Modelling the molecular pathogenesis of triple A syndrome (AAAS) with iPSC-derived neurons and adrenocortical cells.
Lead Research Organisation:
Queen Mary University of London
Department Name: William Harvey Research Institute
Abstract
Oxidative stress (OS) occurs when the body's natural antioxidant defences fail to protect against damage from free radicals, produced by essential processes in the body. OS underlies numerous degenerative conditions, including Triple A syndrome (AAAS). AAAS is a rare genetic disorder, which presents in early childhood. It results in progressive, debilitating problems including adrenal gland failure, which is life threatening if untreated, and severe degeneration of the nervous system.
Treatments for AAAS are aimed at symptomatic relief rather than preventing the devastating impact on quality of life or early death. AAAS results from mutations in the AAAS gene, responsible for the production of ALADIN, a poorly understood protein thought to be critical for many essential cellular processes. A detailed understanding of how mutations in this gene cause AAAS is necessary to aid the development of treatments and potentially slow the adrenal and nervous system damage. Research in this field also has wider implications for other adrenal and degenerative disorders of the nervous system.
The existing disease models used to study AAAS all have significant shortcomings. Obtaining adrenal or brain tissue from patients is not feasible. Most research is performed using cells obtained from patients' skin, which is not typically affected in AAAS. Other studies use cancer cells, which differ significantly from normal and patient cells. The AAAS animal model does not exhibit the classical features of the disease. Therefore in collaboration with the Sanger Institute, I have developed gene-edited human stem-cell models of AAAS. These will be used to generate AAAS adrenal and brain tissue cells, providing near-natural conditions to study AAAS in the tissues primarily affected by the disease.
These novel disease models will be used to uncover the abnormal cell processes and drivers of OS in AAAS. Through a rigorous series of experiments, I will investigate how AAAS affects the normal functioning of adrenal and brain tissue and what abnormalities underlie the disease process. I will use RNA sequencing , a method used to examine any changes in gene expression that result from AAAS disease. By examining the activity of genes in diseased compared to healthy cells, I will be able to identify key difference that can be attributed to AAAS and signpost ALADIN's critical roles within the cell. Another hypothesis is that mutations in the AAAS gene cause problems in the cell's energy generators, called mitochondria. If my experiments support this theory, I will undertake experiments to examine this in detail.
Ultimately, I aim to identify potential therapeutic targets that could be targeted to slow or halt disease progression in AAAS and other degenerative diseases. Adrenal stem cell modelling is a new field of research and information gathered by this project will further advance this field, leading to the use of this technology as a treatment for patients with adrenal failure.
Treatments for AAAS are aimed at symptomatic relief rather than preventing the devastating impact on quality of life or early death. AAAS results from mutations in the AAAS gene, responsible for the production of ALADIN, a poorly understood protein thought to be critical for many essential cellular processes. A detailed understanding of how mutations in this gene cause AAAS is necessary to aid the development of treatments and potentially slow the adrenal and nervous system damage. Research in this field also has wider implications for other adrenal and degenerative disorders of the nervous system.
The existing disease models used to study AAAS all have significant shortcomings. Obtaining adrenal or brain tissue from patients is not feasible. Most research is performed using cells obtained from patients' skin, which is not typically affected in AAAS. Other studies use cancer cells, which differ significantly from normal and patient cells. The AAAS animal model does not exhibit the classical features of the disease. Therefore in collaboration with the Sanger Institute, I have developed gene-edited human stem-cell models of AAAS. These will be used to generate AAAS adrenal and brain tissue cells, providing near-natural conditions to study AAAS in the tissues primarily affected by the disease.
These novel disease models will be used to uncover the abnormal cell processes and drivers of OS in AAAS. Through a rigorous series of experiments, I will investigate how AAAS affects the normal functioning of adrenal and brain tissue and what abnormalities underlie the disease process. I will use RNA sequencing , a method used to examine any changes in gene expression that result from AAAS disease. By examining the activity of genes in diseased compared to healthy cells, I will be able to identify key difference that can be attributed to AAAS and signpost ALADIN's critical roles within the cell. Another hypothesis is that mutations in the AAAS gene cause problems in the cell's energy generators, called mitochondria. If my experiments support this theory, I will undertake experiments to examine this in detail.
