Reversible Inhibition of PSD-95 mRNA Translation by miR-125a ...

Molecular Cell

Article

Reversible Inhibition of PSD-95mRNA Translation by miR-125a,FMRP Phosphorylation, and mGluR SignalingRavi S. Muddashetty,1 Vijayalaxmi C. Nalavadi,1 Christina Gross,1 Xiaodi Yao,1 Lei Xing,1 Oskar Laur,3

Stephen T. Warren,4,5 and Gary J. Bassell1,2,*1Department of Cell Biology2Department of Neurology3Department of Microbiology4Department of Human Genetics5Department of Biochemistry

Emory University School of Medicine, Atlanta, GA 30322, USA*Correspondence: [email protected]

DOI 10.1016/j.molcel.2011.05.006

SUMMARY

The molecular mechanism for how RISC and micro-RNAs selectively and reversibly regulate mRNAtranslation in response to receptor signaling isunknown but could provide a means for temporaland spatial control of translation. Here we showthat miR-125a targeting PSD-95 mRNA allowsreversible inhibition of translation and regulation bygp1 mGluR signaling. Inhibition of miR-125a in-creased PSD-95 levels in dendrites and altereddendritic spine morphology. Bidirectional control ofPSD-95 expression depends on miR-125a andFMRP phosphorylation status. miR-125a levels atsynapses and its association with AGO2 are reducedin Fmr1 KO. FMRP phosphorylation promotes theformation of an AGO2-miR-125a inhibitory complexon PSD-95 mRNA, whereas mGluR signaling oftranslation requires FMRP dephosphorylation andrelease of AGO2 from the mRNA. These findingsreveal a mechanismwhereby FMRP phosphorylationprovides a reversible switch for AGO2 andmicroRNAto selectively regulate mRNA translation at synapsesin response to receptor activation.

INTRODUCTION

MicroRNAs (miRs) are small, conserved, noncoding RNAs that

act in association with the RNA-induced silencing complex

(RISC) to regulate gene expression posttranscriptionally (Bartel,

2004). miR-mediated translational repression may occur at pre-

and/or postinitiation steps (Abu-Elneel et al., 2008; Filipowicz

et al., 2008; Lee et al., 1993; Nottrott et al., 2006; Richter,

2008). Numerous miRNAs are expressed at high levels in the

nervous system and regulate development (Kosik, 2006), spine

morphology, and synapse function (Schratt, 2009b). miRNAs

are implicated in neurological and neuropsychiatric disease

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(Kocerha et al., 2009; Kosik, 2006; Lee et al., 2008). A subset

of miRNAs are localized to dendrites (Kye et al., 2007; Schratt

et al., 2006; Siegel et al., 2009), where local translationmay affect

dendritic spinemorphology (Schratt, 2009b; Schratt et al., 2006).

A critical gap is understanding how the repression of target

mRNA translation by RISC components and miRNAs may be

dynamically regulated by receptor signaling pathways. The

ability of a cell to dynamically regulate RISC interactions with

target mRNAs would allow for more precise temporal and spatial

control of miRNA function than achieved solely by regulation of

miRNA biogenesis. The physiological signals and molecular

mechanisms that promote or inhibit the association of RISC

complexes onto specific mRNAs are unknown.

A major advance in our understanding of the regulation of

miRNA-mediated translation has been the evidence of revers-

ibility in cultured dividing cells deprived of serum. The interaction

of mRNA-binding proteins with cis-acting elements can affect

miRNA activity (Bhattacharyya et al., 2006; Vasudevan et al.,

2007). These studies suggest the hypothesis that mRNA-binding

proteins may act as molecular intermediates downstream of

receptor signaling pathways to modulate the interactions of

RISC-associated miRNAs to target sequences. Since specific

mRNA binding proteins can be localized nonuniformly within

the cytoplasm to influence mRNA localization, they are uniquely

positioned to co-opt RISC as a means to regulate local mRNA

translation in polarized cells.

Dynamic regulation of synaptic protein synthesis in response

to activation of neurotransmitter receptors, such as group 1

metabotropic glutamate receptors (gp1 mGluRs), plays a key

role in long-term synaptic plasticity underlying learning and

memory (Bramham and Wells, 2007; Martin and Ephrussi,

2009). MicroRNAs appear to be ideally suited to reversibly inhibit

mRNA translation at synapses in response to receptor signaling

(Chang et al., 2009; Kosik, 2006). This, in principle, could provide

selective, bidirectional, and spatial control for the regulation of

mRNA translation. We sought to identify a molecular mechanism

whereby a specificmRNAcanbe selectively silencedby amiRNA

through RISC components, and to investigate whether a selec-

tive mRNA-binding protein in response to physiological signals

may reversibly regulate these interactions.

olecular Cell 42, 673–688, June 10, 2011 ª2011 Elsevier Inc. 673

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http://dx.doi.org/10.1016/j.molcel.2011.05.006

Figure 1. Activation of gp1 mGluR Stimulates Translation of a PSD-

95 30UTR Reporter and Its Dissociation from AGO2

(A) Relative luciferase activity (firefly luciferase/renilla luciferase) from cultured

cortical neurons (DIV 14) transfected with either f-luciferase-PSD-95 30UTR or

f-luciferase-b-actin 30UTR (n = 4, p = 0.01, paired t test, ±SD). Values were

normalized to basal levels. gp1 mGluR activation was carried out by treating

neurons with 50 mM DHPG, a gpI mGluR agonist for 15 min.

(B) f-luciferase mRNA ratio (pellet/supernatant) with an eIF2C2 (AGO2)

immunoprecipitate from cultured cortical neurons was analyzed by qPCR

(n = 3, paired t test, ±SD). Values were normalized to basal levels. There was

significant dissociation of f-luciferase-PSD-95 30UTR (p = 0.001) and endog-

enous PSD-95 mRNA (p = 0.004) from AGO2 upon DHPG treatment (also see

Figure S1).

(C) Input and pellet of AGO2 immunoprecipitate from cultured cortical neurons

(basal and DHPG treated) were immunoblotted with eIF2C2 (AGO2) antibody

(also see Figure S1).

Molecular Cell

PSD-95 Translation Regulation by FMRP and miR

Here we elucidate the molecular mechanism of reversible and

selective regulation of postsynaptic density protein 95 (PSD-95)

mRNA translation in response to stimulation of gp1 mGluRs.

miR-125a and fragile X mental retardation protein (FMRP) coop-

erate on the 30UTR of PSD-95 mRNA to enable inhibition and

confer flexibility allowing for translational activation at synapses

in response to receptor activation. Loss of FMRP causes fragile

X syndrome (FXS), the most common monogenetic form of

inherited intellectual disability and autism. FMRP is localized to

dendrites and synapses, where it regulates mRNA transport

and local protein synthesis necessary for neuronal development

and synaptic plasticity (Bassell and Warren, 2008). FMRP

interacts with mammalian eIF2C2 (AGO2) and associates with

miRNAs (Edbauer et al., 2010; Jin et al., 2004b). We show that

FMRP phosphorylation promotes the formation of an AGO2-

miR125a inhibitory complex on PSD-95 mRNA. Conversely,

mGluR stimulation leads to dephosphorylation of FMRP, release

of AGO2 from the mRNA, and activation of translation. The

control of translation by mGluR signaling to FMRP and AGO2

is on the timescale of minutes. This demonstrates that the phos-

phorylation status of an mRNA-binding protein can affect the

rapid and reversible control of miRNA-mediated translational

regulation. This study provides mechanistic insight into the

selective and cooperative interactions that may function as

reversible switches to dynamically regulate miRNA function,

and has important implications for understanding neuropsychi-

atric disorders resulting from altered regulation of miRNA

pathways.

