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NOS1的物种依赖性转录后调控FMRP在开发大脑皮质

Species-Dependent Posttranscriptional Regulation of NOS1by FMRP

in the Developing Cerebral Cortex

Kenneth Y.Kwan,1Mandy https://www.wendangku.net/doc/8615752655.html,m,1Matthew B.Johnson,1Umber Dube,1Sungbo Shim,1Mladen-Roko Ra s in,1,2 Andre′M.M.Sousa,1,3So?a Fertuzinhos,1Jie-Guang Chen,1,4Jon I.Arellano,1Daniel W.Chan,1Mihovil Pletikos,1,5 Lana Vasung,5David H.Rowitch,6Eric J.Huang,7Michael L.Schwartz,1Rob Willemsen,8Ben A.Oostra,8Pasko Rakic,1 Marija Heffer,9Ivica Kostovi c,5Milos Juda s,5and Nenad Sestan1,*

1Department of Neurobiology and Kavli Institute for Neuroscience,Yale University School of Medicine,New Haven,CT06510,USA

2Department of Neuroscience and Cell Biology,University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, Piscataway,NJ08854,USA

3Graduate Program in Areas of Basic and Applied Biology,Abel Salazar Biomedical Sciences Institute,University of Porto,

Porto4099-002,Portugal

4School of Optometry and Ophthalmology,Wenzhou Medical College,Wenzhou,Zhejiang325003,China

5Croatian Institute for Brain Research,University of Zagreb School of Medicine,Zagreb10000,Croatia

6Departments of Pediatrics and Neurosurgery,Eli and Edythe Broad Institute for Stem Cell Research and Regeneration Medicine and Howard Hughes Medical Institute

7Department of Pathology

University of California,San Francisco,San Francisco,CA94143,USA

8Department of Clinical Genetics,Erasmus MC,Rotterdam3000CA,The Netherlands

9Department of Medical Biology,Josip Juraj Strossmayer University of Osijek,Osijek31000,Croatia

*Correspondence:nenad.sestan@https://www.wendangku.net/doc/8615752655.html,

DOI10.1016/j.cell.2012.02.060

SUMMARY

Fragile X syndrome(FXS),the leading monogenic cause of intellectual disability and autism,results from loss of function of the RNA-binding protein FMRP.Here,we show that FMRP regulates transla-tion of neuronal nitric oxide synthase1(NOS1)in the developing human neocortex.Whereas NOS1 mRNA is widely expressed,NOS1protein is tran-siently coexpressed with FMRP during early synapto-genesis in layer-and region-speci?c pyramidal neurons.These include midfetal layer5subcortically projecting neurons arranged into alternating columns in the prospective Broca’s area and orofacial motor cortex.Human NOS1translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1mRNA,which is expressed in mouse pyramidal neurons,but not ef?ciently translated.Correspondingly,neocortical NOS1protein levels are severely reduced in devel-oping human FXS cases,but not FMRP-de?cient mice.Thus,alterations in FMRP posttranscriptional regulation of NOS1in developing neocortical circuits may contribute to cognitive dysfunction in FXS. INTRODUCTION

The development of neural circuits is a precisely regulated process susceptible to genetic alterations that can lead to disor-ders affecting the most distinctively human aspects of cognition, including speech and language,theory of mind,and complex social behavior(Geschwind and Levitt,2007;Lui et al.,2011; Ramocki and Zoghbi,2008;State,2010;Walsh et al.,2008). One such disorder,fragile X syndrome(FXS),is the leading inherited cause of intellectual disability and is often accompa-nied by autistic-like features,aggression,attention de?cits, and delays in speech and language development(Abbeduto et al.,2007;Rogers et al.,2001;Willemsen et al.,2011).FXS is caused by loss of function of the FMR1gene,which encodes an RNA-binding protein(FMRP)involved in mRNA localization, stability,and translation(Ashley et al.,1993;Bagni and Greenough,2005;Bassell and Warren,2008;Zalfa et al., 2007).Many FMRP mRNA targets function in synaptic develop-ment and plasticity(Brown et al.,2001;Darnell et al.,2011). Concordantly,Fmr1-de?cient mice show neural de?cits also found in patients with FXS(The Dutch-Belgian Fragile X Consor-tium,1994).However,FMRP target mRNAs and their role in human neurodevelopment are not as well understood.

The study of human FMRP function may also provide insights into the molecular mechanisms and neural circuits affected in autism spectrum disorders(ASDs),which are highly comorbid with FXS(Rogers et al.,2001).ASDs are a group of complex developmental syndromes characterized by impairments in social communication and language development,and repetitive behaviors.Multiple lines of evidence point to the dysfunction of neocortical circuits involved in social,emotional,and language processing in ASDs(Geschwind and Levitt,2007;State,2010; Walsh et al.,2008).Although no overt neuroanatomical alter-ations have been linked to the autistic brain,there is emerging evidence of abnormal organization of cortical minicolumns Cell149,899–911,May11,2012a2012Elsevier Inc.899

(Casanova et al.,2002;Peters,2010),which are composed of vertically arranged neurons connected into a local network and thought to originate from developmental radial units (Mountcastle,1997;Rakic,1988).Whether the molecular mech-anisms altered in ASDs are associated with the development of speci?c human cortical circuits,including minicolumns,remains unknown.

