Alzheimer disease amyloid ␤-peptide (A␤) is generated via proteolytic processing of the ␤-amyloid precursor protein by ␤-and ␥-secretase. ␥-Secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal antiinflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid A␤ 42 and concomitantly increase the levels of the rather benign A␤ 38 . Here we show that A␤ 42 and A␤ 38 generation occur independently from each other. The amount of A␤ 42 produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of ␥-secretase, appeared to correlate with the respective age of onset in patients. However, A␤ 38 levels did not show a negative correlation with the age of onset. Modulation of ␥-secretase activity by sulindac sulfide reduced A␤ 42 in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of A␤ 42 or only rather subtle effects. Strikingly, even the mutations that showed no effect on A␤ 42 levels allowed a robust increase of A␤ 38 upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase A␤ 42 and to decrease A␤ 38 . For mutants that predominantly produce A␤ 42 , the ability of fenofibrate to further increase A␤ 42 levels became diminished, whereas A␤ 38 levels were altered to varying extents for all mutants analyzed. Thus, we conclude that A␤ 38 and A␤ 42 production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the A␤ 42 -lowering capacity of ␥-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to ␥-secretase modulators.Alzheimer disease is the most abundant form of dementia, and increasing numbers of patients are to be expected in the near future. Amyloid ␤-peptide (A␤) 5 is a central player in the disease pathology. Originally it was purified as the building block of the disease-defining amyloid plaques. Now it is becoming clear that amyloid plaques are probably not the major neurotoxic entity in the disease rather this is an assembly of soluble oligomeric A␤ species (1). These assemblies initiate the so-called amyloid cascade and finally induce abnormal phosphorylation of tau and subsequent formation of paired helical filaments (2). A␤ is generated by proteolytic processing of the ␤-amyloid precursor protein (APP). Two proteases, ␤-secretase and ␥-secretase, perform the cleavages on the N and C termini of the A␤ domain, respectively (3). ␤-Secretase is a conventional aspartyl protease, whereas ␥-secretase i...

To explore membrane-permeable synthetic inhibitors that discriminate between endogenous calpain and proteasome in cells, we examined the inhibition of profiles against calpain and proteasome in vitro and in vivo of peptidyl aldehydes possessing di-leucine and tri-leucine. The tripeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLLal) strongly inhibited calpain and proteasome activities in vitro. The concentration required for 50% inhibition (IC50) of the casein-degrading activity of calpain was 1.25 microM, and the IC50s for the succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide (Suc-LLVY-MCA)- and benzyloxycarbonyl-leucyl-leucyl-leucine-4-methylcoumaryl -7-amide (ZLLL-MCA)-degrading activities of proteasome were 850 and 100 nM, respectively. On the other hand, the synthetic dipeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLal) strongly inhibited the casein degrading activity of calpain (IC50 1.20 microM), but the inhibition of proteasome was weak (IC50S for SucLLVY-MCA- and ZLLL-MCA-degrading activities were 120 and 110 microM, respectively). Thus, while calpain was inhibited by similar concentrations of ZLLal and ZLLLal, the inhibitory potencies of ZLLLal against the ZLLL-MCA- and Suc-LLVY-MCA-degrading activities in proteasome were 1,100 and 140 times stronger than those of ZLLal, respectively. To evaluate the effectiveness of these inhibitors on intracellular proteasome, the induction of neurite outgrowth in PC12 cells caused by proteasome inhibition was examined. ZLLLal and ZLLal initiated neurite outgrowth with optimal concentrations of 20 nM and 10 microM, respectively, again showing a big difference in the effective concentrations for the proteasome inhibition as in vitro. As for the effect on intracellular calpain, the concentration of ZLLLal and ZLLal required for the inhibition of the autolytic activation of calpain in rabbit erythrocytes were 100 and 100 microM or more, respectively. The almost equal inhibitory potencies of ZLLLal and ZLLal were in agreement with the inhibition of calpain in vitro. These differential effects of inhibitors against calpain and proteasome are potentially useful for identifying the functions of calpain and proteasome in cell physiology and pathology.

Calpain has been implicated in excitotoxic neurodegeneration, but its mechanism of action particularly in adult brains remains unclear. We generated mutant mice lacking or overexpressing calpastatin, the only solely calpain-specific inhibitor ever identified or synthesized. Modulation of calpastatin expression caused no defect in the mice under normal conditions, indicating that calpastatin functions as a negative regulator of calpain only under pathological conditions. Kainateevoked excitotoxicity in hippocampus resulted in proteolytic activation of a proapoptotic Bcl-2 subfamily member (Bid), nuclear translocation of mitochondriaderived DNA fragmentation factors (apoptosis-inducing factor and endonuclease G), DNA fragmentation, and nuclear condensation in pyramidal neurons. These apoptotic responses were significantly augmented by calpastatin deficiency. Consistently calpastatin overexpression suppressed them. No evidence of caspase-3 activation was detected. Our results demonstrated that calpain mediates excitotoxic signals through mobilization of proapoptotic factors in a caspase-independent manner. These mutant mice will serve as useful tools for investigating calpain involvement in various diseases.

Proteases are known to be involved in the apoptotic pathway. We report here that benzyloxycarbonyl (Z)-Leu-Leu-leucinal(ZLLLal), a leupeptin analogue, can induce apoptosis in MOLT-4 and L5178Y cells. ZLLLal is a cell-permeant inhibitor of proteasome. Among the protease inhibitors tested, only calpain inhibitor I (acetyl-Leu-Leu-norleucinal) and ZLLLal caused a marked induction of apoptosis in MOLT-4 cells. In contrast Z-Leu-leucinal, a specific inhibitor of calpain, did not induce apoptosis. When MOLT-4 cells were incubated in the presence of ZLLLal, p53 accumulated in the cells. These results strongly suggest that inhibition of proteasome induces p53-dependent apoptosis and that proteasome can protect cell from apoptosis.