DETERMINING THE ROLE FOR EXOSOMES DURING CELLULAR SENESCENCE AND AGEING

Lead Research Organisation: Queen Mary, University of London
Department Name: Blizard Institute of Cell and Molecular

Abstract

The number elderly population in the UK has dramatically increased in the last few decades. As a consequence, conditions associated with ageing, such as cancer, cardiovascular and neurodegenerative diseases, are having a huge impact on the public health system and the UK economy. It is therefore imperative to promote research that will improve our understanding of the mechanisms implicated in the maintenance of health across life. The primarily hallmark of ageing is the decline in the tissue homeostasis. However, the precise basic and cellular mechanisms implicated in the ageing process are not well established, hindering the advances in understanding how healthy ageing happens.

The organs and tissues in our body are formed by a vast number of cells, which altogether co-ordinate their actions for our body to function properly. However, a number of "abnormal" cells have been found in tissues derived from old patients. These particular cells suffer a growth arrest or lack of proliferation termed "senescence", which is thought to affect how the tissue functions. Senescent cells fail to proliferate, but they manage to communicate with their neighbour cells, mainly through the release of inflammatory proteins. In this application we will focus on a different way of intercellular communication during senescence, the release of small extracellular vesicles, called exosomes. Exosomes contain biological material in the form of proteins, RNA or small RNAs called microRNAs. Exosomes can release their inside content into particular cells, which as a consequence induce phenotypic changes in the target cells. Our preliminary data show that senescent cells secrete a high number of exosomes and, as a consequence, induce senescence in neighbouring cells. This application will explore the molecular mechanisms by which exosomes released from senescent cells induce senescence in neighbouring cells. We will also determine the inside content of these particular exosomes, in addition to the inside content of exosomes secreted by cells donated by elderly patients, to study similarities and differences between both models. Finally, we will focus on the functionality of exosomes using three-dimensional models of skin culture. During this application we will use a range of approaches, including human cell culture, high-resolution imaging techniques, state-of-the-art proteomics and genomics and an in vitro human skin model. Altogether, this project will help understand the mechanisms by which exosomes regulate normal ageing and it will allow to improve human health and wellbeing throughout life.

Technical Summary

The overall hypothesis of this project is that exosomes secreted by senescent and ageing cells play a key role in inducing senescence and ageing in a non-cell autonomous fashion. The secretion of inflammatory proteins, termed senescence-associated secretory phenotype or SASP, has been described as the main mechanism for senescent cells to communicate with their environment. However, most cells also signal through the release of extracellular vesicles (EV) termed exosomes, but little is known of their role during senescence or ageing. Exosomes contain a subset of proteins, lipids and nucleic acids that can change in composition depending on the parent cell. Our preliminary data show that senescent cells release a higher number of exosomes than proliferating cells. We show that exosomes from senescent cells express the cell adhesion protein integrin beta 3 and can induce senescence in other cells. This application proposes to study the mechanism(s) by which these exosomes induce senescence by studying the role of integrin beta 3 and investigating the role that other kinases play. We will also determine the cargo of exosomes from senescent and elderly patient cells and study their function using human skin organotypic models. Altogether, we propose a programme of research that aims to determine the role of exosomes secreted by senescent and old patient-derived cells in modulating the behaviour of cells in the surrounding environment. Data obtained from this proposal will give an extensive overview of the basic mechanisms for exosome function during ageing and senescence. In fact, it will advance our knowledge of the biology of normal ageing and will lead to approaches to improve healthy ageing.

Planned Impact

There are currently more than 10 million people in the UK over 65 years old, with an estimation for a significant increase within the next few years. The UK's ageing population has considerable consequences for public spending and the UK economy, due to the development of a number of age-associated diseases, such as cardiovascular, neurodegenerative and cancer. Consequently, there is an urgent need to promote research that will lead to a healthy lifespan across the entire life course.

