Is mental retardation a defect of synapse structure and function?

Review Article

Is Mental Retardation a Defect of Synapse Structure and Function? Magdalena Chechlacz, PhD, and Joseph G. Gleeson, MD Mental retardation is believed to be a result of alterations in molecular pathways underlying neuronal processes involved in cognitive functions. It is not fully understood, however, which molecular pathways are critical for cognitive mechanisms. Furthermore, whether mental retardation is a developmental or ongoing disorder of cognitive functions is unknown. Answering these questions will help elucidate the etiology of mental retardation and possibly lead to new therapies. Several recently published studies suggested that mental retardation might be caused by defects in synapse structure and function. Four genes mutated in families with mental retardation encode proteins known as Rho guanine nucleotide exchange factor 6, oligophrenin-1, p21-activated kinase, and guanine dissociation inhibitor 1. Each of these interacts with various guanine nucleotide-binding proteins involved in signaling pathways that regulate the actin cytoskeleton, neurite outgrowth, neurotransmitter release, and dendritic spine morphology. The goal is to understand the roles of these genes in normal cognitive functions. © 2003 by Elsevier Inc. All rights reserved. Chechlacz M, Gleeson JG. Is mental retardation a defect of synapse structure and function? Pediatr Neurol 2003; 29:11-17.

Introduction Mental retardation (MR), as classified by the Diagnostic and Statistical Manual of Mental Disorders–Fourth Edition (DSM-IV), affects approximately 2-3% of children and young adults. The phenotype of MR is characterized by reduced cognitive functioning defined by intelligence quotient lower than 70 and severe deficits in basic adaptive and social skills (DSM-IV). MR associated with mild

From the Division of Pediatric Neurology, University of California, San Diego, La Jolla, CA.

© 2003 by Elsevier Inc. All rights reserved. doi:10.1016/S0887-8994(03)00152-8 ● 0887-8994/03/$—see front matter

to severe learning and behavioral deficits may be the only manifestation of the disease (nonsyndromic MR) or may be accompanied by other clinical features (syndromic MR). Therefore, MR is an extremely heterogeneous condition that may result from both genetic and non-genetic factors that overall affect cognitive functioning. The environmental factors that may contribute to MR include alcohol exposure, malnutrition and infectious diseases during pregnancy, premature birth, perinatal anoxia, and trauma. Although some cases probably result from a combination of multigenic and environmental causes, MR can also result from mutations in a single gene. Several forms of MR known as X-linked MR (XLMR) are associated with mutation in a single gene on the X chromosome, and MR is consistently found to be more common in boys (20-30% higher incidence) than in girls [1-3].

XLMR Genes Mutations in 11 X-linked genes have been identified in families with nonsyndromic MR. In addition, three other genes that map to chromosome X have been linked to both syndromic and nonsyndromic MR (Table 1). It is difficult to speculate how many novel genes may eventually be identified [4]. Each of the known mutations is evident in only a small number of MR individuals. Although these mutations are infrequent, they provide substantial evidence for the involvement of the single gene in the pathogenesis of the disorder. Identified genes encode diverse proteins that fall into distinct functional subclasses including transcription factors, transmembrane proteins, proteins linked to microtubule-associated protein-2 kinase- and Ras-mediated signaling, G-protein-linked receptors, and proteins involved in fatty-acid metabolism. A possible role of these proteins in the brain and their link to

Communications should be addressed to: Dr. Gleeson; Department of Neurosciences, University of CaliforniaSan Diego & School of Medicine; 9500 Gilman Drive; La Jolla, CA 92093-0624. Received August 22, 2002; accepted February 17, 2003.

Chechlacz and Gleeson: XLMR and Synaptic Function 11

Table 1.

