Download Muddashetty RS, Nalavadi VC, Gross C, Yao X, Xing L, Laur O, Warren ST, and Bassell GJ. Reversible Inhibition of PSD-95 mRNA Translation by miR-125a, FMRP Phosphorylation, and mGluR Signaling. Molecular Cell 42: 673-688 (June 2011).

Molecular Cell
Article
Reversible Inhibition of PSD-95
mRNA Translation by miR-125a,
FMRP Phosphorylation, and mGluR Signaling
Ravi 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 Biology
of Neurology
3Department of Microbiology
4Department of Human Genetics
5Department of Biochemistry
Emory University School of Medicine, Atlanta, GA 30322, USA
*Correspondence: [email protected]
DOI 10.1016/j.molcel.2011.05.006
2Department
SUMMARY
The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA
translation in response to receptor signaling is
unknown but could provide a means for temporal
and spatial control of translation. Here we show
that miR-125a targeting PSD-95 mRNA allows
reversible inhibition of translation and regulation by
gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered
dendritic spine morphology. Bidirectional control of
PSD-95 expression depends on miR-125a and
FMRP phosphorylation status. miR-125a levels at
synapses and its association with AGO2 are reduced
in Fmr1 KO. FMRP phosphorylation promotes the
formation of an AGO2-miR-125a inhibitory complex
on PSD-95 mRNA, whereas mGluR signaling of
translation requires FMRP dephosphorylation and
release of AGO2 from the mRNA. These findings
reveal a mechanism whereby FMRP phosphorylation
provides a reversible switch for AGO2 and microRNA
to selectively regulate mRNA translation at synapses
in 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 preand/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
(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 translation may affect
dendritic spine morphology (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 reversibility 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 specific mRNA can be selectively silenced by a miRNA
through RISC components, and to investigate whether a selective mRNA-binding protein in response to physiological signals
may reversibly regulate these interactions.
Molecular Cell 42, 673–688, June 10, 2011 ª2011 Elsevier Inc. 673
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) cooperate on the 30 UTR 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 AGO2miR125a 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 phosphorylation 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 neuropsychiatric disorders resulting from altered regulation of miRNA
pathways.
RESULTS
mGluR-Mediated Translation of PSD-95 Involves
MicroRNAs
Activation 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 (Bhattacharyya 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 regulation, 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 30 UTR of PSD-95
mRNA is sufficient to elicit activity-mediated regulation of PSD95 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 mGluR674 Molecular Cell 42, 673–688, June 10, 2011 ª2011 Elsevier Inc.
Figure 1. Activation of gp1 mGluR Stimulates Translation of a PSD95 30 UTR 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 30 UTR or
f-luciferase-b-actin 30 UTR (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 30 UTR (p = 0.001) and endogenous 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).
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
PSD-95 Translation Regulation by FMRP and miR
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 stimulated with DHPG (Figure 1B). A similar DHPGinduced decrease in the association with AGO2 was also
observed for endogenous PSD-95 mRNA (61% ± 20%) (Figure 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
30 UTR of PSD-95 mRNA is sufficient to elicit mGluR-mediated
translational activation in neurons. Translational activation coincides with the dissociation of the reporter mRNA (and endogenous PSD-95 mRNA) from AGO2, suggesting a possible role of
microRNAs in this process. This led us to search for a microRNA(s) that regulates PSD-95 mRNA translation.
miR-125a Regulates the Translation of PSD-95 mRNA
Among the few microRNAs predicted to target the 30 UTR 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 30 UTR (John et al., 2004;
and Figure 2A). In neuro2A cells, only the transfection of miR125a had a significant inhibitory effect on expression of f-luciferase-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 (Figure 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-PSD95 UTR (Figure 2E), indicating that the two-nucleotide mutation
rendered the reporter unresponsive to miR-125a. Blocking
endogenous miR-125a by transfecting antimiR-125a significantly 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 luciferase reporter with PSD-95 30 UTR suggest that antimiR-125a
relieved the translational inhibition imposed on the PSD-95
30 UTR containing an intact binding site for miR-125a.The effect
of antimiR-125a was dose dependent (Figure S2C) and specific
to PSD-95 30 UTR, as it had no effect on f-luciferase-b-actin UTR
(Figure S2B). Mutation of the miR-125a-binding site (f-luciferasePSD-95 UTRmt125a) also resulted in a marked reduction of this
mRNA association with AGO2 (59% ± 20) compared to the wildtype UTR, as measured by AGO2 immunoprecipitation and
q-PCR analysis of mRNA (Figure 2G). However, total levels of
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 30 UTR.