Here,we report that FMRP binds human neuronal nitric oxide synthase1(NOS1,also known as nNOS)mRNA and increases its translation in the developing neocortex in a species-dependent manner.NOS1produces the gaseous signaling molecule nitric oxide(NO),which plays important roles in the development and function of the nervous system(Bredt and Snyder,1994; Garthwaite,2008).Our study of NOS1posttranscriptional regu-lation was instigated by our observation of a marked discrep-ancy between the midfetal human neocortex expression patterns of NOS1mRNA,which is widespread,and NOS1 protein,which is restricted to layer-and region-speci?c subpop-ulations of pyramidal neurons.These include layer5(L5) subcortically-projecting neurons with an alternating minicolum-nar arrangement in the frontoparietal operculum(FOp).The FOp encompasses the prospective Broca’s area and orofacial motor cortex,regions involved in speech production and language comprehension(Keller et al.,2009).After our screen for RNA-binding proteins revealed that FMRP is abundantly bound to human NOS1mRNA,we found that FMRP interacts with sequences in the NOS1-coding region that contain G-quartet(GQ)motifs and leads to increased NOS1protein expression.These motifs are absent from mouse Nos1mRNA, and replacing the GQ-containing region of human NOS1with the mouse orthologous sequence abrogates FMRP-dependent activation of translation.Concordantly,neocortical NOS1 protein levels are dramatically reduced in human FXS,but not Fmr1-de?cient mice.Thus,we identi?ed a species-dependent posttranscriptional regulation of human NOS1by FMRP in speci?c neocortical circuits during column development and synaptogenesis,and showed it to be altered in FXS.

RESULTS

NOS1Protein Is Transiently Expressed in Developing Human Pyramidal Neurons

The current research stems from our unexpected observation that strong NADPH-diaphorase(NADPH-d)activity,a reliable histoenzymatic marker of NOS(Dawson et al.,1991;Hope et al.,1991),is transiently present in subpopulations of pyramidal neurons in the developing human neocortex(Sestan and Kostovi c,1994),in addition to its previously reported localization to interneurons and cortical plate(CP)neuropil(Fertuzinhos et al.,2009;Judas et al.,1999).Our comprehensive analysis of pre-and postnatal postmortem brains ranging from8postcon-ceptional weeks(PCW)to adulthood identi?ed transient expres-sion of NOS1/NADPH-d in two layer-and region-speci?c popu-lations of pyramidal neurons with a predominant localization to somata and apical dendrites(Figures1A–1C;see Figure S1A available online).Speci?cally,morphologically immature pyra-midal neurons expressing NOS1were present in the middle of the CP corresponding to the future L5exclusively in the ventro-lateral frontal cortex of the FOp and the dorsal part of the anterior insula starting around15PCW.One week later,NOS1+pyra-midal neurons were also found in the anterior cingulate cortex (ACC)and adjacent dorsolateral frontoparietal cortex in the upper CP corresponding to the future L2and L3.NOS1expres-sion in both of these regions was also temporally regulated.The ACC L2/L3expression of NOS1was maintained at high levels throughout the late fetal ages,and decreased during early infancy(Figures1B and S1A;data not shown).In contrast,L5 expression of NOS1occurred in two waves.First,the L5expres-sion was restricted to the FOp,and started at15PCW,peaked at 18–20PCW,and was rapidly downregulated at approximately23 PCW,after which a small number of NOS1+pyramidal neurons were present in the ventral part of the anterior insula.Second, sparse pyramidal NOS1expression was present throughout neocortical L5in the weeks immediately prior to birth and was progressively downregulated after birth(Figures1B and S1A; data not shown).Thus,in developing pyramidal neurons, NOS1expression is precisely regulated,exhibiting temporal, laminar,and regional speci?city.

Fetal L5NOS1+Pyramidal Neurons Form Alternating Columns

Further analysis of the midfetal FOp L5NOS1+pyramidal neurons revealed that they were arranged vertically into alternating arrays of intensely(NOS1+)and lightly(NOS1à)stained pyramidal neurons(Figures1A and1C)resembling previously described ontogenetic columns(Rakic,1988).In contrast the ACC L2/L3 NOS1+pyramidal neurons were more densely distributed(Fig-ure1D;FOp L5,67.86±5.66cells per1,000m m2;ACC L3, 178.57±27.95cells per1,000m m2;p=4.12310à5)and lacked this alternating columnar arrangement(Figure1E;nearest neighbor distance between cell clusters:FOp L5,27.87±5.26m m;ACC L3,15.75±4.68m m;p=4.24310à9).In contrast to midgestation,perinatal NOS1+L5neurons did not exhibit columnar organization(Figure S1A).

Because the FOp is structurally and functionally lateralized (Keller et al.,2009),we investigated whether L5NOS1+columns exhibited left-right asymmetry in two whole midfetal brains(18 and20PCW).Serial reconstruction con?rmed the two separate domains of NOS1+pyramidal neurons in the FOp and ACC of both hemispheres(Figures1F and S1B)and provided approxi-mate total numbers of FOp NOS1+columns(18PCW:41,380; and20PCW:45,150).Although the number of NOS1+columns was not signi?cantly different between the left and right hemi-spheres(p=0.569),the distribution of NOS1+columns showed an asymmetric trend,peaking more rostrally in the right hemi-sphere,in both brains.Thus,the columnar organization of NOS1+neurons in the midfetal FOp L5is bilaterally present. Molecular and Projectional Identity of NOS1+Pyramidal Neurons

To molecularly characterize the identity of NOS1+neurons,we examined their expression of neuronal subtype markers.In the midfetal FOp L5,markers of subcortically-projecting pyramidal neurons,BCL11B(CTIP2)and FEZF2(FEZL,ZFP312)(Chen et al.,2005;Kwan et al.,2008;Leone et al.,2008;Molyneaux et al.,2007),were selectively coexpressed by L5NOS1+

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neurons,forming an alternating columnar pattern identical to that of NOS1(Figures2A,2B,and2D).NOS1+neurons also coex-pressed FOXP2(Figure2E),a gene altered in a developmental disorder characterized by impaired speech and linguistic de?cits (Lai et al.,2001).In contrast,SATB2,a marker of upper-layer cor-ticocortical pyramidal neurons(Britanova et al.,2008),was highly expressed in NOS1à,but not NOS1+,neurons(Figures 2A and2B),suggesting that NOS1àneurons were later born and likely migrating in between L5NOS1+columns to the upper layers.Consistent with this,we observed vimentin(VIM)-positive radial glial?bers in between but not within NOS1+columns(Fig-ure S2A).This suggests that glial-guided migration of upper-layer neurons occurs via corridors formed between L5neuronal columns.In the ACC,all L2/L3NOS1+neurons coexpressed SATB2(Figure S2B),con?rming their upper-layer identity and distinction from the FOp L5NOS1+neurons.

