A survey of the protective effects of some commercially available antioxidant supplements in genetically and chemically induced models of oxidative stress in Drosophila melanogaster

AbstractOxidative stress remains one of the most well studied, albeit somewhat contentious, causes of age-related changes in humans. Consequently, a large number of putative antioxidant compounds are freely available in myriad formulations that are often not tested for their efficacy or regulated for quality control. Following the development of a Drosophila model of oxidative-stress dependent aging (p38 MAP K (p38K) mutants) in our laboratory, we attempted to test the protective effect of some of these commonly available formulations against oxidative stress, in the p38K model. As environmental exposure to oxidizing toxins has been linked to a variety of human diseases, we also tested the efficacy of these supplements on chemically-induced models of oxidative stress (paraquat and hydrogen peroxide exposure). Our results suggest that when added as a dietary supplement, some of these over-the-counter compounds, notably containing açai extracts, confer significant protection for both the p38K-dependent genetic model as well as the toxin-induced model. These products were also remarkably effective at dampening stress-induced expression of the detoxifying enzyme GSTD1 and eliminating paraquat induced circadian rhythm deficits. Overall, our results suggest potential benefits of dietary supplementation with some of these compounds, especially under conditions of elevated oxidative stress. These findings should be assessed in the context of other studies that seek to identify active principles in these extracts, determine their effective dosage for human consumption and evaluate the safety of long-term prophylactic applications

The Translational Repressor Pumilio Regulates Presynaptic Morphology and Controls Postsynaptic Accumulation of Translation Factor eIF-4E

Translational repression by Drosophila Pumilio (Pum) protein controls posterior patterning during embryonic development. Here, we show that Pum is an important mediator of synaptic growth and plasticity at the neuromuscular junction (NMJ). Pum is localized to the postsynaptic side of the NMJ in third instar larvae and is also expressed in larval neurons. Neuronal Pum regulates synaptic growth. In its absence, NMJ boutons are larger and fewer in number, while Pum overexpression increases bouton number and decreases bouton size. Postsynaptic Pum negatively regulates expression of the translation factor eIF-4E at the NMJ, and Pum binds selectively to the 3′UTR of eIF-4E mRNA. The GluRIIa glutamate receptor is upregulated in pum mutants. These results, together with genetic epistasis studies, suggest that postsynaptic Pum modulates synaptic function via direct control of eIF-4E expression

A Muscle-Specific p38 MAPK/Mef2/MnSOD Pathway Regulates Stress, Motor Function, and Life Span in Drosophila

SummaryMolecular mechanisms that concordantly regulate stress, life span, and aging remain incompletely understood. Here, we demonstrate that in Drosophila, a p38 MAP kinase (p38K)/Mef2/MnSOD pathway is a coregulator of stress and life span. Hence, overexpression of p38K extends life span in a MnSOD-dependent manner, whereas inhibition of p38K causes early lethality and precipitates age-related motor dysfunction and stress sensitivity, that is rescued through muscle-restricted (but not neuronal) add-back of p38K. Additionally, mutations in p38K are associated with increased protein carbonylation and Nrf2-dependent transcription, while adversely affecting metabolic response to hypoxia. Mechanistically, p38K modulates expression of the mitochondrial MnSOD enzyme through the transcription factor Mef2, and predictably, perturbations in MnSOD modify p38K-dependent phenotypes. Thus, our results uncover a muscle-restricted p38K-Mef2-MnSOD signaling module that influences life span and stress, distinct from the insulin/JNK/FOXO pathway. We propose that potentiating p38K might be instrumental in restoring the mitochondrial detoxification machinery and combating stress-induced aging

Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling

FGF signaling in neurons is regulated by Survival Motor Neuron, a component of a complex that regulates snRNP biogenesis and FGF receptor expression

Characterisation and expression of calpain family members in relation to nutritional status, diet composition and flesh texture in gilthead sea bream (Sparus aurata).

Calpains are non-lysosomal calcium-activated neutral proteases involved in a wide range of cellular processes including muscle proteolysis linked to post-mortem flesh softening. The aims of this study were (a) to characterise several members of the calpain system in gilthead sea bream and (b) to examine their expression in relation to nutritional status and muscle tenderisation. We identified the complete open reading frame of gilthead sea bream calpains1-3, sacapn1, sacapn2, sacapn3, and two paralogs of the calpain small subunit1, sacapns1a and sacapns1b. Proteins showed 63-90% sequence identity compared with sequences from mammals and other teleost fishes, and the characteristic domain structure of vertebrate calpains. Transcripts of sacapn1, sacapn2, sacapns1a and sacapns1b had a wide tissue distribution, whereas sacapn3 was almost exclusively detected in skeletal muscle. Next, we assessed transcript expression in skeletal muscle following alteration of nutritional status by (a) fasting and re-feeding or (b) feeding four experimental diets with different carbohydrate-to-protein ratios. Fasting significantly reduced plasma glucose and increased free fatty acids and triglycerides, together with a significant increase in sacapns1b expression. Following 7 days of re-feeding, plasma parameters returned to fed values and sacapn1, sacapn2, sacapns1a and sacapns1b expression was significantly reduced. Furthermore, an increase in dietary carbohydrate content (11 to 39%) diminished growth but increased muscle texture, which showed a significant correlation with decreased sacapn1 and sacapns1a expression, whilst the other calpains remained unaffected. This study has demonstrated that calpain expression is modulated by nutritional status and diet composition in gilthead sea bream, and that the expression of several calpain members is correlated with muscle texture, indicating their potential use as molecular markers for flesh quality in aquaculture production

