Epithelial stress sensors: novel roles for cytochrome P450s and organellar calcium in integrated stress and immune responses.

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences


All cells produce reactive oxygen species (ROS) due to chemical reactions, which also occur in the organelles contained within cells. For example, the production of energy from the mitochondria and chemical reactions in the peroxisomes generate ROS. Cells and tissues deal with the production of ROS using different enzymes and processes, which 'neutralise' ROS. When there is an imbalance between the production of ROS and the ability of the cells or tissues in question to deal with the charged ions, 'oxidative stress' results. Cellular responses to oxidative stress are regulated by signal transduction mechanisms which utilise small signalling molecules including calcium ions (Ca2+). Oxidative stress affects cellular proteins, lipids and DNA, and in humans is associated with neurodegenrative disease, cardiovascular disease including stroke and heart attack, and with the ageing process. However, ROS production can also closely linked to immunity, as ROS can be used in defence against infection. It is increasingly known that mechanisms of oxidative stress and immune function are linked, and these are important areas of research for the basic fundamentals of biological mechanisms, and for human health and ageing. Invertebrate model organisms eg., Drosophila melanogaster, are central for investigating the impact of gene function in stress responses in the organism. Furthermore, studies in Drosophila have also have provided the clearest links between manipulation of stress-associated genes and physiological output. The importance of the tissue- or cell-specific context of stress-response genes in oragnismal responses to stress, makes Drosophila particularly useful, as cell- and tissue-specific targeting of genes in Drosophila is well-established. The Drosophila epithelial Malpighian (renal) tubule is equivalent to vertebrate kidney and liver and plays critical roles in detoxification, fluid and ion transport. Tubules are packed with mitochondria and peroxisomes, and produce ROS as part of their normal function. We have shown that tubules have specific adaptations to counter the high mitochondrial activity and production of ROS, including being enriched for 'anti-oxidant' genes which are also conserved in humans. We have also shown that de-regulation of tubule mitochondrial calcium signalling by excess ROS leads to tubule cell death (apoptosis) and death of the whole fly. Thus, this epithelial tissue has a central role in stress-sensing for the whole organism. The tubule is also a stand-alone immune-sensing tissue which uses 'innate' immune mechanisms to sense bacterial or fungal challenge. Such innate immune mechanisms in epithelia are also conserved in vertebrates, and constitute an increasingly important area of study, as it is now known that diseases including Crohns' Disease and asthma are due to deregulation of innate immune mechanisms in these fluid-transporting epithelial tissues (intestine, and lungs, respectively). Our recent data from survival studies in response to oxidative stress and immune challenge, work on calcium signals in mitochondria and analysis of the ~13500 genes of the fly in response to stress shows that key novel tubule-specific genes involved with the stress/immune responses the fly. These genes encode 'detoxification' proteins-the cytcochrome P450s, normally associated with mitochondria or peroxisomes and here, we have shown involvement with the stress response. We will investigate these genes in the context of calcium signals in the peroxisome and mitochondria, and assess the impact of each of these genes/proteins on survival of the whole organism using functional assays for novel genes, genetics/transgenics with calcium reporters targeted to mitochondria and peroxisomes in the intact tissue. This will allow us to uncover new ways of regulating stress responses in epithelial cells, which may impact on human biology.

Technical Summary

We have identified the Drosophila renal tubule as a key epithelial tissue in organismal stress responses to reactive oxygen species (ROS) and our recent microarray data show that tubule-enriched detoxification genes - cytochrome P450s - are common to stress and immune responses. The interaction between stress and immune response also exists in vertebrate tissues eg., ROS are associated with innate immune responses in lung epithelia. Thus, epithelial tissues play a significant role in organismal stress responses; but due to the high rates of metabolic activity in these tissues, they may also be the major sources of ROS in the organism, and thus must be equipped for stress sensing.We have also demonstrated modulation of organismal responses to stress via mitochondrial and peroxisomal calcium signalling in tubule. Therefore, it is of importance to define ROS production in the tubule, and to define key genes and those calcium signalling pathways which modulate stress sensing and response. ROS production in tubules and midgut from flies exposed to oxidative stress and from flies subjected to immune challenge, will be assessed with a mitochondrial superoxide indicator. This will allow assessment of direct roles of ROS in immune sensing by gut epithelia. As cytochrome P450s (CYPs) are implicated in oxidative stress, we will also investigate specific CYPs by generating transgenic lines for our cyp genes of interest, and assessing the cell-specific role of these CYPs in tubule and midgut on whole organism response to stress and immune challenge. Finally, given CYP localisation in mitochondria and peroxisomes, we will measure calcium signals in these organelles in the intact tubule and midgut, using targeted calcium probes, which we already have (mitochondria) or will generate (peroxisomes). Finally, we will measure ROS in the tubules and midgut from Cyp transgenic lines, thus correlating CYP function, organellar Ca2+ and ROS for the first time in an organotypic context.


