Transcriptional role of cyclin D1 in development revealed by a “genetic-proteomic” screen

Author manuscript: 2010 September 22.Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers[superscript 1, 2]. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas—an organ that critically requires cyclin D1 function[superscript 3, 4]—cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1-/-) retinas. Transduction of an activated allele of Notch1 into Ccnd1-/- retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term ‘genetic–proteomic’, can be used to study the in vivo function of essentially any protein

Novel microwell-based spectrophotometric assay for determination of atorvastatin calcium in its pharmaceutical formulations

The formation of a colored charge-transfer (CT) complex between atorvastatin calcium (ATR-Ca) as a n-electron donor and 2, 3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as a π-electron acceptor was investigated, for the first time. The spectral characteristics of the CT complex have been described, and the reaction mechanism has been proved by computational molecular modeling. The reaction was employed in the development of a novel microwell-based spectrophotometric assay for determination of ATR-Ca in its pharmaceutical formulations. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient (0.9995) was found between the absorbance and the concentration of ATR-Ca in the range of 10-150 μg/well. The limits of detection and quantitation were 5.3 and 15.8 μg/well, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from the drugs that are co-formulated with ATR-Ca in its combined formulations. The assay was successfully applied to the analysis of ATR-Ca in its pharmaceutical dosage forms with good accuracy and precision. The assay described herein has great practical value in the routine analysis of ATR-Ca in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for ATR-Ca, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed

TRAF6 Autoubiquitination-Independent Activation of the NFκB and MAPK Pathways in Response to IL-1 and RANKL

The adapter protein TRAF6 is critical for mediating signal transduction from members of the IL-1R/TLR and TNFR superfamilies. The TRAF6 RING finger domain functions as an ubiquitin E3 ligase capable of generating non-degradative K63-linked ubiquitin chains. It is believed that these chains serve as docking sites for formation of signaling complexes, and that K63-linked autoubiquitination of TRAF6 is essential for formation and activation of a complex involving the kinase TAK1 and its adapters, TAB1 and TAB2. In order to assess independently the E3 ligase and ubiquitin substrate functions of TRAF6, we generated, respectively, RING domain and complete lysine-deficient TRAF6 mutants. We found that while the TRAF6 RING domain is required for activation of TAK1, it is dispensable for interaction between TRAF6 and the TAK1-TAB1-TAB2 complex. Likewise, lysine-deficient TRAF6 was found to interact with the TAK1-TAB1-TAB2 complex, but surprisingly was also found to be fully competent to activate TAK1, as well as NFκB and AP-1 reporters. Furthermore, lysine-deficient TRAF6 rescued IL-1-mediated NFκB and MAPK activation, as well as IL-6 elaboration in retrovirally-rescued TRAF6-deficient fibroblasts. Lysine-deficient TRAF6 also rescued RANKL-mediated NFκB and MAPK activation, and osteoclastogenesis in retrovirally-rescued TRAF6-deficient bone marrow macrophages. While incapable of being ubiquitinated itself, we demonstrate that lysine-deficient TRAF6 remains competent to induce ubiquitination of IKKγ/NEMO. Further, this NEMO modification contributes to TRAF6-mediated activation of NFκB. Collectively, our results suggest that while TRAF6 autoubiquitination may serve as a marker of activation, it is unlikely to underpin RING finger-dependent TRAF6 function

Genome-Wide Analysis of Müller Glial Differentiation Reveals a Requirement for Notch Signaling in Postmitotic Cells to Maintain the Glial Fate

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10–14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle

Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors Restores Retinal Structure and Function in Degenerative Mice

This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs), could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease

Notch and Wnt Signaling Mediated Rod Photoreceptor Regeneration by Müller Cells in Adult Mammalian Retina

Background: Evidence emerging from a variety of approaches used in different species suggests that Müller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched Müller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells. Methodology/Principal Findings: We demonstrate that Notch and Wnt signaling activate Müller cells through their canonical pathways and that a rare subset of activated Müller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated Müller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate Müller cells. The number of activated Müller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception. Conclusion/Significance: In summary, our results provide a proof of principle for non-neurotoxin-mediated activation o

