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Title: Using human induced pluripotent stem cells to reveal astrocyte-neuron interactions in health and disease
Author: Hedegaard, Anne
ISNI:       0000 0004 8507 8535
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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Interactions between neurons and astrocytes underpin normal brain function. Astrocytes fulfil a variety of essential roles, including regulating synapse formation and maintenance, clearing neurotransmitters, and providing homeostatic regulation of the extracellular ionic environment. Evidence for a more active participation is also emerging, in which astrocytes respond to neuronal signals by influencing synaptic strength through gliotransmission. Most of our knowledge regarding neuron-astrocyte interactions stems from animal models, which have been important in dissecting events at a cellular and molecular level. Yet a major question for experimental neuroscience and drug development is how well findings translate from rodents to the human condition. The recent developments in induced pluripotent stem cell (iPSC) technology are providing a valuable tool with which to answer this question. Furthermore, because iPSCs can be derived from clinically diagnosed patients, this technology affords exciting new opportunities to model human disease processes. The objective of this thesis was to establish a human co-culture system in which neuron-astrocyte interactions can be investigated. To this end, the first part of the thesis characterises the functionality of cortical human neurons and astrocytes generated using protocols that recapitulate key aspects of corticogenesis. This characterisation establishes that human iPSC-derived astrocytes satisfy many of the hallmarks of rodent astrocytes. Live cell imaging methods are used to investigate intracellular ion dynamics, which are believed to underpin how astrocytes communicate with neurons and regulate the extracellular environment. This reveals that iPSC-derived astrocytes exhibit intracellular Ca2+ signalling events both spontaneously and in response to neurotransmitters. Furthermore, through a combination of monoculture and xeno-transplantation models, iPSC-derived astrocytes are shown to respond to neuronal activity and extracellular K+ by exhibiting similar membrane depolarisation and H+ dynamics to those observed in rodent astrocytes. The latter part of the thesis focuses on the use of iPSC co-cultures for studying astrocyte-to-neuron signalling. First, I establish that human astrocytes convey pro-maturational effects in terms of enhancing the intrinsic excitability and synaptic activity of co-cultured human neurons. Next, I develop an optogenetic paradigm for directly eliciting membrane depolarisation and Ca2+ events in astrocytes. This leads to glutamate receptor activation in nearby neurons and astrocyte stimulation over a period of days causes a potentiation of the synaptic inputs to co-cultured neurons. Finally, astrocytes are generated from individuals diagnosed with sporadic Alzheimer's disease (sAD), whom are homozygous for the APOE4 allele - the highest genetic risk factor associated with sAD. Compared to controls, APOE4 astrocytes are found to secrete less APOE and fail to generate astrocyte-mediated increases in synaptic inputs. Together, this work advances the use of human iPSC-derived cells for studying neuron-astrocyte interactions. iPSC-derived human astrocytes can recapitulate many of the functional properties of rodent astrocytes and can be used in different co-culture settings to examine multiple forms of astrocyte-to-neuron communication. This represents an important step towards establishing more complex human-based models, which are expected to prove useful for modelling disease processes.
Supervisor: Akerman, Colin Sponsor: Alzheimer's Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available