Molecular Mechanisms of Fragile X Syndrome: A Twenty-Year ......named the Sherman paradox, remained...

PM07CH09-Warren ARI 12 December 2011 9:1

Molecular Mechanismsof Fragile X Syndrome:A Twenty-Year PerspectiveMichael R. Santoro,1 Steven M. Bray,1

and Stephen T. Warren1,2

1Department of Human Genetics and 2Departments of Biochemistry and Pediatrics, EmoryUniversity School of Medicine, Atlanta, Georgia 30322; email: [email protected],[email protected], [email protected]

Annu. Rev. Pathol. Mech. Dis. 2012. 7:219–45

First published online as a Review in Advance onOctober 10, 2011

The Annual Review of Pathology: Mechanisms ofDisease is online at pathol.annualreviews.org

This article’s doi:10.1146/annurev-pathol-011811-132457

Copyright c© 2012 by Annual Reviews.All rights reserved

1553-4006/12/0228-0219$20.00

Keywords

FMR1, FMRP, intellectual disability, autism, synaptic plasticity,trinucleotide repeat expansion

Abstract

Fragile X syndrome (FXS) is a common form of inherited intellectualdisability and is one of the leading known causes of autism. The muta-tion responsible for FXS is a large expansion of the trinucleotide CGGrepeat in the 5′ untranslated region of the X-linked gene FMR1. Thisexpansion leads to DNA methylation of FMR1 and to transcriptionalsilencing, which results in the absence of the gene product, FMRP, aselective messenger RNA (mRNA)-binding protein that regulates thetranslation of a subset of dendritic mRNAs. FMRP is critical for mGluR(metabotropic glutamate receptor)-dependent long-term depression, aswell as for other forms of synaptic plasticity; its absence causes excessiveand persistent protein synthesis in postsynaptic dendrites and dysreg-ulated synaptic function. Studies continue to refine our understandingof FMRP’s role in synaptic plasticity and to uncover new functions ofthis protein, which have illuminated therapeutic approaches for FXS.

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Intellectual disability(ID): characterized byan IQ of less than 70

INTRODUCTION

Clinical diagnoses of common complex disor-ders, such as Alzheimer’s disease and autism,inevitably encompass heterogeneous subsets ofpatients with differing molecular mechanismsof disease. The study of rare Mendelian formsof the broader disease makes it possible to iden-tify a homogeneous group of patients, therebysimplifying molecular analysis. Focusing on themolecular genetics of these single-gene dis-orders has proven valuable in identifying theunderlying molecular pathways of disease, theknowledge of which can then be applied toour general understanding of the broader non-Mendelian forms. This pattern is especially ev-ident in the study of intellectual disability (ID)and autism by the knowledge gained of fragileX syndrome (FXS).

Inherited ID comprises a broad, heteroge-neous group of disorders; with an incidenceof 1 in 5,000 males (1), FXS represents oneof the most common forms of inherited ID.Since the underlying gene was cloned 20 years

Figure 1Eighteen-year-old male with fragile X syndrome.

ago, there has been tremendous progress inour understanding of the neurological deficitsthat contribute to FXS. As expected, researchinto FXS has revealed many of the pathwaysthat are critical to learning and memory forma-tion. Knowledge of these pathways has enabledthe rational design of potential therapeutics forFXS. Additionally, it has opened new avenuesof investigation into the molecular mechanismsbehind other forms of ID and autism.

Identification of Fragile X Syndromeas a Distinct Syndrome

In 1969, Lubs (2) identified a family with fourmale members diagnosed with ID, each ofwhom had an unusual chromosomal gap onhis X chromosome long arm. This observationhad limited clinical utility until 1977, whenSutherland (3) showed that specific cultureconditions were necessary to visualize thegap consistently. Such chromosomal gaps orconstrictions in metaphase spreads are termedfragile sites due to their propensity to breakunder certain conditions (4). It soon becameclear that this cytogenetic marker is diagnosticfor a distinct X-linked form of ID, designatedFXS after the fragile site found in patients.

Individuals with FXS have mild to severe ID,often with autism-like behaviors (5). Other neu-rological symptoms include developmental de-lay and increased susceptibility to seizures (5).Upon postmortem examination, the neurons ofFXS patients are found to have dense, imma-ture dendritic spines (5). The most prominentphysical symptom in males is macroorchidism,which usually develops just before puberty (5).More subtle physical symptoms may include along, narrow face with prominent ears, joint lax-ity, and flat feet (Figure 1) (5). These featurespoint to a potential connective tissue disorderthat has yet to be elucidated in any detail.

Analysis of more FXS pedigrees showed thatthe syndrome does not follow a typical patternof inheritance for an X-linked disease. Mostprominently, the pedigrees contained maleobligate carriers with no symptoms of FXS(6, 7). Furthermore, the grandchildren of these

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Untranslated regions(UTRs): the 5′ and 3′segments of messengerRNA, which are nottranslated into protein

healthy male carriers developed FXS at muchhigher rates than did the siblings of the carri-ers (6). This example of genetic anticipation,named the Sherman paradox, remained a mys-tery until the mutation underlying FXS wasidentified.

Identification of the FMR1 Gene

The gene for FXS was cloned in 1991 andnamed fragile X mental retardation gene 1(FMR1) (8). FMR1 is located at cytogeneticposition Xq27.3, the exact location of the di-agnostic fragile site (9). In almost all cases, thecausative mutation is the expansion of a CGGrepeat located in the 5′ untranslated region(UTR) of FMR1. Although this was a novel mu-tational mechanism at the time of its discovery,such trinucleotide-repeat expansions have sincebeen found in other disease genes (see the side-bar). In FMR1, the length of the CGG repeatis polymorphic in the healthy population andranges from 6 to 54 repeats (Figure 2a) (10).When the number of repeats exceeds 200, theexpansion is referred to as a full mutation alleleand results in FXS (10). At the molecular level,the large number of CGG repeats in the fullmutation leads to marked methylation of boththe CGG repeats and the FMR1 promoter, hy-poacetylation of associated histones, and chro-matin condensation; these epigenetic changesresult in transcriptional silencing of FMR1 andsubsequent loss of its protein product, fragile Xmental retardation protein (FMRP) (Figure 2c)(11–14). Alleles with an intermediate number(55–200) of repeats are referred to as premu-tation alleles (10). Premutation alleles do notcause an FXS phenotype (see the section titledPremutation Allele Phenotypes, below) but areprone to large increases in repeat length duringmeiosis, especially female meiosis (10). Theobservation that premutation alleles are morevulnerable to expansion during meiosis alsohelped resolve the Sherman paradox; we nowunderstand that males with a premutation alleletransmit it intact to their healthy daughters(10). Such premutation alleles in the daughtersoften expand to full mutation alleles during

TRINUCLEOTIDE REPEAT EXPANSIONSAND HUMAN DISEASE

Trinucleotide repeat (TNR) expansions are the causal muta-tion in approximately 20 disorders in addition to FXS, includingHuntington disease, myotonic dystrophy, and several inheritedataxias (44). TNRs are stretches of DNA composed of a three-base sequence (i.e., CGG, CAG, CTG, and GAA) that is repeatedmultiple times in tandem. Repeats of certain trinucleotides tendto expand or contract; if this occurs during gametogenesis, a dif-ferent number of repeats may be passed on to the next generation.The exact mechanisms of repeat instability are unknown; how-ever, it is believed that during DNA replication and repair, theTNR tract forms a hairpin loop that is incorrectly incorporatedinto the genome by DNA polymerase (44). Because longer re-peats are more unstable than short ones, TNR disorders oftendisplay genetic anticipation. TNRs may be located in either thecoding region or the noncoding region of genes. When foundin coding regions, TNRs produce proteins with extended poly–amino acid stretches that affect many aspects of normal function,such as cleavage and aggregate formation. TNRs in the noncod-ing region, as in myotonic dystrophy and FXS, may lead to toxicRNA production or cause transcriptional silencing.

oogenesis, which causes the higher incidenceof FXS observed in the next generation (10).

