Dendritic Spine Structural Anomalies in Scott A. Irwin1,2, Roberto Galvez1 and William T. Greenough1,3,4,5 Fragile-X Mental Retardation Syndrome 1Neuroscience and 2Medical Scholars Programs, Departments of 3Psychology, 4Psychiatry, and 5Cell and Structural Biology, and the Beckman Institute, University of Illinois, Urbana, IL 61801, USA

Fragile-X syndrome is the most common single-gene inherited form associated neural activity (Goodman and Shatz, 1993; Hata and of mental retardation. Morphological studies suggest a possible Stryker, 1994). The strengthening of synapses appears to involve failure of the synapse maturation process. Cerebral cortical spine a transition from small immature synapses associated with morphology in fragile-X syndrome and in a knockout mouse model of long, thin, dendritic spines early in development to larger syn- it appears immature, with long, thin spines much more common than apses associated with more ‘mushroom-shaped’ dendritic spines the stubby and mushroom-shaped spines more characteristic of (Greenough and Chang, 1985, 1988a; Hwang and Greenough, normal development. In human fragile-X syndrome there is also a 1986; Schuz, 1986; Galofre et al., 1987; Horner, 1993; Greenough higher density of spines along dendrites, suggesting a possible failure et al., 1994). Synaptic overproduction, pruning and strengthen- of synapse elimination. While variously misshapen spines are ing may also underlie postdevelopmental experience/learning characteristic of a number of mental retardation syndromes, the associated synapse addition. overabundance of spines seen in fragile-X syndrome is unusual. The synaptic selection/maturation process may be grossly Taken with evidence of neurotransmitter activation of the synthesis impaired in cases of clinical fragile-X syndrome. A qualitative of the fragile-X protein (FMRP) at synapses in vitro and evidence for study of rapid-Golgi-stained human autopsy material from a behaviorally induced FMRP expression in vivo, and with evidence compatible with a role for FMRP in regulating the synthesis of other single fragile-X patient described long, thin, tortuous, dendritic proteins, it is possible that FMRP serves as an ‘immediate early spines with prominent heads and irregular dilations on apical protein’ at the synapse that orchestrates aspects of synaptic dendrites of pyramidal cells in layers III and V of the parieto- development and plasticity. occipital neocortex (Rudelli et al., 1985; Wisniewski et al., 1991). Reduced mean synaptic contact area was also reported using electron microscopy; however, no other major neuro- Introduction pathologies were noted. Two additional fragile-X patients were In investigating molecular mechanisms of synaptic plasticity, added to this study by Hinton et al. (Hinton et al., 1991). Similar it was discovered that the fragile-X mental retardation protein dendritic spine characteristics were noted, and no differences (FMRP), the protein absent in those aff licted with fragile-X in neuronal density between fragile-X patients and controls syndrome, is synthesized in synaptoneurosome preparations in were found, possibly suggesting normal neurogenesis and cell response to neurotransmitter stimulation (Weiler et al., 1997). migration in fragile-X patients; however, a stereological method This was a revolutionary discovery which suggested that this that would have overcounted larger cells was used. protein is made in vivo at synapses in response to afferent Despite a lack of evidence for changes in neuronal number activation, possibly for involvement in synaptic plasticity or other gross structural deficits, fragile-X patients have been processes (Weiler and Greenough, 1999). This chapter briefly shown to display subtle abnormalities in gross brain anatomy. describes fragile-X syndrome, summarizes developmental Two groups (Reiss et al., 1991; Mostofsky et al., 1998), using neuronal plasticity processes, and details dendritic structure magnetic resonance imaging (MRI), showed that fragile-X males anomalies found associated with fragile-X syndrome. It then exhibited a reduction in the size of the posterior cerebellar compares these structural deficits with those of other mental re- vermis and an enlargement of the fourth ventricle. An increased tardation syndromes, suggests a possible therapeutic approach, volume in fragile-X patients as compared with controls has also and reviews possible roles of FMRP in the nervous system and been reported in the hippocampus (Reiss et al., 1994; Kates et other tissues. al., 1997), caudate nucleus (Reiss et al., 1995), and lateral Fragile-X syndrome is a common form of mental retardation ventricles (Reiss et al., 1995). Furthermore, an age-related affecting nearly one in 2000 males and roughly half as many increase in the volume of the hippocampus and an age-related females (Brown, 1996). It is caused by the insertion of extra DNA decrease in the volume of the superior temporal gyrus were into the X chromosome that silences the gene encoding FMRP. found in fragile-X patients (Reiss et al., 1994). It should be noted Phenotypic traits include facial abnormalities, macro-orchidism, that the changes in fragile-X patients found using MRI were developmental delay, mental retardation and autistic-like behav- subtle and not replicated by physical measurement of autopsy iors (Hagerman and Cronister, 1996). material, but this data was derived from only two different fragile-X patients (Reyniers et al., 1999).

