Human Synapses: iPSC-derived neurons as a model to study human synapse formation and function

Synapses are the main contact points in the brain where signals are transmitted between neurons. This signal transduction, called synaptic signaling, is essential for brain function. Synaptic connections are thought to be the physiological basis for long-term memory, which can last throughout life. At the same time, the formation of new synapses, removal of existing synapses, and changes in strength of synaptic signals, allow new memory formation and learning. The mechanisms underlying synapse formation and function are not completely understood. Even less is known about synapse development and function in human neurons, since synapses have mainly been studied in animal models. The overall aim of this thesis was to study the formation and function of human synapses. To this end, we used human skin or blood cells that were “reprogrammed” to induced pluripotent stem cells (iPSCs), a cell type that can be transformed into almost any other cell type, while retaining the genetic information of the human donor. These iPSCs were differentiated into neurons, referred to as iNeurons. Because iNeurons are a relatively new model system, we aimed to characterize their development and function. In Chapter 2 we developed an experimental setup in which iNeurons grow in isolation and form synapses onto themselves. This system allowed us to measure the electrical currents generated by synaptic signaling. We found that iNeurons form functional synaptic connections which develop over time and become stable after 5-6 weeks in culture. Therefore, we propose that this system can be used to study changes in synaptic signaling caused by disease-related mutations. In Chapter 3 we further investigated how iNeurons develop over time by studying the expression of proteins between the start of synapse formation and the stage of stable synaptic transmission. The time between these two stages is longer than in mouse neurons grown in the same system: ~3 weeks instead of ~3 days. We found that most synaptic proteins were already expressed during early development, when most synapses are not yet formed and their expression levels increase over time. Therefore, we propose that it is likely not the expression of synaptic proteins but their transport or recruitment to nascent synaptic sites that determines the relatively slow development of synapses in iNeurons. iNeurons retain the genetic information from the human donor and are therefore uniquely suited to model patient-own neurons in a dish. This allows the investigation of disease phenotypes at the cellular level. However, it was unknown how many cells and how many patients are needed to discover mechanisms underlying a disease of interest. In Chapter 4 we measured the variation between iNeurons derived from different healthy individuals to do statistical power calculations. Based on these calculations, we found that many published disease studies using iPSC-derived neurons are underpowered and would need to increase the number of patients and controls included in the study. Moreover, we found that a change in study design could greatly increase the statistical power without needing to greatly increase the number of individuals. We generated an online tool to allow researchers to perform statistical power analysis before starting a disease modeling study. The relatively slow development in iNeurons is reminiscent of the prolonged neuronal development in humans compared to other species. In Chapter 5 we investigated whether a human-specific gene could be acting as a “break” on neuronal maturation in humans. Contrary to our hypothesis, we found that removing this protein from iNeurons resulted in decreased neuron size, decreased synaptic density and decreased neuronal viability. However, more research is needed to fully understand the function of this protein in human neurons.