The output of this piece of research will benefit a large number of sectors within the UK:

Academic and researcher peers will benefit from this proposal by providing new knowledge and basic scientific advancement of intercellular communication mechanisms during senescence and ageing. This will include not only peers within the immediate professional circle, but also professionals within other research areas such as cancer, immunobiology and fibrosis, where senescent cells have been identified in vivo.

Pharmaceutical companies will benefit from this project if we discover new kinases regulating exosome-induced senescence. We hypothesise that blocking the long-term accumulation of senescent cells induced by exosomes release and spread will improve healthy ageing in humans. In fact, the Van Deursen Lab has successfully demonstrated in animal models of premature ageing, that the elimination of senescence cells alleviates different ageing-related phenotypes.

The general public will also benefit from this research if it leads to the development of therapies improving healthy ageing and increasing the quality of life throughout our lifecourse. It will also benefit by increasing the public awareness and the scientific understanding of how healthy ageing occurs and by explaining how basic research impacts on the UK society and economy. In the event of finding very specific cargo contents in exosomes secreted by senescence cells and age-derived fibroblasts, future investigations will lead to the discovery of specific ageing biomarkers in a relatively short-term.

Our research will provide the UK Life sciences community with highly trained and exceedingly skilled scientists who will have received training in public engagement and have a clear emphasis on keeping the public informed about research discoveries in this field, as this is a QMUL number one priority. This is a benefit to the UK Life sciences sector by increasing UK Life sciences expertise, which will eventually help the UK science economy.

The UK Economy will also greatly benefit from the results obtained from this application. There are currently a large number of elderly people in the UK and, more than 65% of the Department for Work and Pensions benefits expenditure goes to this sector. This poses a huge burden on the UK economy and the NHS sector. Our research will lead to understanding the basic mechanism(s) for how normal and healthy ageing occurs and it will help promote healthy ageing and avoid the overuse of the NHS public spending.

Publications

10 25 50
 
Description Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response termed senescence-associated secretory phenotype (SASP) 1-3. Although senescent cells can stay without proliferating for long periods of time, they are metabolically and transcriptionally active and communicate with their microenvironment through the release of inflammatory proteins. The SASP is important in influencing the behaviour of neighbouring cells and altering the microenvironment and tissue function; yet, this role has been mainly attributed to soluble factors. In this research project, we proposed an alternative way for senescent cells to communicate with other cells. We hypothesised that exosomes or small extracellular vesicles (sEV) released by senescent cells play a key role in inducing senescence and ageing in proliferating cells via a non-cell autonomous fashion 1.

The strong preliminary data from our previous proposal confirmed our initial hypothesis. As proposed in Aim1, we demonstrated that sEV are capable of transmitting paracrine senescence to nearby cells named exosome-induced senescence, ExIS, herein 4. To address this, we isolated sEV using a variety of techniques and characterised the isolated sEV employing a diversity of methods as recommended by the International Society of Extracellular Vesicles guidelines -MISEV2018- to which we contributed 5,6. Confirmation of the senescent phenotype in proliferating cells treated with sEV from senescent cells was determined by transcriptomics analysis and validation. EV internalisation was determined following different techniques including but not limited to confocal microscopy. Importantly, we further confirmed a direct functional correlation between individual cell EV internalization and the establishment of the senescent phenotype using a Cre-loxP reporter system. The use of inhibitors and siRNA targeting neutral sphingomyelinase enzyme, which is important for the release of EV, prevented the transmission of paracrine senescence. As expected, the transmission of paracrine senescence mediated by sEV is dependent on the activation of classical senescence pathways such as p16INK4A and p21CIP. In addition, a MAPK screen to determined signalling pathways implicated in ExIS paracrine senescence identified the p38MAPK (mitogen-activated protein kinases), mTOR (mammalian target of rapamycin) and IKK (I?B kinase) pathways as important in ExIS. In fact, we found that the canonical NF-?B pathway plays a key role in ExIS transmission 7.

