Defects in Mitochondrial Dynamics and Metabolomic Signatures of Evolving Energetic Stress in Mouse Models of Familial Alzheimer's Disease

The identification of early mechanisms underlying Alzheimer's Disease (AD) and associated biomarkers could advance development of new therapies and improve monitoring and predicting of AD progression. Mitochondrial dysfunction has been suggested to underlie AD pathophysiology, however, no comprehensive study exists that evaluates the effect of different familial AD (FAD) mutations on mitochondrial function, dynamics, and brain energetics.We characterized early mitochondrial dysfunction and metabolomic signatures of energetic stress in three commonly used transgenic mouse models of FAD. Assessment of mitochondrial motility, distribution, dynamics, morphology, and metabolomic profiling revealed the specific effect of each FAD mutation on the development of mitochondrial stress and dysfunction. Inhibition of mitochondrial trafficking was characteristic for embryonic neurons from mice expressing mutant human presenilin 1, PS1(M146L) and the double mutation of human amyloid precursor protein APP(Tg2576) and PS1(M146L) contributing to the increased susceptibility of neurons to excitotoxic cell death. Significant changes in mitochondrial morphology were detected in APP and APP/PS1 mice. All three FAD models demonstrated a loss of the integrity of synaptic mitochondria and energy production. Metabolomic profiling revealed mutation-specific changes in the levels of metabolites reflecting altered energy metabolism and mitochondrial dysfunction in brains of FAD mice. Metabolic biomarkers adequately reflected gender differences similar to that reported for AD patients and correlated well with the biomarkers currently used for diagnosis in humans.Mutation-specific alterations in mitochondrial dynamics, morphology and function in FAD mice occurred prior to the onset of memory and neurological phenotype and before the formation of amyloid deposits. Metabolomic signatures of mitochondrial stress and altered energy metabolism indicated alterations in nucleotide, Krebs cycle, energy transfer, carbohydrate, neurotransmitter, and amino acid metabolic pathways. Mitochondrial dysfunction, therefore, is an underlying event in AD progression, and FAD mouse models provide valuable tools to study early molecular mechanisms implicated in AD

Mitochondrial trafficking and distribution in primary embryonic neurons from APP, PS1 and APP/PS1 mice.

<p><b>A–C.</b> Real time imaging of mitochondrial movement within the axon of Hip neuron from PS1 mouse 7 days in culture. <b>A.</b> Phase image of the axon; cell body is in the upper right corner. <b>B.</b> Same axon with mitochondria visualized using TMRM. Scale bar, 10 µm. <b>C (a–c).</b> Recording of mitochondrial movement in live axon: arrow and circle indicate the progress of the same organelle along the axon with time. Images were acquired using LSM 510 laser scanning microscope (Carl Zeiss) with 100× oil DIC (1.4 na) lens. Scale bar, 5 µm. <b>D.</b> Mitochondria in PS1 neurons cover significantly shorter distances between stops in both, anterograde and retrograde directions compared to organelles in NTG, APP or APP/PS1 neurons. Almost no mitochondria in PS1 neurons cover distances longer than 10 µm. Number of organelles taken into analysis is the same as in (<b>F,G</b>). Blue – NTG; Red – APP; Orange – APP/PS1; Green – PS1. *p<0.001. <b>E.</b> Selective analysis of mitochondrial dynamics was done using analytical software (Analyze) that allows to trace each organelle from the first frame (<b>a</b>) through all 600 frames of the movie (stacked movie frames, <b>b</b>) to generate a final profile of movement (<b>c</b>). Resultant kymograph (<b>c</b>) is used to calculate velocities and identify the number of stationary and moving mitochondria. Rates of organelle motility in anterograde (<b>F</b>) and retrograde (<b>G</b>) directions include analysis of movement of 74 to 285 individual mitochondria in 24 to 33 neurons from at least three individual platings for each genotype. *, p<0.001; **, p<0.05. <b>H.</b> Length of 85–156 individual mitochondrion was estimated in five randomly selected axons in E17 neurons. *, p<0.001; **, p<0.0001. <b>I.</b> The number of organelles normalized per axonal length in embryonic neurons from NTG and AD mice used in axonal trafficking analysis. Number of mitochondria increases in APP and PS1 mice, and decreases in APP/PS1 cells comparing to NTG neurons. *, p<0.01. Colors as in <b>D</b>.</p

Mitochondria in AD mice have altered morphology and reduced oxidative activity.

<p><b>A.</b> Progressive age-dependent accumulation of abnormal mitochondria in AD mouse brains with dramatic loss of cristae integrity. Mitochondrion in APP mouse 45 weeks of age is shown. Scale bar, 1 µm. <b>B.</b> Abnormal synaptic mitochondria in APP/PS1 mouse brains were already observed at 8 weeks of age (a–c). Arrow denotes swollen mitochondria with total loss of inner structure. (d) Mitochondria in NTG mouse of the same age. Asterisks indicate the synapses; scale bar, 100 nm. <b>C.</b> Loss of mitochondrial oxidative activity in relationship to total mitochondrial mass in Hip live brain slices detected using MTG and MTO. Mitochondria in NTG brain have extensive colocalization of green (MTG, mitochondrial mass) and red (MTO, oxidative activity) fluorescence with the ratio of MTG/MTO = 0.96. Mitochondria in PS1, APP and APP/PS1 mouse brains have reduced oxidative activity as judged by the loss of red fluorescence intensity. The ratios of MTG/MTO were estimated to be: 0.8 (PS1), 0.6 (APP), and 0.5 (APP/PS1). Images were acquired using LSM 510 with 40× lens. Scale bar, 10 µm.</p

Mitochondria in APP and APP/PS1 mouse brains acquire abnormal shape.

