Multi-level mapping of mitochondrial quality control pathways in Parkinson's dopaminergic neurons
Lead Research Organisation:
University of Oxford
Department Name: Physiology Anatomy and Genetics
Abstract
Parkinson's disease (PD) is a neurodegenerative disorder that affects movement, cognition, and behaviour. One of the causes of these movement problems is the progressive loss of dopamine-producing neurons in the substantia nigra, a region of the brain that is involved in movement control. The cause of PD is not fully understood, in some people it is caused by large changes in a few genes but in most people with Parkinson's it is thought to be caused by a combination of genetic and environmental factors.
Two key features of cells that are lost in PD are the accumulation of clumps of a protein called alpha-synuclein and dysfunction of mitochondria. Mitochondria are the powerhouses of the cell, and they play critical roles in energy production as well as affecting many aspects of how cells work. The dopamine-producing cells have several features including their size and activity that make them more susceptible to mitochondrial damage.
Mitophagy ('eating mitochondria') is a process by which damaged mitochondria are degraded by the cell. It is a tightly controlled process that is essential for maintaining the health of mitochondria and cells. Decreases in mitophagy have been linked to a variety of neurodegenerative diseases, including PD. In particular, changes in genes that control mitophagy cause early-onset PD. We have recently discovered that these clumps of the protein alpha-synuclein can damage mitochondria and activate the process of mitophagy
This study will first understand how the dopamine-producing cells usually perform mitophagy as this appears to be different to other cell types. We will then investigate how clumps of alpha-synuclein or rare changes in genes which control mitophagy affect how cells perform mitophagy.
Using this information, we will then investigate how small changes in genes that are linked to (but don't cause) PD affect mitophagy. To understand how these genes might be involved in changing mitophagy, we will use new technology, called CRISPR activation or CRISPR interference, to mimic the effect of these gene changes by increasing or decreasing how active the gene is- like a volume controller.
To further understand how these genes affect dopamine producing cells, we will measure the levels of lots of proteins in these cells. Using a new technique we can also measure how active the proteins are, giving us a more complete picture of how small gene changes that are more common in PD affect cells. This will be interesting as it may tell us more about changes in mitophagy but it could tell us that other processes in the cell (like alpha-synuclein clumping) are changed. We will also be able to see which gene changes are similar to each other and which are different and may mean we are able to group and treat people with Parkinson's accurately.
This research will tell us about how small gene changes might build up to eventually cause PD in some people and which parts of the cell are most affected. Given mitochondria are key to the dopamine-producing cells, we will first focus on this and get a very detailed picture of this that may lead to identifying which people might benefit the most from mitochondria-targeted treatments.
This research will be used by many PD researchers and will help us understand how common gene changes might mimic the rarer larger gene changes that cause PD, this understanding will lead to better understanding of PD and eventually new treatments.
Two key features of cells that are lost in PD are the accumulation of clumps of a protein called alpha-synuclein and dysfunction of mitochondria. Mitochondria are the powerhouses of the cell, and they play critical roles in energy production as well as affecting many aspects of how cells work. The dopamine-producing cells have several features including their size and activity that make them more susceptible to mitochondrial damage.
Mitophagy ('eating mitochondria') is a process by which damaged mitochondria are degraded by the cell. It is a tightly controlled process that is essential for maintaining the health of mitochondria and cells. Decreases in mitophagy have been linked to a variety of neurodegenerative diseases, including PD. In particular, changes in genes that control mitophagy cause early-onset PD. We have recently discovered that these clumps of the protein alpha-synuclein can damage mitochondria and activate the process of mitophagy
This study will first understand how the dopamine-producing cells usually perform mitophagy as this appears to be different to other cell types. We will then investigate how clumps of alpha-synuclein or rare changes in genes which control mitophagy affect how cells perform mitophagy.
Using this information, we will then investigate how small changes in genes that are linked to (but don't cause) PD affect mitophagy. To understand how these genes might be involved in changing mitophagy, we will use new technology, called CRISPR activation or CRISPR interference, to mimic the effect of these gene changes by increasing or decreasing how active the gene is- like a volume controller.
