The data presented in this study demonstrate that genetic disruption of 5LO results in a significant reduction of Aβ deposition and steady-state levels in the brain of the Tg2576 mouse model of AD and thereby provide the first evidence that 5LO might represent a novel target for modulating amyloidogenesis in AD.
Reports from our laboratory and another group have demonstrated that 5LO expression is strongly increased in aging rats and mice, especially in the hippocampus area, which is more susceptible to neurodegeneration (12
). In the present study, we showed that 5LO is up-regulated in old Tg2576 mice compared to young ones. Taking into account that aging is one of the strongest risk factors for AD development, these data provide an initial support for a possible link between 5LO and this disease. In line with these observations, we found that this enzyme is up-regulated in AD brains compared to controls.
In Tg2576 × 5LO−/− mice, total Aβ immunoreactivity to the 4G8 antibody was remarkably decreased compared with Tg2576 × 5LO+/+ controls. From our data, it appears that the rate of Aβ deposition was considerably slowed, because 15-month-old Tg2576 × 5LO−/− mice showed only a slight increase in amyloid plaque deposition compared to the 12-month-old group.
In our study, we observed that 5LO modulatory effect on Aβ manifests also some region dependency in the brain. Thus, both effects on Aβ deposition and Aβ reduction in 5LO-knockout mice were greater in the hippocampus than in the cortex. These data can be explained by previous findings in rodents, which show that 5LO expression in the hippocampus is higher than in the cortex, especially in older animals (12
). Confirming these previous reports, in the current study we indeed observed that 5LO mRNA levels were higher in the hippocampus of young Tg2576 mice, and there was also a more robust age-associated increase in mRNA levels in this area compared to the cortex.
We performed extensive experiments with three different techniques to investigate the effect of the absence of 5LO on Aβ peptide levels. Thus, all of the data obtained with sandwich ELISA, immunohistochemistry with C-terminal-specific antibodies, and Western blot consistently pointed to the fact that the reducing effect on Aβ42 levels was always stronger than on Aβ40 levels in Tg2576 mice deficient for 5LO. The fact that significant lowering of Aβ42 alone resulted in a remarkable reduction of amyloid plaque deposition in the brain of these animals is not surprising after all, since the central role of Aβ42 in Aβ deposition has been demonstrated in several studies (34
The significant reduction of Aβ42 and the increase in the ratio of Aβ1–38:Aβ1–42 in 5LO knockout mice would suggest the involvement of γ-secretase (37
). Furthermore, the absence of any significant change in sAPPα, sAPPβ, CTF-α, CTF-β, and BACE1 in the 5LO-deficient Tg2576 mice indicates that the involvement of α- and β-secretases is unlikely and is consistent with the hypothesis of an involvement of the γ-secretase. Thus, measurement of γ-secretase activity in brain homogenates of these animals confirmed that there was a significant reduction in the activity of this enzyme in the Tg2576 mice genetically deficient for 5LO, when compared with Tg2576 controls.
In line with the hypothesis of γ-secretase being the mechanism of Aβ reduction in 5LO knockout mice, the activation of 5LO enzyme in APP × 5LO+/+ MEFs led to an increase in γ-secretase activity. This effect could not be induced in APP × 5LO−/−, and could be reversed in APP × 5LO+/+ MEFs in the presence of zileuton, a selective 5LO inhibitor, indicating that 5LO is required for this effect on γ-secretase.
In another set of experiments, we used HEK293 cells that stably express C99 to further study the influence of 5LO on the γ-secretase complex. C99 is the precursor of Aβ and the immediate substrate for γ-secretase (1
); therefore, these cells provide a good model to study this pathway. In these cells, 5LO metabolites induced an increase, and the 5LO inhibition induced a decrease, in Aβ formation and γ-secretase activity, further supporting the involvement of this secretase in the amyloidogenic effect of 5LO. However, differently from the in vivo
studies, we did not find a stronger effect on Aβ1–42 compared with Aβ1–40. This dissimilarity can be explained by the rather complex structure of γ-secretase and the fact that different factors can considerably alter the response of this complex, depending on the in vivo vs
. the in vitro
system used. Different γ-secretase machinery in various species could also be another reason for these differences; in fact, data generated in MEFs obtained from Tg2576 mice, closely mimicked the findings in these mice.
We also sought to rule out other mechanisms that might be responsible for the reduction in Aβ levels in 5LO-deficient Tg2576 mice, such as Aβ-degrading enzymes, including neprilysin and IDE and also apoE alterations. In our study, no significant change was observed in either the protein or mRNA levels. We also assessed markers of inflammation and oxidative stress in Tg2576 × 5LO−/− and Tg2576 × 5LO+/+ and did not find any significant difference. This finding suggests that the antiamyloidogenic effect observed in these mice is not secondary to an anti-inflammatory or anti-oxidative effect resulting from the genetic absence of 5LO.
In conclusion, our studies unveil for the first time a novel functional role for 5LO enzyme in AD-like amyloidosis, since genetic absence of 5LO significantly reduces Aβ pathology in Tg2576 mice. We also provide several lines of evidence suggesting that this effect is mediated through the modulation of the γ-secretase pathway. Thus, 5LO could provide a novel and important target for the purpose of lowering Aβ pathology in AD.