Considering that disturbances in energy metabolism underlie some neurological conditions such as schizophrenia, affective disorders, fragile X-associated tremor and ataxia syndrome (FXTAS) and autism 
, we tested if Pten
haplo-insufficiency in neural tissues resulted in the occurrence of aberrant social and repetitive behavior and/or mitochondrial dysfunction, and evaluated the mechanism for such dysfunction. To this end, we developed a Pten
haplo-insufficient model, similar to the one described by Parada's group 
, but based on a two-step breeding process (to reduce the genetic variability between littermates in the second-generation mice). This approach allowed us to generate a mouse model more representative of the heterozygous germline mutations reported in children with autism 
while minimizing the risk for cancer development. Our results were indicative of a mechanism that entailed a sustained activation of PI3K/Akt pathway, for which Pten is a negative modulator, followed by a negative feedback loop between Pten and p53 resulting in a specific decrease in CCO activity, ensuing in energy stress in cerebellum and hippocampus.
Symptomatic mice presented lower CCO activity in both cerebellum and hippocampus, deficits that paralleled those in p21 and SCO2 protein expression. It is interesting to note the interplay between Pten, p53 and CCO, given that p53 is the only transcription factor that has been found to be directly linked to CCO activity 
. Lower SCO2 (a metallochaperone involved in the synthesis of subunit II of CCO and CCO maturation 
) resulted in impaired CCO activity, along with the occurrence of behavioral abnormalities in 20–29 weeks old mice. It is of interest to note that SCO2 deficiency and/or CCO activity defects have been reported in a number of patients with learning disabilities as well as autism 
and that a number of mutated proteins in ASD had been found associated with p53-signaling pathway 
Considering that mtDNA deletions were significant in Pten
haplo-insufficient mice (20 to 29 weeks old) and that p53 has been implicated in the maintenance of mtDNA (this study and others 
), among other genes 
, it could be argued that clonal expansion of mtDNA with deletions can accumulate over time and outnumber wild-type mtDNA 
. It has been suggested that the replicative advantage of large-scale deletions is due to a faster completion of replication of smaller mtDNA molecules 
. Although we observed that mtDNA deletions accumulate in hippocampus with age in HET and, at a 2-fold increase in HET-CRE mice () suggesting a higher copy-error probability, to evidence a biochemical defect on CCO activity, the level of a mtDNA deletion needs to exceed a critical threshold level of 50% to 60% 
, level only reached at 1 year of age in HET-CRE and close 2 years of age in HET according to our simulation (see limitations of simulation in , legend). Thus, deletions acquired in later adult life of HET do not seem to have sufficient time to express a biochemical defect and/or to reach significant levels by random genetic drift alone, whereas in HET-CRE, and if our model were correct, mtDNA deletions might exacerbate the CCO deficiency initiated earlier by the PTEN-p53-SCO2 axis. Furthermore, it is interesting to note that multiple mtDNA deletions have been reported in association with psychiatric and behavioral disturbances with slow progressive course and adult onset 
and possibly in the observed delayed phenotype of Huntington's disease (Napoli et al., 2012, submitted manuscript).
mtDNA deletions in HET-CRE and HET hippocampus accumulate with age.
Several lines of evidence connect Pten and p53 functionally (reviewed in 
), including Akt-mediated phosphorylation of MDM2 allowing MDM2-mediated ubiquitination and degradation of p53 
(). A feedback loop has also been suggested by the potential of p53 to regulate Pten
. This loop would explain our results obtained with the HCT 116 model in which the sole p53 haplo-insufficiency results in decreased Pten protein levels. Social deficits present in conditional Pten null
mice were ameliorated by treatment with rapamycin 
, indicating the involvement of the mTOR (mammalian target of rapamycin complex) pathway. The latter observations, together with our findings, predict a role for an mTOR-mediated response to the earlier energy stress resulting from MD.
Interplay between Pten and p53 and energy stress response mechanism to mitochondrial dysfunction.
