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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 14, 2005 as doi:10.1096/fj.05-4524fje. |
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* Department of Pathobiology, College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee, USA; and
Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA
1 Correspondence: Department of Pathobiology, College of Veterinary Medicine, The University of Tennessee, 2407 River Dr., Knoxville, TN 37996, USA. E-mail: xmx{at}utk.edu
SPECIFIC AIMS
Calpain inhibitor MDL 28170 has been reported to modulate the production of Aß by influencing the 
-secretase mediated processing of amyloid precursor protein (APP). Our aims are to determine the possible mechanism by which MDL 28170 modulates Aß formation, by examining its effects on the three major intramembrane cleavages of APP, namely the -cleavage, the 
-cleavage, and the newly identified 
-cleavage, all of which are involved in the generation of the C-terminal of Aß40 and Aß42.
PRINCIPAL FINDINGS
1. At low concentrations, MDL 28170 causes accumulation of intracellular Aß46 without inhibiting the formation of the secreted Aß40/42
N2a cells stably expressing both wild type PS1 and myc-tagged Swedish mutant APP were treated with MDL 28170 for 36 h. As shown in Fig. 1
, at low range of concentrations (up to 40 µM), MDL 28170 caused a dose-dependent, but slight increase in the accumulation of intracellular Aß46 (lanes 2–7). In a recent study, we have shown that nontransition state -secretase inhibitors such as DAPT and compound E, cause a dose-dependent increase in the accumulation of intracellular Aß46 and a concomitant decrease in secreted Aß40/42, suggesting a precursor-product relationship between Aß46 and secreted Aß40/42. However, it is notable that the dose-dependent increase in Aß46 caused by MDL 28170 at low concentrations is not associated with a decrease in Aß40/42. Quite the opposite, significant dose-dependent increases in both Aß40 and Aß42 are observed (bottom panel). This observation clearly indicates that the mechanism by which, at low concentration, MDL 28170 causes an increase in Aß46 is different from that of compound E and DAPT that inhibits the formation of Aß40/42. At high concentrations, MDL 28170 caused a decline in both Aß46 and Aß40/42. At 60 µM of MDL 28170, Aß46 declined to the basal level (lane 9).
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2. At early time points, MDL 28170 blocks the formation of Aß40/42 by inhibiting the formation of the intermediate Aß46
To further determine the effect of MDL 28170 on the formation of these Aß species, a time course experiment was performed by treating cells with 30 µM of MDL 28170, at this concentration, both Aß46 and Aß40/42 reached their peaks at 36 h as shown by a dose curve experiment (Fig. 1
, lane 6). In the absence of inhibitor, secreted Aß40/42 becomes detectable by Western blot analysis after 3 h incubation, and it accumulates in the conditioned medium in a time-dependent manner. The basal level of CTFß is not changed during the time course, indicating a constant and steady turnover of CTFß. The basal level of intermediate Aß46 is barely detectable and does not change throughout the time course, indicating a quick turnover of Aß46 into Aß40/42. However, when the cells are cultured in the presence of 3 nM of compound E, which blocks the turnover of Aß46, a significant amount of Aß46 can be observed after 30 min of treatment. The accumulation of Aß46 reaches its peak after 3 h of treatment. In contrast, as shown in Fig. 2
, during the first 24 h incubation, neither Aß46 nor Aß40/42 was detected in cells treated with 30 µM of MDL 28170. Instead, a time-dependent accumulation of CTFß was detected (top and third panels). Beyond the time point of 36 h incubation, CTFß began to quickly decline and the secreted Aß40/42 robustly increased (bottom panel). These observations suggest two possibilities: one is that, at early time points, MDL 28170 inhibits the formation of both Aß46 from CTFß and Aß40/42 from Aß46; the other one is that MDL 28170 inhibits the formation of Aß40/42 by blocking the formation of the intermediate Aß46. Since we found that at 30 µM of concentration, MDL 28170 has not effect on the turnover of Aß46, thus, these results suggest that, at early time points, MDL 28170 inhibits the formation of Aß40/42 by inhibiting the formation of the intermediate Aß46 by -cleavage from CTFß rather than directly inhibiting the formation of Aß40/42 by -cleavage from Aß46.
