Determining the effects of genetic variation and early life stress on the regulation of the galanin gene in fat and alcohol selection.

Lead Research Organisation: University of Aberdeen
Department Name: Sch of Medicine, Medical Sci & Nutrition

Abstract

Overindulgence in high calorie nutrients, which include fat and alcohol, has had a major effect on the health of the UK population where 67% of males and 57% of females are classed as overweight. Because obesity is linked to type 2 diabetes and cardiovascular disease we need to find the causes of this calorie overconsumption and a major source of the calories consumed by the UK population come in the form of fat and alcohol. In addition to cardiovascular disease and cancer, excessive alcohol consumption is also linked to liver cirrhosis and pancreatitis. Many of us are susceptible to over consume fat and alcohol as a result of differences in our DNA but there is also evidence that environmental factors such as early life stress also play a role. However, we still know very little about the mechanisms that influence our intake of fat and alcohol and how these mechanisms are affected by genetic variation and environment. Important clues have emerged. For example, a small neuropeptide, called galanin is expressed in a region of the brain called the hypothalamus that controls fat and alcohol intake. Removal of the gene which encodes galanin reduces fat and alcohol intake and decreases weight gain. A critical aspect of the function of galanin is that it should be expressed in the correct cells, in the correct amount and in response to the correct stimuli. However, nothing was known about the mechanisms controlling the production of galanin in the hypothalamus or how differences in the regions of DNA that control this expression might affect fat and alcohol intake. We first discovered a DNA switch sequence that was an excellent candidate for the undiscovered switch sequence that controls galanin in the hypothalamus. We then found that sequence changes within this switch that exist within the human population, and were linked to alcohol abuse, increased the strength of this switch in the hypothalamus. We also found epigenetic changes within the switch, called DNA methylation, that were altered by stress and seemed to also change the strength of the switch. We hypothesise that these sequence changes and methylation changes within the GAL gene switch work together to alter the production of galanin in the hypothalamus and to influence fat and alcohol intake. To address this hypothesis we used a remarkable new method called CRISPr genome editing to quickly and efficiently delete the switch from the genome. We will initially determine the effects of removing the switch on galanin production in the hypothalamus and determine its effects on fat and alcohol selection. We will use CRISPr technology again to reproduce known human sequence changes within the switch (a process known as humanisation) to model their effects on galanin production and fat and alcohol intake. We will then use these "humanised" models to determine the effects of early life stress on the methylation of the switch sequence and how this methylation affects its function. Using this revolutionary CRISPr technology we will determine how changes that occur naturally in the human population, as a result of genetics or environment, affect our fat and alcohol intake. Considering the huge problem of obesity and excessive alcohol intake in our society these studies will open avenues for not only understanding the role of genetics and environment in excess fat and alcohol intake but will also provide novel opportunities for reducing susceptibility to obesity by controlling excess calorie intake in the human population.

Technical Summary

We will determine the effects of genetics and epigenetics on the mechanisms that control galanin gene (GAL) expression in the paraventricular nucleus (PVN) where galanin controls fat and alcohol intake. Excess fat and alcohol contribute to obesity that is a risk factor in diabetes and cardiovascular disease. Alcohol abuse is also linked to cirrhosis and pancreatitis. We need to know;
1. What happens to the expression of galanin and fat and alcohol intake when you remove the regulatory elements that control GAL expression in PVN.
2. What are the effects of genetic variation on these regulatory elements and in fat and alcohol intake.
3. How are these regulatory elements affected by epigenetic modification as a result of early life stress.
We identified a regulatory element (GAL5.1) that is active in GAL expressing cells of the PVN. Polymorphisms within GAL5.1, associated with alcohol abuse, have a significant effect on its activity. Early life stress was shown to increase GAL mRNA in the PVN and led to changes in GAL5.1 methylation. We hypothesise that GAL5.1 is important in the regulation of GAL in the PVN and that polymorphisms and epigenetic modification influence the expression of GAL that, in turn, influence fat and alcohol intake. We have used CRISPr technologies to knock out GAL5.1 from the mouse genome and will use GAL mRNA expression analysis and fat/alcohol intake tests to determine the effects of GAL5.1 deletion. Humanisation of GAL5.1 in mice using CRISPr technologies to reproduce both human polymorphisms will permit a comparison of alleles on GAL expression and alcohol/fat intake. Humanisation will also permit an analysis of the effects of early life stress on the methylation of the human GAL5.1 in-vivo whose CpG content and distribution differs from mouse. The unique studies described in this proposal will revolutionise our understanding of the roles of gene regulation in nutrition and will have important ramifications for obesity research.

