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To find genes involved in tumorigenesis and the development of esophageal cancer, the suppression subtractive hybridization (SSH) method was used to identify genes that are overexpressed in esophageal cancer tissues compared to normal esophageal tissues. In our SSH library, the forkhead box O3 (FOXO3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and myeloid differentiation primary response 88 (MYD88) genes were the most highly upregulated genes, and they were selected for further studies because of their potential role in the induction of autophagy. Upregulation of these genes was also observed in clinical samples using qRT-PCR. In addition, coexpression analysis of the autophagy-related genes Beclin1, ATG12, Gabarapl, PIK3C3, and LC3 demonstrated a significant correlation between the differentially overexpressed genes and autophagy. Autophagy is an important mechanism in tumorigenesis and the development of chemoresistance in cancer cells. The upregulation of FOXO3, GAPDH, and MYD88 variants in esophageal cancer suggests a role for autophagy and provides new insight into the biology of esophageal cancer. We propose that FOXO3, GAPDH, and MYD88 are novel targets for combating autophagy in esophageal cancer. Show less

Programmed nuclear death (PND) in the ciliate protozoan Tetrahymena thermophila is a novel type of autophagy that occurs during conjugation, in which only the parental somatic macronucleus is destined to die and is then eliminated from the progeny cytoplasm. Other coexisting nuclei, however, such as new micro- and macronuclei are unaffected. PND starts with condensation in the nucleus followed by apoptotic DNA fragmentation, lysosomal acidification, and final resorption. Because of the peculiarity in the process and the absence of some ATG genes in this organism, the mechanism of PND has remained unclear. In this study, we focus on the role of class III phosphatidylinositol 3-kinase (PtdIns3K, corresponding to yeast Vps34) in order to identify central regulators of PND. We identified the sole Tetrahymena thermophila ortholog (TtVPS34) to yeast Vps34 and human PIK3C3 (the catalytic subunit of PtdIns3K), through phylogenetic analysis, and generated the gene knockdown mutant for functional analysis. Loss of TtVPS34 activity prevents autophagosome formation on the parental macronucleus, and this nucleus escapes from the lysosomal pathway. In turn, DNA fragmentation and final resorption of the nucleus are drastically impaired. These phenotypes are similar to the situation in the ATG8Δ mutants of Tetrahymena, implying an inextricable link between TtVPS34 and TtATG8s in controlling PND as well as general macroautophagy. On the other hand, TtVPS34 does not appear responsible for the nuclear condensation and does not affect the progeny nuclear development. These results demonstrate that TtVPS34 is critically involved in the nuclear degradation events of PND in autophagosome formation rather than with an involvement in commitment to the death program. Show less

We evaluated multiple effects of genetic variations of five candidate loci (LEP, LEPR, MC4R, PIK3C3 and VRTN) on four production traits (average daily weight gain (ADG); backfat thickness (BFT); loin eye muscle area (EMA); and intramuscular fat content (IMF)) in a closed nucleus herd of pure Duroc pigs. Polymorphisms in LEPR, MC4R and PIK3C3 had significant single gene effects on ADG and BFT. The additive genetic variance in ADG and BFT (16.99% and 22.51%, respectively) was explained by genetic effects of these three loci. No correlations were observed between the LEP genotype and production traits in this study. Although we detected marginally epistatic interactions between LEPR and PIK3C3 on the eye muscle area, there were no significant epistatic effects on any traits among all loci pairs. These results suggest that LEPR, MC4R, PIK3C3 and VRTN may independently influence growth rate and fat deposition. Furthermore, the statistical models for predicting the breeding values of each trait had the lowest Akaike's information criterion values when considering the effect of the MC4R, LEPR, PIK3C3 and VRTN genotype simultaneously. These results suggest that LEPR, MC4R, PIK3C3 and VRTN are useful markers for accurately predicting breeding values in Duroc pigs. Show less

Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5 (-/-) cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors. Show less