Next, we investigated whether miR-125a had a role in mGluRmediated 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
30 UTR in cultured cortical neurons, but when the reporters were
cotransfected with antimiR-125a this translational activation
was blocked (Figure 2H). Activation of mGluRs with DHPG had no
effect on the translation of mutant f-luciferase-PSD-95mt125a
(Figure 2H) in cultured cortical neurons. DHPG-induced dissociation of PSD-95 UTR from AGO2 was also specifically abolished
by antimiR-125a (Figure 2I). These results indicate miR-125a
specifically targets the 30 UTR of PSD-95 mRNA and is essential
for mGluR-mediated translation in neurons.
To test the effect of miR-125a on the expression of endogenous 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 transfection 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 30 UTR 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 30 UTR there is a ‘‘g instead
of a’’ compared to the seed region of human resulting in a tolerated 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 30 UTR, 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 30 UTR (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 30 UTRs, indicting the
mismatch in the seed region resulting in a G.U wobble behaves
similarly (Figure S3C).
miR-125a Is Localized to Dendrites
Only 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 synaptoneurosomes upon stimulation with DHPG (15 min) indicating possible
mGluR-induced turnover (Figure 3B). Fluorescence in situ
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PSD-95 Translation Regulation by FMRP and miR
Figure 2. miR-125a Targets the 30 UTR of PSD-95 mRNA and Is Necessary for gp1 mGluR-Stimulated Translation
(A) Predicted miR-125a-binding site in the human 30 UTR 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 30 UTR 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, oneway 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). Antisense miR-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).
676 Molecular Cell 42, 673–688, June 10, 2011 ª2011 Elsevier Inc.
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PSD-95 Translation Regulation by FMRP and miR
hybridization (FISH) showed that miR-125a is present in distal
dendrites of cultured hippocampal neurons (Figure 3C). Quantitative 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 (Figure 3E), in contrast to miR-298, which was restricted to the cell
body layer with almost undetectable signal in the dendrites (Figure 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 experiments using LNA probes that carried mismatch mutations 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 (Figure S3F). These results suggest the possibility that miR-125a
may function locally in dendrites and at synapses.
Inhibiting miR-125a Affects Spine Density
and Branching
The localization of miR-125a to dendrites, its abundance in synaptoneurosomes, and its involvement in an mGluR pathway suggest 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 antimiR-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 antimiR125a 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
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 Regulation
of PSD-95 mRNA Translation
FMRP, the deficiency of which causes FXS, is reported to regulate 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 30 UTR of PSD-95 mRNA (Zalfa et al., 2007), we confirmed
that FMRP is associated with the f-luciferase with the 30 UTR 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-PSD95 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 30 UTR of PSD-95 mRNA. In primary
cortical neurons from Fmr1 KO mice, translation of the transfected f-luciferase-PSD-95 UTR was significantly higher than
(+205% ± 50%) wild-type neurons, but coexpression of FMRPWT 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 30 UTR in
cells, as shown by an IP and qPCR assay, was validated by additional experiments. When FMRP and PSD-95 30 UTR were expressed in separate cultures, lysed, and mixed before immunoprecipitation (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 interacts directly with the region of the 30 UTR in vivo, by using a stringent 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 FMRP binding to PSD-95 mRNA in vivo prior to lysis.
(H) Relative luciferase activity from cultured cortical neurons (untreated or stimulated with DHPG) transfected with either f-luciferase-PSD-95 30 UTR or f-luciferase-PSD-95 UTRmt125a. AntimiR-125a was cotransfected with luciferase reporter where indicated. Values were normalized to basal levels and represent the
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 immunoprecipitates from cultured cortical neurons that were cotransfected with
antimiR-125a or antimiR-124 (n = 3, p = 0.03, paired t test, ±SD) with and without DHPG stimulation. The values were normalized to basal levels.
