Interrogation of links between risks and early pathogenesis at single cell resolution in a novel human ALS organoid neuraxis model

Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease, a disabling and fatal neurological condition with rapidly progressing muscle weakness, which is currently untreatable. In the UK, approximately 1 in 400 people are at risk of developing this disease, with an increasing burden on patients, relatives and society. Quality of life is reduced due to loss of muscle strength in the arms and legs, but occasionally also to memory and behavioural problems. Death is most commonly caused by failure of breathing muscles, with survival time as short as three years after diagnosis. Despite recent progress, there are key unresolved questions limiting the development of effective treatments.

We are now beginning to understand that damage in ALS occurs not only in the neurons controlling our muscle movements and thinking, but also in many other affected supporting cell types, including glia and immune cells in the brain and the spinal cord. However, we do not know yet: i) when and how this damage occurs in the cells, ii) how the disease spreads in the nervous system from one cell to another; iii) how further changes can occur to our genetic code, the DNA and whether this could influence our symptoms throughout our lives; and iv) and how environmental risk factors, such as brain injuries, affect the onset and progression of ALS.

Finding answers to these questions will guide the development of therapies to prevent or treat the disease early, before the major damage or cell loss occur. The main problem is that it is difficult to study the initial cell disturbances, as we often cannot identify the patients before the symptoms occur. Nor can we get brain or spinal cord samples from them at this early stage. Although animal and human cell cultures in the dish provided useful insight into these problems, they are not suitable alone to understand the full picture. The reason is that animal and human cells in the dish can behave quite differently when compared to their counterparts in the brain.

To overcome this problem, for the first time, we have created a new model system from patient-derived stem cells, which can be grown as three-dimensional cultures in a dish, acquiring a similar structure, cell-types and connections as seen in the human brain and spinal cord. Because of the similarities to organs, these are called organoids, and often referred to mini-brains or mini-spinal cords with the difference in the wiring and certain cell types, so that they cannot gain consciousness. A major advantage is that our organoid model allows studies on very similar disturbances that occur in ALS, and we can now examine how these would effect the function of brain neurons that control spinal cord neurons that influence muscle movements. Another, important advantage is that we can grow this model from patients who have the most common genetic abnormality causing ALS with broad relevance to patients, and from those cells too in which this genetic problem is corrected, which can ensure that our observations specifically reflect ALS-related problems.

In this proposal, we will now use this accessible experimental human platform and combine it with other innovative tools that help us to tease out molecular changes in individual cells at the same time, providing an immense opportunity to understand the complexity of disease initiation, progression and possible prevention. Our studies may help identify preventable cellular disturbances and those molecular changes, called biomarkers that may pose as specific signatures of early ALS, which may guide us when to treat, how to treat and who to treat in the future. The support by the MRC would provide the foundations for me to continue my international-level leadership in human organoid disease models, to facilitate the career development of my trainees, and ultimately to fully harness our novel human organoid models for discoveries in neurodegeneration research and therapeutics.

Amyotrophic lateral sclerosis (ALS) is a disabling and fatal neurodegenerative disease due to progressive neuronal, glial and immune cell dysfunction, leading to muscle paralysis. In the UK, approximately 1 in 400 people are at risk, with no available treatment.

My overall aim is to elucidate unresolved key issues halting early treatment strategies. My main objectives are to investigate i) initiating cellular disturbances; ii) non-cell autonomous pathogenesis; and iii) the interplay between genetic and environmental risk factors with disease pathways; and iv) the need for targeting multiple pathogenic pathways. However, capturing somatic cell disturbances in preclinical stages is not possible, no animal models faithfully recapitulate ALS pathology, and human two-dimensional cell models lack bona fide cell-cell interactions required to observe precise non-cell autonomous pathogenesis.

To overcome these challenges, we developed the first functionally connected human brain-spinal-cord-muscle organoid axis that recapitulates cell-cell interactions and early molecular ALS pathology. We generate this organoid platform from patient-derived cells harbouring the C9ORF72 mutation, which covers the full pathological spectrum in ALS, and from their genetically-corrected counterparts, allowing mutation-specific observations.

We will exploit our state-of-the-art human platform and ALS post-mortem tissue with innovative single cell-based genomic tools and biological assays to address: i) which cell-type display the earliest molecular changes? ii) how do they affect neighbouring cells? iii) how do genetic variations trigger the vicious cycle of DNA damage, inflammation and proteostasis defects, and can this be potentiated by trauma? iv) can we block these problems early by targeting common or multiple pathways to improve motor network function? Our studies may help identify preventable molecular disturbances and specific biomarkers in ALS to guide when, how, who to treat.