Ren, Yi

ABSTRACT Most brain organoids derived from human induced pluripotent stem cells (iPSCs) lack microglia and thus immune function. Microglia‐like cells (MGCs) can be differentiated from iPSCs, while the characteristics of isogenic MGC‐containing brain organoids in modeling neurodegeneration and cell‐cell communications have not been well investigated. In this study, iPSC‐derived MGCs are co‐cultured with isogenic forebrain cortical organoids (iFCo), which are stimulated with extracellular vesicles (EVs) of brain organoids differentiated from Alzheimer's disease (AD) patient‐derived iPSCs ( APOE ε4/ε4 and presenilin 1). The AD EV‐stimulated co‐culture organoids are treated with EVs from healthy MGCs or co‐culture. Differential responses of the co‐cultured organoids and the MGCs to AD EVs are demonstrated. The co‐cultured organoids mitigated pro‐inflammatory gene expressions. EVs from healthy MGCs or co‐culture reduced the expression of IL‐12β , iNOS , TREM2 , and CASS4 , which are associated with neural inflammation and degeneration, as well as showed regulation on genes involved in microglial activation and carbon metabolism. AD EV cargo analysis by proteomics and microRNA‐sequencing revealed APOE and APP proteins and microRNAs regulated pathways such as mitophagy. This study paves the way for understanding the role of microglia and brain organoids in modeling neural degeneration and the development of EV‐based cell‐free therapeutics for AD treatment.

Abstract Activation of spinal cord microglia contributes to the development of peripheral nerve injury-induced neuropathic pain. However, the molecular mechanisms underlying microglial function in neuropathic pain are not fully understood. We identified that the voltage-gated proton channel Hv1, which is functionally expressed in spinal microglia, was significantly increased after spinal nerve transection (SNT). Hv1 mediated voltage-gated proton currents in spinal microglia and mice lacking Hv1 (Hv1 KO) display attenuated pain hypersensitivities after SNT compared with wildtype (WT) mice. In addition, microglial production of reactive oxygen species (ROS) and subsequent astrocyte activation in the spinal cord was reduced in Hv1 KO mice after SNT. Cytokine screening and immunostaining further revealed that IFN-γ expression was compromised in spinal astrocytes in Hv1 KO mice. These results demonstrate that Hv1 proton channel contributes to microglial ROS production, astrocyte activation, IFN-γ upregulation, and subsequent pain hypersensitivities after SNT. This study suggests Hv1-dependent microglia-astrocyte communication in pain hypersensitivities and identifies Hv1 as a novel therapeutic target for alleviating neuropathic pain.