Transgenic approaches to understanding astrocyte heterogeneity

Lead Research Organisation: University College London
Department Name: The Wolfson Inst for Biomedical Research

Abstract

The cells in our brain are generally divided into two major categories based on their function - neurons (commonly known as nerve cells) and glial cells. Neurons are very important to our body because they process and transmit information to control our actions in daily life, while glial cells are seen as playing supporting roles to neurons. We used to think that our brain was mainly run by neurons with a little help from glia. In recent years, with rapid advances in neuroscience, this view has started to change and we now recognize that brain function is the result of concerted activities of both neurons and glia. Astrocytes are an important class of glial cells defined by their star-like shape and other features. Despite the fact that they are the most abundant cells in the brain making up half of the brain volume, our knowledge about astrocytes is still rudimentary. Astrocytes are thought to be highly diverse, and our previous work found a link between their diversity and developmental origin - that is, we might be able to predict an astrocyte's function according to where it comes from in early development. Our proposed study aims to explore further how astrocytes diversify during development and after settling in their final resting site in the mature brain, where they come under the influence of micro-environmental signals from neurons and other cells in their vicinity. Based on our data so far, we hypothesize that astrocyte diversity is determined at both developmental and micro-environmental levels. We will test this hypothesis by experiments in mice. Through genetic ("transgenic") manipulation of mice we are able to label different populations of astrocytes with green or red fluorescent proteins and also to perform "genetic surgery" to remove particular astrocyte populations of interest. We aim to produce a map of the developmental origins of astrocyte sub-populations and to relate this to the adult functions of the astrocytes in, for example, supporting communication among neurons. In addition, we plan to identify new molecular markers for the different populations of astrocytes. The reason why we need to study astrocyte development and diversity is that different subtypes of astrocytes might be functionally distinct from each other and therefore differentially involved in brain disorders such as autism. Our proposed study will produce direct information about astrocyte functional diversity and provide useful tools for future astrocyte research that can be provided to the neuroscience community at large.

Technical Summary

Astrocytes are a major glial cell type in the central nervous system but, despite their great abundance and widespread distribution, their functional roles are still poorly understood. We recently found evidence (Tsai, Li et al. Science 2012) that the local function of astrocytes, e.g. in synaptogenesis, is influenced by their developmental site of origin in the embryonic neural tube. I now want to build on this work, focussing on the mouse forebrain, to test the idea that astrocyte functional heterogeneity is determined by developmental cues together with local environmental factors. First, I will map the origins of astrocyte sub-populations using the Cre/lox system and will conditionally ablate those populations with Diphtheria toxin A chain (DTA) in transgenic mice; this will allow me to determine whether astrocytes with different developmental origins (i.e. from different regions of the ventricular zone, VZ) are functionally interchangeable with respect to their adult function. Second, I will investigate whether there is further diversification driven by the microenvironment. I recently identified a glial-specific G-protein-coupled receptor, GPR37L1, which is expressed in a subset of mature astrocytes, unrelated to developmental origin. Preliminary evidence indicates that GPR37L1 is involved in glia-synapse interactions. I will examine the role of GPR37L1 expressing glia in synaptogenesis in GPR37L1 knockout mice and after targeted ablation of GPR37L1(+) glia using DTA. Finally, I will look for new markers for astrocyte subtypes by transcriptional profiling of sorted subsets of astrocytes on Affymetrix arrays and by RNA-seq. This project will provide new insights into astrocyte biology and will produce useful new tools for astrocyte research.

Planned Impact

Impacts on academic community. This project will significantly advance the research into astrocyte diversity - a basic topic in brain research. We will present solid evidence to prove that astrocytes, the most abundant cells in human brain, are heterogeneous and not just the brain glue supporting neurons, but actively involved in many brain functions. Our work will contribute new knowledge and research materials to the academic community and inspire other researchers to reevaluate astrocyte' roles in physiological and pathological conditions. The proposed transgenic methods will incentivize researchers to design their own projects for cell lineage studies. In the meantime, the postdoctoral research assistant and technical assistant employed on this project will benefit from excellent training in molecular biology, histology and neurobiology.

Impacts on Business/Industry community. This study will prove that astrocytes are more complex than originally thought. Our findings will help understand the mechanisms at work in neurological disorders and have the potential to attract the interest of pharmaceutical industry in developing/testing new drugs using our models.