Prion protein (PRNP) has been implicated in various types of neurodegenerative diseases. Although much is known about prion diseases, the function of cellular PRNP remains cryptic. Here, we show that PRNP mediates amyloid β1–42 (Aβ42)-induced autophagy activation through its interaction with BECN1. Treatment with Aβ42 enhanced autophagy flux in neuronal cells. Aβ42-induced autophagy activation, however, was impaired in prnp-knockout primary cortical neurons and Prnp-knockdown or prnp-knockout neuronal cells. Immunoprecipitation assays revealed that PRNP interacted with BECN1 via the BCL2-binding domain of BECN1. This interaction promoted the subcellular localization of BECN1 into lipid rafts of the plasma membrane and enhanced activity of PtdIns3K (whose catalytic subunit is termed PIK3C3, mammalian ortholog of yeast VPS34) in lipid rafts by generating PtdIns3P in response to Aβ42. Further, the levels of lipid rafts that colocalized with BECN1, decreased in the brains of aged C57BL/6 mice, as did PRNP. These results suggested that PRNP interacts with BECN1 to recruit the PIK3C3 complex into lipid rafts and thus activates autophagy in response to Aβ42, defining a novel role of PRNP in the regulation of autophagy. Show less

The mammalian homolog of yeast Vps34 (PIK3C3/VPS34) is implicated in the regulation of autophagy, and recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of renal glomerular podocytes. To date, however, the role of PIK3C3 in podocytes has remained unknown. We generated a line of podocyte-specific Pik3c3-knockout (Pik3c3 (pdKO) /mVps34 (pdKO) ) mice and demonstrated an indispensable role for PIK3C3 in the regulation of intracellular vesicle trafficking and processing to protect the normal cellular metabolism, structure and function of podocytes. Show less

Autophagy is a cell 'self-digestion' pathway involving the synthesis, trafficking and delivery of autophagosomes to lysosomes for degradation. Beclin 1 is a core component of the class III phosphatidylinositol 3-kinase (PI3K-III) complex, which plays an important role in membrane trafficking and restructuring involved in autophagy, endocytosis, cytokinesis and phagocytosis. To date Beclin 1 has largely been characterized in the context of autophagy; it modulates the lipid kinase activity of PI3K-III catalytic unit VPS34, which generates phosphatidylinositol 3-phosphate (PI(3)P), enabling the recruitment of a number of autophagy proteins involved in the nucleation of the autophagosome. Beclin 1 seems to function as an adaptor for recruiting multiple proteins that modulate VPS34. The recent identification of Beclin 1 protein modifications has shed light on its regulation in autophagy, and the discovery of non-autophagy functions of Beclin 1 has expanded our view of Beclin 1's involvement in tissue homeostasis and human diseases. Show less

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase. Show less

The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation. Show less

Since RNA expression differences have been reported in autism spectrum disorder (ASD) for blood and brain, and differential alternative splicing (DAS) has been reported in ASD brains, we determined if there was DAS in blood mRNA of ASD subjects compared to typically developing (TD) controls, as well as in ASD subgroups related to cerebral volume. RNA from blood was processed on whole genome exon arrays for 2-4-year-old ASD and TD boys. An ANCOVA with age and batch as covariates was used to predict DAS for ALL ASD (n=30), ASD with normal total cerebral volumes (NTCV), and ASD with large total cerebral volumes (LTCV) compared to TD controls (n=20). A total of 53 genes were predicted to have DAS for ALL ASD versus TD, 169 genes for ASD_NTCV versus TD, 1 gene for ASD_LTCV versus TD, and 27 genes for ASD_LTCV versus ASD_NTCV. These differences were significant at P <0.05 after false discovery rate corrections for multiple comparisons (FDR <5% false positives). A number of the genes predicted to have DAS in ASD are known to regulate DAS (SFPQ, SRPK1, SRSF11, SRSF2IP, FUS, LSM14A). In addition, a number of genes with predicted DAS are involved in pathways implicated in previous ASD studies, such as ROS monocyte/macrophage, Natural Killer Cell, mTOR, and NGF signaling. The only pathways significant after multiple comparison corrections (FDR <0.05) were the Nrf2-mediated reactive oxygen species (ROS) oxidative response (superoxide dismutase 2, catalase, peroxiredoxin 1, PIK3C3, DNAJC17, microsomal glutathione S-transferase 3) and superoxide radical degradation (SOD2, CAT). These data support differences in alternative splicing of mRNA in blood of ASD subjects compared to TD controls that differ related to head size. The findings are preliminary, need to be replicated in independent cohorts, and predicted alternative splicing differences need to be confirmed using direct analytical methods. Show less

