Mental retardation - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2009 | 109 | 1–14

doi: 10.1111/j.1471-4159.2009.05881.x

*De´partement Neurotransmission et Sećre´tion Neuroendocrine, Institut des Neurosciences Cellulaires et Inte´gratives (UPR-3212) CNRS and Universite´ de Strasbourg, Strasbourg, France Institut Cochin, Universite´ Paris Descartes, INSERM, CNRS UMR, Paris, CHU Cochin, Paris, France

Abstract Among mental disorders, mental retardation has been shown to be caused by various factors including a large array of genetic mutations. On the basis of remarkable progress, the emerging view is that defects in the regulation of synaptic activity and morphogenesis of dendritic spines are apparently common features associated with mutations in several genes implicated in mental retardation. In this review, we will discuss X-linked MR-related gene products that are potentially involved in the normal structure and function of the synapses,

with a particular focus on pre- and/or post-synaptic plasticity mechanisms. Progress in understanding the underlying conditions leading to mental retardation will undoubtedly be gained from a closer collaboration of geneticists, physiologists and cognitive neuroscientists, which should enable the establishment of standardized approaches. Keywords: LTP, mental disorder, receptor recycling, synapse, synaptic plasticity. J. Neurochem. (2009) 109, 1–14.

Mental retardation (MR) is a common condition characterized by significant limitation in intellectual function and adaptive behavior that arise during childhood. MR is defined by an overall intelligence quotient lower than 70 associated with deficit in social, daily living and communication skills, and is estimated to affect 1–3% of the population. Causes of MR are extraordinarily heterogeneous, ranging from environmental to chromosomal and monogenetic causes. Conventionally, genetic forms of MR have been subdivided into syndromic and non-syndromic forms, depending on whether MR is associated or not with clinical, radiological, metabolic or biological features, however, the boundaries in this classification are vanishing as several MR-related genes are involved in both syndromic and non-syndromic forms of MR (Frints et al. 2002). To date nearly 300 MR-related genes have been identified and classified according to the associated syndrome (Inlow and Restifo 2004). Many of those genes are involved in brain development, neurogenesis and neuronal migration (Reviewed in Vaillend et al. 2008). However, since some MR brains are normal in terms of size and macro-architecture, it has recently been proposed that cognitive limitations may also be due to synaptic dysfunctions (Chelly et al. 2006). Cognition, as other brain functions, requires an adequate treatment of sensory information, which depends on the

timing of signal transfer within connected brain areas. Along this track, synapses constitute major ‘‘hurdles’’ but also provide an important source of qualitative treatment of neuronal information. Indeed, synapses are able to sense and integrate modulating signals in order to change their response to a given input, a phenomenon called synaptic plasticity. Since the discovery of long-term forms of synaptic plasticity in the 1970s (Bliss and Lomo 1973), these adaptations of synaptic strength to neuronal activity are thought to be cardinal in learning and memory (Sah et al. 2008). Because learning deficit is a constant feature of patients with MR, it is attempting to attribute some of MR traits to alterations in synaptic functions. This hypothesis is supported by histological data. Indeed, post-mortem analysis of human MR brain tissue often shows dendritic spines with altered shapes and densities (Kaufmann and Moser 2000). The degree of Received October 24, 2008; revised manuscript received December 23, 2008; accepted December 30, 2008. Address correspondence and reprint requests to Vitale N: INCI, CNRS UPR-3212. E-mail: [email protected] Abbreviations used: AMPA, a-amino-3-hydroxy-5-methylisoxazole4-propionate; FMRP, fragile X mental retardation protein; LTD, longterm depression; LTP, long-term plasticity; PKA, protein kinase A; PLD, phospholipase D; PPR, paired pulse ratio; XLMR, X-linked mental retardation.

Ó 2009 The Authors Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 1–14

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2 | Y. Humeau et al.

these defects is correlated with the severity of MR (Purpura 1974). Defects in dendritic spine morphology are also consistently found in mouse models with genetically generated-MR (see Table 1), but also for non-genetic MR (review by Newey et al. 2005). Therefore, alterations in the shape of dendritic spines are likely to be associated with or causal in cognitive deficits (Fig. 1a and b). Functionally, much of what we know about synaptic deficits associated with MR comes from in vitro studies of MR mouse brains, i.e. obtained from animals bearing MR genes mutations mimicking the mutations found in humans. These mutations are generally infrequent, but they provide substantial evidence for the involvement of single gene in the pathogenesis of the disorder. Interestingly, over the last 20 years, about 60 X-linked MR genes have been identified (Ropers and Hamel 2005). The apparent excess of X-linked genes involved in MR (XLMR) disorders led to the hypothesis that a disproportionately high density of genes influencing cognitive abilities reside on the human X chromosome. However, recent estimates suggesting a downturn in XLMR prevalence (Mandel and Chelly 2004; Poirier et al. 2006), as well as the emerging high frequency of pathogenic autosomal copy number variations are not in favor of this hypothesis, and its value remains a matter of debate for the moment. With the exception of transcription and chromatin-remodeling factors, it is worth noticing that most of the MR-related proteins are enriched at pre- and/or post-synaptic compartments (Fig. 1c). Some of the neuronal protein complexes involved in human X-linked brain diseases have been recently reviewed (Laumonnier et al. 2007). In this review, based on reported synaptic function deficits in MR mouse models, we will discuss X-linked MRrelated gene products that are potentially involved in the normal structure and function of the synapses, with a particular emphasis on pre- and/or post-synaptic plasticity mechanisms.

