Motor neuron disease (MND) is characterised by the progressive degeneration of upper and lower motor neurons in the central and peripheral nervous system. Approximately 10% of all MND cases are familial and driven by underlying genetic causes. Mutations in Optineurin (OPTN) and TANK-binding kinase 1 (TBK1) are rarer contributors, accounting for ~1% of familial MND cases individually. Both OPTN and TBK1 are multifunctional proteins involved in autophagy, including mitophagy, and immune homeostasis1. However, their role in regulating neuroinflammatory responses and mitochondrial function in resident immune cells of the brain, microglia, remains poorly understood.
The primary objective of this study was to characterise how loss of OPTN or TBK1 affects mitochondrial function in human iPSC-derived microglia under inflammatory stimulation. To address this, we utilised iPSC-derived microglia2,3 carrying OPTN knockout (OPTN-KO) or TBK1 knockout (TBK1-KO) genotypes (JAX/iNDI lines). Cells were treated with lipopolysaccharide (LPS; 100 ng/ml; 24 hours) and the STING pathway agonist cyclic GMP-AMP (cGAMP; 1 µg/ml; 2 hours). Mitochondrial respiration was assessed using the Seahorse XF Mitochondrial Stress Test, while mitochondrial membrane potential and morphology were evaluated using Opera Phenix® high-content imaging.
Seahorse analysis revealed that OPTN-KO microglia exhibited significantly increased maximal respiratory capacity, spare respiratory capacity, and non-mitochondrial respiration following 24-hour stimulation with LPS and interferon gamma (IFNγ), compared to non-treated control microglia (p <0.05[OP1] , n = 3–4, two-way ANOVA with Tukey’s multiple comparisons). In contrast, TBK1-KO microglia did not show significant alterations in mitochondrial respiration under any tested condition (n = 3, two-way ANOVA with Tukey’s multiple comparisons). Preliminary high-content imaging analyses suggest genotype- and stimulus-dependent alterations in mitochondrial membrane potential and mitochondrial network organisation.
Together, these findings indicate that loss of OPTN, but not TBK1, sensitises human microglia to inflammatory stimuli by altering mitochondrial bioenergetic responses. This work highlights distinct roles for OPTN and TBK1 in regulating microglial mitochondrial function during neuroinflammation. Improved understanding of microglial metabolic dysregulation in MND may provide insight into non–cell-autonomous disease mechanisms and identify novel therapeutic targets aimed at modulating neuroinflammatory responses.