Background: TDP-43 is an RNA/DNA-binding protein implicated in the pathogenesis of several neurodegenerative disorders (NDDs), including amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer’s disease. Emerging evidence suggests that TDP-43 loss in microglia alters their functional state, contributing to exaggerated synaptic engulfment. Excessive reliance on glycolysis in microglia has been associated with heightened inflammatory outputs and pathological synaptic pruning, whereas enhanced oxidative phosphorylation (OXPHOS) moderates cytokine release and promotes engulfment of pathological cargoes. Based on these observations, we asked whether metabolic alterations represent the mechanistic link between TDP-43 loss and exaggerated synaptic engulfment.

Study aim: This study aims to demonstrate the mediating role of cellular metabolic pathways in exaggerated synaptic phagocytosis by microglia with TDP-43 loss of function.

Ethical standards and consent to participate: Not applicable.

Materials and methods: To recapitulate TDP-43 loss of function, TDP-43 was knocked down in murine BV2 and human HMC3 microglial cells using RNA interference. The effects of TDP-43 depletion on microglial metabolism were then examined via glucose uptake and lactate, ATP, and reactive oxygen species (ROS) production assays. Furthermore, live-cell metabolic flux analysis (MFA) of the two cell lines was performed after TDP-43 knockdown under glycolytic and mitochondrial stress. Finally, synaptoneurosome uptake was quantified in cells with TDP-43 knockdown after metabolic rerouting.

Statistical analysis: Data from biological triplicates across independent passages were normalised to matched non-silencing controls and pooled. Outliers were excluded and distributional assumptions assessed before hypothesis testing. Two-group comparisons used parametric tests with variance correction when appropriate, or non-parametric alternatives when assumptions were not met; time-course metabolic responses were analysed with repeated-measures models and post hoc multiple-comparisons testing, and viability across time points was assessed by within-group ANOVA with post hoc testing.

Results: TDP-43 depletion was associated with increased microglial glycolysis in both cell lines, without a concomitant decrease in mitochondrial oxidative phosphorylation as measured by metabolic flux analysis. This was corroborated by increased glucose uptake, lactate production, and intracellular ATP and ROS levels. TDP-43 knockdown also led to exaggerated engulfment of synaptoneurosomes by microglia, which was reversed by metabolic rerouting via supplementation with pyruvate-containing, glucose-free medium.

Conclusions: Collectively, our findings identify TDP-43–dependent microglial metabolic rewiring as a potential driver of pathological synapse loss. The mechanistic links between TDP-43 depletion and a hyperglycolytic, pro-phagocytic microglial phenotype define a novel non-cell autonomous route to neurodegeneration. Importantly, pyruvate supplementation’s ability to attenuate microglial metabolic and functional aberrations highlights metabolic rerouting as a promising therapeutic avenue in TDP-43-associated NDDs, warranting validation in vivo.