Fig. 9: AMPK-mediated phosphorylation of ULK2 is crucial for oligomeric Aβ42-induced synaptotoxicity and loss of mitochondrial biomass.
a Schematic of ULK2 domain structure highlighting the four predicted AMPK-mediated phosphorylation sites (see Supplementary Fig. S8) conserved in ULK1 and ULK2. b Flag-mULK2 WT or Flag-mULK2 4SA (the four conserved phosphorylation sites shown in Supplementary Fig. S8b are mutated to alanine) was overexpressed in HEK293T cells co-expressing either GST or GST-ca-AMPKα1(1–312) (constitutively active AMPK). Cells were treated with DMSO or 50 μM of compound 991 for 1 h. Blots were probed with AMPK substrate motif antibody, Flag M2 monoclonal antibody, total ULK2, p-Thr172 AMPK, total AMPK, and actin antibodies. c Representative high-magnification images of secondary dendritic segments showing dendritic spines in the upper panel and mitochondria in the lower panel. ULK2F/F embryos were ex utero electroporated at E15.5 with pCAG-mVenus, pCAG-mito-DsRed, either a scrambled pCAG-Cre (Control, no Cre), or pCAG-Cre recombinase. The pCAG-Cre recombinase conditions in which ULK2 is genetically removed were also co-electroporated with either pCAG-mULK2 WT (Wild type), pCAG-mULK2 KI (K39I) (Kinase inactive), or pCAG-mULK2 4SA (Kinase dead, see Supplementary Fig. S8). Neurons were treated at 20DIV with either a vehicle control or Aβ42o for 24 h. d–f Quantification of d dendritic mitochondrial length, (e) dendritic mitochondrial density, and f spine density. Analyses were done blind to the experimental conditions and done by manual counting using FIJI. For western blot in panel b, the same results were obtained for three independent experiments. In panels d–f, data are represented by box plots displaying minimum to maximum values, with the box denoting 25th, 50th (median), and 75th percentiles from three independent experiments. nULK2WT Control = 32 dendrites, 194 mitochondria; nULK2WT Aβ42o = 30 dendrites, 289 mitochondria; nULK2KI Control = 36 dendrites, 183 mitochondria; nULK2KI Aβ42o = 34 dendrites, 449 mitochondria; nULK24SA Control = 34 dendrites, 198 mitochondria; nULK24SA Aβ42o = 32 dendrites, 472 mitochondria. Statistical analyses were performed using a Mann–Whitney test in (d–f). Exact P values are indicated on the figure when available through Prism software, otherwise, the test significance is provided using the following criteria: *P < 0.001; ****P < 0.0001. Scale bar for magnified dendritic segments = 2 μm.
