JNK Signaling Confers Tolerance to Oxidative Stress and Extends Lifespan in Drosophila

AbstractChanges in the genetic makeup of an organism can extend lifespan significantly if they promote tolerance to environmental insults and thus prevent the general deterioration of cellular function that is associated with aging. Here, we introduce the Jun N-terminal kinase (JNK) signaling pathway as a genetic determinant of aging in Drosophila melanogaster. Based on expression profiling experiments, we demonstrate that JNK functions at the center of a signal transduction network that coordinates the induction of protective genes in response to oxidative challenge. JNK signaling activity thus alleviates the toxic effects of reactive oxygen species (ROS). In addition, we show that flies with mutations that augment JNK signaling accumulate less oxidative damage and live dramatically longer than wild-type flies. Our work thus identifies the evolutionarily conserved JNK signaling pathway as a major genetic factor in the control of longevity

Synaptic and genomic responses to JNK and AP-1 signaling in Drosophila neurons

BACKGROUND: The transcription factor AP-1 positively controls synaptic plasticity at the Drosophila neuromuscular junction. Although in motor neurons, JNK has been shown to activate AP-1, a positive regulator of growth and strength at the larval NMJ, the consequences of JNK activation are poorly studied. In addition, the downstream transcriptional targets of JNK and AP-1 signaling in the Drosophila nervous system have yet to be identified. Here, we further investigated the role of JNK signaling at this model synapse employing an activated form of JNK-kinase; and using Serial Analysis of Gene Expression and oligonucleotide microarrays, searched for candidate early targets of JNK or AP-1 dependent transcription in neurons. RESULTS: Temporally-controlled JNK induction in postembryonic motor neurons triggers synaptic growth at the NMJ indicating a role in developmental plasticity rather than synaptogenesis. An unexpected observation that JNK activation also causes a reduction in transmitter release is inconsistent with JNK functioning solely through AP-1 and suggests an additional, yet-unidentified pathway for JNK signaling in motor neurons. SAGE profiling of mRNA expression helps define the neural transcriptome in Drosophila. Though many putative AP-1 and JNK target genes arose from the genomic screens, few were confirmed in subsequent validation experiments. One potentially important neuronal AP-1 target discovered, CG6044, was previously implicated in olfactory associative memory. In addition, 5 mRNAs regulated by RU486, a steroid used to trigger conditional gene expression were identified. CONCLUSION: This study demonstrates a novel role for JNK signaling at the larval neuromuscular junction and provides a quantitative profile of gene transcription in Drosophila neurons. While identifying potential JNK/AP-1 targets it reveals the limitations of genome-wide analyses using complex tissues like the whole brain

Menin uncouples Elk-1, JunD and c-Jun phosphorylation from MAP kinase activation

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Clastosome: a subtype of nuclear body enriched in 19S and 20S proteasomes, ubiquitin, and protein substrates of proteasome.

Nuclear bodies represent a heterogeneous class of nuclear structures. Herein, we describe that a subset of nuclear bodies is highly enriched in components of the ubiquitin-proteasome pathway of proteolysis. We coined the term clastosome (from the Greek klastos, broken and soma, body) to refer to this type of nuclear body. Clastosomes contain a high concentration of 1) ubiquitin conjugates, 2) the proteolytically active 20S core and the 19S regulatory complexes of the 26S proteasome, and 3) protein substrates of the proteasome. Although detected in a variety of cell types, clastosomes are scarce under normal conditions; however, they become more abundant when proteasomal activity is stimulated. In contrast, clastosomes disappear when cells are treated with proteasome inhibitors. Protein substrates of the proteasome that are found concentrated in clastosomes include the short-lived transcription factors c-Fos and c-Jun, adenovirus E1A proteins, and the PML protein. We propose that clastosomes are sites where proteolysis of a variety of protein substrates is taking place

A Versatile ΦC31 Based Reporter System for Measuring AP-1 and Nrf2 Signaling in Drosophila and in Tissue Culture

This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements (synthetic transcription factor binding sites or natural regulatory sequences), reporter genes (eGFP, or DsRed.T4), and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase

Identification and characterization of proteins with novel functions in Nrf2 signaling

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2015.Oxidative stress causes widespread damage to biomolecules, leads to different pathological conditions and contributes to aging. The Nrf2 transcription factor, a major mediator of oxidative stress responses, controls gene expression programs that protect multiple organs from oxidative damage, delay the onset of some age-associated diseases and promote longevity at least in some organisms. In an unstressed condition, Nrf2 interacts with its cytoplasmic inhibitor Keap1, which targets it for proteasomal degradation. Oxidative stress prevents Keap1-mediated degradation of Nrf2, resulting in its accumulation and nuclear translocation. In the nucleus, Nrf2 dimerizes with a small Maf protein, binds to ‘Antioxidant Response Elements’ and induces multiple antioxidant and detoxification genes. Complete understanding of the molecular mechanisms of Nrf2 regulation is important to assess its role in normal physiology and disease. The key components of Nrf2 signaling are conserved in Drosophila where CncC is the homolog of mammalian Nrf2. In order to study mechanisms of Nrf2 function in Drosophila, cell-based and in vivo transcriptional reporters for Nrf2 were developed. A cell-based dsRNA library screen was carried out to find novel regulators of Nrf2 signaling. Among others we identified Cdk12 and Fs(1)h. Cdk12, a RNA PolII-CTD kinase, was found to be required for CncC target gene expression in a cell-autonomous manner and to be important for oxidative stress resistance. In contrast, Fs(1)h, the sole member of the BET protein family in Drosophila, was identified as an inhibitor of CncC. Bromodomain-containing BET proteins have complex functions in chromosome organization and the control of gene expression. Fs(1)h was found to physically interact with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and induces protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from that of Keap1. Consistent with this, combinations of drugs that can specifically target Keap1 and Fs(1)h cause a synergistic and specific activation of CncC dependent gene expression. This synergism might be exploitable for the design of combinatorial therapeutic approaches, targeting Nrf2 in various diseases

