Ucp2 Induced by Natural Birth Regulates Neuronal Differentiation of the Hippocampus and Related Adult Behavior

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DOI: 10.1371/journal.pone.0042911 · Source: PubMed
Abstract
Mitochondrial uncoupling protein 2 (UCP2) is induced by cellular stress and is involved in regulation of fuel utilization, mitochondrial bioenergetics, cell proliferation, neuroprotection and synaptogenesis in the adult brain. Here we show that natural birth in mice triggers UCP2 expression in hippocampal neurons. Chemical inhibition or genetic ablation of UCP2 lead to diminished neuronal number and size, dendritic growth and synaptogenezis in vitro and impaired complex behaviors in the adult. These data reveal a critical role for Ucp2 expression in the development of hippocampal neurons and circuits and hippocampus-related adult behaviors.
Ucp2
Induced by Natural Birth Regulates Neuronal
Differentiation of the Hippocampus and Related Adult
Behavior
Julia Simon-Areces
1
, Marcelo O. Dietrich
2,3
, Gretchen Hermes
2
, Luis Miguel Garcia-Segura
1
, Maria-
Angeles Arevalo
1
, Tamas L. Horvath
2,3,4
*
1 Instituto Cajal, CSIC, Madrid, Spain, 2 Program in Integrative Cell Signaling and Neurobiology of Metabolism, Section of Comparative Medicine, Yale University School of
Medicine, New Haven, Connecticut, United States of America, 3 Department of Biochemistry, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil,
4 Departments of Neurobiology and Obstetrics, Gynecology and Reproduct ive Sciences, Yale University School of Medicine, New Haven, Connecticut, United States of
America
Abstract
Mitochondrial uncoupling protein 2 (UCP2) is induced by cellular stress and is involved in regulation of fuel utilization,
mitochondrial bioenergetics, cell proliferation, neuroprotection and synaptogenesis in the adult brain. Here we show that
natural birth in mice triggers UCP2 expression in hippocampal neurons. Chemical inhibition or genetic ablation of UCP2 lead
to diminished neuronal number and size, dendritic growth and synaptogenezis in vitro and impaired complex behaviors in
the adult. These data reveal a critical role for Ucp2 expression in the development of hippocampal neurons and circuits and
hippocampus-related adult behaviors.
Citation: Simon-Areces J, Dietrich MO, Hermes G, Garcia-Segura LM, Arevalo M-A, et al. (2012)
Ucp2
Induced by Natural Birth Regulates Neuronal Differentiation
of the Hippocampus and Related Adult Behavior. PLoS ONE 7(8): e42911. doi:10.1371/journal.pone.0042911
Editor: Gemma Casadesus, Case Western Reserve University, United States of America
Received June 24, 2011; Accepted July 16, 2012; Published August 8, 2012
Copyright: ß 2012 Simon-Areces 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: The authors acknowledge support from the NIH Director’s Pioneer Award (DP1OD006850), NIH grants DK080000, DK 060711 and ADA 7-08-MN-25 to
T.L.H; the Ministerio de Ciencia e Innovacio
´
n, Spain (BFU2008-02950-C03- 01) and from Comunidad de Madrid (CCG08-CSIC/SAL-3617). The funders had no role in
study design, data collection and analysis, decision to publish, or preparati on of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tamas.horvath@yale.edu
Introduction
The perinatal environment represents a critical period in brain
development that determines the adult architecture of the central
nervous system and related functions. In vitro approaches to study
aspects of neuronal differentiation and synaptogenesis have long
been used to gain a better understanding of the developmental
processes of neuronal circuits. However, practical aspects of the
successful maintenance of primary neuronal cultures do not
represent close mimicking of the in vivo environment of neurons
during the perinatal period. Specifically, the nutrient composition
of culture media is vastly different from that provided by the
placenta, clostrum and breast milk, the main source of fuel for
developing neurons perinatally. An important characteristic of
breast milk, in contrast to placental blood support, is its high
content of long chain fatty acids besides glucose [1]. We have
identified mitochondrial uncoupling protein 2 (Ucp2) as a critical
determinant of fatty acid utilization by adult neurons [2]. Ucp2
promotes free radical scavenging [3–5], which is critical for
enabling fatty acid beta oxidation in neurons [2]. This mechanism
is also critical for adult synaptogenesis [6]. Ucp2 is also implicated
in protection of adult [7] as well as developing neurons in a febrile
seizure model in rats at a time of breastfeeding [8]. In the present
study, we sought to determine whether Ucp2 induction occurs in
the hippocampus perinatally, and if so, whether Ucp2-associated
cellular mechanisms are involved in the development of neuronal
circuits in vitro with implications for adult behavior.
