Mitochondrial Dysfunction in <em>Pten</em> Haplo-Insufficient Mice with Social Deficits and Repetitive Behavior: Interplay between Pten and p53

<div><p>Etiology of aberrant social behavior consistently points to a strong polygenetic component involved in fundamental developmental pathways, with the potential of being enhanced by defects in bioenergetics. To this end, the occurrence of social deficits and mitochondrial outcomes were evaluated in conditional <em>Pten</em> (Phosphatase and tensin homolog) haplo-insufficient mice, in which only one allele was selectively knocked-out in neural tissues. <em>Pten</em> mutations have been linked to Alzheimer's disease and syndromic autism spectrum disorders, among others. By 4–6 weeks of age, Pten insufficiency resulted in the increase of several mitochondrial Complex activities (II–III, IV and V) not accompanied by increases in mitochondrial mass, consistent with an activation of the PI3K/Akt pathway, of which Pten is a negative modulator. At 8–13 weeks of age, <em>Pten</em> haplo-insufficient mice did not show significant behavioral abnormalities or changes in mitochondrial outcomes, but by 20–29 weeks, they displayed aberrant social behavior (social avoidance, failure to recognize familiar mouse, and repetitive self-grooming), macrocephaly, increased oxidative stress, decreased cytochrome <em>c</em> oxidase (CCO) activity (50%) and increased mtDNA deletions in cerebellum and hippocampus. Mitochondrial dysfunction was the result of a downregulation of p53-signaling pathway evaluated by lower protein expression of p21 (65% of controls) and the CCO chaperone SCO2 (47% of controls), two p53-downstream targets. This mechanism was confirmed in Pten-deficient striatal neurons and, HCT 116 cells with different p53 gene dosage. These results suggest a unique pathogenic mechanism of the Pten-p53 axis in mice with aberrant social behavior: loss of Pten (via p53) impairs mitochondrial function elicited by an early defective assembly of CCO and later enhanced by the accumulation of mtDNA deletions. Consistent with our results, (i) SCO2 deficiency and/or CCO activity defects have been reported in patients with learning disabilities including autism and (ii) mutated proteins in ASD have been found associated with p53-signaling pathways.</p> </div

mtDNA deletions in HET-CRE and HET hippocampus accumulate with age.

<p>Accumulation of mtDNA with deletions (average of deletions at the segments encoded for CYTB, COX3 and ND4) with age per single hippocampal cell from HET (white circles) and HET-CRE (black circles). The mtDNA deletions (represented as the mean ± SEM) values were fitted to a linear regression (equations shown in the figure) and the goodness of the regression was expressed as r². From the slope of each equation, the copy-error probability was estimated by using the following formulae N_del = (N_mtDNA*P_del*t * ln 2)/<i>t</i>_1/2, where N_del is the number of mtDNA with deletions, t is the age of mice in days throughout their lifetime (estimated as 2 years), N_mtDNA = 1,200 mtDNA copy number/cell (experimentally determined in hippocampus by evaluating the ratio of mitochondrial ND1 gene copy number to the nuclear PK gene copy number), and a mtDNA half-life (<i>t</i>_1/2) of 10 d <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#pone.0042504-Elson1" target="_blank">[121]</a>. For HET mice, a low copy-error probability was obtained (9.5×10⁻⁶) associated with a low incidence of accumulation of deletions with age, becoming more significant towards the end of their life. Higher copy-error probabilities, as that calculated with HET-CRE mice (1.9×10⁻⁵) lead to a greater accumulation of mtDNA with deletions throughout a simulated mouse life. A biochemical defect on CCO activity is evidenced when the level of a mtDNA deletion exceeds a critical threshold level of 50%–60% <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#pone.0042504-Hayashi1" target="_blank">[77]</a> (indicated with a dotted line).</p

Protein expression of p53, p21 and SCO2 and CCO activity in Pten-deficient striatal neurons.

<p><b>A.</b> Representative Western blot and densitometry of p-Akt and total Akt protein levels in striatal neurons. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#s2" target="_blank">Results</a> are expressed as the ratio between p-Akt and total Akt and reported as mean ± SEM. <b>B.</b> Representative Western blots of p53, p21 SCO2 and Tubulin. Forty µg of protein were loaded in each lane. Details on the antibodies used are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#s4" target="_blank">Materials and Methods</a>. <b>C</b> Densitometry results for p53, p21, SCO2 protein expression, normalized by Tubulin (loading control). CCO activity was expressed as nmol×(min×10⁶ cells)⁻¹ and reported normalized by citrate synthase. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#s2" target="_blank">Results</a> are mean ± SEM of experiments in triplicates.</p

Characteristics of the mouse groups and tests performed.

<p>The age range represents the 95% Confidence interval age limits in weeks and the age of the group in weeks was expressed as mean ± SEM. Abbreviations: BMB, molecular tests in biochemistry and molecular biology; H, histology; BW, brain weight; B, behavior; F, female, M, male. The <i>p</i>-values were obtained by comparing the age of HET-CRE to that of HET.</p

Changes of Cre-mediated expression of Pten protein and gene with age in brain from <i>Pten</i> haplo-insufficient mice.

