Tunneling nanotubes at the microglia‐neuron interface in neuroinflammation and Alzheimer’s disease

Background Brain‐resident innate immune cells termed microglia can mediate synapse formation and plasticity, but become dysregulated in Alzheimer’s disease (AD), contributing to chronic neuroinflammation and synapse loss (Wu, Dissing‐Olesen et al. 2015, Hong, Beja‐Glasser et al. 2016). Yet, the mechanisms underlying direct microglia‐neuron interactions are poorly defined. Pro‐inflammatory signals can induce extremely long, thin, membranous connections, i.e., tunneling nanotubes (TNTs), in peripheral innate immune cells (Zaccard, Rinaldo et al. 2016). TNTs have been observed in various cell types and can support high‐speed, cell‐cell trafficking of diverse cytoplasmic and membrane‐bound cargoes. Herein, we explore the role of TNTs in microglia‐neuron communication during neuroinflammation and AD. Method We established a co‐culture model of human induced excitatory neurons (iENs) and postmortem elderly adult human microglia. Microglia‐tropic adeno‐associated virus vectors encoding plasma membrane‐targeted tdTomato fluorophores were utilized to label microglia and emerging TNTs. We treated co‐cultures with pro‐inflammatory factors, i.e., oligomeric amyloid β (Aβ) and interferon‐gamma, or anti‐inflammatory factors for 24 hours, and utilized our recently developed enhanced resolution confocal microscopy technique to assess TNTs. Result The number of TNT‐positive microglia and number of TNTs per microglia were increased in live human iEN‐microglia co‐cultures under AD‐relevant pro‐inflammatory conditions compared to anti‐inflammatory or control conditions. Additionally, TNT length and the percentage of TNTs contacting neuronal axons or dendrites were significantly increased under pro‐inflammatory conditions. We used time‐lapse enhanced resolution imaging to capture microglial TNTs interacting with spine‐like dendritic protrusions, which extended finer spinule‐like protrusions. Finally, we visualized direct, rapid, cell‐cell transfer of oligomeric Aβ and enhanced localization of oligomeric Aβ to TNTs under pro‐inflammatory conditions in microglia mono‐cultures. Conclusion Our results in human iEN‐microglia co‐cultures indicate that an AD‐relevant, pro‐inflammatory micro‐environment increases microglial TNT formation and TNT‐driven interactions with neuronal axons, dendrites, and dendritic protrusions. TNTs can provide additional contact points and extend the reach of microglia, facilitating communication with distant target microglia or sub‐neuronal elements. Dur