Impact on funding bodies and charitable organizations. Astrocytes have been reported to be involved in many neurological diseases such as Multiple Sclerosis, Autism and Alzheimer's disease. Moreover, gene mutation in astrocytes may be the cause of some diseases (such as human Rett syndrome) to which primary neuronal dysfunction is generally attributed. Therefore, our findings could open the door to the possibility of astrocyte subset based cell replacement therapy for astrocyte related diseases. Our findings will help raise funding bodies' and charitable organizations' awareness of astrocyte research and attract more funding for astrocyte study.

Impacts on the public. Understanding how our brain works is of general interest to the public. Our research will show how fascinating our brain is in a new light and hopefully our work could inspire more of the younger generation to take up neuroscience research.
 
Description Our research funded on this grant is about finding the reason behind the diverse types and functions of astrocytes - the star-like cells in the brain and spinal cord. We have identified a membrane protein GPR37L1 which is expressed in a subtype of astrocytes. In the past years, we have studied the role that GPR37L1-positive astrocytes play in brain function by nullifying GPR37L1 ( gene knockout) in the mouse. We found that GPR37L1-positive and -negative astrocytes are coupled together into the same nerve network. Our data also show that GPR37L1 is up-regulated in astrocytes after ischemia in the mouse model. We revealed that GPR37L1 can protect neurons after induced ischemia in cultured brain slices. These new data has recently been published in Glia as the cover story (Jolly S, et al. Glia. 2018).
We have also identified 2 new astrocyte specific markers, both transcription factors, each of which is specifically expressed in a subtype of astrocytes. We will characterize these two new transcription factors in the next stage.
Exploitation Route Our data will be valuable for understanding astrocyte biology and astrocyte related neurological conditions. Our data will become part of the new knowledge that will convert into forces of production in the long term. As GPR37L1 is neuron-protective after ischemia, it will be a potential drug target for the treatment of ischemia.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Academy of Medical Sciences Newton International Fellowships (Dr Yajun Wang)
Amount £66,000 (GBP)
Organisation Newton Fund 
Sector Public
Country United Kingdom
Start 03/2017 
End 02/2019
 
Description Towards CRISPR/Cas9-mediated gene correction for inherited retinal disease (for Dr Jing An)
Amount £99,000 (GBP)
Funding ID NIF\R1\181649 
Organisation Academy of Medical Sciences (AMS) 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2019 
End 02/2021
 
Description Wenlin Li's Newton Advanced Fellowship (co-applicant)
Amount £111,000 (GBP)
Funding ID NAF001\1006 
Organisation Academy of Medical Sciences (AMS) 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2015 
End 03/2018
 
Description pilot project
Amount £50,000 (GBP)
Funding ID ARUK-PPG2017B-013 
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2017 
End 08/2018
 
Description researcher links workshops
Amount £20,800 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2016 
End 12/2016
 
Title Aldh1l1-lox-GFP-STOP-lox-DTA 
Description A transgenic mouse line in which astrocytes are ablated after crossbreeding with a specific Cre line 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Not yet. 
 
Title GPR-lox-GFP-STOP-lox-DTA 
Description Transgenic ablation mouse line to kill a group of astrocytes expressing GPR 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Not yet. 
 
Description Retina research with Xi'an Jiaotong University 
Organisation Xi'an Jiaotong University
Department School of Medicine Xi'an Jiaotong
Country China 
Sector Academic/University 
PI Contribution I am currently supervising a visiting fellow, Dr Jing An, from Xi'an Jiaotong University to perform collaborative research on the retina and also study cholesterol function in the retina.
Collaborator Contribution Xi'an Jiaotong University is paying for the living costs and salary of Dr Jing An in 2018.
Impact The visiting fellow has been given a Newton International Fellowship by Academy of Medical Sciences, starting from 01/03/2019.
Start Year 2017
 
Description SMMU 
Organisation Secondary Military Medical University
Country China 
Sector Academic/University 
PI Contribution We provided tissues from our transgenic mice for our collaborator and have been training a visiting fellow in cutting-age transgenic techniques and molecular biology skills
Collaborator Contribution The partner's side is committed to compound screening and iPSC culture
Impact The partner, Dr Wenlin Li, was awarded the Newton International fellowship by Academy of Medical Sciences in 2015, of which I am the co-applicant and the UK lead. We have made a set of BAC reporter constructs which will be used for small molecule screening. We aim to find the compounds that can facilitate stem cell differentiation into oligodendrocyte lineage cells. The visiting fellow has set up a platform for iPSC culture in our lab and is optimizing a protocol for myelinated cortex culture. Wenlin and I are co-organizing a workshop to be held later this month in China.
Start Year 2014
 