Genes mutated in X-linked mental retardation

Nonsyndromic MR AGTR2 ARHGEF6 ARX FACL4 FMR2 GDI1 ILR1RAPL OPHN1 PAK3 TM4SF2 VCX-A Syndromic and nonsyndromic MR ATRX (ATR-X syndrome) MECP2 (Rett syndrome) RSK2 (Coffin-Lowry syndrome) Abbreviations: AGTR2 ⫽ ARHGEF6 ⫽ ARX ⫽ ATRX ⫽ FACL4 FMR2 GDI1

Angiotensin II receptor Rho guanine nucleotide exchange factor 6 Aristaless-related homeobox gene ␣-Thalassaemia/mental retardation syndrome ⫽ Fatty acid-CoA ligase 4 ⫽ Fragile-X mental retardation 2 ⫽ Guanine dissociation inhibitor 1

Encoded Protein and Potential Function

References

Angiotensin II receptor Rho GEF involved in actin cytoskeleton dynamics Homeodomain protein Fatty acid-CoA ligase 4, fatty acid metabolism Transcription factor RabGDP-dissociation inhibitor Member of IL-1 receptor family Rho GAP involved in actin cytoskeleton dynamics Downstream effector of Rho GTPases Tetraspanin involved in integrin network Unknown function

50 34 51 52 53-55 47 56 33 31,32 57 58

DNA-binding helicase, chromatin remodeling Methyl CpG-binding protein Serine/threonine protein kinase

59-61 62,63 64,65

ILR1RAPL MECP2 OPHN1 PAK3 RSK2 TM4SF2 VCX-A X

cognitive function are beginning to be investigated. A considerable number of recent studies indicate that MR may result from deficits in the regulation of the actin cytoskeleton, which controls neuronal connectivity and synaptic function. The Neuronal Cytoskeleton Like all other cell types, neurons contain a specialized structural network called the cytoskeleton. Neurons are polarized cells that have a complex morphology with numerous long dendrites and an axon, which branch and form synaptic junctions with other cells. The neuronal cytoskeleton is composed of three types of filamentous structures: actin microfilaments, microtubules, and neurofilaments. Many important functional properties of mature neurons depend on the association of actin microfilaments with neuronal membrane. The actin-based membrane skeleton is essential for the synaptic localization of N-methyld-aspartate and 2-amino-3-(3-hydroxy-5-methyl-isoxazol4-yl) propanoic acid glutamate receptors [5]. The targeting of glutamate receptors to the cell surface is thought to depend on the function of actin-binding proteins that anchor the receptor subunit proteins in the membrane [6]. The actin cytoskeleton of neurons is crucial for morphologic differentiation, including development of specialized dendritic morphology, neurite outgrowth, cell polarity, synapse formation, synaptic plasticity, and protein transport [7,8]. Neuronal morphogenesis is the process by which the undifferentiated neuroblast with a simple cy-

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⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

Interleukin-1 receptor accessory proteinlike Methyl CpG-binding protein 2 Oligophrenin 1 p21-activated kinase Ribosomal S6 kinase 2 Transmembrane 4 superfamily member 2 Variable charged Chromosome mRNA

toskeleton becomes a fully differentiated neuron with characteristic dendritic and axonal shapes. A complex cytoskeletal network supports these structures throughout life. In addition, the cytoskeleton of fully differentiated neurons must remain modifiable to allow dendritic spines to undergo morphologic changes in response to excitatory synaptic inputs [9]. Rho GTPase Signaling in Axonal Outgrowth and Dendritic Plasticity Rho GTPases are the key components of signaling pathways controlling the organization of the actin cytoskeleton, and in neuronal cells are known to regulate growth cone morphology as well as growth cone guidance [10-13]. During nervous system development, specific connections are formed by dendritic and axonal arbors. The formation of functional neuronal circuits is mediated by a specialized, motile structure that is at the distal tip of growing axon or dendrite and is called the growth cone [7,14]. Neurite outgrowth is driven by cytoskeletal elongation, steering, and branching of the growth cone, and extracellular cues are believed to underlie the cytoskeletal changes that lead to growth cone motility. The most distal part of the growth cone consists of two constantly extending and retracting elements: most distal, thin, and spikelike processes called filopodia; and flattened, broad extensions that are located in the more central region and are known as lamellipodia (Fig 1). Both filopodia and lamellipodia mediate attachment of the growth cone to the substrate and

