AbstractShape and function are intertwined features in every cell, and this link is especially important for cells with complex morphologies. Neurons are amongst the most architecturally intricate cells, with some specific neuronal subtypes featuring extreme arborization and size. Motor neurons in particular feature axons that can reach up to 1m in length in the human body, and much longer in other mammals. The underlying homeostasis and metabolism of these cells need to adapt to this extreme shape and specific mechanisms need to be in place to maintain biological processes across scales. While the fact that these adaptations exist is plain, the details on how these neurons adapt their biological mechanisms to function “at a distance” are lacking and so far, no systematic analysis of axonal length has been conducted. The recent advancements in stem cell modelling allow the generation of nearly any neuronal subtype in vitro and enabled the study of cell-autonomous, but current in vitro platforms either do not allow to model axonal shape faithfully or are not suitable to perform a systematic analysis controlling precisely axonal length in the cultured cells. My research focused on creating an in vitro platform to model axonal length using bioengineering and iPSC-derived motor neurons to investigate the relationship between axonal length and underlying biological processes in the axon.
First, I created an axonal elongation platform that allowed me to systematically investigate axonal length in vitro. I commenced by designing and optimizing the fabrication and culture conditions to generate substrates for axonal elongation. I tested different biofunctionalization complexes to obtain suitable properties for long-term axonal elongation, and I optimized the microfabrication of topographical features on these substrates to provide guidance and alignment and further accelerated axonal elongation. The combination of bioengineering techniques and stem cell modelling allowed me to generate axons in the μm and cm range, which I subsequently used to investigate axonal biology across scales. I used the novel platform to identify fundamental structural and functional changes that occur when axons reach certain sizes in vitro. I identified alterations in neurofilament composition, calreticulin content and spontaneous calcium spike propagation. Remarkably, these alterations appeared at set transition points, which suggests an intrinsic “threshold” length that triggers these adaptations. I then continued to compare axons below and above this length and identified alterations in metabolism and homeostasis in long axons. Overall, I provided evidence that an increase of axonal length above a certain threshold length leads to underlying adaptations of essential biological functions, which highlights that axonal length is a viable feature to model in vitro. During these experiments, I also found an axonal intrinsic symmetry during outgrowth, resulting in a clockwise turn of axons extending from neural aggregates. This feature must be considered when designing axonal outgrowth platforms and can be harnessed for on-chip devices as a robust feature which provides guidance.
Developing and applying new techniques and tools are essential to push the boundaries of experimental setups and analysis. Technological progression and investigation of biological questions are tightly linked and can only progress in synchrony. Following the initial development of my axonal elongation platform and its use in the investigation of axonal length I also further invested in tool development, creating pipelines for image analysis and subsequent statistical evaluation, and well as novel microfabrication pipelines to further improve the axonal elongation studies. In particular, I developed a pipeline to combine soft lithography and volumetric 3D printing based on stereolithographic techniques that allowed me to further develop my own axonal platform and valuable, fast and versatile tools for generating any cell culture device.
|Date of Award||1 Feb 2023|
|Supervisor||Andrea Serio (Supervisor) & Juan Burrone (Supervisor)|