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Brown KM (2013) Phosphodiesterase-8A binds to and regulates Raf-1 kinase. in Proceedings of the National Academy of Sciences of the United States of America

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Cabrero P (2014) Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. in Proceedings of the National Academy of Sciences of the United States of America

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Davies SA (2013) Signaling by Drosophila capa neuropeptides. in General and comparative endocrinology

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Davies SA (2009) Modulation of epithelial innate immunity by autocrine production of nitric oxide. in General and comparative endocrinology

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Davies SA (2009) Organellar calcium signalling mechanisms in Drosophila epithelial function. in The Journal of experimental biology

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Davies SA (2012) Immune and stress response 'cross-talk' in the Drosophila Malpighian tubule. in Journal of insect physiology

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Davies SA (2014) Cell signalling mechanisms for insect stress tolerance. in The Journal of experimental biology

Description We have:
- established the major tissues and pathways for stress sensing in the fly, which can solely determine survival fate of the insect, including novel tissue-specific roles for key CYP450 'detox' enzymes in stress handling, with unexpected roles in insecticide resistance. Thus, epithelial stress handling pathways may provide novel insecticide targets.
- validated and extended novel observations in mammalian stress handling (Brown et. al., PNAS, 2013).
established the first neuroendocrine pathway implicated in desiccation (Terhzaz et al., PLoS One, 2012; Peptides, 2013) and cold tolerance (Terhzaz et al., in prep for PNAS).
We now have unique insights into insect stress handling and from systems biology, which have allowed us to further consolidate an international lead. Additionally, over the grant period, the PIs published 25 peer reviewed publications and attracted further ~£1.7M in funding from BBSRC, other funders and industry, allowing development and potential application of our work to insect pest control.
Objectives achieved:
1. Identify the relative contributions of exogenous vs endogenous ROS to immune sensing by intact midgut and tubules, and their impact on survival of the whole organism.
Achieved - We now know that there is no overlap between oxidative and immune stress in tubules (Overend et al., J Insect Physiol., 2012) although possibly in gut. We have also demonstrated that feeding flies with oxidative stressors increases endogenous ROS levels in dissected tissues and that tubule/midgut communication exists (Terhzaz et al., in prep).
2. Understand the role of specific cytochrome P450 (CYP) genes and one other putative
'detoxification' gene in organismal stress. This will be achieved by specific targeting of key CYPs and CG11897 to specific cells (tubule principal cells) and tissues (tubule, midgut) via the GAL4/UAS system, then investigating organismal survival to oxidative and immune stress in these transgenic Drosophila and in parental lines.
Achieved - paper in prep (Terhzaz et al., Novel role of CYP4e3 in renal ROS scavenging)
3a. Work out the role of peroxisomal calcium to whole organism responses to stress by direct measurement of calcium levels in the peroxisomes. This will be achieved by generating peroxisomal-targeted calcium reporters based on aequorin and fluorescent probes which we have already utilised in previous published work. Targeting of the peroxisomal reporter to tubule and midgut cells will be achieved by GAL4/UAS, which will allow peroxisomal calcium measurements in the intact tubule.
Achieved - Chintapalli et al., PLoS One 2012
3b. Measure changes in peroxisomal calcium upon expression of key CYPs, after localising these CYPs in intact tubule. 4. Measure changes in mitochondrial calcium upon expression of key CYPs in vivo, using targeted mitochondrial probes (aequorin and fluorescent) which we have utilised in previously published studies.
Achieved - Localisation studies with tagged over-expression lines showed most of the CYPs localised to the ER and not peroxisomes or mitochondria. Furthermore, measurements of intracellular calcium in tubules targeted with key CYPs, showed little or no change. Therefore, peroxisomal/mitochondrial calcium was unaffected by these CYPs.

We have published 9 papers including one in PNAS USA; one under revision in PNAS; and one to be submitted in Jan 2015:

1: Davies SA, Cabrero P, Overend G, Aitchison L, Sebastian S, Terhzaz S, Dow JA.
Cell signalling mechanisms for insect stress tolerance. J Exp Biol. 2014 Jan
1;217(Pt 1):119-28. doi: 10.1242/jeb.090571. Review. PubMed PMID: 24353211.

2: Terhzaz S, Overend G, Sebastian S, Dow JA, Davies SA. The D. melanogaster
capa-1 neuropeptide activates renal NF-kB signaling. Peptides. 2014
Mar;53:218-24. doi: 10.1016/j.peptides.2013.08.004. Epub 2013 Aug 14. PubMed
PMID: 23954477.

3: Davies SA, Cabrero P, Povsic M, Johnston NR, Terhzaz S, Dow JA. Signaling by
Drosophila capa neuropeptides. Gen Comp Endocrinol. 2013 Jul 1;188:60-6. doi:
10.1016/j.ygcen.2013.03.012. Epub 2013 Apr 1. Review. PubMed PMID: 23557645.

