Article

miR-153 Regulates SNAP-25, Synaptic Transmission, and Neuronal Development

Department of Biological Sciences, Vanderbilt University and Medical School, Nashville, Tennessee, United States of America.
PLoS ONE (Impact Factor: 3.23). 02/2013; 8(2):e57080. DOI: 10.1371/journal.pone.0057080
Source: PubMed

ABSTRACT

SNAP-25 is a core component of the trimeric SNARE complex mediating vesicle exocytosis during membrane addition for neuronal growth, neuropeptide/growth factor secretion, and neurotransmitter release during synaptic transmission. Here, we report a novel microRNA mechanism of SNAP-25 regulation controlling motor neuron development, neurosecretion, synaptic activity, and movement in zebrafish. Loss of causes overexpression of SNAP-25 and consequent hyperactive movement in early zebrafish embryos. Conversely, overexpression of causes SNAP-25 down regulation resulting in near complete paralysis, mimicking the effects of treatment with Botulinum neurotoxin. -dependent changes in synaptic activity at the neuromuscular junction are consistent with the observed movement defects. Underlying the movement defects, perturbation of function causes dramatic developmental changes in motor neuron patterning and branching. Together, our results indicate that precise control of SNAP-25 expression by is critically important for proper neuronal patterning as well as neurotransmission.

Full-text

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miR-153
Regulates SNAP-25, Synaptic Transmission, and
Neuronal Development
Chunyao Wei
1.
, Elizabeth J. Thatcher
1.
, Abigail F. Olena
1
, Diana J. Cha
1
, Ana L. Perdigoto
2
,
Andrew F. Marshall
1
, Bruce D. Carter
2
, Kendal Broadie
1
, James G. Patton
1
*
1 Department of Biological Sciences, Vanderbilt University and Medical School, Nashville, Tennessee, United States of America, 2 Department of Biochemistry, Vanderbilt
University and Medical School, Nashville, Tennessee, United States of America
Abstract
SNAP-25 is a core component of the trimeric SNARE complex mediating vesicle exocytosis during membrane addition for
neuronal growth, neuropeptide/growth factor secretion, and neurotransmitter release during synaptic transmission. Here,
we report a novel microRNA mechanism of SNAP-25 regulation controlling motor neuron development, neurosecretion,
synaptic activity, and movement in zebrafish. Loss of miR-153 causes overexpression of SNAP-25 and consequent
hyperactive movement in early zebrafish embryos. Conversely, overexpression of miR-153 causes SNAP-25 down regulation
resulting in near complete paralysis, mimicking the effects of treatment with Botulinum neurotoxin. miR-153-dependent
changes in synaptic activity at the neuromuscular junction are consistent with the observed movement defects. Underlying
the movement defects, perturbation of miR-153 function causes dramatic developmental changes in motor neuron
patterning and branching. Together, our results indicate that precise control of SNAP-25 expression by miR-153 is critically
important for proper neuronal patterning as well as neurotransmission.
Citation: Wei C, Thatcher EJ, Olena AF, Cha DJ, Perdigoto AL, et al. (2013) miR-153 Regulates SNAP-25, Synaptic Transmission, and Neuronal Development. PLoS
ONE 8(2): e57080. doi:10.1371/journal.pone.0057080
Editor: Erik C. Johnson, Wake Forest University, United States of America
Received December 1, 2012; Accepted January 16, 2013; Published February 25, 2013
Copyright: ß 2013 Wei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Institutes of Health (NIH) to JGP (GM 075790 and EY019759), KB (GM 54544), and BDC (NS038220)
and by training fellowships to EJT (T32 GM08556) and A LP (F30 NS061403 and T32 GM07347) . Antibodies were obtained from the Zebrafish International
Resource Center NIH-NCRR, grant RR12546. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: James.G.Patton@Vanderbilt.edu
. These authors contributed equally to this work.
Introduction
Trimeric soluble N-ethylmaleimide-sensitive factor attachment
protein
receptor (SNARE) complexes form the core machinery
mediating vesicular exocytosis [1–3]. In the nervous system,
SNARE complexes are involved in membrane addition during
neuronal growth as well as both dense core vesicle (DCV) release
of proteins and synaptic vesicle (SV) release of fast neurotrans-
mitters. At synapses, the core SNARE protein SNAP-25 interacts
with accessory proteins that together regulate SV exocytosis by
linking Ca
2+
sensing to membrane fusion and neurotransmitter
release [4–7]. SNAP-25 is a specific target of Botulinum
neurotoxin proteases that block vesicle release, resulting in rapid
paralysis and death [8,9]. Misregulation of SNAP-25 is associated
with several human diseases and neurodegenerative disorders
including Huntington’s Disease [10], Alzheimer’s Disease [11],
and diabetes [12].
SNAP-25 is required for action potential-evoked glutamatergic,
cholinergic, and glycinergic transmission in neurons [13,14].
Mouse knockouts of SNAP-25 are therefore lethal although
neuronal cultures from SNAP-25 null mutants maintain the ability
to exhibit stimulus-independent transmitter release [13,15].
GABAergic inhibitory synapses express lower levels of SNAP-25
and may be more sensitive to calcium regulation, whereas
glutamatergic excitatory synapses express higher amounts of
SNAP-25 that alters calcium sensitivity [4]. Part of this differential
regulation could be due to accessory proteins that control SNAP-
25 distribution and levels to modulate synaptic activity [16–18].
Transcriptional mechanisms regulating SNAP-25 levels have also
been suggested to play key roles in the dynamic control of synaptic
function [19–23].
Several miRNAs have been shown to regulate synapse
formation or homeostasis, mostly within the post-synaptic dendrite
[22,24,25]. On the presynaptic side, most forms of regulation
center on modulation of calcium channels and calcium-dependent
vesicle release [26,27]. In this study, we show that miR-153 inhibits
SNAP-25 expression in the developing nervous system. Precise
control of SNAP-25 by miR-153 is necessary not only for
presynaptic vesicle release, but also for protein secretion, motor
neuron patterning, and outgrowth.
Results
miR-153 Regulates Embryonic Movement
miR-153 has been proposed to be one of a limited number of
ancient miRNAs that evolved with the establishment of tissue
identity [28]. It is conserved among bilaterians displaying distinct
expression patterns in neurosecretory brain cells of the deutero-
stome marine worm Platynereis dumerilii and the protostome annelid
Capitella [28]. In zebrafish, miR-153 is expressed in distinct regions
of the developing nervous system and brain, including neurose-
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cretory cells of the hypothalamus [29,30]. Using deep sequencing
and in situ localization, we detected robust miR-153 expression in
the developing zebrafish brain and reduced, but detectable levels
in the spinal cord as early as the 18 somite stage, with progressively
increasing expression thereafter [30,31] [32].