Planned Impact

Our research will contribute fundamental new knowledge relating to the mechanisms regulating fat and alcohol intake with a range of potential beneficiaries in the academic, commercial and public sectors. To maximise engagement with these stakeholders and deliver the widest possible impact from our findings, we will pursue the following specific objectives:
1: Impact through training. The current proposal will permit the further training of a highly skilled and motivated post-doctoral scientist; Scott Davidson, in the role of genetics and epigenetics in the control of fat/alcohol intake. Dr. Davidson will receive training in new and powerful technologies such as CAS9/CRISPr genome editing, that Dr. MacKenzie has adopted and mastered in Aberdeen, epigenetic analysis of early life stress, genomics, mRNA expression analysis and in-vivo physiological and metabolic studies. This training will allow Dr. Davidson to eventually progress as an independent researcher within the UK.
2: Expansion of current public engagement activities.
An immediate impact of this project is its opportunity to raise public awareness and understanding of the role of gene regulation in the control of appetite and nutrient selection. Additionally, engaging with school children is vital, not only for educating in the benefits of a healthy diet and the role of genetics and environment in its control but also for inspiring the next generation of researchers in nutrition. Throughout the project, we will engage with the public in the following ways:
(a)To maximise the availability of our research, we will publish the results from this project in high-impact journals with Open Access Compliant policies. We will also promote the findings of our studies on our respective University web pages and in the national and international press.
(b)We will continue to make use of the University of Aberdeen Press Office, as well as the BBSRC media services, to disseminate our findings as widely as possible to the general public.
(c)At least once a year we will continue our outreach activities with school children .
(d)Once a year we will engage the public through public seminars and science festivals.
(e)We will continue to use the University of Aberdeen Public Engagement with Science Unit (PERU) to keep us updated on additional public engagement opportunities to expand our current activities.
3: Establish formal links with clinical science .
Our published work the identification of functional variation within the GAL5.1 enhancer has inspired at least two major patient based association analysis that established a significant association between GAL5.1 haplotypes and increased alcohol consumption in certain populations. We will continue to engage the clinical community by;
(i) Publishing our results in a timely manner and continuing to chair and deliver presentations at national and international conferences attended by both scientists and clinicians.
(ii) Seek formal collaborations with clinicians to explore potential clinical applications of the planned research.
4: Commercial exploitation of research outcomes.
We have had success in collaborating with industrial partners as reflected by our current partnership with GW Pharmaceuticals. We will continue to seek further industrial collaborations and have organised meetings with representatives from Eli Lilly, Alder pharmaceuticals and Pharmnovo who I have invited to speak at Neuropeptides-2015 (a conference organised by AM in Sept 2015) and who have an interest in discussing the implications of genetic and epigenetic variation on appetite and patient drug responses. We will also seek assistance of University bodies such as the Kosterlitz Centre to seek further links with academic and industrial partners. We will also work with the Research and Innovation Department at The University of Aberdeen who will review opportunities for commercialisation throughout the project.

Publications

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MacKenzie A (2017) Neuropeptides-2015, Aberdeen University, Scotland. in Neuropeptides

 
Description Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n=115,865; p=0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans that was consistent with our previous UK Biobank studies. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men. This study has been accepted for publication by the journal Molecular Psychiatry which is one of the top journals in the field.
Exploitation Route Our data suggests that comparative genomics, CRISPR genome editing and mouse behavioural analysis are highly effective methods of determining the role of the non-coding genome in health and disease. In addition, by collaborating with Andrew McIntosh and Toni Clarke at the University of Edinburgh we have found a significant association between increased alcohol intake in human males the GG allele of our enhancer and anxiety. These studies have been published on line (MS ID#: BIORXIV/2019/572065
MS TITLE: CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests its role in anxiety and male alcohol intake.).
We have also been able to complete another manuscript (MS ID#: BIORXIV/2019/544585
MS TITLE: Genetic, epigenetic and pharmacological influences modulating tissue specific regulation of the cannabinoid receptor -1 (CB1) gene; implications for cannabinoid pharmacogenetics.) whose work was also partially funded by the BBSRC grant.
I have also just completed an article in collaboration with The Conversation.
Sectors Agriculture, Food and Drink,Creative Economy,Education,Healthcare,Pharmaceuticals and Medical Biotechnology,Other