To examine whether the midfetal pyramidal neurons of diverse subtypes have distinct cytoarchitectonic arrangements,we?rst measured the nearest neighbor ratio(NNR)and total path length ratio(TPLR)(Buxhoeveden et al.,1996)of NOS1+and NOS1àL5 neurons in the20PCW FOp(Figure2C).This con?rmed that NOS1+L5neurons were signi?cantly closer to being perfectly columnar(1.0)compared to NOS1àL5neurons,as determined by both NNR(NOS1+,1.145±0.146;NOS1à,1.528±0.202; p=2.97310à5)and TPLR(NOS1+,1.122±0.077;NOS1à, 1.740±0.369;p=2.41310à3).Next,we retrogradely traced projection neurons in a postmortem20PCW brain.We labeled subcortical projection neurons with Fast DiI inserted into the internal capsule and corticocortical projection neurons with Fast DiA inserted into the corpus callosum(Figure2H).DiI-labeled subcortical projection neurons in the FOp formed columns similar in organization to the NOS1+columns(Figure2I). In contrast,DiA-labeled callosal neurons in the ACC did not exhibit columnar organization(Figure2J).Collectively,these results indicate that FOp NOS1+neurons exhibit the columnar organization and molecular identity of postmigratory L5sub-cortically-projecting neurons.

Transient NOS1Expression in Pyramidal Neurons

Is Concomitant with Early Synaptogenesis

Previous studies have shown that signi?cant neocortical synap-togenesis starts during midgestation(Molliver et al.,1973).

ACC (20 PCW) NOS1FOp (20 PCW)

NOS1

(

)

100μm

1 cm

100μm

13 PCW

20 PCW

30 PCW

40 PCW

Adult

L2/L3 NOS1/NADPH-d+ pyramidal neurons

L5 NOS1/NADPH-d+pyramidal neurons

L3L5

Frontal

Number of labeled

L5 columns per section

A B

C D

Number of labeled

L5 columns per section

Figure1.Spatiotemporal Dynamics of NOS1Expression in Pyramidal Neurons of the Human Neocortex

(A)NADPH-d histochemistry at18PCW revealed intensely labeled pyramidal neurons in ACC L3and FOp L5,where stained neurons were arranged into alternating vertical columns(open arrow).Interneurons(arrowhead)and blood vessels(asterisks)were also labeled.Boxes represent the25th,50th,and75th percentiles.

(B)Schematic summary of the spatiotemporal dynamics of NOS1/NADPH-d staining in pyramidal neurons of L2/L3(green)and L5(red)in the developing and adult human neocortex.

(C)NOS1immunohistochemistry of radial and tangential sections at20PCW.NOS1+pyramidal neurons exhibited a clear columnar organization in FOp L5,but not ACC L3.

(D and E)Analysis of cell density(D)and cluster spacing(E)revealed that NOS1+neurons in FOp L5were signi?cantly distinct in cytoarchitectonic organization from those in ACC L3.*p<0.05.Boxes represent the25th,50th,and75th percentiles.Error bars represent the5th and95th percentiles of30measurements.

(F)Serial section analysis of NADPH-d+L5columns in two brains at18and20PCW.NADPH-d+columns were present bilaterally.

See also Figure S2.

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data not shown),indicating that pyramidal expression of NOS1is species dependent.

Discordant NOS1mRNA and Protein Expression

Patterns in the Fetal Neocortex

To examine the expression pattern of NOS1mRNA,adjacent tissue sections of the midfetal neocortex were analyzed with NOS1in situ hybridization,NADPH-d,and NOS1immunostain-ing(Figure4A).Surprisingly,NOS1mRNA was abundantly and widely present in the CP in all cortical layers and regions exam-ined,including the great majority of pyramidal neurons that did not express NOS1protein.This striking difference between the highly restricted NOS1protein and widespread NOS1 mRNA expression suggests that NOS1is posttranscriptionally regulated.

Remarkably,Nos1mRNA was also abundantly and widely expressed in the early postnatal mouse neocortex(Figure4B). Pyramidal expression of mouse Nos1mRNA was con?rmed by quantitative RT-PCR of?uorescently sorted pyramidal neurons fate mapped in mice doubly transgenic for Emx1-Cre and a CRE-responsive Gfp(CAG-Cat-Gfp)(Figure4C).Therefore, whereas NOS1mRNA is expressed in pyramidal neurons of both human and mouse neocortex,its ef?cient translation into NOS1protein occurs in subpopulations of human,but not mouse,pyramidal neurons.This indicates that pyramidal NOS1 expression is driven by species-dependent posttranscriptional regulation.

NOS1mRNA Associates with FMRP in Human Fetal Neocortex

To identify potential NOS1mRNA-binding proteins,we used immobilized full-length human NOS1mRNA to pull down candi-date proteins from the human frontal CP at20and21PCW. To facilitate the isolation of sequence-dependent RNA-binding proteins,we used three negative control RNAs(GAPDH, EGFP,and NeoR).NOS1mRNA-interacting proteins showed a distinct enrichment at a molecular weight of approximately 75kDa(Figure5A).To identify the protein present in this band, we analyzed our human brain transcriptome data set(http:// https://www.wendangku.net/doc/8615752655.html,;Johnson et al.,2009; Kang et al.,2011)for RNA-binding proteins near75kDa that are expressed in the midfetal frontal neocortex.Analysis of four candidates(FMRP,FXR1,CPEB3,and EIF2C2)by immuno-blotting of pulled-down proteins revealed that FMRP,but not the others,was strongly and speci?cally associated with NOS1 mRNA(Figures5A and S4A).The presence of FMRP in this NOS1-enriched band was con?rmed by mass spectrometry (data not shown),which also revealed the putative presence of PABPC4,a poly-adenylate-binding protein,and HSPA8,a chap-erone protein.Double-immuno?uorescent staining showed that FMRP was highly coexpressed in NOS1+pyramidal neurons in the midfetal FOp and ACC(Figures5B and S4B).Subcellularly, FMRP and NOS1colocalized to the soma and apical dendrite. Interestingly,most NOS1+interneurons in the SP and CP did not express FMRP at high levels during midgestation.Together, these results suggest a potential role of FMRP in the posttran-scriptional regulation of NOS1in fetal human pyramidal neurons. Species Differences in FMRP-NOS1mRNA Association in the Developing Neocortex