Phosphatidylinositol phosphates are key regulators of vesicle identity, formation and trafficking. In mammalian cells, the evolutionarily conserved class III PtdIns 3-kinase PIK3C3/VPS34 is part of a large multiprotein complex that catalyzes the localized phosphorylation of phosphatidylinositol to phosphatidylinositol-3-phosphate (PtdIns3P). We demonstrate that PIK3C3 has a key function in vesicular trafficking, endocytosis and autophagosome-autolysosome formation in the highly specialized glomerular podocytes. Show less

Degradation of cytoplasmic material by autophagy plays a key role in protein homeostasis and metabolic control, as well as in the removal of intracellular protein aggregates, pathogens and damaged organelles. The concept of up- or down-regulating this pathway pharmacologically in neurodegenerative diseases, infections, inflammation and cancer is therefore attractive. Among the key pharmacological targets in regulation of autophagy are the phosphoinositide 3-kinases (PI3Ks), which mediate the phosphorylation of phosphatidylinositol (PtdIns) or PtdIns 4,5-bisphosphate in the 3-position of the (phospho)inositol headgroup. The catalytic products, PtdIns 3-phosphate (PtdIns3P) and PtdIns 3,4,5-trisphosphate [PtdIns(3,4,5)P3 ], respectively, have opposing roles in autophagy. PtdIns3P, the product of class II and III PI3Ks, mediates the recruitment of specific autophagic effectors to the sites of origin of autophagic membranes and thereby plays an essential role in canonical autophagy. By contrast, PtdIns(3,4,5)P3 , the product of class I PI3Ks, triggers the target of rapamycin signalling pathway, which inhibits autophagy. In this review, we discuss the functions of class I, II and III PI3Ks in autophagy and describe the protein effectors of PtdIns3P and PtdIns(3,4,5)P3 that promote or inhibit autophagy, respectively. We also provide examples of how PI3K-mediated control of autophagy is relevant to an understanding of tumour suppression and progression. Show less

The accumulation of amyloid-β-containing neuritic plaques and intracellular tau protein tangles are key histopathological hallmarks of Alzheimer's disease (AD). This type of pathology clearly indicates that the mechanisms of neuronal housekeeping and protein quality control are compromised in AD. There is mounting evidence that the autophagosome-lysosomal degradation is impaired, which could disturb the processing of APP and provoke AD pathology. Beclin 1 is a molecular platform assembling an interactome with stimulating and suppressive components which regulate the initiation of the autophagosome formation. Recent studies have indicated that the expression Beclin 1 is reduced in AD brain. Moreover, the deficiency of Beclin 1 in cultured neurons and transgenic mice provokes the deposition of amyloid-β peptides whereas its overexpression reduces the accumulation of amyloid-β. There are several potential mechanisms, which could inhibit the function of Beclin 1 interactome and thus impair autophagy and promote AD pathology. The mechanisms include (i) reduction of Beclin 1 expression or its increased proteolytic cleavage by caspases, (ii) sequestration of Beclin 1 to non-functional locations, such as tau tangles, (iii) formation of inhibitory complexes between Beclin 1 and antiapoptotic Bcl-2 proteins or inflammasomes, (iv) interaction of Beclin 1 with inhibitory neurovirulent proteins, e.g. herpex simplex ICP34.5, or (v) inhibition of the Beclin 1/Vps34 complex through the activation of CDK1 and CDK5. We will shortly introduce the function of Beclin 1 interactome in autophagy and phagocytosis, review the recent evidence indicating that Beclin 1 regulates autophagy and APP processing in AD, and finally examine the potential mechanisms through which Beclin 1 dysfunction could be involved in the pathogenesis of AD. Show less

