Using human induced pluripotent stem cells (iPSCs) to study Down syndrome neural pathology

Supervisor: Professor Patrizia Ferretti

Using human induced pluripotent stem cells (iPSCs) to study Down syndrome neural pathology and possible interventions

Background:
Down syndrome (DS) is the most common chromosomal abnormality in humans. Affected children have mental retardation and display premature onset of Alzheimer's disease (AD). DS is a complex disease with several mechanisms underlying impaired brain function. Studies in mouse models of DS have suggested that neuronal loss is due to defective proliferation and/or apoptosis at early stages of neuronal differentiation rather than a reduction in neural stem cells (NSCs) number and also that imbalance between excitatory and inhibitory synapses may underlie some of the cognitive impairment. It has also been suggested that DS brain is more susceptible to damage (e.g. Ca2+ imbalance/oxidative stress).

Aims:
The overall aim of this project is to further develop a human in vitro model of Down Syndrome (DS) to investigate the cellular and molecular pathology of neural development in these patients, and establish a tool to assess novel therapeutic interventions that could ameliorate brain function.

Specific Objectives:

• To better understand the DS brain by assessing differences in neuronal subtypes and glia in sections from different regions of DS brains.
• To investigate neural differentiation in DS and test the hypothesis that there are differences in neuronal subtype production and behaviour between DS and control iPSC-derived neurons.
• To set up robust models of DS in 3-dimensional (3D) cultures to better mimic tissue architecture and high cellularity, and use them to test pharmacological interventions that may normalize cell death/ survival, neuron/glia ratio, or neuronal function.

Experimental design:
1)   Distribution and relative number of different neuronal subtypes in DS brains will be assessed by immunohistochemistry / immunofluorescence and quantified.
2)   Neural stem cells (NSCs) derived from control or DS iPSCs (induced pluripotent stem cells) will be used to monitor proliferation and cell death in DS-NSCs, and in their progeny upon differentiation into neurones and glia. Specific neuronal subtypes will be monitored using reporter NSC lines that the student will generate using lentiviral constructs. Functional differences (e.g. Ca2+ activity and synaptic vesicles trafficking) in control and DS neurones will also be assessed using fluorescent dyes and optical reporters.
3)   DS-NSCs growth and differentiation will be compared in 2D and 3D hydrogel-based culture already established in the laboratory using the reporter lines described above. Depending on the findings in (2), activity of specific neuronal subtypes or/and calcium availability will be modulated pharmacologically to assess whether differentiation and function of DS cells can be normalized and could provide possible avenues for early intervention in DS patients that could improve their brain function.

References:
1. Helguera, P., Pelsman, A., Pigino, G., Wolvetang, E., Head, E., Busciglio, J., 2005. ets-2 promotes the activation of a mitochondrial death pathway in Down's syndrome neurons. J Neurosci 25, 2295-2303.
2.  Hibaoui, Y., Grad, I., Letourneau, A., Sailani, M.R., Dahoun, S., Santoni, F.A., Gimelli, S., Guipponi, M., Pelte, M.F., Bena, F., Antonarakis, S.E., Feki, A., 2014. Modelling and rescuing neurodevelopmental defect of Down syndrome using induced pluripotent stem cells from monozygotic twins discordant for trisomy 21. EMBO Mol Med 6, 259-277.
3.  Kurabayashi, N., Nguyen, M.D., Sanada, K., 2015. DYRK1A overexpression enhances STAT activity and astrogliogenesis in a Down syndrome mouse model. EMBO Rep 16, 1548-1562.
4.  Rueda, N., Florez, J., Martinez-Cue, C., 2013. Apoptosis in Down's syndrome: lessons from studies of human and mouse models. Apoptosis 18, 121-134.
5.  Stagni, F., Giacomini, A., Emili, M., Guidi, S., Bartesaghi, R., 2018. Neurogenesis impairment: An early developmental defect in Down syndrome. Free Radic Biol Med 114, 15-32.
6. U, K. P., Subramanian, V., Nicholas, A. P., Thompson, P. R., Ferretti, P., 2014. Modulation of calcium-induced cell death in human neural stem cells by the peptidylarginine deiminase-AIF pathway. BBA Mol Cell Res. 1843, 1162-1171.