Loss of mTOR signaling affects cone function, cone structure and expression of cone specific proteins without affecting cone survival

Cones are the primary photoreceptor (PR) cells responsible for vision in humans. They are metabolically highly active requiring phosphoinositide 3-kinase (PI3K) activity for long-term survival. One of the downstream targets of PI3K is the kinase mammalian target of rapamycin (mTOR), which is a key regulator of cell metabolism and growth, integrating nutrient availability and growth factor signals. Both PI3K and mTOR are part of the insulin/mTOR signaling pathway, however if mTOR is required for long-term PR survival remains unknown. This is of particular interest since deregulation of this pathway in diabetes results in reduced PR function before the onset of any clinical signs of diabetic retinopathy. mTOR is found in two distinct complexes (mTORC1 and mTORC2) that are characterized by their unique accessory proteins RAPTOR and RICTOR respectively. mTORC1 regulates mainly cell metabolism in response to nutrient availability and growth factor signals, while mTORC2 regulates pro-survival mechanisms in response to growth factors. Here we analyze the effect on cones of loss of mTORC1, mTORC2 and simultaneous loss of mTORC1 and mTORC2. Interestingly, neither loss of mTORC1 nor mTORC2 affects cone function or survival at one year of age. However, outer and inner segment morphology is affected upon loss of either complex. In contrast, concurrent loss of mTORC1 and mTORC2 leads to a reduction in cone function without affecting cone viability. The data indicates that PI3K mediated pro-survival signals diverge upstream of both mTOR complexes in cones, suggesting that they are independent of mTOR activity. Furthermore, the data may help explain why PR function is reduced in diabetes, which can lead to deregulation of both mTOR complexes simultaneously. Finally, although mTOR is a key regulator of cell metabolism, and PRs are metabolically highly active, the data suggests that the role of mTOR in regulating the metabolic transcriptome in healthy cones is minimal

Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells

Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme that targets immunoglobulin (Ig) genes to initiate class switch recombination (CSR) and somatic hypermutation (SHM)(1). Through off-target activity, however, AID has a much broader impact on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in lymphoma development and progression(2). AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation(3). The phosphatidylinositol 3-kinase (PI3K) δ pathway plays a key role in AID regulation by suppressing its expression in B cells(4). Novel drugs for leukemia or lymphoma therapy such as idelalisib, duvelisib or ibrutinib block PI3Kδ activity directly or indirectly(5–8), potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation (SHM) and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both these effects were completely abrogated in AID deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumors in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IgH and AID off-target sites in human chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased SHM in AID off-targets. In summary, we show that PI3Kδ or BTK inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism, an effect that should be carefully considered as such inhibitors are administered for years to patients

The BRAF pseudogene functions as a competitive endogenous RNA and induces lymphoma in vivo

SummaryResearch over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo “CDS” or “3′ UTR” develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer

Espressione e analisi funzionale dei geni Soul/HBP (Heme Binding Protein) durante lo sviluppo renale di Zebrafish

