Supervisors: Dr Giovanni Baranello, Dr Serena Barral
Developing 3D patient-derived neuronal models to elucidate neurodevelopmental defects in Spinal Muscular Atrophy.
Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease due to the lack of SMN protein which leads to progressive degeneration of motor neurons in the spinal cord. Alongside progressive impairment of motor and respiratory function, the majority of SMA 1 children, the more severe end of the SMA spectrum, may exhibit developmental delay and communication difficulties1. A recent study has shown that the expression of SMN protein in the human brain declines substantially between foetal and postnatal stages, indicating that elevated SMN levels are required during foetal and early postnatal typical brain development2. Previous studies in humans have shown early SMN expression in all neurons, with a progressive shift in SMN subcellular localization from mainly nuclear to cytoplasmic and then to axons during CNS maturation. Additionally, animal studies have shown that growth and development of a subset of brain regions are disrupted when SMN levels are reduced, with a particular disruption in neuronal density and proliferation in the hippocampus3. Although novel treatments are becoming increasingly available to patients in recent years, it is still unclear whether the observed brain-related comorbidities including intellectual disability and speech/language/communication impairment may be equally targeted.
Recent advances in induced pluripotent stem cells (iPSCs) and 3D culture systems have led to the generation of “brain” organoids that resemble several areas of the human brain. Organoids can recapitulate aspects of in vivo brain architecture and physiology and therefore offer new possibilities for modelling neurological disorders and for the development of new therapeutic approaches. We can now generate iPSC-derived cortical assembloids4 and hippocampal spheroids5, which resemble tissue structure, cellular composition, and physiology of the developing human cerebral cortex and hippocampus, providing novel tools for the study of disease-related phenotypes affecting these brain areas.
Aims/Objectives:
The overarching objective of this project is to investigate the cellular and molecular mechanisms underlying the disruption in brain development in SMA using patient iPSC-derived neuronal models.
Therefore, the objects of this project are:
1. Generate SMA type 1 patient-derived iPSCs and CRISPR-Cas9 isogenic controls.
2. Develop SMA hiPSC-derived cortical assembloids and hippocampal spheroids from SMA type 1 patient-derived iPSC lines and investigate neurodevelopment disruption.
3. Analyse neuronal maturation and network formation during development using electrophysiology (single-cell patch-clamp and local field potential) in SMA patient-derived cortical assembloids and hippocampal spheroids.
Methods:
1. SMA type 1 skin fibroblasts will be collected from three patients and reprogrammed into pluripotent cells using a Sendai Virus-based protocol. Derived iPSC lines will be fully characterized using a panel of established technologies (Sanger sequencing, single nucleotide polymorphism array, immunofluorescence technology, real time PCR, spontaneous in vitro differentiation). iPSC isogenic control lines will be generated using CRISPR/Cas9 technology and further characterized for chromosomal integrity.
2. SMA patient-derived, healthy age-match control (already available) and isogenic iPSC lines will be differentiated in cortical assembloids and hippocampal spheroids using established protocols. Cellular identity will be investigated using immunofluorescence and quantitative RT-PCR for a panel of neural progenitors and mature neurons markers. SMN protein expression will be analysed via immunofluorescence and western blotting to confirm absence in patient organoids.
3. Developmental defects will be evaluated using immunofluorescence analysis for proliferative and cell death markers, and neural progenitor/neuronal mature specific proteins to evaluate alteration in cortical and hippocampal development. Synaptic maturation and neuronal network formation will be analysed using relevant pre- and postsynaptic proteins (e.g. VGAT, GEPHYRIN) and using promoter-specific lentiviruses (LV-hSyn-RFP and LV-mdlx5/6-GFP) to evaluate neurite arborization complexity. Bulk RNA sequencing will be performed comparing patient to control derived organoids to identify alteration in gene expression underlying the disease phenotype.
3. SMA and control-derived cortical assembloids and hippocampal spheroids will be analysed using electrophysiological analysis in collaboration with UCL Institute of Neurology (Dr Gabriele Lignani). Single cell patch-clamp will be used to measure neuronal excitability, while local field potential will be performed to evaluate neuronal network formation.
Timeline (if applicable):
Months 0-9: Generation of SMA type 1 patient-derived induced pluripotent stem cells (iPSCs) and CRISPR-Cas9 isogenic controls.
Months 10-20: Development of SMA hiPSC-derived cortical assembloids and hippocampal spheroids from SMA type 1 iPSCs and characterization.
Months 20-36: Investigation of neurodevelopment disruption in SMA patient-derived cortical assembloids and hippocampal spheroids.
References:
1. Polido GJ, et al. Cognitive performance of children with SMA. Dem Neuropsychol. 2019;13(4):436-443.
2. Ramos DM, et al. Age-dependent SMN expression. J Clin Invest. 2019 1;129(11):4817-4831.
3. Wishart TM, et al. SMN deficiency disrupts brain development in a mouse model of severe SMA. Hum Mol Genet. 2010 1;19(21):4216-28.
4. Sloan SA, et al. Generation and assembly of human brain region-specific three-dimensional cultures. Nat Protoc 2018 2062-2085.
5. Pomeshchik Y, et al. Human iPSC-derived hippocampal spheroids: An innovative tool for stratifying Alzheimer disease patient-specific cellular phenotypes and developing therapies. Stem Cell Reports 2020 15(1):256-273.