To determine the function of miR-153, we injected either
synthetic miR-153 or antisense morpholinos against miR-153 into
single cell embryos and allowed development to proceed for 1–2
days. Two different morpholinos were used to ensure specificity
and we verified overexpression and knockdown of miR-153 using
northern blots (Fig. S1). No gross morphological changes were
observed in injected embryos and normal localization of neuronal
markers was detected at the midbrain-hindbrain boundary, inner
ear, and retina at 1–2 dpf (data not shown). Despite the lack of
morphological changes, we observed striking behavioral move-
ment defects in injected embryos. To quantify movement,
embryos were recorded over time (Movie S1) with analyses
restricted to embryos within the chorion at 24 hpf. Normal
zebrafish embryos move within the chorion with a characteristic
frequency of ,1 twitch/minute at 24 hpf (Fig. 1). Strikingly,
embryos injected with miR-153 were almost completely motionless,
with little or no spontaneous movement, although their hearts
were beating normally and minimal movement could be elicited
by touch stimulation (Fig. 1). In contrast, knockdown of miR-153
caused a dramatic and significant 7-fold increase in the frequency
of spontaneous movement (Fig. 1). Interestingly, upon touch
stimulation, miR-153 morphants would initially respond with
unusually robust, hyperactive movements after which all motion
would cease altogether for a period of time (whether touched or
not), followed by a resumption of hyperactive movement upon
stimulation. At 52 hpf, miR-153 overexpression fish embryos were
still mostly motionless, while miR-153 knockdown embryos were
still hyperactive (data not shown).
miR-153 Targets snap-25
To identify mRNAs regulated by miR-153, we used target
prediction algorithms, compared the expression patterns of both
potential mRNA targets and miR-153, and assayed phenotypes
from gain and loss of function experiments. Based on these
criteria, snap-25 proved to be a bona fide target for miR-153 based
on the results of reporter silencing experiments (Fig. 2) and
consistent with conservation of miRNA recognition elements
(MREs) from fish to humans (Fig. S2).
There are two SNAP-25 paralogs in zebrafish (a and b isoforms)
with similar, but not identical, 39 UTRs [33,34]. For reporter
assays, we fused the 39 UTR from both snap-25 isoforms to the
GFP reading frame (snap-25a data shown in Fig. 2A; snap-25b
shown in Fig. S3). Synthetic mRNAs prepared from these
reporters were injected into single cell embryos in the presence
or absence of exogenous miR-153 or miR-153 morpholinos (MOs).
Based on fluorescence levels in live embryos at 1 dpf, co-injection
of miR-153 resulted in obvious down-regulation of GFP for both
isoforms (Fig. 2B). To confirm that the loss of GFP was due to
pairing with the predicted MREs, we created deletions of
individual and combinations of MREs in snap-25a and snap-25b.
Deletion of both MREs from snap-25a and all three MREs from
snap-25b abolished the ability of miR-153 to silence expression
(Fig. 2B; Fig. S3B). For snap-25a, we tested each of the individual
MREs and found that deletion of a single MRE resulted in only
modest silencing whereas deletion of both MREs caused a loss of
silencing. We conclude that miR-153 targets both isoforms of snap-
25 in an MRE-dependent manner.
If miR-153 targets snap-25, knockdown of endogenous miR-153
should lead to increased reporter fluorescence. To test this
prediction, antisense morpholinos were co-injected with reporter
mRNAs (Fig. 2). We found that knockdown of miR-153 caused
a significant increase in GFP expression compared to embryos
with wild type levels of endogenous miR-153. Lastly, we performed
western blots using antibodies against GFP and analyzed protein
Figure 1.
miR-153
regulates embryonic movement. Embryonic movement was recorded at 1 dpf for each of the singly and multiple injected
conditions shown (see Movies). The number of twitches per minute was counted and significance determined by comparing the noninjected control
(NIC) embryos to all other conditions using ANOVA with Dunnett’s post-test. *, p,0.05; **, p,0.01. Movements were counted for approximately 60
embryos over 2–5 minutes for each condition.
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miR-153 Regulation of Snap-25
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miR-153 Regulation of Snap-25
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levels in lysates prepared from pools of embryos treated as above
(Fig. 2C,D). The levels of GFP mirrored the effects observed using
fluorescence imaging in live embryos–reduced reporter expression
in the presence of miR-153 and increased reporter expression upon
knockdown of miR-153 (Fig. 2C,D). In all cases, the effects were
dependent on intact MREs. Taken together, the in vivo reporter
assays and western blots support the conclusion that snap-25 is
a target of miR-153.
We next tested whether miR-153 targets endogenous snap-25.
Single cell embryos were injected with either miR-153 or antisense
morpholinos followed by western blots on pooled 1 dpf embryo
lysates using antibodies against SNAP-25. Titration experiments
were performed to optimize the levels of injected reagents (Figs.
S4,S5). After optimization, protein levels were analyzed and fold
changes in expression were determined compared to the amounts
detected in noninjected controls (NIC) (Fig. 3). Under these
conditions, excess miR-153 led to a ,50% decrease in SNAP-25
levels whereas knockdown of endogenous miR-153 increased
SNAP-25 levels ,2-fold. To test for specificity we co-injected
embryos with combinations of miR-153, snap-25a,b mRNAs, or
morpholinos against both (Fig. 3). Injection of mRNAs encoding
snap-25a,b resulted in a 2-fold elevation in SNAP-25 levels whereas
injection of morpholinos that block the translation start site of snap-
25 led to a ,50% decrease in SNAP-25 levels. Importantly, co-
injection of combinations of RNAs and morpholinos could
suppress these effects and rescue SNAP-25 levels (Fig. 3). For
both suppression experiments, the effects were dose dependent.
Even though snap-25a was more effective than snap-25b at rescuing
endogenous SNAP-25 levels, combinations both were the most
effective (Fig. 3). These results indicate specific targeting of snap-25
by miR-153. Although miR-153 is likely to have additional targets,
the ability to specifically rescue the effects of overexpression and
knockdown of both miR-153 and snap-25 indicates that the effects
we observe are specific to targeting of snap-25 by miR-153.
miR-153 Regulates snap-25 to Control Movement
Because we could specifically suppress the effects of over-
expression or knockdown of miR-153 by co-injection of either snap-
25a,b mRNA or morpholinos against snap-25a,b, we next sought to
test whether the movement defects are caused by altered miR-153
levels could likewise be rescued in a snap-25 dependent manner.
Embryonic movements were quantitated at 24 hpf after injection
of antisense morpholinos against snap-25 (snap25
MO
) or with snap-
25a,b mRNAs (Fig. 1; Movie S1). Knockdown of snap-25 resulted
in dramatically decreased embryonic movements, similar to
overexpression of miR-153 (Fig. 1). In contrast, overexpression of
snap-25a,b increased movement approximately 5-fold over control
NIC embryos (Fig. 1). For rescue experiments, co-injection of snap-
25a,b mRNA with miR-153 restored near normal movement
(Fig. 1; Movie S1). Similarly, co-injection of morpholinos against
both snap-25 and miR-153 also restored normal movement (Fig. 1;
Movie S1). Thus, not only were SNAP-25 protein levels restored to
normal, but also movement defects were rescued, demonstrating
specific targeting of snap-25 by miR-153.
