Project description:Cortical dysplasia (CD) is a common cause for intractable epilepsy. Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway has been implicated in CD; however, the mechanisms by which mTOR hyperactivation contribute to the epilepsy phenotype remain elusive. Here, we investigated whether constitutive mTOR hyperactivation in the hippocampus is associated with altered voltage-gated ion channel expression in the neuronal subset-specific Pten knockout (NS-Pten KO) mouse model of CD with epilepsy. We found that the protein levels of Kv1.1, but not Kv1.2, Kv1.4, or Kv?2, potassium channel subunits were increased, along with altered Kv1.1 distribution, within the hippocampus of NS-Pten KO mice. The aberrant Kv1.1 protein levels were present in young adult (?postnatal week 6) but not juvenile (?postnatal week 4) NS-Pten KO mice. No changes in hippocampal Kv1.1 mRNA levels were found between NS-Pten KO and WT mice. Interestingly, mTOR inhibition with rapamycin treatment at early and late stages of the pathology normalized Kv1.1 protein levels in NS-Pten KO mice to WT levels. Together, these studies demonstrate altered Kv1.1 protein expression in association with mTOR hyperactivation in NS-Pten KO mice and suggest a role for mTOR signaling in the modulation of voltage-gated ion channel expression in this model.
Project description:Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients.
Project description:The NS-Pten knockout (KO) mouse exhibits hyperactivity of the mammalian target of rapamycin (mTOR) and is a model of autism spectrum disorder (ASD). ASD presents with marked deficits in communication which can be elucidated by investigating their counterpart in mice, ultrasonic vocalizations (USVs). While USVs have been found to be altered in NS-Pten KO pups, no study has assessed whether this communication deficit persists into adulthood. In the present study, we investigate female urine-induced USVs, scent marking behavior, and open field activity in NS-Pten KO and wildtype (WT) adult male mice. Results showed that there was no difference in the quantity of vocalizations produced between groups, however, there were extensive alterations in the spectral properties of USVs. KO mice emitted vocalizations of a lower peak frequency, shorter duration, and higher peak amplitude compared to WT mice. KO animals also emitted a significantly different distribution of call-types relative to controls, displaying increased complex and short calls, but fewer upward, chevron, frequency steps, and composite calls. No significant differences between groups were observed for scent marking behavior and there was no difference between groups in the amount of time spent near the female urine. Overall, this study demonstrated that mTOR hyperactivity contributes to communication deficits in adult mice.
Project description:PTEN-induced putative kinase 1 (PINK1) is a Parkinson's disease (PD) gene. We examined miRNAs regulated by PINK1 during brain development and neural stem cell (NSC) differentiation, and found that lvels of miRNAs related to tumors and inflammation were different between 1-day-old-wild type (WT) and PINK1-knockout (KO) mouse brains. Notably, levels of miR-326, miR-330 and miR-3099, which are related to astroglioma, increased during brain development and NSC differentiation, and were significantly reduced in the absence of PINK1. Interestingly, in the presence of ciliary neurotrophic factor (CNTF), which pushes differentiation of NSCs into astrocytes, miR-326, miR-330, and miR-3099 levels in KO NSCs were also lower than those in WT NSCs. Furthermore, mimics of all three miRNAs increased expression of the astrocytic marker glial fibrillary acidic protein (GFAP) during differentiation of KO NSCs, but inhibitors of these miRNAs decreased GFAP expression in WT NSCs. Moreover, these miRNAs increased the translational efficacy of GFAP through the 3'-UTR of GFAP mRNA. Taken together, these results suggest that PINK1 deficiency reduce expression levels of miR-326, miR-330 and miR-3099, which may regulate GFAP expression during NSC differentiation and brain development.
Project description:Monoamine oxidase A (MAOA) is a mitochondrial enzyme, which degrades monoamine neurotransmitters and dietary amines and produces H2O2. Recent studies have shown increased MAOA expression in prostate cancer (PCa), glioma, and classical Hodgkin lymphoma. However, the biological function of MAOA in cancer development remains unknown. In this study, we investigated the role of MAOA in the development of prostate adenocarcinoma by creating a prostate-specific Pten/MAOA knockout (KO) mouse model, in which MAOA-floxP mouse was crossed with the conditional Pten KO PCa mouse that develops invasive PCa. In contrast to Pten KO mice, age-matched Pten/MAOA KO mice exhibited a significant decrease in both prostate size and the incidence of invasive cancer. We observed a significant decline in AKT phosphorylation and Ki67 expression in Pten/MAOA KO mice, which reduced epithelial cell growth and proliferation. As cancer stem cells (CSCs) are required for tumor initiation and growth, we investigated expression of OCT4 and NANOG in the setting of decreased MAOA expression. We found that both OCT4 and NANOG were significantly attenuated in the prostate epithelia of Pten/MAOA KO mice compared to Pten KO mice, which was confirmed with targeted knockdown of MAOA with a short-hairpin(sh) vector targeting MAOA compared to cells transfected with a control vector. Expression of other markers associated with the a stem cell phenotype, including CD44, ?2?1, and CD133 as well as HIF-1?+CD44+ stem cells were all decreased in shMAOA PCa cells compared with empty vector-transfected control cells. We also found spheroid formation ability in PCa cells was decreased when endogenous MAOA was suppressed by siRNA or MAOA inhibitor clorgyline in a colony formation assay. Using the TCGA database, elevated MAOA expression was associated with reduced Pten levels in high Gleason grade in patient samples. Further, we found that Pten-positive PCa cells were more resistant to clorgyline treatments than Pten-null cells in tumorigenicity and stemness. Taken together, these studies suggest that MAOA expression promotes PCa development by increasing cell proliferation and CSCs and highlights the potential use of MAOA inhibitors for the treatment of PCa.