It is now the standard of care to measurethe FMR1 CGG repeat size in all childrenwho present with developmental delay, ID, orautism (5). Given the broad heterogeneity ofsuch patients, typically only 1% to 3% of suchchildren tested have the full mutation that leadsto a diagnosis of FXS (15). The full mutation isalmost completely penetrant in males, but only50% of females with a full mutation show FXSsymptoms, probably because of random X inac-tivation. Although most known FXS cases arecaused by the expansion of the CGG repeat to afull mutation, a small number of deletions andmissense mutations in the FMR1 gene have alsobeen reported (16–18). Recent studies suggestthat more comprehensive screening for suchmutations may increase the diagnostic yield by30% compared with screening for repeat lengthalone (17). In particular, there remains an

www.annualreviews.org • Fragile X Syndrome 221

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FXS

FXTAS

FXPOI

FMRP

DNA methylation andchromatin condensation

Transcription

a Normal

b Premutation

c Full mutation

55–200 CGG repeats

ATG

< 55 CGG repeats

ATG

> 200 CGG repeats

ATGX

Figure 2(a) Normal alleles (<55 repeats) allow for physiologically appropriate transcription of FMR1 and translation of FMRP. (b) Premutationalleles (55–200 repeats) cause significantly increased transcription of FMR1. However, levels of FMRP are actually lower than in healthyindividuals. Higher levels of FMR1 messenger RNA (mRNA) cause fragile X–associated tremor/ataxia syndrome (FXTAS) and fragileX–related primary ovarian insufficiency (FXPOI), probably because of the expanded CGG repeats in the mRNA that sequester certainmRNA-binding proteins. (c) Full mutation alleles (>200 repeats) lead to epigenetic changes in the CGG repeats and in the promoter ofFMR1, as well as transcriptional silencing of the gene. Symptoms of fragile X syndrome (FXS) are caused by the lack of FMRP.

Primary ovarianinsufficiency (POI):the onset ofmenopause at orbefore the age of 40

unexplained deficit of FMR1 missense muta-tions; only two have been reported thus far(17, 18).

Premutation Allele Phenotypes

Although premutation alleles do not causeFXS, they can lead to two other distinct dis-orders: fragile X–related primary ovarian in-sufficiency (FXPOI) and fragile X–associatedtremor/ataxia syndrome (FXTAS). FXPOI af-fects approximately 20% of female premutationcarriers (19); some of these women experience

primary ovarian insufficiency as early as theirteenage years. The number of CGG repeatscorrelates with the penetrance and age of onset,albeit in a nonlinear fashion (20). There havealso been conflicting reports concerning thepresence of a paternal parent-of-origin effectin FXPOI (21–23). Very little is known aboutthe molecular mechanism behind FXPOI, al-though differing hormone levels may play a role(24, 25).

FXTAS is a late-onset neurodegenerativedisease that manifests as progressive-actiontremor and ataxia; some cases also show


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progressive cognitive decline (26). Post-mortem examinations reveal ubiquitin-positiveintranuclear inclusions throughout the neuronsand astrocytes of FXTAS patients (27, 28).Penetrance in male premutation carriers olderthan age 70 is greater than 50% (29), whereaspenetrance in females is lower (30). LargerCGG repeats confer an increased risk of de-veloping FXTAS (31).

Investigations into the effect of the premu-tation allele on FMR1 processing have revealedmuch about the pathogenesis of FXTAS. In-stead of being methylated and silencing tran-scription, as in the full mutation, CGG re-peats in the premutation range lead to increasedlevels of FMR1 messenger RNA (mRNA)(32–34); however, FMRP levels are mod-estly lower than levels in normal individuals(Figure 2b) (33–35). The expanded CGG re-peats cause FMR1 mRNA transcripts to formintranuclear aggregates that grow larger overtime and probably correlate with the inclusionsobserved in FXTAS patients (36). The molec-ular basis for these observations is as yet un-known, although the presence of the expandedCGG repeat itself seems to be responsible(37, 38). Furthermore, several RNA-bindingproteins, including Purα, CUGBP1, heteroge-neous ribonucleoprotein (hnRNP) A2/B1, andSam68, interact with CGG-repeat RNA (39–41) and may be involved in the formation ofaggregates.

One hypothesis to explain the pathogeniceffect of the premutation allele is that theexpanded CGG repeats drive elevated FMR1transcript levels, which then recruit RNA-binding proteins to intranuclear aggregates,thereby titrating the proteins away from theirnormal function in other cellular locationsand leading to neurodegeneration. Remark-ably, overexpression of Purα, hnRNP A2/B1,or CUGBP1 can rescue the neurodegener-ative phenotype observed in the Drosophilamelanogaster model of FXTAS (39, 40). Addi-tional support for this hypothesis comes fromthe findings that Sam68 can be sequestered byCGG-repeat transcripts in cell culture (41) andthat knockout (KO) of Sam68 as well as Purα

in mice causes motor-coordination deficits(42, 43). These findings suggest that reducedlevels of Sam68 and/or Purα may be responsi-ble for some of the FXTAS phenotypes.

Animal Models and Stem Cells

FMR1 is highly conserved across species (8),which has allowed for the development ofseveral animal models of FXS. The mostwidely used models have been generated in themouse and fruit fly. The FMR1 ortholog inthe mouse, Fmr1, is located on the murine Xchromosome, and its amino acid sequence has97% homology to FMRP (45). Unfortunately,there is no relevant animal model of the fullCGG expansion, as mice engineered with anexpanded number of repeats fail to recapitulatethe methylation and transcriptional silencingfound in humans (34). However, a targeteddeletion of exon 5 created a KO mouselacking FMRP, the functional equivalent ofthe full mutation in humans (46). These micerecapitulate many of the phenotypes observedin FXS patients, including disrupted learn-ing and memory, increased susceptibility toseizures, and large testes (46). They also showan abundance of dense, immature dendriticspines, as observed in FXS patients (47). Morerecently, a mouse line was engineered in whichloxP sites flank the promoter and exon1 ofFmr1, which allowed for conditional KO ofthe gene (48). This latest mouse model willenable the creation of null alleles in specificcell types and at specific stages of development,thereby providing new ways to explore FMRP’sfunction in vivo. Another mouse model wascreated in which one of the rare missensemutations observed in patients was introducedinto the endogenous murine Fmr1 gene (49).These mice phenocopy the deficits observed inFmr1 KO mice (49), thereby allowing a betterunderstanding of the effects of this unusualmutation.

D. melanogaster models of FXS have alsobeen created by making null mutations in thefly ortholog of FMR1, dFmr1. These flies haveabnormal neuronal architecture and synaptic


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γ-Aminobutyric acid(GABA): an aminoacid neurotransmitter

function (50–52), impairment of long-termmemory (53), and reduced courtship interest(a model of behavioral abnormalities) (54). TheD. melanogaster model is more amenable thanthe mouse model in some aspects of neurobi-ological dissection and screening for geneticinteractions. For example, Chang et al. (55)discovered that dFmr1 mutant flies die duringdevelopment if reared on food containing in-creased levels of glutamate. This observationformed the basis for a small-molecule screenthat identified several compounds that couldrescue the lethality (55). These compounds,including γ-aminobutyric acid (GABA) andacetylcholine receptor agonists, represent po-tential therapeutic agents for FXS and provideinsights into the pathways involved in diseasepathogenesis.

Advances in embryonic and inducedpluripotent stem (iPS) cell technology are alsoallowing investigations into aspects of FXS thatare difficult to study in animal models. Mostnotably, both the timing and the mechanismbehind the methylation and gene silencingobserved in full mutation alleles remain poorlyunderstood, and as noted above, mouse modelsusing a CGG expansion cannot replicate themethylation or gene silencing found in humans(34). Human embryonic stem cell lines derivedfrom embryos diagnosed with a full mutationshowed that FMR1 is unmethylated and ex-pressed in these cells, but upon differentiationof the cells FMR1 undergoes methylation,histone modifications, and silencing (56). Thisfinding is consistent with those from studies ofhuman chorionic villi samples, which indicatethat FMR1 is expressed early in developmentand is silenced between 10 and 12.5 weeks ofgestational development (57). In contrast, iPScells generated from the fibroblasts of FXSpatients show that FMR1 remains methylatedand transcriptionally silenced in these celllines (58). Understanding the differencesbetween the human embryonic stem cells andthe iPS cells may help unlock key aspectsof the methylation and could lead to novelapproaches to demethylate the full mutationallele in FXS patients.

FMRP FUNCTION

When FMR1 was cloned in 1991, nothing wasknown about the function of its protein prod-uct, FMRP. Research from the past 20 yearshas led to the understanding that FMRP playsa critical role in synaptic plasticity. FMRP lo-calizes to the postsynaptic spaces of dendriticspines, where it binds to and represses transla-tion of a targeted subset of dendritic mRNAs.Upon receipt of the appropriate synaptic sig-nals, FMRP derepresses translation, allowingsynthesis of key synaptic plasticity proteins tooccur at a specific time and location. Despitethe progress made to date, only a small num-ber of FMRP’s target mRNAs have been ver-ified. Furthermore, the molecular mechanismthat FMRP uses to inhibit translation is unclear,and there is only a small amount of evidence forthe model of how FMRP is regulated.

