Astrocytes and microglia play orchestrated roles and respect phagocytic territories during neuronal corpse removal in vivo

Abstract

Cell death is prevalent throughout life; however, the coordinated interactions and roles of phagocytes during corpse removal in the live brain are poorly understood. We developed photochemical and viral methodologies to induce death in single cells and combined this with intravital optical imaging. This approach allowed us to track multicellular phagocytic interactions with precise spatiotemporal resolution. Astrocytes and microglia engaged with dying neurons in an orchestrated and synchronized fashion. Each glial cell played specialized roles: Astrocyte processes rapidly polarized and engulfed numerous small dendritic apoptotic bodies, while microglia migrated and engulfed the soma and apical dendrites. The relative involvement and phagocytic specialization of each glial cell was plastic and controlled by the receptor tyrosine kinase Mertk. In aging, there was a marked delay in apoptotic cell removal. Thus, a precisely orchestrated response and cross-talk between glial cells during corpse removal may be critical for maintaining brain homeostasis.

Fig. 1. Live imaging of astrocytes and microglia during cell corpse removal.

(A) Photochemical ablation and induction of single-cell apoptosis via 2Phatal in live mouse cortex. (B) Hoechst 33342 dye labeling of cell nuclei (blue), fluorescently labeled neurons (white), and microglia (green) before 2Phatal. The boxed region is magnified, and time-lapse imaging shows targeting of a single neuron for 2Phatal (blue arrow) followed by a single microglial cell occupying the territory of the ablated neuron 24 hours later. (C) Time-lapse imaging showing microglia engulfment of a dying neuron with a condensed nucleus (red arrow). (D) Traces depicting 21 cell death events, showing the timing of microglia arrival and engagement with the dying cells from four mice. (E) Time-lapse imaging detailing microglia engulfment of apoptotic neurites (red arrows) over 6 hours. (F) Time-lapse imaging showing subtle astrocyte polarization around a cell targeted with 2Phatal (green arrowheads). (G) Timing of astrocyte engagement with 15 single dying cells collected from four mice. (H) Time-lapse imaging showing the dynamic formation of astrocyte polarization around apoptotic neurites over 6 hours (green arrowheads).

Fig. 2. Territorial segregation of glial processes during cell corpse engulfment.

(A) Confocal fluorescence image showing division of labor between microglia (green) and astrocytes (red) during elimination of a neuron (white, yellow arrow) and its apical dendrite, after cell death induction by high-titer adeno-associated virus-9 (AAV9)–green fluorescent protein (GFP) infection. The microglia has converged on the soma and apical dendrite (yellow arrowheads), while the astrocyte surrounds the distal dendrite (white arrowheads), with boundaries observed between processes of these glial cells. (B) Confocal fluorescence image of spontaneous cell death during postnatal development in mouse cortex. The soma (yellow arrow) and neurites (blue arrows), labeled via caspase-3, are engulfed by microglia (green), while the distal dendritic processes are engulfed by astrocytes (red). Intermingling of both glia processes is observed in the middle portion of the dendrite. (C and D) Examples of cells labeled by caspase-3 immunofluorescence showing astrocytes (red) and microglia (green) polarized toward different parts of the dying cell with territorial boundaries (white arrowheads). (E) Quantification of the relative engulfment of cell bodies and dendrites by astrocytes and/or microglia following cell death by high-titer AAV9-GFP infection (n = 3 mice per group, greater than 50 apoptotic neurites and 20 cell bodies per mouse). Statistics with two-way analysis of variance (ANOVA) with Holm Sidak’s multiple comparisons test.

Fig. 3. Deletion of Mertk, but not Axl, leads to a delay in microglial detection and clearance of apoptotic cells.