Description Stem cell research with Northwest University (China) 
Organisation Northwest University
PI Contribution I have been acting as a visiting professor in the college of life sciences, Northwest University (China)
Collaborator Contribution Northwest University (China) is planning to send visiting scholars to my lab in the coming year
Impact Northwest University (China) agrees to send visiting scholars to my lab and will pay for research costs of the visiting scholars
Start Year 2017
 
Description White Matter Diseases With GMC 
Organisation Institute of Aging Research
Country China 
Sector Academic/University 
PI Contribution I received a visiting fellow from the partner's institute in my lab who has since been trained in cutting-edge neuroscience research.
Collaborator Contribution Two visiting fellows whose salaries were paid by the partner institution has been working in my lab on a collaborative research project in 2016.
Impact We have been awarded a conference grant "collaborative strategies to study myelin and fight against multiple sclerosis and other white matter diseases" from British Council. This conference will help raise awareness of WMDs and revamp traditional clinical perceptions of WMDs in China and in the UK to the benefit of both WMD patients and WMD research. The workshop was co-organized by me and Prof Bin Zhao of GMC was taken place on 11-13 October 2016, Swindon UK. (1) In this workshop, participants from both China and the UK covered leading topics in the field of white matter diseases. Our workshop provided a chance for Chinese neurologists/neuroscientists to brainstorm and compare notes with their peers in Brain on MS and other white mater diseases. (2) This workshop provided an opportunity for early career researchers from both countries to interact with senior figures in the field. The career development session was well received among young participants. Heated discussions on how to become an independent researcher, apply for research grants, manage a research team and set up collaboration were carried out in the workshop. Two special guests with recent success in securing prestigious fellowships were invited to talk about their career paths. (3) Three special guests from the British Council, Multiple Sclerosis Society and Science and Technology Section of Chinese Embassy in London gave talks about available funding for China-UK joint research, encouraging participants to set up collaborations. (4) Several special guests/participants gave presentations on cutting-edge techniques for neuroscience research, which have the potential to be applied in MS/myelin research for new breakthroughs. Dr Yajun Wang of GMC was awarded for a Newton International Fellowship under my supervision starting from 01/09/2018
Start Year 2015
 
Description A lecture at Haberdasher's Aske's School for Boys 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact About 30 pupils attended this lecture. There were encouraging comments and enthusiastic questions on our work and research techniques used after the talk.
Year(s) Of Engagement Activity 2015
 
Description A lecture at North London Collegiate School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact About 40 pupils attended this lecture. There were encouraging comments and enthusiastic questions after the talk on the scientific content and on the research methods used.
Year(s) Of Engagement Activity 2015
 
Description A talk or presentation - London PhD network 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I gave a talk on the history of model neuroscience to the PhD students and postdocs in non-neuroscience field
Year(s) Of Engagement Activity 2017
 
Description Chinese embassy education sector - discussion 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact 20 scientists from British Universities were invited by the education sector of the Chinese embassy in London to discuss how to facilitate scientific collaboration between the two countries. The Chinese officials at the meeting pledged more funds for young scientist exchanging programs from both sides.
Year(s) Of Engagement Activity 2015
 
Description Chinese embassy scientific sector - discussion 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 40 scientists from British Universities were invited by the scientific sector of the Chinese embassy in London to discuss how to facilitate scientific collaboration between the two countries. It was also during Chinese New Year holiday season
Year(s) Of Engagement Activity 2017
 
Description Chinese embassy scientific sector - discussion 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact about 50 scientists from British Universities were invited by the scientific sector of the Chinese embassy in London to discuss how to facilitate scientific collaboration between the two countries. It was also during Chinese New Year holiday season
Year(s) Of Engagement Activity 2018,2019
 
Description Interview with Chinese News Agency 
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 Media (as a channel to the public)
Results and Impact Interview was published in several Chinese Newspapers.

After the interview, I received a number of emails asking for further questions.
Year(s) Of Engagement Activity 2014
URL http://news.xinhuanet.com/tech/2014-10/20/c_1112895163.htm
 
Description Interview with Chinese News Agency 
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 Media (as a channel to the public)
Results and Impact Interview was published in several Chinese Newspapers.
Year(s) Of Engagement Activity 2016
 
Description London PhD network 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact The aim of London PhD Network is to give London-based young Chinese scientists an opportunity to network and exchange ideas for cross-disciplinary collaborations between China and the UK. It also provides an interface for young PhD students to interact with senior researchers for inspiration and advice on career development. I coordinate/chair this Network.
Year(s) Of Engagement Activity 2015,2016
URL http://www.londonphd.org