Pulimood, N. S. | Search bioRxiv

bioRxiv2025

Transcriptomic analysis of C9orf72-mutated iPSC-derived microglia implicates cell-autonomous upregulation of selected NLRP3 inflammasome genes in motor neuron degeneration

Hexanucleotide repeat expansion (HRE) in the non-coding region of the gene C9orf72 is the most prevalent mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 HRE contributes to neuron degeneration in ALS/FTD through both cell- autonomous mechanisms and non-cell autonomous disease processes involving glial cells such as microglia. The molecular mechanisms underlying the contribution of C9orf72-HRE microglia to neuron death in ALS/FTD remain to be fully elucidated. In this study, we generated microglia from human C9orf72-HRE and isogenic iPSCs using three different microglia derivation methods. RNA sequencing analysis reveals a cell-autonomous dysregulation of extracellular matrix (ECM) genes and genes involved in pathways underlying inflammasome activation in C9orf72-HRE microglia. In agreement with elevated expression of inflammasome components, conditioned media from C9orf72-HRE microglia enhance the death of C9orf72- HRE motor neurons implicating microglia-secreted molecules in non-cell autonomous mechanisms of C9orf72 HRE pathology. These findings suggest that aberrant activation of inflammasome-mediated mechanisms in C9orf72-HRE microglia results in a pro-inflammatory phenotype that contributes to non-cell autonomous mechanisms of motor neuron degeneration in ALS/FTD. SummaryThis study describes phenotypic alterations in C9orf72-ALS/FTD microglia implicating extracellular matrix remodeling and inflammasome activation in microglia-mediated neurodegeneration in ALS/FTD.

neuroscience↗

bioRxiv2022

Comparing the potential of microglia preparations generated using different human iPSC-based differentiation methods to model microglia-mediated mechanisms of amyotrophic lateral sclerosis pathophysiology

Microglia are the resident immune cells of nervous system. In healthy conditions, microglia actively patrol neural tissues in a homeostatic state, which can rapidly change to an activated state in response to local injury/disease. Dysregulated microglia activation is a hallmark of disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The elucidation of the roles of microglia in human biology and disease has recently benefitted from the development of human induced pluripotent stem cell (iPSC)-based approaches to generate microglia-like cells. Microglia represent a heterogeneous group of cells with spatial diversity in both health and disease. This situation poses a considerable challenge along the path towards establishing the most pathologically-relevant human iPSC-derived microglia preparations to investigate the complex roles of microglia in ALS and other neurological diseases. The success of these approaches must account for microglia diversity in different regions of the brain and spinal cord. In this study, we compared the transcriptomes of human iPSC-derived microglia generated using different methods to determine whether or not separate strategies can be used to generate microglia with distinct transcriptional signatures in vitro. We show that microglia derived using two different methods display distinct in vitro maturation characteristics. We also reveal that different derivation methods give rise to preparations comprising human microglia with distinct transcriptomic signatures resembling the gene profiles of specific microglia subpopulations in vivo. These findings suggest that a careful, and coordinated, implementation of multiple microglia differentiation methods from human iPSCs can be an effective approach towards the goal of generating multiple microglia subtypes that will offer enhanced model systems to account for microglia heterogeneity in vivo. Spatially-defined human iPSC-derived microglia would represent an enhanced tool to study the multiple levels of involvement of microglia in mechanisms of motor neuron degeneration in ALS, as well as other neurological diseases and disorders.

neuroscience↗