Pre-synaptic function and plasticity The pre-synaptic compartment ensures neurotransmitter release in response to an incoming action potential. This requires the fusion of pre-synaptic synaptic vesicles containing neurotransmitter with plasma membrane, a phenomenon called exocytosis (Fig. 1c). As these vesicular fusion events are universal in eukaryotic cells, the molecular basis of vesicular trafficking and fusion have been studied in various cellular systems including neuronal and neuroendocrine cells (Bader et al. 2004). However, neurons are particular in their ability to adapt release in response to the presence of chemical signals provided by surrounding cells, allowing more qualitative information to reach post-synaptic cells. Hence, at a given pre-synapse, the plasticity ‘repertoire’ is particular, because of the heterogenous expression of ionotropic receptors (Pinheiro and Mulle 2008), Ca2+ channels or

Ca2+-binding proteins. A synapse ‘‘at work’’ is usually characterized by three quantal parameters, namely the quantum of neurotransmission Q, the probability of release P and the number of active release sites N. Thus, every incoming action potential leads to a post-synaptic response corresponding to the actual N.P.Q product. A change in synaptic strength – i.e. synaptic plasticity – is due to the change of one or more of these quantal parameters. It is usually difficult to attribute a change of Q or N to a particular synaptic compartment, whereas P is purely determined by pre-synaptic factors. For years, paired pulse ratio (PPR) was used to assess for changes in P, because reliably affected by changes in external Ca2+ concentrations triggering release (but see also Heine et al. 2008). As shown in Table 1, changes in PPR were reported after mutations of several MR genes, namely, GDI1, OPHN1 and MeCP2, pointing to a presynaptic deficit. However, it is of note that despite the important body of work on MeCP2, a synaptic target for MeCP2 controlling neurotransmitter release and responsible for this PPR change has not been clearly identified to date (Chahrour and Zoghbi 2007). The following paragraphs discuss the potential molecular cascades underlying these changes. A key step: the fusion of synaptic vesicles In neurons, vesicles appear morphologically segregated close to an electron-dense structure of the plasma membrane. This area is called the active zone and physiologically corresponds to sites of synaptic vesicle docking and fusion, which allow neurotransmitter release. The active zone can be dissected into three morphologically and functionally distinct components: (i) the plasma membrane juxtaposed to the postsynaptic density where synaptic vesicle fusion occurs, (ii) the cytomatrix immediately internal to the plasma membrane where synaptic vesicles dock, and (iii) the electron-dense projections extending from the cytomatrix into the cytoplasm on which synaptic vesicles are tethered. All active zones have these three components, although they vary in appearance, especially in size and shape of dense projections (review by Schoch and Gundelfinger 2006). The fusion of synaptic vesicle with plasma membranes requires the breakdown of large energy barriers created by the local dehydration of polar phospholipid headgroups and membrane deformation. Surmounting these barriers requires the action of proteins with the special capacity to lower the energy costs. The important role of SNARE proteins (Syntaxin, Synaptobrevin/VAMP and SNAP25) has been well documented concerning neurotransmitter release, as well as other exocytotic processes, although the precise mechanism of action of SNARE proteins complex in membrane merging remains elusive (Rizo and Rosenmund 2008). The lipid composition of membranes at fusion sites is also crucial. It is controlled by the activity of lipid-modifying enzyme such as phospholipase D (PLD), which has been


Locus

Xq21.3

Xq12

Xq13

Genes

PAK3

OPHN1

NLGN3


Autism

S-XLMR

NS-XLMR

Phenotypes

Postsynaptic

Presynaptic Postsynaptic

Localization

NLGN3 R451C KI mouse

OPHN1 KO by siRNA OPHN1 constitutive and conditionnal KO mouse

Forebrain-specific PAK dominant negative mouse

Expression of PAK3 R419X (inactivation of kinase activity) PAK3 KO by siRNA Expression of PAK3 A365E (inactivation of kinase activity) Expression of PAK3 R67C (defective in Cdc42 binding) PAK3 KO mouse

MR model

ND

Unchanged I/O curve Unchanged AMPA/ NMDA currents

Rat hippocampal organotypic culture

Hippocampal acute slice Hippocampal neuronal culture Neocorical acute slice

Neocortical acute slice

Rat hippocampal organotypic culture Hippocampal acute slice Hippocampal neuronal culture

ND


ND

Reduced PPF Unchanged LTP (HFS,CA1) Unchnged LTD (DHPG,CA1)

ND

Increased mIPSC freq. Unchanged excitatory I/O curve Increased inhibitory I/O curve

ND

Increased LTP (HFS,TBS) Increased LTD (LFS)

Unchanged PPF/PTP/PTD Reduced LTP (HFS,CA1) Unchanged LTD (LFS,CA1)

ND

ND

Unchanged PPF Reduced LTP (PP,CA1)

Synaptic plasticity

ND

Unchanged I/O curve Increased mEPSC freq./amp

Reduced mEPSC freq. Reduced AMPA/NMDA ratio

Synaptic transmission


Experimental model

Table 1 Review of synaptic plasticity and spine morphology phenotypes in model of XLMR

Reduced spine density Increased shorter and larger spines Reduced spine length Reduced spine density in slice Increased spine density in culture ND

Reduced mature spines Increased filipodialike spines Reduced spine density Unchanged spine morphology Unchanged in culture

Reduced mature spines Increased filipodialike spines

Dendritic spines

Tabuchi et al. 2007

Khelfaoui et al. 2007

Govek et al. 2004

Hayashi et al. 2004

Meng et al. 2005

Kreis et al. 2008

Boda et al. 2004

References

X-linked mental retardation | 3

Xp22.2 Coffin-Lowry syndrome

RSK2

NS-XLMR

FMR2

Xq28

Rett syndrome

MECP2 Xq28

NS-XLMR

Xq28

GDI1

Phenotypes

Locus

Genes

Table 1 Continued

MR model

Nuclear

Hippocampal acute slice Neocortical neuronal culture

Hippocampal acute slice

Experimental model

Reduced mEPSC freq.