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3. The accumulation of CTF caused by MDL 28170 at high concentration is a result of the inhibition of CTF degradation
At high concentrations MDL 28170 causes a dose-dependent increase in CTF as well as CTFß and CTF
with concomitant decline in Aß46 and Aß40/42 (Fig. 1)
. Since both CTF and Aß (Aß46 and Aß40/42) are produced from CTFß by -secretase, it is impossible that MDL 28170 inhibits only the N-terminal product Aß, but not the C-terminal product CTF of the same -secretase-mediated processing of CTFß. One possibility is that at high concentrations, MDL 28170 may have inhibitory effects on both the -secretase mediated cleavage of CTFß and also proteases, including calpain, mediated degradation of CTF. To determine whether the accumulation of CTF by MDL 28170 at high concentrations is due to the protection of CTF from degradation rather than a failure to inhibit CTF formation, a cell-free system was used to assay the inhibitory effect of MDL 28170 on the formation of CTF. Since the residual soluble CTF produced during the culture was washed away during the preparation of the membrane, CTF was not detected in the control of membrane incubated at 0°C for 2 h. When the membrane was incubated at 37°C for 2 h, de novo generation of CTF with a concomitant decreases in CTFß and CTF was detected. When the membranes were incubated at 37°C in the presence of MDL 28170 for 2 h, a dose-dependent decrease in the de novo generation of CTF with a concomitant increase in the unprocessed CTFß and CTF was observed. At 100 µM, MDL 28170 almost completely abolished the formation of CTF. This result clearly indicates that, although it may be less specific, MDL 28170 does have an inhibitory effect on -secretase mediated -cleavage. Thus, the increase in CTF caused by MDL 28170 at high concentrations is not due to the inability of MDL 28170 to inhibit the formation of CTF by -cleavage. Rather, the increase in CTF is due to the ability of MDL 28170 to inhibit the degradation of CTF produced by the residual -secretase activity after the addition of MDL 28170, as well as the CTF produced before the addition of MDL 28170.
CONCLUSIONS AND SIGNIFICANCE
First, as illustrated in Fig. 3
, our data suggest that, at low concentrations and early time points, MDL 28170 blocks the formation of Aß40/42 by inhibiting the formation of the intermediate Aß46, indicating that -cleavage, which produces the intermediate Aß46, plays an important role in the generation of secreted Aß40/42.
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Second, our observation that the effect of MDL 28170 on the formation of Aß is completely dose- and time-dependent may provide an answer to the controversial results reported by previous studies, some of which show that MDL 28170 inhibits the formation of both Aß40 and Aß42 and the others show that MDL 28170 increases both Aß40 and Aß42 and has a stronger enhancing effect on the formation of Aß42. The conflicting results reported by previous studies may be due to the differences in the dose used or differences in duration of treatment with the inhibitor. In contrast to the 2- to 3-fold increase in Aß42 and < 1.5-fold increase in Aß40 reported in the previous studies, our data reveal that MDL 28170 can dramatically increase Aß40 formation by 18-fold and increase Aß42 by 20-fold (Fig. 1)
.
Third, and more important, our data strongly suggest that MDL 28170 inhibits the degradation of Aß species and other APP derivatives produced by -secretase, and this inhibition of the degradation of Aß is the major factor contributing to the increase in secreted Aß40/42 caused by MDL 28170. The observation that, at low range of concentrations, MDL 28170 causes increase in both the product Aß40/42 and its CTFß, which is the initial substrate of -secretase, suggests a possibility that MDL 28170 may inhibit the degradation of CTFß. This inhibition of the degradation of CTFß may in turn contribute to the increase in Aß40/42 by increasing the concentration of the substrate of -secretase, as suggested by a previous study. Nevertheless, since a significant amount of CTFß was detected in the absence of any inhibitors and under the same condition only a trace amount of Aß46 was detected (Figs. 1
, 2)
, it is unlikely that the formation of CTFß is the rate-limiting step in the formation of secreted Aß. Therefore, inhibiting the degradation of CTFß may not be the major means by which MDL 28170 causes an increase in secreted Aß40/42. The other possibility, and a more likely one, is that MDL 28170 may also cause an increase in secreted Aß40/42 by directly inhibiting its degradation. In this regard, our data strongly suggest that MDL 28170 also protects other APP derivatives, including CTF, from degradation. The knowledge obtained from this study would contribute to a better understanding of the mechanism by which -secretase produces Aß from APP and the mechanism underlying the effect of calpain inhibitor MDL 28170 on the formation of Aß. The information obtained from this study would also be important for development of -secretase inhibitor and strategy of Aß clearance.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4524fje;
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