RESULTS

mGluR-Mediated Translation of PSD-95 InvolvesMicroRNAsActivation of gp1 mGluRs rapidly stimulates the translation of

PSD-95 mRNA (Muddashetty et al., 2007). PSD-95 is a key

component of the postsynaptic molecular architecture whose

levels at the synapse are dynamically regulated (Bingol and

Schuman, 2004; Colledge et al., 2003). PSD-95 controls AMPAR

endocytosis, synaptic strength, and spine stabilization (Bhatta-

charyya et al., 2009; Xu et al., 2008; De Roo et al., 2008). Gp1

mGluRs are implicated in different forms of mGluR-mediated

synaptic plasticity that depend on new protein synthesis (Anwyl,

2009; Auerbach and Bear, 2010; Ronesi and Huber, 2008). In

addition, mGluR-driven synthesis of postsynaptic components,

such as PSD-95, may play an important role in the growth and/or

stabilization of dendritic spines (Schutt et al., 2009; Vanderklish

and Edelman, 2002).

To investigate the molecular mechanism of translational regu-

lation, a firefly luciferase reporter (f-luciferase) with the 30

untranslated region (UTR) of PSD-95 mRNA was expressed in

cultured cortical neurons. The reporter responded to brief

(15 min) mGluR activation, indicating the 30UTR of PSD-95

mRNA is sufficient to elicit activity-mediated regulation of PSD-

95 mRNA translation (Figure 1). (S)-3,5-dihydroxyphenylglycine

(DHPG), a specific agonist of group I mGluRs (gpI mGluR),

induced a rapid and significant increase in the expression of

f-luciferase-PSD-95 UTR (93% ± 32%) in neurons (Figure 1A).

To study the involvement of the microRNA pathway in mGluR-

674 Molecular Cell 42, 673–688, June 10, 2011 ª2011 Elsevier Inc.

mediated translation, we measured the association of luciferase

reporter mRNAs with eIF2C2 (mammalian homolog of AGO2,

henceforth referred to as AGO2), a major functional component

Molecular Cell


of the RISC. Quantitative measurement of f-luciferase mRNA in

the AGO2 immunoprecipitate using qPCR showed a significant

(44% ± 13%) decrease of f-luciferase-PSD-95 UTR in the pellet

from neurons stimulatedwith DHPG (Figure 1B). A similar DHPG-

induced decrease in the association with AGO2 was also

observed for endogenous PSD-95 mRNA (61% ± 20%) (Fig-

ure 1B), while endogenous b-actin mRNA was unaffected (see

Figure S1C available online). Treatment with DHPG had no

apparent effect on the stability of b-actin or PSD-95 UTR

reporters or endogenous mRNAs, as all mRNA levels tested

were unchanged (Figures S1A and S1B). In summary, the

30UTR of PSD-95 mRNA is sufficient to elicit mGluR-mediated

translational activation in neurons. Translational activation coin-

cides with the dissociation of the reporter mRNA (and endoge-

nous PSD-95 mRNA) from AGO2, suggesting a possible role of

microRNAs in this process. This led us to search for a micro-

RNA(s) that regulates PSD-95 mRNA translation.

miR-125a Regulates the Translation of PSD-95 mRNAAmong the few microRNAs predicted to target the 30UTR of

PSD-95 mRNA (TargetScan 4.1), we initially chose miR-1 and

miR-103 because their target site was conserved among species

and possessed a higher percent complementarity with PSD-95

mRNA. We also chose to test miR-125a, which was previously

predicted to target PSD-95 mRNA 30UTR (John et al., 2004;

and Figure 2A). In neuro2A cells, only the transfection of miR-

125a had a significant inhibitory effect on expression of f-lucif-

erase-PSD-95 UTR (�21% ± 6%), while miR-1 and miR-103

had no effect (Figure S2A); hence we pursued only miR-125a

for further studies. In cultured cortical neurons, overexpression

of miR-125a significantly reduced (�32%± 4%) the endogenous

PSD-95 levels as measured by quantitative immunoblots.

Antagonization of miR-125a by transfecting locked nucleic acid

(LNA)-antisense RNA oligonucleotides (antimiR-125a) led to

2.7 (±1)-fold increase in endogenous PSD-95 (Figures 2B and

2C). To further validate whether miR-125a targets PSD-95

mRNA, we generated f-luciferase-PSD-95 UTRmt125a by

mutating two nucleotides in the seed region of the UTR which

was expected to disable miR-125a binding to this mRNA (Fig-

ure 2D). In neuro2A cells miR-125a transfection had no effect

on the expression of f-luciferase-PSD-95 UTRmt125a, while

it significantly reduced the translation of f-luciferase-PSD-

95 UTR (Figure 2E), indicating that the two-nucleotide mutation

rendered the reporter unresponsive to miR-125a. Blocking

endogenous miR-125a by transfecting antimiR-125a signifi-

cantly increased expression of f-luciferase-PSD-95UTR (94% ±

47%), while it had no effect on f-luciferase-PSD-95 UTRmt125a

(Figure 2F). These data on the endogenous PSD-95 and lucif-

erase reporter with PSD-95 30UTR suggest that antimiR-125a

relieved the translational inhibition imposed on the PSD-95

30UTR containing an intact binding site for miR-125a.The effect

of antimiR-125a was dose dependent (Figure S2C) and specific

to PSD-95 30UTR, as it had no effect on f-luciferase-b-actin UTR

(Figure S2B). Mutation of themiR-125a-binding site (f-luciferase-

PSD-95 UTRmt125a) also resulted in a marked reduction of this

mRNA association with AGO2 (59% ± 20) compared to the wild-

type UTR, as measured by AGO2 immunoprecipitation and

q-PCR analysis of mRNA (Figure 2G). However, total levels of

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the mutant mRNA reporter did not differ from wild-type (data

not shown). This suggests that a single miR125a target site

may be largely responsible for AGO2-mediated translational

regulation in the context of the PSD-95 30UTR.Next, we investigated whether miR-125a had a role in mGluR-

mediated activation of PSD-95 mRNA translation in primary

neurons. DHPG treatment induced a significant increase in

the relative luciferase activity of a reporter with the PSD-95

30UTR in cultured cortical neurons, but when the reporters were

cotransfected with antimiR-125a this translational activation

wasblocked (Figure 2H). Activation ofmGluRswithDHPGhadno

effect on the translation of mutant f-luciferase-PSD-95mt125a

(Figure 2H) in cultured cortical neurons. DHPG-induced dissoci-

ation of PSD-95 UTR from AGO2 was also specifically abolished

by antimiR-125a (Figure 2I). These results indicate miR-125a

specifically targets the 30UTR of PSD-95 mRNA and is essential

for mGluR-mediated translation in neurons.

To test the effect of miR-125a on the expression of endoge-

nous PSD-95 protein in dendrites, cultured hippocampal

neurons were transfected with antisense oligonucleotides

against miR-125a (antimiR-125a) at DIV 10, and cells were fixed

and immunostained for PSD-95 protein after 48 hr (Figure 2J).

There was a significant increase (45% ± 13%) in the expression

of endogenous PSD-95 immunofluoresent signal in the distal

dendrites of neurons exposed to antimiR-125a, while transfec-

tion with scrambled sequence oligonucleotides or nonspecific

antimiR-124 did not cause any significant changes (Figure 2K).