ND

ND


Hippocampal acute slice Hippocampal acute slice

MECP2 ± mouse FMR2 KO mouse

MECP2 308/y mouse

Neocortical acute slice Hippocampal acute slice Neocortical acute slice

MECP2)/y mouse

ND

ND

ND

ND

ND

ND

ND

Dendritic spines

Increased PPF Increased LTP (HFS,CA1) Unchanged PTP ()/y,Tg1)

Increased PPF Increased PTP Unchanged LTP (HFS,CA1, CA3) Reduced LTP (HFS,CA1) Unchanged LTP (TBS,CA1) ND

Synaptic plasticity

Reduced PPF (CA1) ND Unchanged PTP (CA1) Reduced LTP (HFS,TBS,CA1) Reduced LTD (LFS,CA1) ND ND Reduced spine density Decreased spine length Increased I/O curve (CA1) Reduced PPF (CA1) Reduced PSD length (CA1) Unchanged I/O curve (Cx) Reduced LTP (HFS,TBS, Unchanged dendritic CA1) arborization (Cx) Reduced LTP (TBS,Cx) Reduced LTD (LFS,CA1) Unchanged LTD (DHPG, CA1) ND Reduced LTP (HFS,TBS, ND CA1) Unchanged I/O curve (CA1) Unchanged PPF (CA1) ND Increased LTP (HFS,CA1)

Hippocampal acute Unchanged I/O curve (CA1) slice Autaptic hippocampal Reduced mEPSC freq./ neurons eEPSC amp. ()/y) Increased mEPSC freq./ eEPSC amp. (Tg1) MECP2 )/y mouse Neocortical acute Reduced mEPSC charge slice Increased mIPSC charge MECP2)/y mouse Hippocampal acute Unchanged I/O curve (CA1) slice

GDIalpha KO mouse Expression of RSK2 dominant negative MECP2 Tg1 mouse (overexpression) MECP2)/y and Tg1 mice

Presynaptic GDIalpha KO Postsynaptic mouse

Localization

Guy et al. 2007 Gu et al. 2002

Fukuda et al. 2005 Moretti et al. 2006

Dani et al. 2005 Asaka et al. 2006

Collins et al. 2004 Chao et al. 2007

D’Adamo et al. 2002 Thomas et al. 2005a,b

Ishizaki et al. 2000

References




Xq27.3

Xq26

FMR1

ARGHEF6

NS-XLMR

Fragile-X syndrome

Phenotypes

Localization

ARGHEF6 KO by siRNA

FMR1 KO mouse

MR model

Unchanged I/O curve

Neocortical acute slice


Cerebellar acute slice

ND

Neocortical neurons

ND

ND

Unchanged mEPSC freq./amp. Unchanged AMPA /NMDA ratio

Unchanged I/O curve (CA1)


Hippocampal acute slice

Experimental model

ND

Reduced STDP–LTP (PP) Unchanged STDP–LTD (PP) Unchanged PPF/PTP/PTD Increased LTD (PP,PC)

Unchanged PPF/PPD/ PTP/PTD Reduced LTP (HFS, 10TBS) Reduced LTP (PP)

Unchanged LTP (HFS,10TBS,CA1) Reduced LTP (5TBS,CA1) Increased LTD (DHPG,PP–LFS,CA1) Unchanged LTD (LFS,CA1) ND

Unchanged PPF

Synaptic plasticity

Unchanged spine density Increased longer spines Reduced mushroom spines Increased filipodia-like spines

Increased spine density Increased longer spines ND

ND

Dendritic spines

Node´-Langlois et al. 2006

Koekkoek et al. 2005

Larson et al. 2005 Zhao et al. 2005 Desai et al. 2006 Wilson and Cox 2007

Comery et al. 1997 Nimchinsky et al. 2001 Li et al. 2002

Godfraind et al. 1996 Paradee et al. 1999 Huber et al. 2002 Lauterborn et al. 2007 Li et al. 2002

References

S/NS-XLMR: syndromic/non-syndromic XLMR; KI/KO: knock in/out; siRNA: small interfering RNA; mEPSC/mIPSC: miniature excitatory/inhibitory postsynaptic current; freq.: frequency; amp.: amplitude; I/O curve: input/output curve; ND: not documented. LTP/LTD: long-term potentiation/depression; PPF/PPD: paired-pulsed facilitation/depression; PTP/PTD: post-tetanic potentiation/ depression; STDP: spike-timing-dependent plasticity. Into brackets are given either the experimental protocol used to induce synaptic plasticity (DHPG: group I mGluR agonist dihydroxyphenylglycine; HFS: high-frequency stimulation; LFS: low-frequency stimulation; PP: pairing pre-synaptic stimulus and post-synaptic depolarization protocol; TBS: theta-burst stimulation) or the brain areas studied (CA1-3: hippocampal areas CA1-3; Cx: cortex; PC: Purkinje cell).

Locus

Genes

Table 1 Continued



(a)

(b)

Fig. 1 Alterations in MR synapses. (a) Dendritic spines observed in MR mouse models are often dysmorphic, with elongated spine neck and in some cases decreased spine volume with associated loss of AMPARs. (b) Some of the documented or postulated deficits in synaptic physiology are presented in the scheme, including a lack of neurotransmitter release, a decrease in the number and/or the quality of AMPARs, and dendritic filtering introduced by the spine morphology. As a consequence, the synaptic strength is modified (here schematized as a decrease of the excitatory current recorded in the postsynaptic neuron. (c) A typical excitatory synapse is illustrated. At pre-synaptic and post-synaptic level, vesicular trafficking allows the emission and the reception of the chemical (i.e., the neurotransmitter) signal. Some of the identified MR gene products potentially or admitted to be involved in one or more of the key steps of the vesicular cycle are presented. In dark gray are synaptic proteins postulated but yet not undoubtedly confirmed to cause MR.