Methods
Animals
CD1 mice were raised in the Cajal Institute and all procedures
for handling and killing the animals used in this study were in
accordance with the European Commission guidelines (86/609/
CEE) and were approved by the animal care and use committee of
the Cajal Institute. Vaginal birth occurs at around E18 in the CD1
mouse colony of the animal facility of the Cajal Institute. Ucp2
gene knockout and wild type littermates C57BL6 mice were
generated as described previously (20) All procedures were
approved by the Institutional Animal Care and Use Committee
of Yale University.
Hippocampal Neuronal Cultures and Incubation
Conditions
The hippocampus was dissected out from embryonic day 18
(E18) mouse embryos and dissociated to single cells after digestion
with trypsin (Worthington Biochemicals, Freehold, NJ) and DNase
I (Sigma-Aldrich). Neurons were plated on 6-wells plates or glass
coverslips coated with poly-L-lysine (Sigma-Aldrich) at a density of
200–600 neurons/mm
2
, and they were cultured in Neurobasal
supplemented with B-27 and GlutaMAX I (Invitrogen, Crewe,
United Kingdom). Under the conditions used, our cultures were
nearly devoid of glia. After the indicated time in vitro, the cultures
were fixed for immunostaining (4% paraformaldehyde/4%
sucrose in PBS) or harvested for real-time PCR. Parallel cultures
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were prepared by adding to the cultures medium 20 mM genipin
(Wako Chemicals USA) before plating cells.
Quantitative real time polymerase chain reaction (real
time PCR)
Total RNA was extracted from cultures at different stages of
neuron development with illustra RNAspin Mini RNA isolation kit
from GE Healthcare (Buckinghamshire, UK). On the other hand,
hippocampi were dissected from E18, delivered with Caesarian
section (CS) or vaginal birth (VB), P10 and adult mouse, disrupted
and homogenized in lysis buffer and total RNA extracted with the
same kit. First strand cDNA was prepared from 2
mg RNA using
the RevertAid
TM
H Minus First Strand cDNA Synthesis Kit (MBI
Fermentas, Bath, UK) according to the supplied protocol. After
reverse transcription, the cDNA was diluted 1:3 and 5
ml were
amplified by real-time PCR in 20
ml using SYBR Green Master
Mix or TaqMan Universal PCR Master Mix (Applied Biosystems,
AB, Foster City, CA) in a ABI Prism 7000 Sequence Detector
(AB), with conventional AB cycling parameters (40 cycles of 95uC,
15 s, 60uC 1 min). Primer sequences were designed using Primer
Express (AB) and were for Ucp2: forward, 5 9-ACAAGACCATTG-
CACGAGAG-39 and reverse, 59-ATGAGGTTGGCTTTCAG-
GAG-39; for Nrf1: forward, 59-CGCAGCACCTTTGGAGAA-39
and reverse, 59-CCCGACCTGTGGAATACTTG-39; for Tfam:
forward, 59-GGAATGTGGAGCGTGCTAAAA-39 and reverse,
59-TGCTGGAAAAACACTTCGGAATA-39. Ngn3 and Glycer-
aldehyde 3-phosphate dehydrogenase (Gapdh), which was selected
as control housekeeping gene, were analyzed using Assay-on-
Demand gene expression products (AB). After amplification, a
denaturing curve was performed to ensure the presence of unique
amplification products. For visualizing and sequencing the PCR
products, each mixture was electrophoresed in 2% (w/v) ethidium
bromide-stained agarose gels. Then, bands were excised and
cDNA was purified using the QIAquick PCR purification Kit
(Qiagen, GmbH, Germany). One hundred nanograms of each
sample were sequenced (Automatic Sequencing Center, CSIC,
Madrid, Spain) with the corresponding forward or reverse primer.