Figure 1. Actin cytoskeleton and neuronal morphogenesis. (1-3) Plastic changes in neuronal cytoskeleton are required for differentiation of neuroblasts into mature neurons and development of specialized neuronal circuits. The formation of neuronal networks during neuronal differentiation is mediated by the distal tip of the growing axon, the growth cone supported by the actin filament network. The central region of the growth cone forms flattened, broad extensions called lamellipodia as well as thin, spikelike processes called filopodia. Lamellipodia and filopodia are rich in actin and involved in detecting external environmental cues used for the navigation of growing axon, dendritic branching, and, finally, synapse formation. F, filopodia; L, lamellipodia. (4) Growth cone guidance, axonal outgrowth, and dendritic branching depend on the signaling pathways activated by extracellular cues that mediate rapid remodeling of actin cytoskeleton. Dynamic changes of actin filaments (polymerization/depolymerization) in response to environmental stimuli are regulated by members of a family of small GTP-binding proteins (Rho GTPases), primarily RhoA, Rac1, and Cdc42.

are used to detect environmental cues [15]. When a positive guidance cue is detected, filopodia induce extension of the growth cone toward the signal, and lamellipodia provide the material to expand growth [7,16]. Growth cone motility depends on cytoskeletal reorganization [7,15,17]. Dynamic changes of actin filaments and microtubules (polymerization/depolymerization) occur in the growth cone before any visible changes in the growth cone shape and motility are observed [7]. The guiding cues most likely work through Rho GTPases, a family of small guanosine 5⬘-triphosphate (GTP)-binding proteins that induce changes in the actin cytoskeleton [7,18-20]. Studies examining expression of different members of Rho GTPase family in cultured cortical neurons demonstrated that expression of Rac1 is up-regulated during early stage of neurite outgrowth, and expression of RhoA is upregulated during later stages, possibly during synaptogenesis [21]. Furthermore, both axonal and dendritic branching depend on Rho GTPase expression [11,22-25]. During neuronal morphogenesis, the number of dendrites and dendritic branching depend on the expression of RhoA, Rac1 and Cdc42 [26]. During brain development, the initial dendritic arborization and outgrowth of dendritic spines precede synapse formation. The structural plasticity of dendritic spines remains throughout life and is essential for the function of

mature synapses as well as establishing new ones. The plastic changes in the neuronal cytoskeleton are triggered in mature neurons in response to excitatory neurotransmission [27]. These activity-dependent rearrangements of the actin network mediate structural changes of dendritic spines and are mediated by Rho GTPases. Dendritic spines are tiny elements localized at the postsynaptic sites of excitatory synapses. As sites of axonal-dendritic contacts, they are potential mediators of the connective plasticity underlying learning, memory, and cognition [28]. The present understanding is that synaptic remodeling and dendritic plasticity (changes in dendritic spine morphology, shape, and number) are the anatomic basis for learning and memory formation. Excitatory transmission controls molecular events that modulate spine structure, and dendritic plasticity depends on morphologic changes that follow rearrangements of the cytoskeleton in response to neural activity at the synaptic sites [27,29,30]. XLMR Genes and Rho GTPases Signaling Recent discovery of XLMR genes that encode PAK3 (p21-activated kinase), OPHN1 (oligophrenin 1), and ARHGEF6 (Rho guanine nucleotide exchange factor 6), all linked to Rho GTPase signaling suggests that formation of neuronal processes and synaptic plasticity are critical for cognitive functions [31-34]. All three genes are functionally related to the Ras superfamily of GTP-binding proteins, also known as small GTPases. PAK3, OPHN1, and ARHGEF6 interact with Rho GTPases, such as RhoA, Rac1, and Cdc42, that primarily regulate the actin cytoskeleton. The function of all the small GTPases is regulated by their GTP/GDP-bound state: active when they bind GTP and inactive when GTP is hydrolyzed to guanosine 5⬘-diphosphate (GDP) (Fig 2). The ratio of the two forms determines their activity and is regulated in turn by two groups of proteins: GTPase-activating proteins (GAPs) that increase hydrolysis of bound GTP, and guanine exchange factors (GEFs), which enhance the exchange of bound GDP to GTP [13]. The domain structure of OPHN1 indicates its role as a RhoGAP and ARHGEF6 as a RhoGEF. The third protein PAK3 is a downstream component of the Rho GTPase signaling pathways, which provides a link to downstream effector pathways. The discoveries that proteins involved in Rho GTPase signaling are associated with XLMR lead to hypotheses that MR may result from altered development or plasticity of neuronal networks. The assumption that Rho GTPase signaling may play an essential role in cognitive functions is based on their role in modulating neuronal cytoskeleton dynamics. The neuronal processes potentially controlled by Rho GTPases and their regulatory proteins, including PAK3, ARHGEF6, and OPHN1, are believed to be critical for both neuronal morphogenesis and neuronal plasticity in the mature nervous system. Because the Rho GTPases likely function to regulate neuronal development and