To further understand how these genes affect dopamine producing cells, we will measure the levels of lots of proteins in these cells. Using a new technique we can also measure how active the proteins are, giving us a more complete picture of how small gene changes that are more common in PD affect cells. This will be interesting as it may tell us more about changes in mitophagy but it could tell us that other processes in the cell (like alpha-synuclein clumping) are changed. We will also be able to see which gene changes are similar to each other and which are different and may mean we are able to group and treat people with Parkinson's accurately.
This research will tell us about how small gene changes might build up to eventually cause PD in some people and which parts of the cell are most affected. Given mitochondria are key to the dopamine-producing cells, we will first focus on this and get a very detailed picture of this that may lead to identifying which people might benefit the most from mitochondria-targeted treatments.
This research will be used by many PD researchers and will help us understand how common gene changes might mimic the rarer larger gene changes that cause PD, this understanding will lead to better understanding of PD and eventually new treatments.
Technical Summary
Background
The factors governing the preferential loss of the A9 dopaminergic neurons in Parkinson's disease are multifactorial and centre around a number of physiological characteristics of the neurons intersecting with dysfunction in cellular processes identified by genetics and exposure to toxins. These dopaminergic neurons are particularly susceptible to disruption of mitophagy, as evidenced by post-mortem pathology of patients with mutations in PINK1 and PRKN.
Research goals
1) To understand disease-relevant mitophagy in dopaminergic neurons and how PINK1 mutations impair this.
2) To profile the effects of disease-linked mitochondrial genes on mitophagy and wider cellular health.
Key methods
1) Generation of physiologically-relevant iPSC-derived dopaminergic neurons from engineered and patient lines.
2) Manipulation of pathway and disease-relevant genes using CRISPRi/a.
3) Profiling of mitophagy using mtKeima and immunofluorescence assays.
4) Induction of disease-relevant stress using pre-formed alpha-synuclein fibrils.
5) Profiling the post-translational proteome using mass-spec based PTMomics.
Anticipated outcomes
1) A greater understanding of (patho)physiologically-relevant mitophagy in dopaminergic neurons.
2) Understanding how patients lacking PINK1 activity maintain mitochondrial function.
3) Identification of Parkinson's associated genes that regulate mitophagy.
4) Global proteome profiling of Parkinson's associated genes allowing unbiased mechanistic insights.
5) Validation of an approach to assess disease-associated risk genes at scale.
The factors governing the preferential loss of the A9 dopaminergic neurons in Parkinson's disease are multifactorial and centre around a number of physiological characteristics of the neurons intersecting with dysfunction in cellular processes identified by genetics and exposure to toxins. These dopaminergic neurons are particularly susceptible to disruption of mitophagy, as evidenced by post-mortem pathology of patients with mutations in PINK1 and PRKN.
Research goals
1) To understand disease-relevant mitophagy in dopaminergic neurons and how PINK1 mutations impair this.
2) To profile the effects of disease-linked mitochondrial genes on mitophagy and wider cellular health.
Key methods
1) Generation of physiologically-relevant iPSC-derived dopaminergic neurons from engineered and patient lines.
2) Manipulation of pathway and disease-relevant genes using CRISPRi/a.
3) Profiling of mitophagy using mtKeima and immunofluorescence assays.
4) Induction of disease-relevant stress using pre-formed alpha-synuclein fibrils.
5) Profiling the post-translational proteome using mass-spec based PTMomics.
Anticipated outcomes
1) A greater understanding of (patho)physiologically-relevant mitophagy in dopaminergic neurons.
2) Understanding how patients lacking PINK1 activity maintain mitochondrial function.
3) Identification of Parkinson's associated genes that regulate mitophagy.
4) Global proteome profiling of Parkinson's associated genes allowing unbiased mechanistic insights.
5) Validation of an approach to assess disease-associated risk genes at scale.