FMRP: Role in Synapse Maturation and Pruning? During normal brain development, forebrain sensory systems Neuronal Morphology in the Fragile-X Brain generate more neuronal processes and connections than will In order to examine the effects of a deficiency of FMRP on ultimately survive. This is followed by an activity-mediated dendritic structure, a series of quantitative anatomical studies competition process in which some synapses are eliminated was undertaken by the Dutch–Belgian Fragile-X Consortium of while others are stabilized and strengthened (LeVay et al., both human autopsy material from fragile-X patients and of 1980), so that patterned neural circuitry arises from experience- transgenic mice in which the FMR-1 gene was knocked out

© Oxford University Press 2000. All rights reserved. Cerebral Cortex Oct 2000;10:1038–1044; 1047–3211/00/$4.00 Figure 2. Schematic of dendritic spine morphologies defined as immature or mature.

exhibited significantly more spines with immature shapes than did controls on apical dendritic shafts, apical oblique and basilar dendrites (Figures 1 and 2). These two findings suggest an impairment of normal spine maturation in fragile-X syn- drome that is persistent throughout the entire life span of these patients. Fragile-X patients also exhibited a significantly greater spine density on distal segments of apical dendritic shafts and on apical oblique and basilar dendrites (Figure 1), suggesting a persistent failure of normal synapse pruning processes as well. No differences in dendritic diameter were found for any dendritic branch type. Increased spine density and increased numbers of long and immature spines are the most salient neuropathologies de- scribed in association with fragile-X syndrome thus far and are likely to play a role in the cognitive deficits associated with it. This is the first finding of increased dendritic spine density in fragile-X patients. Furthermore, these findings of higher numbers of longer and more immature type spines in fragile-X patients agree with the qualitative neuronal morphological findings of other groups (Rudelli et al., 1985; Wisniewski et al., 1991; Hinton et al., 1991), and extend them to the entire dendritic tree of cortical layer V pyramidal neurons. These findings suggest that synaptic pruning and maturation may be grossly impaired in cases of fragile-X syndrome, and further imply a role in these processes for FMRP. Further support for a role of FMRP in synaptic plasticity comes from in vitro studies suggesting that this protein is made at synapses in response to synaptic activation (Weiler et al., 1997) (Figure 3) and in vivo studies demonstrating increased FMRP immuno- reactivity in brain regions known to be undergoing: (i) active synaptogenesis in response to motor-skill learning or complex environment rearing or (ii) increased activation in response to repetitive motor activity (Irwin et al., 1998, 2000) (S.A. Irwin et al., in preparation) (Figure 4). Figure 1. Examples of typical spine morphologies on Golgi-impregnated dendrites from A mouse model of fragile-X syndrome (FraX mice) was devel- (A) a human afflicted by fragile-X syndrome and (B) an unaffected control. Dendrites are oped by the Dutch–Belgian Fragile-X Consortium (Consortium, at extremes of density differences and not intended to depict the norm. 1994). These mice initially exhibited macro-orchidism, mild learning deficits on a reversal condition in the Morris water-maze (Consortium, 1994). It should be noted that this knockout model task and hyperactivity. More extensive studies of the FraX is less susceptible to a common criticism of knockout models, as mice were carried out by Kooy et al. (Kooy et al., 1996), which both fragile-X patients and these transgenic mice undergo again demonstrated macro-orchidism [caused by an increase in development without a functional FMR-1 gene. the rate of Sertoli cell proliferation from postnatal day 12 to 15 A quantitative neuroanatomical study of Golgi–Kopsch- (Slegtenhorst-Eegdeman et al., 1998)] and subtle learning deficits stained human autopsy material from three male adult fragile-X in the Morris water-maze; however, there was no indication of patients and three male age-matched controls was carried out in hyperactivity upon retesting. No impairment of short- or long- order to quantify and further describe dendritic spine character- term potentiation was found in hippocampal slices from these istics in fragile-X patients by comparing dendritic spine length, mice (Godfraind et al., 1996). Furthermore, despite