SNAP-25 is a known target of Botulinum neurotoxin (BoNT)
proteases A and E [8,9]. If miR-153 is targeting snap-25, the effects
of increased miR-153 should mimic the effects of BoNT A. To test
this prediction, injected zebrafish were exposed to BoNT A for 30
minutes at 27 hpf. One hour later, western blots were performed
on pooled protein samples to determine whether it was possible to
rescue SNAP-25 over-expression phenotypes associated with miR-
153 knockdown or injection of snap25a,b mRNAs. Exposure to
BoNT A dramatically reduced SNAP-25 levels, recapitulating the
effects of miR-153 knockdown and over-expression (Fig. 4A,B). For
movement, exposure to BoNT A rescued the hyperactive
phenotypes observed after injection with MOs against miR-153
or overexpression of snap-25a&b mRNAs (Fig. 4C; Movie S1).
Together, these experiments strongly support the conclusion that
miR-153 specifically targets snap-25 to regulate embryonic move-
ment.
miR-153 Regulation of Motor Neuron Development
SNAP-25 is a well-characterized t-SNARE protein, with an
established function in vesicular exocytosis [1–3]. In the de-
veloping nervous system, the SNARE complex mediates vesicular
membrane addition driving neurite outgrowth and morphological
patterning [1–3,35]. Moreover, DCV-mediated release of signal-
ing proteins and growth factors is important for axon guidance,
path finding, and morphological development [36–39]. We
therefore sought to determine whether snap-25 regulation by
miR-153 would alter neuronal morphogenesis. Because zebrafish
motor neuron development is well characterized [40–45], we
decided to focus on the effects of miR-153 on motor neurons
during early zebrafish development.
We first injected miR-153 or morpholinos against miR-153 to
observe the effects on the development and morphology of motor
neurons in a transgenic zebrafish line in which motor neurons are
specifically labeled with RFP (Tg(mnx1:TagRFP-T) [46]. Perturba-
tion of miR-153 levels caused striking changes in motor neuron
structure and branching (Fig. 5A,B). Compared with NICs,
overexpression of miR-153 dramatically changed the axonal
architecture with significant decreases in branch numbers and
length (Fig. 5C, D). Knockdown of miR-153 resulted in completely
opposite effects with increased motor projection architectural
complexity, increased axonal length, and increased branch
numbers (Fig. 5B–D). To test whether the effects were specific,
we conducted rescue experiments, as above. Injection of snap-25a,b
mRNA or morpholinos against snap-25a/b produced virtually the
same phenotypes observed in embryos subjected to miR-153
knockdown or overexpression, respectively. In contrast, co-
injection of miR-153 and snap-25a,b mRNAs or morpholinos
against miR-153 and snap-25a,b almost completely restored the
normal patterning and branching of motor neurons (Fig. 5B–D).
These results indicate that miR-153 regulates motor neuron
development via control of snap-25a,b.
To further dissect the function of miR-153 on motor neuron
development, immunofluorescence was performed on whole-
mount zebrafish embryos (55 hpf) with antibodies that label
Figure 2.
miR-153
targets
snap-25a
. (A) GFP reporter constructs were created by fusing the reading frame of GFP to the snap-25a 39UTR. Two
predicted miRNA recognition elements (MREs) were identified in the snap-25a 39 UTR. The miR-153 sequence is indicated in red and the
corresponding snap-25a UTR sequence is shown in green. (B) Single cell zebrafish embryos were injected with mRNAs derived from GFP reporters
lacking a UTR (GFP), fused to the full length snap-25a UTR (+snap-25), or mutant versions of the snap-25a UTR lacking individual MREs (snap-
25aDMRE1 and snap-25aDMRE2) or both MREs (snap-25aDMRE1&2). Embryos were injected in the presence or absence of exogenous miR-153 or
morpholinos against miR-153 (miR-153
MO
). Fluorescence levels were examined at 1 dpf. Clusters of embryos (,60) are shown as well as a high
magnification image of a single representative embryo. (C) Lysates from ,100 embryos were prepared from embryos treated as in B and GFP protein
levels were determined by western blotting using antibodies against GFP or control antibodies against a-tubulin. (D) Quantitation of westerns was
performed with a paired Student’s t-test (n = 5).
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miR-153 Regulation of Snap-25
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primary (Znp-1 or anti-synaptotagmin 2) or secondary (Zn-8 or
Alcama) motor neurons [47]. Compared to NIC embryos,
a striking difference in primary motor neuron axon architecture
was observed with both miR-153 overexpression (miR-153) and
knockdown (miR-153
MO
)(Fig. 6). A significant decrease in branch-
ing was observed in miR-153 injected embryos whereas knock-
down of miR-153 caused a dramatic increase in branching.
Likewise, injection of snap-25a,b mRNA led to increased axonal
growth and branching in primary motor neurons whereas
knockdown of snap-25a,b caused decreased outgrowth and
branching (Fig. 6). Co-injection experiments showed that snap-
25a,b mRNA and morpholinos against snap-25 could partially
counteract the effects of the corresponding gain and loss of miR-
153.
For secondary motor neurons, rostral axon outgrowth was
similarly stunted and/or irregularly spaced by miR-153 over-
expression and slightly elongated by miR-153 knockdown (Fig. S6).
Differences in the caudal region were minimal compared to earlier
developing rostral neurons, possibly reflecting temporal limitations
to injection experiments or perhaps increased vulnerability of
rostral motor neurons to altered SNAP-25 levels. Focusing on
rostral effects, injection of snap-25a,b mRNA phenocopied miR-153
knockdown and injection of morpholinos against snap-25 resulted
in patterns that closely resembled miR-153 overexpression. Co-
injection of morpholinos against both miR-153 and SNAP-25
largely restored normal secondary motor neuron patterning,
although the injection of snap-25a,b mRNAs was not as effective
at rescuing the defects that resulted from miR-153 overexpression
(Fig. S6). This may indicate a possible additional function for miR-
153 in regulating axonal growth and patterning during secondary
motor neuron development.
Expression of miR-153 in Mo tor Neurons
To ensure that the effects of miR-153 on motor neuron
patterning were due to expression of miR-153 in these cells, we
FACS sorted cells from the trunks of 52 hpf (Tg(mnx1:TagRFP-T)
embryos and conducted RT/qPCR. As shown in Fig. 7, there was
a greater than 10-fold enrichment for miR-153 in RFP+ cells
compared to RFP- cells. Prior work had shown that miR-153 is
expressed in the brain and spinal cord but these results show that
miR-153 is expressed in developing motor neurons.
Figure 3.
miR-153
regulates endogenous
snap-25a
expression. (A) Embryo lysates were prepared from either NIC embryos or embryos injected
with miR-153, miR-153
MO
, mRNAs encoding snap-25a and snap-25b, morpholinos against snap-25, or combinations thereof, as indicated. Western
blots were performed using antibodies against SNAP-25 and a–tubulin. (B) Quantification of SNAP-25 levels from the western blots (n = 3) shown in A.
Significance was determined by a two-tailed Student’s t-test. Error bars show s.e.m.
