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ARTICLE IN PRESS Model
SL 29697 1–5
Neuroscience Letters xxx (2013) xxx– xxx
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters
jou rn al hom epage: www.elsev ier .com/ locate /neule t
he role of p38 in mitochondrial respiration in male and female mice
iaohua Ju, Yi Wen, Daniel Metzger, Marianna Jung ∗
epartment of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, United States
i g h l i g h t s
p38-Knockout creates a sex difference in mitochondrial respiration.p38-Knockout suppresses mitochondrial respiration in male mice and increases COX expression in female mice.Estrogen protects against mitochondrial respiratory impairment.
a r t i c l e i n f o
rticle history:eceived 4 March 2013eceived in revised form 29 March 2013ccepted 10 April 2013
eywords:38-knockouturkinje neuronitochondrial respiration
a b s t r a c t
p38 is a mitogen-activated protein kinase and mediates cell growth, cell differentiation, and synapticplasticity. The aim of this study is to determine the extent to which p38 plays a role in maintainingmitochondrial respiration in male and female mice under a normal condition. To achieve this aim, wehave generated transgenic mice that lack p38 in cerebellar Purkinje neurons by crossing Pcp2 (Purkinjecell protein 2)-Cre mice with p38loxP/loxP mice. Mitochondria from cerebellum were then isolated fromthe transgenic and wild-type mice to measure mitochondrial respiration using XF24 respirometer. ThemRNA and protein expression of cytochrome c oxidase (COX) in cerebellum were also measured usingRT-PCR and immunoblot methods. Separately, HT22 cells were used to determine the involvement of
17�-estradiol (E2) and COX in mitochondrial respiration. The genetic knockout of p38 in Purkinje neu-rons suppressed the mitochondrial respiration only in male mice and increased COX expression only infemale mice. The inhibition of COX by sodium azide (SA) sharply suppressed mitochondrial respiration ofHT22 cells in a manner that was protected by E2. These data suggest that p38 is required for the mitochon-drial respiration of male mice. When p38 is below a normal level, females may maintain mitochondrialrespiration through COX up-regulation.© 2013 Published by Elsevier Ireland Ltd.
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. Introduction
Mitochondrial oxidative phosphorylation is essential for aero-ic organisms, as it provides the bulk of usable energy in the formf ATP. Mitochondrial ATP production is driven by transferringlectrons across mitochondrial electron transfer complexes (ETC–IV). In humans or experimental animals, mitochondrial defectsre often observed in a variety of CNS disorders and brain aging [26],artly due to mitochondrial and nuclear gene mutations [5,6]. A sexifference in mitochondrial function has been determined, with
Please cite this article in press as: X. Ju, et al., The role of p38 in mitochhttp://dx.doi.org/10.1016/j.neulet.2013.04.004
emale rat brain maintaining mitochondrial function to a greateregree than males [8].
∗ Corresponding author at: Department of Pharmacology and Neuroscience, Uni-ersity of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fortorth, TX 76107-2699, United States. Tel.: +1 817 735 0132; fax: +1 817 735 2091.
E-mail address: [email protected] (M. Jung).
304-3940/$ – see front matter © 2013 Published by Elsevier Ireland Ltd.ttp://dx.doi.org/10.1016/j.neulet.2013.04.004
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p38 is a mitogen-activated protein kinase and is involved ininflammation, cell growth, and cell death [20,21]. There are fourisoforms of p38: �, �, �, and �. p38� and p38� are expressed inneurons and glial cells, whereas p38� and p38� are exclusivelyexpressed in immune cell types [28,29]. Of the p38 isoforms, theactivities of both p38� and p38� are remarkably high in normaladult mouse brain including cerebral cortex, hippocampus, cere-bellum, and few nuclei of the brainstem. This indicates that �and � isoforms of p38 are primarily responsible for most of theconstitutive p38 activity in brain [18]. The excessive p38 activa-tion occurs during the stress of alcohol withdrawal and stroke,whereas the inhibition of p38 promotes neuronal survival [10,16].While the aberrant activation of p38 is associated with patho-logical conditions, a certain level of p38 appears essential forcellular and neuronal survival. For instance, p38 mediates synaptic
ondrial respiration in male and female mice, Neurosci. Lett. (2013),
plasticity, memory formation, gene regulation, and brain-derivedneurotrophic factor production [25]. p38 also mediates the stim-ulating effect of Purkinje cell protein 2 (Pcp2) on the cellulardifferentiation [8]. Although p38 is mainly located in cytosol,
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tudies have demonstrated the effects of p38 on mitochondria.38 activation is associated with free radical production [22].38 inhibits the adverse effect of a nuclear receptor (PPAR�)oactivator on mitochondrial respiration in muscle [7]. These stud-es suggest that there is a crosstalk between cytosolic p38 and
itochondria.In the current study, we intend to determine whether p38 plays
positive or negative role in maintaining mitochondrial respirationnder a normal condition and whether there is a sex difference inhis effect of p38. To reach this aim, we have generated transgenic
ice that lack p38 in cerebellar Purkinje cells and assessed mito-hondrial respiration, cytochrome c oxidase [(COX) ETC IV], and theffect of estrogen on these parameters.