(J) Immunofluorescence localization of endogenous PSD-95 protein in cultured hippocampal neurons transfected with antimiR-125a or scrambled oligonucleotides as a negative control. Cultured neurons were transfected at DIV 10 and fixed at DIV 12. Immunofluorescence was performed using anti-PSD 95 antibody
and Cy3- fluorochrome-conjugated anti-mouse antibody.
(K) Quantification of fluorescent intensities measured in distal dendrites of hippocampal neurons (>40 mm from cell body) that were transfected with scrambled,
antimiR-125a, or antimiR-124 oligonucleotides (n = 23 neurons from three independent experiments, p = 0.042, one-way ANOVA, Bonferroni’s test, ±SEM).
Values were normalized to the scrambled (also see Figures S2 and S3).
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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) Quantitative measurement of the changes in miR-125a and miR-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).
Surprisingly, we observed that the two-nucleotide mutation
inserted in the PSD-95 30 UTR to disable the miR-125a interaction
had no significant effect on FMRP binding (Figure 5A); however,
678 Molecular Cell 42, 673–688, June 10, 2011 ª2011 Elsevier Inc.
this mutation completely removed the effect of FMRP on translational inhibition of the 30 UTR reporter. While FMRP knockdown in neuro2A cells resulted in a significant increase in the
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PSD-95 Translation Regulation by FMRP and miR
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 arrowheads (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).
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 30 UTR of PSD-95 mRNA is sufficient to elicit FMRP mediated regulation of translation, but it
requires a functional miR-125a interaction. These data suggest
that FMRP binding to the 30 UTR on its own is insufficient to
regulate translation. The mGluR and miR-125a mediated regulation 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 Fmr1 KO mice, unlike WT 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
which is executed by relieving miR-125a imposed inhibition on
the 30 UTR.
To further explore the overlap in the FMRP- and miR-125amediated 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). In WT synaptoneurosomes, activation of gpI mGluR by DHPG resulted in significant shift (fractions 7–10) of endogenous PSD-95 mRNA into polysomes
(Figure 5F). DHPG treatment also led to a decrease in the miR125a 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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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 significantly in polysomal fractions (fraction 5–10) compared to WT at
basal state (Figure 5F and Muddashetty 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 PSD95 mRNA is absent in Fmr1 KO synaptoneurosomes and
neurons (Muddashetty et al., 2007; Todd et al., 2003). Interestingly, the distribution of both PSD-95 mRNA and miR-125a on
linear sucrose gradients from unstimulated KO synaptoneurosomes 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 PSD95 mRNA translation, and FMRP seems to control the execution
of miR-125a inhibition on PSD-95 mRNA translation in a reversible manner.
FMRP Is Essential for the Interaction of AGO2
with PSD-95 mRNA at Synapses
The 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 PSD95 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 miR125a, since there was no difference in the miR-124 precipitated
by AGO2 between WT and Fmr1 KO, both in total extract and
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 (Figure 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-125a
Regulation of PSD-95 mRNA Translation
Thus 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 regulation, 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 FMRPWT in Fmr1 KO neurons and murine L-M(TK) cells in which
have low endogenous FMRP levels (Ceman et al., 1999). Comparable 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 transfected f-luciferase-PSD-95 UTR constructs (Figure S7F). In Fmr1
KO neurons, overexpression of FMRP-WT and FMRPS499D
Figure 5. miR-125a and FMRP Are Essential for the gp1 mGluR-Dependent Regulation of PSD-95 mRNA Translation
(A) The ratio of f-luciferase mRNA in FMRP immunoprecipitates from neuro2A cells transfected with either f-luciferase-PSD-95 UTR, f-luciferase-PSD95 UTRmt125a, or f-luciferase-PSD-95 UTRDGR (deletion of 667–835 of PSD-95 30 UTR including G-rich miR-125a-binding region) and analyzed by qPCR (n = 3,
p = 0.01,one-way ANOVA, ±SD). Values are normalized to f-luciferase-PSD-95 UTR (see Figure S5).