We note that the miR-125a target site on the human 30UTR of

PSD-95 mRNA does not comply with the strict ‘‘seed rule,’’ as

there is a single nucleotide bulge on the microRNA and only five

out of six matches in the two to seven seed region (Figure 2A,

Figure S3A). Such bulges are reported in functional miR-mRNA

interactions previously (Brodersen and Voinnet, 2009; Ha et al.,

1996). Also of note, in the mouse 30UTR there is a ‘‘g instead

of a’’ compared to the seed region of human resulting in a toler-

ated G.U wobble (Figure S3A). The luciferase reporter bearing

the mouse PSD-95 UTR responded similarly to the luciferase

reporter with the human PSD-95 30UTR, both showing increased

translation in primary neurons in response to either antimiR-125a

or DHPG stimulation (Figure S3B). We also tested the effect of

restoring the perfect seed match for miR-125a in the mouse

PSD-95 30UTR (Figure S3C). The construct with a perfect seed

match responded to antimiR-125a and DHPG stimulation similar

to wild-type mouse and human PSD-95 30UTRs, indicting the

mismatch in the seed region resulting in a G.U wobble behaves

similarly (Figure S3C).

miR-125a Is Localized to DendritesOnly a small subset of neuronal microRNAs are localized to

dendrites and synapses (Kye et al., 2007). miR-125a is highly

expressed in neurons and abundant in synaptoneurosomal

fractions from mouse cerebral cortex (Figure 3A) compared

to several other microRNAs which are previously reported

in dendrites and at synapses (Figure 3A and Kye et al.,

2007; Schratt et al., 2006). There was a small but significant

reduction in miR-125a levels (�37% ± 23%) from synaptoneur-

osomes upon stimulation with DHPG (15min) indicating possible

mGluR-induced turnover (Figure 3B). Fluorescence in situ


Figure 2. miR-125a Targets the 30UTR of PSD-95 mRNA and Is Necessary for gp1 mGluR-Stimulated Translation

(A) Predicted miR-125a-binding site in the human 30UTR of PSD-95 RNA.

(B) Immunoblots depicting the effect of miR-125a overexpression (lanes 4 and 5) and antagonization (lanes 2 and 3) on the levels of endogenous PSD-95 in

primary cortical neurons. miR-124a is the negative control. Tubulin (lower panel) was the loading control.

(C) Quantification of endogenous PSD-95 levels in primary cortical neurons, normalized to tubulin in each lane (n = 3, p = 0.003, one-way ANOVA, Dunnett

test ±SD).

(D) The 30UTR PSD-95 mRNA reporter (FL, full length) was mutated in the seed region by site-directed mutagenesis to generate PSD-95-UTR-mt125a which

would be insensitive to miR-125a (f-luciferase-PSD-95 UTRmt125a).

(E) Relative luciferase activity from neuro2A cells transfected with either f-luciferase-PSD-95 UTR or f-luciferase-PSD-95 UTRmt125a (n = 3, p = 0.01, one-

way ANOVA, Dunnett’s test ±SD). miR-125a or miR-124 was cotransfected with luciferase reporters; values are normalized to the control (no miR

overexpression).

(F) Relative luciferase activity from neuro2A cells transfected with either f-luciferase-PSD-95 UTR or f-luciferase-PSD-95 UTRmt125a (n = 3, p = 0.02, one-way

ANOVA, Dunnett’s test ±SD). AntisensemiR-125a or miR-124 oligonucleotides were cotransfected with luciferase reporters; values are normalized to the control

(no antimiR).

(G) f-luciferase mRNA ratios of pellet/supernatant from the AGO2 immunoprecipitate of neuro2A cells that were transfected with either f-luciferase-PSD-95 UTR

or f-luciferase-PSD-95 UTRmt125a, analyzed by qPCR. Values are normalized to f-luciferase-PSD-95-UTR (n = 3, p = 0.01, paired t test, ±SD).

Molecular Cell



Molecular Cell


hybridization (FISH) showed that miR-125a is present in distal

dendrites of cultured hippocampal neurons (Figure 3C). Quanti-

tative in situ hybridization also revealed a reduced miR125a

signal in dendrites after 15 min stimulation of the neurons with

DHPG (Figure 3D).

Dendritic localization of miR-125a was further validated by

FISH on sections of mouse brain. miR-125a was visualized

clearly in the dendritic layers of hippocampus in CA1 region (Fig-

ure 3E), in contrast to miR-298, which was restricted to the cell

body layer with almost undetectable signal in the dendrites (Fig-

ure 3E). Our results are consistent with a previous report that

miR-298 is highly expressed in neurons, but almost absent

from dendrites (Kye et al., 2007). The specificity of the

miR125a signal in dendrites was demonstrated by FISH experi-

ments using LNA probes that carriedmismatchmutations at only

two positions (miR-125a-MM2) within the 125a-specific probe

(Figure S3D). This led to significant decrease (�50% ± 5%) in

fluorescent intensity compared to miR-125a probe (Figure S3E).

Mismatch probes showed strong reduction of dendritic signal in

cultured cells (Figure S3D) and on brain slices, respectively (Fig-

ure S3F). These results suggest the possibility that miR-125a

may function locally in dendrites and at synapses.

Inhibiting miR-125a Affects Spine Densityand BranchingThe localization of miR-125a to dendrites, its abundance in syn-

aptoneurosomes, and its involvement in anmGluR pathway sug-

gest miR-125a might function to regulate local protein synthesis

affecting dendritic spine-synapse morphology (Schratt, 2009b;

Schratt et al., 2006). Blocking miR-125a by transfecting anti-

miR-125a had a marked effect on the spine morphology of

cultured hippocampal neurons (Figure 4A). Analysis of dendritic

spines in cultured hippocampal neurons (transfected at DIV 10,

fixed, and immunostained at DIV 14) showed a 33% (±3%)

increase in spine density (Figure S4A) and a 3-fold increase in

spine branches (300.9% ± 14.0%) in neurons transfected with

antimiR-125a compared to neurons transfected with scrambled

RNA (Figures 4D and 4E). Neurons transfected with antimiR-

125a also showed increased number of synapsin punctae

compared to scrambled RNA transfected neurons, suggesting

increased synapse number in these neurons (Figure S4C).

Since miR-125a targets PSD-95 mRNA and transfection of

antimiR-125a leads to increased endogenous PSD-95 (Figures

2A–2C), we tested the effect of PSD-95 knockdown on the spine

phenotype observed by antagonization of miR-125a. Specific

siRNAs against PSD-95 mRNA (Fan et al., 2009) significantly

reduced PSD-95 protein levels in hippocampal neurons (Figures

(H) Relative luciferase activity from cultured cortical neurons (untreated or stimu

erase-PSD-95 UTRmt125a. AntimiR-125a was cotransfected with luciferase repo

mean of three independent experiments (p = 0.01, paired t test, ±SD).

(I) The ratio of f-luciferase-PSD-95 UTR mRNA, analyzed by qPCR, in AGO2 imm

antimiR-125a or antimiR-124 (n = 3, p = 0.03, paired t test, ±SD) with and withou

(J) Immunofluorescence localization of endogenous PSD-95 protein in cultured

cleotides as a negative control. Cultured neuronswere transfected at DIV 10 and fi

and Cy3- fluorochrome-conjugated anti-mouse antibody.

(K) Quantification of fluorescent intensities measured in distal dendrites of hippoc

antimiR-125a, or antimiR-124 oligonucleotides (n = 23 neurons from three inde

Values were normalized to the scrambled (also see Figures S2 and S3).

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4B and 4C), while the control (scrambled) siRNA had no effect.

The reduced PSD-95 completely rescued the increased spine

branching phenotype caused by antimiR-125a (Figures 4B, 4D,

and 4E). It also significantly lowered the spine density increase

caused by antimiR-125a (Figure S4B). Control siRNA had no

effect on either spine density or branching. These data suggest

that the excessive spine density and spine branching observed

by antagonization of miR-125a is mediated by the increased

synthesis of PSD-95.