To con?rm the putative FMRP-NOS1mRNA interaction,we per-formed RNA-binding protein immunoprecipitation(RIP)using 21PCW frontal CP lysate.RNAs coimmunoprecipitated with FMRP were analyzed using quantitative RT-PCR(Figure5C). Compared to rabbit immunoglobulin(IgG)control,anti-FMRP antibodies immunoprecipitated 6.8-±0.8-fold more NOS1 mRNA,a level of enrichment similar to MAP1B mRNA(7.1-±0.6-fold),a well known target of FMRP(Darnell et al.,2011), and signi?cantly higher than GAPDH mRNA(1.5-±0.4-fold), a negative control.In contrast in the early postnatal mouse neocortex(Figure5D),Nos1mRNA was enriched only2.7-±0.3-fold by anti-FMRP immunoprecipitation,markedly lower than the8.6-±0.5-fold enrichment for Map1b mRNA and comparable to Gapdh mRNA(2.0-±0.1-fold).Consistent with

ACC FOp

ACC Lateral frontal neocortex

L2/L3L5L2/L3L5

L2/L3L5

L5 L5

https://www.wendangku.net/doc/8615752655.html,parative Analysis of NADPH-

d Activity in Mouse,Macaque,and Human

Neocortex at Equivalent Developmental

Ages

In the P4mouse ACC and lateral frontal cortex(A),

intense NADPH-d staining was restricted to inter-

neurons,with neuropil staining in ACC L2/L3.In

the E73macaque neocortex(B),intense NADPH-d

activity was present in ACC L2/L3pyramidal

neurons similar to those labeled in the human18

PCW ACC(C).In the macaque FOp,NADPH-d+L5

pyramidal neurons were arranged into vertical

columns similar in organization to the human

FOp columns(C).Strong interneuronal and weak

neuropil NADPH-d staining was present in all

cortical areas in mouse,macaque,and human

neocortex.See also Figure S3.

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this,Nos1was absent from the FMRP targets identi?ed in a recent,comprehensive HITS-CLIP analysis of the mouse brain (Darnell et al.,2011).Thus,FMRP strongly associates with human but not mouse NOS1mRNA in the developing neocortex, suggesting that FMRP may underlie species differences in NOS1 translation.

FMRP Binds GQ-Containing Sequences in the Human NOS1-Coding Region

FMRP can interact with speci?c mRNA sequences including GQ structures(Darnell et al.,2001;Schaeffer et al.,2001)and poly-uridine stretches(Chen et al.,2003).Analysis of human NOS1 mRNA revealed three putative GQ motifs and six poly-uridine stretches(Figure6A).RNA pull-down assays from21PCW frontal CP lysate revealed that FMRP had strong af?nity for each of the two NOS1-coding region GQs(GQ1and GQ2),but not GQ3or the U-rich regions(UR1–UR6)(Figure6B).To con?rm this,we synthesized a fragment of RNA representing both GQ1 and GQ2and performed an electrophoretic mobility shift assay (EMSA;Figure6C).In the presence of FMRP,this RNA exhibited a signi?cant shift that was abolished by the addition of excess nonbiotinylated‘‘cold’’RNA or a neutralizing FMRP antibody. To determine whether human GQ1and GQ2form RNA G-quad-ruplex structures,we used a reverse-transcription termination assay(Figure6D).Reverse-transcriptase activity pauses at sites of GQ structures in a cation-dependent manner(Schaeffer et al., 2001).Reverse transcription from both GQ1and GQ2RNA exhibited a signi?cant pause at the expected GQ site in the

NOS1 protein NADPH-diaphorase

**********

***

50 μm

Nos1 mRNA NOS1 protein NADPH-diaphorase

Emx1Non-Emx1

FACS + Nos1 qRT-PCR

Figure4.Discordant NOS1mRNA and NOS1Protein Expression Patterns in the Developing Human and Mouse Neocortex

(A)Nissl staining,FEZF2and NOS1in situ hybridization,NOS1immunohistochemistry,and NADPH-d histochemistry in adjacent sections from18PCW FOp, ACC,and dorsal lateral prefrontal cortex(PFC).NOS1mRNA was abundantly present in all cortical regions and layers examined.Intense NOS1and NADPH-d labeling in L5pyramidal columns(asterisks)were present in the FOp.In all examined cortical regions,NOS1and NADPH-d were present in interneurons(open arrowheads)and neuropil.Red boxes represent areas enlarged.

(B)Nos1in situ hybridization,NOS1,and NADPH-d staining in adjacent sections from P3mouse frontal neocortex.Nos1mRNA was widely present;intense NOS1 and NADPH-d stainings were exclusively present in interneurons(open arrowheads).Neuropil was weakly stained.MZ,marginal zone;SP,subplate.

(C)Pyramidal neurons of the Emx1lineage were isolated from the P3mouse neocortex by?uorescent cell sorting(FACS)and analyzed by quantitative(q) RT-PCR.Nos1mRNA was abundantly present in pyramidal neurons.Error bars represent the5th and95th percentiles of four measurements.

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presence of potassium,which facilitates GQ formation,but not lithium,which abrogates it.Therefore,FMRP interacts with GQ-forming sequences found within the coding region of human NOS1mRNA.