FMRP Expression and FunctionalProtein Domains

FMRP is widely expressed in mammaliantissues (59), but it is especially abundantin the brain and testes (59, 60), consistentwith the predominant phenotypes (ID andmacroorchidism) observed in patients withFXS. In the brain, FMRP is expressed primar-ily in neurons, where it is mostly cytoplasmic,being found in the cell body, dendrites, andsynapses (59, 61, 62). The pattern of FMRP ex-pression seems to begin early in developmentand continue throughout life (60). Because ofthe focus on patients’ cognitive deficits, thefunction of FMRP in nonneuronal cells has re-ceived scant attention.

Multiple alternatively spliced isoforms ofFMRP exist in humans (45, 63), mice (45), andfruit flies (Figure 3a) (64); there is some ev-idence that individual isoforms may have im-portant differences in expression and function.For example, in the recently characterized longand short isoforms of D. melanogaster dFmr1,the short dFmr1 lacks a glutamine-asparagine(QN)-rich protein interaction domain in the Cterminus of the protein; deletion of the longisoform revealed that the short isoform, without


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2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1

0 100 200 300 400 500 600

Amino acid

NLS KH1 KH2 NES RGGAgenet 1

CGGrepeat

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

I304N S500

71 kDa

R138Q

Agenet 2

a

b

c

Figure 3FMR1 gene, messenger RNA (mRNA), and protein. (a) FMR1 gene. Coding exons (dark green), untranslated regions (light green), andintrons (black lines) are shown. Blue lines denote alternative splicing. (b) FMR1 mRNA. Exons (light blue) are positioned above theircorresponding amino acids. The variable CGG repeat is shown in yellow in DNA and mRNA. (c) FMRP. Abbreviations: Agenet 1 andAgenet 2, tandem Agenet domains, chromatin binding; NLS, nuclear localization signal; KH1 and KH2, K homology domains 1 and 2;NES, nuclear export signal; RGG, arginine-glycine-glycine box, RNA-binding; S500, primary phosphorylated serine. R138Q is anaturally occurring mutation in a patient with developmental delay; I304N is a naturally occurring mutation that abolishespolyribosome association.

the QN domain, is insufficient to properly formshort- or long-term memories (64). The corre-sponding C-terminal region in human FMRPmediates interaction with kinesin and dendritictransport (65), which suggests that it is also im-portant in humans.

In mammals, the main isoform of FMRP is a71-kDa protein composed of several conservedfunctional domains (Figure 3c). FMRP hasthree RNA-binding motifs, including two Khomology domains (KH1 and KH2) and thearginine-glycine-glycine (RGG) box. FMRPbinds RNAs in a sequence-specific mannermediated by these domains. In particular,the methylation status of the arginines in theRGG box seems to regulate FMRP’s affinityfor certain RNAs (66). FMRP also containsnuclear localization and export signals (67),which facilitate its shuttling into and out of thenucleus (61). A recent study identified a patientwith developmental delay who harbored anovel R138Q mutation in the nuclear local-ization signal (17). The functional significanceof this mutation is unclear, but it points to the

importance of the domain. Although not fullyappreciated until recently (see the section titledFMRP Nuclear Function, below), FMRP alsocontains two tandem Agenet domains at its Nterminus (68). The Agenet domains are part ofa proposed “royal family” of protein domainsthat also includes the Tudor, MBT, andChromo domains (68). The Agenet domainsbind trimethylated lysine residues and arestructurally similar to the UHFR1 protein,which is believed to interact with methylatedhistone H3K9 (69). In addition to theseconserved domains, other regions of FMRPhave also been implicated in protein-proteininteractions that are important for its function.

FMRP Nuclear Function

Although FMRP is predominantly cytoplasmic,it can shuttle between the cytoplasm and thenucleus (61). Models of FMRP function sug-gest that FMRP enters the nucleus to bind itsmRNA targets and then chaperone them outof the nucleus (67). For many years, it was be-lieved that export of FMRP from the nucleus


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Chromatinimmunoprecipitationfollowed bymassively parallelsequencing(ChIP-seq): genome-wide method to mapDNA/chromatin-binding sites for agiven protein

was dependent on mRNA synthesis. In sup-port of this idea, a recent report demonstratedthat knockdown of the bulk mRNA exporterTap/NXF1 increased the nuclear localizationof FMRP and that FMRP physically associateswith Tap/NXF1 in an RNA-dependent man-ner (70). Also, FMRP associates with the activetranscription units of lampbrush chromosomesin amphibian oocytes (70). These new observa-tions offer the first experimental evidence thatFMRP binds mRNA in the nucleus, indicatingthat FMRP may enter the nucleus to bind RNAand facilitate trafficking and/or regulation of itscargo.

FMRP may also have other importantfunctions in the nucleus, according to anotherrecent study (R. Alpatov, U. Wagner, M.Nakamoto-Kinoshita, Z. Ye, Y. Luu, E.L.Greer, Z. Wang, G. Hu, W.N. Beavers, P.T.Morrison, M.D. Simon, C.R. Vakoc, K. Zhao,B. Ren, S.T. Warren & Y. Shi, manuscript in re-vision). These authors showed for the first timethat FMRP uses its Agenet domains to bindmethylated H3K79 chromatin and to mediatethe DNA-damage response pathway). A ChIP-seq (chromatin immunoprecipitation followedby massively parallel sequencing) assay revealedthat nearly one-third of FMRP’s chromatin-binding sites are associated with commonfragile sites. Interestingly, only half of theFMRP-binding sites overlapped binding sitesidentified by ChIP-seq of Dot1, the H3K79methyltransferase (R. Alpatov, U. Wagner,M. Nakamoto-Kinoshita, Z. Ye, Y. Luu, E.L.Greer, Z. Wang, G. Hu, W.N. Beavers, P.T.Morrison, M.D. Simon, C.R. Vakoc, K. Zhao,B. Ren, S.T. Warren & Y. Shi, manuscript inrevision). This finding suggests that FMRPmay play additional roles in regulating chro-matin structure outside of H3K79 binding.How these potential roles would correlate withother known functions of FMRP is still unclear.

FMRP Is a MessengerRNA–Binding Protein

FMRP is a selective RNA-binding protein; itbinds to as much as 4% of the mRNA in

the mammalian brain (71). Microarray andyeast three-hybrid assays have identified morethan 400 putative mRNAs that associate withFMRP (72–75), although only 14 of thesehave been validated by showing direct bio-chemical interaction (Table 1). A recent studyused high-throughput sequencing of RNAs iso-lated through cross-linking immunoprecipita-tion (HITS-CLIP) to expand the list of possibleFMRP target mRNAs (76). The mRNAs iden-tified were significantly enriched for proteinsinvolved in neuronal and synaptic transmission.Strikingly, 28 of the FMRP target genes iden-tified in the new study were candidate genes forautism (76).

There is evidence that FMRP interacts withits target mRNAs through adapter molecules.The most intensely studied putative adapter isbrain cytoplasmic 1 (BC1), a small, noncod-ing RNA. Investigations by Zalfa et al. (77, 78)indicated that BC1 directly binds FMRP and,through base-pairing, interacts with mRNAtargets of FMRP. However, this issue is con-troversial, and a recent report from five labsfound no specific BC1/FMRP interactions invivo or in vitro and showed that the asso-ciation of FMRP to its target mRNAs doesnot require BC1 (79). This research suggeststhat the FMRP/BC1/mRNA interactions pre-viously observed were nonspecific. Further-more, double-KO mice lacking both FMRPand BC1 showed increased audiogenic seizuresusceptibility and more severe place-learningdeficits than did single-KO animals lacking onlyFMRP or BC1 (80). This finding implies thatboth FMRP and BC1 act in similar neurolog-ical pathways but do so independently of oneanother. In contrast, yet another study foundthat BC1 increases the affinity of FMRP forone of its protein-binding partners (81), whichdemonstrates that this issue remains open.