FMRP Controls miR-125a-Mediated Regulationof PSD-95 mRNA TranslationFMRP, the deficiency of which causes FXS, is reported to regu-

late the mGluR-mediated translation of postsynaptic proteins

(Schutt et al., 2009), including PSD-95 (Muddashetty et al.,

2007). Based on previous reports that FMRP directly binds to

the 30UTR of PSD-95 mRNA (Zalfa et al., 2007), we confirmed

that FMRP is associated with the f-luciferase with the 30UTR of

PSD-95 mRNA (Figure S5F). We then tested the effect of

FMRP on the translation of this reporter by depleting FMRP in

neuro2A cells by specific siRNA (Figure S5J). FMRP knockdown

significantly increased the translation of f-luciferase-PSD-

95 UTR (Figure S5K), while it had no effect on f-luciferase-b-actin

UTR. These data show that FMRP is necessary for translational

inhibition imposed on the 30UTR of PSD-95 mRNA. In primary

cortical neurons from Fmr1 KO mice, translation of the trans-

fected f-luciferase-PSD-95 UTR was significantly higher than

(+205% ± 50%) wild-type neurons, but coexpression of FMRP-

WT in KO neurons sharply reduced the translation of the reporter

(Figure S5E). This indicates an increased translation of PSD-95

mRNA in Fmr1 KO neurons on transient transfection of the

f-luciferase-PSD-95 UTR reporter, an effect that can be rescued

by coexpression of FMRP-WT.

The interaction between FMRP and PSD-95mRNA 30UTR in

cells, as shown by an IP and qPCR assay, was validated by addi-

tional experiments. When FMRP and PSD-95 30UTR were ex-

pressed in separate cultures, lysed, and mixed before immuno-

precipitation (Figure S5G), their interaction was significantly

lower (�78% ± 12%) compared to when they are expressed

together in the same cells. We also tested whether FMRP inter-

acts directly with the region of the 30UTR in vivo, by using a strin-

gent UV-crosslink-IP (CLIP) method (Figures S5H and S5I) (Ule

et al., 2005). A significant reduction (�64% ± 5%) in the binding

of f-luciferase mRNA where the FMRP binding region is deleted

(f-luci-PSD-95 UTRDGR) compared to full-length UTR indicates

that this region (which includes the miR-125a binding site) is

essential for FMRPbinding to PSD-95mRNA in vivo prior to lysis.

lated with DHPG) transfected with either f-luciferase-PSD-95 30UTR or f-lucif-

rter where indicated. Values were normalized to basal levels and represent the

unoprecipitates from cultured cortical neurons that were cotransfected with

t DHPG stimulation. The values were normalized to basal levels.

hippocampal neurons transfected with antimiR-125a or scrambled oligonu-

xed at DIV 12. Immunofluorescencewas performed using anti-PSD 95 antibody

ampal neurons (>40 mm from cell body) that were transfected with scrambled,

pendent experiments, p = 0.042, one-way ANOVA, Bonferroni’s test, ±SEM).


Figure 3. miR-125a Is Present in Dendrites and Synapses

(A) Quantitative measurement of selected microRNAs in mouse cortical synaptoneurosomes by qPCR. Values are expressed as 2-difference in the Ct between

synaptoneurosomes to total cortical extract (n = 3, ±SD).

(B) Quantitativemeasurement of the changes inmiR-125a andmiR-124 levels in cortical synaptoneurosomes in response to 150 DHPG stimulation (n = 4, p = 0.02,

paired t test, ±SD).

(C) FISH for miR-125a using a digoxigenin labeled LNA probe in cultured hippocampal neurons. Inset of a selected dendritic segment is enlarged (top) to illustrate

punctate signal. The right-side panel is an overlay of fluorescent and DIC (differential interference contrast) images. Bottom panels depict digoxigenin labeled

LNA probes specific for the U6-RNA and scrambled probe as a negative control.

(D) Quantitative analyses of miR125a-specific FISH experiments on hippocampal neurons (DIV 14) after DHPG treatment (n = 38 [basal], n = 39 [DHPG],

p = 0.0016, Mann-Whitney U test, ±SEM).

(E) FISH analysis of hippocampal slices from mice (P21) using DIG-labeled LNA-probes. Middle panel is the magnification of the selected region shown in upper

panel.

(F) FISH analysis with U6 RNA and scrambled probe (bottom panels) (also see Figure S3).

Molecular Cell


Surprisingly, we observed that the two-nucleotide mutation

inserted in the PSD-95 30UTR to disable themiR-125a interaction

had no significant effect on FMRP binding (Figure 5A); however,


this mutation completely removed the effect of FMRP on trans-

lational inhibition of the 30UTR reporter. While FMRP knock-

down in neuro2A cells resulted in a significant increase in the

Figure 4. miR-125a Affects Dendritic Spine Density and Branching

(A) Upper panels show low-magnification images of phalloidin-stained cultured hippocampal neurons transfected with scrambled (left) or antimiR-125a (right).

Boxed insets are enlarged in lower panels showing higher magnification of distal dendritic segments. Arrows (yellow) indicate unbranched spines, and arrow-

heads (white) indicate branched spines.

(B) Phalloidin-stained cultured hippocampal neurons transfected with siRNA against PSD-95 mRNA (left panel) or scrambled siRNA (right panel) prior to

transfection with antimiR-125a. PSD-95 knockdown rescued the spine phenotype caused by antimiR-125a. Lower panels show the immunofluorescence for

PSD-95 proteins in the same neurons indicating the levels of PSD-95 with siRNA transfection.

(C) Immunoblot showing the knockdown of PSD-95 protein in neurons transfected with specific siRNA against PSD-95 mRNA. The lower panel is tubulin protein

(loading control).

(D) Spine branches were measured as a ratio of branches per mm of dendritic segment (p < 0.0001, n = 3, one way ANOVA, Bonferroni’s test, ±SEM).

(E) Scatter plot of the data from (D) showing the distribution of individual data points for spine branching of each neuron with mean ± SEM (also see Figure S4).

Molecular Cell


expression of f-luciferase-PSD-95 UTR (59% ± 16%), it had no

effect on f-luciferase-PSD-95 UTRmt125a (Figure 5B). These

experiments indicate that the 30UTR of PSD-95 mRNA is suffi-

cient to elicit FMRP mediated regulation of translation, but it

requires a functional miR-125a interaction. These data suggest

that FMRP binding to the 30UTR on its own is insufficient to

regulate translation. The mGluR and miR-125a mediated regula-

tion of PSD-95 translation was also disrupted in the absence

of FMRP in a mouse model of FXS (Figures 5D and 5E). In

cultured cortical neurons from Fmr1KOmice, unlikeWT neurons

(Figure 5D), treatment with DHPG or antimiR-125a did not

increase the translation of f-luciferase-PSD-95 UTR (Figure 5E).