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miR-153 Regulates Vesicular Exocytosis to Control
Signaling
Since SNAP-25 has a well-established function in the fusion and
release of numerous vesicle types, we next examined the role that
miR-153 plays in modulating exocytosis. Owing to the core role of
miR-153 in movement control, we first focused on synaptic activity
at the neuromuscular junction (NMJ) in zebrafish embryos. For
this analysis, we measured synaptic vesicle (SV) cycling using the
styryl dye, FM1-43 [48,49]. At 55 hpf, embryonic NMJs were
imaged with Alexa 594-conjugated a-bungarotoxin (a-Btx) to label
postsynaptic acetylcholine receptor (AChR) clusters, while mon-
itoring FM1-43 uptake into NMJ presynaptic boutons (Fig. 8). The
terminals were acutely depolarized for 5 minutes with high [K
+
]
saline (45 mM) to drive the SV cycle and load FM1-43, whereas
only weak loading was evident in low [K
+
] conditions. In non-
injected controls, fluorescence was observed along terminal axon
branches with intense staining at individual synaptic varicosity
boutons (Fig. 8A). Compared to NIC labeling, miR-153 over-
expression resulted in a significant decrease in FM1-43 loading in
presynaptic terminals, indicating slowing of the SV cycle (Fig. 8B).
In sharp contrast, knockdown of miR-153 showed a significant
increase in FM1-43 loading, indicating an elevated SV cycling rate
(Fig. 8C). The significant difference between miR-153 knockdown
and overexpression conditions indicates that miR-153 plays an
important role in controlling the rate of vesicle cycling (Fig. 8D).
Together, these results reveal a key function for miR-153 in the
control of presynaptic vesicle release at the embryonic NMJ,
consistent with a role for miR-153 in the regulation of embryonic
movement. The overall effects on movement are therefore
a combination of effects on motor neuron development and
patterning as well as overall exocytic activity.
SNAP-25 has a highly conserved role mediating vesicular fusion
in both neurons and other neurosecretory cells where it is critical
for DCV release [50]. To test whether miR-153 plays a role in this
secretory context, we examined exocytosis in a rat neuroendocrine
pituitary cell line (GH4C1) expressing human growth hormone
(hGH) [51]. Release of hGH in these cells provided a functional
readout of exocytic activity (Fig. 9). GH4C1 cells were therefore
transfected with miR-153, morpholinos against miR-153/snap-25,
or vectors expressing snap-25a,b, followed by determination of
hGH levels in the media by ELISA. Overexpression of miR-153
and knockdown of snap-25a,b (snap-25a,b
MO
) reduced the levels of
hGH to below the amount detected in culture media from mock
transfected cells (Fig. 9). In sharp contrast, knockdown of miR-153
and overexpression of snap-25 both significantly increased the
amount of secreted hGH 8–10 fold over the mock transfected
control (Fig. 9). The differences observed due to perturbation of
miR-153 levels in the GH4C1 cell line compared to embryonic
NMJs are most likely due to differences in the efficiency of miR-
153/miR-153
MO
delivery between the two experiments, as well as
developmental differences. Nevertheless, the effects in this case
were fully suppressed by co-expression of either miR-153/snap-
25a,b mRNA or MOs against miR-153/snap-25a,b, demonstrating
specific regulation of snap-25 by miR-153. These data strongly
support the conclusion that miR-153 functions to precisely control
SNAP-25 levels to regulate vesicle exocytosis.
Discussion
In this study, we show that miR-153 regulates the critical core
SNARE component, SNAP-25, to modulate exocytosis and
neuronal development. Increased miR-153 levels cause decreased
SNAP-25 expression resulting in decreased embryonic movement,
decreased neuronal secretion, and decreased neuronal growth/
Figure 4.
miR-153
mimics the effects of BoNT A. (A) Single cell
embryos were injected as indicated and then at 27 hpf, exposed to
Botulinum neurotoxin A (BoNT) for 30 minutes. After recovery for 1
hour, western blots were performed on emb ryo lysates using
antibodies against SNAP-25 or a–tubulin. (B) Quantitation of SNAP-25
levels from A, n = 3. **, p,0.01 (C) Embryonic movement in the
presence or absence of BoNT A. The number of twitches per minute
was counted as in Fig. 1 for embryos treated as indicated. Significance
was determined by comparing mock embryos to all other conditions
using ANOVA with Dunnett’s post-test, n = 15. *, p,0.05.
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branching. Conversely, miR-153 knockdown causes elevated
SNAP-25 expression resulting in hyperactive movement, increased
neuronal secretion, and increased neuronal growth/branching.
Accumulating evidence suggests that SNAP-25 misregulation plays
a role in numerous human disease states including ADHD,
schizophrenia, bipolar I disorder, Huntington’s disease, Alzhei-
mer’s disease, and diabetes [52]. Regulated expression of miR-153
provides an attractive model to mechanistically explain tight
control of SNAP-25 levels.
SNAP-25 Functions during Development
It is well established that axon outgrowth during neuronal
development occurs via SNARE-dependent addition of membrane
for growth cone extension [35,53]. Axonal growth, pathfinding,
and target recognition are secondarily modulated by SNARE-
dependent release of developmental signals via dense core vesicle
(DCV) exocytosis [54–59]. The outgrowth of both axons and
dendrites is blocked by Botulinum neurotoxins A and C1,
proteases specific for SNAP-25, demonstrating a direct role of
SNAP-25 in neuronal morphogenesis [55,56,60]. Likewise, in-
Figure 5.
miR-153
regulates the morphology and structure of motor neurons. (A) A transgenic zebrafish line, Tg(mnx1:TagRFP-T), that
expresses RFP in motor neurons was used to monitor the effects of altered levels of miR-153 and snap-25 at 55 hpf. For all confocal images,
developing motor neurons were examined from the same somites, as indicated. (B) Morphology of developing motor neurons under each of the
indicated conditions. Arrows indicate increased branching after knockdown miR-153 (miR-153
MO
) or overexpression snap-25a,b mRNA. Arrowheads
indicate the structural defects after miR-153 overexpression or knockdown of snap-25a,b (snap-25a,b
MO
). Scale bar: 20 mm. (C) Quantification of motor
neuron axonal branch number under the different conditions shown in (B). Error bars show s.e.m. Significance was determined using ANOVA with
Dunnett’s post-test, n = 5. *, p,0.01; **, p,0.005. (D) Quantification of motor neuron axon length relative to uninjected control under the different
conditions shown in (B). Error bars show s.e.m. ANOVA with Dunnett’s post-test, n = 5. *, p,0.05; **, p,0.01.