Direct binding of FMRP to its target RNAsis mediated by the presence of RNA secondarystructures (Figure 4), the best studied of whichis known as the G quadruplex. A G quadruplexconsists of two to four G quartets or tetradsstacked on top of each other; each G quartet ismade of four guanines in a planar conformation


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Table 1 Validated FMRP target messenger RNAs (mRNAs)a

mRNADendritic

localizationG quadruplex–like

structurebmGluR

stimulated Methodc Reference(s)App − + + Co-IP 87Arc + − + Co-IP 149—151CamKIIα + − + Co-IP 77, 96, 97, 122eEF1A + − + Co-IP, in vitro 94, 152Fmr1 + + + In vitro 62, 84, 95GABAAδ + − + APRA, in vitro 65, 73GluR1/2 + − + Co-IP 97hASH1 − −d − In vitro 90Map1b + + + Co-IP, in vitro, biophysical 72, 83, 85, 95, 122, 153Psd95 + + + Co-IP, in vitro, CLIP 88, 97, 154Rgs5 + − + APRA, in vitro 65, 73Sapap3/4 + − + Co-IP 65, 72, 93, 117Sema3F − + − Co-IP, biophysical 83, 86Sod1 − −e − In vitro 92

aAt least 14 mRNAs have been validated as FMRP targets through biochemical methods. Many colocalize with FMRP in dendrites, and most aretranslationally upregulated in response to metabotropic glutamate receptor (mGluR) stimulation. Several contain G quadruplex structures necessary forFMRP binding. Others contain different secondary structures, such as superoxide dismutase 1 (Sod1) stem loops interacting with FMRP (SoSLIP) orU-rich regions, or their secondary structures have not been investigated. Pluses indicate direct experimental evidence; minuses indicate lack of directexamination, unclear results, or evidence against.bSome targets contain putative G quadruplexes or G-rich regions lacking the canonical G quadruplex consensus sequence; they are referred to as Gquadruplex–like structures.cSeveral in vivo methods have been used to validate FMRP-mRNA interactions, namely coimmunoprecipitation (co-IP), cross-linkingimmunoprecipitation (CLIP), antibody-positioned RNA amplification (APRA), and so on. In vitro methods include filter binding, gel shift, affinitycapture, and UV cross-linking assays.dhASH1 contains a U-rich region that mediates its binding to FMRP.eSod1 contains the first example of the SoSLIP structure, which mediates its binding to FMRP. Abbreviations: App, amyloid precursor protein;GABA, γ-aminobutyric acid.

that interact via cyclic Hoogsteen-type hy-drogen bonds (82). FMRP’s C-terminal RGGbox recognizes the G quadruplex in vitro (83),and several of FMRP’s target mRNAs possessputative G quadruplex structures (Table 1).Because G quadruplexes are hard to predictbioinformatically, only biochemical or bio-physical experiments can verify their actualformation. Accordingly, biochemical assaysconfirmed that G quadruplexes mediate theinteraction of FMRP with Fmr1, MAP1b,and Sema3F mRNAs (84–86). FMRP alsocoimmunoprecipitates with a G-rich re-gion in amyloid precursor protein (APP)mRNA (87), which could potentially form aG quadruplex–like structure. However, thebinding of FMRP to a putative G quadruplex

in the 3′ UTR of PSD-95 mRNA occurredeven in the presence of lithium (88), which candisrupt FMRP/G quadruplex interactions (83);therefore, whether this mRNA forms a truequadruplex remains a mystery.

Other RNA secondary motifs have alsobeen identified in FMRP targets. MultipleU-rich pentamers were found in both codingand 3′ UTR regions of some FMRP targetmRNAs (89), and a recent study used UVcross-linking and mutagenesis assays to showthat FMRP binds to a U-rich region in the 5′

UTR of hASH1 (90). The U-rich structure stillneeds further characterization, and we do notknow which domain of FMRP may associatewith these U-rich regions. In addition, anothersecondary structure referred to as the kissing


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GG

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a

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Figure 4RNA secondary structures recognized by FMRP. (a) G quadruplex in parallel configuration. (b) SoSLIP(superoxide dismutase 1 stem loops interacting with FMRP). (c) So-called kissing complex.

complex has been reported to bind FMRP’sKH2 domain in vitro (91). Importantly, thekissing complex was formed by a selection ofrandomly synthesized RNA, and this structurehas not been observed in endogenous mRNA.One explanation may be that the kissingcomplex forms in vivo only when two RNAmolecules interact and, therefore, may berelevant for the association of target mRNAstogether with adapter RNAs or microRNAs(miRNAS; see the section titled Mechanismsof Translational Repression, below). Finally, arecent study also showed that FMRP binds tosuperoxide dismutase 1 (Sod1) mRNA througha novel RNA structure termed Sod1 stem loopsinteracting with FMRP (SoSLIP) (92). SoSLIPconsists of three stem-loop structures separatedby short stretches of single-stranded RNA andacts as a translational activator (92). SoSLIPinteracts with FMRP’s C-terminal region,which includes the RGG box, and competes

for binding with the G quadruplex structure(92).

FMRP Regulates DendriticMessenger RNAs

The cognitive deficits suffered by FXS patients,along with the dense, immature dendritic spinesobserved in the brains of both FXS patientsand Fmr1 KO mice, indicated that FMRP playsa role in dendritic development and function.Indeed, many of the mRNAs identified as tar-gets of FMRP localize to dendrites. In situhybridization demonstrated the dendritic lo-calization of RGS5, GABAAδ , SAPAP3/4, andeEF1A mRNAs (73, 93, 94). Through FISH,Map1b mRNA was proven to be dendriticallylocalized (95); both it and Arc/Arg3.1 mRNAcoprecipitate with FMRP in brain extracts(77). In addition, Arc/Arg3.1 and CamKIIαmRNAs are present in dendrites (96). Finally,


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Polyribosome: acytoplasmic structureconsisting of multipleribosomes, all attachedto a single mRNAmolecule

Synaptoneurosome:a purified synapsecontaining intact pre-and postsynaptictermini

PSD-95 mRNA directly associates with FMRPin dendrites both in vitro and in vivo (88, 97).Together, these findings support a model inwhich FMRP binds and regulates a subset ofdendritic mRNAs.

Although it is clear that FMRP interactswith mRNAs in dendrites, other studies indi-cate that FMRP is not required for the con-stitutive localization of these mRNAs. In situhybridization in wild-type and Fmr1 KO neu-rons revealed no differences in the dendriticlocalization of MAP2, CamKIIα, and dendrinmRNAs (98). This study also showed that therapid transport of Arc/Arg3.1 mRNA to den-drites following seizures is unaffected in Fmr1KO mice (98). Furthermore, FISH detectedno changes in the localization of CamKIIα orPSD-95 mRNAs in brain sections or culturedneurons of Fmr1 KO mice (97). In contrast,one study found a reduction of RGS5 mRNA indendrites of hippocampal neurons from Fmr1KO mice (73), which could be explained bya decrease of mRNA stability. In support ofthis explanation, FMRP increases the stabil-ity of PSD-95 mRNA in hippocampal neurons(88). The effect on PSD-95 mRNA stabilityis cell-type specific, and no evidence for in-creased stability was found for 10 other FMRP-associated mRNAs (88). Overall, these resultsindicate that FMRP is not essential for the den-dritic localization or stability of the majorityof FMRP-associated mRNAs. Most recently,Buckley et al. (99) presented data suggest-ing that retained intronic sequence–containingretrotransposon ID elements target mRNAs,including FMR1 mRNA, to the dendrite.

FMRP Is a Translational Repressor

In subcellular fractionation experiments,FMRP cosediments with polyribosomes inboth neuronal and nonneuronal cells (100–102). FMRP’s association with polyribosomessupports the hypothesis that FMRP acts as atranslational regulator of its mRNA targets.This association is disrupted by puromycin,which disrupts actively translating polyribo-somes, indicating that FMRP is associated

with actively translating polyribosomes (102).In an FXS patient with an I304N missensemutation, FMRP’s interaction with activelytranslating polyribosomes was abolished (100),which indicates that this association is crucialto FMRP’s normal function. In addition tointeracting with polyribosomes, FMRP is alsofound in stress granules, which are presumedto sequester mRNAs whose translation isbeing suppressed, in mRNA-protein (mRNP)complexes (103). Together, these observationsimplicate FMRP in dynamic translationalregulation of its mRNA partners.