The obtained sequence was aligned with the expected sequence of
each transcript obtained from the GenBank. All reactions were
performed in triplicate and the quantities of target gene expression
were normalized to the corresponding Gapdh expression in test
samples and plotted.
Western blot analysis of UCP2 protein expression
Lysate from hippocampal samples of animals at the time of birth
with VB, CS or at postnatal day 10 (born naturally) or adulthood
(born naturally) were processed for Western blot analyzes using
UCP2 antisera and procedures as described in Horvath et al. 2002
[9]. Mouse hippocampi were homogenized in 20 mM Tris/HCl
(pH 7.4), 10 mM potasium acetate, 1 mM DTT, 1 mM EDTA,
0,25% NP-40 and an anti-protease cocktail (Roche Diagnostics)
and centrifuged at 700 g (10 min). The supernatant was then
centrifuged at 10 000 g (15 min), and the pellets were resuspended
in SDS-PAGE loading buffer.
Proteins were resolved by SDS-PAGE and transferred onto
nitrocellulose membranes (Millipore). The membranes were
blocked in Tris-buffered saline containing 0.1% Tween 20 and
2% ECL advance blocking reagent (Amersham) and incubated
first with rabbit anti-UCP2 polyclonal antibody (1:2000) [9] and
rabbit anti-aralar antibody (loading control, 1:1000; a gift from
doctor Araceli del Arco) and then with horseradish peroxidase-
conjugated goat anti-rabbit and goat anti-mouse secondary
antibodies (1:10000; Jackson Immuno Research). Specific proteins
were visualized with enhanced chemiluminescence detection
reagent according to the manufacturer’s instructions (Amersham).
Densitometry and quantification of the bands were carried out
using the Quantity One software (Bio-Rad). Statistical analysis of
the data was performed using an unpaired t-test.
Immunocytochemis try, image acquisition and
morphometric analysis
The following primary antibodies were used: chicken anti-bIII
tubulin (1:1000; Abcam, Cambridge, UK) and mouse anti-
synaptophysin I (1:500; Progen, Heidelberg, Germany). To verify
that the labeling was caused specifically by the primary antibodies,
it was either omitted or replaced by similarly diluted normal serum
from the same species. Secondary antibodies were donkey anti-
chicken-FITC (1:500) and goat anti-mouse-Cy3 (1:1000), both
from Jackson Immuno Research (West Grove, PA). For the
evaluation of soma size, dendritic morphology and presynaptic
terminal identification in dissociated cell cultures, labeled neurons
were visualized by standard epifluorescence under a 406 oil
objective under a Leica microscope. Images were captured with a
Leica digital camera controlled by the Leica software (Leica,
Heidelberg, Germany). Somas size was evaluated using ImageJ
1.38 (NIH). Primary dendrite number i.e., the number of dendrites
associated with the soma, and terminal counts were performed
manually. A circular region of interest (ROI) with a diameter of
100
mm was projected onto the bIII- Tubulin labeled neuron, its
center roughly coinciding with the center of the soma. Synaptic
terminals contacting somata or dendrites were counted within the
circular ROI.
To determine the number of neurons in the cultures, at least
fifteen culture fields of 600
mm
2
were photographed per stage
using a reverse microscope equipped with phase contrast, at
1.5 hours after seeding and at the time corresponding to different
stage of neuron development. The number of cell was counted
manually.
To identify neuronal cells, rabbit anti-Tau (1:20; Abcam,
Cambridge, UK) as axonal marker and mouse anti-MAP2 (1:500;
Sigma-Aldrich) as dendrite marker were used. Secondary
antibodies: goat anti-rabbit-Alexa488 (1:500) and goat anti-
mouse-Cy3 (1:1000), both from Jackson Immuno Research (West
Grove, PA).
Open-Field Testing
The open field test apparatus was a square, polyurethane arena
(36.5 cm636.5 cm630 cm, Plexiglas). The animal was placed in
corner of the apparatus locomotion speed, distance traveled,
entries into the central zone, and time spent in contact with the
outer walls, were recorded for 5 minutes. Behavioral testing took
place from 1000 to 1400 h (i.e. in the light phase of the light-dark
cycle). The apparatus was cleaned with 10% ethanol after each
animal exposure. ANY-Maze Software
TM
(Stoelting Company,
Wood Dale, IL) was used to record and analyze behavioral data.