"Omics" analysis of the sEV content described in Aim2 showed differential proteomics and transcriptomics expression between sEV isolated from proliferating and senescent cells. Importantly, bioinformatics analysis comparing mass spectrometry analyses of a previously published soluble fraction and the sEV fraction released by senescent cells in this project show little correlation suggesting that a particular sEV-specific secretome might exist 4,8. These findings have been further confirmed by a recent independent report 9. The top proteins and microRNA found to be enriched in sEV from senescent cells have been further validated. In addition, by combining functional assays manipulating the sEV protein content with functional siRNA screens we identified two proteins implicated in the interferon pathway (IFITM3 and MX1) as being partially responsible for the transmission of paracrine senescence to proliferating cells 4.

Regarding the physiological relevance of sEV in senescence and ageing described in Aim3 we have shown an increase in the number of sEV released in the plasma of healthy old human donors (?80 years) in comparison with young donors (?33 years) 4. Interestingly, we found an increase in the protein expression levels of IFITM3 in these sEV isolated from the old human donors versus sEV from young donors 4. We have also confirmed that sEV isolated from human primary fibroblasts cultures derived from elderly donors (?80 years) transmit paracrine senescence to human primary fibroblasts cultures derived from young donors (?2 years) suggesting that sEV could play a role in the transmission of ageing 7.




Objectives achieved beyond the described aims

We found an increase in the number of multivesicular bodies (MVB) within senescent cells by electron microscopy in a pancreatic mouse model of senescence in collaboration with the University of Jerusalem and King's College. Importantly, we established a new collaboration with the University of Cambridge where we found that the cells staining positive for senescence-associated-?-galactosidase (SA-?? -Gal), a biomarker for senescence, by immunohistochemistry in lung fibrotic lesions from human patients co-stained with CD63, a tetraspan biomarker for EV 4.

In addition, as an increase in the release of sEV during senescence independent of the trigger occurs, we further hypothesised that the lipid biosynthesis pathway could be altered during senescence. In fact, we found that a main regulator of lipid synthesis, the fatty acid synthase enzyme (FASN) was an important regulator of senescence. Thus, blocking FASN activity not only prevented senescence via p53 activation but also blocked the release of sEV 10.

We have also unveiled a role for sEV in delaying ageing both in aged human cell cultures and naturally aged mice 11,12. Thus, sEV have the potential to be considered as rejuvenation therapy 11,13. Aged human cell cultures and mice tissue contain a mixed population of both proliferative and senescent cells. While we have previously unveiled the effect that sEV from senescent cell have on proliferating cells 4, here we have found that sEV from proliferative cells also influence senescent cells. We have found that sEV from proliferative cells contain a glutathione-S-transferase enzyme (GST) conferring sEV with GST activity. As a consequence, sEV from proliferative cells confer GST activity to human aged cell cultures and mice aged tissues such as liver, kidney, adipose tissue and serum. These changes in the glutathione metabolism pathway influence the oxidation of lipids and induce cellular and tissue rejuvenation 12.