Most evidence is consistent with FMRPinhibiting translation of most of its targetmRNAs. FMRP reduces translation of variousmRNAs in rabbit reticulocyte lysate, Xenopuslaevis oocytes (104), and immortalized cellsfrom an Fmr1 KO mouse (105). In the retic-ulocyte assay, removal of the FMRP-bindingsite from MBP mRNA abolished FMRP’sability to repress its translation, confirmingthat FMRP binding is necessary for translationregulation (106). Biochemical and geneticassays also indicate that D. melanogaster dFmr1represses translation of the Map1B orthologfutsch (51). In vivo assays further demonstratedthat the target proteins Map1B, Arc/Arg3.1,and CamKIIα are overexpressed in the brainsof Fmr1 KO mice, which is consistent with theloss of FMRP-mediated repression (77, 107).To specifically interrogate FMRP’s effect ontranslation at synapses, synaptoneurosomesfrom Fmr1 KO mice were examined; theyshowed increased levels of MAP1B, CamKIIα,and Arc/Arg3.1 proteins (77). Subcellularfractionation of Fmr1 KO synaptoneurosomesalso revealed a shift of CamKIIα, PSD-95,and GluR1/2 mRNAs to actively translatingpolyribosomes, which is consistent with thesemRNAs being derepressed (97). Surprisingly,FMRP seems to upregulate the translation ofSod1 mRNA by strengthening SoSLIP’s abilityto activate translation (92) and to increasetranslation of hASH1 through an unknownmechanism (90). These findings signify thatFMRP may also activate translation of sometranscripts.


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(S)-3,5-Dihydroxyphenyl-glycine (DHPG): ametabotropicglutamate receptor 1and 5 agonist

Mechanisms of TranslationalRepression

One model of how FMRP regulates thetranslation of mRNAs proposes that it inhibitsthe initiation of translation (Figure 5a). In

AGO2/RISC

AAAAA

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Figure 5Models of translation inhibition. (a) FMRP recruits CYFIP1 to blockformation of the eIF4A-eIF4G-eIF4E (eIF4F) complex and prevent translationinitiation. (b) FMRP causes ribosomes to stall during the elongation phase oftranslation. (c) FMRP recruits the RNA-induced silencing complex (RISC) toinhibit translation.

cap-dependent translation, which is importantin neurons, initiation requires the eIF4A-eIF4G-eIF4E (eIF4F) complex to associatewith the 5′ m7GTP cap of the mRNA template.4E-binding proteins (4E-BP) interfere with theeIF4E-eIF4G interaction, thereby regulatingthe formation of the eIF4F complex. Recently,cytoplasmic FMRP-interacting protein (CY-FIP1), a known protein-binding partner ofFMRP, was discovered to be a 4E-BP (81).Coimmunoprecipitation confirmed that bothCYFIP1 and eIF4E are associated with FMRPin vivo (81). Interestingly, the formation of theFMRP/CYFIP1/eIF4E complex was increasedby the presence of capped-Arc mRNA, a knownFMRP target, but not capped-luciferase mRNA(81). These data have led to a model in whichFMRP recruits CYFIP1 to specific mRNAs,subsequently associates with eIF4E, andblocks recruitment of the translation-initiationmachinery. Consistent with this model, levelsof MAP1B, CamKIIα, and APP proteins,other known FMRP targets, are increased inmice that express half of the normal CYFIP1levels (81). Furthermore, activation of proteinsynthesis by brain-derived neurotrophic factoror (S)-3,5-dihydroxyphenylglycine (DHPG)reduces the amount of eIF4E associatedwith the FMRP/CYFIP1 complex (81), whichdemonstrates that binding of eIF4E to CYFIP1is inversely correlated with translation levels.

An alternate model for how FMRP inhibitstranslation posits that FMRP causes ribosomesto stall during the elongation phase of trans-lation (Figure 5b). This model is supportedby data showing that some FMRP cosedi-ments with polyribosomes, even after treatmentwith puromycin, meaning these polyribosomesprobably represent stalled ribosomes (102). Ri-bosome runoff experiments in which cells weretreated with sodium azide, a nonspecific in-hibitor of translation initiation that does notaffect elongation, also revealed that a portionof FMRP was associated with stalled ribosomes(108). These data have been reinforced by arecent HITS-CLIP experiment that suggestedthat FMRP binds to the coding sequencesof mRNA transcripts that are associated with


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Synaptic plasticity:the ability of synapsesto change in strengthin response tostimulation; is criticalfor learning andmemory

stalled ribosomes (76). Ribosome stalling hasnot been thoroughly characterized, but it is pre-sumed to be influenced by transfer RNA avail-ability, subcellular localization, folding dynam-ics of the nascent protein, and RNA secondarystructure (109). Although this hypothesis hasnot yet been specifically tested, it is reasonableto suppose that FMRP binding to secondarystructures in target mRNAs, such as G quadru-plexes, may stabilize such structures and act asa steric block for ribosome elongation.

Data also indicate that FMRP repressestranslation of its target mRNAs through theRNA interference (RNAi) pathway (Figure 5c).Both D. melanogaster dFmr1 and mammalianFMRP associate with Argonaute 2 and Dicer,critical components of the RNA-inducedsilencing complex (RISC) (110–112), as wellas with specific miRNAs (113). Experimentsin D. melanogaster further demonstrated thatArgonaute2 is necessary for dFmr1-dependentsynaptic plasticity (113). In vitro, FMRP canhelp assemble miRNAs on target RNAs, anactivity directed by its KH domains (112).In addition, mouse embryonic fibroblasts(MEFs) from Fmr1 KO mice showed impairedRNAi compared with wild-type MEFs (112),although another study found that FMRPand the RISC associate with different pools ofmRNAs and that FMRP-deficient cells havenormal RISC activity (103). Nevertheless,a more recent study showed that FMRPselectively associates with several miRNAs inthe mouse brain (114). Overexpression of twoof these miRNAs, miR-125b and miR-132,in cultured rat hippocampal neurons resulted indendritic spine defects; knockdown of FMRP inthese cells rescued both phenotypes (114). Thesynaptic mRNA NR2A is also a target of bothmiR-125b and an FMRP ligand (114). Simi-larly, in another study, the 3′ UTR of PSD-95bound both miR-125a and FMRP, and bothwere required for translation inhibition (115).

Taken together, these genetic and bio-chemical data support a mechanism inwhich FMRP binds to the 3′ UTR of targetmRNAs, where it then mediates or sta-bilizes the binding of the complementary

miRNA-RISC complex to block translation.Whether the miRNA-RISC/FMRP complexblocks translation initiation or elongation isunknown, and both mechanisms have beenimplicated for miRNAs in general (116). Inter-estingly, one miRNA, miR-125a, shifted fromthe mRNP fraction into polyribosomes uponactivation of synaptoneurosomes with DHPG(115), which is consistent with the miR-125a–RISC/FMRP complex primarily blockingtranslation initiation prior to activation.

Importantly, these three models of FMRP-regulated translation inhibition are not neces-sarily mutually exclusive, and different mecha-nisms could apply at different times in the lifeof an mRNA molecule. For example, FMRPmay inhibit translation initiation of its targetmRNAs during transport to dendritic spines,but once the mRNAs reach the synapse, FMRPmay repress translation via ribosome stallingor recruitment of an miRNA-RISC complex.Likewise, synaptic activation may change theinhibitory mechanism to allow repeated trans-lation starting and stopping. In addition, giventhat putative binding sites for FMRP are dis-tributed among the 5′ UTR, 3′ UTR, andprotein-coding regions of mRNAs, distinctsubsets of mRNA targets may be regulated dif-ferently by FMRP on the basis of the relativeposition of FMRP’s binding. For instance, ifFMRP binds in the 3′ UTR, it may coordi-nate primarily with miRNAs, but if it bindsthe 5′ UTR or within the gene body, it mayform a steric block and cause ribosome stalling.Further work will help unravel these nuances.

