Altered brain energetics induces mitochondrial fission arrest in Alzheimer's Disease - PubMed (original) (raw)

Sergey Trushin 1, Trace A Christensen 2, Benjamin V Bachmeier 1, Benjamin Gateno 1, Andreas Schroeder 1, Jia Yao 3, Kie Itoh 4, Hiromi Sesaki 4, Wayne W Poon 5, Karen H Gylys 6, Emily R Patterson 7, Joseph E Parisi 7, Roberta Diaz Brinton 3 8 9, Jeffrey L Salisbury 2 10, Eugenia Trushina 1 10

Affiliations

Altered brain energetics induces mitochondrial fission arrest in Alzheimer's Disease

Liang Zhang et al. Sci Rep. 2016.

Abstract

Altered brain metabolism is associated with progression of Alzheimer's Disease (AD). Mitochondria respond to bioenergetic changes by continuous fission and fusion. To account for three dimensional architecture of the brain tissue and organelles, we applied 3-dimensional electron microscopy (3D EM) reconstruction to visualize mitochondrial structure in the brain tissue from patients and mouse models of AD. We identified a previously unknown mitochondrial fission arrest phenotype that results in elongated interconnected organelles, "mitochondria-on-a-string" (MOAS). Our data suggest that MOAS formation may occur at the final stages of fission process and was not associated with altered translocation of activated dynamin related protein 1 (Drp1) to mitochondria but with reduced GTPase activity. Since MOAS formation was also observed in the brain tissue of wild-type mice in response to hypoxia or during chronological aging, fission arrest may represent fundamental compensatory adaptation to bioenergetic stress providing protection against mitophagy that may preserve residual mitochondrial function. The discovery of novel mitochondrial phenotype that occurs in the brain tissue in response to energetic stress accurately detected only using 3D EM reconstruction argues for a major role of mitochondrial dynamics in regulating neuronal survival.

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Figures

Figure 1

Figure 1. Mitochondrial morphology in CA1 hippocampi of NTG and FAD mice visualized using standard TEM and super-resolution immunofluorescence.

(a) Mitochondrion in a neuropil in brain tissue of a NTG mouse. (b–f) Micrographs of mitochondrial profiles in the brain tissue of APP (b), 3xTgAD (c,d), and APP/PS1 (e,f) mice. (d) Consecutive serial sections of hippocampi from 3xTgAD mouse showing mitochondrial fission. (g–j) Membrane connections that contain mitochondrial matrix (g,h) or are devoid of matrix components (i,j) observed in APP/PS1 mice. (k,l) Mitochondria in brain tissue of NTG (k) or APP/PS1 (l) mice observed using Tom20 antibody (green) and a super-resolution fluorescence microscopy. Low magnification images on the right are co-stained with Hoechst (blue) to define nuclei. Scale bar, 2 μm. Three to five mice per each genotype were examined. All mice were females 40 weeks of age. Ten random sections with ~100 mitochondria were examined by blinded investigator for each brain tissue.

Figure 2

Figure 2. 3D EM reconstruction of mitochondria in CA1 hippocampi of NTG and APP/PS1 mice.

TEM images from ~28 serial sections 0.09 μm thick were stacked, aligned, and reconstructed using 3D reconstruction software in NTG (a) and APP/PS1 (b,c) mice 40 weeks of age (n = 3–5 per each group, all females). Scale bars, 2 μm. (d) Blind morphometric analysis of randomly selected EM of MOAS in CA1 brain region of FAD mice compared to NTG age-matched control at different ages (n = 3–9 mice per group, all females). Ten random sections with ~100 mitochondria were taken into analysis for each brain tissue. Data represent average ± SEM. NTG mice in the graph include NTG1 and NTG2 that did not have MOAS at 40 or 60 weeks of age.

Figure 3

Figure 3. MOAS phenotype in hippocampi and entorhinal cortices of AD patients.

The hippocampi, entorhinal cortices and cerebella were collected and fixed 6 – 24 hrs postmortem. (a) Low magnification representative survey image of hippocampus from an AD patient illustrating the overall degree of tissue preservation. The box contains teardrop shaped mitochondrion with membrane extension. (b) Representative EM micrograph of mitochondrial profiles in cerebellum from control individual. (c,d) Representative micrographs of teardrop shaped mitochondria with tubular membrane extensions observed in hippocampi from individuals diagnosed with AD (c,d). (e,f) Serial sections demonstrating MOAS phenotype in the hippocampus of an AD patient. Scale bars, 0.5 μm. Demographic data is presented in Supplementary Table S1.

Figure 4

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

(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.

Figure 5

Figure 5. Acute hypoxia induces MOAS in WT mice.

(a,c) Elongated mitochondria were observed using TEM (a) or super-resolution fluorescence microscopy with Tom20 (red) and Drp1 (green) antibodies (c) in the CA1 hippocampi of a WT mouse 10 weeks of age sacrificed by cervical dislocation without prior use of anesthetics. (b,d) Exposure of a WT mouse 10 weeks of age to CO2 for 5 min produced MOAS observed by TEM (b) or super-resolution fluorescence microscopy with Tom20 (red) and Drp1 (green) antibodies (d). (e) MOAS in the CA1 hippocampi in a WT mouse 88 weeks of age sacrificed by cervical dislocation. (f) Exposure of a WT mouse 88 weeks of age to CO2 for 5 min caused MOAS formation. Exposure of a WT mouse 10 weeks of age to CO2 for 5 min produced mitochondria with increased size (g, FSC histogram, median intensity of control 109.19 and hypoxia 203.0) and granularity (h, SSC histogram, median intensity of control 138.56 and hypoxia 268.44), and resulted in enhanced mitochondrial recruitment of Drp1 phosphorylated at S616 (53.8%, S616-Tom20 plots) in the CA1 hippocampal region compared to age-matched control. (h–j) Flow cytometry analysis of mitochondria isolated from hippocampi of mice exposed to hypoxic conditions compared to control mice (g,h) confirmed that enhanced recruitment of the activated Drp1 to mitochondria results in elongated organelles (i,j). Scale bars, 2 μm (a,e,b,f). Scale bars, 1 μm (c,d). Three to four female mice per each group were examined.

Figure 6

Figure 6. MOAS formation is associated with fission delay.

(a) Real-time imaging (t, seconds) of mitochondrial axonal movement in live cortical neurons (E17) from WT (left panel), APP mice (middle panel), and WT neurons treated with 2 μM PGJ2 for 30 min prior to imaging (right panel). Arrows indicate progress along the axon of the same daughter mitochondrion produced after fission (asterisk) from parental organelle. Thirty individual mitochondria were examined from each of the three movies generated for each condition. Experiments were repeated in three independent platings. (b) Model of two-step reaction leading to fission arrest where the first step (_k_1) represents Drp1 recruitment to a mitochondrion and subsequent self-assembly resulting in formation of early fission intermediates. The second step (_k_2) consists of either a rapid GTP hydrolysis-driven constriction resulting in mitochondrial membrane scission (_k_2 ≫ _k_1) or a decrease in the membrane constriction/scission rate resulting in MOAS formation (_k_2 ≪ _k_1). (c) Kinetic model of MOAS accumulation as an intermediate product during the reaction formula image calculated as formula image for three different scenarios. First: _k_2 ≪ _k_1 (green line) where formula image and formula image. Second: _k_2 ≫ _k_1 (red line) where formula image and formula image. Third: formula image (blue line) where formula image and formula image. Fission rate of mitochondria in cortical neurons used for calculations for formula image fission/mitochondria/s−1.

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