Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses (2021)
Figure 3

Figure 3. Neuronal Cdc50a deletion induces PS exposure preferentially in the cell soma

A. Schematic diagram of the in vitro CDC50A-mCherry expression experiment. After 6 days in vitro (DIV), cortical neurons were transfected with CAG-GFP and CAG-mCherry or CAG-GFP and CAG-Cdc50a-mCherry. Representative confocal z-stack images of the rER (Nissl staining, cyan) with GFP (green) and mCherry/CDC50A-mCherry (red). The dashed line represents where the high magnified insert was taken from. Scale bars, 50 (left), 20 (middle), and 5 μm (right).

B. Schematic diagram of in vivo CDC50A-mCherry expression experiment. Neonatal (P0) wild-type mice were injected with AAV9-CAG-Cdc50a-mCherry. Representative confocal z-stack images of the rER (Nissl staining, green) and CDC50A-mCherry (red) in the CTX (cortex). The dashed line represents where the high magnified insert was taken from. Scale bars, 20 μm (upper) and 5 μm (lower).

C. Schematic diagram of the experiment in which PS exposure was detected in control and Cdc50a cKO neurons in vitro. After 6 DIV, cortical Cdc50afl/fl neurons were transfected with either hSyn-mCherry or hSyn-Cre-p2a-dTomato to generate control and Cdc50a cKO neurons, respectively. Representative live confocal z-stack images showing PS exposure (pSIVA, green) in mCherry/dTomato (red)-expressing neurons. Scale bar, 20 μm.

D. The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice.

E. Schematic diagram of the method in which PS exposure was detected in vivo using secreted Annexin V (secA5) fused with mCherry (red). Representative confocal z-stack images of secA5-mCherry (red) expression in the brains of wild-type mice using AAV9-GFAP-secA5-mCherry injection. The white circles indicate PS exposure detected by secA5. Scale bar, 10 μm.

F. Schematic diagram of the method in which PS exposure was detected in the brains of control and Cdc50a cKO mice in vivo after AAV9-GFAP-secA5-mCherry was injected in the neonatal stage (P0). Representative confocal single-plane images showing PS exposure (secA5-mCherry, red) in the somas of Thy1-CreERT2, -EYFP-expressing HP (hippocampus) CA1 neurons or IC (inferior colliculus) neurons (green) from control and Cdc50a cKO mice. Scale bar, 5 μm.

G. Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP. Representative confocal z-stack images showing Gephyrin-positive inhibitory post-synapses in the somas (red) and proximal (green) and distal dendrites (cyan) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice. Scale bar, 20 μm.

H. Bar graphs showing the number of Gephyrin-positive inhibitory post-synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice (n = 4 for each group, P*** = 0.0002, P**** < 0.0001).
Data information: The individual dots indicate experimental replicates (3 to 5 images were taken from 2 brain slices per an animal, H). The data are the mean ± s.e.m. and were analyzed by two-tailed unpaired t-test .