An Investigation on the Molecular Pathogenic Mechanism Underlying Mutant Pten-Induced Autism Using Knockin Mouse Model.

Autism spectrum disorders (ASDs) affect about 0.6-1 % of children and represent a heterogeneous group of neurodevelopmental disorders, which share common behavioral features including defective social interactions, impaired verbal and non-verbal communication, limited range of interests and stereotyped and repetitive behaviors. ASDs are highly heritable neuropsychiatric disorders, and a complex genetic basis is involved in the pathogenesis of ASD. Several monogenic hereditary disorders also exhibit typical autistic symptoms and meet diagnostic criteria for ASD. PTEN (phosphatase and tensin homolog deleted on chromosome ten) was originally identified as a tumor suppressor gene, and germline mutations in PTEN cause inherited disorders collectively known as PTEN hamartoma tumor syndromes, including Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS). Initial studies reported that children with CS or BRRS displayed autistic behaviors, macrocephaly and delayed cognitive development. Subsequent molecular genetic studies identified heterozygous missense or nonsense PTEN mutations in ASD individuals with macrocephaly. A major portion of autistic children with PTEN mutation also exhibit cognitive impairment. Most of ASD PTEN mutations were not observed in CS or BRRS, indicating a specific pathogenic role of PTEN mutations in causing ASD. PTEN mutations play a pathogenic role in 1-1.5 % of total ASD patients. Up to now, the pathogenic mechanism underlying mutant PTEN-induced ASD has not been investigated in detail. PTEN contains two main functional domains, the phosphatase domain and the C2 domain. PTEN is expressed in the cerebral cortex, amygdala and hippocampus of human and mouse brain, and a high level of PTEN expression is observed in the cell body, dendrites and spines of pyramidal neurons. PTEN antagonizes PI3K signaling by dephosphorylating PIP3 and acts the major negative regulator of the PI3K-AKT-mTOR pathway in the brain. PI3K-AKT-mTOR signaling, which promotes local protein synthesis in the dendrites and spines, is required for dendritic arborization, spine formation/morphogenesis and synaptic plasticity including late-long-term potentiation (LTP) and mGluR-dependent long-term depression (mGluR-LTD). Thus, PTEN, an inhibitor of PI3K-AKT-mTOR pathway, plays a crucial role in regulating dendritic development, spine formation/maturation, late-LTP and mGluR-LTD in the in the cerebral cortex, amygdala and hippocampus. PTEN also directly or indirectly via PI3K-AKT pathway regulates glutamatergic or GABAergic transmission of pyramidal neurons by downregulating membrane expression of GABAA-R and AMPA-R. All of heterozygous ASD PTEN mutations, including (R130X), (I135R), (F241S) or (D252G) mutation, are highly conserved and found in two major functional domains, the phosphatase domain and the C2 domain. In the present study, we hypothesized that ASD mutant PTEN is defective in dephosphorylating PIP3 and repressing activation and phosphorylation of AKT. In accordance with this hypothesis, our result showed that overexpression of wild-type PTEN in HEK 293 cells decreased the protein expression of active and phospho-AKTThr308. In contrast, ASD mutant (R130X), (I135R), (F241S) or (D252G) PTEN failed to inhibition the activation and phosphorylation of AKT. Thus, ASD PTEN mutation causes the loss of PTEN functions including PTEN-mediated PIP3dephosphorylation and inhibition of PI3K-AKT-mTOR pathway. As a result, expression level of functional PTEN is decreased in the brain of ASD patients with heterozygous PTEN mutation, leading to an impaired PTEN dephosphorylation of PIP3 and resulting upregulated PIP3 level and AKT-mTOR pathway activity. Previous neuroimaging studies revealed that aberrant structural and functional connectivity and abnormal neuronal activity in the amygdala, anterior cingulate cortex and medial prefrontal cortex contribute to the pathogenesis of autism and resulting behavioral deficits of ASD patients. Therefore, we hypothesize that reduced level of functional PTEN and resulting PTEN haploinsufficiency, caused by heterozygous ASD (R130X), (I135R), (F241S) or (D252G) PTEN mutation, induces aberrant development of dendrites and spines, synaptic transmission and synaptic plasticity in the amygdala, frontal lobe and hippocampus, resulting in ASD behavioral deficits and cognitive dysfunction. In the present study, animal model of mutant PTEN-induced autism is prepared by generating knockin mice expressing ASD mutant (R130X), (I135R), (F241S) or (D252G) PTEN. Subsequently, we will investigate molecular pathogenic mechanisms of mutant PTEN-induced ASD by performing the following experiments using PTENR130X/+, PTENI135R/+, PTENF241S/+ or PTEND252G/+ heterozygous knockin mice, which displayASD behavioral deficits and cognitive impairment: (1) It is hypothesized that ASD (R130X), (I135R), (F241S) or (D252G) PTEN mutation causes the loss of PTEN phosphatase activity and defective dephosphorylation of PIP3, leading to accumulation of PIP3 and overactivation of AKT-mTOR signaling pathway. Biochemical and Western blot assays are performed to visualize elevated PIP3 level and upregulated AKT-mTOR signaling in the amygdala, frontal cortex and hippocampus of PTENR130X/+, PTENI135R/+, PTENF241S/+ or PTEND252G/+ knockin mice. (2) It is possible that ASD mutation-induced loss of PTEN function and resulting dysregulated glutamatergic and/or GABAergic transmission causes aberrant neuronal activity and dysfunction of pyramidal cells in the anterior cingulate cortex (ACC) or amygdala, leading to autistic behavioral deficits. To test this hypothesis, we will study possible alteration of glutamatergic and/or GABAergic neurotransmission in ACC or amygdalar pyramidal neurons of PTENR130X/+, PTENI135R/+, PTENF241S/+ or PTEND252G/+ knockin mice. (3) It is hypothesized that ASD PTEN mutation-induced loss of PTEN function and resulting dysregulation of PI3K-AKT-mTOR pathway causes aberrant late-LTP and/or mGluR-dependent LTD in amygdalar, neocortical or hippocampal pyramidal neurons, resulting in autistic symptoms and cognitive dysfunction. To test this hypothesis, we will study possible dysregulation of late-LTP and/or mGluR-LTD in amygdalar, ACC, mPFC or hippocampal pyramidal neurons of PTENR130X/+, PTENI135R/+, PTENF241S/+ or PTEND252G/+ knockin mice. (4) It is possible that ASD PTEN mutation-induced dysregulation of PI3K-AKT-mTOR pathway in neocortical or amygdalar pyramidal neurons causes aberrant dendritic arborization and alteration in morphology and number of dendritic spines, leading to malfunction of frontal cortex and amygdala and autistic phenotypes. To test this hypothesis, imaging analysis is performed to visualize possible abnormal dendritic arbors and altered spine morphology and number of ACC, mPFC or amygdalar pyramidal neurons of PTENR130X/+, PTENI135R/+, PTENF241S/+ or PTEND252G/+ knockin mice. Our study should shed light on molecular pathogenic mechanisms of mutant PTEN-induced ASD and physiological functions of PTEN in the brain. The results obtained from the present investigation could also lead to the development of possible therapeutic strategy ASD.

Project ID:PC10007-1161
External Project ID:NSC100-2320-B182-017