(c)

proposed to provide key fusogenic lipids at the site of neurotransmitter (Humeau et al. 2001, 2002) and hormonal release (Zeniou-Meyer et al. 2007). A likely possibility is that phosphatidic acid locally produced by PLD1 activity may play a decisive role in the late stages of membrane fusion by changing the membrane curvature required for membrane merging (Zeniou-Meyer et al. 2007). Interestingly, two MR gene products, oligophrenin-1 (OPHN1) and ribosomal protein S6 kinase (RSK2) are known or predicted to be potential modulators of PLD1 activity (Zeniou-Meyer et al. 2008). Mutations in RSK2 are associated with the Coffin Lowry syndrome, which is a rare X-linked disorder characterized by short stature, distinctive facieses, skeletal abnormalities and severe mental retardation (Trivier et al. 1996). The RSK family proteins are serine/threonine kinases, of which four members have been identified. RSK proteins are composed of two functional kinase catalytic domains, linked together by a regulatory linker region. The carboxyl-terminal kinase domain is related to the calcium/calmodulin-dependent kinase, whereas the amino-terminal kinase domain shares common structural features with the AGC (cAMP-dependent

protein kinase/protein kinase G/protein kinase C) kinases. The amino-terminal kinase domain is responsible for the phosphotransferase activity towards substrate, whereas the carboxyl-terminal kinase domain is the site of phosphorylation by extracellular signal-regulated kinases that leads to autophosphorylation (Fisher and Blenis 1996). RSKs have been involved in several important cellular events, mainly through transcriptional control of gene expression. Indeed, RSKs are involved in processes enabling the opening of chromatin structure through histone acetylation. However, in addition to transcription regulators, a few other RSK2 specific physiological substrates have been recently identified. Among these, PLD1 has been recently shown to be phosphorylated on threonine 147 by RSK2 in chromaffin cells, which argues for a potential role of RSK2 in presynaptic exocytosis (Zeniou-Meyer et al. 2008). Indeed, in chromaffin cells depleted for RSK-2 by siRNA, overexpression of a phosphomimetic PLD1 mutant rescued exocytotic function (Zeniou-Meyer et al. 2008). In agreement with this idea, significant learning and memory impairments have recently been reported in a mouse model invalidated for RSK2 (Poirier et al. 2007), suggesting altered



synaptic function (Fig. 1c). In addition, the phosphorylation of post-synaptic PDZ-containing proteins by RSK2 also appears to control mEPSC (miniature excitatory post-synaptic current) frequency (Thomas et al. 2005a,b). A clear understanding of RSK2 function in neurons awaits the use of simplified cell culture models that will allow discrimination between pre- and post-synaptic dysfunction. Rho GTPases are key regulators of cytoskeleton dynamics that have been identified in pre-synaptic nerve terminals, and are important for neurotransmitter release. For example, in neurons, Rac1 and Rho were shown to positively control neurotransmitter release (Humeau et al. 2002; McMullan et al. 2006). In pre-synapses, Rho GTPases cycle between active (GTP bound) and inactive (GDP bound) forms because of the activity of Guanine nucleotide exchange factor and GAP (GTPase-activating protein). As a consequence, Rho guanine nucleotide exchange factor 6 (ARHGEF6) and OPHN1 GAP activity are important in determining the balance between the active and inactive forms of the Rho GTPases (Fig. 1c). This cycle appears to be crucial to maintain synaptic functions as decreasing activated Rac1 level limited the number of pre-synaptic active release sites (Humeau et al. 2002, 2007). Interestingly, mutations in ARHGEF6 and OPHN1 lead to changes in PPR (Table 1), though additional genetic studies are required to validate implication of ARHGEF6 in mental retardation. Because Rac1 controls PLD1 activity (Hammond et al. 1997), these genetic defects may be linked to the presence of inappropriate lipids at fusion sites. As for other MR mouse models, physiological data are still preliminary, and direct input/output assessment of synapse efficacy will be necessary to distinguish between defects in neurotransmitter release and other deficits associated with guanine nucleotide-binding proteins associated with MR (Chechlacz and Gleeson 2003). Trafficking and docking of synaptic vesicles The GTPase Rab3 also organizes neurotransmitter release at the synaptic terminals in neurons (reviewed by Sudhof 2004). Rab3 seems to play an important role in the priming of synaptic vesicle at the release sites (Schlu¨ter et al. 2006). Like most GTPase, Rab3 activity is regulated by GDP/GTP cycling and requires recycling of GDP-bound Rab3 by the guanine dissociation inhibitor GDIa encoded by the GDI1 gene. Three mutations in the GDI1 gene in patients with XLMR have been identified, suggesting the importance of GDIa in proper synaptic function (Bienvenu et al. 1998; D’Adamo et al. 1998). Surprisingly, however, in mice model lacking GDIa, an enhancement of excitatory neurotransmission during repetitive stimulation in CA1 region of the hippocampus (post-tetanic potentiation), Ishizaki et al. 2000; Table 1), associated with a change in PPR were reported. These two synaptic parameters suggested a change in the dynamics of Ca2+ in pre-synapses, and are consistent