Chechlacz and Gleeson: XLMR and Synaptic Function 13

Figure 2. Three of the recently cloned X-linked mental retardation genes, ARHGEF6, OPHN1, and PAK3, encode proteins involved in the regulation of small GTP-binding protein (Rho GTPase) activity and downstream signaling pathways. Members of the family of small GTPbinding proteins play an important role in the control of axonal and dendritic development by regulating actin cytoskeleton dynamics in response to environmental cues. Function of the small GTP-binding proteins depends on the GTP versus GDP-bound state: active when they bind GTP and inactive when they bind GDP. Various extracellular cues are transmitted to Rho GTPase-signaling pathways through two classes of regulatory factors: guanine nucleotide exchange factors (Rho GEFs) and GTPase-activating proteins (Rho GAPs). Guanine nucleotide exchange factors including ARHGEF6 promote the exchange of bound GDP for GTP, thereby activating Rho GTPases, which in the GTP-bound form can stimulate various downstream effectors such as PAK3, regulating actin cytoskeleton. By contrast, GTPase-activating proteins including OPHN1 inactivate Rho GTPases through facilitating hydrolysis of bound GTP to GDP.

synaptic function, the newly discovered XLMR genes provide a good model to study the cellular and molecular mechanisms of MR. Dendritic Spines, Synaptic Function, and MR Because three recently discovered XLMR-linked genes, OPHN1, ARHGEF6, and PAK3, encode proteins involved Table 2.

Genetic disorders associated with MR and dendritic spine pathology

Disorder Down’s syndrome

Fetal alcohol syndrome Fragile-X syndrome Patau syndrome Phenylketonuria Rett syndrome

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in Rho GTPase signaling, MR may arise from altered neuronal network organization and function. How mutations in OPHN1, ARHGEF6, and PAK3 could result in defects of neuronal network is unknown. Although the link between these three genes and Rho GTPase signaling is documented, the exact function of proteins encoded by these genes is unknown. Given that the neuronal cytoskeleton is regulated by Rho GTPases and loss-of-function mutations in OPHN1, ARHGEF6, and PAK3 would result in either increased or decreased Rho GTPase signaling, deficits in proteins encoded by these genes should affect actin cytoskeleton dynamics. But how could this action result in MR? Besides the evidence that the actin cytoskeleton plays important roles in neuronal outgrowth and therefore may contribute to neuronal network formation, Rho GTPases also modulate the formation of dendritic spines. Although there is no information about abnormalities in neuronal connectivity and synaptic function in XLMR, dendritic spine pathology associated with MR is well documented. Several genetic disorders associated with MR are characterized by abnormalities in dendritic branching as well as in shape and density of dendritic spines (Table 2). Most observations came from studies on postmortem material, and similar patterns of dendritic alterations were demonstrated in studies of Down’s syndrome, fragile-X syndrome, fetal alcohol syndrome, and nonsyndromic MR [35-41]. The pioneering work used Golgi stain in material from Down’s syndrome and Patau syndrome patients and demonstrated different types of dendritic abnormalities: reduction in overall density and giant spines, reduction of spine size, or increase in spine density associated with abnormal shape [37,42]. Later studies revealed that increase in spine density and distortion of spine shape, mostly long and tortuous spines exhibiting immature spine shapes, are the most common pathology in different conditions associated with MR. Such abnormalities were found in fragile-X syndrome patients and fragile-X knockout mice [41,43-45]. The similarity of dendritic spine alterations in different disorders associated with MR suggests that genetic defects in MR may result in disruption of common signaling pathways most likely controlling the actin cytoskeleton underlying dendritic morphology and plasticity. Dendritic

