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The Proton-Pump Inhibitor Lansoprazole Enhances Amyloid Beta Production
Nahuai Badiola, Victor Alcalde, Albert Pujol, Lisa-Marie Munter, Gerd Multhaup, Alberto Lleo,
Mireia Coma, Montserrat Soler-Lopez, Patrick Aloy
Paper Review by Nisha Crouser
In this study, researchers wanted to look at the effects of protein pump inhibitors (PPIs), specifically the drug lansoprazole, on the accumulation of amyloid-beta (AB) species. They chose to focus on AB accumulation because it is a key event in the pathogenesis of Alzheimer’s disease. The researchers explored the effects of these PPIs in both animal and cellular models. In the end, they found that overall lansoprazole enhances the production of AB37, AB40, and AB42 and lowers the levels of AB38 in cellular models.
In the first experiment, the researchers treated PS70 Chinese hamster ovary (CHO) cells with increasing amounts of lansoprazole over the course of 24 hours and measured the amounts of AB40 and AB42 produced over time.They found that lansoprazole treatment increased AB levels at commonly used concentrations. Next they wanted to see if other PPIs had the same effect and treated CHO cells with other widely used drugs. The researchers found that these PPIs also increased AB production in a similar way to lansoprazole. In the cellular model, the researchers also wanted to better understand the mechanism by which lansoprazole increased AB production. For this reason, they looked at whether or not lansoprazole modulates the gamma-secretase complex and the ways in which lansoprazole interacts with certain proteases. They found evidence to suggest that lansoprazole does in fact modulate gamma-secretase and may increase BACE1 activity (protease).
In the second set of experiments, the researchers used an animal model to see how lansoprazole effects AB production specifically in the brain. They conducted short-term intraperitoneal administration of lansoprazole in wt and AD- triple transgenic mouse models and found that AB40 levels increased significantly in both cases (although AB40 production was much higher in non-transgenic mice). This is the first study to present data showing that PPIs can affect amyloid production both in vitro and in vivo. Future research could evaluate whether the treatment with PPIs has an effect on Alzheimer’s pathology.
Overall, I thought the paper was easy to follow and presented very interesting findings. The figures are straightforward and visually appealing. All of the bar graphs are complete with error bars and p-values, providing the reader with the necessary information to understand their results. Also the bar graphs were a good choice because they easily illustrate the dose-dependent increases in AB production.
There were times when I felt that the figures were not very compelling. For example in Figure 2B, the figure legend states that there was a decrease in AB38 and an increase in AB42 in those cells treated with lansoprazole; but this is not clear from the Western blot. It is not obvious in the short or long exposure images that there is any difference between the control and the lansoprazole treated cells. The blots look very similar and without being told their results, you would most likely not be able to tell the difference. Also where you are supposed to notice there is a decrease in AB38 there is big smear in the corner of the long exposure Western blot, so its hard to tell what is going on there.
The researchers used several Western blots to support their claims, but many of these were taken from a “representative experiment”. This means the researchers chose one of the Western blots to use as an example of several other experiments. Consequently, there is no way of knowing if these Western blots were representative of all of the experiments or not. The researchers could have easily chosen the one Western blot that best demonstrated what they were trying to argue. For this reason, I think these figures aren’t as compelling as the other graphs.
On the otherhand, I think the rest of the figures did a good job of presenting the researcher's data. Figure 2A clearly shows how treatment with lansoprazole effects the levels of various AB products. For example, it is very clear that AB42 increases in the presence of lansoprazole because there wasn't even a peak for AB42 in the MS data for the untreated cells. Also Figure 4 was helpful in bringing the findings of the paper all together by showing the mechanism by which lansoprazole increases AB production. The figure is relatively simplistic, but still does a good job of showing how various parts of the system are amplified in the presence of lansoprazole. The figure does not show exactly how lansoprazole modulates the gamma-secretase complex, but this could be because they do not know the exact process. For the most part, the researchers did a good job of representing their data, with the exception of the Western blot images.
Figures- permission pending
A) This figure shows the effect of lansoprazole on AB production in PS70 Chinese hamster ovary cells which stably expressed wild-type human APP and presenilin 1. These two graphs show that levels of AB40 and AB42 increased in the PS70 cells when treated with increasing concentrations of lansoprazole (250nM- 50mM) for 24 hours. Researchers used ELISA immunoassays to determine these results. More specifically it can be noted that the AB40 levels increased after treatments of about 10mM, with up to a two-fold increase in the amount of AB40 with respect to the vehicle-treated cultured cells. Also it appears that there is a dose-dependent increase in AB42 between 5mM and 50mM. The DAPT column showed neglible levels of AB in both graphs, which was expected because these cells were treated with a gamma-secretase inhibitor and were acting as a control. The (+) and (*) above the bars indicate p values of <0.05 and <0.01 respectively.