with data previously obtained following introduction of RaB3A mutants into pre-synaptic neurons (Doussau et al. 1998). Interestingly, in human cases mutations in GDI1 gene causes XLMR associated with epileptic seizures, suggesting that GDIa might be involved in suppressing synaptic hyperexcitability, possibly by controlling synaptic homeostasis and therefore the inhibitory/excitatory balance onto neurons. Ca2+-signaling and short-term plasticity IL1RAPL1 Interleukin 1 receptor accessory protein-like 1 (IL1RAPL1) encodes a 696 amino acid protein that has homology to IL-1 receptor accessory proteins and is mainly expressed in the brain (Born et al. 2000). IL1RAPL1 was first identified as a MR-related gene through a positional cloning approach based on the investigation of continuous gene deletions in families with MR, adrenal hypoplasia, Duchenne muscular dystrophy and glycerol kinase deficiency (Carrie´ et al. 1999). It was later shown that deletion, inversion, and mutation of this gene were found in brains of patients with XLMR (Lepreˆtre et al. 2003; Sasaki et al. 2003; Wheway et al. 2003; Tabolacci et al. 2006) and Autistic spectrum disorders (AST) (Piton et al. 2008), indicating that functional incapacitation or abnormal expression of IL1RAPL1 was associated with cognitive functions. Further studies revealed that IL1RAPL1 is most abundantly expressed in memory/concentration-associated brain areas such as hippocampus, dendate band, osmesis endothelial layer, parietal cortex and piriform cortex in rat and mouse (Carrie´ et al. 1999; Gao et al. 2008). Albeit most physiological and biological properties of IL1RAPL1 remain largely unknown, IL1RAPL1 was found to interact with the neuronal calcium sensor-1 protein (NCS-1), a protein widely expressed in neurons and the related chromaffin and PC12 cells (Bahi et al. 2003). NCS-1 belongs to a large family of calciumbinding protein that regulates calcium-dependent exocytosis through its activation of phosphoinositol 4-kinase (de Barry et al. 2006), as well as calcium channel trafficking (Weiss and Burgoyne 2002). Interestingly, we recently demonstrated that stable expression of IL1RAPL1 in PC12 cells induces a specific and NCS-1-dependent silencing of N-type voltagegated calcium channels activity that explains a secretion deficit observed in these IL1RAPL1 cells (Gambino et al. 2007). Further, IL1RAPL1 expression prevented nerve growth factor-induced neurite elongation in PC12 cells, a phenotype rescued by NCS-1 knockdown (Gambino et al. 2007). Since NCS-1 has been proposed to contribute to the activity-dependent synaptic facilitation at many synapses, it is tempting to speculate that the balance between IL1RAPL1 and NCS-1 levels may be critical for proper synaptic function (Fig. 1c).



Long-term pre-synaptic plasticity The discovery of long-term plasticity (LTP) by Bliss and Lomo in 1973 is a milestone in our understanding of the relationship between behavioral changes and underlined cellular processes. If the concept is solid, in most of the cases, the link between synaptic plasticity and behavioral learning remains difficult to demonstrate, mainly because of the variety of LTP forms. Contributing to the uncertainty of this concept is the existence of pre-synaptic synaptic plasticity in the mammalian brain (Krueger and Fitzsimonds 2006). The expression of pre-synaptic protein kinase A (PKA)-dependent LTP appears to be a general feature, occurring at mossy fiber synapses in the hippocampus, at cerebellar parallel fiber synapses, cortico-thalamic synapses, parabrachial afferents to the central amygdale, and cortical afferent to the lateral amygdala (Salin et al. 1996; CastroAlamancos and Calcagnotto 1999; Nicoll and Schmitz 2005; Lopez de Armentia and Sah 2007; Fourcaudot et al. 2008). Probably because this form of LTP is not usually tested in rapid physiological screenings, no deficits in pre-synaptic PKA-dependent LTP have been reported yet. However, one can expect to find deficits in pre-synaptic LTP at some MR synapses. Indeed, one of the key PKA targets in the active zone is the scaffolding protein RIM1a that interacts with a number of proteins involved in neurotransmitter release including Munc13-1, a-liprins, synaptotagmin 1, and presynaptic voltage-dependent Ca2+ channels (VDCCs), but also Rab3A, controlled by the XLMR GDIa gene. In addition, synapsin 1 also possesses a PKA phosphorylation site and it was recently shown to be a major PKA effector in the modulation of activity-dependent synaptic vesicle exocytosis (Menegon et al. 2006). Therefore, deficits are likely in pre-synaptic function and plasticity, and these may play a role in the cognitive impairments present in MR patients. To date, their contribution may be underestimated because the focus of physiological studies has been limited to the CA1 area of the hippocampus (Table 1). Hence, the systematic examination of a pre-synaptic index such as PPR or other forms of shortand PKA-dependent LTP now need to be performed at synapses in MR mouse models, in order to establish as extensively as possible a repertoire of synaptic plasticity in the absence of MR proteins. Since all pre-synaptic MR proteins appear to regulate vesicular trafficking and fusion, these physiological events need to be considered as key determinants in some of the physiological aspects of synaptic function linked to cognitive functions.