Regulatory Interactions between Fibroblast Growth Factor, a Matrix Metalloproteinase and a Proteoglycan in the Control of Branching Morphogenesis

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biomedical Genetics, 2010.Fibroblast Growth Factor (FGF) is a central regulator of branching morphogenesis processes, such as angiogenesis or the development of branched organs including lung, kidney and salivary gland. The formation of the air sac during the development of the Drosophila tracheal system is a powerful and genetically accessible model to investigate how FGF signaling patterns such emerging structures. In this study, I characterized the Drosophila matrix metalloproteinase 2 (Mmp2) as an extracellular inhibitor of FGF function during tracheal development. RNAi mediated knock-down of Mmp2 disrupts the FGF-dependent patterning and morphogenesis of the air sac. Mmp2 expression in the developing air sac is induced by the Drosophila FGF homolog Branchless, and then feeds back on FGF signaling in a lateral inhibition mechanism that refines the precise air sac patterning. The inhibitory effect of Mmp2 on FGF signaling might be mediated by the proteolysis of Dally, a Drosophila Heparan Sulphate Proteoglycan (HSPG) and a known co-receptor of FGF receptor (FGFR). This thesis describes the identification of Dally as the first known substrate of Mmp2 and presents evidence in support of a mechanism by which the proteolysis of Dally mediates a spatial refinement of FGF-dependent patterning

Characterization of the Drosophila Tor Complex II Subunit Sin1 in Growth and Cellular Signaling

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biomedical Genetics, 2008.Growth is an integral part of development in higher eukarya. It is a tightly controlled process regulated by both environmental and nutritional cues. Tor is a highly conserved kinase that interprets these cues and acts as a sensor to either promote, or shut down growth. In diseases, such as cancer and diabetes, this function of Tor is deregulated and the normal growth program becomes perturbed. Tor is present within two highly conserved complexes termed Tor Complex I (TORC1) and Tor Complex II (TORC2). The latter can specifically phosphorylate the oncogene-encoded kinase Akt. Interestingly, the phosphorylation of Akt by TORC2 is marker of poor prognosis in several forms of cancer, and conversely, the inhibition of Akt is thought to contribute the pathology of type II diabetes. Sin1 is a recently characterized subunit of mammalian TORC2 that is required for the phosphorylation of Akt by Tor. Mammalian Sin1 is also reported to regulate the stress-inducible kinase JNK. This is interesting, as JNK, like Akt, contributes to the pathology of type II diabetes. Therefore, Sin1 may be a potential therapeutic target for the treatment of both cancer and diabetes. However, the role of Sin1 in neither Akt, nor JNK signaling is well understood. The goal of the work presented here was to use the power of Drosophila genetics to characterize the role of Sin1 in Akt and JNK regulation, specifically in relation to cell signaling and growth. The work presented here demonstrates that Drosophila Sin1 is functionally conserved, in relation to its mammalian counterpart, in TORC2-mediated Akt phosphorylation. However, in contrast to reports on its mammalian homolog, Drosophila Sin1 was not involved in stress mediated JNK activation, nor JNK regulated growth. Interestingly, the loss of Sin1 did not generally impair signaling downstream of Akt in Drosophila. Global Akt substrate phosphorylation, as well as the phosphorylation of two specific and previously verified Akt targets, was unaffected in Sin1 loss-of-function conditions. However, ablation of Sin1 in Drosophila not only decreased TORC2 sensitive phosphorylation of Akt, but also correlated with suppressed growth. Although the kinase activity of Akt towards its substrates was unaltered by the loss of Sin1, growth inhibition was partially dependent on the Akt target and transcription factor FoxO. This suggests that Sin1 may regulate Akt substrate specificity rather than global kinase activity. The results presented here demonstrate that Sin1 regulates growth by a mechanism that requires the transcription factor FoxO. FoxO is known to be a negative regulator of growth when transcriptionally active, which can be repressed by Akt, and presumably Sin1-dependent Akt phosphorylation. This is significant because FoxO activation is associated with growth inhibition within the pancreas of diabetics and its inactivation by Akt may to contribute to cancer development. Therefore, Sin1 may be a therapeutic target for treating both diseases. Although chemicals that block Akt kinase activity are potent in retarding tumor growth, they are also highly toxic. The loss of Sin1, unlike the ablation of Akt, is non-lethal in Drosophila. Thus, ablating Sin1 function may be a less toxic strategy for targeting Akt in diseases in which FoxO contributes to deregulated growth