signaling or development have not been reported. However, given the tight coupling between many components of the Rho signaling network, any deletion or mutation of crucial components is likely to shift the balance in the network to a suboptimal state, locking actin in a certain configuration (i.e. polymerized or depolymerized, contracted or relaxed). Consequently, neurons might be less responsive to environmental cues, giving rise to suboptimal neuronal connectivity and/or plasticity. In this scenario, it is not important whether a mutation stimulates or inhibits Rho signaling or which Rho GTPase is affected. A specific hypothesis

If and how the mutations in OPHN1, PAK3 and ARHGEF6 result in the presumed deficiencies in neuronal connectivity still has to be established. However, limited evidence suggests that oligophrenin 1 acts preferentially on RhoA, suggesting that activation of Rho causes MR [21]. Inactivation of p190 Rho–GAP in mice causes defects in axon guidance and fasciculation, whereas effects on dendritic properties have not been reported [69]. The mutations in PAK3 probably cause loss of function and thus decreased signaling downstream of Rac/Cdc42 [22,70]. Similarly, loss-of-function of FGD1 should specifically decrease Cdc42 activity [36]. Mutations in ARHGEF6 result in loss-of-function or deletion of 28 amino acids within the calponin homology domain [23], which might impair interaction with filamentous actin [71]. Thus, if any common principle can be detected at this stage, it would be increased activation of RhoA or decreased signaling via Rac1/Cdc42 and PAK3, possibly also involving LIMK1 [38]. Given the antagonism between RhoA and Rac1, these alterations could produce similar morphological changes consistent with decreased dendritic branching and spine densities.

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Acknowledgements I gratefully thank J. Chelly (Paris), H. van Bokhoven (Nijmegen), J. Collard, C. Levelt, G. Boer and R. Swaab (Amsterdam) for their useful comments, G. Boer for help with the figures, and P. van Hulten for support in the lab. I apologize to the many investigators who are not directly cited here for reasons of limited space. This review is dedicated to Jo Ramakers, who made me see the beauty of nature.

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However, increased RhoA activity does not appear to be compatible with long, thin, tortuous spines as seen in MR. To clarify this issue, it is essential to know the effects of the mutations on neuronal morphology and spines. For the time being, a more specific working hypothesis (Fig. 2) assumes that loss-of-function mutations in PAK3 or ARHGEF6 result in decreased Rac1/Cdc42 signaling, and loss-of-function mutation in OPHN1 increases signaling through RhoA. By decreasing actin filament assembly or increasing actomyosin contraction, the responsiveness and dynamics of growth cones and filopodia would be reduced, resulting in decreased or abnormal connectivity. In adulthood, a decrease in spine responsiveness could reduce synaptic plasticity. Any of these consequences would be likely to limit information processing and cognitive function. Conclusions and outstanding questions

Mutations in Rho-linked MRX genes have outlined how deficient cellular information processing could give rise to the abnormal neuronal connectivity and impaired information processing that underlie MR. Single-gene mutations that cause purely cognitive deficit now provide an excellent opportunity to

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address many remaining questions directly. Detailed cognitive testing, functional imaging and quantitative analysis of the neuronal microanatomy of human brains with known mutations are necessary to expand our limited knowledge on the neuropathology of MR. Studies in genetically modified mice should further our understanding of Rho signaling in cognition and its neuroanatomical substrate. Cell biological studies on cultured neurons should reveal the developmental processes by which the mutated genes cause abnormal connectivity. Here, an important challenge lies in establishing which extracellular signals are coupled to which Rho proteins by the different regulators, in unraveling the links with the actin cytoskeleton by the intervening kinases and effector proteins, and in their relevance for neuronal morphology, plasticity and function. Answers to these questions will shed light on the cellular functions crucial to appropriate cognitive functioning (i.e. intelligence). It is surprising that no mutations in NMDA receptors or other genes associated with long-term potentiation have turned up in MR [72]. Although most mutations will impair cognition, it is tempting to speculate that some might actually enhance cognitive function.

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Integration of sodium and osmosensory signals in vasopressin neurons Daniel L. Voisin and Charles W. Bourque Vasopressin (antidiuretic hormone) release has been thought to be controlled by interacting osmoreceptors and Na+-detectors for >20 years. Only recently, however, have molecular and cellular advances revealed how changes in the external concentration of Na+ and osmolality are detected during acute and chronic osmotic perturbations. In rat vasopressin-containing neurons, local osmosensitivity is conferred by intrinsic stretch-inactivated cation channels and by taurine release from surrounding glia. Na+ detection is accomplished by acute regulation of the permeability of stretch-inactivated channels and by changes in Na+ channel gene expression. These features provide a first glimpse of the integrative processes at work in a central osmoregulatory reflex.

The constancy of the milieu intérieur, a prerequisite for life [1], involves a rigorous defence of the tonicity of extracellular fluids [1–4]. Indeed, because biological membranes are generally permeable to http://tins.trends.com

water, but not to solutes, centrally controlled mechanisms that stabilize fluid osmolality have evolved to prevent water shifts that might perturb cell or tissue function [2,4]. In mammals, small (±1%) changes in plasma osmolality or extracellular Na+ concentration {[Na+]} induce physiological (neuroendocrine) and behavioural responses that maintain systemic osmotic pressure close to a defined set-point [2–8] (Fig. 1). Physiological responses to hyperosmolality include increased water retention and increased Na+ excretion (natriuresis), which are evoked by increases in the rates of release of antidiuretic (vasopressin) and natriuretic (e.g. oxytocin) hormones from the neurohypophysis [2,6], respectively. Behavioural

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