. Materials and methods
.1. Chemicals
Analytic grade reagents were purchased from IDT Company (Sanose, CA), the Jackson Laboratory (Bar Harbor, Maine), Sigma AldrichSt. Louis, MO), Cellsignaling Technology (Danvers, MA), Seahorseioscience (North Billerica, MA), Invitrogen (Grand Island, NY), andbcam (Cambridge, MA).
.2. Purkinje cell-specific deletion of p38
Among p38 isoforms, we selected p38� because it is the mostbundant isozyme in the brain and the best characterized isoform9,24]. To avoid lethality, we employed a conditional transgenic
ouse system to down-regulate Purkinje p38 genes using there/loxP system and Pcp2 promoter (Purkinje neuron specificarker) [8]. Transgenic mice (Pcp2-Cre mice) that express Cre
ecombinase under the control of the Pcp2 (Jackson Laboratory)ere cross-mated with floxed-p38� mice to generate the Pcp2-re+/−/p38loxP/loxP mice. The mice with floxed-p38 were kindlyrovided by Boehringer Ingelheim Inc. The p38 floxed allele wasenerated by homologous recombination of embryonic stem cellsn which two sites of ATG containing Purkinje p38 sequence wereanked by loxP [3,23] and excised in the presence of Pcp2-Cre.hen pups reached 21 days old, the tips of the tail were collected
or identification of genotype. All mice were sacrificed at the age ofwo months.
.3. Genotyping procedure for Pcp2 and p38
DNA was isolated by incubating tail samples overnight at 55 ◦Cn proteinase K buffer. Primer sequences were as follows: for Pcp2-re transgene forward, 5′-GCG GTC TGG CAG TAA AAA CTA TC-3′;
or Pcp2-Cre transgene reverse, 5′-GTG AAA CAG CAT TGC TGTAC TT-3′; for Pcp2-Cre internal positive control forward, 5′-CTAGC CAC AGA ATT GAA AGA TCT-3′; for Pcp2-Cre internal posi-
ive control reverse, 5′-GTA GGT GGA AAT TCT AGC ATC ATC C-3′;oxP-flanked p38� allele: 5′-TCCTACGAGCGTCGGCAAGGTG-3′ and′-ACTCCCCGAGAGTTCCTGCCTC-3′. Sequential denaturing (96 ◦C,0 s), annealing (52 ◦C, 1 min) and extension (72 ◦C, 1 min) wereepeated 35 times for genotyping the Pcp2-Cre transgene. The pro-ram of 30 cycles of denaturing (94 ◦C for 30 s), annealing (58 ◦C,0 s), and extension (72 ◦C, 45 s) was used to genotype the p38�lleles.
.4. Immunohistochemical detection of p38 in cerebellum
Please cite this article in press as: X. Ju, et al., The role of p38 in mitochhttp://dx.doi.org/10.1016/j.neulet.2013.04.004
Mice were anesthetized with isoflorane and perfused with.9% saline. The formalin-fixed and paraffin-embedded left hemi-pheres were cut into 8 �m-thick slices on a microtome. The
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slices were deparaffinized in xylene, rehydrated through a seriesof graded ethanol solutions, and washed with PBS. The slices weresubsequently moisturized at 95 ◦C and incubated with primaryantibody, polyclonal rabbit anti-p38� (1:50) overnight at 4 ◦C.The slices were then incubated with broad spectrum poly HRPconjugate for 40 min at room temperature. The antigen-antibodybindings were visualized with a diaminobenzidine color reactionfor 10 min. The slides were further rinsed, dehydrated througha series of graded ethanol and xylene, and mounted with Per-mount. All photographs were taken with a compatible Zeiss digitalcamera.