Y-Maze Testing
Spatial memory was assessed using the two-trial Y-maze task. A
single Y-maze was made of black Plexiglas and consisted of three
arms with an angle of 120u between each of the two arms. Each
arm was 8 cm630 cm615 cm (width6length6height). The three
arms were randomly designated: start arm, in which the mouse
started to explore (always open), novel arm, in which the mouse
started to explore (always open), novel arm, which was blocked
during the first trial but open during the second trial.
The maze was placed on a flat surface within the behavioral
testing room. Proximal visual cues (pictures within the arms of the
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apparatus) and distal visual cues (the configuration of the room,
curtain, wall art) remained constant throughout testing. The floor
of the maze was covered with white chip bedding. Between each
trial the apparatus was cleaned with 10% ethanol and new
bedding was added. Behavioral testing took place from 1000 to
1400 h (i.e. in the light phase of the light-dark cycle).
The Y-maze test consisted of two trials separated by an inter-
trial interval (ITI) of 60 minutes to assess spatial memory. The first
trial had a five-minute duration and allowed the mouse to freely
explore only two arms (start arm and other arm) while the third
arm was blocked. After a 60 min ITI, the second trial also of five
minutes duration was conducted during which all three arms were
accessible and novelty vs. familiarity was compared in all three
arms. ANY-Maze Software
TM
(Stoelting Company, Wood Dale,
IL) was used to record and analyze behavioral data.
Figure 1. Time course changes in
Ucp2
,
Nrf1
and
Tfam
mRNA expression in the mouse hippocampal development. (a and c) Total RNAs
were extracted from the hippocampal tissue (a) or cultured hippocampal neurons (c), retrotranscribed to cDNA and specific mRNA expression of each
gene analyzed by real time PCR (see supplementary material for methods). Gapdh gene expression was used as internal standard. Data represent the
mean+SEM (n = 4 for each). ANOVA revealed significant differences among experimental groups (a): Ucp2:F
3,11
= 20.96; p,0.0001; Nrf1:F
3,45
= 28.64;
p,0.0001; Tfam:F
3,11
= 202.5; p,0.0001; (c): Ucp2:F
5,72
= 61.79; p,0.0001; Nrf1:F
5,34
= 20.42; p,0.0001; Tfam:F
5,34
= 39.35; p,0.0001. (b) Total RNAs
were extracted from the hippocampal tissue from wild type and Ucp2 KO mice, retrotranscribed to cDNA and specific mRNA expression of each gene
analyzed by real time PCR. Gapdh gene expression was used as internal standard. Data represent the mean+SEM (n = 4 for each) and are expressed as
relative of the wild type Caesarea delivered fetal value. Asterisks indicate statistical differences between the data sets connected by horizontal lines
(*, p,0.05; **, p,0.01; ***, p,0.001), as determined by using the Student’s t-test. CS, Caesarian section; VB, vaginal birth; G, genipin.
doi:10.1371/journal.pone.0042911.g001
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Statistical Analysis
All data are expressed as the mean + SEM or mean 6 SEM
from at least 3 experiments. Data was analyzed using the Graph
Pad Prism 5.0 program (GraphPad Software, Inc., San Diego,
CA). The means between two samples were analyzed by student t-
test and between more than two samples by one-way ANOVA
followed by Bonferroni post hoc. Significance was taken at
p,0.05.
Results
UCP2 mRNA is induced by birth
We analyzed Ucp2 expression in the hippocampus of mice
delivered with Caesarian section (CS), vaginal birth (VB) and in
various postnatal ages from animals born naturally. Ucp2 mRNA
levels were induced in newborns by vaginal birth compared to age-
matched mice that were delivered with Cesarean section (Fig. 1a).
