Cullin-4 Regulates Wingless and JNK Signaling-Mediated Cell Death in the Drosophila Eye.

In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. Here we show that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. We found that Wingless (Wg) and c-Jun-amino-terminal-(NH2)-Kinase (JNK) signaling are ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. Here we present evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, our studies provide insights into a novel role of cul-4 in promoting cell survival in the developing Drosophila eye

Characterization of LP-Z Lipoprotein Particles and Quantification in Subjects with Liver Disease Using a Newly Developed NMR-Based Assay

Background: Lipoprotein particles with abnormal compositions, such as lipoprotein X (LP-X) and lipoprotein Z (LP-Z), have been described in cases of obstructive jaundice and cholestasis. The study objectives were to: (1) develop an NMR-based assay for quantification of plasma/serum LP-Z particles, (2) evaluate the assay performance, (3) isolate LP-Z particles and characterize them by lipidomic and proteomic analysis, and (4) quantify LP-Z in subjects with various liver diseases. Methods: Assay performance was assessed for linearity, sensitivity, and precision. Mass spectroscopy was used to characterize the protein and lipid content of isolated LP-Z particles. Results: The assay showed good linearity and precision (2.5-6.3%). Lipid analyses revealed that LP-Z particles exhibit lower cholesteryl esters and higher free cholesterol, bile acids, acylcarnitines, diacylglycerides, dihexosylceramides, lysophosphatidylcholines, phosphatidylcholines, triacylglycerides, and fatty acids than low-density lipoprotein (LDL) particles. A proteomic analysis revealed that LP-Z have one copy of apolipoprotein B per particle such as LDL, but less apolipoprotein (apo)A-I, apoC3, apoA-IV and apoC2 and more complement C3. LP-Z were not detected in healthy volunteers or subjects with primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, or type 2 diabetes. LP-Z were detected in some, but not all, subjects with hypertriglyceridemia, and were high in some subjects with alcoholic liver disease. Conclusions: LP-Z differ significantly in their lipid and protein content from LDL. Further studies are needed to fully understand the pathophysiological reason for the enhanced presence of LP-Z particles in patients with cholestasis and alcoholic liver disease

Activation of JNK Signaling Mediates Amyloid-ß-Dependent Cell Death

Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of Aß42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood.We employed a Drosophila eye model of AD to study how Aß42 causes cell death. Misexpression of higher levels of Aß42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. We found that blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH(2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of Aß42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of Aß42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in Aß42 retina as compared to the wild-type retina. When we blocked both caspases and JNK signaling simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone.Our data suggests that (i) accumulation of Aß42 plaques induces JNK signaling in neurons and (ii) induction of JNK contributes to Aß42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies

Characterization of Microtubule Depolymerization by the HIV Protein Rev

The HIV-1 Rev protein enables the nucleocytoplasmic export of unspliced or partially spliced mRNAs that is required for the synthesis of structural proteins. By doing so, it regulates the switch to the late phase of the viral replication cycle (Cullen, 1992). This regulatory control over viral replication makes Rev an attractive target for anti-viral intervention. The development of anti-viral remedies is hindered because the three-dimensional structure of Rev has not yet been solved by X-ray crystallography and NMR. Rev, which polymerizes into regular hollow filaments at high concentrations, forms side-to-side and end-to-side interactions making it prone to aggregation and precipitation (Wingfield et al., 1991). Watts et al. (2000) in an attempt to solve the solubility of Rev discovered a novel interaction between Rev and tubulin. They observed that Rev filaments react with microtubules (MTs) to form Rev-tubulin toroidal (RTT) complexes showing that Rev is a microtubule depolymerizing agent that possibly mimics the mechanism used by Kinesin-13 proteins, themselves potent microtubule depolymerases. The first goal of the experiments conducted here was to develop a sedimentation assay capable of measuring Rev stimulated microtubule depolymerization. Under the conditions employed here, Rev tubulin toroidal complexes (RTTs) were not formed due to limiting concentrations of magnesium ions so that the amount of tubulin released from microtubule polymers would not reform high molecular weight complexes that would sediment in our assays. Initial experiments determined that bacterial expressed Rev was capable of depolymerizing GMPCPP stabilized microtubules. Depolymerization was not affected by the oligomeric state of Rev. Rev polymerized into filaments or maintained as monomers by the addition of high salt concentrations were equally able to depolymerize microtubules. Microtubule depolymerization appears to be partially dose dependent and occurs at concentrations as low as 300 nM. At low concentrations of Rev, more tubulin is released from the microtubule polymer than there is Rev present in the reaction. This suggests that Rev either has higher affinity for microtubule ends in the lattice or that Rev multimerization is important for depolymerization activity. Depolymerization occurs quickly which is consistent with the findings of Watts et al. (2000). In contrast to the findings of Watts et al. (2000) who demonstrated a complete disappearance of Taxol stabilized microtubules when treated with Rev, Rev was unable to completely depolymerize microtubule polymers stabilized by GMPCPP