Interestingly, DHPG had no effect on the interaction of FMRP

with endogenous PSD-95 mRNA (Figure 5C and Figure S5L), in

contrast to the DHPG-induced dissociation of endogenous

PSD-95 mRNA from AGO2 shown earlier (Figure 1B). Taken

together, these data show that FMRP association with the

mRNA is essential for mGluR-mediated regulation translation

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which is executed by relieving miR-125a imposed inhibition on

the 30UTR.To further explore the overlap in the FMRP- and miR-125a-

mediated regulation of PSD-95 mRNA translation, we studied

the polysomal distribution of endogenous PSD-95 mRNA and

miR-125a from WT and Fmr1 KO synaptoneurosomes. Actively

translating polysomes in the synaptoneurosomes can be clearly

distinguished by separating them on linear sucrose gradient and

analysis of the differential effects of cyclohexamide, puromycin

and EDTA treatment (Figures S5A–S5D and Muddashetty

et al., 2007; Stefani et al., 2004). InWT synaptoneurosomes, acti-

vation of gpI mGluR by DHPG resulted in significant shift (frac-

tions 7–10) of endogenous PSD-95 mRNA into polysomes

(Figure 5F). DHPG treatment also led to a decrease in the miR-

125a in fraction 1, which represents the mRNPs (Figure 5G).

The reason for this shift is not entirely clear, but we speculate

that the translational activation induced by mGluR stimulation

leads to the dissociation of PSD-95:miR-125a inhibitory complex


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Molecular Cell


and a concomitant shift of PSD-95 mRNA into polysomes and

also a resultant decrease in miR-125a in the mRNP fraction. In

Fmr1 KO synaptoneurosomes, PSD-95 mRNA is elevated signif-

icantly in polysomal fractions (fraction 5–10) compared to WT at

basal state (Figure 5F andMuddashetty et al., 2007). This may be

due to the inability to form a miR-125a-mediated inhibitory

complex on PSD-95 mRNA in the absence of FMRP (discussed

further below). mGluR-mediated translational activation of PSD-

95 mRNA is absent in Fmr1 KO synaptoneurosomes and

neurons (Muddashetty et al., 2007; Todd et al., 2003). Interest-

ingly, the distribution of both PSD-95 mRNA and miR-125a on

linear sucrose gradients from unstimulated KO synaptoneuro-

somes resembled that of DHPG treated synaptoneurosomes

from WT (Figures 5F and 5G). In summary, both FMRP and

miR-125a are essential for mGluR-mediated regulation of PSD-

95 mRNA translation, and FMRP seems to control the execution

of miR-125a inhibition on PSD-95 mRNA translation in a revers-

ible manner.

FMRP Is Essential for the Interaction of AGO2with PSD-95 mRNA at SynapsesThe microRNA-mediated translational inhibition occurs by

formation of a RISC on mRNAs (Bartel, 2004). The components

of this inhibitory complex are reported to be present at synapses

(Lugli et al., 2005). Here we show dendritic localization of AGO2

in cultured hippocampal neurons and their colocalization with

piccolo, a synaptic marker (Figure S4D). The necessity of

FMRP and miR-125a for mGluR-mediated translation of PSD-

95 mRNA and its dysregulation in Fmr1 KO synaptoneurosomes

opens the possibility of failure to form RISC on PSD-95 mRNA at

synapses in the absence of FMRP. Supporting this hypothesis, in

Fmr1 KO synaptoneurosomes, a significantly reduced (�66% ±

12.2%) amount of miR-125a was found in precipitated AGO2

(Figure 5H) compared to WT. This effect was specific to miR-

125a, since there was no difference in the miR-124 precipitated

by AGO2 between WT and Fmr1 KO, both in total extract and

Figure 5. miR-125a and FMRP Are Essential for the gp1 mGluR-Depen

(A) The ratio of f-luciferase mRNA in FMRP immunoprecipitates from neuro2

95 UTRmt125a, or f-luciferase-PSD-95 UTRDGR (deletion of 667–835 of PSD-95

p = 0.01,one-way ANOVA, ±SD). Values are normalized to f-luciferase-PSD-95 U

(B) Relative luciferase activity from neuro2A cells transfected with f-lucifease-PSD

For FMRP knockdown (FMRP-KD), neuro2A cells were transfected with Fmr1 siRN

with scramble siRNA) (also see Figures S5J and S5K).

(C) The ratio of PSD-95 mRNA in FMRP immunoprecipitates from cultured cortic

(D) Relative luciferase assay fromWT cortical neurons (DIV 14) transfectedwith f-lu

luciferase reporters as indicated. Effects of DHPG on control and antimiR-125a-tr

neurons) (n = 4, p = 0.0012,one-way ANOVA, Dunnett’s test, ±SD).

(E) Relative luciferase assay from Fmr1 KO-cortical neurons (DIV 14) transfected w

ANOVA, Dunnett’s test±SD) (also see Figure S5E).

(F) Distribution of PSD-95 mRNA on a linear sucrose gradient (15%–45%) from t

state and after DHPG stimulation is compared to Fmr1 knockout (KO) at basal s

(G) Distribution of miR-125a on a linear sucrose gradient from cortical synapton

Figures S5A–S5D).

(H) The ratio of miR-125a in AGO2 immunoprecipitate in WT and Fmr1 KO (KO) fr

WT (n = 4, p = 0.004, paired t test, ±SD).

(I) Quantitative measurement of miR-125a and miR-124 from WT and Fmr1 K

expressed as two-difference in the Ct values between synaptoneurosomes to to

(J) The ratio of PSD-95mRNA in AGO2 immunoprecipitate inWT and Fmr1 KO (KO

to WT (n = 3, p = 0.009, paired t test, ±SD) (also see Figure S6).

M

synaptoneurosomes (Figure S6A). Interestingly, miR-125a itself

was significantly decreased in Fmr1 KO synaptoneurosomes

compared to WT (Figure 5I). The ratio of miR-125a in Fmr1 KO

synaptoneurosomes (S) to the total (T) extract (Figure 5I) was

less than 50% of WT (�53.1% ± 10.4). This effect was specific

to miR-125a, while miR-124, another synaptic microRNA (Fig-

ure 5I), and several other microRNAs tested (data not shown)

were unaffected. Endogenous PSD-95 mRNA precipitated with

AGO2 was also significantly reduced (�58% ± 13%) in Fmr1

KO synaptoneurosomes (Figure 5J) compared to WT, while

precipitation of b-actin mRNA was unaffected (Figure S6B).

These data suggest that FMRP is necessary to recruit AGO2

complexes containing miR-125a onto PSD-95 mRNA at

synapses.

Phosphorylation of FMRP Modulates miR-125aRegulation of PSD-95 mRNA TranslationThus far our results indicate that FMRP acts as a modulator

between the mGluR signaling pathway and miR-125a-mediated

regulation of PSD-95 mRNA translation. To understand how

FMRP provides flexibility to miR-mediated translational regula-

tion, we investigated the phosphorylation status of FMRP as

a likely mode of control. FMRP is phosphorylated primarily on

the conserved S499 (human S500) (Ceman et al., 2003), which

is thought to be critical for regulating the translation of its target

mRNAs (Ceman et al., 2003; Narayanan et al., 2007, 2008). In

our study, we used the overexpression of phosphomutants

S499D (mimics the phosphorylated form) S499A (mimics

the dephosphorylated form) (Narayanan et al., 2007) or FMRP-

WT in Fmr1 KO neurons and murine L-M(TK�) cells in which

have low endogenous FMRP levels (Ceman et al., 1999). Compa-

rable amounts of FMRP-WT, FMRP-S499D, and FMRP-S499A

were expressed in these cells (Figure 6A and Figure S7E), and

the phosphomutants had no effect on the mRNA levels of trans-

fected f-luciferase-PSD-95 UTR constructs (Figure S7F). In Fmr1

KO neurons, overexpression of FMRP-WT and FMRPS499D

dent Regulation of PSD-95 mRNA Translation

A cells transfected with either f-luciferase-PSD-95 UTR, f-luciferase-PSD-

30UTR including G-rich miR-125a-binding region) and analyzed by qPCR (n = 3,

TR (see Figure S5).

-95 UTR or f-luciferase-PSD-95 UTRmt125a (n = 3, p = 0.03, paired t test, ±SD).