Subsequently, Ucp2 mRNA levels began to decrease and reached
levels below the fetal ones. Ucp2 related genes involved in
mitochondrial biogenesis, Nrf1 and Tfam, showed a similar
expression profile (Fig. 1a). Differences in the level of the
expression of these latter genes were observed between Ucp2 KO
mice and their wild type littermates in each stage of development
(Fig. 1b). Nrf1 mRNA expression level was decreased in the Ucp2
KO mice compared to wild type mice at E18 delivered with
Caesarian section; Tfam mRNA expression level was smaller in the
Ucp2 KO mice than in wild type mice at all developmental stages
studied. The expression of Ngn3 mRNA, a proneural transcription
factor related with hippocampal neuron development [10] was
decreased in Ucp2 KO mice compared to wild type mice at P10
and in adults.
UCP2 mRNA expression is induced by culture conditions
We next used cultured hippocampal neurons from day E18 that
show a transition through a sequence of five morphological stages
[11]. The level of Ucp2 mRNA was the highest at stage 1 (Fig. 1c),
which corresponds to in vivo stage of birth. Levels of Ucp2 mRNA
decreased at stages of neuron differentiation (2 and 3) and showed
a small increase at stage 4, when the dendrites have already been
defined and neurons start to establish its synaptic contacts. Nrf1
and Tfam mRNA levels showed shifts in expression that was not
always similar to changes in Ucp2 (Fig. 1c). Thus, in vitro culturing
of neurons taken from mice that were surgically delivered has
resemblance to the mimics the effect of vaginal delivery on Ucp2
expression.
UCP2 protein expression
We observed a significantly higher level of UCP2 protein
expression at the day of delivery in animals that were born via VB
compared to those with CS (Fig. 2). In naturally born mice, UCP2
protein remained elevated early post-nataly (P10) as well as in
adulthood (Fig. 2).
Effect of chemical or genetic suppression of UCP2 activity
on hippocampal cultures
To test the effect of Ucp2 induction on neuronal differentiation,
cultures first were treated with the Ucp2 inhibitor, genipin [12]
(Fig. 3). We have extensively analyzed the effect of genipin on
mitochoindrial functions and how they relate to UCP2’s effect [2].
In those studies, we confirmed genipin’s brain effects but we also
showed that genipin has a more broad action on mitochondrial
metabolism [2]. Thus, we concluded that while genipin is not
UCP2-specific, it does have an overall effect on mitochoindrial
and cellular function that is consistent with an effect that opposes
UCP2 action. Between 0.5 and 4 DIV, the number of neurons was
significantly decreased by genipin treatment compared to control
cultures (Fig. 3). Furthermore, genipin caused a decrease of the
neuronal soma size, the number of primary neurites and dendrites
and the number of synaptophysin positive presynaptic clusters on
Figure 2. UCP2 protein levels at E18, delivered with Caesarian
section (CS) or vaginal birth (VB), P10 and adult mice. Mouse
hippocampi were homogenized and mitochondrial proteins were
purified and separated by SDS-PAGE. Upper panel, representative
Western blot for UCP2 and Aralar (loading control). Bottom panel, graph
representing the mean + SEM of densitometric signal of UCP2 versus
Aralar protein. The levels of significance were denoted as *p,0.05,
***p,0.001 for the data sets (n = 3) connected by horizontal lines as
determined by an unpaired t-test.
doi:10.1371/journal.pone.0042911.g002
Figure 3. Cultured hippocampal neurons. Cultured hippocampal
neurons from wild type mice were treated or untreated with genipin
(20
mM). Cells were fixed at 4 DIV and at 7 DIV and immunostained with
antibodies against MAP2 (red) and Tau (green) to label dendrites and
axons, respectively.