Ultimately, I aim to identify potential therapeutic targets that could be targeted to slow or halt disease progression in AAAS and other degenerative diseases. Adrenal stem cell modelling is a new field of research and information gathered by this project will further advance this field, leading to the use of this technology as a treatment for patients with adrenal failure.
Technical Summary
Triple A syndrome (AAAS), a rare disorder, presents with tissue-specific degeneration. Encoded by the AAAS gene, ALADIN is a nuclear pore complex (NPC) protein necessary for nuclear import of DNA protective molecules and is important for redox homeostasis.
ALADIN's role is not fully characterised: its discovery at the centrosome and the endoplasmic reticulum suggests a role outside the NPC. Mitochondrial dysfunction may be implicated by disruption of mitochondrial steroidogenic enzymes, enlarged mitochondrial mass and increased mitochondrial superoxide species. My preliminary data shows ALADIN localisation to mitochondria in SHSY5Y cells. Investigation of AAAS is marred by suboptimal models. In collaboration, I have generated novel induced pluripotent stem cell (iPSC) models of disease using CRISPR-Cas9 gene-editing: 1) Homozygous knock out (AAAS-KO) and 2) Homozygous hotspot mutation (AAAS-mutant). The unedited iPSC will serve as an isogenic control.
Aim: To differentiate the AAAS-iPSC and controls towards adrenal-like and neuronal phenotypes, for use in:
1. OS profiling of AAAS, including mitochondrial phenotyping.
2. Identification of gene expression in AAAS, signposting ALADIN's non-canonical functions.
Method
Differentiation: Through established in-house pipelines.
OS profiling: Susceptibility to OS/bioenergetic response in the AAAS-mutant, AAAS-KO and control cells will be assessed by XFe96 extracellular flux analyser. If mitochondrial dysfunction is confirmed, mitochondrial phenotyping will ensue.
RNA Seq: AAAS-KO neurons (clones, n=10) and controls (clones, n=10) will be sent for RNA seq. Bioinformatics will be performed with in-house expertise. Recapitulation in adrenal cells will identify pathways common to both tissues. Functional experiments will target genes/pathways of interest to reverse the AAAS phenotype.
Impact: Insight into the pathogenesis of AAAS has therapeutic development potential for AAAS/degenerative diseases.
ALADIN's role is not fully characterised: its discovery at the centrosome and the endoplasmic reticulum suggests a role outside the NPC. Mitochondrial dysfunction may be implicated by disruption of mitochondrial steroidogenic enzymes, enlarged mitochondrial mass and increased mitochondrial superoxide species. My preliminary data shows ALADIN localisation to mitochondria in SHSY5Y cells. Investigation of AAAS is marred by suboptimal models. In collaboration, I have generated novel induced pluripotent stem cell (iPSC) models of disease using CRISPR-Cas9 gene-editing: 1) Homozygous knock out (AAAS-KO) and 2) Homozygous hotspot mutation (AAAS-mutant). The unedited iPSC will serve as an isogenic control.
Aim: To differentiate the AAAS-iPSC and controls towards adrenal-like and neuronal phenotypes, for use in:
1. OS profiling of AAAS, including mitochondrial phenotyping.
2. Identification of gene expression in AAAS, signposting ALADIN's non-canonical functions.
Method
Differentiation: Through established in-house pipelines.
OS profiling: Susceptibility to OS/bioenergetic response in the AAAS-mutant, AAAS-KO and control cells will be assessed by XFe96 extracellular flux analyser. If mitochondrial dysfunction is confirmed, mitochondrial phenotyping will ensue.
RNA Seq: AAAS-KO neurons (clones, n=10) and controls (clones, n=10) will be sent for RNA seq. Bioinformatics will be performed with in-house expertise. Recapitulation in adrenal cells will identify pathways common to both tissues. Functional experiments will target genes/pathways of interest to reverse the AAAS phenotype.
Impact: Insight into the pathogenesis of AAAS has therapeutic development potential for AAAS/degenerative diseases.
Planned Impact
Degenerative Disease
Degenerative diseases affect all body systems and represent a major global health burden in the developed world. These are frequently driven by oxidative stress. This project is aimed at clarifying the molecular pathways underlying Triple A syndrome. As this work will investigate oxidative stress and mitochondrial physiology there is potential relevance to numerous other debilitating degenerative diseases. Clarification of the cellular pathways affected in these disorders will identify putative therapeutic targets for the treatment of AAAS and other oxidative stress driven degenerative diseases. Although not immediately commercially exploitable, it is expected this will be of interest to the pharmaceutical industry and ultimately lead to improvements for patients living with degenerative diseases. Ultimately, novel therapies will aim to halt disease progression, reduce disability, improve quality of life and function in society. This will be of wide economic benefit to the NHS. Securing funding for this project will ensure its success and promote world-leading molecular biology research in our University and the UK.