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hibition of SNAP-25 by antisense oligonucleotides blocks axonal
outgrowth [54]. In stark contrast, neuronal outgrowth was
surprisingly not inhibited in SNAP-25 null mice [13]. The
explanation for this inconsistency is not clear. Our results show
a clear requirement for SNAP-25 in motor neuron outgrowth and
branching in zebrafish. It is possible that the requirement for
SNAP-25 may be species specific but we found that altered levels
of miR-153 caused similar branching defects in rat PC12 cells as
observed in zebrafish motor neurons, strongly arguing against this
(data not shown). Perhaps the differences are due to cell-specific
requirements for SNAP-25. In the retina, for example, SNAP-25 is
expressed in a dynamic spatiotemporal pattern and such
differential expression may underlie specific development of
cholinergic amacrine cells and photoreceptors [61]. An intriguing
possibility based on the results presented here is that develop-
mental, stage-specific and/or cell-specific expression of miR-153
Figure 6.
miR-153
regulates primary motor neuron development. (A) Immunofluorescence performed on whole mount zebrafish embryos at
55 hpf using Znp-1 antibodies to label primary motor neurons. Confocal images were acquired from the same somites for all embryos, as indicated.
(B) Effects on primary motor neuron structure and branching under the indicated conditions. Scale bar: 40 mm.
doi:10.1371/journal.pone.0057080.g006
miR-153 Regulation of Snap-25
PLOS ONE | www.plosone.org 8 February 2013 | Volume 8 | Issue 2 | e57080
Page 8
may similarly regulate SNAP-25 levels, which then drives de-
velopmental and cell-specific effects.
SNAP-25 in Synaptic Vesicle Exocytosis
SNAP-25 is one of three SNARE proteins that contribute a-
helices that mediate fusion between synaptic vesicles and pre-
synaptic membranes [1,3]. Blockage of synaptic transmission by
Clostridium and Botulinum neurotoxins first established that
SNARE proteins are critical for neurotransmitter release [62].
Cleavage of SNAP-25 by Botulinum neurotoxin A causes
a paralytic phenotype that resembles the loss of movement we
observe in zebrafish embryos expressing excess miR-153. SNAP-25
haploinsufficient mice show no observable phenotypic defects but
complete loss of SNAP-25 blocks evoked synaptic transmission
[13]. Moreover, overexpression of SNAP-25 inhibits normal
calcium responsiveness and can impair memory-associated syn-
aptic plasticity [63]. These findings suggest that modulation of
SNAP-25 levels are important for overall SNARE function,
especially in generating differences in calcium dependence
between neuronal and non-neuronal secretory vesicular fusion
events. Matteoli and colleagues (2009) have shown that SNAP-25
is differentially expressed between excitatory glutamatergic and
inhibitory GABAergic neurons in a developmental-specific man-
ner [4]. These results remain controversial, as earlier studies did
not observe this difference, but the data are consistent with an
important role for SNAP-25 as a required component for both
glutamatergic and GABAergic transmission [64,65]. Mechanisms
for how SNAP-25 levels might be regulated in a development-
and/or cell-specific manner are uncertain, but our data strongly
support miRNA regulation as a likely candidate and a critical
mechanism controlling SNAP-25 levels. A recent report describing
the effects of chronic overexpression of SNAP-25 in the rat dorsal
hippocampus demonstrated the critical importance of controlling
SNAP-25 levels [63]. Elevated expression of SNAP-25 produced
increased levels of secreted glutamate with cognitive deficits similar
to those observed in ADHD and schizophrenia. We propose that
miR-153 control of SNAP-25 levels allows for precise regulation of
SNAP-25 during development and exocytosis.
miRNAs Regulation of Neuronal Morphogenesis and
Synaptic Activity
Localized translation control in synaptic dendrites is common,
requiring repression of mRNA translation during transport.
miRNA mediated inhibition of translation is an attractive
mechanism that can precisely control gene expression in neurons.
Consistent with this hypothesis, many miRNAs are neuron or
brain specific [66]. Moreover, the effector complexes that carry
out repression of translation (RNA Induced Silencing Complexes;
RISCs) are composed of several subunits that have been
implicated in both neuronal function and disease [22,24,67]. For
example, nervous system specific miRNAs have been shown to
regulate the maturation of dopamine neurons in the midbrain as
well as control serotonin transport by regulating the serotonin
transporter [68,69]. Likewise, miR-1, miR-124, miR-125b, miR-132,
bantam, miR-34 and the miR-310 cluster have all been implicated in
the modulation of synaptic homeostasis [70–76]. Similarly,
synaptic plasticity is reportedly regulated by miR-134 through
targeting of SIRT1 or Limk1, which control dendritic spine
morphogenesis [77,78]. In addition, miR-124 in retinal ganglion
cell growth cone was shown to act through CoREST to regulate
the intrinsic temporal sensitivity to Sema3A, a guide cue during
axonal pathfinding and morphogenesis [79]. Our work demon-
strates that miR-153 is a member of this subset of miRNAs
implicated in neuronal function but by a distinctly different
mechanism through targeting of snap-25. miR-153 also likely
targets other mRNAs [80], but SNAP-25 regulation alone is
required and sufficient to explain the role of miR-153 regulation of
movement, motor neuron morphogenesis, and SNARE-mediated
secretion.
Materials and Methods
Ethics Statement
The Animal Care and Use Committee monitors all animal care
and research at Vanderbilt. Vanderbilt University has on file with
the Office for Protection from Research Risks of the NIH an
Assurance of Compliance with Public Health Service regulations
and requirements and provisions of the Animal Welfare Act. All
zebrafish experiments in this paper were approved by the
Vanderbilt University Institutional Animal Care and Use Com-
mittee (IACUC) under protocol M-09-398. In accordance with
that protocol, all necessary means were taken to avoid pain. For
any manipulations that might induce pain, animals were
anesthetized with a 0.15% solution of Tricaine (3-amino-benzoic
acidethylester). The approved method for euthanizing zebrafish is
incubation in ice water.
Microinjections
Single cell zebrafish male and female embryos were injected
with 200 pg of miR-153, 5 ng each of miR-153
MO
and miR-
153
loopMO
and/or 100 pg of in vitro-transcribed, capped GFP
reporter mRNA with or without the snap-25a or b 39UTR.
Zebrafish snap-25a,b 39 UTR sequences were amplified by PCR
and subcloned downstream of the GFP ORF in pCS2+ [81].
Figure 7.
miR-153
is expressed in motor neurons. To enrich for
motor neurons, heads were removed from 52 hpf embryos just
posterior to the otic vesicle and trunks were dissociated to facilitate
sorting of RFP+ and RFP- cells. RNA was isolated from these cell
fractions and RT/PCR was performed to determine miR-153 levels
relative to U6 snRNA. Significance was determined by a two-tailed
Student’s t-test with the error bars representing s.e.m.; p,0.02.
doi:10.1371/journal.pone.0057080.g007
miR-153 Regulation of Snap-25
PLOS ONE | www.plosone.org 9 February 2013 | Volume 8 | Issue 2 | e57080
Page 9
miR-153 Regulation of Snap-25
PLOS ONE | www.plosone.org 10 February 2013 | Volume 8 | Issue 2 | e57080
Page 10
Rescue experiments used injections of 3 ng of snap-25
StartMO
and
snap-25
5’UTRMO
, 150 pg of snap-25a,b mRNA, 250 pg of snap-25a
mRNA, or 300 pg of snap-25b mRNA without 39UTRs.
