Mitochondrial dysfunction and oxidative stress are known to play a role in Alzheimer’s disease (AD) pathogenesis. In human AD brains, the mitochondrial α-ketoglutarate dehydrogenase (α-KGDHC) activity is markedly reduced in either damaged or relatively undamaged areas [17
]. These changes occur predominantly in cortical regions of the brain [18
]. α-KGDHC is a key enzyme of the tricarboxylic acid cycle (TCA) that is composed of three subunits: a thiamine pyrophosphate-dependent dehydrogenase enzyme (E1), dihydrolipoyl succinyltransferase enzyme E2 (DLST) and dihydrolipoamide dehydrogenase enzyme (E3). Gibson et al. have previously reported that in frontal and temporal cortices from AD patients bearing the Swedish APP670/671 mutation, levels of El and DLST declined, whereas levels of E3 were unchanged [20
]. It is important to note that only E1 and DLST are unique to α-KGDHC. There is a large body of evidence demonstrating that α-KGDHC and DLST participate in ROS formation and oxidative stress [16
]. Thus, we asked whether DLST could accelerate the onset of AD pathogenesis in vivo by examining the effects of its partial genetic deletion in Tg19959 mice. At about 4 months of age, Tg19959 mice develop amyloid plaques in the cortex, the hippocampus and the amygdala, together with progressive cognitive deficits, as those found in TgCRND8 mice [23
In our study, Tg19959 mice were crossbred with DLST+/− mice and offspring were tested at 2–3 months of age. At this age, amyloid pathology normally begins and cognitive impairment is not present yet (personal observations). Since, in AD pathogenesis, memory deficit is the most important clinical feature, we assessed the effects of DLST partial deficiency on spatial learning and memory in the Morris water maze. In both the acquisition period and the probe trial, which measures spatial learning and memory retention respectively, Tg19959 mice did not show any impairment in comparison with DLST+/− and wild-type littermates. However, Tg19959-DLST+/− female mice displayed spatial learning and memory retention deficits compared to wild-type and DLST+/− female littermates. In addition to our comparative data, we found that Tg19959-DLST+/− female mice were unable to learn the location of the hidden platform over the course of the acquisition period, as shown by their flat learning curve. Furthermore, they were unable to recall the location of the platform during the probe trial. Tg19959-DLST+/− mice spent only about 25 % of the time in the NW quadrant, which reflects a random search.
The second important pathological feature in AD is the formation of amyloid plaques. In Tg19959 mice, DLST deficiency increased amyloid plaque burden especially in female animals. In addition, we found that A11-positive Aβ oligomers were increased in the cortex and the hippocampus of Tg19959-DLST+/−
female mice. It should be noted that the detection of Aβ oligomers is complex and highly dependent on technical factors including the antibody used. The A11 antibody is commonly used to assay Aβ oligomers, though it also recognizes oligomers of other proteins [26
]. Taken together, these data revealed that DLST partial deficiency in vivo exacerbated AD-related phenotype mostly in female Tg19959 mice. In fact, it has been previously reported that in transgenic mouse model of amyloid deposition, female mice were affected more severely by amyloid pathology [24
]. This gender difference could be explained by increased γ-secretase activity in aged female mice, which can lead to increased APP processing and Aβ production [29
]. In our model, it is possible that the effect of DLST deficiency was enhanced by a higher APP and Aβ toxicity in females.
To further investigate the mechanism by which DLST affected AD-related phenotype, we measured levels of SDS-soluble Aβ species. Even though, there was an increase of amyloid plaques and oligomers with partial DLST deletion, levels of Aβ1–42 and Aβ1–40 were unchanged. These data suggest that partial genetic deletion of DLST could decrease resistance to Aβ toxicity. Partial DLST deficiency could also act downstream of the APP processing and Aβ production, by accelerating the rate of plaque formation and Aβ oligomerization.