Evasion of apoptosis, which enables cells to survive and proliferate under metabolic stress, is one of the hallmarks of cancer. We have recently reported that SH3GLB1/Bif-1 functions as a haploinsufficient tumor suppressor to prevent the acquisition of apoptosis resistance and malignant transformation during Myc-driven lymphomagenesis. SH3GLB1 is a membrane curvature-inducing protein that interacts with BECN1 though UVRAG and regulates the post-Golgi trafficking of membrane-integrated ATG9A for autophagy. At the premalignant stage, allelic loss of Sh3glb1 enhances Myc-induced chromosomal instability and results in the upregulation of anti-apoptotic proteins, including MCL1 and BCL2L1. Notably, we found that Sh3glb1 haploinsufficiency increases mitochondrial mass in overproliferated prelymphomatous Eμ-Myc cells. Moreover, loss of Sh3glb1 suppresses autophagy-dependent mitochondrial clearance (mitophagy) in PARK2/Parkin-expressing mouse embryonic fibroblasts (MEFs) treated with the mitochondrial uncoupler CCCP. Interestingly, PARK2-expressing Sh3glb1-deficient cells accumulate ER-associated immature autophagosome-like structures after treatment with CCCP. Taken together, we propose a model of mitophagy in which SH3GLB1 together with the class III phosphatidylinositol 3-kinase complex II (PIK3C3CII) (PIK3R4-PIK3C3-BECN1-UVRAG) regulates the trafficking of ATG9A-containing Golgi-derived membranes (A9(+)GDMs) to damaged mitochondria for autophagosome formation to counteract oncogene-driven tumorigenesis. Show less

The class III phosphatidylinositol (PtdIns)-3 kinase, PIK3C3/VPS34, forms multiple complexes and regulates a variety of cellular functions, especially in intracellular vesicle trafficking and autophagy. Even though PtdIns3P, the product of PIK3C3, is thought to be a critical membrane marker for the autophagosome, it is unclear how PIK3C3 is regulated in response to autophagy-inducing stimuli. A complexity of PIK3C3 biology is due in part to the existence of multiple complexes, of which the ATG14- or UVRAG-containing complexes play important roles in autophagy. We recently discovered differential regulation of distinct PIK3C3 complexes in response to energy starvation and showed a mechanism by which AMPK directly phosphorylates PIK3C3 and BECN1 to regulate non- and pro-autophagic PIK3C3 complexes, respectively. Show less

The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knock-out mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knock-out mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but it leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively. Show less

Fetal alcohol spectrum disorders (FASD) results from ethanol exposure to the developing fetus and is the leading cause of mental retardation. FASD is associated with a broad range of neurobehavioral deficits which may be mediated by ethanol-induced neurodegeneration in the developing brain. An immature brain is more susceptible to ethanol neurotoxicity. We hypothesize that the enhanced sensitivity of the immature brain to ethanol is due to a limited capacity to alleviate cellular stress. Using a third trimester equivalent mouse model of ethanol exposure, we demonstrated that subcutaneous injection of ethanol induced a wide-spread neuroapoptosis in postnatal day 4 (PD4) C57BL/6 mice, but had little effect on the brain of PD12 mice. We analyzed the expression profile of genes regulating apoptosis, and the pathways of ER stress response (also known as unfolded protein response, UPR) and autophagy during these ethanol-sensitive and resistant periods (PD4 versus PD12) using PCR microarray. The expression of pro-apoptotic genes, such as caspase-3, was much higher on PD4 than PD12; in contrast, the expression of genes that regulate UPR and autophagy, such as atf6, atg4, atg9, atg10, beclin1, bnip3, cebpb, ctsb, ctsd, ctss, grp78, ire1α, lamp, lc3 perk, pik3c3, and sqstm1 was significantly higher on PD12 than PD4. These results suggest that the vulnerability of the immature brain to ethanol could result from high expression of pro-apoptotic proteins and a deficiency in the stress responsive system, such as UPR and autophagy. Show less