Evolution of NOS1mRNA GQ-Containing Sequences

To investigate whether GQ motifs are present in other mammals,we analyzed the 21species for which NOS1mRNA sequence was available.Highly stable tetrads at both GQ1and GQ2positions were predicted only in the great apes and macaque monkey (Figure S5A).Among great apes,which otherwise have perfectly conserved GQs,only orangutan has a point mutation that leads to a less stable two-stack GQ1quartet,but a fully conserved GQ2quartet.In marmoset,a New World monkey,and nonprimate mammals,with the excep-tion of the guinea pig that exhibited one quartet,they are absent from both positions.Further analysis of the entire NOS1coding region in nine placental mammals revealed a very high degree of conservation (Figure S5B),with the vast

majority of substitutions being synonymous.The few nonsynon-ymous substitutions,however,were selectively clustered in the GQ region.This marked reduction in amino acid identity in an otherwise highly conserved protein is consistent with the hypothesis that the sequences containing the GQ motifs evolved and made possible posttranscriptional regulation by FMRP.Furthermore,these sequences have remained quite stable since their emergence in catarrhine primates,which is consistent with the expression of NOS1in human and macaque pyramidal neurons.

FMRP Increases NOS1Expression via Interaction with a GQ-Containing Sequence

To test the functional consequences of FMRP on NOS1transla-tion,we cotransfected human expression constructs of FMRP (CAG-h FMR1)and NOS1(CAG-h NOS1)into Neuro-2a cells and quanti?ed NOS activity (Figure 6E).With CAG-h FMR1co-transfection,NOS1activity was increased in a dose-dependent manner,by up to 3.6-±0.9-fold (p =0.043),indicating that

N O S 1G A P D H G F P

N e o R

250

1309572553628

NOS1-enriched band (~75kDa)mRNA pull-down (human 21 PCW neocortex lysate)

Silver staining

Western blot (αFMRP)

N O S 1G A P D H G F P N e o R FMRP RIP

I n p u t

FMRP (75kDa)

P e r c e n t a g e o f i n p u t m R N A c a p t u r e d

k D a GAPDH

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MAP1B

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Gapdh

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Map1b

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I g G αF M R P P e r c e n t a g e o f i n p u t m R N A c a p t u r e d

NOS1FMRP DAPI Merge

F O p (20 P C W )

L 5 p y r a m i d a l n e u r o n s

S u b p l a t e i n t e r n e u r o n s Figure 5.FMRP Binds NOS1mRNA in the Human Fetal Neocortex

(A)Proteins eluted from an mRNA pull-down assay using lysates of a 21PCW human neocortex were analyzed by silver staining and immunoblotting.NOS1,but not control (GAPDH ,GFP ,and NeoR ),mRNA speci?cally associated with an $75kDa protein that was immunopositive for FMRP.Asterisk indicates an artifact of gel transfer.

(B)NOS1(green),FMRP (red),and DAPI (blue)staining of 20PCW FOp.FMRP was coexpressed by L5columnar NOS1+pyramidal neurons (solid arrowheads),but not most interneurons (arrows).

(C and D)FMRP immunoprecipitated mRNAs from a 21PCW human and P0mouse CP were analyzed by quantitative RT-PCR.Relative to control GAPDH and MAP1B mRNAs,FMRP strongly associated with NOS1mRNA (green bar)in human,but not mouse.Error bars represent the 5th and 95th percentiles of four measurements.See also Figure S4.

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FMRP acts as a positive regulator of NOS1expression.No increase in NOS1activity occurred when a mouse Nos1construct (CAG-m Nos1)or a human FMR1construct harboring the I304N mutation (CAG-h FMR1[I304N])(Siomi et al.,1994)was used,or when the GQ-containing sequence of the human NOS1was replaced with the orthologous sequence from mouse Nos1(CAG-murinized-h NOS1).Therefore,the FMRP-mediated increase in NOS1expression is dependent on the species of the NOS1sequence,the intact KH2domain of FMRP,and the

presence of GQ-containing sequences in the NOS1mRNA.To speci?cally examine the GQ region,we cloned the NOS1sequences containing GQ1and GQ2into the 30UTR of SV40-GL3and performed luciferase assays in Neuro-2a cells (Figure 6F).The inclusion of the human NOS1GQs (SV40-GL3-h NOS1-GQs)led to signi?cant dose-dependent increases in luciferase activity in response to CAG-h FMR1,indicating that FMRP increases NOS1translation via binding to these sequences.Importantly,a mouse FMRP expression

construct

GAT GGGG CCTC GGG TCCC GGG AAT GGG CCTCA AGT GGG AGCAGA GGGG TCAA GGG A GGGG CACC GAT GGGG CCTC GGG TCCC GGG AAT GGG CCTCA AGT GGG AGCAGA GGGG TCAA GGG A GGGG CACC GAT GGGG CCTC GGG TCCT GGG AAT GGG CCTCA AGT GGG AGCAGA GGGG TCAA GGG A GGGG CACC

GACA G A G TCACA GG TCTG GG TAAT GG CCCTCA AGT GGG AGCAAA G CCACCAACA G A GGGGG ACC GACA GGG TCCCA GG TCCCA G TAAC GG ACCTCA GGT GGGGG AAAA G CA G TCAACA G A GGGGG ACC ** * * * * **** * ** ** ***** ***** * * ** *** ****** ***

GQ1

GQ2

GQ1

GQ2

GQ3

UR1

UR2

UR3

UR4

Western blot

αFMRP

NOS1 RNA pull-down

UR6

UR5

Reverse transcription

termination assay GQ1GQ2

K +

Li +

10bp

Full-length

RT bands

NOS1 RNA EMSA

+ + + + + + + + +

D E

79bp 63bp

Colorimetric NOS assay

Luciferase assay

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Figure 6.FMRP Binds Human NOS1GQ-Containing Sequences and Enhances Human,but Not Mouse,NOS1mRNA Translation

(A)Prediction of putative FMRP-binding GQ and U-rich (UR)motifs in the human NOS1mRNA sequence and alignment of GQ1and GQ2.GQ1and GQ2were highly conserved in primates,but not rodents.