<p><b>A.</b> 2D EM micrograph of mitochondrion with abnormal shape in Hip tissue of APP mouse 40 weeks of age. <b>B.</b> 3D reconstruction of ten serial sections of consecutive EM micrographs of the same tissue as in (<b>A</b>). <b>C.</b> 3D reconstruction of mitochondrial structure in Hip tissue of APP/PS1 mouse 24 weeks of age. Note the dramatic elongation of mitochondrion in APP/PS1 tissue compared to the length and shape of the organelle in the brain of NTG mouse of the same age (<b>D</b>). Scale bar, 1 µm.</p

Brain tissue of APP, PS1, and APP/PS1 mice has distinct metabolomic profiles compared to NTG mice.

<p><b>A.</b> PLS-DA score plot showing distinct metabolomic profiles of Hip brain tissue from PS1, APP and APP/PS1 mice compared to NTG mice. <b>B.</b> PLS-DA score plot demonstrating a significant gender effect on metabolomic profiles in APP/PS1 mice. Metabolomic alterations associated with mitochondrial dysfunction were more pronounced in female than in male APP/PS1 mice. Note significant differences in metabolomic profiles between APP/PS1 males and females and much smaller variation between NTG males and females. Each group included 3 mice 16 week of age.</p

Mitochondrial distribution is altered in hippocampus of AD mice.

<p><b>A.</b> Neurons in APP/PS1 and PS1 mice exhibit increased sensitivity to NMDA treatment. Open circles – NTG; Open squares – APP; Close circles – APP/PS1; Triangles – PS1. <b>B.</b> Electron micrographs of an altered mitochondrial distribution in brain of APP/PS1 mouse 8 weeks old compared to NTG mouse of the same age. Asterisk denotes mitochondria with altered shape; arrowheads denote accumulation of mitochondria in neuropils. Scale bar, 5 µm (APP/PS1), 2 µm (NTG). <b>C.</b> Accumulation of normal and degenerating (asterisks) mitochondria in brain of APP mouse 12 weeks old. Scale bar, 500 nm.</p

Relative changes of 30 most important metabolites in APP, PS1 and APP/PS1 transgenic mice compared to aged and gender matched NTG littermates.

<p>For each group, the relative values of each metabolite (mean±SD) are the average obtained from three mice. The average basal metabolite values of NTG group were arbitrarily set at 1 for each group. The value of fold change (FC, log2 of fold change) for each metabolite is relative to the value in aged and gender matched NTG mice. Metabolites were selected based on VIP values; p values were estimated using Student's t-test.</p

Expression of mitochondrial fusion and fission proteins is not altered in brain tissue from AD mice.

<p>Representative immunoblot (<b>A</b>) revealed no differences in expression of fusion and fission proteins in brain tissue from 12 months old APP/PS1, PS1 and NTG mice. <b>B.</b> No changes in expression of fission/fusion proteins were found in different brain regions (hippocampus, Hip, cortex, Ctx, and cerebellum, Cer) in APP mouse 13 months old compared to NTG mouse of the same age. Each sample was loaded twice with second sample having 2× concentration.</p

Comparison of individualized metabolomic profiles and affected metabolic pathways in FAD mouse models.

<p><b>A, C and E.</b> PLS-DA score plots showing distinct metabolomic profiles of PS1 (<b>A</b>), APP (<b>B</b>) and APP/PS1 (<b>C</b>) female mice compared to NTG littermates. <b>A, B and C, middle panels.</b> Panels of specific biomarkers as a plot of variable importance in the projection (VIP) indicating the 15 most significant metabolites in discriminating between metabolomic profiles of NTG and Tg groups in the PLS-DA model. <b>A, B and C, right panels.</b> Metabolic pathways specifically affected in each FAD mouse model. APP and PS1 mice were 36 weeks old, APP/PS1 – 16 weeks old.</p

Inhibition of axonal trafficking in neurons from PS1 and APP/PS1 mice is not specific to mitochondria.

<p><b>A.</b> Analysis of real time movement of round-shaped vesicles (endosomes or lysosomes, “v”) that were not stained with TMRM was done using same movies that were acquired to study mitochondrial (‘m’) trafficking. Scale bar, 4 µm. <b>B.</b> Similar to mitochondria, round-shaped vesicles move with reduced velocities in both, anterograde and retrograde directions, travel shorter distances between stops in neurons from APP, PS1 and APP/PS1 mice (<b>C</b>), and stop more frequently in neurons from PS1 and APP/PS1 mice (<b>D</b>). Analysis was done in randomly selected neurons (13–19 for every genotype); pattern and rate of motion of 10 to 58 individual vesicles was analyzed. *p<0.001.</p