Investigating the role of neuroinflammation in Amyotrophic Lateral Sclerosis using patient stem cell-derived brain cells

BACKGROUND:

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Amyotrophic lateral sclerosis (ALS), also known as Motor Neuron disease, is a rare and progressive neurodegenerative disorder affecting motor neurons, the nerve cells that connect the brain to muscles, in the brain and spinal cord. ALS leads to muscle weakness, twitching, and eventually paralysis. Cognitive function typically remains intact. The exact cause is unclear, with some cases having a genetic link, but most are sporadic. Neuroinflammation plays a significant role in the pathogenesis of ALS, although the exact mechanisms are still the subject of ongoing research. Microglia, the resident immune cells of the brain, and astrocytes, another type of glial cell, are central to this neuroinflammatory response. Dysregulation of these cells can lead to the release of pro-inflammatory molecules and toxic factors that harm motor neurons. Understanding the precise relationships between immune system activation, glial cell dysfunction, and motor neuron death is crucial for the development of potential therapies that could modulate the immune response and reduce inflammation to slow down the disease's progression.

In my PhD, I am investigating whether neuroinflammation and reactive microglia in ALS could be an important driver for disease onset or progression. Specifically, we intend to uncover a) the effect of ALS-linked mutations on microglia inflammatory states and homeostatic functions, b) the molecular basis for pathological microglia activation and c) the effect of reactive microglia on motor neuron function.

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Overall, my research will provide a deeper understanding of the role of microglia at the cellular level of ALS and is expected to unveil microglia heterogeneity and impact on motor neuron survival, leading towards the development of therapies rooted in knowledge of ALS disease mechanisms.

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Figure 1: Microglia activation in ALS (created in BioRender.com).

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METHODOLOGY:

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I am using ALS induced Pluripotent Stem Cells (iPSCs) derived from patients with ALS to generate microglia, motor neurons and astrocytes in cell culture. I have genetically engineered the iPSCs to overexpress Transcription Factors that allow their rapid and effective differentiation by forward programming. Subsequently, cell types are cultured together at defined ratios to establish a tri-culture model of neuroinflammation. Using methods such as Live Cell Imaging, Confocal Microscopy, Electrophysiology and Transcriptomics, I aim to uncover the complex interplay of processes that contribute to motor neuron damage and death.

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RESULTS:

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ALS microglia exhibited a significant impairment in homeostatic functions such as phagocytosis, release of inflammatory molecules and reactive morphologies consistent with a neuroinflammatory phenotype. RNA sequencing revealed consistent alterations in the expression of genes involved in relevant pathways such as apoptosis, neurodegeneration and cytokine signalling. Finally, ALS-microglia recapitulated pathological hallmarks of ALS previously observed in motor neurons. Co-cultures of microglia and motor neurons on astrocytes were successfully established and characterised.

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Figure 2: Microglia interacting with motor neurons in a co-culture (left). Healthy and ALS microglia phagocytosing (eating) fluorescent bioparticles (right). Scale bar: 10μm.

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