a possible morphology and density along apical shaft, apical oblique and role for FMRP in the transport of mRNA to synapses (see below), basilar dendrites on layer V pyramidal cells in the temporal no disruptions in the dendritic localization of the mRNAs for cortex of these subjects (Irwin et al., 1999) (S.A. Irwin et al., MAP2, CaMKII or dendrin, or in the rapid dendritic transport of submitted for publication). The results showed that fragile-X the mRNA for activity-regulated cytoskeletal protein (ARC) were patients had significantly more long spines and significantly found in FraX mice (Steward et al., 1998). fewer short spines than controls on apical shaft, apical oblique The results of Comery et al. (Comery et al., 1997) in these and basilar dendrites (Figure 1). Likewise, fragile-X patients mice support the involvement of FMRP in normal spine

Cerebral Cortex Oct 2000, V 10 N 10 1039 Figure 3. Western blot of protein from synaptoneurosomes illustrating increased FMRP immunoreactivity in synaptoneurosomes after neurotransmitter stimulation. GFAP was used as a control (Weiler et al., 1997). Reprinted with permission from Proceedings of the National Academy of Sciences, USA. development, as they demonstrated an increase in spine density and spine length on layer V pyramidal cells in the visual cortex of FraX mice. This has been the only brain abnormality so far reported in these mice (Consortium, 1994; Godfraind et al., 1996; Kooy et al., 1996); however, it was later determined that an unknown number of the mice used in that study may have been homozygous for a recessive retinal degeneration gene that would have been likely to affect cell morphology in the visual Figure 4. FMRP immunohistochemistry illustrating increased FMRP immunoreactivity cortex of these animals. A quantitative neuroanatomical study of in (A) an animal receiving motor-skill training for 7 days compared with (B) a control Golgi–Cox-stained material from ten young-adult male Frax mice animal and in (C) an animal exposed to a complex environment for 20 days compared and ten young-adult wild-type control mice was carried out in with (D) another control animal. Animals in A and C are known to exhibit synapto- genesis in motor and visual cortex, respectively (Irwin et al., 2000). Reprinted with order to characterize the structure of dendrites and dendritic permission from Academic Press. spines of layer V pyramidal cells in the visual cortex of non-blind FraX mice (Irwin et al., 1999) (S.A. Irwin et al., in preparation). The purpose of these investigations was: (i) to confirm the fragile-X syndrome; however, the differences may be more results of Comery et al. (Comery et al., 1997); (ii) to determine subtle. These findings also partially replicate those of Comery et if abnormalities similar to those of fragile-X patients were pres- al. (Comery et al., 1997), but the absence of a significant spine ent; (iii) to examine dendritic-tree characteristics [as dendrites of density difference suggests that the mice in that study may have some cell populations also undergo an overproduction and been affected by the retinal degeneration mutation. The fact that pruning developmental process (Falls and Gobel, 1979; Brunjes dendritic spine abnormalities are found in FraX mice suggests et al., 1982; Murphy and Magness, 1984; Greenough and Chang, that they are a viable model for the study of the human condition; 1988b)]; and (iv) to further evaluate these mice as a model of the however, these mice, unlike the fragile-X patients and the human condition. Comery et al. mice, did not exhibit statistically significant ab- FraX mice were found to have longer, more immature den- normalities in spine density. It should be noted that both apical dritic spines than wild-type control mice. These mice exhibited shafts and apical oblique dendrites of these FraX mice exhibited more longer and fewer shorter spines than controls on segments a tendency toward increased spine density. The abnormal spines of apical dendritic shafts, apical oblique dendrites and basilar found in these mice might be associated with their behavioral dendrites. Similarly, FraX mice exhibited more immature spine deficits, and the fact that the spine abnormalities were more shapes and fewer mature shapes on segments of apical dendritic subtle in mice than in humans might explain why this subtlety shafts and apical oblique dendrites. No differences in the fre- appears to be so, thus far, for the behavioral deficits found in quencies of spine shapes along basilar