B) In this figure, researchers wanted to see if other protein-pump inhibitors (PPIs) showed the same results as lansoprazole. They investigated the effect of omeprazole, pantoprazole and esomeprazole within concentrations of 5mM-50mM on AB production in PS70 cells. As seen in the two graphs, both AB40 and AB42 increased in response to all three drugs. Furthermore, the AB42 increase appeared to be dose-dependent.
A) This figure was created to gain a better understanding of the relative AB species induced by lansoprazole. This figure shows the results from an AB- immunoprecipitation which was then analyzed using MALDI-MS. This spectrometry method is ideal for analyzing large molecules, such as proteins. Researchers found that different AB species are generated because gamma-secretase has multiple cleavage sites as indicated by the multiple peaks in the figure; the main product is AB40 (large peak) and to a lesser extent AB38 and AB42. The interesting result researchers found, is seen in the second row of the figure when the cells were treated with 50 mM of lansoprazole for 24 hours. In this case, the relative levels of AB42 and AB37 increased while AB38 decreased. DAPT was again used as a negative control and as seen in the figure showed no AB peaks.
B) In this figure, researchers wanted to confirm the previous results that AB42 increased and AB38 decreased when treated with lansoprazole (50mM for 24 hours). In this case, they used a Western blot to analyze the species present in the treated PS70 supernatants. The figure shows Western blots for both short and long exposure times. A decrease in AB38 and increase in AB42 were detected.
C) In this figure, researchers wanted to look at how R-flurbiprofen, a non-steroidal anti-inflammatory drug, affected production of AB42. AB42 levels were measured using ELISA immunoassays. The graph shows that lansoprazole (50mM) increased AB42, while R-flurbiprofen (200mM) decreased AB42 production. When cells were treated with both lansoprazole and R-flurbiprofen AB42 still increased although not as much as with the lansoprazole alone. This suggests that lansoprazole is able to overcome the inhibitory effects of R-flurbiprofen. DAPT was used as a negative control and the vehicle represents the untreated cells.
D) This figure shows the Western blot analysis of APP and BACE1 protein levels in total lysates. The Western blot shows there was no difference in protein levels between the vehicle (untreated) and lansoprazole (treated) cells. This figure shows a representative experiment, in total three experiments were done.
E) In this figure, researchers wanted to see if lansoprazole could enhance BACE1 activity so they measured the amount of sAPPB (a BACE1 cleavage product) produced. The immunoblot shows that more sAPPB was produced when treated with lansoprazole. Meanwhile, sAPPa levels were the same in the treated and untreated cells. This is a representative experiment, a total of three experiments were done.
A) This figure shows the effects of lansoprazole on wild-type mice. Non-transgenic mice were intraperitoneally administered 100kg/mg of lansoprazole for 5 consecutive days. Soluble AB40 and AB42 from brain extracts were measured by ELISA immunoassays (n=10). The graph shows that soluble AB40 levels increased significantly (p<0.05) when treated with lansoprazole. AB42 also appears to increase slightly, but the results were not significant.
B) This graph shows the effects of lansoprazole on AD triple-transgenic (3xTg-AD) mice models. Mice were intraperitoneally administered 20 mg/kg/day or 100mg/kg/day for 5 consecutive days. As seen in the graph, AB40 levels increased in a dose-dependent manner. AB42 levels also increased but not significantly. Comparing figures A and B, it is clear that AB40 production was much higher in the 3xTg-AD mice compared to the wt mice when treated with lansoprazole.
This figure shows the hypothetical mechanism the researchers developed for how lansoprazole affects AB production based on the results they gathered from their experiments. This mechanism shows that in basal conditions (left) a variety of different AB species are formed. On the contrary, when cells are treated with lansoprazole, BACE1 activity is increased and as a result more sAPPB and C99 fragments are generated. This increases the AB production overall. The figure also attempts to show that lansoprazole could shift the gamma-secretase cleavage, which would increase AB42 and decrease AB38 levels. In total, lansoprazole appears to increase AB42, AB40, and AB37 levels while simultaneously decreasing AB38 levels.
Badiola et. al 2013. The Proton-Pump Inhibitor Lansoprazole Enhances Amyloid Beta Production. PLoS One 8(3): e58837
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