Identifying mechanisms underlying forebrain pathology in human PAX6 haploinsufficiency
Lead Research Organisation:
University of Edinburgh
Department Name: Centre for Discovery Brain Sciences
Abstract
The human brain is a complex structure comprised of billions of neurons which must connect to each other in a precise manner to form functional neural circuits and enable normal functioning of the brain. To make this possible, the correct numbers and types of neurons must be generated, mature, then form appropriate connections with one another. Much of this normally occurs during foetal brain development. Instructions for these developmental processes are provided by genes. One gene well known to regulate early neurodevelopment is PAX6. PAX6 protein acts by turning on or off other genes needed for normal brain development. In PAX6 haploinsufficiency, one of the two copies of PAX6 is mutated. Patients mainly suffer from congenital aniridia, a rare eye disease characterised by total or partial loss of the iris. However, they also suffer from brain malformations such as a decrease in brain areas, connectivity defects between different brain regions, and behavioural impairments, including impaired social cognition, autism and intellectual disability. As yet, the brain defects described in PAX6 haploinsufficiency patients remain mostly understudied.
Previous work in animal models indicated that PAX6 is important for ensuring that the brain forms the correct numbers and types of neurons needed, and that neurons connect with their appropriate targets. We hypothesize that human PAX6 mutations disrupt these processes early in development, leading to brain malformations and behavioural symptoms found in PAX6 haploinsufficiency patients.
We propose to identify the underlying causes of the symptoms displayed by PAX6 haploinsufficiency patients by investigating the effects of PAX6 mutations on the processes that we believe underlie the brain malformations found in PAX6 haploinsufficiency patients. This includes: the number and types of neurons generated, brain connectivity and neuronal circuits that result from neuronal connections. Practical and ethical constraints on use of human embryonic tissue makes studying the early actions of human PAX6 difficult. We shall therefore use advanced stem cell culture techniques to generate 3D embryonic brain tissue, termed 'cerebral organoids', which recapitulate many features of the developing human brain.
We have engineered PAX6 haploinsufficient stem cell lines and will use cerebral organoids grown from these to determine how PAX6 mutations affect production of the correct numbers and types of neurons in the embryonic brain. We shall do this by identifying sets of genes expressed in individual cells in our organoids, which will tell us whether the correct number and type of neurons are being produced. By fusing organoids together in pairs, we shall how PAX6 haploinsufficiency affects the formation of neural connections between them. Tracing the neural connections using dyes will tell us whether connections are being made to the correct targets. In addition, we shall determine whether PAX6 mutations cause defective connections by staining for synaptic proteins, which are involved in these neural connections, and analyse them using an automated image analysis pipeline. This will reveal how PAX6 mutations affect the characteristics of the neural connections made. Finally, we shall study the effects of PAX6 mutations on neural circuits by recording neural activity with electrophysiological techniques. We shall dissect the characterisation of these neural circuits using pharmacological drugs that specifically affect excitatory or inhibitory neurons, to reveal how PAX6 mutations affect the state of neural circuits.
By taking this multifaceted approach to identify the underlying causes of the brain malformations suffered by PAX6 haploinsufficiency patients, we can help inform further studies that target these causes, ultimately to ameliorate patient symptoms.
Previous work in animal models indicated that PAX6 is important for ensuring that the brain forms the correct numbers and types of neurons needed, and that neurons connect with their appropriate targets. We hypothesize that human PAX6 mutations disrupt these processes early in development, leading to brain malformations and behavioural symptoms found in PAX6 haploinsufficiency patients.
We propose to identify the underlying causes of the symptoms displayed by PAX6 haploinsufficiency patients by investigating the effects of PAX6 mutations on the processes that we believe underlie the brain malformations found in PAX6 haploinsufficiency patients. This includes: the number and types of neurons generated, brain connectivity and neuronal circuits that result from neuronal connections. Practical and ethical constraints on use of human embryonic tissue makes studying the early actions of human PAX6 difficult. We shall therefore use advanced stem cell culture techniques to generate 3D embryonic brain tissue, termed 'cerebral organoids', which recapitulate many features of the developing human brain.