2.5. Mitochondrial respiration
Mitochondria were isolated from cerebellum by conventionaldifferential centrifugation [15], diluted with assay solution (KCL,KH2PO4, MgCL2, HEPES, EGTA, and FA-free BSA), and trans-ferred into XF24 cell culture plate. The samples were centrifuged(4 ◦C, at 3000 × g), added with succinate (substrate) and rotenone(ETC I inhibitor), incubated (37 ◦C), and loaded into the XF24respirometer. ADP and oligomycin were then injected into theXF injection ports. State II and III mitochondrial respirations aredefined as oxygen consumption rate (OCR) before and after ADP isadded to stimulate respiration, respectively. State IV respiration isdefined as OCR in the absence of ADP or any metabolic inhibitors[2].
2.6. Real-time PCR to measure COX subunit IV (COXIV) mRNA
COX is composed of 13 subunits. Subunit IV was measuredbecause of its abundant expression and sensitivity [14,27]. TotalRNA was isolated from cerebellum using Trizol reagent follow-ing the manufacturer’s instructions. RNA was converted to cDNAby adding random primers and Superscript III reverse transcrip-tase. Real-time PCR was conducted to analyze gene expressionusing an ABI PRISM 7000 sequence detection system with Taq-Man primers. Primer sequences were as following: for COXIVforward, 5′-GTTCAGTTGTACCGCATCCA-3′; for COXIV reverse, 5′-TTGTCATAGTCCCACTTGGC-3′.
2.7. Immunoblotting
Cerebellar mitochondrial lysate was used to detect COX subunitIV protein. Protein samples were loaded into 12% polyacrylamidegel and then transferred onto polyvinylidene fluoride membranes.Blots were probed overnight with mouse monoclonal primaryantibody against COX subunit IV (1:1000) and incubated withhorseradish peroxidase-conjugated anti-mouse secondary anti-body for 1 h. Bands were detected using UVP bioimaging system andquantified by the image densitometer. Voltage dependent anionchannel1 (VDAC1) was used as a positive loading control for themitochondrial protein level.
2.8. Mitochondrial respiration in HT22 cells
HT22 cells were seeded into XF24 plates at the density of 15,000per well, and divided into the control group, the sodium azide (SA)(a COX inhibitor) group, and the SA + E2 group. HT22 cells weretreated with 0.5 mM SA and/or E2 ranging from 0.01 to 1.0 �M for5 h. OCR was then measured as mentioned above.
ondrial respiration in male and female mice, Neurosci. Lett. (2013),
3. Statistical analysis
All numerical data are expressed as mean ± SEM. Results ofXF24 assay and immunoblots were analyzed by t-test or two-way
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Fig. 1. Purkinje cell-specific deletion of p38. Pcp2-Cre mice were cross-mated with floxed-p38� mice. Animal tail samples and PCR method were used to identify genotypes.(A) illustrates the DNA images of Pcp2-Cre, homozygous p38loxP/loxP, and an internal positive control (DNA quality control) at 100, 414, and 324 base pair (bp), respectively. In(B), the cerebellar sections of these mice were immunostained using p38� antibody. Wild-type mice (C57BL/6 mice) and transgenic mice (Pcp2-Cre−/−/p38loxP/loxP) withoutP uronse tationQ4v
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cp2-Cre (A-b) show p38 positive stains (dark brown deposits) in the Purkinje nexpress both Pcp2-Cre and p38loxP/loxP(Pcp2-Cre+/−/p38loxP/loxP) (A-a). (For interpreersion of this article.)
NOVA. Post hoc Tukey’s test was then conducted to detect an indi-idual group difference. p value was set less than 0.05 to indicatetatistical significance.
