Modelling peripheral axonal pathology of ALS in vitro - Assembly of optogenetic neuromuscular circuits from pluripotent stem cells

Amyotrophic Lateral Sclerosis (ALS) is a medical condition which affects motor neurons, the type of nerve cell which connects the brain to muscle. ALS leads to paralysis, so that affected individuals can no longer walk, speak or breathe. The disease is both quite common and currently incurable, and therefore many scientists study its causes and possible therapies. As many manipulations required to investigate ALS cannot be carried out in patients, experimental animals, mostly mice and rats, are used in most studies. These animals are often genetically modified such that they develop a disease which is similar to the one seen in humans. This approach uses a large number of animals and is expensive and time-consuming, and therefore not suitable to systematically study hereditary traits which may cause ALS.
We propose to develop an alternative way of investigating ALS, which will be based on complex, life-like artificial tissues grown directly from cultured human stem cells. The artificial tissues will resemble nerve tissue (motor neurons and accessory cells called 'glia') and muscle. They will be cultured in a compartmentalized biochip designed and manufactured for this project. In this device, the two tissues will be segregated - as they are in humans and animals-, and the motor neurons will connect to muscle through nerve fibres and control muscle contractions. The cells in the device will contain biological optical probes that allow us to feed instructions into the cells on the biochip and read information out though fluorescent light signals. We will test the validity of the new experimental system by first modelling the degeneration of nerves and the loss of communication between nerves and muscle. Then, we will use this 'ALS in a dish' model to investigate why different forms of ALS result in different patterns of degeneration, how genetic traits protect some people from developing ALS, and how such protection may be mimicked with chemical compounds.
We think that this tissue culture model could allow scientist to discover ALS drugs faster, cheaper and without the use of animal experimentation.

We propose to develop an in vitro model of Amyotrophic Lateral Sclerosis (ALS) and neuromuscular circuitry, based on sorted pluripotent stem cell-derived motor neurons, astrocytes and myofibres, which are co-cultured in a compartmentalized tissue culture device. We plan to investigate the disease mechanism of ALS, with a focus on peripheral synaptic and axonal cellular pathology. To this end, we will equip human induced pluripotent stem cells carrying disease-associated mutations in the genes SOD1, TARDBP or C9ORF72 with genetically encoded optogenetic actuators and optical probes which will allow us to stimulate the cells, read their electrical activity and trace cellular features with live imaging. The optogenetic stem cell-motor neurons will be activated with light and used to control the myofibres connected to them through neuromuscular junctions (NMJs). We will map different ALS-associated mutations to specific patterns of early cellular degeneration, which may be initiated either centrally or peripherally. We will use this in vitro model to study the impact of different neural activity patterns, modifier genes, and small molecule compounds on early pathological features, in particular abnormal NMJs and defective axonal transport. This may enable us to pinpoint potential therapeutic pathways, and, in the long-term, develop ALS drugs which are specifically customized to the individual genotypes and phenotypes of ALS patients.

Exploitation and Application (year 3-4):
We intend to develop a new type of stem cell-based in vitro model of neuromuscular circuits and neuromuscular disease, which, long-term, may be used for drug discovery. This research promises to create valuable intellectual property (IP) as such a tissue culture system that would be highly desirable for pharmaceutical companies interested in developing treatments for neuromuscular disease, such as ALS. The technology transfer teams of KCL and National University of Singapore (NUS) will engage and liaise with the PIs to identify new IP should it arise. King's Commercialisation Institute has extensive experience in IP management and exploitation and has established working relationships with reputable IP law firms. IP arising shall be owned by the host institutions of the applicants and project partners. Commercial partners will be sought at an early stage following the filing of any patent applications.

Advancing Training (year 1-4):
The proposed grouping (PIs and project partners) have considerable expertise in the molecular, cellular and morphological analyses of neural and muscle tissue, which will provide excellent training opportunities for the named postdoctoral researcher, Dr Machado, in a variety of skill sets. Dr Machado will be involved in the supervision of lab projects that are part of the Ph.D. programs at KCL. In this way, we will provide an environment that will contribute to the training of scientists, and which will facilitate the development of skills to acquire her ability to manage and supervise lab projects. Furthermore, Dr Lieberam is co-supervising a graduate student, Miss Perrine Pluchon, for a joined (separately funded) Ph.D. thesis in his group and the group of Dr Virgile Viasnoff, NUS. Miss Pluchon is working with Dr Viasnoff on optimizing the material science aspects of the compartmentalized tissue culture device, and will move to Dr Lieberam's group in London in November 2015 to help adapt the system to cultures of sorted ESC-derived neural cells and myofibres. Participating in this project provides a unique training opportunity from her, and might allow her to learn a wide variety of scientific concepts, experimental methods and manufacturing techniques in stem cell biology and engineering.

Communication and Dissemination of Data (year 3-4):
We support the RCUK's policy to make the research data generated via their funding to be available to the community in as timely a manner as possible. The proposed project will generate a number of different data types, which will all stored in industry standard formats to maximise data portability. Data will be automatically archived and centrally collected in Dr Lieberam's group (see Data Management Plan). Our intention is to make our data available primarily through peer-reviewed scientific journals that can be accessed through PubMed, allowing our main findings to be monitored for accuracy and content. In line with current policy we will also ensure that data are published in journals with an Open-Access policy of not more than 12 months ('Green' option). Whenever possible, we will choose immediate unrestricted access ('Gold' option). Any reagents produced will be made freely available to the scientific community immediately on publication through repositories such as Addgene. We plan to publish and/or present our data at meetings.

Public Understanding of Science (year 1-4):
Throughout the project we will engage with school students and support teachers. Dr. Lieberam supports the educational charity In2ScienceUK and is accepting school students from low-income backgrounds for a week of work experience in his laboratory to encourage them pursue a career in science. We are committed to the idea that an engagement in teaching and education at different levels is an important aspect of scientific practice.