doi:10.1371/journal.pone.0042911.g003
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Figure 4. Effect of Ucp2 inhibition and
Ucp2
genetic deletion on morphology and synaptic inputs on cultured hippocampal
neurons. (a) Cultures were untreated or treated whit genipin (20
mM) and images were taken under a reverse microscope equipped with phase
contrast (see supplementary material for methods). Graph represents the number of cell per field at different days in vitro (DIV). (b) Representative
images of neurons at 4 DIV. Scale bar 100
mm. (c) Immunofluorescence images of neurons at different stages of development marked with anti bIII-
tubulin antibody. Scale bar 10
mm. (d,e) Graphs representing the quantitative analysis of neuronal soma size in different preparations. (f,g)
Histograms representing the number of primary neurites. (h,j) Representative immunofluorescence images of cultured hippocampal neurons fixed at
4 DIV immunostained with antibodies against bIII-tubulin (green) and synaptophysin (red). (i, k) Counts of synaptophysin immunoreactive terminals
in contact with a neuron per ROI. ROI diameter: 100
mm. Typically 60–75 neurons were evaluated in each condition ((n = 3). Bar represents the mean
6 SEM. The level of significance was denoted as ***p,0.001 *p,0.05, **p,0.01 for the data sets connected by horizontal lines as determined by
using the Student’s t-test.
doi:10.1371/journal.pone.0042911.g004
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Figure 5. Open field studies. Open field studies revealed significant differences across genotype; ucp2
2/2
mice were significantly more anxious
with fewer entries into the center of the apparatus, greater time in contact with walls of the apparatus and shorter distance travelled. As has been
reported elsewhere, ucp2
2/2
mice were significantly slower than controls. (** p,0.001; *** p,0.0001).
doi:10.1371/journal.pone.0042911.g005
Figure 6. Spatial memory task. In an assessment of hippocampal-based spatial memory, ucp2
2/2
mice demonstrated an impairment relative to
controls with reduced travel distance and time spent in the novel arm.
doi:10.1371/journal.pone.0042911.g006
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the soma and dendrites (Fig. 4). Next we analyzed, cultures from
Ucp2 KO mice and their wild type littermates. Similar to the effect
of chemical inhibition of UCP2, Ucp2 KO cultures showed
decreased neuronal soma size, decreased number of primary
neurites and decreased number of synaptophysin positive termi-
nals compared to the cultures from their wild type littermates
(Fig. 4).
Behavioral tests
Across a test of unconditioned response to potential threat,
ucp2
2/2
mice displayed significantly more thigmotaxis, fewer trips
to the center of the testing arena, shorter distance travelled and a
slower rate of locomotion. (Fig. 5a–e).
Open Field Testing (Fig. 5)
WT, N = 12; KO N = 10. In this measure of exploration stress,
ucp2
2/2
mice travelled significantly less distance that WT (KO
distance, 4.960.8 m; WT distance,1061.0 m; t(22) = 4.0,
p,0.001). As compared to WT mice, ucp2
2/2
made significantly
fewer trips to the center of the arena (KO trips to cen-
ter = 2.160.80; WT trips to center = 13.0861.54, t(16) = 6.34,
p,0.0001). Time spent in contact with the walls of the apparatus
was also significantly different across genotype; ucp2
2/2
mice
displayed high levels of thigmotaxis relative to wild type animals
(KO contact with wall = 419.7623 s; WT contact with
wall = 281.4622.51 s, t(19) = 4.3, p,0.001). As previously report-
ed, ucp2
2/2
mice were significantly slower than age-matched WT
controls (KO average speed m/s = 0.01760.003; WT average
speed m/s = 0.03460.002, t(22) = 4.8, p,0.0001).
Spatial Memory Task (Fig. 6)
The distance traveled in the novel arm, a proxy for exploratory
behavior related to recognition of novelty differed significantly
(KO travel in novel arm 2.3860.24 m; WT travel in novel arm
3.4260.37 m, t(11) = 2.36, p,0. 04). Time spent in the novel arm
also differed (KO time in novel arm 58.3468.60 s; WT time in
novel arm 95.08610.14 s, t(12) = 2.76, p,0. 02).
Discussion
In adult mice, under conditions of synaptogenesis, such as
triggered by voluntary exercise, the absence of Ucp2 not only
blocks synaptogenesis, but decreases spine synapse number on
granule cells as well as on CA1 pyramidal neurons [6]. The role of
UCP2 in promotion of scavenging of reactive oxygen species
(ROS) in neurons [2,5,13] is a likely contributor for the promotion
of dendritic growth and synaptogenezis: ROS were found to be a
critical negative regulator of hippocampal circuit development
[14] and, they are important contributors to neuronal lipid
metabolism [2] a process important for membrane fluidity.
