Oxidative stress induced regulation of synaptic growth in the nervous system - dissection of genetic and cellular mechanisms.

Lead Research Organisation: University of York
Department Name: Biology

Abstract

Brains are very sensitive to ageing and most of us have experience of ageing relatives with faulty memories. The brain requires high levels of food and oxygen to function effectively. By using a lot of oxygen to generate energy, the brain produces a by-product. This by-product is toxic forms of oxygen and is termed Reactive Oxygen Species or ROS. Normally the brain can cope with low levels of ROS that are generated as a by-product of normal metabolism, but as the brain ages, the self-repair mechanisms become less effective and ROS become excessive. ROS are destructive to cells by a self-perpetuating cycle of damage. Primarily, ROS generation occurs in the structure within the cell responsible for producing energy from food and oxygen called the mitochondria. We term ROS generated by the mitochondria, mitochondrial ROS (m-ROS). An ageing brain struggles to clear itself of cellular material damaged by ROS. As waste material accumulates, it can also generate a second source of ROS, generated by metals within the accumulated waste material reacting with oxygen to produce more ROS. We term these cytoplasmic ROS (c-ROS). Both sources of ROS now contribute to the increasing cycle of damage as neurons age. We found that the connections between nerve cells, called synapses, grow excessively when ROS are excessive. Synapses are normally known to grow while the brain carries out learning and memory functions and the connections between nerve cells improve their communication efficiency. We therefore find it surprising to see synapses growing during a period when we would expect a decline in the efficiency of neuronal communication. In this proposal we will examine and uncover the processes in nerve cells that react to ROS to cause synapse growth. We have already found that nerve cells activate a process of self-renewal when ROS are present in the brain and suspect that this may be inducing synapse growth. Exactly how this happens we aim to determine. The changes that we have observed are very likely of critical importance to our understanding of the decline in brain function as we age. This work will help us to understand the mechanisms, events and molecules that cause failure in nerve cell function in the ageing brain. The results of this work have every potential to aid the discovery of drugs and treatments to alleviate adverse effects of ageing and will thus, in time, benefit society as a whole.

Technical Summary

Oxidative stress is a hallmark of ageing and neurodegenerative diseases. This proposal seeks to investigate how reactive oxygen species (ROS) impinge on the nervous system, specifically the growth of synaptic terminals, where increases in ROS unexpectedly cause overgrowth. Using the genetically tractable fruitfly, Drosophila, as a model we will address key issues. 1. We will determine the effects of different sources of oxidative stress on presynaptic terminals, at late larval neuromuscular junction (NMJ) and in the central nervous system (CNS) as well as postsynaptic dendritic arbors of central neurons. 2. We found that different sources of ROS, cytoplasmic versus mitochondrial, induce synaptic terminal growth via different Jun-N-terminal Kinase (JNK) pathway components and will now identify the relevant upstream components and downstream transcriptional codes. 3. We will establish cellular processes and underlying mechanisms mediating ROS-induced synaptic growth, investigating the role of autophagy and associated genes. We will use genetics to identify interactions between genes and recently developed mosaic and intersectional expression systems to target expression of dominant negative, fluorophore tagged and RNAi transgenes to specific neurons. We will visualize pre- and postsynaptic terminals by immunofluorescence antibody staining and targeted expression of fluorophore tagged reporter constructs, imaging these with point and field scanning confocal systems. Live imaging will inform us about changes to the dynamics of ROS induced growth, T.E.M. about the role of autophagy. We will apply 3-D reconstruction algorithms for quantitative analysis of complex branched dendrites. Given the high conservation of the signaling pathways and cellular processes associated with oxidative stress, this proposal is likely to identify new candidate genes as potential targets for therapeutic strategies aimed at ameliorating the effects of ageing in the nervous system.

Planned Impact

See submission by M.L., Cambridge.