(B) Relative luciferase activity from neuro2A cells transfected with f-lucifease-PSD-95 UTR or f-luciferase-PSD-95 UTRmt125a (n = 3, p = 0.03, paired t test, ±SD).
For FMRP knockdown (FMRP-KD), neuro2A cells were transfected with Fmr1 siRNA or scrambled siRNA. Values were normalized to the control (cells transfected
with scramble siRNA) (also see Figures S5J and S5K).
(C) The ratio of PSD-95 mRNA in FMRP immunoprecipitates from cultured cortical neurons (n = 3, paired t test, ±SD).
(D) Relative luciferase assay from WT cortical neurons (DIV 14) transfected with f-luciferase-PSD-95 UTR. AntimiR-125a oligonucleotides were cotransfected with
luciferase reporters as indicated. Effects of DHPG on control and antimiR-125a-treated neurons were examined. Values are normalized to the control (untreated
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 with f-luciferase-PSD-95 UTR and treated similar to WT neurons (n = 4, one-way
ANOVA, Dunnett’s test±SD) (also see Figure S5E).
(F) Distribution of PSD-95 mRNA on a linear sucrose gradient (15%–45%) from the lysate of cortical synaptoneurosomes (representative graph) of WT at basal
state and after DHPG stimulation is compared to Fmr1 knockout (KO) at basal state. Values shown represent the percentage of mRNA in each fraction.
(G) Distribution of miR-125a on a linear sucrose gradient from cortical synaptoneurosomes of WT (basal and DHPG stimulated) and Fmr1 KO mice (also see
Figures S5A–S5D).
(H) The ratio of miR-125a in AGO2 immunoprecipitate in WT and Fmr1 KO (KO) from total (T) and synaptoneurosomal (S) preparations. Values are normalized to
WT (n = 4, p = 0.004, paired t test, ±SD).
(I) Quantitative measurement of miR-125a and miR-124 from WT and Fmr1 KO (KO) lysates of mouse cortical synaptoneurosomes by qPCR. Values are
expressed as two-difference in the Ct values between synaptoneurosomes to total cortical extract (n = 4, p = 0.005, paired t test, ±SD).
(J) The ratio of PSD-95 mRNA in AGO2 immunoprecipitate in WT and Fmr1 KO (KO) from total (T) and synaptoneurosomal (S) preparations. Values are normalized
to WT (n = 3, p = 0.009, paired t test, ±SD) (also see Figure S6).
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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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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-luciferasePSD-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 significantly 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 in mediating
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-luciferasePSD-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 fractions (Figures 6E and 6F) when compared to cells overexpressing FMRP-WT (47% ± 1.6%) or FMRP-S499A. The overexpression of FMRP-S499D also resulted in a significant increase
of miR-125a in fraction 1–2 (mRNP), compared to FMRPS499A, 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 activation because of the constitutively phosphorylated form of
FMRP (Figures 6A–6D). Further evidence for an inhibitory complex is suggested by increased association of FMRP-S499D
with miR-125a (Figure S7H) and PSD-95 mRNA in neurons,
both endogenous and 30 UTR 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
dissociation of endogenous PSD-95 mRNA from AGO2 was
occluded (Figure 7A). Similarly, FMRP-S499D (mimics phospho-FMRP) overexpression in neurons occluded the DHPGinduced 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 transfected with FMRP-WT were stimulated with DHPG, 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 FMRPS499D (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-95 mRNA and activates mRNA translation. The interaction between AGO2 and
FMRP appears to be RNA dependent, as RNase treatment
significantly reduced the myc-FMRP precipitated with FlagAGO2 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 Translation
in the mGluR Signaling Pathway
This 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 miR125a-mediated inhibition and mGluR-induced translation of
PSD-95 mRNA in neurons. Conversely, miR-125a binding to
the 30 UTR of PSD-95 is necessary for FMRP-mediated
(B) Quantification of effect of overexpression of FMRP-WT and its phosphomutants (FMRP-S499D and FMRP-S499A) on level of endogenous PSD-95 protein in
cultured cortical neurons from Fmr1 KO; values were normalized to tubulin (n = 4, p = 0.007, one-way ANOVA, ±SD).