Project description:To investigate the underlying mechanisms mediating resistance to NOTCH inhibition in Pten-null T-ALL tumor cells we performed gene expression profiling of isogenic Pten-positive and Pten-deleted leukemia lymphoblasts after acute treatment with DBZ in vivo. This analysis revealed that, while direct NOTCH1 target genes (such as Hes1, Dtx1, PtcrA, HeyL and Notch3) are effectively downregulated in both Pten-positive and Pten-deleted tumors, genetic ablation of Pten elicits a global reversal of much of the transcriptional effects of NOTCH inhibition. We performed microarray gene expression analysis of GSI treatment in isogenic Pten KO or WT NOTCH1 induced leukemias
Project description:To understand the mechanism behind aberrant Akt activation in T-ALL, PIK3CA, PTEN and SHIP1 expression and genotype were assessed. No cell lines or primary ALLs harbored PIK3CA mutations. PTEN was expressed in just one-third of the cell lines, but in two-thirds of the primary ALLs, though in the inactivated (phosphorylated) form. SHIP1 was undetectable in most primary ALL and in the T-ALL cell line Jurkat, which harbored a bi-allelic null mutation and a frame-shift deletion; primary ALL harbored the frame-shift as well as other translationally-inactivating deletions and insertions. The inactivation of SHIP1 could play a central role in the deregulation of Akt pathway and tumorigenesis, perhaps in conjunction with PTEN inactivation.
Project description:BACKGROUND:The prostate-specific phosphatase and tensin homolog deleted on chromosome 10 (Pten) gene-conditional knockout (KO) mouse carcinogenesis model is highly desirable for studies of prostate cancer biology and chemoprevention due to its close resemblance of primary molecular defect and many histopathological features of human prostate cancer including androgen response and disease progression from prostatic intraepithelial neoplasia to invasive adenocarcinoma. Here, we profiled the proteome and transcriptome of the Pten-KO mouse prostate tumors for global macromolecular expression alterations for signaling changes and biomarker signatures. METHODS:For proteomics, four pairs of whole prostates from tissue-specific conditional knockout Pten-KO mice (12-15 weeks of age) and their respective wild-type littermates housed in the same cages were analyzed by 8-plex isobaric tags for relative and absolute quantitation iTRAQ. For microarray transcriptomic analysis, three additional matched pairs of prostate/tumor specimens from respective mice at 20 to 22 weeks of age were used. Real-time quantitative reverse transcription-polymerase chain reaction was used to verify the trends of protein and RNA expression changes. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out for bioinformatic characterizations of pathways and networks. RESULTS:At the macromolecular level, proteomic and transcriptomic analyses complement and cross-validate to reveal overexpression signatures including inflammation and immune alterations, in particular, neutrophil/myeloid lineage suppressor cell features, chromatin/histones, ion and nutrient transporters, and select glutathione peroxidases and transferases in Pten-KO prostate tumors. Suppressed expression patterns in the Pten-KO prostate tumors included glandular differentiation such as secretory proteins and androgen receptor targets, smooth muscle features, and endoplasmic reticulum stress proteins. Bioinformatic analyses identified immune and inflammation responses as the most profound macromolecular landscape changes, and the predicted key nodal activities through Akt, nuclear factor-kappaB, and P53 in the Pten-KO prostate tumor. Comparison with other genetically modified mouse prostate carcinogenesis models revealed notable molecular distinctions, especially the dominance of immune and inflammation features in the Pten-KO prostate tumors. CONCLUSIONS:Our work identified prominent macromolecular signatures and key nodal molecules that help to illuminate the patho- and immunobiology of Pten-loss driven prostate cancer and can facilitate the choice of biomarkers for chemoprevention and interception studies in this clinically relevant mouse prostate cancer model.
Project description:The goal of this study is to compare gene expression profiles of PTEN WT and KO GBM cells when they are growing in vivo. Isogenic PTEN WT or KO LN229 GBM cells were subcutaneously implanted into Balb/c nude mice (two in each group) to form tumor. At the 30th day tumors were harvested and total RNA was isolated. RNA-seq analysis was performed to examine the differential express pattern of the two type of xenografts. Overall design: Xenograft GBM tumor mRNA profiles of PTEN wild type (WT) and KO were generated by deep sequencing, in triplicate, using BGISEQ-500.