The post-synaptic compartment Post-synaptic structures are designed to receive the chemical signal emitted by pre-synaptic afferent synapses. In the case of glutamatergic transmission, the post-synaptic compartment is often a dendritic spine, a very well organized

structure, which allows the concentration of glutamatergic ionotropic and metabotropic receptors. Some observations indicate that post-synaptic compartments are likely to be affected in MR brains. Dysmorphic dendritic spines are often found in post-mortem human MR brains and in neurons of MR animal models (Table 1, Kaufmann and Moser 2000). In mice, this morphological phenotype correlates well with perturbed LTP, classically tested at the Schaeffer collaterals to CA1 pyramidal cell synapses (Table 1). Here, associative LTP is mostly expressed as an increase in the number of aamino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors at synapses, because of changes in post-synaptic trafficking of AMPA receptors (Malinow and Malenka 2002). This suggests that activity-induced changes in AMPA receptor trafficking may be altered at excitatory synapses in some MR brains. This is reinforced by the fact that the reverse phenomenon – i.e. increased endocytosis of AMPA receptors leading to long-term depression (LTD) – is also affected in some MR mouse models (Table 1). However, from a physiological point of view, alternative explanations can be envisioned: Indeed, the observed morphological changes in dendritic spine shape and density in MR neurons may have strongly modified the associative learning rules at synaptic contacts, leading to a lack of LTP induction instead of LTP expression deficits. Some supporting data are discussed in the last section. Post-synaptic trafficking of AMPA receptors in the expression of LTP The trafficking of post-synaptic receptors is central to plasticity of excitatory synapses (Malinow and Malenka 2002; Isaac et al. 2007). The presence of post-synaptic AMPA receptors at the plasma membrane is tightly controlled by the ambient glutamate (Turrigiano et al. 1998), but also by iterative activities of pre-synaptic structures. The intensity and frequency of these activities determine the positive or negative effect on synaptic strength (Malinow and Malenka 2002) possibly guided by the amount of Ca2+ entering into the dendritic spines (Bienenstock et al. 1982; Nevian and Sakmann 2006; Humeau et al. 2005). Transmembrane AMPA receptor regulatory proteins control AMPA receptor maturation, trafficking, and function (reviewed by Isaac et al. 2007). Importantly, the trafficking of AMPA receptors is a bi-dimensional process, consisting of the lateral diffusion within the plasma membrane and constant vesicular recycling. Both processes can be controlled by the same TARP (Transmembrane AMPA receptor Regulatory Proteins). For example, Stargazin concentrates membranous AMPA receptor at synaptic sites by interacting with both AMPA receptor and PSD-95 (Bats et al. 2007), and also controls the ratio between internalized and externalized AMPA receptors (Chen et al. 2000). Interestingly, this last feature appears to be comparable to pre-synaptic trafficking of synaptic vesicles, implicating SNARE-depen-



dent exocytosis (Lledo et al. 1998) and dynamin-dependent endocytosis (Carroll et al. 1999). The sequence of physiological events leading to LTP has been characterized: the arrival of new AMPA receptors by vesicular fusion (reviewed by Malinow and Malenka 2002; Turrigiano 2000) is triggered by Ca2+ entry into the postsynaptic compartment, principally due to the depolarizationdependent unblocking of synaptic NMDA receptors. The subsequent cascade was recently dissected in two main steps: The early phase of LTP (< 3 min after induction) is mediated by the arrival of GluR1/GluR1 AMPA receptors permeable to calcium ions, already present in dendritic spines. Their presence at synapses leads to continuous Ca2+ entry which induces the subsequent fusion of vesicles containing GluR2/ GluR1 receptors, and this is responsible for the late phase of the excitatory response increase (review in Isaac et al. 2007; but see Adesnik and Nicoll 2007). A similar sequence, but slightly different in timing, generates homeostatic plasticity following modulations of background glutamatergic activity (review in Isaac et al. 2007). Conversely, mGluR-dependent LTD, mediated by rapid endocytosis of AMPA receptor has been described in cerebellum and hippocampus (Bellone et al. 2008) following activation of post-synaptic metabotropic glutamate receptors. Abnormalities in AMPA receptor trafficking can be tested either by looking at miniature excitatory currents, which are bi-directionally affected by manipulating background network activity (Turrigiano et al. 1998), or by acutely inducing associative LTP or LTD. Mouse lines bearing MR gene mutations have been extensively tested for classical associative plasticity in the CA1 area of the hippocampus, but mostly inconsistent results were obtained, even in comparable experimental situations (Table 1). The archetype being the study of LTP and LTD in mice mutated in the FMR1 gene responsible for the fragile X syndrome. FMR1 FMR1 product, fragile X mental retardation protein (FMRP), binds mRNA and allows local translation of dendritic mRNAs in response to synaptic activity (Antar and Bassell 2003). The dendritic localization of FMRP mRNA is linked to the activation of metabotropic glutamatergic receptors (mGluRs), and subsequent Ca2+ entry leading to PKC activation (Weiler et al. 1997; Antar et al. 2004). In addition, the absence of FMRP increases the mGluR5-induced internalization of AMPA receptor in cultured hippocampal neurons (Nakamoto et al. 2007), suggesting that FMRP could participate to the expression of mGluR-dependent plasticity (Fig. 1c). Indeed, mGluR-dependent LTP (Desai et al. 2006) and LTD (Huber et al. 2002; Koekkoek et al. 2005) of glutamatergic transmission were reported in FMRP mouse model, eventually associated with deficits in associative learning (Koekkoek et al. 2005). While the FMR1 gene was identified 17 years ago (Verkerk et al. 1991) and the mouse model has been available