dendrites were found. these mice as well. The fact that FraX mice do not show any FraX and wild-type mice did not exhibit statistically significant dendritic arborization abnormalities suggests that the role of spine density or dendritic diameter differences along any of the FMRP in neurons is limited to the spine/synaptic structure and branch types; however, a trend toward a higher spine density in does not play a crucial regulatory role affecting overall neuronal FraX mice along both apical shaft and apical oblique dendrites morphology of pyramidal neurons in the cerebral cortex. was evident. For characteristics of apical and basilar dendritic A lack of FMRP leads to abnormal dendritic spine structure trees, no significant differences in the number of branches at and number on layer V pyramidal cells in the temporal cortex of each order, the chance of bifurcation at each order or the humans and abnormal dendritic spine structure on layer V pyra- amount of dendritic material at constant intervals away from the midal cells in the visual cortex of FraX mice, and the neuronal soma were found between FraX and wild-type mice for layer V structural abnormalities in fragile-X appear to be limited to pyramidal visual cortical cells. dendritic spines. This presents strong evidence for the involve- These findings indicate that FraX mice exhibited similar ment of FMRP in synaptic structural transformation processes dendritic spine abnormalities to those of humans afflicted with across brain regions and points to a cause of the behavioral

1040 Dendritic Spine Structural Anomalies in Fragile-X • Irwin et al. deficits observed in both humans and mice lacking FMRP. What on the nervous system, and both may be involved in a vicious role FMRP plays in this process remains unknown. A lack of spiral leading to aberrant neuronal structure. a ‘housekeeping’ protein that would lead to altered metabolism It is perhaps a little curious that FraX mice show more subtle could certainly affect neuronal morphology, as could the lack and slightly different abnormalities relative to the fragile-X of a cytoskeletal protein involved in synaptic shape. Where patients. It must be kept in mind that mice can never be a perfect FMRP fits into this spectrum of proteins affecting neuronal human model, and that the mice used in this study were young morphology also remains a mystery. Comparisons with other adults as opposed to the middle- to old-aged subjects used in forms of mental retardation demonstrate that FMRP is not alone the human study. However, a closer examination of both the in having morphological effects, as several syndromes have been human and mouse data side by side might suggest another reported to be associated with neuronal structural abnormal- explanation for these phenomena. The data from the control ities. However, the deficits associated with these syndromes mice appear more similar than those from the knockout mice to have not been limited to dendritic spines, nor have any of these fragile-X syndrome humans. Perhaps housing mice in standard syndromes been found to exhibit an increase in dendritic spine laboratory cages makes them more like a ‘mental retardation’ number. model, whereas animals raised in toy and object filled comp- lex environments, which exhibit increased brain connectivity (Turner and Greenough, 1985) and learning capacity (Hebb, Neuronal Morphological Abnormalities in Other Forms of Mental 1947; Greenough et al., 1970; Black et al., 1998) when Retardation compared with animals raised in standard laboratory cages, are Several other disorders are known to exhibit aberrations in more like ‘normal’ mice. It may be the case that complex both dendritic arbor and dendritic spine structure. In the most environment rearing may produce a more profound difference common form of genetically related mental retardation, Down between control and FraX mice. Evidence for this comes from syndrome, increases in dendritic arbor and dendritic length on longitudinal studies that described declines in IQ and in adaptive visual cortical pyramidal cells were found in infants (Becker behavior of fragile-X patients. These scores were reported not et al., 1986; Becker, 1991), but decreases in both of these to reflect a decline in intellectual or social skills, but rather a parameters have been found in juveniles and up through widening gap with age between these fragile-X and normally adulthood (Becker et al., 1986; Becker, 1991; Takashima et al., developing individuals (Fisch et al., 1996). Although experience 