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Spine Morphology

References

Giant spines, increased spine density and abnormal spine shape, decreased spine density Abnormally thin and long spines Increase in spine density, abnormally thin and long spines Abnormal shape of dendritic spines Increased spine density Decreased spine density

37,40,42,66

39 41,43,44 42 67 68

Figure 3. The hypothesized effects of loss-of-function mutation in ARHGEF6, OPHN1, PAK3, and GDI1 during brain development and adulthood. Recent studies indicate that mental retardation may result from various defects in molecular pathways underlying neuronal morphologic development or normal cognitive function in postnatal life. Although the central question whether mental retardation is a developmental disorder or ongoing defect in cognitive function remains unanswered, all findings indicate some sort of synaptic and neuronal connectivity deficits as the major underlying cause of mental retardation. Synapses are the major sites of neuronal signaling and both defects at the presynaptic and postsynaptic site may result in impaired synaptic structure and function. Mutations in ARHGEF6, OPHN1, and PAK3 during brain development may cause defective synaptic structure because of impaired growth cone guidance, axonal outgrowth, and dendritic branching. These mutations may also lead to impaired formation of new synapses and deficits in dendritic spine plasticity in postnatal life. GDI1 is believed to play an important role in neuronal vesicle transport, and mutations in this gene are likely to result in defects in neurotransmitter release and synaptic transmission and therefore continuously altered synapse function. Abbreviations: ARHGEF6, Rho guanine nucleotide exchange factor 6; GDI1, guanine dissociation inhibitor 1; OPHN1, oligophrenin 1; PAK3, p21-activated kinase; NT, neurotransmitter.

spines are the major postsynaptic sites of excitatory synapses, and any abnormalities in actin cytoskeleton dynamics may affect the structure and function of developing and mature synapses (Fig 3). MR and Deficits in Synaptic Vesicle Transport The transfer of information in the nervous system, which is essential for cognitive functions, depends on intercellular communication through synapses. Neurotransmitter release at the synaptic terminals in neurons is regulated by Rab3, a neuronal member of Rab family of GTP-binding proteins (Rab GTPases). This protein is implicated in synaptic vesicle fusion with the synaptic terminal membrane (i.e., release of neurotransmitter). Rab3 activity is regulated by GTP/GDP binding (active GTP-bound and inactive GDP-bound state) and requires recycling of GDP-bound Rab3 by GDI␣ encoded by the GDI1 (guanine dissociation inhibitor 1) gene (Fig 4). Therefore it is likely that GDI␣ deficits will result in alterations of synaptic transmission and subsequent im-

Figure 4. Recent studies demonstrated that mutations in guanine dissociation inhibitor 1 (GDI1) gene are associated with mental retardation. GDI1 gene encodes GDI␣ protein involved in Rab3 regulatory cycle at the presynaptic site. Rab3 is the member of the Rab family of GTP-binding proteins that regulates neurotransmitter release from the presynaptic site of neuronal synapses. Active GTP-bound Rab3 mediates neurotransmitter release from the synaptic vesicle (v) by mediating membrane fusion. Once neurotransmitter is released, GTP is hydrolyzed to GDP, and GDI␣ retrieves GDP-bound Rab3 from the membrane and deposits it into cytosol where the exchange of GDP to GTP on Rab3 occurs and the new cycle begins. Therefore, mutations in GDI1 may result in impairment of neurotransmitter release and subsequent defects in synaptic transmission.