Two different morpholinos against miR-153 were utilized. One
was perfectly complementary to the mature sequence; the second
was complementary to a portion of the mature sequence and then
extending into the precursor loop. Targeting of snap-25a,b mRNAs
was performed using morpholinos against the region including the
start codon.
Botulinum Toxin Analysis
Embryos injected at the 1-cell stage were treated with purified
Botulinum neurotoxin A (Metabiologics, Inc., Madison, WI).
Initial titration experiments were performed testing a range of
BoNT A concentrations with final selection of 1 ng per 10 ml of
water for 30 minutes at either 24-hpf or 48-hpf. Embryos were
washed 10 times in fresh water and then allowed to recover for 1
hour prior to protein extraction or video capture to monitor
movement.
qRT-PCR and Northern Blots
Total RNA extracted from both RFP+ and RFP- cells was
reverse transcribed and qPCR reactions were carried out using
Taqman miRNA assays (Life Technologies, NY) using the CFX96
Real-time PCR system (Bio-Rad), as previously described [32].
Northern blots were also performed as described [82,83].
Western Blots
Embryos were dechorionated, deyolked, and sonicated in lysis
buffer as described [83]. Approximately 100 embryos were pooled
and one-tenth of the resulting samples were loaded into each lane.
Membranes were probed with antibodies against a-tubulin
(Abcam, ab15246), GFP (Torrey Pines, TP401) or SNAP-25
(Alomone Labs). For detection, anti-rabbit or anti-mouse HRP-
conjugated secondary antibodies were used, followed by visuali-
zation with ECL.
GFP Reporter Analyses
Reporter analyses and western blots were as described [83]. To
generate the snap-25a,b GFP reporters, the GFP ORF was fused to
the 39 UTR sequence of zebrafish snap-25a or b. snap-25a,b UTRs
were cloned from zebrafish whole embryo RNA preparations
using oligo d(T) primed reverse transcription followed by PCR
amplification with gene specific primers. Images were acquired
with a Leica MZFIII dissecting scope equipped with a fluorescent
laser using a Qimaging camera with Qimaging software and
imported into Adobe Photoshop for orientation and cropping.
Immunofluorescence
Embryos were fixed in 4% PFA overnight at 4uC and then
permeabilized in 0.5% TritonX-100 for 60 minutes followed by
treatment with protease K (20 mg/ul) for 10 minutes at room
temperature. Samples were washed in PBT-DMSO before
blocking overnight at 4uC (PBT-DMSO, 2% BSA, 5% goat
serum). Primary antibodies (SNAP-25, 1:1000; SV-2, 1:300; ZNP-
Figure 8.
miR-153
regulates synaptic activity at the neuromuscular junction. (A) FM1-43 loading of neuromuscular junction (NMJ) boutons
in 55 hpf fish embryos. (B) Postsynaptic clusters of AChRs were labeled with Alexa 594-conjugated a-bungarotoxin. Overexpression of miR-153
caused decreased FM1-43 loading, indicating down-regulation of the synaptic vesicle cycle within NMJ boutons (arrowheads). (C) Knockdown of miR-
153 (miR-153
MO
) promoted greater uptake of FM1-43 dye, indicating increased synaptic vesicle cycling. Scale bar: 10 mm. (D) Quantification of FM1-43
fluorescent intensity with a paired Student’s t-test. Error bars show s.e.m. *p,0.01; **p,0.02.
doi:10.1371/journal.pone.0057080.g008
Figure 9.
miR-153/snap-25
regulates vesicular exocytosis. GH4C1 cells stably expressing human growth hormone (hGH) were transfected, as
indicated. The effects of exogenous expression on hGH levels secreted into the culture media were determined by ELISA using hGH antibodies.
Significance was determined by comparing mock transfected to all other treatments using ANOVA with Dunnett’s post-test. Error bars show s.e.m. *,
p,0.01.
doi:10.1371/journal.pone.0057080.g009
miR-153 Regulation of Snap-25
PLOS ONE | www.plosone.org 11 February 2013 | Volume 8 | Issue 2 | e57080
Page 11
1, 1:2000; ZN-8, 1:25) were incubated overnight at 4uC, washed
with PBT-DMSO, and then embryos were incubated with Cy5 or
Cy3-conjugated donkey anti-mouse or rabbit antibodies (Jackson
Immuno) for 4 hrs at room temperature. Before mounting and
visualization, embryos were washed with PBT-DMSO. PC12 cells
were fixed in 4% PFA for 15 mins, washed in PBS before
incubating with primary antibodies for 1 hr, washed, incubated
with secondary antibodies for 1 hr, Hoechst dye for 5 mins,
washed, and visualized.
Tissue Dissociation and Motor Neuron Isolation
Tg(mnx1:TagRFP-T) zebrafish embryos of 52 hpf were dechor-
ionated, deyolked, and then dissected just posterior to the otic
vesicle to collect trunks (excluding the hearts). Tissues were kept in
buffer (16PBS, pH 6.4, 1%BSA) and then dissociated using
16 U/ml papain and 0.2 U/ml Dispase (Worthington, NJ) for
30 mins at 28uC on a rotator. After complete dissociation of the
tissue by careful pipetting up and down, cells were pelleted at
80006g for 2 mins. Resuspended cells were then treated with
1 mg/ml leupeptin (Worthington, NJ) and 100 U/ml DNaseI
(Sigma-Aldrich) in PBS at pH 7.4 containing 2 mg/ml MgCl
2
for
10 mins at room temperature and then kept on ice for RFP+ and
RFP- cell isolation. Gating was based on cell size and fluorescence
intensity, determined by the control sample of dissociated cells
from WT fish at the same developmental stage.
FM1-43 Dye Labeling
Embryos at 55 dpf were incubated in HBSS (137 mM NaCl,
5.4 mM KCl, 1 mM MgSO
4
, 0.44 mM KH
2
PO
4
, 0.25 mM
Na2HPO4, 4.2 mM NaHCO3, 1.3 mMCaCl2, 5 mM Na-
HEPES) containing 0.2% Tricaine and glued onto sylgard coated
glass chambers before removing the skin using a glass needle.
FM1-43 and a-bungarotoxin (a-Btx) labeling procedures were as
previously published [49], except the preloading incubation of
FM1-43 dye was omitted and the Advasep incubation period was
elongated to 15 mins. For data analysis, axons with puncta labeled
with a-Btx were considered as synaptic boutons. FM1-43 puncta
with sizes of 0.5–2 mm were collected for analysis using Image J.
Cell Culture and ELISA
PC12 cells (ATCC CRL-1721) were maintained using Ham’s
F12K media with 15% horse serum and 5% FBS, and transfected
individually or in combination with miRNAs, mRNAs, and
morpholinos. Transfections were performed with 300 nM miR-
153, biotinylated snap-25 MOs and miR-153 MOs and 1.5 m gof
snap-25a,b using Lipofectamine 2000 [84]. Co-transfection of
a GFP plasmid was used to determine transfection efficiencies.