The induction of Ucp2 mRNA by natural birth is consistent with
previous findings that correlated the beneficial effects of Ucp2 and
high fat content of breast milk with protection of febrile seizures in
early postnatal animals [8]. It is reasonable to suggest that Ucp2
mRNA induction may be associated with hypoxia/ischemia that
accompanies vaginal birth. An intracellular mechanism that is
known to be activated by hypoxia/ischemia is AMPK [15], which
is an upstream inducer of Ucp2 induction in neurons [2]. AMPK
activation suppresses acetyl CoA carboxylase (ACC) activity
eliminating the inhibitory effect of malonyl-CoA on carnitine
palmitoyl transferase 1 (CPT1) activity. CPT1 activation enhances
long chain fatty acid oxidation, which leads to generation of
reactive oxygen species (ROS). ROS together with long chain fatty
acid availability promotes Ucp2 transcription and activity [16,17].
Ucp2 activity neutralizes ROS [2–5] allowing continuous CPT1-
promoted fatty acid oxidation and transcription of genes
promoting mitochondrial proliferation (such as Nrf1 and Tfam as
described in the present study) enabling continuous support of the
bioenergetics needs of sustained neuronal firing and synaptic
plasticity [2]. This intracellular signaling should be supportive of
neuronal activation and decreased vulnerability of neurons to
cellular stress in any region where they are activated. Indeed, mice
with enhanced Ucp2-expression in the hippocampus, substantia
nigra, or striatum, resisted to neurodegeneration in models of
epilepsy [7], Parkinson’s disease [13,18,19], and global ischemia
[20].
The current data suggests that the induction of Ucp2 by birth -
associated physiological stress enables metabolic adaptation to a
switch available nutrient utilization that is critical for proper
survival and development of hippocampal and other brain
neurons. Whether impaired Ucp2 induction by non-natural birth
or by chemical interference could have long lasting effects on the
functioning of the brain is an intriguing and potentially clinically
relevant question. Our observation of adult UCP2 KO animals in
open field and Y maze tests argue that long lasting effects of
impaired UCP2 activity during development may in fact affect
complex adult behaviors.
Acknowledgments
We thank Maria Garcia-Maurin˜o and Marya Shanabrough for technical
assistance.
Author Contributions
Conceived and designed the experiments: TLH MAA LMGS GH.
Performed the experiments: JSA MOD GH. Analyzed the data: JSA MOD
MAA GH. Wrote the paper: TLH MAA JSA.
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    • "Their sequences were 5 -CTT GCA AGA CAG AGA GCC G-3 (forward) and 5 -CTG GGT GTC TGA TTG CTG TTC-3 (reverse), which resulted in a 210-bp PCR product. The sequences of the primers amplifying the murine TFAM cDNA were 5 -GGA ATG TGG AGC GTG CTA AAA-3 (forward) and 5 -TGC TGG AA A AAC ACT TCG GAA TA-3 (reverse); these were chosen from a pub- lication [17], and they produced a 118-bp amplicon. The ACTB gene was used as an internal control for quantifying the transcription levels of these genes, and the primers were designed based on GeneBank sequence NM 007393.3, "
    [Show abstract] [Hide abstract] ABSTRACT: The cytotoxicity of quercetin is not well understood. Using an ICR murine model, we unexpectedly found that mice exposed to 7 Gy total body irradiation (TBI) exhibited general in vivo toxicity after receiving quercetin (100 mg/kg PO), whereas this result was not observed in mice that received TBI only. In order to understand the involvement of alterations in mitochondrial biogenesis, we used a real-time qPCR to analyze the mitochondrial DNA copy number (mtDNAcn) by amplifying the MTRNR1 (12S rRNA) gene in murine bone marrow. We also utilized reverse transcription qPCR to determine the mRNA amounts transcribed from the polymerase gamma (POLG), POLG2, and mammalian mitochondrial transcription factor A (TFAM) genes in the tissue. In the mice exposed to TBI combined with quercetin, we found: (1) the radiation-induced increase of mtDNAcn was inhibited with a concurrent significant decrease in POLG expression; (2) TFAM expression was significantly increased; and (3) the expression of POLG2 was not influenced by the treatments. These data suggest that the overall toxicity was in part associated with the decrease in mtDNAcn, an effect apparently caused by the inhibition of POLG expression and overexpression of TFAM; unaltered POLG2 expression did not seem to contribute to toxicity.
