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Retinal neovascularization is a leading cause of severe visual loss in humans, and molecular mechanisms of microglial activation-driven angiogenesis remain unknown. Using single-cell RNA sequencing, we identified a subpopulation of microglia named sMG2, which highly expressed necroptosis-related genes Rip3 and Mlkl. Genetic and pharmacological loss of function demonstrated that hypoxia-induced microglial activation committed to necroptosis through the RIP1/RIP3-mediated pathway. Specific deletion of Rip3 gene in microglia markedly decreased retinal neovascularization. Furthermore, hypoxia induced explosive release of abundant FGF2 in microglia through RIP3-mediated necroptosis. Importantly, blocking signaling components of the microglia necropotosis–FGF2 axis largely ablated retinal angiogenesis and combination therapy with simultaneously blocking VEGF produced synergistic antiangiogenic effects. Together, our data demonstrate that targeting the microglia necroptosis axis is an antiangiogenesis therapy for retinal neovascular diseases.

The primate neocortex exerts high cognitive ability and strong information processing capacity. Here, we establish a single-cell RNA sequencing dataset of 133,454 macaque visual cortical cells. It covers major cortical cell classes including 25 excitatory neuron types, 37 inhibitory neuron types and all glial cell types. We identified layer-specific markers including HPCAL1 and NXPH4, and also identified two cell types, an NPY-expressing excitatory neuron type that expresses the dopamine receptor D3 gene; and a primate specific activity-dependent OSTN + sensory neuron type. Comparisons of our dataset with humans and mice show that the gene expression profiles differ between species in relation to genes that are implicated in the synaptic plasticity and neuromodulation of excitatory neurons. The comparisons also revealed that glutamatergic neurons may be more diverse across species than GABAergic neurons and non-neuronal cells. These findings pave the way for understanding how the primary cortex fulfills the high-cognitive functions.

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Purpose Neurons are the bricks of the neuronal system and experimental access to certain neuron subtypes will be of great help to decipher neuronal circuits. Here, we identified trophoblast glycoprotein (TPBG)-expressing GABAergic amacrine cells (ACs) that were selectively labeled in DAT-tdTomato transgenic mice. Methods Retina and brain sections were prepared for immunostaining with antibodies against various biomarkers. Patch-sequencing was performed to obtain the transcriptomes of tdTomato-positive cells in DAT-tdTomato mice. Whole-cell recordings were conducted to identify responses to light stimulation. Results Tyrosine hydroxylase immunoreactive cells were colocalized with tdTomato-positive cells in substantia nigra pars compacta, but not in the retina. Transcriptomes collected from tdTomato-positive cells in retinas via Patch-sequencing exhibited the expression of marker genes of ACs ( Pax6 and Slc32a1 ) and marker genes of GABAergic neurons ( Gad1 , Gad2 , and Slc6a1 ). Immunostaining with antibodies against relevant proteins (GAD67, GAD65, and GABA) also confirmed transcriptomic results. Furthermore, tdTomato-positive cells in retinas selectively expressed Tpbg , a marker gene for distinct clusters molecularly defined, which was proved with TPBG immunoreactivity in fluorescently labeled cells. Finally, tdTomato-positive cells recorded showed ON–OFF responses to light stimulation. Conclusions Ectopic expression occurs in the retina but not in the substantia nigra pars compacta in the DAT-tdTomato mouse, and fluorescently labeled cells in the retina are TPBG-expressing GABAergic ACs. This type of transgenic mice has been proved as an ideal tool to achieve efficient labeling of a distinct subset of ACs that selectively express Tpbg .