(B)FMRP association with each putative binding motif was analyzed by an mRNA pull-down assay using 21PCW human neocortex lysates.FMRP selectively associated with GQ1and GQ2.

(C)EMSA of GQ1and GQ2.RNA containing both GQs exhibited a signi?cant shift in mobility in the presence of FMRP.This shift was abolished by addition of excess unbiotinylated (‘‘cold’’)RNA or a neutralizing anti-FMRP antibody.

(D)Reverse-transcription termination assay.Reverse transcription paused at the expected GQ sites for both GQ1and GQ2in the presence of K +,which facilitates GQ formation,but not Li +,which disrupts it.

(E)Colorimetric NOS assays in Neuro-2a cells cotransfected with CAG-h FMR1or CAG-h FMR1(I304N)and one of CAG-h NOS1,CAG-m Nos1,or CAG-murinized-h NOS1.NOS activity from h NOS1,but not m Nos1or murinized h NOS1,increased dose dependently with increasing wild-type FMRP.The I304N mutation in FMRP abolished its activation of h NOS1translation.*p <0.05.Error bars represent the 5th and 95th percentiles of four measurements.

(F)Luciferase assays in Neuro-2a cells transfected with an empty reporter construct (GL3-empty),or constructs containing the human NOS1GQs (GL3-h NOS1[GQs])or the orthologous sequence in mouse Nos1(GL3-m NOS1[GQortholog]).Luciferase activity in cells transfected with GL3-h NOS1(GQs)increased dose dependently with cotransfection of human CAG-h FMR1(solid blue line)or mouse CAG-m Fmr1(broken blue line).Luciferase activity from the GL3-m Nos1(GQortholog)decreased with increasing amounts of CAG-h FMR1(solid red line).Error bars represent the 5th and 95th percentiles of six measurements.See also Figure S5.

906Cell 149,899–911,May 11,2012a2012Elsevier Inc.

(CAG-m Fmr1)dose dependently increased luciferase expres-sion in a manner highly similar to CAG-h FMR1.However,when the human NOS1GQ sequences were replaced with the orthologous region of the mouse Nos1(SV40-GL3-m Nos1-GQortholog),FMRP failed to enhance luciferase activity.These results indicate that FMRP activates NOS1protein expression via binding to a sequence containing GQ motifs and that this interaction exhibits species differences.Together,these data strongly support a scenario wherein FMRP activation of NOS1translation evolved through NOS1nucleotide substitu-tions that gave rise to a GQ-containing sequence targeted by FMRP.Mouse Pyramidal Neurons Ef?ciently Translate Human NOS1in an FMRP-Dependent Manner

Because mouse FMRP is able to enhance human NOS1expres-sion,we hypothesized that exogenous human NOS1mRNA can be ef?ciently translated in mouse pyramidal neurons,likely in an Fmr1-dependent manner.To test this in vivo,we introduced a NOS1expression construct with the CAG-Gfp reporter into mouse neocortical ventricular zone (VZ)using in utero electro-poration (IUE)at E13.5to target L5pyramidal neurons.At P0,the majority of CAG-h NOS1-electroporated L5pyramidal neurons expressed NOS1protein at high levels (Figures 7A–7C).In contrast those electroporated with CAG-m Nos1or

NOS1

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Figure 7.Ef?cient Translation of Human NOS1in Pyramidal Neurons Requires FMRP and Is Severely Reduced in Fetal FXS Neocortex

(A–C)Neocortex of wild-type or Fmr1KO mouse electroporated in utero at E13.5and immunostained for NOS1(red)at P0.In wild-type neocortex the majority of

pyramidal neurons transfected with h NOS1expressed high levels of NOS1properly localized to the soma and apical dendrite.NOS1protein expression from m Nos1or murinized-h NOS1in wild-type and from h NOS1in Fmr1KO neocortex was dramatically reduced in comparison.The ?uorescent intensity of NOS1staining normalized to GFP (B)and the proportion of GFP +cells expressing NOS1(C)were quanti?ed.*p <0.05.Boxes represent the 25th ,50th ,and 75th percentiles.Error bars represent the 5th and 95th percentiles of at least four animals.

(D)Immunoblots of human fetal FXS and P0mouse Fmr1KO neocortex.Normalized to GAPDH levels,the neocortical expression of NOS1protein was severely reduced in both human fetal FXS cases.In neonatal mouse neocortex,loss of Fmr1did not alter neocortical NOS1expression.See also Figure S6.

Cell 149,899–911,May 11,2012a2012Elsevier Inc.907

CAG-murinized-h NOS1expressed only low levels of NOS1in a minority of neurons,indicating reduced protein expression ef?-ciency consistent with our in vitro assays.Human and mouse NOS1exhibited similar somatodendritic localization(Figures 7A and7B).Therefore,our results show that mouse pyramidal neurons possess all of the cellular machinery necessary for the translation of human NOS1protein and suggest that their dimin-ished expression of endogenous NOS1is a result of differences in the NOS1mRNA sequence between human and mouse.

To assess FMRP dependence,we further electroporated Fmr1knockout(KO)mice with CAG-h NOS1and CAG-Gfp (Figure7A).Both the number of GFP+neurons expressing NOS1and the levels of NOS1protein decreased signi?cantly compared to wild-type(Figures7B and7C).In addition, neurons cultured from E14.5Fmr1KO and transfected with CAG-h NOS1also exhibited a signi?cant reduction in NOS1 levels compared to control(Figure S6A;43.8%±7.8%reduc-tion;p=0.0065).These data show that FMRP is required for the ef?cient expression of human NOS1protein in pyramidal neurons.

