The optimisation of an iPSC-derived cortical neuronal platform for the study of excitatory synaptic transmission and plasticity in neurological disorders

Many neurological disorders affecting the central nervous system exhibit alterations in glutamatergic synapse function. One such disorder is Alzheimer’s disease (AD), which affects 5 % of the world’s population. With an aging population and a lack of disease-modifying therapeutics, the prevalence of this disease is only expected to increase. Therefore, it is essential that we gain a greater understanding of AD pathogenesis in order to develop successful therapeutics. In AD, synapse loss is the best correlate of cognitive decline, and evidence from rodent studies suggests that this is preceded by synaptic dysfunction, primarily manifesting as alterations in glutamatergic synaptic plasticity. The advent of induced pluripotent stem cell (iPSC) technology has made it possible to investigate disease pathophysiology in patient-derived cells. Biochemical alterations in line with AD pathology have been described in AD-patient iPSC-derived neurons, but changes in synaptic transmission are yet to be investigated. The functional properties described for iPSC-derived neurons are often immature, and it is unclear whether they exhibit the pre- and post-synaptic properties required for the induction and expression of glutamatergic synaptic plasticity. A limited number of reports have described synaptic plasticity-like phenomena in iPSC-derived neurons. However, these studies use non-physiological methods, often show poor reproducibility and lack direct evidence for an activity-dependent change in synaptic efficacy.
The objective of this thesis was to determine methods to enhance neuronal maturity, and to generate a well-characterized platform for the study of physiological forms of synaptic plasticity. To this end, I first used whole-cell patch clamp electrophysiology to extensively characterize the functional properties of healthy control and familial AD patient iPSC-derived cortical neurons and found them to be immature. From several strategies tested, only the co-culture of iPSC-derived cortical neurons with rat astrocytes was found to robustly enhance intrinsic neuronal maturity and produce modest increases in excitatory synaptic transmission. Taking an alternative approach, I refined a transcription factor-based neuronal differentiation protocol and found that the exogenous expression of neurogenin-2 in iPSC-derived neural progenitor cells generated highly active excitatory synaptic networks.
Physiological synaptic plasticity assays require the isolation and manipulation of monosynaptic connections. Dual-patch recordings in the neurogenin-2 iPSC-derived cortical cultures revealed a low probability (10 %) of detecting an excitatory monosynaptic response. However, the expression of the optogenetic tool, channelrhodopsin-2 (ChR2), in a subset of neurons enabled the generation of light-evoked monosynaptic responses, which were three-fold more frequent. Excitatory monosynaptic responses exhibited similar properties to connections in rodent cortex, and demonstrated robust expression of functional synaptic AMPA and NMDA receptors. Monosynaptic connections failed to exhibit spike-timing dependent plasticity or classical long-term potentiation (LTP) using a pairing-protocol, and similarly, no changes were exhibited in spontaneous excitatory synaptic transmission following a chemical LTP protocol. However, prolonged optogenetic activation of pre-synaptic ChR2-expressing neurons for 2-4 days during cell culturing induced an activity-dependent potentiation of spontaneous excitatory current amplitude. This was primarily expressed by a subset post-synaptic neurons with mature intrinsic properties and thereby suggests that the degree of neuronal maturity is likely to be critical factor when exploring synaptic plasticity. This work provides a foundation for future investigations into synaptic plasticity and extends the range of experimental assays that can be applied to human iPSC-derived cortical neurons for the study of excitatory synaptic transmission.

Keywords: Stem cell
Advisor: Akerman, C
Wade-Martins, R