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. 2015 Aug 7:1616:101-11.
doi: 10.1016/j.brainres.2015.04.059. Epub 2015 May 8.

Estrogen amelioration of Aβ-induced defects in mitochondria is mediated by mitochondrial signaling pathway involving ERβ, AKAP and Drp1

Saumyendra Sarkar  1 , Sujung Jun  1 , James W Simpkins  2
Affiliations

Estrogen amelioration of Aβ-induced defects in mitochondria is mediated by mitochondrial signaling pathway involving ERβ, AKAP and Drp1

Saumyendra Sarkar et al. Brain Res. .

Abstract

Perturbations in dynamic properties of mitochondria including fission, fusion, and movement lead to disruption of energy supply to synapses contributing to neuropathology and cognitive dysfunction in Alzheimer׳s disease (AD). The molecular mechanisms underlying these defects are still unclear. Previously, we have shown that ERβ is localized in the mitochondria and ERβ knock down disrupts mitochondrial functions. Because a selective ERβ modulator (DPN) can activate PKA, and localized PKA signaling in the mitochondrial membrane regulates mitochondrial structure and functions, we reasoned that ERβ signaling in the mitochondrial membrane rescues many of the mitochondrial defects caused by soluble Aβ oligomer. We now report that DPN treatment in primary hippocampal neurons attenuates soluble Aβ-oligomer induced dendritic mitochondrial fission and reduced mobility. Additionally, Aβ treatment reduced the respiratory reserve capacity of hippocampal neuron and inhibited phosphorylation of Drp1 at its PKA site, which induces excessive mitochondrial fission, and DPN treatment ameliorates these inhibitions. Finally, we discovered a direct interaction of ERβ with a mitochondrial resident protein AKAP1, which induces the PKA-mediated local signaling pathway involved in increased oxidative phosphorylation and inhibition of mitochondrial fission. Taken together, our findings highlight the possibility that ERβ signaling pathway may be a useful mitochondria-directed therapeutic target for AD.

Keywords: AKAP1; Alzheimer׳s disease; Estrogen receptor β; Mitochondrial Fission And Fusion; Mitochondrial Movement; PKA.

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Conflict of interest statement

The Authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Estrogens rapidly enhance phosphorylation of Drp1 (ser637), rescue Aβ-induced inhibition of Drp1 phosphorylation and reduce mitochondrial fission in dendrites of primary rat hippocampal neuron. A) Effects of DPN (50 nM) on Drp1 phosphorylation. Expression levels were normalized to actin and presented as mean ± SD compared to the vehicle (**p< 0.01 versus vehicle; n = 4). B) Effects of Aβ (Aβ1-42 oligomers 200 nM) and DPN (50 nM) on Drp1 phosphorylation. Expression levels were normalized to actin (mean ± SD, ***p< 0.001 DPN versus vehicle; and ***p< 0.001 versus the connected group, n= 4). C–H) Effects of Aβ, PPT, DPN and their combination on mitochondrial length. I) Lengths of dendritic mitochondria. **p< 0.01 and ***p< 0.001 versus the connected groups. N = 600 (Vehicle), 450 (Aβ), 400 (PPT), 480 (Aβ + PPT), 550 (DPN) and 500 (Aβ + DPN).
Fig. 2
Fig. 2
Ameliorating effects of DPN on Aβ-induced defects in mitochondrial movement in dendrites of primary rat hippocampal neuron. A–D). Representative snapshot images from time lapse video showing mitochondrial movement in vehicle (A) Aβ (B), DPN (C), and Aβ + DPN (D) treatment groups. (E) Percentage of stationary and moving mitochondria. Depicted are mean ± SD. *p< 0.05 vs. the connected groups, **p< 0.01 vs. the connected groups. N = 600 (Vehicle), 450 (Aβ), 550 (DPN) and 500 (Aβ + DPN) mitochondria. (F) Analysis of mean anterograde and retrograde velocity of movable mitochondria (μm/sec) is shown. Depicted are mean ± SD. ***p< 0.001 vs. the groups connected. N= 58 (Vehicle), 61 (Aβ), 62 (DPN) and 62 (Aβ + DPN) dendrites.
Fig. 3
Fig. 3
Localization of AKAP1 and ERβ in the mitochondria. HEK 293 cells were transfected with equimolar concentrations of C-terminal DDK fused to AKAP1 and C-terminal HA fused to ERβ expression vector (both GeneCopoeia) plasmid DNA, using lipofectamine 2000 reagent (Invitrogen, CA, USA). Forty eight hours after transfection, cells were immunostained with anti-DDK mouse and anti-HA rabbit primary antibody followed by Alexa 633 anti-mouse and Alexa 488 anti-rabbit secondary antibody. Cells were then visualized by Zeiss LSM510 confocal microscope. Photomicrographs were analyzed for colocalization of AKAP1 and ERβ, using Zeiss LSM software. A) Red, DDK antibody, B) Green, HA antibody, C) Merged, and D) Marked cell used for co-localization analysis as shown in (E) and in the tabular form in F. For the localization of endogenous AKAP1 and ERβ, rat primary neurons were sequentially immunostained with anti-AKAP1 and anti-ERβ primary antibody. Photomicrographs were shown in blue for DAPI (G), in red for ERβ (H), in green for AKAP1 (I) and merged (J).
Fig. 4
Fig. 4
Mitochondrial resident AKAP1 protein (AKAP149) interacts with ERβ protein. (A) Top panel, immunoblot of pull-down assay with anti-DDK antibody; bottom panel, same blot after stripping and probing with anti-HA antibody. (B) AKAP1 protein interacts with arginine methylated domain containing ERβ peptide with high affinity. Top panel, immunoblot of pull-down assay with anti-DDK antibody; bottom panel same blot without stripping and probing with anti-Avidin-HRP antibody. (C) Aβ treated hippocampal neurons exhibit decreased respiration and DPN ameliorates Aβ-induced inhibition of respiration. Arrows indicate time of addition of oligomycin (1 μM), FCCP (1 mM), and rotenone (1 μM). Oxygen consumption rates (OCR) in Aβ treated primary neurons are lower than control and DPN treatment ameliorates both basal and maximal OCR. N= 4 and data are expressed as mean ± SD.
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