The mitogen-activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here, we report the discovery of irreversible inhibitors of JNK1/2/3. We describe two JNK3 cocrystal structures at 2.60 and 2.97 Å resolution that show the compounds form covalent bonds with a conserved cysteine residue. JNK-IN-8 is a selective JNK inhibitor that inhibits phosphorylation of c-Jun, a direct substrate of JNK, in cells exposed to submicromolar drug in a manner that depends on covalent modification of the conserved cysteine residue. Extensive biochemical, cellular, and pathway-based profiling establish the selectivity of JNK-IN-8 for JNK and suggests that the compound will be broadly useful as a pharmacological probe of JNK-dependent signal transduction. Potential lead compounds have also been identified for kinases, including IRAK1, PIK3C3, PIP4K2C, and PIP5K3. Show less

A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34(f/f) mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34(f/f) mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34(f/f) mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function. Show less

Metastasis is the spread of cancer cells from their primary location to other parts of the body. Metastatic cancer is responsible for most cancer deaths. Increasing evidence indicates that epithelial-mesenchymal transition (EMT), a crucial developmental program, contributes to control cancer invasion and metastasis. We recently reported that death effector domain-containing DNA-binding protein (DEDD), a key effector molecule for cell death signaling receptors, attenuates EMT and acts as an endogenous suppressor of tumor growth and metastasis. We found that DEDD physically interacts with the class III PtdIns 3-kinase complex containing PIK3C3 and BECN1, which controls critical aspects of autophagy; this interaction activates autophagy and induces the autophagy-mediated lysosomal degradation of SNAI/Snail and TWIST, two master inducers of the EMT process. Further study reveals that the DEDD-PIK3C3 interaction can support the stability of PIK3C3 to maintain autophagic activity and promote the degradation of SNAI and TWIST. Our finding indicates that DEDD is a prognostic marker and a potential therapeutic target for the prevention and treatment of cancer metastasis. Moreover, regulation of the DEDD-PIK3C3 interaction may serve as an entry point to translate modifiers of this interaction into clinical endpoints. Show less

PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function. Show less

The mammary gland is key to all mammal species; in particular in multiparous species like pigs the number and the shape of functional mammary gland complexes are major determinants of fitness. Accordingly, we aimed to catalog the genes relevant to mammogenesis in pigs. Moreover, we aimed to address the hypothesis that the extent and timing of proliferation, differentiation, and maturation processes during prenatal development contribute to postnatal numerical, morphological and functional properties of the mammary gland. Thus we focused on differentially expressed genes and networks relevant to mammary complex development in two breeds that are subject to different selection pressure on number, shape and function of teats and show largely different prevalence of non-functional inverted teats. The expression patterns of fetal mammary complexes obtained at 63 and 91 days post conception (dpc) from German Landrace (GL) and Pietrain (PI) were analyzed by Affymetrix GeneChip Porcine Genome Arrays. The expression of 11,731 probe sets was analysed between the two stages within and among breeds. The analysis showed the largest distinction of samples of the breed GL at 63 dpc from all other samples. According to Ingenuity Pathways Analysis transcripts with abundance at the four comparisons made (GL63-GL91, PI63-PI93, GL63-PI63 and GL91-PI91) were predominantly assigned to biofunctions relevant to 'cell maintenance, proliferation, differentiation and replacement', 'organismal, organ and tissue development' and 'genetic information and nucleic acid processing'. Moreover, these transcripts almost exclusively belong to canonical pathways related to signaling rather than metabolic pathways. The accumulation of transcripts that are up-regulated in GL compared to PI indicate a higher proliferating activity in GL, whereas processes related to differentiation, maturation and maintenance of cells are more prominent in PI. Differential expression was validated by quantitative RT-PCR of five genes (GAB1, MAPK9, PIK3C2B, PIK3C3 and PRKCH) that are involved in several relevant signaling pathways. The results indicate that mammary complex development in PI precedes GL. The differential expression between the two breeds at fetal stages likely reflects the prenatal initiation of postnatal phenotypes concerning the number and shape as well as functionality of teats. Show less