Identification of Novel Ligands and Structural Requirements for Heterodimerization of the Liver X Receptor Alpha

LXRs, LXRa (NR1H3) and LXRß (NR1H2), are ligand-activated transcription factors that are members of the nuclear receptor superfamily. Oxysterols and nonsteroidal synthetic compounds bind directly to LXRs and influence the expression of LXR dependent genes. The use of murine models and LXR-selective agonists have established the important role of LXRs as sterol sensors that govern the absorption, transport, and catabolism of cholesterol. Upon activation, these receptors have been shown to increase reverse cholesterol transport from the macrophage back to the liver to aid in the removal of excess cholesterol. Not surprisingly, LXR dysregulation is a feature of several human diseases, including metabolic syndrome. Due to their roles in the regulation of lipid and cholesterol metabolism, LXRs are potentially attractive pharmaceutical targets. As ligand binding and dimerization play pivotal roles in modulating LXR activity, the identification of novel ligands and requirements for LXR dimerization can potentially aid the drug development process. Herein, using a variety of biophysical assays, including fluorescence based assays coupled with in silico molecular modeling, I have identified medium chain fatty acids and/or their metabolites as the novel endogenous agonists of LXRa. There is mounting evidence that ligand induced dimerization regulates the transcriptional output of nuclear receptors. Thus, it is important to identify factors that modulate protein-protein interactions. This work demonstrated that (a) LXRa binds PPARa with a high affinity (low nanomolar concentration), (b) ligands for LXRa alter the binding dissociation constant values of LXRa-PPARa interaction, and (c) ligand binding induces conformational changes in the dimer secondary structure. Furthermore, site-directed mutagenesis investigated the strength of individual contributions of residues located in the ligand binding domain to dimerization and transactivation properties of LXRa. Data herein highlight the importance of hydrophobic interactions and salt bridges at the interface, and suggest that key interface residues are required for the ligand-dependent activation of LXRa in a promoter specific manner. Mutagenesis of LXRa L414 to an arginine revealed the importance of this site in dimerization, specifically with RXRa. This work showed that this particular mutation specifically abolished dimerization with RXRa. Taken together, this study provided insights into the functional roles of fatty acids as novel LXRa ligands and the effects mutations may have in modulating molecular interactions and activity profile of LXRa

Hippo Signalling Controls Dronc Activity to Regulate Organ Size in Drosophila

The Hippo pathway controls organ size by multiple mechanisms that ultimately regulate the transcriptional co-activator Yorkie (Yki). Downregulation of Hippo signalling leads to tissue overgrowths due to Yki-mediated activation of target genes, whereas overexpression of the pathway triggers apoptosis in developing tissues. However, the mechanism underlying cell death induced by Hippo (Hpo)-activation is not understood. We found that overexpression of Hpo leads to induction of Dronc (Drosophila Caspase-9 homologue) expression and downregulation of dronc can suppress/block Hpo-mediated apoptosis. Furthermore, upregulation of Dronc activity strongly suppressed the overgrowth caused by Yki overexpression thereby suggesting that Hippo signalling restricts Dronc activity. Hippo-mediated cell death requires the activity of the initiator caspase Dronc. Loss-of-function of dronc in genetic mosaics leads to cell survival and increased cell proliferation in imaginal discs. dronc is transcriptionally suppressed in Yki overexpressing cells or cells mutant for other Hippo pathway components like warts (wts). We propose that Dronc is a transcriptional target of the Hippo signalling pathway. The Hippo–Dronc connection has implications in control of overall organ size and other growth regulatory mechanisms like compensatory proliferation and cell competition

Misexpression of Aß42 triggers cell death in the differentiating neurons.