Over-expression of Pten in embryonic fibroblasts of transgenic mice showed increased mitochondrial biogenesis and OXPHOS capacity via PGC1α pathway activation 
. Conversely, decreases in Pten would be expected to result in decreased mitochondria biogenesis and lower MnSOD (PGC1α is a positive regulator of MnSOD transcription 
). However, our model of Pten haplo-insufficiency seem to point to the activation of a mechanism that does not necessarily involve PGC1α, i.e., tissue-specific regulation of Pten and p53 resulting in lower mitochondrial OXPHOS without significant changes in mitochondrial mass (as judged by CS activity) and increased levels of MnSOD. This apparent discrepancy can be bridged considering the differences in biological models (generalized overexpression of Pten in utero
vs. specific knock-down of Pten in cerebellum and hippocampus at postnatal stages).
haplo-insufficient mice aged 20–29 weeks showed abnormal social and repetitive behavior with some features similar to those present in ASD, i.e.
, limited preference for social novelty, failure to habituate to a familiar stimulus, social avoidance behavior and repetitive or stereotyped behavior. Of note, atypical social interactions are observed in many psychiatric disorders besides autism, including depression and schizophrenia 
. In this regard, an argument could be made that Pten
haplo-insufficient mice would serve as a better model for schizophrenia than autism given that (i) the atypical social behavior observed in this study was not observed until post adolescence when an autistic-like phenotype would require a model in which the behavioral and anatomical changes are present before adolescence (autism requires a diagnosis before 3 years of age 
) and (ii) mitochondrial dysfunction is present in individuals with schizophrenia 
. However, (i) schizophrenia is characterized by reduced brain size 
whereas a subset of individuals with autism presents macrocephaly 
; (ii) schizophrenia tends to be associated with reduced function of genes involved in the up-regulation of the PI3K/Akt pathway 
whereas autism has been associated with loss of function of genes acting as negative regulators of the PI3K/Akt/mTOR pathway 
; (iii) in this regard, de novo Pten
mutation had been found strongly associated with increased risk of developing ASD not schizophrenia 
; and (iv) the difference in the onset and full penetrance of aberrant social behavior in our study vs. those utilizing a null
(i.e., our study 8–13 and 20–29 weeks vs. 6–8 and 8–12 weeks for null models 
, respectively) and the phenotypic characteristics (Lhermite-Duclos disease, Cowden syndrome, and ASD, seizures, death vs. aberrant social and repetitive behavior) seem consistent with a Pten
gene dose effect (i.e., this study, Cre−/+
were haploid for Flox-Pten as compared to previous studies, in which Flox-Pten was homozygous) more than a set of developmental processes that result in a post-adolescence behavioral phenotype.
Finally, it is worth mentioning the role of oxidative stress in Pten
haplo-insufficient mice also observed in CCO-deficient cybrids constructed with mtDNA from patients with sporadic AD 
. In addition, increased oxidative/nitrative stress might arise from increased production of NO by eNOS, activated by the Akt pathway, which is normally negatively regulated by PTEN; SCO2 deficiency resulting in oxidative DNA damage 
; and loss/decrease in assembled CCO - as it has been observed with mutations in SCO2 
- could also affect the activity of Complex III, the major site for ROS production in mitochondria considering that the respiratory chain can exist as a supercomplex 
. Although we have not observed changes in SCCR activity in Pten
haplo-insufficiency, this biochemical assay does not evidence relatively small changes in Complex III activity 
, which could be significant in terms of ROS production.
This study underlines the importance of MD as a contributor to either the development of symptoms and/or severity of the phenotype. The final response to energy stress could be dependent on a combination of genetic (Pten
, other genes) and/or environmental factors 
increasing the predisposition to abnormal neurodevelopment 
and resulting in a convergent phenotypic characterized by aberrant social and repetitive behavior. Indeed, energy-requiring processes that rely heavily on mitochondrial OXPHOS and are necessary for typical neurodevelopment, such as neuronal migration in the amygdala-hippocampal region, have been found deficient in individuals with ASD 
. Alternatively, symptoms attributed to autism as well as a number of neurological disorders 
, may result from an imbalance of excitatory and inhibitory neurotransmission produced by an abnormal GABA catabolism which is linked to mitochondrial function (“GABA shunt”; 
), possibly compounded by accumulation of mtDNA deletions.