1994). In addition, neurons of Down syndrome patients were may widen the gap between normally developing and fragile-X found to exhibit decreased spine density in a number of brain affected humans or animals, it may also serve to ameliorate the regions (Marin-Padilla et al., 1972; Marin-Padilla, 1976; Suetsugu deficits. Success along these lines has been found in a rat model and Mehraein, 1980; Takashima et al., 1981; Ferrer and Gullotta, of fetal alcohol syndrome (Klintsova et al., 1997, 1998). Simi- 1990). Decreases in spine number and/or dendritic length have larly, others have reported amelioration of genetically related similarly been found in cases of general mental retardation behavioral defi-ciencies by exposing mentally retarded children (Huttenlocher et al., 1974; Purpura, 1974), Rett syndrome to an ‘enriched’ toy and object filled environment (Horner, 1980) (Armstrong, 1992; Belichenko et al., 1994; Armstrong et al., or rearing animals in a more ‘normal’ environment (Caston et al., 1995) and autism (Williams et al., 1980). Various forms of 1999; Rampon et al., 2000). Studies are underway in our metabolic storage diseases due to inborn (genetic) errors of laboratory to investigate how experience affects neuronal metabolism have also been found to exhibit decreases in den- structure and learning performance in FraX mice, and these dritic material and/or decreases in spine density; syndromes with studies may give us a better model and understanding of the such findings include Type C Niemann-Pick disease (Braak et al., human condition and how experience may affect it. 1983), neuronal ceroid lipofusicosis (Purpura and Suzuki, 1976), It should be mentioned that FMRP is expressed in a number of infantile Tay-Sachs B variant (Purpura and Suzuki, 1976; tissues, especially early in development, and may play a similar Takashima et al., 1985), Hurler’s syndrome (Takashima et al., role in structural transformations occurring in tissues other than 1985), infantile type 2 sialidosis (Takashima et al., 1985) and the brain. Evidence for this comes from studies demonstrating Sanfilippo type A syndrome (Takashima et al., 1985). Thus, high levels of FMRP expression in a number of tissues during while dendritic spine numbers were found to be lower and/or embryonic development (Abitbol et al., 1993; Hinds et al., dendritic tree characteristics were found to be affected in many 1993), a period of massive cellular proliferation. A role for FMRP mental retardation disorders, fragile-X syndrome appears to be in cellular remodeling is further supported by its increased levels the only form of mental retardation that exhibited dendritic after quiescent mouse kidney cells were stimulated to resume spine abnormalities and increased numbers of dendritic spines; proliferation (Khandjian et al., 1995). In addition, Devys et al. unlike many other syndromes, there are no alterations in the (Devys et al., 1993) found that dividing layers of epithelial tissues dendritic arbor. This contrast with other syndromes further as well as cells during wound healing and pathogenesis that supports the hypothesis that FMRP is somehow necessary for involved active cellular remodeling, such as in heart myocytes developmental maturation and pruning of dendritic spines. of patients with ischemic cardiopathy, exhibited strong FMRP expression. In non-neuronal tissue FMRP may be involved in Other Evidence Regarding FMRP Function cellular structural transformations accompanying cellular prolif- While it may be that the dendritic spine abnormalities seen in eration, which may have mechanisms in common with neuronal fragile-X syndrome are due directly to the absence of FMRP, structural transformations, such as synaptogenesis and/or spine particularly at synapses, it must be kept in mind that altered maturation and pruning. afferent input can also lead to abnormal neuronal structure. For example, it is possible that the lack of this protein leads to abnormal afferent input by way of corrupting a neurotransmitter Molecular Roles of FMRP system, which in turn would lead to these structural abnorm- Clearly FMRP plays a role in the central nervous system and is alities. It may prove very difficult to disentangle the effects of likely to play an important one that affects synaptic plasticity. this (or any) genetic defect from those of altered afferent input Since FMRP was found to bind mRNA and associate with ribo-

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1044 Dendritic Spine Structural Anomalies in Fragile-X • Irwin et al.