Phosphorylation RegulatesFMRP-Mediated TranslationInhibition

In vivo, FMRP exists in both phosphory-lated and unphosphorylated forms, althoughphosphorylated FMRP is the predominantform in dendritic granules (117). FMRPcontains a serine residue that is conserved fromDrosophila to humans (human Ser500, murineSer499, Drosophila Ser406) and is the primaryphosphorylated residue in FMRP (108). The


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Metabotropicglutamate receptor(mGluR): a synapticreceptor involved insynaptic plasticity;group 1 mGluRsinclude mGluR1 andmGluR5

Long-termdepression (LTD):an activity-dependentdecrease in theeffectiveness ofsynaptic transmissionthat lasts for hours ordays

AMPAR: α-amino-3-hydroxyl-4-isoxazolepropionic acidreceptor; itsinternalization is a keystep in hippocampalLTD

phosphorylation status of this key serine isbelieved to control the functionality of FMRP.Although phosphorylation of FMRP doesnot affect its ability to bind RNA, it doesaffect its association with polyribosomes andits ability to inhibit translation (108, 115).Using constitutively phosphorylated andunphosphorylated mimics of murine FMRPcontaining the S499D and S499A mutations,respectively, Ceman et al. (108) showed thatonly the S499D-FMRP is still associated withpolyribosomes after sodium azide–inducedpolyribosome runoff. This finding suggeststhat phosphorylated FMRP is associated withstalled ribosomes, whereas unphosphorylatedFMRP allows translation to proceed. Thissuggestion is consistent with the observationby Mudashetty et al. (115) that overexpressionof S499D-FMRP, but not S499A-FMRP,could inhibit translation of a construct con-taining the 3′ UTR of PSD-95. Furthermore,these authors showed that S499D-FMRPshifted this construct into mRNP fractionson sucrose gradients (115), which argues thatthe phosphorylation of FMRP suppresses thetranslation initiation of PSD-95. Interestingly,Dicer associates with unphosphorylated butnot phosphorylated FMRP (118), indicatingthat phosphorylation of FMRP may alsoregulate the RNAi pathway by blocking theprocessing by Dicer of premiRNAs (precursormiRNAs). These data support a model in whichphosphorylated FMRP inhibits translation ofits target mRNAs while unphosphorylatedFMRP allows translation to proceed.

ROLE OF FMRP INNEUROLOGICAL PATHWAYS

Researchers have put a great deal of effortinto elucidating the specific neurologicalpathways in which FMRP plays a role. Group1 metabotropic glutamate receptor (mGluR)-dependent long-term depression (mGluR-LTD) has received the most attention, and thedetails of how FMRP interacts with this path-way are well understood. It is widely believedthat the majority of neurological symptoms ob-

served in FXS patients stem from the dysregula-tion of the mGluR-LTD pathway caused by theabsence of FMRP. FMRP is also important inother pathways and other regions of the brain.Much less is known about FMRP’s role in thesepathways, but it is believed that FMRP acts asa translational repressor in these instances aswell. A major focus of the field is determiningwhich pathways are crucial for the symptomsobserved in patients and which of these areamenable to pharmaceutical intervention.

mGluR Theory of Fragile X Syndrome

Deficits in synaptic plasticity correlate withlearning and memory impairment in the brain,so it was expected that such deficiencies maybe at the core of FXS. mGluR-LTD is a majorform of synaptic plasticity and depends on thelocal protein synthesis of postsynaptic, dendrit-ically localized mRNAs in response to synapticstimulation. This local protein synthesis resultsin the internalization of α-amino-3-hydroxyl-4-isoxazole propionic acid receptors (AMPAR),a key step in mGluR-LTD. The mGluR the-ory of FXS proposes that FMRP acts in thispathway downstream of mGluRs and upstreamof local protein synthesis. As such, this theorypredicts that FMRP represses translation of itsmRNA targets in the normal basal state but thatupon mGluR activation FMRP repression is re-leased, allowing the burst of local protein syn-thesis necessary for AMPAR internalization andLTD (Figure 6a) (119). When FMRP is ab-sent, as in FXS, protein synthesis should be con-stitutively elevated, which would lead to over-active AMPAR internalization and exaggeratedLTD, even in the absence of mGluR activation(Figure 6b).

In support of this theory, mGluR-LTD isenhanced in the hippocampus of Fmr1 KOmice and does not require new protein synthesis(120, 121). In addition, dephosphorylation ofFMRP following mGluR activation coincidesclosely with the release of translation inhibitionof FMRP target mRNAs, such as PSD-95 (117).In agreement with this evidence, the levels ofseveral FMRP target mRNAs associated with


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mRNA Ribosome FMRP Phosphate New protein P

a Normal b Fragile X syndrome

P

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AMPARmGluR1/5 AMPARmGluR1/5

Figure 6Metabotropic glutamate receptor (mGluR) theory of fragile X syndrome. (a) FMRP binds its target messenger RNAs (mRNAs) in thenucleus and is cotransported with them to dendrites. Upon mGluR activation, FMRP is dephosphorylated and allows translation toproceed. This protein synthesis results in the internalization of α-amino-3-hydroxyl-4-isoxazole propionic acid receptor (AMPAR) andlong-term depression (LTD). The same pool of FMRP may be rephosphorylated, or a new pool of phosphorylated FMRP may betransported to dendrites. (b) In neurons lacking FMRP, a subset of dendritic proteins are constitutively overexpressed even in theabsence of mGluR stimulation, which causes excessive AMPAR internalization and exaggerated LTD.

2-Methyl-6-(phenylethynyl)-pyridine (MPEP): anmGluR5 antagonist

actively translating polyribosomes are increasedin response to DHPG, an mGluR agonist, inwild-type mouse synaptoneurosomes, but notin Fmr1 KO synaptoneurosomes (97). Further-more, metabolic labeling with [35S] methionineshows that the DHPG-induced synthesis ofPSD-95 and CamKIIα proteins observed inwild-type synaptoneurosomes is absent inFmr1 KO synaptoneurosomes (97). Levelsof the FMRP protein itself rise rapidly uponmGluR stimulation (62, 122) and then quicklyfall as the protein is ubiquitinated and degraded(122). Remarkably, blocking this degradationwith proteasome inhibitors abolishes mGluR-LTD (122). Both the persistently enhancedmGluR-LTD and the inability to furtherincrease protein synthesis in response to new

synaptic stimuli are the likely culprits behindID in FXS.

The mGluR theory also predicts that antag-onizing the mGluR pathway may lead to a re-duction of FXS phenotypes. The mGluR5 an-tagonist 2-methyl-6-(phenylethynyl)-pyridine(MPEP) can indeed rescue behavioral andcognitive deficits in the fruit fly, zebrafish, andmouse models of FXS (123–126). MPEP alsorescues the altered dendritic spine morphologyobserved in Fmr1 KO neurons and restoresproper AMPAR internalization (127). Further-more, genetic reduction of mGluR5 in Fmr1KO mice rescues many of the disease-relatedphenotypes (128, 129). Pharmacological rescueof FXS phenotypes has now opened many newavenues for potential therapeutic intervention


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in FXS and has spawned several clinical trialsof drugs that target the mGluR pathway.

mGluR-LTD Signaling Pathways:mTOR and ERK

mGluR-LTD requires a cascade of signalingevents that culminate in increased proteinsynthesis and AMPAR internalization. Bothmammalian target of rapamycin (mTOR)and extracellular signal–related kinase (ERK)signaling downstream of mGluR activation

are required for formation of normal LTD(Figure 7a). Establishing FMRP’s role in thesepathways is important for our understandingof FXS and for further development of rationaltherapeutic interventions. Because the phos-phorylation status of FMRP is a key step inthe mediation of mGluR-LTD, it makes sensethat it would be regulated by the mTOR orERK pathway. Studies have identified proteinphosphatase 2A (PP2A) and ribosomal proteinS6 kinase 1 (S6K1) as FMRP’s primary phos-phatase and kinase, respectively (117, 130).

Synaptic protein synthesis

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Figure 7Metabotropic glutamate receptor (mGluR) signaling through mammalian target of rapamycin (mTOR) and extracellularsignal–regulated kinase (ERK) pathways. (a) Interactions between FMRP and the mTOR and ERK pathways. Blue ovals, componentsof the mTOR pathway; green ovals, components of the ERK pathway; orange ovals, FMRP targets. (b) Regulation of FMRP. Less than1 min after mGluR stimulation, protein phosphatase 2A (PP2A) dephosphorylates FMRP, derepressing protein synthesis. More than5 min after mGluR stimulation, mTOR inhibits PP2A and activates ribosomal protein S6 kinase 1 (S6K1), phosphorylating FMRP andrerepressing protein synthesis. Abbreviations: 4E-BP, 4E-binding protein; eIF4F, eIF4A-eIF4G-eIF4E; LTD, long-term depression;MEK, mitogen-activated protein kinase kinase; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C.


