From Gene to Glia: Unravelling microglia mechanisms behind ALS using human brain organoids

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive loss of upper and lower motor neurons, leading to muscle weakness, paralysis, and death typically within 3–5 years after onset. Although ALS primarily affects motor function, it shares clinical, genetic, and pathological features with frontotemporal dementia (FTD), and both disorders are now considered part of an ALS–FTD disease spectrum. Most ALS cases are sporadic, but 5–10% are familial, with the hexanucleotide repeat expansion in C9ORF72 (C9-HRE) representing the most common genetic cause of ALS/FTD.
Increasing evidence implicates immune dysregulation and microglial dysfunction in ALS pathogenesis, particularly in C9-ALS/FTD. However, the precise contribution of microglia remains unclear, in part due to limitations of model systems that fail to capture the complex multicellular environment of the human brain. To address this, my thesis developed and applied human iPSC-derived cerebral organoids in which microglia emerge intrinsically alongside neurons and astrocytes, enabling investigation of microglial dysfunction in a physiologically relevant 3D context.
In Chapter 2, I generated cerebral organoids from iPSCs derived from C9-ALS/FTD patients and healthy controls, allowing microglia to develop endogenously. Organoid-derived microglia (oMGs) from C9-ALS/FTD patients exhibited hallmark C9-HRE molecular pathologies, including RNA foci and C9ORF72 haploinsufficiency. Morphological analyses revealed C9-dependent alterations in microglial structure, indicating early microglial abnormalities that may contribute to disease pathogenesis.
Chapter 3 focused on transcriptional profiling of oMGs. RNA sequencing revealed broad downregulation of genes associated with microglial homeostasis, phagocytosis, lysosomal function, and immune signaling in C9-oMGs. Pathway analyses showed impaired lysosomal organization, phagocytosis, and NF-κB signaling, consistent with dysfunctional microglial states. In contrast, upregulated pathways were linked to synapse assembly and axon development, potentially reflecting compensatory responses. Transcription factor analysis identified dysregulation of key regulators, particularly the master microglial transcription factor PU.1 (SPI1), highlighting disrupted transcriptional control as a central disease mechanism.
In Chapter 4, I translated these transcriptional changes into functional outcomes. Live imaging assays demonstrated reduced phagocytic capacity of C9-oMGs, which was further validated by impaired clearance of synaptic proteins within organoids. Microglia stimulation experiments revealed defective cytokine responses in C9 microglia specifically in the 3D organoid context, underscoring the importance of the multicellular environment for revealing disease-relevant phenotypes.
Chapter 5 I investigated mechanisms underlying these defects. Given the suppression of PU.1 and its downstream targets (including TREM2), I overexpressed PU.1 in C9 iPSC-derived microglia. This intervention restored phagocytic capacity and increased expression of key phagocytosis-related genes, with a stronger rescue effect in C9 microglia than in controls, identifying PU.1 as a potential therapeutic target.
In summary, this thesis demonstrates that C9-HRE induces profound transcriptional and functional impairments in human microglia that are only fully revealed in a 3D multicellular context. These findings highlight the central role of microglial dysfunction in ALS/FTD and establish human brain organoids as a powerful platform for studying disease mechanisms and therapeutic strategies.