Project Information
Functional and metabolomic analysis of iPSC-derived Purkinje neurons from A-T patients
SCIENTIFIC LEAD: Dr Marco Foiani & Dr Domenico Delia: FIRC Institute of Molecular Oncology, Milan,
Italy
LENGTH: Concluded
COSTS: £80,000
STUDY: This project involved cutting-edge science and involved addressing what is arguably the biggest question in AT research: Why do some brain-cells die off in the absence of the ATM protein, particularly the Purkinje cells and granule neurons. Purkinje cells are large neurons with many branching extensions found in the cortex of the cerebellum of the brain and they play a fundamental role in controlling motor skills.
Before answering the question, due to the impossibility of removing Purkinje cells from the brains of living people, the scientists had to focus on growing Purkinje cells in the laboratory from induced pluripotent stem cells, developed from the cells of people with AT, and then turn these into Purkinje cells (PC). Once they had achieved this, they carried out a series of analyses of the metabolic processes at work in the cells, to determine what factors and alterations make these cells hypersensitive to the absence of ATM.
OUTCOME: The project concluded in 2023 and achieved two major results. The first was in regards to the development of a reproducible protocol for the generation of cerebellar PCs from cells derived from people with AT and people without. Importantly, this protocol, unlike others, does not make use of mouse cerebellar granule cells in co-culture with the human cells to achieve PCs maturation.
The second results concerns the characterization at single cell level of the metabolic profile (mitochondrial dependence, glycolytic capacity, glucose dependence, fatty acid and amino acid oxidation capacity) of normal and AT PCs. Using novel approaches, and finding that it is possible to assess the metabolic profile of PCs relative to other cells present in the culture, this can now be exploited to set up high-throughput differential
screenings to identify pharmacological drugs or biological compounds capable of antagonizing or preventing the death of AT PCs.
PROGRESS: The team have succeeded in creating the first ATM-deficient Purkinje cells, a significant development in itself. They have obtained mature Purkinje neurons without needing to involve coculture with mouse cerebellar granule cells. It is the first time this has ever been achieved with cells from people with AT. The cells are now being produced in sufficient numbers so they can then be used to screen potential drugs and in further experiments to understand why it is that these cells die off when other neurons don’t. So far, imaging analysis has been performed in order to compare the mitochondrial content, structure and activity, and an analysis undertaken to determine the response to drugs that induce metabolic stress by targeting the glycolysis pathway. Mitochondria are the powerhouse of the cell. They produce the energy required for the cell’s function but can produce by-products that can damage cellular components. An assessment of the expression of certain mitochondrial proteins which are defective as a consequence of ATM-deficiency, on cerebellar biopsies from AT patients, is also being undertaken.