Mitochondrial bioenergetic deficit precedes Alzheimer's pathology in female mouse model of Alzheimer's disease

Abstract

Mitochondrial dysfunction has been proposed to play a pivotal role in neurodegenerative diseases, including Alzheimer's disease (AD). To address whether mitochondrial dysfunction precedes the development of AD pathology, we conducted mitochondrial functional analyses in female triple transgenic Alzheimer's mice (3xTg-AD) and age-matched nontransgenic (nonTg). Mitochondrial dysfunction in the 3xTg-AD brain was evidenced by decreased mitochondrial respiration and decreased pyruvate dehydrogenase (PDH) protein level and activity as early as 3 months of age. 3xTg-AD mice also exhibited increased oxidative stress as manifested by increased hydrogen peroxide production and lipid peroxidation. Mitochondrial amyloid beta (Abeta) level in the 3xTg-AD mice was significantly increased at 9 months and temporally correlated with increased level of Abeta binding to alcohol dehydrogenase (ABAD). Embryonic neurons derived from 3xTg-AD mouse hippocampus exhibited significantly decreased mitochondrial respiration and increased glycolysis. Results of these analyses indicate that compromised mitochondrial function is evident in embryonic hippocampal neurons, continues unabated in females throughout the reproductive period, and is exacerbated during reproductive senescence. In nontransgenic control mice, oxidative stress was coincident with reproductive senescence and accompanied by a significant decline in mitochondrial function. Reproductive senescence in the 3xTg-AD mouse brain markedly exacerbated mitochondrial dysfunction. Collectively, the data indicate significant mitochondrial dysfunction occurs early in AD pathogenesis in a female AD mouse model. Mitochondrial dysfunction provides a plausible mechanistic rationale for the hypometabolism in brain that precedes AD diagnosis and suggests therapeutic targets for prevention of AD.

Fig. 1.

Age-related increase in Aβ-IR in female 3xTg-AD mice. Representative images show Aβ-IR in hippocampus CA1 region from female 3xTg-AD mice at 3, 6, 9, and 12 months and female nonTg mice at 12 months (Scale bar, 100 μm).

Fig. 2.

3xTg-AD female mice have decreased PDH E1α and COX IV protein levels relative to age-matched nonTg female mice. Equal amount of hippocampal (for PDH E1α) and mitochondrial (for COXIV) samples from both 3xTg-AD and nonTg mice of different age groups were loaded onto the gel. Expression of (A) PDH E1α subunit and (B) COX IV subunit were determined by western blot analysis. Bars represent mean relative expression ± SEM (*, P < 0.05 compared with age-matched nonTg group; n = 6).

Fig. 3.

3xTg-AD female mice exhibit higher oxidative stress than age-matched nonTg female mice. Whole brain mitochondria were isolated from intact female mice, and rates of hydrogen peroxide production were determined in state 4 respiration by Amplex-Red hydrogen peroxide assay. Bars represent mean hydrogen peroxide production rates ± SEM (*, P < 0.05 compared with age-matched nonTg group; #, P < 0.05 compared between different age group within the same genotype; n = 6).

Fig. 4.

3xTg-AD female mice exhibit higher lipid peroxidation than age-matched nonTg female mice. Whole brain mitochondria were isolated from intact female mice, and lipid peroxide levels were determined by the leucomethylene blue assay. Bars represent mean ± SEM (*, P < 0.05 compared with age-matched nonTg group; n = 6).

Fig. 5.

3xTg-AD female mice exhibit decreased mitochondrial respiration relative to age-matched nonTg female mice. Whole brain mitochondria were isolated from intact female mice, and state 3:state 4 respiration was determined. Oxygen electrode measurements of respiration using isolated brain mitochondria from 3xTg-AD and nonTg mice were conducted in the presence of 5 mM L-malate, 5 mM L-glutamate, 410 μM ADP to initiate state 3 respiration, and atractyloside to induce state 4₀ respiration. Bars represent the mean ± SEM of the RCR (state3:state 4 respiration) (*, P < 0.05 as compared with age-matched nonTg group; #, P < 0.05 compared between different age group within the same genotype; n = 6).

Fig. 6.

Hippocampal neurons derived from 3xTg-AD mouse brain exhibit decreased mitochondrial respiration and increased glycolysis. Primary embryonic neurons derived from both 3xTg-AD and nonTg mice were cultured in Neurobasal medium plus B27 supplement for 7 days. OCR and ECAR were determined using Seahorse XF-24 Metabolic Flux analyzer. (A) OCRs in primary neurons from 3xTg-AD mice (black) have lower basal rates of mitochondrial respiration than primary neurons derived from nonTg mice (gray). Vertical lines indicate time of addition of mitochondrial inhibitors (A) oligomycin (1 μM), (B) FCCP (1 μM), or (C) rotenone (1 μM). The maximal respiratory capacity (FCCP) is significantly lower in neurons from 3xTg-AD mice than those from nonTg mice. (B) Percent change in mitochondrial respiration in response to mitochondrial inhibitors. Bars represent the mean change in OCR from baseline ± SEM (*, P < 0.05 as compared with age-matched nonTg group; n = 3). (C) Temporal bioenergetic profiling (ECAR vs. OCR) of primary hippocampal neurons after exposure to mitochondrial inhibitors (A) oligomycin, (B) FCCP, and (C) rotenone (*, P < 0.05 as compared with nonTg cultures; n = 3).

Fig. 7.

Female 3xTg-AD mice have increased ABAD and mitochondrial Aβ protein levels relative to age-matched nonTg female mice. Equal amount of hippocampal (for ABAD) and mitochondrial (for Aβ) samples from both 3xTg-AD and nonTg mice of different age groups were loaded onto the gel. Expression of ABAD and 16 kDa mitochondrial Aβ oligomer were determined by western blot analysis. (A) 3xTg-AD mice have increased ABAD level than age-matched nonTg mice; (B) increased mitochondrial 16 kDa Aβ oligomer in the 3xTg-AD mice at 9 months. Bars represent mean relative expression ± SEM (*, P < 0.05 compared with age-matched nonTg group; n = 6).