(C) Relative luciferase activity from primary WT cortical neurons in which f-luciferase-PSD-95 UTR was cotransfected with FMRP-WT or FMRP-S499D (n = 3,
p = 0.01, one-way ANOVA, Bonferroni’s test ±SD). antimiR-125a or antimiR-124 was cotransfected where indicated.
(D) Relative luciferase activity from WT cortical neurons in which f-luciferase-PSD-95 UTR was cotransfected with FMRP-WT or FMRP-S499D before DHPG or
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%) from L-M(TK) cells transfected with FMRP-WT or FMRP-S499D or FMRPS499A. Values shown represent the percentage of the total f-luciferase-PSD-95 UTR mRNA in each fraction (representative graph).
(F) Percentage of f-luciferase-PSD-95 UTR and b-actin mRNA in the polysomal fraction of the linear sucrose gradient from the lysate of L-M(TK) cells transfected
with FMRP-WT or FMRP-S499D or FMRP-S499A (n = 3, p = 0.005, one-way ANOVA, Bonferroni’s test ±SD).
(G) Ratio of miR-125a in AGO-2 pellet to supernatant from Fmr1 KO cortical neurons which were transfected with FMRP-WT, FMRP-S499D, or FMRP-S499A
(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 neurons which were transfected with FMRP-WT, FMRP-S499D, or FMRP-S499A
(n = 3, p = 0.02, one-way ANOVA, Dunnett test ±SD) (also see Figure S7).
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Figure 7. Dephosphorylation of FMRP Is Essential for Dissociation of PSD-95 mRNA from AGO2
(A) Ratio of PSD-95 mRNA 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 luciferase mRNA in AGO2 immunoprecipitate from cortical neurons which were cotransfected with f-luciferase-PSD-95 UTR and FMRP-WT or FMRPS499D (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.
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PSD-95 Translation Regulation by FMRP and miR
repression. FMRP binding to the 30 UTR 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 synaptoneurosomes, 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 AGO2 with both PSD-95 mRNA 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, miR125a is likely degraded, hence the reduced levels in synaptoneurosomes 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 translation of target mRNAs using microRNAs.
Phospho-FMRP Forms the Inhibitory Complex
on PSD-95 mRNA with miR-125a and AGO2
Reversible 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-mediated 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 inhibition. 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 phosphorylation status of FMRP may determine the accessibility of
miR-125a to PSD-95 mRNA, thus conferring additional specificity 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 rerecruitment 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 NMDAreceptor-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
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 microRNA 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 Morphology
Have Implications for FXS
The discovery of several localized miRNAs in neurites of cultured
neurons (Kye et al., 2007) and their enrichment in synaptic fractions (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 widespread 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 endogenous 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 inhibition of PSD-95 mRNA translation may restrain spine growth and/
or branching. This is consistent with observations that overexpression 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 contribution 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 welldescribed 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 miR125a-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
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PSD-95 Translation Regulation by FMRP and miR
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 microRNA function.
Polysome Assay
Synaptoneurosomes were prepared from P21 WT and Fmr1 knockout mice as
described previously (Muddashetty et al., 2007).
EXPERIMENTAL PROCEDURES
Luciferase Assay
Neuro2A and L-M(TK) cells were transfected with Lipofectamine 2000
(Invitrogen), cultured neurons were transfeted by magnetofection (Oz Bioscience), and luciferase activity was measured by dual luciferase assay (Promega).
Cell Culture
Primary neuronal cultures were prepared from cerebral cortices and hippocampi 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 30 UTR of mouse and human PSD-95 mRNA was amplified from cDNA.
Human PSD-95 30 UTR 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 30 UTR 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 (Invitrogen) 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 rhodamine-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) antibodies were used for western blots.
686 Molecular Cell 42, 673–688, June 10, 2011 ª2011 Elsevier Inc.
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).
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 (Emory University) for preparing constructs; and Andrew Swanson,
Tamika Malone, and Yukio Sasaki for technical assistance. This work was supported by FRAXA and NFXF postdoctoral fellowships (R.S.M.); National Institutes 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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