Rare variants are believed to play an important role in disease etiology. Recent advances in high-throughput sequencing technology enable investigators to systematically characterize the genetic effects of both common and rare variants. We introduce several approaches that simultaneously test the effects of common and rare variants within a single-nucleotide polymorphism (SNP) set based on logistic regression models and logistic kernel machine models. Gene-environment interactions and SNP-SNP interactions are also considered in some of these models. We illustrate the performance of these methods using the unrelated individuals data from Genetic Analysis Workshop 17. Three true disease genes (FLT1, PIK3C3, and KDR) were consistently selected using the proposed methods. In addition, compared to logistic regression models, the logistic kernel machine models were more powerful, presumably because they reduced the effective number of parameters through regularization. Our results also suggest that a screening step is effective in decreasing the number of false-positive findings, which is often a big concern for association studies. Show less

Phosphoinositide-3-kinase, class III (PIK3C3) is a member of the phosphoinosite-3-kinases family, involved in cell signaling, membrane trafficking, and neurodevelopment. Previous studies have indeed shown an association between PIK3C3 gene variants and both bipolar disorder (BD) and schizophrenia (SZ). Brain-derived neurotrophic factor (BDNF) is a neurodevelopmental factor, which can regulate the PI3K signaling pathway. Associations have been reported between BDNF gene polymorphisms and affective and psychotic disorders. The aim of the present study was to replicate an association between PIK3C3 and BDNF gene variants in SZ and BD and a putative epistasis between the two genes. Patients meeting the DSM-IV criteria of BD and SZ were included in this study (98 BD and 79 SZ) as well as 158 healthy controls. Blood DNA was extracted and genotyping was performed either by the polymerase chain reaction (PCR) technique followed by enzymatic digestion or by the high-resolution melt (HRM) method. Genotype and haplotype association was assessed with the UNPHASED statistical program.The results showed one nominal association with BD (P < 0.02) and two risk haplotypes in both SZ (P < 0.001) and BP (P < 0.0005), which survived multiple testing correction. A modest interaction between a BDNF variant and PI3KC3 polymorphism was observed (P < 0.04).These preliminary results confirm the genetic association of PI3K gene variants with both SZ and BD, and support the hypothesis that SZ and BD share a genetic background. Show less

The Pik3c3 gene encodes an 887 amino acid lipid kinase, phosphoinositide-3-kinase class 3 (PIK3C3). PIK3C3 is known to regulate various intracellular membrane trafficking events. However, little is known about its functions during early embryogenesis in mammals. To investigate the function of PIK3C3 in vivo, we generated Pik3c3 null mice. We show here that Pik3c3 heterozygous are normal and fertile. In contrast, Pik3c3 homozygous mutants are embryonic lethal and die between E7.5 and E8.5 of embryogenesis. Mutant embryos are poorly developed with no evidence of mesoderm formation, and suffer from severely reduced cell proliferations. Cell proliferation defect is also evident in vitro, where mutant blastocysts in culture fail to give rise to typical colonies formed by inner cell mass. Electron microscopic analysis revealed that epiblast cells in mutant embryos appear normal, whereas the visceral endoderm cells contain larger vesicles inside the lipid droplets. Finally, we provide evidence that mTOR signaling is drastically reduced in Pik3c3 null embryos, which could be a major contributor to the observed proliferation and embryogenesis defects. Show less

A C↔T single nucleotide polymorphism (SNP) on exon 24 of the porcine class 3 phosphoinositide-3-kinase (PIK3C3) gene is considered a possible genetic marker for selecting backfat (BF) thickness and carcass fat, although only one study has published results on its effects by performing experiments on a single resource family. We analyzed the association of this PIK3C3 polymorphism with production traits in 739 Duroc pigs. The C allele frequency was 67.9% in our study population. PIK3C3 polymorphism showed significant effects on average daily weight gain (ADG), BF thickness, intermuscular fat content (IMF), and the size of the loin eye muscle area (EMA). The C alleles increased ADG, BF and IMF, and decreased EMA. The predicted differences in traits between the homozygous pigs of the C and T alleles were 40 g/day for DG, 1.2 mm for BF, 0.44% for IMF, and 1.6 cm(2) for EMA. Furthermore, the statistical models for estimating the breeding values of each trait had lower Akaike's information criterion values when adding PIK3C3 genotype information. We therefore confirmed that the polymorphism in PIK3C3 (C2604T) has the potential to be a genetic marker for production traits in Duroc pigs. Show less

Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery. Our analyses reveal four core eukaryotic PIKs which are type III PIK4A and PIK4B, and at least one homolog each from PI3K (possibly PIK3C3 as the ancestor) and PIP5K families. We also applied evolutionary analyses to PIK disease ontology and drug discovery. Mutated PIK3CA are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. We found conservation of activating mutations of PIK3CA in paralogous isoforms suggesting specific functional constraints on these residues. By mapping published compound inhibition data (IC50s) onto a phylogeny of PI3Ks, type II PI4Ks and distantly related, MTOR, ATM, ATR and PRKDC kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. Finally, we extended the rationale for drugs targeting PIKs of malarial Plasmodium falciparum, and the parasites, Leishmania sp. and Trypanosoma sp. by identifying those PIKs highly divergent from human homologs. Our phylogenomic analysis of PIKs provides new insights into the evolution of second messenger signaling. We postulate two waves of PIK diversification, the first in metazoans with a subsequent expansion in cold-blooded vertebrates that was post-emergence of Deutrostomia\Chordata but prior to the appearance of mammals. Reconstruction of the evolutionary relationships among these lipid kinases also adds to our understanding of their roles in various diseases and assists in their development as potential drug targets. Show less

The lipid kinase PIK3C3 (also known as VPS34) regulates multiple aspects of endo-membrane trafficking processes. PIK3C3 is widely expressed by neurons in the CNS, and its catalytic product PI3P is enriched in dendritic spines. Here we generated a line of conditional mutant mouse in which Pik3c3 is specifically deleted in hippocampal and in small subsets of cortical pyramidal neurons using the CaMKII-Cre transgene. We found that Pik3c3-deficiency initially causes loss of dendritic spines accompanied with reactive gliosis, which is followed by progressive neuronal degeneration over a period of several months. Layers III and IV cortical neurons are more susceptible to Pik3c3-deletion than hippocampal neurons. Furthermore, in aged conditional Pik3c3 mutant animals, there are extensive gliosis and severe secondary loss of wild type neurons. Our analyses show that Pik3c3 is essential for CNS neuronal homeostasis and Pik3c3flox/flox; CaMKII-Cre mouse is a useful model for studying pathological changes in progressive forebrain neurodegeneration. Show less

Identification and characterization of kinase inhibitor potency and selectivity is often an iterative process in which a library of compounds is first screened against a single kinase, and hits from that screen are then profiled against other kinases to determine specificity. By developing kinase assays that employ either a terbium- or a europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) readout, one can take advantage of the distinct emission properties of these labels to develop assays for 2 kinases that can be performed simultaneously in the same well. This not only increases the information content provided per assay well but can immediately provide information on compound specificity. The authors have applied this strategy to the development of multiplexed assays for 2 examples systems: EGFR and IKKbeta, as well as lipid kinase family members mTOR and PIK3C3. They demonstrate the ability of these multiplexed assays to characterize selective kinase inhibitors in a dose-response mode, with no difference in results obtained from traditional single kinase assays performed separately. Show less

Phosphoinositide 3 kinase Class III (PIK3C3) or VPS34-Beclin 1 complex plays a key role in the autophagy-lysosome pathway. Previous identification of numerous binding partners for VPS34-Beclin 1 suggested a complex scheme of the autophagy control mechanism. Recent large-scale screening of autophagy network and signaling pathways in mammalian cells not only confirms the previous binding partners, but also reveals additional interactors and intricate connections of VPS34-Beclin 1 complex to other functional groups of autophagy, yielding a wealth of information that will direct future detailed study of the central control mechanism of autophagy mediated by VPS34-Beclin 1 and other regulators. Show less