Posterior segment of the Drosophila Larval Heart

<p>Shown here is a triple-stained image of the <em>Drosophila melanogaster</em> larval heart tube. The posterior third of the heart (shown here) is contractile in nature and contains the pacemakers that regulate hemolymph pumping in response to both glutamate and CCAP (crustacean cardioactive peptide) (Dulcis and Levine, 2005). The larval heart is stained with FITC conjugated Phalloidin (green) to mark Actin filaments in both the heart muscles and the abdominal body wall muscles, an antibody against the <em>Drosophila</em> homolog of the SERCA protein (red; sarco-endoplasmic reticulum Calcium ATPase) (Sanyal, et. al., 2005; Sanyal et. al., 2006), and an antibody MAb3 that labels pericardial cells surrounding the heart tube (blue) (Yarnitzky and Volk, 1995).</p> <p> </p> <p>References</p> <p> </p> <p>1. Dulcis D, Levine RB. Glutamatergic innervation of the heart initiates retrograde contractions in adult <em>Drosophila melanogaster</em>. J Neurosci. 2005 ;25(2):271-80.</p> <p>2. Sanyal S, Consoulas C, Kuromi H, Basole A, Mukai L, Kidokoro Y, Krishnan KS,Ramaswami M. Analysis of conditional paralytic mutants in <em>Drosophila</em> sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability. Genetics. 2005;169(2):737-50.</p> <p>3. Sanyal S, Jennings T, Dowse H, Ramaswami M. Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in <em>Drosophila</em>. J Comp Physiol B. 2006;176(3):253-63.</p> <p>4. Yarnitzky T, Volk T. Laminin is required for heart, somatic muscles, and gut development in the <em>Drosophila</em> embryo. Dev Biol. 1995;169(2):609-18.</p

A method to visualize different bouton types at the Drosophila larval NMJ

<p>Shown are the neuromuscular junctions (NMJs) at muscle 6/7 in abdominal segment 2 in a third instar <em>Drosophila</em> larva. Panel A shows an NMJ from an animal expressing membrane bound Green Fluorescent Protein (mcd8::GFP) from the <em>futsch</em>[C380]-GAL4 (C380-GAL4 or BG380-GAL4) driver that expresses in all motor neurons (Sanyal, 2009; Budnik et. al., 1996). Accordingly, the two motor axons that innervate this muscle (MN6/7-Ib and MNSNb/d-Is; Hoang and Chiba, 2001) contain GFP label and all boutons on muscle 6/7 are labeled with GFP fluorescence (green in panel A, anti-synaptotagmin staining is used for independent visualization of these synaptic boutons, in red). In panel B, GFP is expressed from the same <em>futsch</em>[C380]-GAL4 but along with GAL80 expressed from the ChAT promoter (Cha-GAL80; Kitamoto, 2002). This leads to an appreciably weaker expression of GFP in type Ib boutons, and by extension, MN6/7-Ib. As a result, following anti-synaptotagmin staining, type Ib and Is boutons (and, therefore, contributions to this synapse from MN6/7-Ib and MNSNb/d-Is) can be discriminated quite easily. Note that only the type Is boutons are double labeled with GFP and synaptotagmin (double arrows), whereas the type Ib boutons are only seen from synaptotagmin staining (double arrowheads). This reagent might be a useful tool to easily discriminate between these two bouton types at a widely studied model synapse.</p> <p>References:</p> <p>1. Budnik V, Koh YH, Guan B, Hartmann B, Hough C, Woods D, Gorczyca M. Regulation</p> <p>of synapse structure and function by the <em>Drosophila</em> tumor suppressor gene dlg. Neuron. 1996 Oct;17(4):627-40.</p> <p>2. Sanyal S. Genomic mapping and expression patterns of C380, OK6 and D42 enhancer trap lines in the larval nervous system of <em>Drosophila</em>. Gene Expr Patterns. 2009 Jun;9(5):371-80.</p> <p>3. Hoang B, Chiba A. Single-cell analysis of <em>Drosophila</em> larval neuromuscular synapses. Dev Biol. 2001 Jan 1;229(1):55-70.</p> <p>4. Kitamoto T. Conditional disruption of synaptic transmission induces male-male</p> <p>courtship behavior in <em>Drosophila</em>. Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):13232-7.</p