. Results
.1. Purkinje cell-specific deletion of p38
The results from genotyping reveal the DNA images of Pcp2-re at 100 base pair, homozygous p38loxP/loxP at 414 base pair,nd an internal positive control (DNA quality control) at 324 bpFig. 1A). Immunohistochemistry results reveal that wild-type mice
Please cite this article in press as: X. Ju, et al., The role of p38 in mitochhttp://dx.doi.org/10.1016/j.neulet.2013.04.004
C57BL/6 mice) and transgenic mice (Pcp2-Cre−/−/p38loxP/loxP)ithout Pcp2-Cre (Fig. 1A-b) show p38 positive stains (dark browneposits) in the Purkinje neurons along the Purkinje layer. Byomparison, the p38 markers do not appear in transgenic mice
ig. 2. Mitochondrial respiration in wild-type and p38-knockout mice. Mitochondria isoF respirometer. OCR did not differ between wild-type and p38-knockout female mice buuccinate, ADP, and oligomycin were added to mitochondrial samples to determine th38-knockout did not affect the mitochondrial respiration states. All data are represented
along the Purkinje layer. The p38 markers do not appear in transgenic mice that of the references to color in this figure legend, the reader is referred to the web
(Fig. 1A-a) that express both Pcp2-Cre and homozygous p38loxP/loxP,proving that p38 has been successfully knocked out in Purkinjeneurons.
4.2. Mitochondrial respiration
p38-knockout did not significantly alter mitochondrial res-piration in female mice (Fig. 2A) but significantly suppressedmitochondrial respiration in male mice (p < 0.0001) (Fig. 2B). p38-knockout did not alter different states (II–IV) of mitochondrialrespiration. For wild-type mice, the mitochondrial respiration
ondrial respiration in male and female mice, Neurosci. Lett. (2013),
(state II) was higher in female (937.00 ± 62.17 pMoles/min) thanmale mice (625.20 ± 10.90 pMoles/min) (p < 0.0001) (figure notshown). These results indicate that p38-knockout suppresses themitochondrial respiration only in male mice.
lated from cerebellum were used to assess mitochondrial respiration (OCR) usingt was lower in p38-knockout male mice than wild-type male mice (***p < 0.0001).
e effect of p38-knockout on a different state (II–IV) of mitochondrial respiration. as mean ± SEM. N = 5 or 6 mice/group.
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Fig. 3. mRNA and protein expressions of COX subunit IV. Cerebellar mitochondriawere used to assess the levels of COX subunit IV mRNA and protein using RT-PCRand immunoblot, respectively. Compared to wild-type mice, a significant increase inthe levels of COX-IV mRNA (***p < 0.001) and protein (*p < 0.05) were found in p38-knockout female mice but not in male mice. Voltage dependent anion channel1(A
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VDAC1) was used as a positive loading control for the mitochondrial protein level.ll data are represented as mean ± SEM. N = 5 or 6 mice/group.
.3. COX subunit IVmRNA and protein expression
The effect of p38-knockout on COX expression was tested inale and female mice. Both mRNA and protein levels of COX sub-
nit IV were increased in p38-knockout female mice comparedo wild-type female mice (Fig. 3A and B). By comparison, nei-her mRNA nor protein content of COX subunit IV was altered in38-knockout male mice. These results indicate that p38-knockout
ncreases COX only in female mice.
.4. The effect of E2 and COX on mitochondrial respiration
Please cite this article in press as: X. Ju, et al., The role of p38 in mitochhttp://dx.doi.org/10.1016/j.neulet.2013.04.004
Mitochondrial respiration was suppressed (p < 0.01) when cellsere treated with a COX inhibitor (SA) alone (Fig. 4). This effect
f SA was blunted in the presence of E2, suggesting that E2 pro-otes mitochondrial respiration through COX activation. Among
ig. 4. Effects of E2 and COX on mitochondrial respiration. HT22 cells were treatedith a COX inhibitor (0.5 mM sodium azide (SA)) with or without E2 (0.1 �M or
.0 �M). SA sharply decreased mitochondrial respiration (**p < 0.01) and this effectf SA was blunted by E2 (*p < 0.01, **p < 0.01). N = 5 wells/group.
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test doses (0.01–1.0 �M), the lowest dose of E2 that was able toexert the protection was 0.1 �M (p < 0.0001). E2 treatment aloneat 0.1 and 1.0 �M did not increase mitochondrial respiration (datanot shown).