<p>(A, A′) Wild-type third instar larval eye imaginal disc displaying randomly distributed TUNEL positive dying cells (A, A″) shown in red channel (arrow). TUNEL staining marks the fragmented DNA within the nuclei of dying cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Singh1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-White1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-McCall1" target="_blank">[61]</a>. (B) Wild-type adult eye. (C, C′) Misexpression of Aß42 (GMR>Aß42) in differentiating neurons of the eye show elevated levels of TUNEL positive cells (C′ arrows). The increased frequency of cell death in neurons can be directly correlated to the misexpression of the Aß42 peptide. Note that misexpression of Aß42 does not affect the differentiation process as the distribution of Elav positive cells is the same in both control and Aß42 third instar eye imaginal discs. (D) GMR>Aß42 results in a strong neurodegenerative phenotype in adult eye. Baculovirus P35 has been shown to block the caspase dependent cell death <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Hay1" target="_blank">[36]</a>. (E, E′, F) Misexpression of P35 along with Aß42 in differentiating neurons (GMR>Aß42+P35) shows significant reduction of dying cells based on number of TUNEL positive cells (red channel) in the larval eye field. However, this rescue is not as strong in (F) adult eye phenotype. (E′) Note that the eye field displays reduced number of TUNEL positive cells (arrow) compared to GMR>Aß42 eye field (C′). It is important to note that Aß42 peptide production is not affected. Elav marks the photoreceptor fate (C′″). Puckered (Puc), a dual phosphatase, is downstream target of JNK signaling pathway and forms a feedback loop to negatively regulate the pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Stronach1" target="_blank">[20]</a>. (G, G′, H) Misexpression of <i>puc</i> along with Aß42 in the differentiating neurons (GMR>Aß42+<i>puc</i>) results in significant suppression of cell death as evident from reduced number of TUNEL positive cells in the third instar larval eye imaginal disc as well as in the (H) adult eye. Note that there is a significant rescue of (D) GMR>Aß42 adult eye phenotype in (H) GMR>Aß42+<i>puc</i> background. These results suggest that JNK signaling might be responsible for neurodegeneration seen in amyloid plaque mediated cell death. (I) Quantification of the number of dying cells in eye imaginal discs based on TUNEL staining in wild-type (served as control), GMR>Aß42, GMR>Aß42+P35 and GMR>Aß42+<i>puc</i>. Note that blocking JNK signaling (GMR>Aß42+<i>puc</i>) exhibit strong rescue of the neurodegenerative phenotype of GMR>Aß42 and GMR>Aß42+P35. This rescue is significant (**) as seen by calculation of P-values based on one-tailed <i>t</i>-test using Microsoft Excel 2007.</p

JNK signaling is activated upon misexpression of Aß42 in the eye.

<p>(A) Schematic representation of hierarchy of Jun-kinase signaling pathway members. (B, B′) Wild-type expression of <i>puc</i> in the developing third instar larval eye imaginal disc using lacZ reporter where reporter (red channel) is restricted only to the developing photoreceptors in the eye imaginal disc proper and in the peripodial membrane cells on the margin of the antennal disc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-MartinBlanco1" target="_blank">[43]</a>. (C, C′) GMR>Aß42 eye imaginal disc exhibits ectopic upregulation of <i>puc</i>-lacZ reporter. (C′) Split channel showing ectopic <i>puc</i>-lacZ expression in the photoreceptor neurons of the eye imaginal disc. (D) Activation of JNK signaling in GMR>Aß42 was detected by checking phospho-Jun levels. Levels of JNK signaling pathway increases three fold in GMR>Aß42 as compared to the wild-type eye imaginal disc.</p

Ectopic upregulation of JNK signaling induces cell death in the GMR>Aß42 eye imaginal disc.