since 1994 (The Dutch-Belgian Fragile X Consortium 1994), more than 15 years of research focusing on LTP has only demonstrated that LTP is inconsistently affected in the different brain areas tested (Zhao et al. 2005; Desai et al. 2006; Li et al. 2002; Larson et al. 2005; Godfraind et al. 1996; Paradee et al. 1999; Lauterborn et al. 2007; see also Table 1). The inconsistency in LTP results may in fact result from differences between induction protocols, a constant problem in the LTP field. For example, Lauterborn et al. investigated LTP in the CA1 area of the hippocampus using different induction protocols in FMR1 mutant mice (Table 1). Very strong induction paradigms applied in both wild-type and FMR1 mutant animals produced to similar extents LTP of the AMPA receptor (Lauterborn et al. 2007), whereas decreasing the intensity of the induction protocol resulted in significantly less LTP in FMR1 mutant mice. Thus, if the overall capacity of cells to generate LTP was maintained in absence of FMRP, the threshold for its induction was apparently higher in FMR1 mutant mice (Lauterborn et al. 2007). These data illustrate the fact that even if defects in LTP have indeed been reported in different MR mouse models, comparative analysis may be impossible because of the variations in the protocols used. Finally, the estimation of the meaningfulness of the studied forms of LTP at behavioral levels remains a difficult task. In FMR1 KO mice, a nice exception exists. There is direct evidence that perturbing LTD at parallel fibers to Purkinje neurons was enough to modify the acquisition of the eye-blink conditioning in mice when the FMR1 gene was mutated only in the post-synaptic compartment (Koekkoek et al. 2005). These effects on synapse activity and plasticity are coherent with a local synaptic function of FMRP, acting as a regulator of activitydependent translation of mRNA-encoding proteins involved in actin/microtubule-dependent synapse growth, remodeling and function (Bagni and Greenough 2005). Interestingly, using the Drosophila model, it was shown that FMRP function is perhaps regulated through a RhoGTPase subfamily member, Rac1-dependent signaling pathway (Billuart and Chelly 2003; Schenck et al. 2003). The importance of synaptic structure and function and their control through RhoGTPase signaling pathways in the pathophysiology of MR are also supported by compelling evidence that include the identification of MR genes that encode regulators and/or effecters of RhoGTPases, such as OPHN1, PAK3, ARHGEF6 and FGD1, together with the recent implication of OPHN1 and PAK3 in the regulation of dendritic spine morphology and/or synaptic plasticity (Boda et al. 2004; Govek et al. 2004; Meng et al. 2005; Khelfaoui et al. 2007). PAK3 PAKs are serine/threonine kinases activated by small GTPases of the Rho-family, namely Rac1 and Cdc42. Among the family (PAK 1–4), mutations in the X-linked PAK3 gene have been associated with non-syndromic MR cases (Allen



et al. 1998; Bienvenu et al. 2000). Interestingly, their contribution to synapse formation and plasticity has been clearly demonstrated by two groups (Boda et al. 2004; Meng et al. 2005). In the first study, Boda et al. used transient over-expression in hippocampal organotypic slice cultures, and showed that PAK3 is localized at dendritic spines, and that PAK3 inactivation results in the formation of abnormal dendritic spine morphology and function (see Table 1). Reported abnormalities include a reduction in the number of mature spines and an increase in filopodia-like structures (Fig. 1c), as well as reduced spontaneous synaptic activity and defective long-term potentiation (LTP). The underlying cascades are not fully understood. The debate is centered on the role of Rac versus that of Cdc42 in post-synaptic dendrites and spines. On the one hand, blocking the function of either Rac1 or the PAKs generated long, thin spines and inhibited spine head growth, morphing and stability (Boda et al. 2004; Tashiro and Yuste 2004), whereas activation of PAKs had the opposite effect (Zhang et al. 2005). On the other hand, a recent study focusing on the three PAK3 mutations found in MR patients demonstrated that PAK3 is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific activator of Cdc42 (Kreis et al. 2008). In another study, Meng et al. (2005), generated a knockout mouse model (deficient) for PAK3 which exhibited significant abnormalities in synaptic plasticity, especially hippocampal late-phase LTP, and deficiencies in learning and memory. Surprisingly, in this knockout model, neither ex vivo cultures of hippocampal neuronal cells, nor Golgi staining of fixed brain sections showed significant alterations in spine morphology, density or length. Although the mechanisms by which PAK3, an effector of Rac1 and Cdc42, produces these effects remain an intriguing issue, one cannot exclude synaptic defects through de-regulation of signaling transcription cascades involved in the expression of factors that are crucial for synaptic morphogenesis, activity and plasticity. In favor of this hypothesis is the dramatic reduction of the phosphorylated active form of the transcription factor cAMP-response element-binding protein observed in knockout mice deficient for PAK3.

Can dendritic spine morphology affect LTP, and vice versa? In various animal models, MR neurons display dysmorphic dendritic spines (Table 1). Whether this phenotype leads to alterations in local dendritic integrative properties of these neurons, or represents a consequence of functional synaptic defects, is still an unresolved issue. Here, we will focus our discussion on the theoretical core of the LTP induction, namely the coupling between afferent activity and coincident post-synaptic detection. Cellular processes leading to neuronal plasticity vary from one neuron to another, and can be different for the same types

