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Individuals with germline mutations in the gene encoding phosphatase and tensin homolog on chromosome ten (PTEN) are diagnosed with PTEN hamartoma tumor syndrome (PHTS) and are at high risk for developing breast, thyroid and other cancers and/or autoimmunity or neurodevelopmental issues including autism spectrum disorders. Although well recognized as a tumor suppressor, involvement of PTEN mutations in mediating such a diverse range of phenotypes indicates a more central involvement for PTEN in immunity than previously recognized. To address this, sequencing of the T-cell receptor variable-region β-chain was performed on peripheral blood from PHTS patients. Based on patient findings, we performed mechanistic studies in two Pten knock-in murine models, distinct from each other in cell compartment-specific predominance of Pten. We found that PTEN mutations in humans and mice are associated with a skewed T- and B-cell gene repertoire, characterized by increased prevalence of high-frequency clones. Immunological characterization showed that Pten mutants have increased B-cell proliferation and a proclivity towards increased T-cell reactivity upon Toll-like-receptor stimulation. Furthermore, decreases in nuclear but not cytoplasmic Pten levels associated with a reduction in expression of the autoimmune regulator (Aire), a critical mediator of central immune tolerance. Mechanistically, we show that nuclear PTEN most likely regulates Aire expression via its emerging role in splicing regulation. We conclude that germline disruption of PTEN, both in human and mouse, results in compromised central immune tolerance processes that may significantly impact individual stress responses and therefore predisposition to autoimmunity and cancer.

Germline mutations in PTEN account for ~10% of cases of autism spectrum disorder (ASD) with coincident macrocephaly. To explore the importance of nuclear PTEN in the development of ASD and macrocephaly, we previously generated a mouse model with predominantly cytoplasmic localization of Pten (Ptenm3m4/m3m4).Cytoplasmic predominant Pten localization results in a phenotype of extreme macrocephaly and autistic-like traits. Transcriptomic analysis of the Ptenm3m4/m3m4 cortex found upregulated gene pathways related to myeloid cell activation, myeloid cell migration, and phagocytosis. These transcriptomic findings were used to direct in vitro assays on Pten wild-type and Ptenm3m4/m3m4 microglia. We found increased Iba1 and C1q expression with enhanced phagocytic capacity in Ptenm3m4/m3m4 microglia, indicating microglial activation. Moreover, through a series of neuron-microglia co-culture experiments, we found Ptenm3m4/m3m4 microglia are more efficient at synaptic pruning compared with wild-type controls. In addition, we found evidence for neuron-microglia cross-talk, where Ptenm3m4/m3m4 neurons elicit enhanced pruning from innately activated microglia. Subsequent in vivo studies validated our in vitro findings. We observed a concurrent decline in the expression of Pten and synaptic markers in the Ptenm3m4/m3m4 cortex. At ~3 weeks of age, with a 50% drop in Pten expression compared with wild-type levels, we observed enhanced activation of microglia in the Ptenm3m4/m3m4 brain. Collectively, our data provide evidence that dysregulated Pten in microglia has an etiological role in microglial activation, phagocytosis, and synaptic pruning, creating avenues for future studies on the importance of PTEN in maintaining microglia homeostasis.

There is a strong genetic association between germline PTEN mutation and autism spectrum disorder (ASD), making Pten-mutant models exemplary for the study of ASD pathophysiology. We developed the Ptenm3m4 mouse, where Pten is largely restricted from the nucleus, which recapitulates patient-like, autism-related phenotypes: behavioral changes, macrocephaly, and white matter abnormalities. This study aimed to investigate the contribution of oligodendrocyte (OL) lineage differentiation and functional changes in myelination to the white matter phenotype. OL lineage differentiation and myelination in Ptenm3m4 mice was studied using immunohistochemical and electron microscopic analyses. We also used primary oligodendrocyte progenitor cells (OPCs) to determine the effect of the Ptenm3m4 mutation on OPC proliferation, migration and maturation. Finally, we assessed the myelinating competency of mutant OLs via co-culture with wildtype dorsal root ganglia (DRG) neurons. The in vivo analyses of Ptenm3m4/m3m4 murine brains showed deficits in proteolipid protein (Plp) trafficking in myelinating OLs. Despite the increased expression of myelin proteins in the brain, myelin deposition was observed to be abnormal, often occurring adjacent to, rather than around axons. Mutant primary OPCs showed enhanced proliferation and migration. Furthermore, mutant OPCs matured precociously, exhibiting aberrant myelination in vitro. Mutant OPCs, when co-cultured with wildtype DRG neurons, showed an inability to properly ensheath axons. Our findings provide evidence that the Ptenm3m4 mutation disrupts the differentiation and myelination programs of developing OLs. OL dysfunction in the Ptenm3m4 model explains the leukodystrophy phenotype, a feature commonly associated with autism, and highlights the growing importance of glial dysfunction in autism pathogenesis.