<p>(A) Wild-type eye imaginal disc showing cell death in random cells, which serve as controls. (B) GMR>Aß42 eye imaginal disc showing ectopic upregulation of <i>puc</i> lacZ in a large number of dying retinal cells as evident from TUNEL positive staining. In comparison to the (C) wild-type adult eye, (D) GMR>Aß42 adult eye are highly reduced due to neurodegeneration. (E–L) Increasing level of JNK signaling in GMR>Aß42 by misexpressing (F, H) activated <i>hemipterous</i> (GMR>Aß42+<i>hep^Act</i>) and (J, L) activated <i>Djun</i> (GMR>Aß42+<i>jun^aspv7</i>) results in (F, J) dramatic increase in dying cell population in the eye imaginal disc, leading to a (H, L) “no-eye” phenotype in the adult fly. Increased levels of (E, G) activated <i>hemipterous</i> (GMR><i>hep^Act</i>), (I, K) activated <i>Djun</i> (GMR><i>jun^aspv7</i>) served as controls and result in some dying cells in the (E, I) eye imaginal disc and (G, K) a small adult eye. However, reducing level of JNK signaling in GMR>Aß42 background by misexpressing (N, P) Dominant negative <i>basket</i> (GMR>Aß42+<i>bsk^DN</i>) results in significant reduction to near complete absence of dying cell population (N) in the eye imaginal disc, leading to a (P) strong rescue of the adult eye phenotype as compared to GMR>Aß42 adult eye. (M, O) Increased levels of Dominant negative <i>basket</i> (GMR><i>bsk^DN</i>) in (M) eye imaginal disc and (O) adult eye served as controls. Note that increased levels of dominant negative <i>basket</i> alone (GMR><i>bsk^DN</i>) does not affect the size of eye imaginal disc and the adult eye.</p

JNK signaling is responsible for cell death in GMR>Aß42 background.

<p>(A, A′) Misexpression of both P35 and <i>puc</i> along with Aß42 (GMR>Aß42+P35<i>+puc</i>) results in strong rescue of cell death as evident from (A′) dramatically reduced TUNEL positive cells. However, the rescue of the phenotype was not significantly stronger than with blocking JNK signaling pathway alone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone-0024361-g005" target="_blank">Figure 5G′</a>). (B) Misexpression of Aß42 (GMR>Aß42) in pupal retina showing cell death as evident from TUNEL positive cells (red channel). Blocking simultaneously both caspase-dependent cell death and caspase-independent JNK signaling mediated cell death in pupal retina (GMR>Aß42+P35<i>+puc</i>) showed a strong rescue in (C, C′, C″) pupal retina and (D) adult eye as compared to (B) GMR>Aß42 pupal retina, (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone-0024361-g003" target="_blank">Figure 3D</a>) GMR>Aß42 adult eye. The cell death is detected by TUNEL staining (red channel), which is (C′, C″) restricted to the periphery of the pupal retina. Note that dying cells on the periphery of the pupal retina corresponds to the programmed cell death as seen in the wild-type pupal retina too <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Brachmann1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024361#pone.0024361-Lin1" target="_blank">[28]</a>. (E) Quantification of the number of dying cells in eye imaginal discs based on TUNEL staining in different genetic combinations. The frequency of cell death in wild-type eye imaginal disc served as a control. Note that blocking JNK signaling (GMR>Aß42+<i>puc</i>) or blocking JNK signaling along with caspase–dependent cell death (GMR>Aß42+P35+<i>puc</i>) exhibit strong rescue of the neurodegenerative phenotype of GMR>Aß42. This rescue is significant (**) as seen by calculation of P-values based on one-tailed <i>t</i>-test using Microsoft Excel 2007.</p