A or scrambled siRNA. Values were normalized to the control (cells transfected

al neurons (n = 3, paired t test, ±SD).

ciferase-PSD-95 UTR. AntimiR-125a oligonucleotideswere cotransfectedwith

eated neurons were examined. Values are normalized to the control (untreated

ith f-luciferase-PSD-95 UTR and treated similar to WT neurons (n = 4, one-way

he lysate of cortical synaptoneurosomes (representative graph) of WT at basal

tate. Values shown represent the percentage of mRNA in each fraction.

eurosomes of WT (basal and DHPG stimulated) and Fmr1 KO mice (also see

om total (T) and synaptoneurosomal (S) preparations. Values are normalized to

O (KO) lysates of mouse cortical synaptoneurosomes by qPCR. Values are

tal cortical extract (n = 4, p = 0.005, paired t test, ±SD).

) from total (T) and synaptoneurosomal (S) preparations. Values are normalized


Figure 6. FMRP Phosphorylation Inhibits PSD-95 mRNA Translation through miR-125a

(A) Immunoblot showing the effect of overexpressing FMRP-WT, FMRP-S499D (mimics constitutively phosphorylated FMRP), or FMRP-S499A (constitutively

dephosphorylated FMRP) in cultured cortical neurons from Fmr1 KO mice. Upper panel shows levels of endogenous PSD-95 protein, middle panel is tubulin

(loading control), and the lower panel is Flag-FMRP.

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Molecular Cell


significantly reduced the endogenous PSD-95 protein levels,

while overexpression of FMRP-S499A had no effect (Figures

6A and 6B). Coexpression of FMRP-S499D with f-luciferase-

PSD-95 UTR in neurons significantly reduced (�74% ± 14%)

the relative luciferase activity compared to coexpression with

FMRP-WT (Figure S7B). Coexpression of FMRP-S499D had

no effect on f-luciferase-PSD-95UTR mt125a. Antagonizing

miR-125a by antimiR-125a blocked the inhibitory effect of

FMRP-S499D on f-luciferase-PSD-95 UTR in cortical neurons

(Figure 6C). Overexpression of FMRP-S499D leads to signifi-

cantly reduced expression of f-luciferase-PSD-95 UTR in

primary cortical neurons at basal state, and the DHPG-induced

increase in the luciferase activity was also abolished (Figure 6D).

These data indicate the critical role of S499 of FMRP inmediating

the inhibition and mGluR-mediated activation of PSD-95 mRNA

translation involving miR-125a.

Overexpression of FMRP-S499D in L-M(TK�) cells had a

significant effect on the distribution of transfected f-luciferase-

PSD-95 UTR mRNA on a linear sucrose gradient (Figure 6E).

There was a significant shift of the reporter mRNA into the

mRNP fraction and a resultant decrease in the polysomal frac-

tions (Figures 6E and 6F) when compared to cells overexpress-

ing FMRP-WT (47% ± 1.6%) or FMRP-S499A. The overexpres-

sion of FMRP-S499D also resulted in a significant increase

of miR-125a in fraction 1–2 (mRNP), compared to FMRP-

S499A, when lysates from L-M(TK�) cells were separated on

a linear sucrose gradient (Figure S7G). The sucrose gradient

and luciferase activity experiments taken together suggest that

FMRP-S499D forms an inhibitory complex on PSD-95 mRNA

involving miR-125a which cannot be relieved by mGluR activa-

tion because of the constitutively phosphorylated form of

FMRP (Figures 6A–6D). Further evidence for an inhibitory com-

plex is suggested by increased association of FMRP-S499D

with miR-125a (Figure S7H) and PSD-95 mRNA in neurons,

both endogenous and 30UTR reporter (Figure 6H and Figure S7A)

compared to FMRP-WT or FMRP-S499A. Finally, miR-125a was

significantly enriched in an AGO2 pellet from Fmr1 KO neurons

overexpressing FMRP-S499D compared to FMRP-WT and

FMRP-S499A (Figure 6G).

Stimulation of mGluRs leads to the dephosphorylation of

FMRP by the specific phosphatase PP2A, which is inhibited by

okadaic acid (Narayanan et al., 2007). When PP2A was inhibited

in cultured cortical neurons by okadaic acid, DHPG-induced

(B) Quantification of effect of overexpression of FMRP-WT and its phosphomutan

cultured cortical neurons from Fmr1 KO; values were normalized to tubulin (n = 4

(C) Relative luciferase activity from primary WT cortical neurons in which f-lucife

p = 0.01, one-way ANOVA, Bonferroni’s test ±SD). antimiR-125a or antimiR-124

(D) Relative luciferase activity from WT cortical neurons in which f-luciferase-PSD

mock treatment (n = 3, p = 0.017, one-way ANOVA, Bonferroni’s test ±SD).

(E) Distribution of f-luciferase-PSD-95 UTR on linear sucrose gradients (15%–45%

S499A. Values shown represent the percentage of the total f-luciferase-PSD-95

(F) Percentage of f-luciferase-PSD-95 UTR and b-actinmRNA in the polysomal fra

with FMRP-WT or FMRP-S499D or FMRP-S499A (n = 3, p = 0.005, one-way AN

(G) Ratio of miR-125a in AGO-2 pellet to supernatant from Fmr1 KO cortical neu

(n = 3, p = 0.001, one-way ANOVA, Dunnett test ±SD).

(H) Ratio of PSD-95 mRNA in Flag-FMRP pellet to supernatant from cortical neu

(n = 3, p = 0.02, one-way ANOVA, Dunnett test ±SD) (also see Figure S7).

M

dissociation of endogenous PSD-95 mRNA from AGO2 was

occluded (Figure 7A). Similarly, FMRP-S499D (mimics phos-

pho-FMRP) overexpression in neurons occluded the DHPG-

induced dissociation of f-luciferase mRNA with PSD-95 UTR

from AGO2 (Figure 7B), while overexpression of FMRP-WT had

no effect on this process. mGluR-mediated dissociation of

PSD-95 mRNA from AGO2 was also reflected in the decreased

interaction between FMRP and AGO2. When neurons trans-

fectedwith FMRP-WTwere stimulatedwithDHPG, a significantly

reduced amount (�48% ± 28%) of AGO2 was present in FMRP

immunoprecipitates (Figures 7C and 7D). In contrast, DHPG had

no effect on the interaction of AGO2 with overexpressed FMRP-

S499D (phospho-FMRP mimic). We showed earlier that DHPG

dissociates AGO2 (Figure 1B) but not FMRP (Figure 5C) from

PSD-95 mRNA. Taken together, these results suggest that

dephosphorylation of FMRP is the essential step for subsequent

dissociation of RISC from FMRP-bound PSD-95mRNA and acti-

vates mRNA translation. The interaction between AGO2 and

FMRP appears to be RNA dependent, as RNase treatment

significantly reduced the myc-FMRP precipitated with Flag-

AGO2 expressed in neuro2A cells (Figure 7E). Further work is

needed to elucidate the molecular details of this interaction. In

summary, the phosphorylated form of FMRP preferentially forms

the inhibitory complex on PSD-95 mRNA involving miR-125a,

whereas dephosphorylation of FMRP is the critical switch

required for relieving miR-mediated inhibition of translation.