URL https://www.biorxiv.org/content/10.1101/572065v2
 
Description My findings have been reported online in The Conversation and I have given public talks in the Aberdeen Science centre (12th March 2019) as part of British Science Week. I have also been booked to deliver a talk at Mayfest on Saterday 25th of May 2019.
First Year Of Impact 2019
Sector Agriculture, Food and Drink,Education,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Societal

 
Description A novel in-vivo CRISPR enhancer screen to identify polymorphic regulatory regions controlling the hypothalamic expression of appetite regulating neuropeptides.
Amount £25,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 04/2018
 
Description Characterisation of a novel selectable cell-surface regulator of cardiovascular progenitors for stem cell-mediated regeneration.
Amount £196,608 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 11/2022
 
Description Determining how the anxiety and alcohol intake modulating GAL5.1 enhancer works
Amount £12,000 (GBP)
Organisation Tenovus Cancer Care 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2019 
End 12/2020
 
Description Determining the effects of genetic variation and early life stress on the regulation of the galanin gene in fat and alcohol selection.
Amount £418,914 (GBP)
Funding ID BB/N017544/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2019
 
Title Production of ECR1 enhancer knockout mice using CRISPR genome editing 
Description We have used CRISPR genome editing to produce a knockout mouse lacking the polymorphic ECR1 enhancer that resides in intron 2 of the cannabinoid-1 receptor gene (CNR1). Analysis of this line demonstrates a reduction of CNR1 expression in the hippocampus and hypothalamus with concurrent changes in ethanol intake and hypothermia response following agonism of the CB1 receptor. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? Yes  
Impact We are in the process of completing a manuscript describing our findings to two journals; Neuropsychopharmacology and Neuroinflammation. 
 
Title Production of GAL5.1 enhancer knockout mice using CRISPR genome editing 
Description We used CRISPR genome editing to delete an enhancer that controls the expression of the galanin gene in the PVN of the mouse. Analysis of this knock out line shows a decrease in GAL expression in the PVN and a significant reduction in fat and alcohol intake. A manuscript describing these findings is being revised with a view to publication in Cell Reports. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? Yes  
Impact A manuscript describing these findings is being revised with a view to publication in Cell Reports. 
 
Title Transgenic animal models 
Description Transgenic animal lines containing reporter constructs driven by enhancers of the TAC1, GAL and CNR1 genes 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact Expression data produced using these transgenic models have contributed to a great many research papers (see outputs). 
 
Description Contributed an article in "The Conversation" about human evolution 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The title of the piece was " Its time we stopped human evolution" and it recieved 71,617 reads and 120 contacts.
Year(s) Of Engagement Activity 2019
URL https://theconversation.com/its-time-we-stopped-human-evolution-geneticist-116544#comment_1945368
 
Description Contributed an article in "The Conversation" about the genome. 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was asked to produce an article for "The Conversation" by the Scotland editor Steve Vass.
Year(s) Of Engagement Activity 2019
URL https://theconversation.com/human-genome-project-new-alcohol-abuse-study-could-help-us-finally-unloc...
 
Description Interview on Gaelic Radio about bacteria and human health 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I was interviewed about the role of bacteria in health and disease. The programme has yet to be scheduled.
Year(s) Of Engagement Activity 2019
 
Description Interview on SHMU radio 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact An interview on SHMU FM about my career as a scientist and the subject of my current studies
Year(s) Of Engagement Activity 2017
 
Description Talk at Cafe Scientifique entitled "Health and the disposable cave man; understanding disease using evolution." 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A talk on my research for a general audience. Attended by nearly 100 members of the public who took a great interest. My work on CRISPR genome editing forged a collaboration with a Prof of Rice genomics who was in the audience (Prof Alan Price) and we wrote a grant together to the BBSRC. The outcome is pending.
Year(s) Of Engagement Activity 2017
 
Description Talk at the the Aberdeen Science centre as part of British science week entitled "Tales form the dark side". 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 60 members of the general public attended a talk to introduce the subject and justification for my research. I was told afterwards that the talk was pitched at the right level and a great deal of discussion followed from interested members of the general public.
Year(s) Of Engagement Activity 2019
URL https://www.facebook.com/events/248617896013636/