Expression pattern of the Alhambra/Dalf gene from an enhancer trap in the larval CNS of Drosophila

<p>Shown is GFP expression from a GFP enhancer trap near the <em>Alhambra</em> (<em>alh/dalf</em>) locus (FlyTrap collection) – the <em>Drosophila</em> homolog of human AF10/AF17 leukemia fusion genes (Linder et. al., 2001). Robust GFP expression is observed in the whole CNS including the brain lobes and both lateral (arrowheads) and dorso-medial motor neurons (arrows). This expression pattern is reminiscent of Alh/Dalf expression in the embryonic and larval nerve cord seen with RNA<em> in situ</em> and antibody staining (Bahri et. al., 2001). <em>Alh/dalf</em> is required in the nervous system for the normal development of <em>eve</em> expressing RP2 motor neurons and was also isolated in a screen for modifiers of the heterodimeric transcription factor AP-1 (Bahri, et. al., 2001; Franciscovich et. al., 2008).</p> <p><br></p> <p>References</p> <p><br></p> <p>1. Linder B, Gerlach N, Jäckle H. The <em>Drosophila</em> homolog of the human AF10 is an HP1-interacting suppressor of position effect variegation. EMBO Rep. 2001; 2(3):211-6.</p> <p>2. Bahri SM, Chia W, Yang X. The<em> Drosophila</em> homolog of human AF10/AF17 leukemia fusion genes (<em>Dalf</em>) encodes a zinc finger/leucine zipper nuclear protein required in the nervous system for maintaining EVE expression and normal growth. Mech Dev. 2001; 100(2):291-301.</p> <p>3. Franciscovich AL, Mortimer AD, Freeman AA, Gu J, Sanyal S. Overexpression screen in Drosophila identifies neuronal roles of GSK-3 beta/<em>shaggy</em> as a regulator of AP-1-dependent developmental plasticity. Genetics. 2008; 180(4):2057-71.</p

Inhibition of Adf-1 in Drosophila motor neurons leads to severely impaired motor function

<p>This video demonstrates the compromised motor performance in adult <em>Drosophila</em> upon the expression of a Ser64/184Ala substituted Adf-1 transcription factor. When Adf-1[S64/184A] was expressed in all motor neurons throughout development using the C380-GAL4 driver (Sanyal S., Gene Expression Patterns, 2009) adult flies could not inflate their wings, nor could the majority of them stand upright or initiate locomotion. This behavioral outcome is consistent with a role for Adf-1 in the regulation of motor neuron development and plasticity as reported in Timmerman et. al., J Neurosci., 2013. We acknowledge help from Dr. Amanda Freeman in video editing.</p> <p> </p> <p> </p> <p>References</p> <p><br></p> <p>1. Sanyal S. Genomic mapping and expression patterns of C380, OK6 and D42 enhancer trap lines in the larval nervous system of <em>Drosophila</em>. Gene Expr Patterns. 2009 Jun;9(5):371-80. PubMed PMID: 19602393.</p> <p> </p> <p>2. Timmerman C, Suppiah S, Gurudatta BV, Yang J, Banerjee C, Sandstrom DJ, Corces VG, Sanyal S. The <em>Drosophila</em> Transcription Factor Adf-1 (nalyot) Regulates Dendrite Growth by Controlling FasII and Staufen Expression Downstream of CaMKII and Neural Activity. J Neurosci. 2013 Jul 17;33(29):11916-31. PubMed PMID: 23864680.</p

Evidence for cell autonomous AP1 function in regulation of Drosophila motor-neuron plasticity

BACKGROUND:The transcription factor AP1 mediates long-term plasticity in vertebrate and invertebrate central nervous systems. Recent studies of activity-induced synaptic change indicate that AP1 can function upstream of CREB to regulate both CREB-dependent enhancement of synaptic strength as well as CREB-independent increase in bouton number at the Drosophila neuromuscular junction (NMJ). However, it is not clear from this study if AP1 functions autonomously in motor neurons to directly modulate plasticity.RESULTS:Here, we show that Fos and Jun, the two components of AP1, are abundantly expressed in motor neurons. We further combine immunohistochemical and electrophysiological analyses with use of a collection of enhancers that tightly restrict AP1 transgene expression within the nervous system to show that AP1 induction or inhibition in, but not outside of, motor neurons is necessary and sufficient for its modulation of NMJ size and strength.CONCLUSION:By arguing against the possibility that AP1 effects at the NMJ occur via a polysynaptic mechanism, these observations support a model in which AP1 directly modulates NMJ plasticity processes through a cell autonomous pathway in the motor neuron. The approach described here may serve as a useful experimental paradigm for analyzing cell autonomy of genes found to influence structure and function of Drosophila motor neurons.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]