Training in hIPSC differentiation protocols to generate motor neuron-muscle cultures to replace rat models in study of mitochondria on axon physiology

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Neurons are the main computational units in the brain. They form a network of interconnected cells that pass information to each other at specialised sites called synapses. Electrical signals known as action potentials, the main currency of information in the neuron, travel down cable-like thin structure called the axon to reach the synapse. Here, they cause the release of chemicals that then result in electrical signals in the receiving neuron. The study of the spreading of electrical signals down axons has been of interest to many researchers over the past 100 years, and Hodgkin and Huxley won the Nobel Prize in 1963 for their identification of the mechanisms that underlie the propagation of these electrical signals. Though their model has been the cornerstone of electrical signal propagation ever since, the equations rely viewing the axon on a relatively large, macroscopic scale. Here we provide preliminary evidence that when viewed on a more microscopic scale, that previously unidentified factors have important influences on how electrical signals are spread down the axon.Mitochondria are structures that reside within cells to produce energy and soak up excess calcium. Mitochondria occur at regular points along an axon, and due to the relative thin diameter of axons mitochondria almost fill the entire cable when looked at a cross section. According to the Hodgkin-Huxely model this would increase the resistance in the spread of electrical signals, much like a pipe with a blockage reduces the flow of water. However, by using the very latest techniques in microscopy that allow us to track the spread of voltage as a change in intensity of light, we observed that mitochondria increase the transmission of electrical signals along the axon. This surprising discovery seems to result from the interaction between the electrical voltage generated by mitochondria and the electrical voltage of the axon.The ability to expand on these results is relatively limited in our current model. The axons of rat neurons that are dissected from the hippocampus region of the brain form complex patterns when they are cultured, weaving in random directions and overlapping with each other. In this proposal Dr Rigby wishes to be trained in the culturing of human induced pluripotent stem cell (hIPSC) derived motor neurons, which have very straight axons that infrequently branch and allow for much simpler acquisition and analysis of the spread of electrical and calcium signals.hIPSCs are the result of some extraordinary discoveries that skin cells from humans can be turned back into a stem cell-like state, from which they can then be turned into any cell type. Motor neurons are the cells which reside in the spine and cause the contraction and relaxation of muscle. Over the past 10 years the protocols to transform hIPSCs into motor neurons and muscle have been refined so that now researchers, including Dr Rigby's collaborator Dr Ivo Lieberam, can reliably produce human neuromuscular cultures without the need of using embryos or animals. It is these protocols that Dr Rigby wishes to learn.The use of hIPSCs confer many advantages. Firstly, Dr Rigby has the opportunity to completely cease animal usage in his work and thus address the 'Replacement' aspect of the 3Rs. In addition, by growing the neurons and muscle on microfluidic devices he will be able to make a customizable and transferable model of the human neuromuscular system, that can be of benefit to other researchers, and thereby further reduce the use of animals in neuroscience research. hIPSCs can also be sourced from human patients with particular neurodegenerative disorders. In our proposal we also plan to benefit from this versatility of the hIPSC model by comparing the spread of voltage along axons in hIPSC-derived motor neurons from healthy patients with neurons derived from patients with Amyotrophic Lateral Sclerosis (ALS), the most common form of motor neuron disease