Severe Reduction in NOS1Protein Levels in Developing Human FXS but Not Mouse Fmr1KO Neocortex

To determine whether NOS1protein levels are altered in human FXS cases,we performed immunoblotting of neocortex from con?rmed midfetal and postnatal FXS cases(15and18PCW; and9,22,and85years)and age-matched controls.Neocortical lysates normalized to GAPDH levels were immunoblotted for NOS1,FMRP,and GAPDH(Figure7D).Remarkably,in both fetal cases of FXS,neocortical NOS1protein levels were severely reduced compared to matched controls.Furthermore,this de?cit was age dependent,being very dramatic in the fetal cases,less so in the cases aged9and22years,and absent in the85years’specimen(Figure S6C).These results indicate that NOS1protein expression is greatly reduced in the devel-oping human FXS neocortex.Notably,neocortical NOS1levels were not affected in early postnatal Fmr1KO mice(Figures7D and S6B),indicating that the requirement of FMRP for NOS1 expression is species dependent.

DISCUSSION

In this study we demonstrate that human neocortical NOS1 expression is posttranscriptionally regulated by FMRP in a species-dependent manner.Molecular analyses revealed that FMRP binds GQ motif-containing sequences present in the coding region of human,but not mouse,NOS1mRNA and facil-itates NOS1protein expression.Concordantly,NOS1expres-sion is severely reduced in the developing FXS human,but not FMRP-de?cient mouse,neocortex.In the human neocortex, NOS1and FMRP are transiently coexpressed during synapto-genesis in subpopulations of pyramidal neurons in regions involved in speech,language,and complex social behaviors. Together,these?ndings provide a novel candidate mechanism and insights into the potential connectional pathology of FXS and possibly ASD.

Our analyses indicate that the FMRP-NOS1interaction emerged as result of closely clustered nucleotide substitutions within the otherwise highly conserved coding sequence of NOS1that gave rise to the GQ-containing motifs,occurring at the potential expense of protein integrity.FMRP binding to GQ motifs has been associated with translational repression (Bechara et al.,2009;Schaeffer et al.,2001).There is,however, a precedent for positive,activity-dependent posttranscriptional regulation in PSD-95(DLG4),which has an FMRP-binding GQ motif(Todd et al.,2003;Zalfa et al.,2007).Interestingly,NOS1 and PSD-95are functionally related.NOS1is anchored to the synaptic membrane via a physical interaction with PSD-95 (Brenman et al.,1996)and its enzymatic product,NO,S-nitrosy-lates PSD-95(Ho et al.,2011).Although the abundant presence of NOS1mRNA in pyramidal neurons suggests that translational regulation is involved,FMRP may also control the stability of the NOS1transcript in a manner similar to its control of PSD-95 mRNA stability(Zalfa et al.,2007).Furthermore,the GQ motif has been shown to mediate the dendritic localization of PSD-95(Dictenberg et al.,2008)and may also play a role in NOS1mRNA targeting.The possibility that NOS1and PSD-95 are similarly regulated by FMRP is consistent with their shared postsynaptic localization,physical interaction,and related func-tions.The binding of FMRP to GQs has been demonstrated both in vitro(Bagni and Greenough,2005;Bassell and Warren,2008) and in vivo(Rackham and Brown,2004;Iioka et al.,2011). Recently,however,it was shown that the presence GQ motifs is not predictive of FMRP binding(Darnell et al.,2011).Therefore, the context dependence of FMRP interactions with GQs remains to be fully elucidated,and individual potential interactions should be validated empirically.

Animal models of FXS exhibit multiple phenotypes present in human FXS,indicating that many aspects of FMRP function are well conserved.Therefore,any contribution of NOS1to the FXS phenotype would likely involve the higher cognitive func-tions that are absent from mouse.This possibility is supported by the coexpression of NOS1and FMRP in projection neurons of the FOp and the ACC and adjacent dorsal frontoparietal neocortex.The FOp encompasses the future Broca’s area and its contralateral hemisphere equivalent,as well as the orofacial motor cortex,regions involved in speech production,language comprehension,and action recognition(Keller et al.,2009). The ACC is involved in decision making,attention,emotional processing,and social awareness(Devinsky et al.,1995). NOS1expression in these regions is also temporally regulated from midgestation to early infancy,a developmental period crit-ical for early synaptogenesis,dendritic spine formation,and ingrowth of cortical afferents(Kang et al.,2011).Therefore,the neuroanatomical localization and timing of the FMRP-NOS1 interaction are consistent with a putative role in the development of neocortical circuits,including those involved in linguistic and social functions likely affected in FXS and ASD.

This potential role of NOS1in the development and function of human neural circuits is further supported by studies of a human NOS1hypomorphic allele,which has been associated with attention de?cit hyperactivity disorder(ADHD),impulsivity,and aggression(Reif et al.,2009),behavioral features often comorbid with FXS(Rogers et al.,2001).This NOS1hypomorphism has also been associated with hypoactivity in the ACC(Reif et al., 2009),a cortical region with prominent midfetal pyramidal

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expression of NOS1protein.Functional imaging studies re-vealed a similar reduction in ACC activation in patients with FXS and ADHD during attentional-processing tasks(Bush et al.,1999;Menon et al.,2004)and in autistic children in response to a familiar face(Pierce and Redcay,2008).The over-lapping de?cits between NOS1hypomorphism,FXS,and FXS comorbidities are consistent with a functional role of NOS1in human brain circuitry related to FXS and ASD.Furthermore, the two midfetal neocortical regions with prominent pyramidal NOS1expression,the ACC and FOp,exhibit highly coordinated resting state activity,suggesting functional connectivity between the two areas and the presence of a cingulo-opercular cognitive network(Power et al.,2011).Interestingly,the connectivity of this cingulo-opercular network has been reported to be impaired in schizophrenia(Tu et al.,2012).Remarkably,multiple sequence variations in NOS1have been associated with schizophrenia (Cui et al.,2010;Reif et al.,2006;Shinkai et al.,2002),supporting a potential role of NOS1in the formation of cognitive circuits and in disorders that affect cognition.

