Inflammatory regulation of cholesterol biosynthesis in a monogenic model of intracerebral haemorrhage

Intracerebral haemorrhage (ICH) is a type of stroke caused by bursting of blood vessels within the brain which can lead to significant disability and/or death. Unfortunately, no medications are currently available for patients. A number of risk factors exist which can increase the likelihood of a person experiencing an ICH. One of these risk factors is low cholesterol levels, but how and why this may be associated with ICH is poorly understood. Cholesterol is a fatty substance that is necessary for several essential bodily functions, such as providing structural support for blood vessels. Too much cholesterol can lead to an accumulation of fatty deposits within vessels which can cause blockages and serious health conditions such as heart disease and ischaemic stroke. However, in ICH, it has been suggested that a reduction in cholesterol may lead to a weakening of the blood vessels in the brain, making them vulnerable to rupturing and bleeding. However, to date, this has not been investigated experimentally. The aim of this research proposal is to study the molecules that are responsible for producing cholesterol in brain blood vessels by using zebrafish models and human cells.

Aicardi-Goutieres syndrome (AGS) is an inherited disease that occurs in children and young adults. AGS patients suffer from brain abnormalities and over-stimulation of an inflammatory molecule called type I interferon - which is normally only activated following a viral infection. Defects in 7 different genes (segments of DNA that encode specific proteins necessary for various body functions) can cause AGS. All of these patients experience the same brain and type I interferon features. However, one subgroup of AGS patients who carry defects in a gene called SAMHD1 also suffer from ICH. We do not know why ICH occurs specifically in these patients and not other AGS subgroups, but it suggests that SAMHD1 may have a previously unrecognised role in stabilising brain blood vessels. To help us try to understand this mechanism, animal models of the disease are required. Zebrafish are commonly used by scientists to model human conditions because they are 'see-through' and it is easy to watch how cells in the brain behave using powerful microscopes. We have made a zebrafish model which also have a defect in SAMHD1. Like the patients, these fish also have increased type I interferon levels and are susceptible to ICH. Genetic analysis has also revealed that these fish have a reduction in 14 genes that control the production of cholesterol. Remarkably, we have also shown that 8 of these genes are significantly reduced in ICH patient blood samples. These findings suggest that the development of ICH in this model is associated with increased type I interferon and a reduction in cholesterol production, and may also indicate a new biological function for SAMHD1 in AGS. Importantly, use of this model may provide essential clues into how and why low cholesterol may lead to ICH. This research proposal will allow us to continue to study these fish, alongside human brain blood vessel cells, so we can answer the following questions:

1: Does a SAMHD1 defect cause a reduction in cholesterol genes specifically in blood vessels in the brain?
2: Does a reduction in cholesterol genes and susceptibility to ICH occur in the SAMHD1 zebrafish model because of over-stimulation of type I interferon?
3: Does interference of the other 6 AGS-related genes result in a reduction in cholesterol genes?

To help answer these questions, we have designed a series of exciting experiments which will allow us to measure cholesterol genes and manipulate type I interferon levels, whilst visualising living brain blood vessels in fish and human cells. Answering these questions will provide new insight into SAMHD1-related AGS whilst also delivering important experimental evidence to support our understanding of how low cholesterol levels may lead to ICH.

Technical Summary
Hypocholesterolemia is a clinical risk factor for intracerebral haemorrhage (ICH). It has been postulated that as cholesterol is an essential membrane component, a reduction in levels may lead to neurovascular instability and proneness to rupture. However, this potential mechanism has not been investigated to date and will be addressed in this proposal. Mutation of the SAMHD1 gene causes one of the seven genetic subtypes of Aicardi-Goutières syndrome (AGS), an inherited childhood brain disorder characterised by upregulation of type I interferon (IFN). Clinically, SAMHD1-patients differ from other AGS subtypes in that they carry a high risk of ICH, suggesting a discrete role for SAMHD1 in neurovascular homeostasis exists. We have generated a mutant samhd1 zebrafish line which exhibit a significant upregulation of type I IFN and susceptibility to ICH. Furthermore, transcriptomic analyses indicate that samhd1 mutant larvae exhibit a significant reduction in expression of 14 cholesterol biosynthesis genes. Analysis of the Genotype-Tissue Expression database reveals that 8 of these genes are also significantly downregulated in ICH patients. Taken together these data suggest that ICH occurrence in this model may be associated with increased type I IFN signalling and an impairment in de novo cholesterol synthesis. The aim of this proposal is to use this model to (i) determine whether the cholesterol biosynthesis defect occurs specifically within brain endothelial cells and (ii) whether vulnerability to ICH is dependent on type I IFN signalling. Furthermore, we will utilise an in vitro approach to determine if disruption of other AGS genes causes a cholesterol synthesis defect in human brain endothelial cells. This work will reveal a previously unrecognised role for SAMHD1 function in the context of AGS and will highlight the need to consider anti-viral signalling and cholesterol biosynthesis as possible intervention targets for future ICH prevention.

Planned Impact
Who might benefit from this research?

The immediate beneficiaries of this research will be the Faculty of Biology, Medicine and Health and the Stroke Research Group here at the University of Manchester. The development of methodologies and the postdoctoral researcher will bring many benefits that will ultimately enrich the research environment. This project will benefit both basic science and clinical researchers working within the fields of intracerebral haemorrhage (ICH) and cerebrovascular disease, as well as those studying inflammation and/or lipid metabolism in health and disease. Furthermore, this work will benefit researchers working within the fields of Aicardi Goutières syndrome (AGS) and/or SAMHD1 biology. Most importantly, individuals at risk of ICH will ultimately benefit from this research in the future, which will also have a beneficial consequence on healthcare systems. Furthermore, AGS patient/carers will benefit from an increased understanding of the underlying pathophysiology associated with the disease, which may also offer therapeutic avenues in the future. We will present/publish our work at scientific conferences and in international high impact peer reviewed journals, which will benefit the wider scientific community and the University in general. The results of this proposal may ultimately contribute to inform strategic funding boards on the growing recognition of the importance of immune-lipid regulation in health and disease. We will continue to utilise public engagement to disseminate our discoveries to a wider audience. As such, the wider public will also benefit from education and exposure to science via activities run by my lab and other members of the Manchester Stroke Research Group.

How might they benefit from this research?

Researchers working within related disciplines (in Manchester and beyond) will benefit most immediately from the results generated through this study. Our data will inform the research community on novel mechanisms of neurovascular stability and inflammatory regulation of cholesterol synthesis, in the context of ICH and AGS. The innovative experiments we have designed, and the discoveries we make, will inspire other researchers to study comparable mechanisms in other related disease conditions, therefore our understanding of these biological processes will expand significantly. The award of this grant will employ a post-doctoral researcher who will develop their skills and experience under my supervision, which will also have a beneficial impact for other members of my lab and within the Stroke Research Group. As our work will improve our understanding of mechanisms that underlie stability of brain blood vessels, it holds the potential to contribute to a reduction in ICH rates on a global scale - by highlighting the need to monitor cholesterol levels more closely in individuals at risk of ICH. No treatments are available for AGS patients and our understanding of the disease pathophysiology is limited. Therefore, this work will help patients/carers understand their condition better and offer hope for potential new therapeutic avenues in the future. The wider pubic will benefit from this research as we hope our results will influence their perception of the role of cholesterol in health and disease.