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. 2019 Jul 29;9:10919. doi: 10.1038/s41598-019-47227-z

Figure 1.

Figure 1

Alpha-synuclein forms discrete nuclear foci that colocalize with known DDR components. (A) Top: A representative image shows that endogenous alpha-synuclein (αSyn) forms discrete foci in HAP1 cell nuclei that are localized within the nucleus. Scale bar 20 μm, inset 10 μm. Middle & Bottom: No such similar staining is seen with a secondary antibody-only control or in SNCA knock-out (αSyn KO) cells. (B) Top: A representative image shows that intranuclear αSyn foci colocalize with DDR components, including the DNA repair factors γH2AX and PAR. Inset shows region shown at higher magnification below. Scale bar 5 μm. Bottom: Quantification of colocalization between αSyn, γH2AX and PAR compared to what would be expected with the same foci density at random (αSyn-γH2AX = 0.136 ± 0.005, random translation = 0.023 ± 0.002, N = 322 nuclei, paired t-test p < 0.0001; αSyn-PAR = 0.165 ± 0.008, random translation = 0.022 ± 0.001, N = 325 nuclei, paired t-test p < 0.0001; γH2AX-PAR = 0.141 ± 0.006, random translation = 0.024 ± 0.001, N = 325 nuclei, paired t-test p < 0.0001). (C) Endogenous mouse αSyn staining in WT animals is specific and can be found as discrete foci within the nucleus of cortical neurons. Top: Images demonstrate a single plane from a three dimensional z-stack showing αSyn foci. Middle: Projection of three dimensional z-stack in 3 different planes shows that αSyn foci are within the nucleus. Bottom: αSyn staining is absent in SNCA KO mouse tissue, since it is completely abolished in all (>200) αSyn KO mouse cortical neurons analyzed, as shown in this representative image. Scale bar 4 μm. (D) Top: A representative image shows that endogenous mouse αSyn forms discrete intranuclear foci in mouse cortical neurons that colocalize with DDR components, including the DNA repair factors γH2AX and PAR. Inset shows region shown at higher magnification below. Scale bar 5 μm. Bottom: Quantification of colocalization between αSyn, γH2AX and PAR compared to what would be expected with the same foci density at random (αSyn-γH2AX = 0.121 ± 014, random translation = 0.011 ± 0.002, paired t-test p < 0.0001; αSyn-PAR = 0.076 ± 0.002, random translation = 0.019 ×  ± 0.002, paired t-test p < 0.0001; γH2AX-PAR = 0.121 ± 0.014, random translation = 0.011 ± 0.002, paired t-test p < 0.0001; N = 79 nuclei, 4 animals).