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Within 30 s of mGluR activation, PP2Adephosphorylates FMRP (117). PP2A activitydeclines approximately 2 min after stimulation,and by 5 min poststimulation, normal levels ofphosphorylated FMRP again become evident(117). However, it is unclear whether the ex-isting FMRP molecules are rephosphorylatedor whether a new pool of phosphorylatedFMRP is transported into the dendritic spines.PP2A and S6K1 are controlled by the mTORpathway. Given the time required for mTOR’sactivation and the immediate response ofPP2A to mGluR stimulation, it is unlikely thatmTOR controls the initial activation of PP2A;rather, mTOR is probably only a negativeregulator of PP2A. Thus, PP2A may be cou-pled directly to mGluR receptors or may haveanother activating mechanism that quicklydephosphorylates FMRP, whereas the mTORpathway inhibits PP2A and concurrently acti-vates S6K1 to restore FMRP phosphorylation(Figure 7b).

In addition to mTOR regulating FMRPphosphorylation, recent evidence indicatesthat the mTOR pathway is also negativelyregulated by FMRP, which places mTORdownstream as well as upstream of FMRP.Consequently, Fmr1 KO mice show a generalexaggeration of mTOR signaling, includingincreased association of raptor with mTOR,higher levels of mTOR kinase activity, in-creased phosphorylation of the mTOR targetsS6 kinase and 4E-BP, and elevated levels ofthe eIF4F complex (131). Phosphatidylinositol3-kinase (PI3K) plays a key role in activatingthe mTOR pathway (Figure 7a); PI3K’s cat-alytic subunit p110β and its upstream activatorPIKE are putative targets of FMRP, and bothare upregulated in Fmr1 KO mice (131). Inter-estingly, the PI3K inhibitor LY294002 reduceslevels of phosphorylated mTOR in Fmr1 KOmice to wild-type levels (131). These resultssuggest that FMRP normally downregulatesthe mTOR pathway by repressing PIKE andPI3K and that, in the absence of FMRP,mTOR signaling is at or near saturation andtherefore insensitive to mGluR activation.

They also imply that FMRP controls its ownregulation by derepressing mTOR, therebyallowing mTOR to phosphorylate FMRP.

Further work has shown that FMRP regu-lates PI3K activity by controlling the synthesisand synaptic localization of p110β (132). Treat-ment of cortical synaptoneurosomes from Fmr1KO mice with LY294002 reversed the pheno-types of increased protein synthesis, AMPARinternalization, and spine density normally ob-served in Fmr1 KO neurons (132). In contrast,treatment with the ERK1/2 antagonist U0126showed no effect on the dysregulated pro-tein synthesis found in FMRP-deficient neu-rons (132). These results suggest that PI3K in-hibitors may hold promise as FXS therapeutics.

In disagreement with the above results, how-ever, a recent study found that increased proteinsynthesis in hippocampal slices from Fmr1 KOmice is rescued by inhibition of ERK1/2, butnot by inhibition of mTOR (133). This studyrevealed that the ERK pathway is not upreg-ulated in Fmr1 KO neurons (133), which sug-gests that in the absence of FMRP, the processof local protein synthesis is hypersensitive tonormal levels of ERK signaling. Another reportfound that mGluR stimulation of Fmr1 KOsynaptoneurosomes results in dephosphoryla-tion of ERK, whereas in wild-type synaptoneu-rosomes, ERK remains phosphorylated (134).Although some differences in tissue preparationbetween studies may have partly contributed tothe divergent results, it is not clear how thedifferent findings concerning the mTOR andERK pathways will be reconciled.

OTHER mGluR SYNAPTICPLASTICITY

Early studies were unable to identify anydisruptions in hippocampal long-term poten-tiation (LTP) in Fmr1 KO mice (135, 136).Recently, though, a new method of inducingLTP by bath application of glycine showedthat this form of LTP was reduced in Fmr1KO hippocampal tissue slices (137). Further-more, in wild-type mice, the group 1 mGluR


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Long-termpotentiation (LTP):an activity-dependentincrease in theeffectiveness ofsynaptic transmissionthat lasts for hours ordays

antagonist DL-AP3 and the NMDA recep-tor antagonist AP5 independently blockedthis glycine-induced LTP (137). However,application of AP5 did not further decreaseglycine-induced LTP in Fmr1 KO mice,whereas application of DL-AP3 resulted ina slight decrease (137). These results suggestthat glycine-induced LTP is mediated by bothNMDA receptors and group 1 mGluRs, butthat FMRP only plays a role in mGluR-LTP.

Much of the research into the function ofFMRP has focused on its role in the hippocam-pus, but additional evidence shows that FMRPaffects mGluR synaptic plasticity in other areasof the brain as well. The emotional symptomsobserved in FXS patients point to possibleinvolvement of the amygdala. Consistentwith evidence from the hippocampus, thereis a reduction of AMPAR surface expressionin the amygdalas of Fmr1 KO mice (138).Interestingly, although this phenomenon leadsto increased mGluR-LTD in the hippocam-pus, it causes impaired mGluR-LTP in theamygdala (138). A reduction in miniatureexcitatory synaptic current frequency was alsoobserved, which indicates possible presynapticdefects (138). Treatment with MPEP doesnot rescue either the mGluR-LTP deficien-cies or the reduction in surface AMPARsin the amygdalas of Fmr1 KO mice; how-ever, it does reverse the changes observedin presynaptic release, suggesting that somesymptoms associated with the amygdala maybe amenable to pharmacological intervention(138).

The anterior cingulate cortex (ACC) is alsoinvolved in learning and memory. Stimulationof group 1 mGluRs in the ACC with DHPGupregulates the expression of FMRP via Ca2+-dependent signaling pathways (139). Suchupregulation is absent in Ca2+/calmodulin-stimulated adenylate cyclase 1 (AC1) KO miceas well as in calcium/calmodulin-dependentkinase IV (CaMKIV) KO mice (139, 140),indicating that AC1 and CaMKIV mediate theregulation of FMRP by group 1 mGluRs in theACC. The increase in FMRP is abolished byactinomycin D (139), a transcription inhibitor,

which suggests that this regulation occurs atthe transcriptional level.

Alternate Neurological Pathways

In addition to mGluR synaptic plasticity,FMRP also affects dopaminergic and GABAer-gic brain function. Dopamine (DA) plays amajor role in neurological pathways activein the prefrontal cortex. DA can bind to fivedifferent G protein–coupled receptors (GRKs),named D1–D5. Upon activation of the D1receptors, prefrontal cortex neurons normallyexhibit an increase in surface expressionand phosphorylation of AMPARs, but suchincreases are absent in neurons from Fmr1KO mice (141, 142). FMRP-deficient neuronsalso show impairment of DA-dependent LTP(141). Treatment of Fmr1 KO mice with a D1receptor agonist partially reduced their hy-peractivity and improved their motor function(141), which may represent another potentialavenue of treatment for FXS patients.

Finally, GABAA synaptic transmission is im-paired in models of FXS. GABAA receptors arethe major inhibitory receptors in the brain andhave been implicated in learning and memory.These findings have motivated research intothe possible role of GABAergic dysregulationin FXS. Indeed, the mRNA for the δ subunit ofthe GABAA receptor is a ligand of FMRP (143);it and several other subunits of GABAA arereduced in the brains of Fmr1 KO mice (143,144). Remarkably, all three subunits of the in-vertebrate GABA receptor are underexpressedin a D. melanogaster model of FXS (143).Neurophysiological experiments in brain slicesprovided direct evidence of abnormal GABAtransmission in KO mice (145, 146). These dataclearly suggest that GABAA agonists could beeffective in treating FXS. In fact, the aforemen-tioned D. melanogaster–based screen uncoveredthree compounds involved in GABAergicsignaling, including GABA itself, that rescuedthe dFmr1 KO phenotype (55). GABAA

agonists are some of the most promisingcompounds for FXS currently used in clinicaltrials.


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CONCLUSION AND FUTUREDIRECTIONS

The year 2011 marks the twentieth anniversaryof the cloning of FMR1. Since then, tremen-dous progress has been made in our under-standing of FMRP’s normal function and howits absence leads to the symptoms observed inFXS patients. Most research has focused onFMRP’s function as a translational repressorin hippocampal mGluR-LTD. The resultingmGluR theory of FXS is supported by a greatdeal of evidence and can account for many of theneurological symptoms observed in patients.

Despite these advances, many lines ofinquiry remain open. Animal models cou-pled with pharmacological assays may allowinvestigators to correlate specific FXS phe-notypes with the dysregulation of specifictarget mRNAs. Also, there are conflicting dataconcerning FMRP’s mechanism of translation

repression. It may directly interact with itstarget mRNAs, or it may rely on adaptermolecules such as BC1. Recent evidence hasreinforced the idea that FMRP acts through themiRNA pathway, but the possibility remainsthat it may inhibit translation initiation orelongation through more direct means.