Efficiencies less than 50% were discarded. One day after
transfection, 50 ng/ml nerve growth factor was added to media
to induce differentiation. Neurite outgrowth was assayed at day 5
by immunostaining with antibodies against acetylated a-tubulin.
Stably transfected GH4C1 cells were a gift from Dr. K.
Kannenberg [51]. ELISAs were performed after 5 days of
transfection and human growth hormone was assayed following
the Diagnostic Systems ELISA kit.
Supporting Information
Figure S1 Northern blot of miR-153 overexpression and
knockdown. Perturbation of miR-153 expression levels by
injection of miR-153 or MOs against different regions of pre-
miR-153 was verified by northern blot. U6 served as a loading
control.
(TIF)
Figure S2 Conservation of snap-25 39 UTR sequences.
The 39 UTRs from mouse, human and zebrafish snap-25a (A) and
snap-25b (B) are shown with the MREs that pair with miR-153
boxed in green. Conserved nucleotides are marked by an asterisk.
The exact pairings between the MREs and miR-153 are shown in
Figure 2 and Figure S3. Despite different levels of conservation,
both MREs in snap-25a pair extensively with miR-153 in the seed
region.
(TIF)
Figure S3 miR-153 targets snap-25b. (A) GFP reporter
constructs were created by fusing the reading frame of GFP to the
snap-25b 39UTR. Three predicted miRNA recognition elements
(MREs) were identified in the snap-25b 39 UTR. The miR-153
sequence is indicated in red and the corresponding snap-25a UTR
sequence is shown in green. (B) Single cell zebrafish embryos were
injected with mRNAs derived from GFP reporters lacking a UTR
(GFP), fused to the full length snap-25b UTR (GFP+snap-25b), or
mutant version of the snap-25b UTR lacking all MREs (GFP+snap-
25bDMRE1, 2&3). Embryos were injected in the presence or
absence of exogenous miR-153 or morpholinos against miR-153
(miR-153
MO
). Fluorescence levels were examined at 1 dpf. Clusters
of embryos (,30) are shown. (C)Lysates from ,100 embryos were
prepared from embryos treated as in B and GFP protein levels
were determined by western blotting using antibodies against GFP
or control antibodies against a-tubulin.
(TIF)
Figure S4 Dose-dependent rescue of miR-153 knock-
down. (A) Single cell embryos were injected with a constant level
of miR-153
MO
and increasing amounts (increments of 2 ng) of snap-
25
MOs
. Embryo lysates from ,60 embryos in each group were
prepared and SNAP-25 protein levels determined by western
blotting. (B) Quantitation of westerns (n = 3) from A. The grey
circle represents the amount of snap- 25
MO
(10 ng) used in co-
injection rescue experiments.
(TIF)
Figure S5 Dose-dependent rescue of miR-153 over-
expression. (A) Single cell embryos were injected with a constant
level of miR-153 and increasing amounts (increments of 50 pg) of
snap-25a, snap-25b,orsnap-25a&b mRNA. Embryo lysates from
,60 embryos were prepared from embryos in each treatment
group and SNAP-25 protein levels were determined by western
blotting. (B) Quantitation of westerns (n = 3) from A. The grey
circles represent the amounts used in co-injection rescue experi-
ments (75 pg each of snap-25a and b, 250 pg of snap-25a, and
300 pg of snap-25b).
(TIF)
Figure S6 miR-153 regulates secondary motor neuron
development. (A) Immunofluorescence was performed on whole
mount zebrafish embryos at 55 hpf using Zn-8 antibodies to label
secondary motor neurons. Confocal images were acquired from
the same somites for all embryos, as indicated. (B) miR-153
knockdown (miR-153
MO
) and snap-25a,b overexpression signifi-
cantly increased the growth of secondary motor neuron axons
(arrows). Overexpression of miR-153 or knockdown of snap-25a,b
(snap-25a,b
MO
) caused severe defects in axon development and
architecture (asterisks). Scale bar: 40 mm.
(TIF)
Movie S1 Embryo Movements in different conditions.
0:00–0:11. NIC Embryo Movements at 24 hpf Noninjected
control (NIC) zebrafish embryos at 24 hpf were filmed for one
minute. Twitching was counted from individual embryos over
multiple movies, as quantitated in Figure 1. 0:11–0:21. Effects
miR-153 Regulation of Snap-25
PLOS ONE | www.plosone.org 12 February 2013 | Volume 8 | Issue 2 | e57080
Page 12
of miR-153 Overexpression on Movement at 24 hpf Single
cell zebrafish embryos were injected with miR-153 and filmed for
one minute at 24 hpf. Twitching was counted from individual
embryos over multiple movies, as quantitated in Figure 1. 0:22–
0:32. Effects of Knockdown of miR-153 on Movement at
24 hpf Single cell zebrafish embryos were injected with miR-
153
MOs
and filmed for one minute at 24 hpf. Twitching was
counted from individual embryos over multiple movies, as
quantitated in Figure 1. 0:33–0:42. Effects of Decreased
SNAP-25 Expression on Movement at 24 hpf Single cell
zebrafish embryos were injected with snap-25a,b
MO
and filmed for
one minute at 24 hpf. Twitching was counted from individual
embryos over multiple movies, as quantitated in Figure 1. 0:42–
0:52. Effects of Increased SNAP-25 Expression on
Movement at 24 hpf Single cell zebrafish embryos were
injected with snap-25a,b mRNA and filmed for one minute at
24 hpf. Twitching was counted from individual embryos over
multiple movies, as quantitated in Figure 1. 0:52–1:02. Effects
of co-Injection of miR-153 and snap-25a,b on Movement
at 24 hpf Single cell zebrafish embryos were co-injected with
miR-153 and snap-25a,b mRNA and filmed for one minute at
24 hpf. Twitching was counted from individual embryos over
multiple movies, as quantitated in Figure 1. 1:02–1:12. Effects
of co-Injection of miR-153
MO
and snap-25a,b
MO
on
Movement at 24 hpf Single cell zebrafish embryos were co-
injected with miR-153
MO
and snap-25a,b
MO
and filmed for one
minute at 24 hpf. Twitching was counted from individual embryos
over multiple movies, as quantitated in Figure 1. 1:12–1:22. NIC
Embryo Movements at 28 hpf Noninjected control (NIC)
zebrafish embryos at 28 hpf were filmed for one minute at the
same time that the following Movies were created. Twitching was
counted from individual embryos, as quantitated in Figure 4C.
1:22–1:32. Effects of Botulinum Toxin Treatment on
Movement at 28 hpf Single cell zebrafish embryos were
injected with injection dye and treated with Botulinum toxin A
at 27 hpf. After a 30 min treatment, embryos were washed and
allowed to recuperate for 1 hour before being filmed. Twitching
was counted from individual embryos, as quantitated in Figure 4C.