    Full-text · Article · Dec 2014
    • "Interestingly, data show that UCP2 induction in mice hippocampus also occurs at time of birth, a process that reveals to be essential for the development of hippocampal neurons and circuits and hippocampus-related adult behaviors (Simon-Areces et al. 2012). On the other hand, the chemical inhibition or genetic ablation of UCP2 led to diminished neuronal number and size, dendritic growth and synaptogenesis in vitro and impaired complex behaviors in the adult (Simon-Areces et al. 2012). Besides neuronal expression, a recent work also demonstrated UCP2 presence in astrocytes, where it was responsible for modulating endoplasmic reticulum stress and neuroinflammation markers in an animal model for Parkinson disease (PD) (Lu et al. 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: The integrity of mitochondrial function is essential to cell life. It follows that disturbances of mitochondrial function will lead to disruption of cell function, expressed as disease or even death. Considering that neuronal uncoupling proteins (UCPs) decrease reactive oxygen species (ROS) production at the expense of energy production, it is important to understand the underlying mechanisms by which UCPs control the balance between the production of adenosine triphosphate (ATP) and ROS in the context of normal physiological activity and in pathological conditions. Here we review the current understanding of neuronal UCPs-mediated respiratory uncoupling process by performing a survey in their physiology and regulation. The latest findings regarding neuronal UCPs physiological roles and their involvement and interest as potential targets for therapeutic intervention in brain diseases will also be exploited.
    Full-text · Article · Sep 2014
    • "In order to evaluate prefrontal cortex activity, tissue from separate subregions was obtained by selectively punching samples of prelimbic, infralimbic, and pregenual anterior cingulate cortex (Harris Uni-Core, Ted Pella, Redding, California, US) from 50 to 200 mm slices obtained on a cryostat (Leica CM3050S) covering the extent of each subregion according to atlas landmarks (Paxinos and Watson, 1997). Mitochondrial, cytoplasmic and nuclei-enriched fractions were obtained using an adaptation of a previously published differential centrifugation protocol (Simon-Areces et al., 2012). Sample lysates were obtained using a Teflon pestle in ice-cold IM homogenization buffer 20 mM Tris HCl, 10 mM KOAc, 1 mM EDTA, 0.25% NP40, containing freshly added 1 mM dithiothreitol (DTT) and a proteinase and phosphatase inhibitor cocktail (Complete EDTAfree, Roche Diagnostics GmbH, Rotkreuz, Switzerland). "
    [Show abstract] [Hide abstract] ABSTRACT: Adolescence is increasingly recognized as a critical period for the development of the social system, through the maturation of social competences and of their underlying neural circuitries. The present study sought to test the utility of resveratrol, a dietary phenol recently reported to have mood lifting properties, in modulating social interaction that is deficient following early life adversity. The main aims were to 1) pharmacologically restore normative social investigation levels dampened by peripubertal stress in rats and 2) identify neural pathways engaged by this pharmacological approach. Following peripubertal (P28-42) stress consisting of unpredictable exposures to fearful experiences, at adulthood the subjects’ propensity for social exploration was examined in the three-chamber apparatus, comparing time invested in social or non-social investigation. Administered intraperitoneally thirty minutes before testing, resveratrol (20 mg/kg) normalized the peripubertal stress-induced social investigation deficit seen in the vehicle group, selectively altering juvenile but not object exploration. Examination of prefrontal cortex subregion protein samples following acute resveratrol treatment in a separate cohort revealed that while monoamine oxidase A (MAOA) enzymatic activity remained unaltered, nuclear AKT activation was selectively increased in the infralimbic cortex, but not in the prelimbic or anterior cingulate cortex. In contrast, androgen receptor nuclear localization was increased in the prelimbic cortex, but not in the infralimbic or anterior cingulate cortex. This demonstration that social contact deficits are reversed by resveratrol administration emphasizes a prosocial role for this dietary phenol, and evokes the possibility of developing new treatments for social dysfunctions.
    Full-text · Article · Jun 2014
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