Microglia Mitochondria Support Neuronal Maturation via Metabolic and Transcriptional Reprogramming in Human 3D In Vitro Brain Model

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by disrupted neuronal circuit maturation. Emerging evidence implicates microglial function and mitochondrial regulation as contributors to ASD-associated biology, yet the mechanisms linking these processes to neuronal development remain poorly defined. Neuronal maturation requires tightly coordinated metabolic and transcriptional remodeling, in which mitochondria play a central role in regulating the developmental tempo and metabolic identity, while microglia modulate neuronal synaptic network maturation; however, whether microglia influence neuronal development through direct mitochondrial contributions remains unknown. Here, using a 3D human in vitro brain model, we show that microglial mitochondria can act as transferable cues that promote metabolic, mitochondria-dynamic, and transcriptional aspects of neuronal maturation. Neurons treated with microglial mitochondria exhibited enhanced oxidative metabolism, improved mitochondrial dynamics, and activation of gene programs associated with nervous system development and neurogenesis. These effects were accompanied by increased expression of dendritic maturation markers, supporting the view that transferred mitochondria can contribute to the regulation of neuronal state. However, full structural and synaptic maturation required the combined action of microglia-derived mitochondria and secreted signaling factors. Together, this study identified microglial mitochondrial transfer as a contributor to neuronal maturation with potential relevance to developmental trajectories disrupted in ASD. Overall design: RNA-seq profiling of NM (Neurons and Microglia co-cultures), NTr (microglia mitochondria-treated neurons), and NUnTr (untreated neurons). (N refers to mt-DsRed2 Induced Human Neural Stem Cells (hiNSCs) and M refers to -mt-EBFP2 HMC3 Microglia Cell Line )