Overactivation of Notch1 Signaling Induces Ectopic Hair Cells in the Mouse Inner Ear in an Age-Dependent Manner

Background: During mouse inner ear development, Notch1 signaling first specifies sensory progenitors, and subsequently controls progenitors to further differentiate into either hair cells (HCs) or supporting cells (SCs). Overactivation of NICD (Notch1 intracellular domain) at early embryonic stages leads to ectopic HC formation. However, it remains unclear whether such an effect can be elicited at later embryonic or postnatal stages, which has important implications in mouse HC regeneration by reactivation of Notch1 signaling. Methodology/Principal Findings: We performed comprehensive in vivo inducible overactivation of NICD at various developmental stages. In CAG CreER+; Rosa26-NICD loxp/+ mice, tamoxifen treatment at embryonic day 10.5 (E10.5) generated ectopic HCs in the non-sensory regions in both utricle and cochlea, whereas ectopic HCs only appeared in the utricle when tamoxifen was given at E13. When tamoxifen was injected at postnatal day 0 (P0) and P1, no ectopic HCs were observed in either utricle or cochlea. Interestingly, Notch1 signaling induced new HCs in a non-cell-autonomous manner, because the new HCs did not express NICD. Adjacent to the new HCs were cells expressing the SC marker Sox10 (either NICD+ or NICDnegative). Conclusions/Significance: Our data demonstrate that the developmental stage determines responsiveness of embryonic otic precursors and neonatal non-sensory epithelial cells to NICD overactivation, and that Notch 1 signaling in the wild type, postnatal inner ear is not sufficient for generating new HCs. Thus, our genetic mouse model is suitable to test additiona

Finding the engram.

Many attempts have been made to localize the physical trace of a memory, or engram, in the brain. However, until recently, engrams have remained largely elusive. In this Review, we develop four defining criteria that enable us to critically assess the recent progress that has been made towards finding the engram. Recent \u27capture\u27 studies use novel approaches to tag populations of neurons that are active during memory encoding, thereby allowing these engram-associated neurons to be manipulated at later times. We propose that findings from these capture studies represent considerable progress in allowing us to observe, erase and express the engram

CD133+ adult human retinal cells remain undifferentiated in Leukaemia Inhibitory Factor (LIF)

<p>Abstract</p> <p>Background</p> <p>CD133 is a cell surface marker of haematopoietic stem and progenitor cells. Leukaemia inhibitory factor (LIF), sustains proliferation and not differentiation of embryonic stem cells. We used CD133 to purify adult human retinal cells and aimed to determine what effect LIF had on these cultures and whether they still had the ability to generate neurospheres.</p> <p>Methods</p> <p>Retinal cell suspensions were derived from adult human post-mortem tissue with ethical approval. With magnetic automated cell sorting (MACS) CD133⁺retinal cells were enriched from post mortem adult human retina. CD133⁺retinal cell phenotype was analysed by flow cytometry and cultured cells were observed for proliferative capacity, neuropshere generation and differentiation with or without LIF supplementation.</p> <p>Results</p> <p>We demonstrated purification (to 95%) of CD133⁺cells from adult human postmortem retina. Proliferating cells were identified through BrdU incorporation and expression of the proliferation markers Ki67 and Cyclin D1. CD133⁺retinal cells differentiated whilst forming neurospheres containing appropriate lineage markers including glia, neurons and photoreceptors. LIF maintained CD133⁺retinal cells in a proliferative and relatively undifferentiated state (Ki67, Cyclin D1 expression) without significant neurosphere generation. Differentiation whilst forming neurospheres was re-established on LIF withdrawal.</p> <p>Conclusion</p> <p>These data support the evidence that CD133 expression characterises a population of cells within the resident adult human retina which have progenitor cell properties and that their turnover and differentiation is influenced by LIF. This may explain differences in retinal responses observed following disease or injury.</p