REFERENCES

1 O'Loghlen, A. Role for extracellular vesicles in the tumour microenvironment. Philos Trans R Soc Lond B Biol Sci 373, doi:10.1098/rstb.2016.0488 (2018).
2 Fafian-Labora, J. A. & O'Loghlen, A. Classical and Nonclassical Intercellular Communication in Senescence and Ageing. Trends Cell Biol 30, 628-639, doi:10.1016/j.tcb.2020.05.003 (2020).
3 Prasanna, P. G. et al. Therapy-Induced Senescence: Opportunities to Improve Anticancer Therapy. J Natl Cancer Inst 113, 1285-1298, doi:10.1093/jnci/djab064 (2021).
4 Borghesan, M. et al. Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3. Cell Rep 27, 3956-3971 e3956, doi:10.1016/j.celrep.2019.05.095 (2019).
5 Carpintero-Fernandez, P., Fafian-Labora, J. & O'Loghlen, A. Technical Advances to Study Extracellular Vesicles. Front Mol Biosci 4, 79, doi:10.3389/fmolb.2017.00079 (2017).
6 Thery, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7, 1535750, doi:10.1080/20013078.2018.1535750 (2018).
7 Fafian-Labora, J. A. & O'Loghlen, A. NF-kappaB/IKK activation by small extracellular vesicles within the SASP. Aging cell 20, e13426, doi:10.1111/acel.13426 (2021).
8 Acosta, J. C. et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Biol 15, 978-990, doi:10.1038/ncb2784 (2013).
9 Basisty, N. et al. A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol 18, e3000599, doi:10.1371/journal.pbio.3000599 (2020).
10 Fafian-Labora, J. et al. FASN activity is important for the initial stages of the induction of senescence. Cell Death Dis 10, 318, doi:10.1038/s41419-019-1550-0 (2019).
11 Rodriguez-Navarro, J. A. et al. Extracellular vesicles as potential tools for regenerative therapy. Mol Cell Oncol 7, 1809958, doi:10.1080/23723556.2020.1809958 (2020).
12 Fafian-Labora, J. A., Rodriguez-Navarro, J. A. & O'Loghlen, A. Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage. Cell Metab 32, 71-86 e75, doi:10.1016/j.cmet.2020.06.004 (2020).
13 O'Loghlen, A. The potential of aging rejuvenation. Cell Cycle, 1-6, doi:10.1080/15384101.2021.2013612 (2022).
Exploitation Route We believe, the identification of a role for small extracellular vesicles during cellular senescence, ageing and age-related diseases has opened new lines of research into the files in addition to the opportunity to take the results of this project to pharmaceutical companies.
Sectors Pharmaceuticals and Medical Biotechnology

URL https://sites.google.com/view/theologhlenlab
 
Description MISEV guidelines
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Becas postdoctorales
Amount € 120,000 (EUR)
Organisation Government of Galicia 
Sector Public
Country Spain
Start 06/2017 
End 06/2020
 
Description Collaboration with an industrial partner
Amount £84,000 (GBP)
Organisation Reneuron 
Sector Private
Country United Kingdom
Start 01/2017 
End 01/2018
 
Description DMM Conference Travel Grant - CTG-DMM181175 - 20th International AEK Cancer Congress
Amount £600 (GBP)
Funding ID CTG-DMM181175 
Organisation Company of Biologists 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2019 
End 03/2019
 
Description Extracellular vesicles rejuvenation potential in ageing
Amount £38,000 (GBP)
Funding ID MGU0497 
Organisation Barts Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2020 
End 01/2021
 
Description Life Science Initiative - Centre for Genomics and Child Health
Amount £5,000 (GBP)
Organisation Queen Mary University of London 
Sector Academic/University
Country United Kingdom
Start 06/2018 
End 12/2018
 
Description Papel de la autofagia glial en neurodegeneracio´n y envejecimiento
Amount € 150,000 (EUR)
Organisation Ministry of Economy, Industry and Competitivity, Spain 
Sector Public
Country Spain
Start  
 
Description Royal Society of London
Amount £15,000 (GBP)
Funding ID RG170399 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 04/2019
 
Description The potential of extracellular vesicles as a therapeutic approach to prevent ageing
Amount £282,000 (GBP)
Funding ID G-002158 
Organisation Barts Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2022 
End 12/2025
 
Title Proteomics for exos from senescent cells 
Description Proteomic analyses of exosomes derived from cell undergoing senescence induce by different triggers. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact Not yet as it hasn't beed released to the public 
 
Title RNA seq of exoPS 
Description Database on RNA seq of exosome-induced senescence 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact No impact yet as it has not been released 
 
