Recent studies have elucidated roles for neural miRNAs at various stages of neuronal development and maturation, including neurite outgrowth and spine ...
miRNA-326 Functions in Neuronal Development and Synaptic Plasticity N. Olde 1Department
1 Loohuis ,
A.
1 Kos ,
W. van
1 Boekel ,
H. van
1,2 Bokhoven ,
A.
1 Aschrafi
of Cognitive Neuroscience, 2Department of Human Genetics, Radboud University Nijmegen Medical Centre, The Netherlands
Introduction MicroRNAs (miRNAs) have emerged as an important class of small noncoding RNAs which act to fine tune the expression of sets of genes and entire pathways, and are thus thought of as master regulators of gene expression. Recent studies have elucidated roles for neural miRNAs at various stages of neuronal development and maturation, including neurite outgrowth and spine formation [1-4]. Studies targeting specific miRNAs convincingly revealed the roles of these miRNAs in synaptic development (Figure 1) [4-11]. In this study, we want to assess the role of miR-326 in neuronal development and synaptic plasticity.
Figure 1. Overview of miRNAs involved in postsynaptic spine growth or shrinkage by interfering with several implicated downstream pathways [1].
Results – Endogenous expression patterns of mature miR-326 In hippocampal slices, miR-326 is enriched at the synaptic compartments compared to the cytoplasm. The cytoplasmically enriched miR126 was chosen as a control.
In the human brain, miR-326 is highly expressed in the hippocampus compared to other brain regions.
*
2,5
100
2 1,5 1 0,5
80 60
miR-126 miR-326
40 20 0
0 Cb
Hip
Str
FCTX
Tot
Tha
Cyt
0,025
Relative expression level
120
3
Relative expression level (%)
Relative expression level
Cb: cerebellum, Hip: hippocampus, Str: striatum, FCTX: frontal cortex, Tha: thalamus
In primary hippocampal neurons, miR-326 levels are increased during the second week of in vitro development.
SyNeu
*
0,02
* *
0,015 0,01 0,005 0 day 0
day 3
day 6 day 10 day 14 day 18 day 21
Results – miR-326 affects neurite length and branching
mCherry A
mCherry-miR-326
A
C
B
Outgrowth analysis revealed a decrease in neurite length upon miR-326 nucleofection at DIV6 (A). In addition, at DIV3, the number of secondary branches was decreased and at DIV6, the amount of both secondary and tertiary branches were decreased (B,C).
DIV1
*
250 200 150 100 50 0
mCherry mCherry-miR-326
DIV3 E
F G
DIV6
C
DIV3 Number of neurites
D
B
DIV6
5 4 3 2 1 0
*
Prim
Sec
Ter
Number of neurites
Representative pictures show neurons transfected with mCherry (A,D) or mCherry-miR-326 (B,E). Neurons were fixed between 1 day in vitro (DIV1) and DIV6, and traced using NeuronJ (C, F).
Mean neurite length (pixels)
The mCherry vector was used to succesfully overexpress miR-326 in primary neurons using nucleofection.
DIV6 *
10 8 6 4 2 0
*
Prim
Sec
Ter
Quar
Results – miR-326 expression is transiently increased during hippocampal LTD
ERK 0 min
10 min
pERK 0 min
10 min
44 42
B
200
*
LTD causes a transient increase in miR-326 expression levels.
*
150 100
0 min 10 min
50 0 pERK/ERK pERK/ERK 42kDa 44kDa
Conclusions miR-326 is abundantly present in the hippocampus and highly enriched at hippocampal synaptic compartments During in vitro development, miR-326 levels are increased in the second week of culturing miR-326 overexpression leads to a decrease of total neurite length and a decrease in the number of branches Upon chemical LTD induction, miR-326 levels transiently increase Our data suggests that miR-326 is a potential regulator of neuronal maturation and that this miRNA may be involved in the process of synaptic activity
Relative expression level
A
pERK/ERK ratio (%)
In acute hippocampal slices long term depression (LTD) was induced using the metabolic glutamate receptor agonist DHPG. LTD induction was confirmed by an increase in pERK/ERK ratios, as shown by Western blot (A,B).
0,025 0,02 0,015 0,01 0,005 0 0 min
15 min
60 min
References 1. Olde Loohuis N., Kos A., et al., 2011, Cellular and Molecular Life Sciences, accepted 2. Davis T., Cuellar T., et al., 2008, The Journal of Neuroscience 28:4322-4330 3. DePietri Tonelli D., Pulvers J., et al., 2008, Development 135:3911-3921 4. Schaefer A., O'carroll D., et al., 2007, Journal of experimental medicine 204:1553-1558 5. Cheng L.C., Pastrana E., et al., 2009, Nature Neuroscience 12:399-408 6. Edbauer D., Neilson J.R., et al., 2010, Neuron 65:373-384 7. Vo N., Klein M.E., et al., 2005, PNAS 102:16426-16431 8. Schratt G.M., Tuebing F. et al., 2006, Nature 439:283-289 9. Gao J., Wang W.Y., et al., 2010, Nature 466:1105-1109 10. Smrt R.D., Szulwach K.E., et al., 2010, Stem cells 28:1060-70 11. Siegel G., Obernosterer G., et al., 2009, Nature cell biology 11:705-716