of neuron in different brain structures, depending on their surrounding partners. However, one model appears quasiuniversal, namely associative plasticity or hebbian plasticity (Cooper and Donald 2005), first postulated by Donald Hebb in the 1950s, and demonstrated in the 1970s (Bliss and Lomo 1973). Since then, molecular and cellular characterization of the mechanism underlying associative plasticity has been achieved. Briefly, glutamate released by cooperative inputs depolarizes the post-synaptic neuron thereby unblocking post-synaptic NMDA receptors, which therefore detect the coincident activity of neurons. The resulting Ca2+ ion influx into the post-synaptic dendritic spine will lead, depending on the final Ca2+-concentration, to a potentiation or a diminution of the AMPA response, or no effect on pre-synaptic glutamate release, because of modulation of the AMPA receptor trafficking (Bienenstock et al. 1982). The best illustration of the ‘Bienenstock Cooper Munro’ or ‘BCM’ law is Spike-timing, dependent plasticity. This plasticity is characterized by the back-propagation of action potentials (APs) that trigger either LTD or LTP when associated with immediately -preceding or –following pre-synaptic glutamate release (reviewed by Sjo¨stro¨m and Nelson 2002). Some of the morphological deficits observed in the dendrites of MR neurons may interfere with rules governing synaptic integration in neuron. In some MR mouse models, diminished dendritic spine densities have been observed in conjunction with alterations of dendritic spine morphology (Table 1). Together these parameters may alter the ability of the post-synaptic cell to detect synchronous activities governing hebbian plasticity. Indeed, in a number of brain areas, including the popular hippocampal CA1 area, synaptic cooperativity is required to unblock NMDA receptors (Sjo¨stro¨m et al. 2001). A decrease in the density of dendritic spines, associated with a decrease in the diameter of the spine neck, would tend to increase the longitudinal electrical resistance and diminish diffusion (filopodia-like dendritic spines). This synergistic effect could alter the threshold required for LTP to occur. Taken to an extreme, this simplification of the dendritic arbor would prevent the excitatory charge to induce post-synaptic cell spiking. Finally, most of the MR mice harbor morphological abnormalities of dendritic spine, which probably dramatically restrict both the transfer of molecules and ions between the spine head and the parent dendrite (Sabatini et al. 2002; Alvarez and Sabatini 2007), and possibly limit the presence of some other spine specific helpers, such as voltage-gated Ca2+-channels (Yasuda et al. 2003; Humeau et al. 2005). This also applies to post-synaptic AMPA receptors because their number is directly proportional to the extent of the postsynaptic density and their accumulation is regulated by PSD95 (Be´¨ıque and Andrade 2003). Therefore, space reduction could also participate to synaptic strength limitation. In conclusion, spine morphology has been linked to synaptic plasticity repertoire (Bonhoeffer and Yuste 2002; Humeau



et al. 2005), which is in favor of the idea that key players in LTP induction and/or expression may be disrupted (or deregulated) in MR dendritic spines, possibly leading to local alterations of synaptic plasticity. Are alterations in dendritic spines intrinsic to the postsynaptic neuron or do they results from alterations in the surrounding tissue? Some of the identified MR genes encode modulators of the Rho GTPases, a family of proteins largely responsible for the dynamics of the actin cytoskeleton, in particular controlling dendritic spine morphology (Matus 2000; Tada and Sheng 2006). The recent convergence between synaptic activity and post-synaptic dendritic spines implicating Rac1 adds more fuel to this discussion (Saneyoshi et al. 2008). Relevant publications discussing the role of small GTPases in the control of spine morphogenesis in relation to MR can be found in recent reviews (Newey et al. 2005; Nadif Kasri and Van Aelst 2008). At present it appears very difficult to isolate alterations in the morphology of dendritic spines from their activity-dependent dynamics. The LTP induction protocol has recently been shown to induce a strong and rapid increase in the head diameter of active spines (Matsuzaki et al. 2004). Alternatively, a decrease in spine size following LTD protocol induction has also been reported (Na¨gerl et al. 2004; Zhou et al. 2004). However, dissociation between dendritic spine morphology and synaptic strength in the cerebellum has also been described (Sdrulla and Linden 2007). Importantly, glutamate releaseinduced dendrite increased size is associated with an increase in the AMPA response, suggesting an accumulation of newly formed AMPA receptors (Matsuzaki et al. 2004; Isaac et al. 2007). Therefore, if altered dendritic spine morphology is a constant feature in MR neurons, modifications of glutamatergic synapse neurotransmission would be expected, and this has been reported in some cases (Table 1). Lastly, it is interesting to note that in two mouse models of X-linked mental retardation, namely mutations of mecp2 and fmr1 genes, neuronal phenotypes could be reversed by increasing BDNF levels (Chang et al. 2006; Lauterborn et al. 2007). Recently, dendritic secretion of BDNF was reported to be of crucial importance for the increased size of dendritic spines, which is normally induced in response to pairing protocols triggering hebbian plasticity (Tanaka et al. 2008). Despite 20 years of research, a formal link between LTP and mental retardation is still not well established. This may be the consequence of the great variety of LTP forms within the brain and the relatively recent availability of MR mouse models. However, several recent studies have uncovered deficits in LTP in numerous MR mouse models (Table 1) and this number will undoubtedly increase with ongoing studies using new X-linked MR mouse models. The main point that remains to be addressed is to determine the exact nature of the observed deficit. At a first glance, it may appear surprising that key proteins of the synaptic architecture, such as PSD95, as well as

essential components of the exocytotic machinery, such as the calcium channels and the SNARE proteins have not been identified in MR diseases. However, given the fundamental importance of neurotransmitter release, the disruption of any one of these essential components is likely to be lethal. Hence the MR-related genes are most probably involved in subtle regulation of synaptic organization and function. By understanding at the molecular level the consequences of a mutation or the deletion of the various MR-related genes described here, we will undoubtedly improve our knowledge of the finely tuned machinery that represents a synapse at work.

Acknowledgments This work was supported by CNRS, INSERM, Paris Descartes and Louis Pasteur Universities, and contracts ANR-06-003-Neuro-01 to YH, ANR-Neuro 2005 to JC, ANR-05-Blan-0326-01 to NV, the European Neuroscience Institutes Network (ENINET, Project Contract N° LSHM-CT-2005-019063), the ‘‘Fondation pour la Recherche Me´dicale’’ and the ‘‘Fondation Je´roˆme Lejeune’’. We wish to thank Dr. N. Grant for critical reading of the manuscript.

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