5. Discussion
The key finding of the current study is that p38 influencesmitochondria differently between male and female mice. p38-knockout suppresses mitochondrial respiration only in male miceand increases COX expression only in female mice. These data sug-gest that a certain level of p38 is required for the mitochondrialrespiration of male mice. When p38 is below a necessary level formitochondrial respiration, females may have capacity to maintainmitochondrial respiration through up-regulating COX.
Sex difference in mitochondria has been reported in previousstudies in which females have age-associated oxidative damageto a smaller degree than males due to more efficient mitochon-drial respiration. The sex difference is also reportedly attributedto a higher level of glutathione peroxidase (a H2O2 quencher) anduncoupling protein 5 (free radical inhibitor) in females than males.The disruption of ETC III impairs mitochondrial respiration of malemice and shortened their life-span compared to female mice [12].Compared to males, mitochondria from female rats generate lesshydrogen peroxide, more reduced glutathione (antioxidant), andmore 16S rRNA of which expression is inversely correlated withaging [1]. Ovariectomy abolishes whereas E2 replacement rein-states the sex differences [1]. These studies suggest that femalemitochondria might be equipped with more effective protectivemechanisms than male mitochondria. Our current observations ofgreater mitochondrial respiration in wild-type female mice thanmale mice essentially agree with these studies.
p38 is a signaling protein kinase and transduces neuronal sig-nals. Accordingly, p38 is abundantly expressed in neurons andsynapsis, mediating synaptic plasticity and gene regulation [25].While excessive p38 activation is associated with pathological con-ditions, a certain level of p38 might be required for maintainingmitochondrial respiration. Our transgenic mice hardly show p38 inPurkinje neurons, certainly indicating that their p38 is well belowa normal level. It is of interest that p38 deletion suppresses mito-chondrial respiration only in male mice and increases COX onlyin female mice. A potential explanation is that p38 deletion maytrigger a compensatory mechanism in female mitochondria to up-regulate COX and subsequently enhance mitochondrial respiration.E2 may play a role in this process by increasing COX as E2 increasesCOX activity [14]. With this scenario, male mitochondria in theabsence of p38 may be less able to respire in part due to insufficientE2 for COX up-regulation. Alternatively, females might be generallymore resistant to p38 alteration than males as hypoxia increasedp38 activation in male but not in female cardiac fibroblasts[30].
The up-regulating effect of E2 on COX has been demon-strated in our previous study in which E2-implantation increasedCOX activity in ovariectomized rats [14,15]. We conducted anin vitro experiment to test whether and how E2 plays a rolein sex-dependent mitochondrial respiration. When cells wereexposed to a COX inhibitor, they lost mitochondrial respirationthat was restored in the presence of E2 (0.1 and 1.0 �M). Thesedata suggest that COX activation mediates mitochondrial respi-ration and E2 acts as a COX activator. However, E2 lost suchprotective effect at a low physiologically relevant concentration
ondrial respiration in male and female mice, Neurosci. Lett. (2013),
(0.01 �M). These results are somewhat puzzling because E2 at aphysiological concentration protects COX from stress associatedwith alcohol withdrawal in rats [14,15]. This discrepancy mayresult from a difference between in vivo and in vitro conditions.
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or instance, when E2 concentration is below a certain level, thisell line may become less sensitive to E2’s protection for COX. Alter-atively, the protective dose of E2 may differ depending upon theature of stress. Others also report that E2 reverses age-related
oss of COX cofactors [13]. E2 protects mitochondria from trauma-emorrhage through COX up-regulation [11]. Although there maye a differential dose effect of E2, these studies strengthen the ideahat E2’s protection for COX helps female mice to maintain mito-hondrial respiration. How cytosolic p38 affects mitochondria istill an open question. One possibility is that p38 activation inducests substrate (p53) to translocate into mitochondria [17]. p53 inurn eliminates unhealthy mitochondrial proteins [17,19], therebynhancing mitochondrial functions. If E2 increases p53 mRNA level6], E2 in female mice might enable such p53’s mito-protection.
In conclusion, our findings provide empirical evidence that38-knockout in the major neurons of cerebellum sex-specificallyegulates mitochondrial respiration of this brain area. The currenttudy may provide a new mechanistic insight involving p38 intoex-dependent mitochondrial respiration.
ncited reference
[4].
cknowledgement
We wish to thank Wenjun Li for his technical support.
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