Description Amaia Lujambio collaboration 
Organisation Mount Sinai Hospital (USA)
Country United States 
Sector Hospitals 
PI Contribution We provided intelectual input on a project and the publication of a manuscript.
Collaborator Contribution Provided a novel experimental approach for the publication of a paper and contacts to obtain a particular cell line.
Impact 10.1016/j.celrep.2017.02.012
Start Year 2016
 
Description Anna Vossenkamper 
Organisation Queen Mary University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimentla design
Collaborator Contribution Provided samples and performed experiments
Impact Manuscript in preparation
Start Year 2018
 
Description CNIO proteomics unit - Javier Munoz 
Organisation Spanish National Cancer Research Center
Country Spain 
Sector Public 
PI Contribution Expertise and intelectual contribution
Collaborator Contribution Expertise in mass spectometry analysis and intelectual input in data analysis
Impact Exosomes are key regulators of non-cell autonomous intercellular communication in senescence (submitted manuscript)
Start Year 2017
 
Description Daniel Munoz-Espin 
Organisation University of Cambridge
Department Department of Oncology
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input onthe experimental design
Collaborator Contribution This collaboration provided access to human samples and technical help by staining and analysing the samples.
Impact A joint publication is currently under preparation
Start Year 2018
 
Description Genomics Centre Bart Cancer Institute 
Organisation Queen Mary University of London
Department Barts Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input in the design of the experiments
Collaborator Contribution Collaboration finding a transcriptional profile in primary cells treated with the exosomes derived from a variety of control and senescent-derived exosomes
Impact Identification of a new trigger of senescence involving non-cell autonomous signaling.
Start Year 2017
 
Description Hector Peinado 
Organisation Spanish National Cancer Research Center
Country Spain 
Sector Public 
PI Contribution Provided intellectual input into novel findings regarding exosome biogenesis
Collaborator Contribution Intellectual contribution into exosome biogenesis
Impact We currently have a manuscript in preparation
Start Year 2017
 
Description Ittai Ben-Porath 
Organisation Hebrew University of Jerusalem
Country Israel 
Sector Academic/University 
PI Contribution Intellectual input and helped perform, analyse and take electron microscopy images.
Collaborator Contribution Provided pancreatic mouse tissue and contributed to discuss the data obtained
Impact We currently have a publication under preparation.
Start Year 2017
 
Description Jose Antonio Rodriguez Navarro 
Organisation Hospital Ramón y Cajal
Country Spain 
Sector Hospitals 
PI Contribution I provided staff and expertise in cellular senescence
Collaborator Contribution Expertise in determining ageing in vivo
Impact Publication under preparation Patent under-revision
Start Year 2019
 
Description Proteomics Francis Crick London 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Intelectual input that led to a publication
Collaborator Contribution Provided expertise in mass spectometry and data analysis
Impact 10.1016/j.celrep.2017.02.012
Start Year 2013
 
Description Rob Lowe 
Organisation Queen Mary University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided RNA seq data and samples
Collaborator Contribution Provided data from RNA sequencing samples and experimental design
Impact Manuscript in preparation
Start Year 2018
 
Description Roland Fleck - KCL 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided samples for analyses and contributed to the staining of samples
Collaborator Contribution Performed, in collaboration with members of my group, staining of samples and helped with the imaging of the samples
Impact Manuscript in preparation
Start Year 2017
 
Description Centre of the Cell 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Discussion aboutn my line of work with Sedondary School pupils
Year(s) Of Engagement Activity 2018,2019,2020
 
Description Interview for MedicalResearch.com 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview by Medical Research.com
Year(s) Of Engagement Activity 2017
URL https://medicalresearch.com/author-interviews/why-cells-fall-apart-protein-regulates-cell-aging/3270...
 
Description Interview for the New Channel CCN 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview by the television news channel CCN
Year(s) Of Engagement Activity 2017
URL http://en.cncnews.cn/news/v_show/63929_Study_makes_Anti-ageing_breakthrough.shtml