<p>HET and HET-CRE groups were sex- and age matched and at each age and each group had 7 to 11 animals (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#pone-0042504-t001" target="_blank"><b>Table 1</b></a>). All values were expressed as mean ± SEM. <b>Panels A–D</b>: HET, white bars; HET-CRE, black bars. <b>Panel A</b>: <i>Pten gene deletion in various brain regions from HET-CRE mice aged 8–13 weeks</i>. Pten gene deletion was calculated as the ratio of the truncated band over that of the normal allele for Pten exon 5 determined as described in the <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#pone.0042504.s001" target="_blank">Methods S1</a></b>. <b>Panels B–D: </b><i>Pten protein expression in brain regions from HET and HET-CRE mice aged 4–6, 8–13, and 20–29 weeks</i>. The densitometry of the Pten band obtained by Western blots was normalized to that of actin for each brain region. The <i>p-</i>values were obtained by using Student's <i>t</i>-test. <b>Panel E: </b><i>Pten gene deletion and Pten protein expression in liver and heart from HET and HET-CRE mice aged 8–13 weeks</i>. <i>Pten</i> gene deletion was obtained as described under <b>1A</b> legend. Pten protein expression was calculated as described under <b>Panel B–D</b> legend but expressed as percentage of age-matched HET values. <b>Panel F</b>: <i>Assessment of macrocephaly in Pten haplo-insufficient mice</i>. Mean values for HET mice at 20–29 weeks were 322±15 mg brain wet weight; body weight 24±3 g; 1.7±0.2 mg cerebellum/g body weight; 1.4±0.4 mg cortex/g body weight; 0.66±0.06 mg hippocampus/g body weight. * All values were significantly different from HET with <i>P</i><0.05.</p

Behavioral tests performed on HET and HET-CRE mice aged 20–29 weeks.

<p>HET and HET-CRE values were expressed as mean ± SEM. The fold increase represents the ratio between HET-CRE and HET means.</p>*<p><i>p</i>-value calculated using the one-tailed <i>t</i>-test between two independent means. For all comparisons, significance was set at <i>p</i>≤0.050.</p>†<p>Normalized by total trial time.</p>‡<p>Normalized by total trial frequency.</p

Changes in ETC activities in cerebellum, hippocampus and cortex in HET and HET-CRE mice.

<p>Enzymatic activities were evaluated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#s4" target="_blank">Materials and Methods</a> section and originally expressed as nmol×(min×mg protein)⁻¹ and normalized to citrate synthase activity for each brain region and age. Complex I activity was evaluated by measuring either NFR (cerebellum and hippocampus) or NQR (cortex) activities. Data were expressed as percentage of HET values (mean ± SEM) and analyzed by unpaired <i>t</i> test. * <i>p</i><0.05 compared with HET.</p

p53 and p53-downstream targets, CCO activity, mtDNA copy number and deletions in <i>Pten</i> haplo-insufficient mice aged 20–29 weeks.

<p>All outcomes were expressed as percentages of HET values. The values for the outcomes from HET mice were the following: Hippocampus: mtDNA copy number 1214±178; mtDNA gene ratios for ND4, CYTB and COX3 = 0.86±0.05, 1.22±0.02 and 1.65±0.3, respectively; CCO 235±26 and CS 144±14 nmol×(min×mg protein)⁻¹. Cortex: mtDNA copy number 1563±177; mtDNA gene ratios for ND4, CYTB and COX3 = 0.92±0.06, 1.12±0.01 and 1.5±0.2, respectively; CCO 245±19 and CS 170±9 nmol×(min×mg protein)⁻¹. Cerebellum: mtDNA copy number 2600±17; mitochondrial gene ratios for ND4, CYTB and COX3 = 0.83±0.06, 1.02±0.002 and 1.1±0.1, respectively; CCO 60±6 and CS 150±4 nmol×(min×mg protein)⁻¹.</p>(a)<p>Percentage of mtDNA deletions were calculated as follows for each of the three tissues: 100−(100×average of mitochondrial gene ratio HET-CRE/HET).</p>(b)<p>Protein expression was performed by western blot and by normalizing the intensity of the band of the loading control (actin) and expressed as percentage of control values.</p>*<p><i>p</i><0.05;</p>**<p><i>p</i><0.01;</p>***<p><i>p</i><0.001. Representative western blot images and densitometry results are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#pone.0042504.s004" target="_blank">Figures S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042504#pone.0042504.s005" target="_blank">S4</a></b>.</p

Additional file 1: of Development of waist circumference percentiles for Japanese children and an examination of their screening utility for childhood metabolic syndrome: a population-based cross-sectional study

Table S1. Box–Cox values for power (L), adjusted mean (M), and coefficient of variation (S) of WC for the Japanese children of boys and girls from 4.50 to 12.75 years of age. Table S2. Box–Cox values for power (L), adjusted mean (M), and coefficient of variation (S) of WC/Ht for the Japanese children of boys and girls from 4.50 to 12.75 years of age. (DOCX 179 kb