pairment of synaptic function underlying learning and memory. Mutations in the GDI1 gene in patients with XLMR also suggest the importance of GDI␣ in proper synaptic function and that neurotransmitter vesicle transport may play an essential role in human cognition (Fig 3) [46,47]. Repetitive stimulation of CA1 pyramidal hippocampal neurons in Rab3 mutant mice leads to a decrease in facilitation of excitatory neurotransmission [48]. Although deficits in Rab3 and GDI␣ were expected to reduce neurotransmitter release and subsequent synaptic depression, Ishizaki surprisingly reported an enhancement of excitatory neurotransmission during repetitive stimulation in CA1 region of the hippocampus in GDI␣ knockout mice [49]. Even though the electrophysiologic phenotype of GDI␣ knockout mice is opposite to the phenotype of Rab3-deficient mice, it resembles to some extent human GDI1 mutations. In known human cases mutations in GDI1 gene cause XLMR associated with epileptic seizures. Similarly, GDI␣ knockout mice exhibited hypersensitivity to epileptic seizure. Therefore, Ishizaki and colleagues suggested that GDI␣ might be involved in suppressing synaptic hyperexcitability. In summary, the link between GDI1 and XLMR indicates that the proper regulation of neurotransmitter release that seems to be critical for cognitive functions may be affected in mentally retarded patients. Interestingly, GDI1 deficits in cultured hippocampal neurons significantly reduce axonal outgrowth. Therefore, it seems that defects in neuronal network formation are

Chechlacz and Gleeson: XLMR and Synaptic Function 15

also a possible cause of MR in patients carrying GDI1 mutations [47].

Conclusions Whether abnormalities in neuronal networks in mentally retarded patients result from deficits in axonal outgrowth, dendritic spine formation during brain development, or deficits in synaptic function in postnatal life is unknown. The morphology of dendritic spines and presumably synaptic structure and function depends on Rho GTPase signaling, and therefore recently discovered Rho GTPase-related genes OPHN1, ARHGEF6, and PAK3 mutated in XLMR patients are of particular interest. Understanding the cellular and biochemical steps in the development of cognitive deficits in mentally retarded patients might allow pharmacologic intervention with drugs that improve cognitive function, as has been suggested in newborns predicted to develop MR on the basis of neonatal genetic screening [69]. However, there are several limitations in the understanding of the cellular and molecular mechanisms underlying this poorly characterized brain disorder. Therefore, recent discoveries indicating some of the possible mechanisms underlying cognitive deficits suggest that it may be indeed possible to use cognitive-enhancing drugs as a treatment in mentally retarded children. Such drugs could promote proper axonal outgrowth, normal dendritic spine morphology, normal spine number, and functional synaptic connections. Further studies examining the role of OPHN1, ARHGEF6, PAK3, GDI1, and other XLMR-linked genes in neurons should clarify whether XLMR is indeed a developmental disorder or rather an ongoing disorder of neuronal network organization and function. Information that can be obtained from human patients is limited to the results of cognitive testing and postmortem brain analysis. However, detailed neuroanatomic and neuroelectrophysiologic studies in mouse transgenic models should provide knowledge necessary to understand the pathogenesis of MR. Studies with genetically generated mouse models should provide an understanding of the complex mechanisms underlying formation of neuronal networks and indicate which aspects of neuronal morphology, synaptic structure, and function are essential for human cognition. Furthermore, if the genes mutated in families with MR are required throughout life for ongoing neuronal functions, then gene replacement should be taken into consideration as a treatment that might correct some of the cognitive defects in affected individuals. The actin skeleton and related signaling networks represent only a small potion of mechanisms that control the properties and function of synapses, and therefore the known number of genes that when mutated contribute to cognitive deficits underlying MR may increase significantly in the near future.

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