A major goal of FXS research has been thedevelopment of therapies to improve the qual-ity of life of those who suffer from FXS. Thisgoal may soon be within reach, as investiga-tions into the function of FMRP have identi-fied several promising pharmacological agents,including mGluR antagonists, PI3K inhibitors,and GABAergic signaling agonists. Clinical tri-als have already begun to assess the efficacy ofsome of these compounds in humans (147, 148).Although these trials have not yet been com-pleted, there is now justifiable optimism thatan effective treatment for FXS may finally beon the horizon.

SUMMARY POINTS

1. FXS is a very common form of inherited ID. FXS patients often display symptoms ofautism.

2. FXS is caused by a large expansion of the CGG repeat in the 5′ UTR of FMR1, whichcauses methylation and silencing of FMR1 and the lack of FMRP.

3. FMRP is a selective mRNA-binding protein that represses translation of a subset ofdendritically localized mRNAs. FMRP derepresses translation in response to synapticactivation.

4. FMRP plays a critical role in dendritic local protein synthesis and synaptic plasticity.In its absence, mGluR-LTD signaling is overactive, and synaptic plasticity is reduced.This molecular dysregulation is the major cause of the cognitive deficits observed in FXSpatients.

5. The phosphorylation status of FMRP regulates its function. Phosphorylated FMRP re-presses translation of its target mRNAs, and unphosphorylated FMRP allows translationto proceed.

6. FMRP may inhibit translation by blocking initiation of translation, causing ribosomestalling, interacting with the RNAi pathway, or a combination of the three.

7. Investigators have used their knowledge of FMRP’s molecular function for the rationaldesign of potential therapeutics, including mGluR antagonists, PI3K inhibitors, andGABAergic agonists. Several of these therapeutics are currently in clinical trials.


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FUTURE ISSUES

1. Now that the identities of many FMRP ligands are known, investigators may be able tocorrelate specific target mRNAs with specific FXS phenotypes.

2. More work is needed to determine the mechanism by which FMRP inhibits translation.Data exist to support three distinct models; FMRP’s function may involve one or moreof them.

3. A novel nuclear role for FMRP has been discovered. This function needs to be moreextensively characterized, and its role, if any, in FXS must be determined.

4. Molecular studies of FMRP have led to the rational design of novel therapeutics. Clinicaltrials will establish the efficacy of known compounds in treating FXS as researchers workto discover and refine new therapies.

DISCLOSURE STATEMENT

S.T.W. is chair of the scientific advisory board of Seaside Therapeutics, Inc. The other authors arenot aware of any affiliations, memberships, funding, or financial holdings that might be perceivedas affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank Cheryl Strauss for editing and Jeannie Visootsak for the patient photograph. Our workis supported in part by National Institutes of Health grants HD020521 and HD024064.

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71. Ashley CT, Wilkinson KD, Reines D, Warren ST. 1993. FMR1 protein: conserved RNP familydomains and selective RNA binding. Science 262:563–66

72. Identified putativeFMRP-associated brainmRNAs.

72. Brown V, Jin P, Ceman S, Darnell JC, O’Donnell WT, et al. 2001. Microarray identification ofFMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.Cell 107:477–87

73. Miyashiro KY, Beckel-Mitchener A, Purk TP, Becker KG, Barret T, et al. 2003. RNA cargoes associatingwith FMRP reveal deficits in cellular functioning in Fmr1 null mice. Neuron 37:417–31

74. Zou K, Liu J, Zhu N, Lin J, Liang Q, et al. 2008. Identification of FMRP-associated mRNAs using yeastthree-hybrid system. Am. J. Med. Genet. B 147:769–77

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76. Darnell JC, Van Driesche SJ, Zhang C, Hung KYS, Mele A, et al. 2011. FMRP stalls ribosomal translo-cation on mRNAs linked to synaptic function and autism. Cell 146:247–61

77. Zalfa F, Giorgi M, Primerano B, Moro A, Di Penta A, et al. 2003. The fragile X syndrome protein FMRPassociates with BC1 RNA and regulates the translation of specific mRNAs at synapses. Cell 112:317–27

78. Zalfa F, Adinolfi S, Napoli I, Kuhn-Holsken E, Urlaub H, et al. 2005. Fragile X mental retardationprotein (FMRP) binds specifically to the brain cytoplasmic RNAs BC1/BC200 via a novel RNA-bindingmotif. J. Biol. Chem. 280:33403–10

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96. Bramham CR, Wells DG. 2007. Dendritic mRNA: transport, translation and function. Nat. Rev. Neurosci.8:776–89

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98. Steward O, Bakker CE, Willems PJ, Oostra BA. 1998. No evidence for disruption of normal patterns ofmRNA localization in dendrites or dendritic transport of recently synthesized mRNA in Fmr1 knockoutmice, a model for human fragile X mental retardation syndrome. Neuroreport 9:477–81

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114. Edbauer D, Neilson JR, Foster KA, Wang C-F, Seeburg DP, et al. 2010. Regulation of synaptic structureand function by FMRP-associated microRNAs miR-125b and miR-132. Neuron 65:373–84

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PM07-FrontMatter ARI 15 December 2011 8:19

Annual Review ofPathology:Mechanisms ofDisease

Volume 7, 2012Contents

Instantiating a Vision: Creating the New Pathology Departmentat Stanford Medical SchoolDavid Korn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

The Life and Death of Epithelia During Inflammation:Lessons Learned from the GutStefan Koch and Asma Nusrat � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �35

The Cell Biology of PhagocytosisRonald S. Flannagan, Valentin Jaumouille, and Sergio Grinstein � � � � � � � � � � � � � � � � � � � � � � � �61

Human Microbiome in Health and DiseaseKathryn J. Pflughoeft and James Versalovic � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �99

Merkel Cell Carcinoma: A Virus-Induced Human CancerYuan Chang and Patrick S. Moore � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 123

Molecular Pathogenesis of Ewing Sarcoma: New Therapeuticand Transcriptional TargetsStephen L. Lessnick and Marc Ladanyi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 145

Mechanisms of Function and Disease of Naturaland Replacement Heart ValvesFrederick J. Schoen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Pathology of Demyelinating DiseasesBogdan F.Gh. Popescu and Claudia F. Lucchinetti � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 185

Molecular Mechanisms of Fragile X Syndrome:A Twenty-Year PerspectiveMichael R. Santoro, Steven M. Bray, and Stephen T. Warren � � � � � � � � � � � � � � � � � � � � � � � � � � 219

Pathogenesis of NUT Midline CarcinomaChristopher A. French � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

Genetic Variation and Clinical Heterogeneity in Cystic FibrosisMitchell L. Drumm, Assem G. Ziady, and Pamela B. Davis � � � � � � � � � � � � � � � � � � � � � � � � � � � � 267

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PM07-FrontMatter ARI 15 December 2011 8:19

The Pathogenesis of Mixed-Lineage LeukemiaAndrew G. Muntean and Jay L. Hess � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 283

ATM and the Molecular Pathogenesis of Ataxia TelangiectasiaPeter J. McKinnon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 303

RNA Dysregulation in Diseases of Motor NeuronsFadia Ibrahim, Tadashi Nakaya, and Zissimos Mourelatos � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 323

Tuberculosis Pathogenesis and ImmunityJennifer A. Philips and Joel D. Ernst � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 353

PsoriasisGayathri K. Perera, Paola Di Meglio, and Frank O. Nestle � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 385

Caveolin-1 and Cancer Metabolism in the Tumor Microenvironment:Markers, Models, and MechanismsFederica Sotgia, Ubaldo E. Martinez-Outschoorn, Anthony Howell,

Richard G. Pestell, Stephanos Pavlides, and Michael P. Lisanti � � � � � � � � � � � � � � � � � � � � � � � 423

Pathogenesis of Plexiform Neurofibroma:Tumor-Stromal/Hematopoietic Interactions in Tumor ProgressionKarl Staser, Feng-Chun Yang, and D. Wade Clapp � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 469

Indexes

Cumulative Index of Contributing Authors, Volumes 1–7 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 497

Cumulative Index of Chapter Titles, Volumes 1–7 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 500

Errata

An online log of corrections to Annual Review of Pathology: Mechanisms of Disease articlesmay be found at http://pathol.annualreviews.org

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