DISCUSSION

MicroRNA and FMRP Partner to Control Translationin the mGluR Signaling PathwayThis study reveals a molecular mechanism for the cooperative

involvement of miR-125a and FMRP in the reversible regulation

of PSD-95 mRNA translation at synapses downstream of gp1

mGluR signaling. Translational inhibition of PSD-95 mRNA by

miR-125a is relieved by mGluR activation, which rapidly induces

the dissociation of the RISC containing AGO2 from PSD-95

mRNA. Inhibition of PSD-95 mRNA by miR-125a was shown to

have bidirectional control of endogenous PSD-95 expression in

dendrites and at synapses. FMRP is necessary for both miR-

125a-mediated inhibition and mGluR-induced translation of

PSD-95 mRNA in neurons. Conversely, miR-125a binding to

the 30UTR of PSD-95 is necessary for FMRP-mediated

ts (FMRP-S499D and FMRP-S499A) on level of endogenous PSD-95 protein in

, p = 0.007, one-way ANOVA, ±SD).

rase-PSD-95 UTR was cotransfected with FMRP-WT or FMRP-S499D (n = 3,

was cotransfected where indicated.

-95 UTR was cotransfected with FMRP-WT or FMRP-S499D before DHPG or

) from L-M(TK�) cells transfected with FMRP-WT or FMRP-S499D or FMRP-

UTR mRNA in each fraction (representative graph).

ction of the linear sucrose gradient from the lysate of L-M(TK�) cells transfected

OVA, Bonferroni’s test ±SD).

rons which were transfected with FMRP-WT, FMRP-S499D, or FMRP-S499A

rons which were transfected with FMRP-WT, FMRP-S499D, or FMRP-S499A


Figure 7. Dephosphorylation of FMRP Is Essential for Dissociation of PSD-95 mRNA from AGO2

(A) Ratio of PSD-95mRNA in AGO2 immunoprecipitate from cortical neurons (n = 3, p = 001, one-way ANOVA, Bonferroni’s test, ±SD) at basal state or after DHPG

treatment. Cells were preincubated with okadaic acid where indicated.

(B) Ratio of luciferasemRNA in AGO2 immunoprecipitate from cortical neuronswhichwere cotransfectedwith f-luciferase-PSD-95UTR and FMRP-WT or FMRP-

S499D (n = 3, p = 0.005, paired t test, ±SD) and analyzed at basal state or after DHPG stimulation.

(C) Immunoblots depicting the AGO2 and Flag-FMRP immunoprecipitated with anti-Flag antibody. Upper panel represents the input and the lower panel the

precipitate. Lanes 1 and 2 are cells transfected with FMRP-WT (1-basal, 2-DHPG treated), lanes 3 and 4 are cells transfected with FMRP-S499D (3-basal,

4-DHPG treated).

(D) Quantification of AGO2 and FMRP immunoprecipitated with anti-Flag antibody. Cortical neurons were either transfected with Flag-tagged FMRP-WT or

FMRP-S499D followed by IP with Flag antibody (n = 3, p = 0.04, paired t test, ±SD) at basal state or after DHPG stimulation.

(E) Immunoblot showing the effect of RNase treatment on the FMRP-AGO2 interaction in neuro2A cells. Upper panel shows the expression of myc-FMRP.Middle

panel shows AGO2 and FMRP in Flag-AGO2 pellet. Bottom panel is the RNA gel (ethidium bromide staining) showing the effect of RNase treatment. (�, +)

indicates RNase treatment. First two lanes are from the cells not expressing Flag-AGO-2.

(F) Model depicting the role of FMRP phosphorylation and miR-125a in the reversible regulation of PSD-95 mRNA translation in response to mGluR activation at

dendritic spines.

Molecular Cell



Molecular Cell


repression. FMRP binding to the 30UTR of PSD-95 mRNA is

insufficient to regulate translation on its own, but was enabled

by the recruitment of RISC (AGO2) and miR-125a. Taken

together, these data suggest that FMRP co-opts RISC

complexes containing miR-125a onto PSD-95 mRNA at

synapses. Devoid of this mechanism in the absence of FMRP,

translation of PSD-95 mRNA at synapses is dysregulated at

basal state and occludes activation of translation by mGluRs.

miR-125a is significantly reduced in Fmr1 KO synaptoneuro-

somes, a possible outcome due to its inability to form and/or

stabilize an inhibitory complex on PSD-95 mRNA in the absence

of FMRP. The association of AGO2with both PSD-95mRNA and

miR-125a is significantly reduced in Fmr1 KO, which further

supports the model that FMRP is essential for the formation of

a RISC-mediated inhibitory complex on PSD-95 mRNA and its

regulation at synapses. With the lack of such a complex, miR-

125a is likely degraded, hence the reduced levels in synapto-

neurosomes from the Fmr1 KO. Future work may uncover

whether this is a general mechanism for FMRP to guide specific

miRNAs onto target mRNAs at synapses. A recent study has

identified that many miRNAs in the brain including miR-125a

are associated with FMRP and demonstrated that FMRP is

necessary for miR-125b and miR-132 to affect dendritic spine

morphology (Edbauer et al., 2010). Further work may reveal

a common molecular mechanism for FMRP to regulate transla-

tion of target mRNAs using microRNAs.

Phospho-FMRP Forms the Inhibitory Complexon PSD-95 mRNA with miR-125a and AGO2Reversible inhibition of PSD-95 mRNA was shown to involve the

phosphorylated form of FMRP forming an inhibitory mRNP

complex with miR-125a and AGO2. Phosphorylation of FMRP

has been previously shown to play a critical role in mGluR-medi-

ated translation, although the mechanism was not known

(Narayanan et al., 2007, 2008). Based on our data, we propose

a model (Figure 7F) whereby phosphorylated FMRP recruits

RISC-miR-125a onto PSD-95 mRNA and inhibits translation.

Dephosphorylation of FMRP at a single serine appears to be

the critical step to reverse this miR-mediated translation inhibi-

tion. Activation of gp1 mGluR induces the dissociation of RISC

from PSD-95 mRNA at synapse and initiates rapid translation

in a process mediated by FMRP dephosphorylation. The phos-

phorylation status of FMRP may determine the accessibility of

miR-125a to PSD-95 mRNA, thus conferring additional speci-

ficity to their interaction. We hypothesize that phosphorylation of

FMRP exposes the miR-125a binding site on PSD-95 mRNA,

while dephosphorylation destabilizes this interaction and thus

leads to miR-125a dissociation from PSD-95 mRNA. Since

FMRP remains bound to the mRNA following stimulation, we

speculate that FMRP rephosphorylation would lead to rerecruit-

ment of RISC and inhibition of translation. There has been prior

evidence for activity-regulated removal of RISC components.

Degradation of MOV10 (a RISC protein) occurs in NMDA-

receptor-mediated activity-dependent manner at synapses,

which may be a general translational control point (Banerjee

et al., 2009). While this study shows the importance of MOV10

as a general checkpoint for translation, it does not decode the

mRNA specificity of receptor-induced translation. Our findings

M

suggest that involvement of RNA-binding proteins such as

FMRP provides specificity and flexibility to this process. The

actual influence of FMRP on the molecular interactions of micro-

RNA with its target mRNAs and its possible direct interaction

with AGO2 or other components of RISC, such as GW182 and

MOV10, needs to be further explored.

