Extended Data Fig. 9: DHHC5 regulates Aβ deposition in 5×FAD mice through autophagy.

a, b, Confocal image (a) and quantification (b) of RFP-EGFP-LC3 in hippocampal neurons of tfLC3;Dhhc5^f/f;Camk2a-Cre mice three days after intraperitoneally treated with 5.3 μmol/kg Tat-scramble or Tat-Beclin 1 peptide. A focus was made on the CA1, CA2 and CA3 regions of the hippocampus. Quantification of LC3-positive dots presented in individual neurons were expressed as a percentage of cell with LC3 dots (minimum three dots per cell) (b, left). RFP puncta per cell were measured to calculate LC3 flux (b, right). Bars represent mean ± s.e.m., each dot represents average quantification from one individual mouse (n = 6 mice for each group). Unpaired two-tailed t-tests. c, Fluorescent microscopy images of hippocampus two months after local stereotactic injections with AAV-hSyn-shmiR-Atg5 and AAV-hSyn-shmiR-NC. The construct expresses EGFP, which allows for verification of the site and efficacy of infection. Similar results were observed in n = 3 independent experiments. d, Confirmation of ATG5 expression by western blots in isolated hippocampal neurons of Dhhc5^f/f mice two months after local stereotactic injections with AAV-hSyn-shmiR-Atg5 and AAV-hSyn-shmiR-NC. Similar results were observed in n = 3 independent experiments. e, f, Confocal image (e) and quantification (f) of RFP-EGFP-LC3 in neurons of CA1, CA2 and CA3 regions of mice with indicated genotypes two months after local stereotactic injections with AAV-hSyn-shmiR-Atg5 or AAV-hSyn-shmiR-NC. Quantification of LC3-positive dots presented in individual neurons were expressed as a percentage of cell with LC3 dots (minimum three dots per cell) (f, left). RFP puncta per cell were measured to calculate LC3 flux (f, right). Bars represent mean ± s.e.m., each dot represents average quantification from one individual mouse (n = 6 mice for each group). Two-way ANOVA, post hoc Bonferroni’s test.

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