Le proteine Soul sono espresse nel corso di una vasta gamma di programmi cellulari inerenti allo sviluppo, compresa la differenziazione di cellule neuronali e eritroidi, dove fungono da trasportatori o amplificatori citosolici per l’eme e le porfirine. Due omologhi di geni Soul di zebrafish sono stati clonati. In tutti i cordati, messaggeri materni di Soul sono stati osservati nell’ooplasma, con crescente affinità per le membrane cellulari durante l’oogenesi. In embrioni di zebrafish, sia zSoul1 che zSoul2 sono trascritti nel sistema nervoso centrale (SNC), nei somiti e nelle cellule del sangue, mentre ogni gene si identifica specificamente nell'estensione del vitello (zSoul1) e nei dotti pronefrici (zSoul2). Le proteine Soul consistono di fattori tetrapirrolici con alta affinità per le porfirine e l’eme, ma il loro ruolo è ancora poco chiaro. E’ stato proposto che queste proteine possano fungere da amplificatori citosolici contro l'intossicazione delle cellule dai prodotti del ferro (Taketani et al., 1998; Zylka e Reppert, 1999; Blackmon et al., 2002; Sato et al., 2004, Babusiak et al., 2005). Recentemente è stato dimostrato come il peptide derivante dal “cleavage” della proteina umana HBP1/SOUL1, funga da chemioattrattivo naturale per le cellule dendritiche e i monociti (Migeotte et al.,2006), mentre altri dati suggeriscono che, oltre a legare l’eme, HBP2/SOUL2 promuove la morte cellulare inibendo il potenziale di membrana mitocondriale (Szigeti et al., 2006). Omologhi di SOUL/HBP sono presenti dagli archeobatteri al riso ed all'uomo, e tutti mostrano un alto livello di identità (Oliveira et al., 1995; Taketani et al., 1998; Zylka e Reppert, 1999; Blackmon et al., 2002; nostri dati). Le proteine SOUL/HBP sono coinvolte in una gamma di processi embrionali, cioè oogenesi, come nel caso dell'omologo dell'insetto, RHBP (Oliveira et al., 1995; Braz et al., 2001). Fin qui, la dinamica spazio-temporale dell'espressione dei gene Soul durante l'embriogenesi è poco chiara. Per studiare la dinamica dei fattori SOUL/HBP, abbiamo clonato e cercato di caratterizzare i modelli strutturali ed embrionali di due ortologhi di Soul in zebrafish. Le informazioni sulle dinamiche trascrizionali sono state evinte via RT-PCR e ibridazione in situ dall’ oogenesis a 3 giorni di sviluppo. I rapporti filogenetici dedotti dal confronto di tutti gli omologhi di entrambi i geni, suggeriscono che la nomenclatura corrente andrebbe riorganizzata come segue: SOUL (sensu Zylka e Reppert, 1999) in Soul1 e p22 HBP in Soul2. Entrambi i geni sono di origine materna. Durante la tarda somitogenesi, Soul1 è espresso nell'estensione del vitello, nei precursori delle cellule eritroidi, nel sistema nervoso centrale e nei somiti posteriori. Allo stesso stadio di sviluppo, trascritti di Soul2 sono stati rilevati nei dotti pronefrici, nei precursori delle cellule eritroidi, nei somiti e nel sistema nervoso centrale. In conclusione, domini comuni e complementari di espressione dei geni Soul durante l’ematopoiesi e la nefrogenesi dello zebrafish rispettivamente, così come nel sistema nervoso centrale e nei somiti, sostengono l'ipotesi di interazioni funzionali

Retinal gene delivery by rAAV and DNA electroporation

Ocular gene therapy is a fast-growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged and easily accessible, and direct viral delivery results primarily in local infection. Because the eye is not a vital organ, mutations in eye-specific genes tend to be more common. To date, over 40 eye-specific genes have been identified harboring mutations that lead to blindness. Gene therapy with recombinant adeno-associated virus (rAAV) holds the promise to treat patients with such mutations. However, proof-of-concept and safety evaluation for gene therapy remains to be established for most of these diseases. This unit describes the in vivo delivery of genes to the mouse eye by rAAV-mediated gene transfer and plasmid DNA electroporation. Advantages and limitations of these methods are discussed, and detailed protocols for gene delivery, required materials, and subsequent tissue processing methods are described

MOESM4 of Expression of meis and hoxa11 in dipnoan and teleost fins provides new insights into the evolution of vertebrate appendages

Additional file 4: Fig. 4 meis3 expression in developing pectoral fins of Neoceratodus. Transversal sections of in situ results at st. 42 and 47

MOESM2 of Expression of meis and hoxa11 in dipnoan and teleost fins provides new insights into the evolution of vertebrate appendages

Additional file 2: Fig. 2 Alignment and phylogenetic analysis of Neoceratodus proteins. Partial protein-translated alignment and molecular phylogeny in ortholog identification of Neoceratodus meis1, meis3 and hoxa11

MOESM3 of Expression of meis and hoxa11 in dipnoan and teleost fins provides new insights into the evolution of vertebrate appendages

Additional file 3: Fig. 3 Position of lungfish riboprobes. WISH riboprobes spanning mature mRNAs

MOESM5 of Expression of meis and hoxa11 in dipnoan and teleost fins provides new insights into the evolution of vertebrate appendages

Additional file 5: Fig. 5 hoxa11 expression in lungfish. In situ results in nervous system, tail and digestive tract