Structural alteration in the organization of minicolumns has been reported in autism and other psychiatric disorders(Casa-nova et al.,2002).In this study we found that within the midfetal FOp,alternating L5columns coexpress FMRP and NOS1,as well as FOXP2,which is implicated in the development of speech,language,and cognition(Lai et al.,2001),functions affected in FXS and ASD.We also showed that neurons within the same column have a shared subcortical molecular identity and connectivity.Positioned in between the columns are migra-tory corridors containing radial glial?bers and corticocortical projection neurons en route to the super?cial layers.Thus,this fetal organization may have implications for the developmental basis of normal minicolumns(Rakic,1988),as well as columno-pathies(Casanova et al.,2002).Interestingly,the NOS1+ columnar neurons of the fetal FOp share some areal and projec-tion properties with adult mirror neurons,which are present in macaque area F5(Rizzolatti and Craighero,2004),an area equiv-alent to the human Broca’s area,and project subcortical axons (Kraskov et al.,2009).Mirror neurons,which are activated during both the observation and execution of a particular goal-directed action,are thought to contribute to theory of mind and language abilities(Rizzolatti and Craighero,2004),and in autistic children the mirror neuron activity that is normally observed in the FOp is absent(Dapretto et al.,2006).Therefore,the molecular pro?le of NOS1+columns,as well as their shared location and connec-tivity with mirror neurons,is consistent with a potential role in motor and cognitive development.

The synthesis of NO,a short-lived gas that cannot be stored or transported,must be precisely regulated and amenable to rapid, localized activation.Because FMRP controls both the dendritic localization and translation of target mRNAs,it is well suited to contribute to the dynamic regulation of NOS1activity.It should be noted,however,that NOS1mRNA may also be under addi-tional,perhaps negative,posttranscription control,as suggested by the lack of NOS1protein expression in the majority of NOS1 mRNA-expressing pyramidal neurons.The modulation of neuronal function by NO in the brain has been widely studied, and postsynaptic NO is thought to represent a retrograde signal that promotes presynaptic differentiation(Bredt and Snyder,1994;Garthwaite,2008).Blockade of NOS1function has been shown to disrupt synapse formation and result in spine loss (Nikonenko et al.,2008).Given the potential role of NO in synapse development,the loss of NOS1expression in the fetal FXS brain during early synaptogenesis may contribute to the dendritic spine phenotype of human FXS(Irwin et al.,2000). Studies have also shown that NO mediates neuronal synchroni-zation(O’Donnell and Grace,1997)and can modulate protein function via S-nitrosylation(Jaffrey et al.,2001),including that of histones(Nott et al.,2008),which can mediate transcriptome changes.In future studies it will be important to characterize the mechanisms of NO function in the developing human neocortex and their potential contribution to FXS.

EXPERIMENTAL PROCEDURES

Human Brain Tissue Processing

The sources and methods for the collection,dissection,and?xation of control and fragile X postmortem human tissues are described in the Extended Exper-imental Procedures.All specimens were collected under guidelines approved by institutional review boards and anonymized prior to our receipt.Fixed tissues were sectioned by vibratome or cryostat.For NADPH-d staining, sections were incubated in b-NADPH,nitro blue tetrazolium,and Triton X-100.Sections were preincubated in hydrogen peroxide for immunohisto-chemistry or directly preblocked in blocking solution for immuno?uorescent staining prior to incubation with primary antibodies followed by biotinylated or?uorophore-conjugated secondary antibodies.For immunohistochemistry, sections were further incubated in avidin-biotin-peroxidase complex and visualized using DAB.

RNA Pull-Down Assay and RIP

For pull-down assays,RNAs were transcribed from cDNA or PCR products, biotinylated,and captured using streptavidin beads.Human midfetal CP lysates were added,and bound proteins were analyzed by SDS-PAGE,silver staining,and immunoblotting.For RIP,FMRP-bound mRNAs were immuno-precipitated from midfetal human and neonatal mouse CP lysates and analyzed using quantitative RT-PCR.

Expression Assays and IUE

The generation of DNA constructs is described in the Extended Experimental Procedures.Neuro-2a cells were transfected by lipofection.Luciferase or NOS activity was assayed48hr after transfection and normalized to transfection ef?ciency controls.For electroporation,DNA was injected into the lateral ventricles of embryonic mice and transferred into VZ cells by40V pulses.Elec-troporated brains were analyzed at P0by immunostaining.All experiments using animals were carried out in accordance with a protocol approved by Yale University’s Committee on Animal Research and National Institutes of Health guidelines.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures and six?gures and can be found with this article online at doi:10.1016/j.cell. 2012.02.060.

ACKNOWLEDGMENTS

We thank D.Budin s c ak,Z.Cmuk,Z.Krsnik,S.Liu-Chen, B.Popovi c, B.Poulos,and B.Sajin for help with tissue acquisition;M.Nakane,W.Sessa, and I.Grkovic for antibodies and reagents;F.Cheng and M.Li for analyzing transcriptome data; E.Gulcicek for help with mass spectrometry;and M.Brown,L.Kaczmarek,A.Louvi,M.Gu¨nel,M.State,and members of the N. https://www.wendangku.net/doc/8615752655.html,boratory for discussions and comments.E.J.H.and D.H.R.received technical support from the Pediatric Neuropathology Research Lab at Cell149,899–911,May11,2012a2012Elsevier Inc.909

University of California,San Francisco(UCOP Award#142675).D.H.R.is a HHMI Investigator.A.M.M.S.is supported by a fellowship from the Portu-guese Foundation for Science and Technology.This work was supported by grants from National Institutes of Health(to M-R.R.,K99NS064303;to P.R., DA023999;to N. S.,MH081896,MH089929,and NS054273),ZonMw912-07-022(to R.W.),Kavli Foundation,NARSAD,and James S.McDonnell Foun-dation Scholar Award(to N. S.).

Received:September9,2011

Revised:December19,2011

Accepted:February15,2012

Published:May10,2012

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