(A and B) AAV neuronal labeling and time-lapse imaging detailing the timing of corpse removal after 2Phatal in wild-type, Axl^−/−, Mertk^−/−, and Axl^−/−Mertk^−/− single- or double-knockout mice. Neuronal corpses were cleared within 24 to 48 hours after 2Phatal in wild-type and Axl^−/−, but remained longer in Mertk^−/− and Axl^−/−Mertk^−/− mice. (C) Quantification detailing the time to corpse removal after 2Phatal, revealing clearance defects in Mertk^−/− and Axl^−/−Mertk^−/− mice but not in Axl^−/−, and no significant additive effects of the double knockout [time to cell clearance: wild type = 41 hours; Axl^−/− = 37 hours; Mertk^−/− = 86 hours; Axl^−/−Mertk^−/− = 96 hours; P values as indicated for each comparison, log-rank (Mantel-Cox) test, n = 3 mice per group]. (D) Visualization of microglia in wild-type and Mertk^−/− mice revealed that the delayed corpse removal in Mertk^−/− was caused by delayed microglia engagement with the dying cell. (E) Traces depicting 16 wild-type and Mertk^−/− cells, indicating the timing of cell condensation, microglia engagement, and corpse clearance. (F) Average time for initial microglia engagement comparing wild-type and Mertk^−/− cells (time to engagement: wild type = 6 hours, Mertk^−/− = 55 hours; P < 0.0001, unpaired t test, n = 16 cells per group).

Fig. 4. Mertk knockout disrupts astrocyte lysosome polarization toward dying cells.

(A) Labeling of astrocyte lysosomes after injection of AAV5.LAMP1-GFP into the subarachnoid space in the adult mouse cortex. (B) Astrocyte response to dying cells in the presence of microglia in adult wild-type mice. Under normal conditions, astrocyte lysosomes exhibited little to no polarization around the dying cell with condensed nuclei (white arrow). (C) After PLX3397-mediated microglia ablation, astrocyte processes polarized lysosomes to gradually digest dying cells (white arrows). (D) Astrocytes failed to polarize lysosomes around dying cells in Mertk^−/− mice. KO, knockout. (E) When Mertk is deleted exclusively from microglia (Csf1r-cre:Mertk^fl/fl), astrocytes polarized lysosomes around the cell corpses, consistent with their response when microglia were completely absent. (F) Graphical representation and quantification of lysosome-filled thick astrocyte processes polarized around cell corpses in all groups (control, PLX3397-treated, germline Mertk deletion, and microglia-specific Mertk deletion, n = 3 mice per group).

Fig. 5. A multicellular astrocytic reaction mediates phagocytosis after microglia elimination.

(A) Time-lapse imaging of virally labeled neuron after 2Phatal induction (yellow arrow), showing cell death and corpse clearance occurring over 24 hours. (B) Time-lapse imaging of a neuron (yellow arrow) after induction of apoptosis in a PLX3397-treated mouse. In the absence of microglia, the cytoplasm of the apoptotic neuron remained stable for an additional 24 to 48 hours, with corpse removal occurring 48 to 78 hours after 2Phatal induction. (C) The relative change in cell body size and timing of apoptotic cell clearance in control- and PLX3397-treated cohorts [time to cell clearance: control = 28.9 hours; PLX3397 = 55.1 hours; P = 0.0246, log-rank (Mantel-Cox) test, 15 single cells per mouse, n = 3 mice per group]. (D and E) Delayed corpse removal in the absence of microglia resulted in astrocyte polarization around the dying cell (white arrows and arrowheads). Neuron and astrocyte labeled via AAV infection or with SR101 fluorescent dye for astrocytes (red) in a Thy1-YFP mouse neuron labeling (green). In the absence of microglia, astrocytes did not move their cell bodies during the formation of the barrier. (F) AAV labeling of lysosomes reveals astrocytic lysosome polarization (arrowheads) around a cell corpse in a PLX3397-treated animal.

Fig. 6. Aging disrupts the timing of cell corpse removal.

(A) Time-lapse images detailing: before, 6 hours after, and 24 hours after 2Phatal induction of apoptotic cell death in a 4-month-old mouse. Cells targeted for ablation labeled via Hoechst dye (cyan with yellow arrows). (B) Time-lapse images detailing: before, 6 hours after, 24 hours after, and up to 72 hours after 2Phatal induction of 2Phatal apoptotic cell death in a 26-month-old mouse. Cells targeted for ablation labeled via Hoechst dye (cyan with yellow arrows). (C) Significant difference in the time course of apoptotic cell removal in 4-month-old versus 26-month-old mice [time to clearance: 4 months = 26.2 hours; 26 months = 57.7 hours, P = 0.0069, n = 4 mice per group, log-rank (Mantel-Cox) test].