Localized MicroRNAs Affecting Spine MorphologyHave Implications for FXSThe discovery of several localized miRNAs in neurites of cultured

neurons (Kye et al., 2007) and their enrichment in synaptic frac-

tions (Lugli et al., 2005; Siegel et al., 2009), followed by the

demonstrated role of miR-134 in the local translation of Limk1

in vitro (Schratt et al., 2006), led to studies showing the wide-

spread presence of a localized system of microRNAs affecting

synaptic structure and function (Schratt, 2009a; Siegel et al.,

2009). Here we visualize the dendritic localization of miR-125a

in vitro and in vivo and demonstrate its abundance in synaptic

fractions. We observed a role for miR-125a in regulating the

morphology and density of dendritic spines. Inhibition of endog-

enous miR-125a function resulted in a marked increase in spine

density and branching. We also demonstrated that the spine

phenotype observed by miR-125a inhibition is due to excess

PSD-95 mRNA translation and could be rescued by PSD-95

knockdown. These data suggest that miR-125a-mediated inhibi-

tion of PSD-95mRNA translation may restrain spine growth and/

or branching. This is consistent with observations that overex-

pression of PSD-95 resulted in the formation of branched

and multi-innervated spines (Nikonenko et al., 2008), which

bears some similarities to the spine phenotype observed by

miR-125a inhibition. Further work is needed to assess the contri-

bution of localized miR-125a and PSD-95 mRNA on spine

morphology. Additional work is also needed to assess how spine

phenotypes in FXS may be due to loss of microRNA-mediated

control of local mRNA translation. An exuberant excess of spines

having an immature, long and thin morphology is a well-

described phenotype of human FXS patients and the Fmr1 KO

mouse model (Bagni and Greenough, 2005). We speculate that

the spine phenotype in FXS may be partly due to excess basal

expression of PSD-95 and other mRNAs, which have lost

their regulation by localized microRNAs. FMRP- and miR-

125a-mediated translation of PSD-95 mRNA at synapses, in

response to mGluR activation, may normally regulate formation

of the PSD and stabilization of newly formed dendritic spines.

It is known that mGluRs stimulate protein-synthesis-dependent

growth of spines (Vanderklish and Edelman, 2002) and that

PSD-95 is important for stabilization of newly formed spines

(De Roo et al., 2008). Recent work shows that spines in Fmr1

KO mice have increased turnover and delayed stabilization

(Pan et al., 2010), which could be due to impaired synthesis of

PSD-95 and other PSD proteins (Muddashetty et al., 2007;

Schutt et al., 2009). Since FMRP associates with many miRNAs,

and this association is necessary for FMRP’s role on spine

morphology (Edbauer et al., 2010), it will be interesting to

assess a possible general role for FMRP-miRNA interactions in

local protein synthesis underlying spine stabilization. The

present study provides further motivation to investigate whether

miRNA-mediated regulation of mRNA translation through FMRP


Molecular Cell


and/or other mRNA binding proteins may represent a general

mechanism to achieve dynamic and bidirectional control of local

protein synthesis affecting synapse structure and function. This

work provides mechanistic insight into the role that dysregulated

miRNAs play in FXS, which has broader implications for the role

of miRNAs in other neurological diseases (Kocerha et al., 2009)

and autism spectrum disorders (Abu-Elneel et al., 2008). Future

research may consider therapeutic strategies to restore micro-

RNA function.

EXPERIMENTAL PROCEDURES

Cell Culture

Primary neuronal cultures were prepared from cerebral cortices and hippo-

campi of E18 mouse (C57BL/6 and Fmr1 knockout, backcrossed in C57BL/

6J) or rat (Sprague dawley) as described previously (Kaech and Banker,

2006). High-density cultures were used for biochemical experiments, while

low-density cocultures with glia were used for imaging experiments. Neuro2A

and L-M(TK�) cells were cultured in DMEM (Hyclone) with 10% FBS (Sigma).

DNA Constructs, Transfection, and MicroRNA Inhibitors

The 30UTR of mouse and human PSD-95 mRNA was amplified from cDNA.

Human PSD-95 30UTR was used for all the experiments unless specified.

F-luci-PSD-95 UTRmt-125a and other mutant constructs were obtained by

site-directed mutagenesis of the hPSD-95 30UTR using overlapping primers.

Pre-microRNA (Invitrogen) and GFP-miR expression vectors (GeneCopoeia)

were used for the overexpression of microRNAs. LNA antisense-RNA

oligonucleotides (Ambion) were used for inhibiting/antagonizing microRNAs.

Double-stranded stealth siRNA (Invitrogen) was used for knocking down

Fmr1 mRNA, and a pool of three target-specific siRNAs (Santa Cruz

BioTechnology) were used for PSD-95 mRNA knockdown. DNA and RNA

were transfected to neuro2A and L-M(TK�) cells by Lipofectamine2000 (Invi-

trogen) and primary cultured neurons by magnetofection using Neuromag

(OZBioscience) according to manufacturer’s instructions.

Fluorescence In Situ Hybridization and Immunocytochemistry

Mature (DIV 14–21) hippocampal neurons were fixed and processed for in situ

hybridization and immunohistochemistry as described before (Schratt et al.,

2006).

Spine and Synapsin Analysis

Cultured hippocampal neurons at DIV 10 were transfected with LNA-anti-miR

oligonucleotides or scrambled RNA (Ambion) by magnetofection using

neuromag reagent (OZBioscience) according to manufacturer’s protocol.

For PSD-95 knockdown, siRNAs against PSD-95 mRNA were cotransfected

with antimiRs at DIV 10. At DIV 14, neurons were stained to excess rhoda-

mine-phalloidin to label spines and anti-synapsin antibody to label synapses.

Synaptoneurosome Preparation

Cortical synaptoneurosomes were prepared either by differential filtration

(Muddashetty et al., 2007) (Millipore) or separation on density gradient as

described earlier (Gardoni et al., 1998). Stimulation was carried out as

described previously (Muddashetty et al., 2007).

Immunoprecipitation and Western Blot

Immunoprecipitation was performed as described previously (Muddashetty

et al., 2007) using protein G Sepharose (Roche). Monoclonal antibody 7G1

for FMRP (Developmental Studies Hybridoma Bank) and AGO2 (Abnova)

were used. Flag-FMRP immunoprecipitation was performed using anti-Flag

agarose (Sigma). Samples were processed for quantitative real time PCR or

western blotting following the immunoprecipitation. PSD-95 (Chemicon),

FMRP (Sigma), Flag (Sigma), AGO-2 (Abnova), and a-tubulin (Sigma) anti-

bodies were used for western blots.


Polysome Assay

Synaptoneurosomes were prepared from P21WT and Fmr1 knockout mice as

described previously (Muddashetty et al., 2007).

MicroRNA Isolation and Quantification

microRNAs were isolated using miRvana kit (Ambion) according to the

manufacturer’s protocol. Quantification was carried out by real-time PCR

by either TaqMan (Applied Biosystem) probes (Kye et al., 2007) or a simpler

SYBR-based detection method (Roche) as described earlier (Ro et al.,

2006).

Luciferase Assay

Neuro2A and L-M(TK�) cells were transfected with Lipofectamine 2000

(Invitrogen), cultured neurons were transfeted by magnetofection (Oz Biosci-

ence), and luciferaseactivitywasmeasuredbydual luciferaseassay (Promega).

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures

and seven figures and can be found with this article online at doi:10.1016/

j.molcel.2011.05.006.

ACKNOWLEDGMENTS

The authors thank Stephanie Ceman for FMRP constructs; Custom Cloning

Core facility (EmoryUniversity) for preparing constructs; andAndrewSwanson,

TamikaMalone, and Yukio Sasaki for technical assistance. This work was sup-

ported by FRAXA and NFXF postdoctoral fellowships (R.S.M.); National Insti-

tutes of Health grants MH085617 (G.J.B.), DA027080 (G.J.B.), and HD020521

(S.T.W.); Baylor-Emory Fragile X Research Center (P30HD024064); and Emory

Neuroscience–National Institute of Neurological Disorders and Stroke Core

Facilities Grant P30NS055077.

Received: October 21, 2010

Revised: February 22, 2011

Accepted: March 25, 2011

Published: June 9, 2011

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