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. 2017 Nov:112:515-523.
doi: 10.1016/j.freeradbiomed.2017.08.021. Epub 2017 Aug 31.

Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer's disease and a novel protective role for ascorbate

Shilpy Dixit  1 , Joshua P Fessel  2 , Fiona E Harrison  3
Affiliations

Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer's disease and a novel protective role for ascorbate

Shilpy Dixit et al. Free Radic Biol Med. 2017 Nov.

Abstract

Mitochondrial dysfunction is elevated in very early stages of Alzheimer's disease and exacerbates oxidative stress, which contributes to disease pathology. Mitochondria were isolated from 4-month-old wild-type mice, transgenic mice carrying the APPSWE and PSEN1dE9 mutations, mice with decreased brain and mitochondrial ascorbate (vitamin C) via heterozygous knockout of the sodium dependent vitamin C transporter (SVCT2+/-) and transgenic APP/PSEN1 mice with heterozygous SVCT2 expression. Mitochondrial isolates from SVCT2+/- mice were observed to consume less oxygen using high-resolution respirometry, and also exhibited decreased mitochondrial membrane potential compared to wild type isolates. Conversely, isolates from young (4 months) APP/PSEN1 mice consumed more oxygen, and exhibited an increase in mitochondrial membrane potential, but had a significantly lower ATP/ADP ratio compared to wild type isolates. Greater levels of reactive oxygen species were also produced in mitochondria isolated from both APP/PSEN1 and SVCT2+/- mice compared to wild type isolates. Acute administration of ascorbate to mitochondria isolated from wild-type mice increased oxygen consumption compared with untreated mitochondria suggesting ascorbate may support energy production. This study suggests that both presence of amyloid and ascorbate deficiency can contribute to mitochondrial dysfunction, even at an early, prodromal stage of Alzheimer's disease, although occurring via different pathways. Ascorbate may, therefore, provide a useful preventative strategy against neurodegenerative disease, particularly in populations most at risk for Alzheimer's disease in which stores are often depleted through mitochondrial dysfunction and elevated oxidative stress.

Keywords: Alzheimer’s disease; Ascorbate; Energy production; Mitochondria; Oxidative stress.

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Figures

Figure 1
Figure 1. ASC deficiency and APP/PSEN1 affect oxygen consumption in mitochondria
A) SVCT2 expression is decreased in mitochondrial isolates from mice heterozygous for SVCT2 compared to isolates with wild-type SVCT2 expression (p = 0.03, n = 5–6 per group). B) ASC content is significantly lower in SVCT2+/− mitochondria at 4 months (p = 0.024, n = 5 per group). C) Qualitative western blot analysis demonstrates detectable amyloid β expression in APP/PSEN1 and SVCT2+/−; APP/PSEN1 mice at 4 months of age, that already appears more severe in the compound mutant mice. D) SVCT2+/− mitochondrial isolates consume less oxygen (p=0.04), while mitochondrial isolates from APP/PSEN1 transgenic mice consume more oxygen when at full respiratory capacity (p = 0.005). WT n = 6, SVCT2+/− n = 5, APP/PSEN1, SVCT2+/−;APP/PSEN1 n = 6. Letters indicate a main effect of SVCT2 (a,b) and APP/PSEN1 (c,d).
Figure 2
Figure 2. APP/PSEN1 genotype affects mitochondrial membrane potential and increases oxidative stress
A) Mitochondrial isolates from SVCT2+/− mice show decreased mitochondrial membrane potential measured by relative fluorescence from accumulated TMRE (p = 0.049, n = 5–6 per group). B) Mitochondrial isolates from APP/PSEN1 transgenic mice show increased membrane potential measured by relative fluorescence from accumulated TMRE (p = 0.0085, n = 7–8 per group). C) The ATP/ADP ratio in SVCT2+/− isolates were not significantly different from WT (p= 0.195). D) The ATP/ADP ratio in APP/PSEN1 transgenic isolates was significantly lower than in WT isolates (p= 0.0299). E) Mitochondrial isolates from SVCT2+/− mice had significantly greater relative fluorescence from oxidized dihydrofluorescein (p = 0.03) than did mice that were wild-type for SVCT2. F) Mitochondrial isolates from APP/PSEN1 transgenic mice also show increased levels of ROS as measured by relative fluorescence from oxidized dihydrofluorescein (p = 0.038).
Figure 3
Figure 3. ASC increases oxygen consumption in isolated mitochondria and decreases ROS production
A) The oxygen consumed by mitochondrial isolated from WT cortex was significantly increased with the addition of 100μM ASC (p = 0.0017) compared to vehicle following establishment of baseline. Oxygen consumption within groups increased with the addition of each new substrate compared to the prior condition (p < 0.0001). Asterices (*) indicate a main effect of ASC following addition of ASC and of each substrate B) SVCT2 xpression is only slightly increased, (not significant), in mitochondria isolated from SVCT2Tg mice compared to WT mitochondria (p = 0.55, n= 5 per group). C) ASC content is significantly elevated,, in SVCT2Tg mitochondria at 4 months (p = 0.05, n = 5–9 per group). D) Mitochondrial isolates from SVCT2Tg mice show no difference in mitochondrial membrane potential measured by relative fluorescence from accumulated TMRE (p = 0.3409, n = 5–7 per group). E) Mitochondrial isolates from SVCT2Tg mice show a significant decrease in levels of ROS as measured by relative fluorescence from oxidized dihydrofluorescein (p = 0.0008, n = 6–8).
Figure 4
Figure 4. Intracellular ASC concentration is altered in SVCT2Tg; APP/PSEN1 mice
A) Amyloid β 42/40 ratio is significantly greater at 12 months compared with 5 months in APP/PSEN1 and SVCT2Tg;APP/PSEN1 mice (p< 0.0001) but does not differ according to genotype with each age group. B) Cortical ASC concentration is significantly elevated in SVCT2Tg mice compared to mice with normal SVCT2 expression (p=0.005) at 5 months, but at 12 months a significant difference is observed between SVCT2Tg and SVCT2Tg;APP/PSEN1 only (p=0.0002).
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