1:33–1:42. Effects of Botulinum Exposure and co-In-
jection of miR-153
MO
on Movement at 28 hpf Single cell
zebrafish embryos were injected with miR-153
MOs
and treated with
Botulinum toxin A at 27 hpf. After a 30 min treatment, embryos
were washed and allowed to recuperate for 1 hour before being
filmed. Twitching was counted from individual embryos, as
quantitated in Figure 4C. 1:42–1:52. Effects of Botulinum
Exposure and co-Injection of snap-25a,b mRNA on
Movement at 28 hpf Single cell zebrafish embryos were
injected with snap-25a,b mRNA and treated with Botulinum toxin
A at 27 hpf. After a 30 min treatment, embryos were washed and
allowed to recuperate for 1 hour before being filmed. Twitching
was counted from individual embryos, as quantitated in Figure 4C.
(MOV)
Acknowledgments
We thank Drs. Sarah Kucenas, Bruce Appel, and Victor Ambros for
critical comments and suggestions and Dr. Jeff Rohrbough and Dr.
Ricardo Pineda for help with the FM1-43 experiments. We also thank Drs.
Li-En Jao and Susan Wente for providing the mnx1:TagRFP-T fish.
Author Contributions
Conceived and designed the experiments: CW EJT JGP. Performed the
experiments: CW EJT AFO DJC ALP AFM. Analyzed the data: CW EJT
AFO BDC KB JGP. Contributed reagents/materials/analysis tools: CW
EJT ALP. Wrote the paper: CW KB JGP.
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miR-153 Regulation of Snap-25
PLOS ONE | www.plosone.org 14 February 2013 | Volume 8 | Issue 2 | e57080
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    • "These data suggest that loss of miR-153 may explain some, but not all, ethanol effects on NSCs. Moreover, these data are collectively consistent with recent evidence that miR-153 prevents the development and maturation of motor neurons (Wei et al., 2013), and serves as a translational repressor for synaptic and signaling proteins that are important for neuronal maturation and function (Doxakis, 2010; Long et al., 2012; Wei et al., 2013). While current analysis focused on miR-153-repressed transcripts, potential direct effects on induced transcripts cannot be discounted. "
    [Show abstract] [Hide abstract] ABSTRACT: Ethanol exposure during pregnancy is an established cause of birth defects, including neurodevelopmental defects. Most adult neurons are produced during the second trimester-equivalent period. The fetal neural stem cells (NSCs) that generate these neurons are an important but poorly understood target for teratogenesis. A cohort of miRNAs, including miR-153, may serve as mediators of teratogenesis. We previously showed that ethanol decreased, while nicotine increased miR-153 expression in NSCs. To understand the role of miR-153 in the etiology of teratology, we first screened fetal cortical NSCs cultured ex vivo, by microarray and quantitative RT-PCR analyses, to identify cell-signaling mRNAs and gene networks as important miR-153 targets. Moreover, miR-153 over-expression prevented neuronal differentiation without altering neuroepithelial cell survival or proliferation. Analysis of 3'UTRs and in utero over-expression of pre-miR-153 in fetal mouse brain identified Nfia (nuclear factor-1A) and its paralog, Nfib, as direct targets of miR-153. In utero ethanol exposure resulted in a predicted expansion of Nfia and Nfib expression in the fetal telencephalon. In turn, miR-153 over-expression prevented, and partly reversed, the effects of ethanol exposure on miR-153 target transcripts. Varenicline, a partial nicotinic acetylcholine receptor agonist that, like nicotine, induces miR-153 expression, also prevented and reversed the effects of ethanol exposure. These data collectively provide evidence for a role for miR-153 in preventing premature NSC differentiation. Moreover, they provide the first evidence in a preclinical model that direct or pharmacological manipulation of miRNAs have the potential to prevent or even reverse effects of a teratogen like ethanol on fetal development.
    Full-text · Article · Jul 2014 · Biology Open
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    • "In the current study, we have determined a previously unknown mechanism of PQ-mediated neurotoxicity with respect to increased miR153 and altered Nrf2 system suggesting miR153 targeting could be beneficial. However, a recent report showed that miR153 is essential for proper neuronal patterning and associated functions during early zebrafish development (Wei et al., 2013). This raises an important issue as to the need to identify specific functions of this miR within the context of species and specific cell types. "
    [Show abstract] [Hide abstract] ABSTRACT: Epidemiological and animal studies suggest that environmental toxins including paraquat (PQ) increase the risk of developing Parkinson's disease (PD) by damaging nigrostriatal dopaminergic neurons. We previously showed that overexpression of a group of microRNAs (miRs) affects the antioxidant promoting factor, Nrf2 and related glutathione-redox homeostasis in SH-SY5Y dopaminergic neurons. Although, dysregulation of redox balance by PQ is well documented, the role for miRs and their impact have not been elucidated. In the current study we investigated whether PQ impairs Nrf2 and its related cytoprotective machinery by misexpression of specific fine tune miRs in SH-SY5Y neurons. Real time PCR analysis revealed that PQ significantly (p<0.05) increased the expression of brain enriched miR153 with an associated decrease in Nrf2 and its function as revealed by decrease in 4x ARE activity and expression of GCLC and NQO1. Also, PQ and H2O2-induced decrease in Nrf2 3' UTR activity was restored on miR153 site mutation suggesting a 3' UTR interacting role. Overexpression of either anti-miR153 or Nrf2 cDNA devoid of 3' UTR prevented PQ and H2O2-induced loss in Nrf2 activity confirming that PQ could cause miR153 to bind to and target Nrf2 3' UTR thereby weakening the cellular antioxidant defense. Adenovirus mediated overexpression of cytoplasmic catalase (Ad cCAT) confirmed that PQ induced miR153 is hydrogen peroxide (H2O2) dependent. In addition, Ad cCAT significantly (p<0.05) negated the PQ induced dysregulation of Nrf2 and function along with minimizing ROS, caspase 3/7 activation and neuronal death. Altogether, these results suggest a critical role for oxidant mediated miR153-Nrf2/ARE pathway interaction in paraquat neurotoxicity. This novel finding facilitates the understanding of molecular mechanisms and to develop appropriate management alternatives to counteract PQ-induced neuronal pathogenesis.
    Full-text · Article · May 2014 · Toxicology Letters
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    [Show abstract] [Hide abstract] ABSTRACT: MicroRNAs are small noncoding RNAs involved in various biological processes. We characterized the expression of miR-344-3p during mouse embryonic development. At E9.5-E10.5 and E15.5, in situ hybridization detected strong miR-344-3p signal in the central nervous system, including the cerebral cortex, hindbrain, cerebellum, thalamus, hindbrain, medulla oblongata, spinal cord, and dorsal root ganglia. Further, qRT-PCR analysis identified miR-344-3p expression at E15.5, with expression stably maintained in the brain from E12.5 to E18.5 before decreasing to relatively low levels postnatally. We also analyzed miR-344-3p expression using immunofluorescence in situ hybridization at E18.5 and within the adult brain. miR-344-3p signal was mainly detected in cortical regions surrounding the ventricular system, choroid plexus, glomerular layer of the olfactory bulb, and granular cell layer of the cerebellar cortex. Altogether, our results indicate miR-344-3p may play an important role in morphogenesis, nervous system development in the brain.
    Full-text · Article · Nov 2013 · Journal of molecular histology
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