Skip to main content
. 2016 Jan 5;6:18725. doi: 10.1038/srep18725

Figure 4. Activated Drp1 phosphorylated at S616 is localized to mitochondria in FAD animals.

Figure 4

(a) Levels of Drp1 phosphorylated at S616 and S637 detected in the whole brain extracts from NTG1, APP, PS1, APP/PS1, NTG2 and 3xTgAD mice using Western blot analysis. Total Drp1 was used as loading control. Each lane represents individual mouse with three to four mice per group (all females, 40–60 weeks of age). Blots were not cropped. (b) Enhanced recruitment of Drp1 and Drp1 S616 to mitochondria isolated from hippocampi of 3xTgAD mice compared to age-matched NTG2 controls (female mice 60 weeks of age). CN – crude nuclear fraction; CY – cytoplasmic fraction; MT – mitochondria-enriched fraction. Heat shock protein 60 (Hsp60) confirmed mitochondrial enrichment and was used as loading control. Crude nuclear and cytoplasmic fractions were probed for nucleoporin 98 (Nu98) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cropped blots are represented; full-length blots are provided in Supplementary Fig. S3. (c) Densitometry analysis of Drp1 S616 and total Drp1 levels in mitochondrial fractions normalized to Hsp60 levels. Experiments were as in (b). Data represent average ± SEM of three independent experiments. RLU – relative light units. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus NTG. (d) Recruitment of activated Drp1 phosphorylated at S616 to mitochondria increases in hippocampi of 3xTgAD mice compared to age-matched NTG2. Freshly isolated mitochondria from hippocampi (50 μg) were stained with either Drp1 S616 or Drp1 S637 antibodies together with Tom20 (mitochondrial marker) antibody. Mitochondria were gated based on light-scattering properties in the SSC and FSC modes and 20,000 events per sample were collected. To establish gating parameters, isotype non-specific IgG and the appropriate secondary antibodies conjugated with either Alexa 488 or 647 (top panels) were used. An increased phosphorylation of Drp 1 at S616 on mitochondria from AD (53%) vs. NTG (19.5%) animals indicates enhanced translocation (compare middle and bottom left panels). No changes in the level of Drp1 phosphorylated at S637 was found in mitochondrial fractions (compare middle and bottom right panels). Experiments were repeated three times.