Primary Adrenal Insufficiency
This project will contribute to advances in stem cell technology as potential therapies for primary adrenal insufficiency. Currently this is an incurable illness with significant impact on quality of life and risk of death. Treatment entails replacement with exogenous steroids, which cannot suitably mimic diurnal variations seen in health and so can incur life altering side-effects such as mood disturbance, obesity, osteoporosis and diabetes. Adrenal stem cell therapy will provide these individuals with a potential cure, reduced morbidity and improvements in quality of life. Therefore, research in this field has economic repercussions for the wider society. Pioneering this technology within our department will ensure that both this Centre, and the UK, will continue to be at the forefront of stem cell research.
Basic Science
Detailed knowledge about subcellular organization and cellular pathways is essential for scientific research in numerous biomedical fields. This project will yield information about the relationships between the nucleoporin ALADIN, mitochondrial physiology and cell division, which is likely to be of relevance to molecular biologists across disciplines. This will continue to develop knowledge and momentum in these fields, promoting both the UK's academic standing and translational research.
Public Health
Oxidative stress and mitochondrial imbalances are thought to contribute to the development of many diseases including, but not limited to, cancer, cardiovascular disease, macular degeneration and neurodegenerative disorders such as Alzheimer's. Environmental factors, such as pollutants driving oxidative stress are likely to remain elusive without better understanding the genetic and cellular processes behind oxidative stress metabolism. We hope our research will clarify some of the cellular processes implicated in AAAS and therefore inform the investigation of environmental causes of oxidative stress.
Degenerative diseases affect all body systems and represent a major global health burden in the developed world. These are frequently driven by oxidative stress. This project is aimed at clarifying the molecular pathways underlying Triple A syndrome. As this work will investigate oxidative stress and mitochondrial physiology there is potential relevance to numerous other debilitating degenerative diseases. Clarification of the cellular pathways affected in these disorders will identify putative therapeutic targets for the treatment of AAAS and other oxidative stress driven degenerative diseases. Although not immediately commercially exploitable, it is expected this will be of interest to the pharmaceutical industry and ultimately lead to improvements for patients living with degenerative diseases. Ultimately, novel therapies will aim to halt disease progression, reduce disability, improve quality of life and function in society. This will be of wide economic benefit to the NHS. Securing funding for this project will ensure its success and promote world-leading molecular biology research in our University and the UK.
Primary Adrenal Insufficiency
This project will contribute to advances in stem cell technology as potential therapies for primary adrenal insufficiency. Currently this is an incurable illness with significant impact on quality of life and risk of death. Treatment entails replacement with exogenous steroids, which cannot suitably mimic diurnal variations seen in health and so can incur life altering side-effects such as mood disturbance, obesity, osteoporosis and diabetes. Adrenal stem cell therapy will provide these individuals with a potential cure, reduced morbidity and improvements in quality of life. Therefore, research in this field has economic repercussions for the wider society. Pioneering this technology within our department will ensure that both this Centre, and the UK, will continue to be at the forefront of stem cell research.
Basic Science
Detailed knowledge about subcellular organization and cellular pathways is essential for scientific research in numerous biomedical fields. This project will yield information about the relationships between the nucleoporin ALADIN, mitochondrial physiology and cell division, which is likely to be of relevance to molecular biologists across disciplines. This will continue to develop knowledge and momentum in these fields, promoting both the UK's academic standing and translational research.
Public Health
Oxidative stress and mitochondrial imbalances are thought to contribute to the development of many diseases including, but not limited to, cancer, cardiovascular disease, macular degeneration and neurodegenerative disorders such as Alzheimer's. Environmental factors, such as pollutants driving oxidative stress are likely to remain elusive without better understanding the genetic and cellular processes behind oxidative stress metabolism. We hope our research will clarify some of the cellular processes implicated in AAAS and therefore inform the investigation of environmental causes of oxidative stress.
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| Alexandra Rodrigues Da Costa (Principal Investigator / Fellow) |