We have engineered PAX6 haploinsufficient stem cell lines and will use cerebral organoids grown from these to determine how PAX6 mutations affect production of the correct numbers and types of neurons in the embryonic brain. We shall do this by identifying sets of genes expressed in individual cells in our organoids, which will tell us whether the correct number and type of neurons are being produced. By fusing organoids together in pairs, we shall how PAX6 haploinsufficiency affects the formation of neural connections between them. Tracing the neural connections using dyes will tell us whether connections are being made to the correct targets. In addition, we shall determine whether PAX6 mutations cause defective connections by staining for synaptic proteins, which are involved in these neural connections, and analyse them using an automated image analysis pipeline. This will reveal how PAX6 mutations affect the characteristics of the neural connections made. Finally, we shall study the effects of PAX6 mutations on neural circuits by recording neural activity with electrophysiological techniques. We shall dissect the characterisation of these neural circuits using pharmacological drugs that specifically affect excitatory or inhibitory neurons, to reveal how PAX6 mutations affect the state of neural circuits.
By taking this multifaceted approach to identify the underlying causes of the brain malformations suffered by PAX6 haploinsufficiency patients, we can help inform further studies that target these causes, ultimately to ameliorate patient symptoms.
Technical Summary
PAX6 haploinsufficiency patients suffer from brain malformations including corpus callosum agenesis or dysgenesis, anterior commissure hypoplasia, and thinner cortices. These structural malformations are likely to underlie neuropsychiatric symptoms in patients, including impaired social cognition, autism and intellectual disability. However, brain defects in PAX6 haploinsufficiency patients remain mostly unstudied.
Studies in mice revealed that Pax6 is required for multiple aspects of brain development, including proliferation, neural differentiation, cell fate determination and synaptogenesis. We hypothesise that early defects in these processes underlie the brain abnormalities described in PAX6 haploinsufficiency patients. We aim to identify underlying causes of the brain defects found in these patients via a multifaceted approach by investigating the effects of PAX6 haploinsufficiency on the processes most likely to underlie the brain malformations found in patients. These include proliferation, neural differentiation, cell fate determination, synaptogenesis, neural connectivity, and functional neural circuitry. As there are important differences between rodents and humans, such as progenitor subpopulations that are scarce in the rodents and proliferation dynamics, we shall use cerebral organoids for our study.
We have engineered iPSC lines with heterozygous PAX6 mutations and isogenic controls. Using these, we shall generate cerebral organoids to interrogate processes listed above using immunohistochemistry and single cell RNA sequencing. We shall study neural connectivity using axonal tracing to assess axon guidance defects and synaptic staining for synaptic defects on assembloids, made by fusing organoids in pairs. Assembloids will also be used to study functional neural circuitry via calcium imaging and multielectrode array recordings. This multifaceted approach will reveal key processes that underlie the pathophysiology of PAX6 haploinsufficiency.
Studies in mice revealed that Pax6 is required for multiple aspects of brain development, including proliferation, neural differentiation, cell fate determination and synaptogenesis. We hypothesise that early defects in these processes underlie the brain abnormalities described in PAX6 haploinsufficiency patients. We aim to identify underlying causes of the brain defects found in these patients via a multifaceted approach by investigating the effects of PAX6 haploinsufficiency on the processes most likely to underlie the brain malformations found in patients. These include proliferation, neural differentiation, cell fate determination, synaptogenesis, neural connectivity, and functional neural circuitry. As there are important differences between rodents and humans, such as progenitor subpopulations that are scarce in the rodents and proliferation dynamics, we shall use cerebral organoids for our study.
We have engineered iPSC lines with heterozygous PAX6 mutations and isogenic controls. Using these, we shall generate cerebral organoids to interrogate processes listed above using immunohistochemistry and single cell RNA sequencing. We shall study neural connectivity using axonal tracing to assess axon guidance defects and synaptic staining for synaptic defects on assembloids, made by fusing organoids in pairs. Assembloids will also be used to study functional neural circuitry via calcium imaging and multielectrode array recordings. This multifaceted approach will reveal key processes that underlie the pathophysiology of PAX6 haploinsufficiency.