4: Brown KM, Day JP, Huston E, Zimmermann B, Hampel K, Christian F, Romano D,
Terhzaz S, Lee LC, Willis MJ, Morton DB, Beavo JA, Shimizu-Albergine M, Davies
SA, Kolch W, Houslay MD, Baillie GS. Phosphodiesterase-8A binds to and regulates
Raf-1 kinase. Proc Natl Acad Sci U S A. 2013 Apr 16;110(16):E1533-42. doi:
10.1073/pnas.1303004110. Epub 2013 Mar 18. PubMed PMID: 23509299; PubMed Central
PMCID: PMC3631629.

5: Cabrero P, Richmond L, Nitabach M, Davies SA, Dow JA. A biogenic amine and a
neuropeptide act identically: tyramine signals through calcium in Drosophila
tubule stellate cells. Proc Biol Sci. 2013 Feb 27;280(1757):20122943. doi:
10.1098/rspb.2012.2943. Print 2013 Apr 22. PubMed PMID: 23446525; PubMed Central
PMCID: PMC3619477.

6: Chintapalli VR, Terhzaz S, Wang J, Al Bratty M, Watson DG, Herzyk P, Davies
SA, Dow JA. Functional correlates of positional and gender-specific renal
asymmetry in Drosophila. PLoS One. 2012;7(4):e32577. doi:
10.1371/journal.pone.0032577. Epub 2012 Apr 4. PubMed PMID: 22496733; PubMed
Central PMCID: PMC3319558.

7: Davies SA, Overend G, Sebastian S, Cundall M, Cabrero P, Dow JA, Terhzaz S.
Immune and stress response 'cross-talk' in the Drosophila Malpighian tubule. J
Insect Physiol. 2012 Apr;58(4):488-97. doi: 10.1016/j.jinsphys.2012.01.008. Epub
2012 Jan 28. Review. PubMed PMID: 22306292.

8: Terhzaz S, Cabrero P, Robben JH, Radford JC, Hudson BD, Milligan G, Dow JA,
Davies SA. Mechanism and function of Drosophila capa GPCR: a desiccation
stress-responsive receptor with functional homology to human neuromedinU
receptor. PLoS One. 2012;7(1):e29897. doi: 10.1371/journal.pone.0029897. Epub
2012 Jan 11. PubMed PMID: 22253819; PubMed Central PMCID: PMC3256212.

9: Terhzaz S, Finlayson AJ, Stirrat L, Yang J, Tricoire H, Woods DJ, Dow JA,
Davies SA. Cell-specific inositol 1,4,5 trisphosphate 3-kinase mediates
epithelial cell apoptosis in response to oxidative stress in Drosophila. Cell
Signal. 2010 May;22(5):737-48. doi: 10.1016/j.cellsig.2009.12.009. Epub 2010 Jan
11. PubMed PMID: 20060894.
Exploitation Route Findings on insect stress tolerance will be key in developing peptide analogs for insect control. This work is being developed with industry and collaborators (e.g. PNAS 2014 - under revision). Collaborator networks have also been developed as a result of our work in insect stress tolerance. Also, our work in Drosophila has directly benefited cell signalling studies for new therapeutics for human disease (e.g. PNAS 2013)
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

URL https://www.ncbi.nlm.nih.gov/pubmed
Description Please see section on key findings - we are keen to develop peptide analogues as novel 'green' biocontrol agents to disrupt physiological process for insect control with key collaborators.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Environment
Impact Types Societal,Economic

Description H2020 SFS-03a - 2014 Native and Alien pests in Agriculture, Horticulture and Forestry
Amount € 7,000,000 (EUR)
Funding ID 634361 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 04/2015 
End 05/2019
Title Cyp transgenic lines 
Description Transgenic lines for multiple Cyt P450 genes 
Type Of Material Biological samples 
Provided To Others? No  
Impact Will have academic/industry impact when used in insecticide resistance research 
URL http://www.ncbi.nlm.nih.gov/pubmed/26073628
Description Peptide analogues 
Organisation U.S. Department of Agriculture USDA
Country United States 
Sector Public 
PI Contribution Devised physiological and functional assays for neuropeptide analogues developed by USDA.
Collaborator Contribution Provision of neuropeptide analogues.
Impact Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2882-7. doi: 10.1073/pnas.1501518112. Epub 2015 Feb 17. Insect capa neuropeptides impact desiccation and cold tolerance. Terhzaz S1, Teets NM2, Cabrero P3, Henderson L3, Ritchie MG4, Nachman RJ5, Dow JA3, Denlinger DL6, Davies SA1.
Start Year 2013
Description Singapore schools research placement 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact 1 week research placement using Drosophila melanogaster. Remote supervision of A level projects.
Participation (and awards gained) in Singapore Science and Engineering Fair
Year(s) Of Engagement Activity 2010,2011,2012,2013
Description Sixth form school research visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 1 week lab placement for sixth form pupils from Moreton House School, Shropshire.
Provision and supervision of 6th form projects using Drosophila to model human disease.
Year(s) Of Engagement Activity 2013,2014,2015