There is a large body of evidence demonstrating a link between Aβ toxicity and oxidative stress. Several oxidative markers are markedly increased in transgenic mouse model of amyloid deposition, such as protein carbonyls, nitrotyrosine and 4-HNE, even at early stage [11
]. It has been shown that Aβ directly generates reactive oxygen species (ROS) in the presence of iron or copper ions [30
] via methionine-35 [31
]. Aβ oligomers also induce neuronal oxidative stress through N-methyl-D-aspartate receptors and calcium influx [32
]. Mitochondria mediated oxidative stress can also influence Aβ toxicity. Overexpression of MnSOD increased resistance to Aβ-induced toxicity in vitro [33
] and in vivo [15
]. On the other hand, partial deletion of MnSOD resulted in increased amyloid pathology [12
]. Recently, Karuppagounder et al. (2008) reported that in Tg19959 mice, thiamine deficiency induced oxidative stress and increased amyloid plaque deposition [21
]. In cells, DLST deficiency increased ROS production after H2
-induced oxidative stress [34
]. Thus, we postulate that DLST deficiency could reduce resistance to APP or Aβ-induced toxicity in vivo through an increase of oxidative stress. In fact, we found that Tg19959-DLST+/−
female mice had increased oxidative stress as shown by elevated nitrotyrosine levels as compared to Tg19959 female littermates.
The detrimental effect of DLST deficiency on oxidative stress was not seen in male mice. This gender effect could be explained by an overall higher APP and Aβ toxicity in female mice. An alternative and potentially complementary explanation for the gender effect could be a differential response towards oxidative stress and mitochondrial function. Even though there is not a clear consensus on gender effect on mitochondrial function, Ali et al. have reported that female wild-type mice had a greater increase of ROS production and a lower level of antioxidant enzymes during aging than male wild-type mice [35
]. Once given a superoxide dismutase mimetic, age-induced ROS production was reduced in both genders but with more efficacy in females [35
]. These results suggest that female mice may be more susceptible to mitochondrial oxidative stress, which could enhance the effect of DLST deficiency.
It should be noted that in contrast to human AD brains, we did not observe any change of α-KGDHC activity at baseline in Tg19959 mouse brains compared to wild-type mouse brains. The reasons for this difference are not certain, but there may be several potential explanations. In human AD brain, reductions in KGDHC activity are region specific [19
], but in our mouse study, KGDHC activity was measured on total brain homogenates. In addition, considering that α-KGDHC is constituted by multiple copies of the three subunits, it is possible that the activity of the complex is maintained by compensatory mechanisms.
In our model, partial genetic deletion of DLST diminished the activity of α-KGDHC in both DLST+/−
mice, and the detrimental effects reported in this paper could be due to this reduction of α-KGDHC activity. However, according to Shi et al., DLST can have effects on ROS production and cell death independent of α-KGDHC activity [34
]. Therefore, in our model, DLST deficiency may have had other effects besides reduction in α-KGDHC activity.
As previously mentioned, oxidative stress may promote Aβ aggregation or fibrillarization. The lipid oxidation product 4-HNE from polyunsaturated lipids is able to modify three histidine residues of Aβ by Michael addition, a reaction that causes protein misfolding and accelerates fibril formation at low protein concentrations [36
]. In addition, oxidized cholesterol products can also modify Aβ by Schiff base formation and accelerate Aβ aggregation [37
]. Thus, by increasing oxidative stress, partial deletion of DLST could elevate the rate of plaque formation and Aβ oligomerization. This mechanism of action has been proposed to explain the beneficial role of the mitochondrial antioxidant response in AD pathogenesis. Overexpression of manganese superoxide dismutase in Tg19959 mice reduced amyloid plaques, without affecting APP processing or Aβ production [15
Both DLST and overall α-KGDHC are crucial in bioenergetic processes within mitochondria. In the central nervous system, the high metabolic demand can lead to a higher level of oxidative stress via the production of free radicals. Under pathological conditions such as AD, oxidative stress can enhance the progression of the disease. To our knowledge, we are the first to demonstrate in vivo that the partial genetic deletion of mitochondrial DLST enzyme can accelerate the appearance of AD-like phenotype in a transgenic mouse model of amyloid deposition.