Publications

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Cagin U (2015) Mitochondrial retrograde signaling regulates neuronal function. in Proceedings of the National Academy of Sciences of the United States of America

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Milton VJ (2011) Oxidative stress induces overgrowth of the Drosophila neuromuscular junction. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We have found that forms of oxygen that are damaging (reactive oxygen species, ROS) to cells can induce changes, notably an increase in size to the connections between a neuron and a muscle (the synapse). We first observed these changes in a form of childhood neurodegeneration, lysosomal storage disease (LSDs) where overgrowth of synapses is observed in the central brain. We showed in our model system that some of this synaptic growth was caused by excessive levels of ROS (oxidative stress). We find that inducing oxidative stress in neurons can cause synaptic overgrowth and we have identified some of the signals that regulate this. We have since gone on to observe that synapses may be using low levels of ROS to regulate their size and possibly their activity. This may arise as the cell produces energy in the mitochondria, a compartment in the cell where sugars are converted into cellular energy using oxygen to do so, ROS are produced as a byproduct. More energy consumption in a neuron would lead to a greater production of ROS. We therefore think that detection of ROS may be a general signal in the neuron to indicate levels of activity, leading to the appropriate regulation of synaptic growth.
Exploitation Route We have secured follow-on funding from the BBSRC (grant reference BB/M002322/1) to expand on these findings. Our findings suggest a novel but widely used mechanism for neuronal homeostasis that has profound implications for our understanding of neuronal function and neurodgeneration where reactive oxygen species are in excess.
Sectors Healthcare

 
Description Project Grant
Amount £261,733 (GBP)
Funding ID BB/M002322/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 10/2017
 
Description Oxidative Stress and Synapse Growth 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution A jointly held BBSRC grant with the lead PI in Cambridge. The study follows up our 2011 PNAS paper on oxidative stress driving synapse growth, the STS group is carrying out proteomics and generating transgenes to support the Cambridge work.
Collaborator Contribution The main investigative role is carried out in Cambridge looking at the role of oxidative stress in dendrite growth.
Impact Lu, Y., Zhang, Z., Sun, D., Sweeney, S.T. and Gao, F.B. (2013) Syntaxin 13, a genetic modifier of mutant CHMP2B in frontotemporal dementia, is required for autophagosome maturation. Molecular Cell, doi: 10.1016/j.molcel.2013.08.041.
Start Year 2011
 
Description Article in Yorkshire Post 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Interview with the regional newspaper, The Yorkshire Post. The YP is partnering the Alzheimer's Society as their charity for Christmas '13. STS was interviewed resulting in a front page article on Frontotemporal Dementia, and accompanying online videos.

Received a call from BBC-Leeds on possibility of participating in a programme on dementia and the creation of the 'Dementia friendly York' scheme by the Joseph Rowntree Foundation. Also direct emails from affected individuals.
Year(s) Of Engagement Activity 2013
URL http://www.yorkshirepost.co.uk/news/main-topics/general-news/struck-by-dementia-at-37-but-hope-lies-...
 
Description BBSRC Schools Regional Champion 2011-2013 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact See other impact activities, school talks, practicals for schools and University alumni.

See other listed activities
Year(s) Of Engagement Activity 2011,2012,2013
 
Description Public engagement activity - Participation in The Brain Box, Manchester Town Hall, 19th June 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I contributed a stall/activity to the Brain Box public engagement day, part of Manchester Day in 2016. I produced an activity for the understanding of temperature sensation in Drosophila that involved the public testing responses of larvae to temperature, against a mutant defective for temperature sensation. 4500 members of the general public attended and I had great than 100 public members engaged in the activity. The discussion was lively and thoroughly engaged across all age groups, I received many interested and informed questions, particularly from children, there was a clear enjoyment. What was particular about this day was the broad make-up of the public, there were many people who would not normally have thought of participating in a science related event who wandered in from the parade outside Manchester Town Hall, and became thoroughly engaged and spent a couple of hours with the exhibits and events.
Year(s) Of Engagement Activity 2016
URL https://mcrbrainbox.wordpress.com
 
Description Schools and alumni outreach 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A practical based on our work was held three times in the Department. Twice to sixth-formers (local) and the third time to alumni of the University of York (all disciplines, 30 participants) during the weekend of the University 50th Anniversary Celebrations.

Contacts from alumni requesting visits for talks and to take the practical to schools.
Year(s) Of Engagement Activity 2013