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Endoplasmic reticulum (ER) stress elicits protective responses of chaperone induction and translational suppression and, when unimpeded, leads to caspase-mediated apoptosis. Alzheimer's disease-linked mutations in presenilin-1 (PS-1) reportedly impair ER stress-mediated protective responses and enhance vulnerability to degeneration. We used cleavage site-specific antibodies to characterize the cysteine protease activation responses of primary mouse cortical neurons to ER stress and evaluate the influence of a PS-1 knock-in mutation on these and other stress responses. Two different ER stressors lead to processing of the ER-resident protease procaspase-12, activation of calpain, caspase-3, and caspase-6, and degradation of ER and non-ER protein substrates. Immunocytochemical localization of activated caspase-3 and a cleaved substrate of caspase-6 confirms that caspase activation extends into the cytosol and nucleus. ER stress-induced proteolysis is unchanged in cortical neurons derived from the PS-1 P264L knock-in mouse. Furthermore, the PS-1 genotype does not influence stress-induced increases in chaperones Grp78/BiP and Grp94 or apoptotic neurodegeneration. A similar lack of effect of the PS-1 P264L mutation on the activation of caspases and induction of chaperones is observed in fibroblasts. Finally, the PS-1 knock-in mutation does not alter activation of the protein kinase PKR-like ER kinase (PERK), a trigger for stress-induced translational suppression. These data demonstrate that ER stress in cortical neurons leads to activation of several cysteine proteases within diverse neuronal compartments and indicate that Alzheimer's disease-linked PS-1 mutations do not invariably alter the proteolytic, chaperone induction, translational suppression, and apoptotic responses to ER stress.

The Alzheimer's disease amyloid peptide Abeta has a heterogeneous COOH terminus, as variants 40 and 42 residues long are found in neuritic plaques and are secreted constitutively by cultured cells. The proteolytic activity that liberates the Abeta COOH terminus from the beta-amyloid precursor protein is called gamma-secretase. It could be one protease with dual specificity or two distinct enzymes. By using enzyme-linked immunosorbent assays selective for Abeta40 or Abeta42, we have measured Abeta secretion by a HeLa cell line, and we have examined the dose responses for a panel of five structurally diverse gamma-secretase inhibitors. The inhibitors lowered Abeta and p3 secretion and increased levels of the COOH-terminal 99-residue beta-amyloid precursor protein derivative that is the precursor for Abeta but did not alter secretion of beta-amyloid precursor protein derivatives generated by other secretases, indicating that the inhibitors blocked the gamma-secretase processing step. The dose-dependent inhibition of Abeta42 was unusual, as the compounds elevated Abeta42 secretion at sub-inhibitory doses and then inhibited secretion at higher doses. A compound was identified that elevated Abeta42 secretion at a low concentration without inhibiting Abeta42 or Abeta40 at high concentrations, demonstrating that these phenomena are separable pharmacologically. Using either of two methods, IC50 values for inhibition of Abeta42 and Abeta40 were found to have the same rank-order and fall on a trend line with near-unit slope. These results favor the hypothesis that Abeta variants ending at residue 40 or 42 are generated by a single gamma-secretase.

The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid beta-peptide (Abeta) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human Abeta. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPPalpha, sAPPbeta, Abeta1-40, and Abeta1-42, while having little to no effect on cell-associated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and Abeta in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and Abeta can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.

The 4-kDa beta-amyloid peptide (Abeta), a principal component of parenchymal amyloid deposits in Alzheimer's disease, is derived from amyloid precursor proteins (APP). To identify potential intracellular compartments involved in Abeta production, we expressed human APP-695 (APPwt) and APP-695 harboring the Swedish double mutation (APPswe) associated with familial early-onset Alzheimer's disease, in mouse N2a cells. We demonstrate that cells expressing APPswe secrete high levels of Abeta peptides and beta-secretase-generated soluble APP derivatives (APP s beta) relative to cells expressing APPwt. In addition, we observed a concomitant diminution in the levels of alpha-secretase-generated soluble APP derivatives (APP s alpha). Our interpretation of these findings is that beta-secretase cleavage occurs in an intracellular compartment and disables those substrates which would normally be cleaved by alpha-secretase. As anticipated, the levels of APPswe are diminished relative to the steady-state levels of surface-bound APPwt; moreover, surface-bound APPswe and APPwt molecules are released from the plasma membrane after cleavage by alpha-secretase, but not by beta-secretase. Finally, by examining the rate of appearance of specific APP metabolites generated by beta-secretase, we now unequivocally demonstrate that beta-secretase cleavage of APPswe occurs within the Golgi apparatus, as early as the medial compartment.

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2 is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-d,l-Phe-CH2F], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain I (with a second-order rate constant for inactivation of 276 000 M^-1 s^-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-d,l-Phe-CH2F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 μM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.

Muscarinic acetylcholine receptor activation induces a loss of muscarinic receptors from cultured neuroblastoma and embryonic chicken cerebrum cells. As measured by specific binding of [3H]quinuclidinyl benzilate, steady-state receptor concentrations decrease 75% in response to receptor-saturating concentrations of cholinomimetic drugs. Both the degree and duration of activation determine the extent of receptor loss. A method for analyzing receptor turnover, which does not rely upon protein synthesis inhibitors, shows that activated receptors have a half-life of 1.6 hr. The regulated rate of receptor disappearance begins as soon as activators are added, and the rate is maintained as long as activators are present. The receptor blocker atropine causes an increase in receptor levels in central nervous system cultures but has no effect on receptors in cultures of adrenergic neuroblastoma cells. Because spontaneous cholinergic activity is expected only in the central nervous system cultures, the increase likely reflects blockade of endogenous regulation. Cytochalasin B blocks receptor regulation, suggesting that regulation may be mediated by a process involving microfilaments.

The calcium-activated thiol-protease calpain I, which is present in cytosolic and membrane preparations from rat brain, was tested for its capacity to degrade the neuronal spectrin-like protein fodrin. In the presence of micromolar calcium concentrations purified calpain I degraded both purified fodrin and the fodrin present in hippocampal and cerebellar membranes. Fodrin was identified as a high molecular weight protein present in brain membranes by the following criteria: (i) comigration on NaDodSO4/polyacrylamide gels with purified fodrin, (ii) reactivity with antibodies to purified fodrin, and (iii) a proteolytic map following calpain activation comparable to that found after calpain-mediated degradation of purified fodrin. The fodrin breakdown was selective in that calpain I did not affect at least 15 other membrane-associated polypeptides. Fodrin degradation by the protease was rapid and was accompanied by the appearance of a lower molecular weight breakdown product. Calpain I had a high affinity for fodrin, with a Km for degradation of about 50 nM. Purified calpain I also degraded purified spectrin and the spectrin present in erythrocyte membranes. Calpain I-mediated degradation of spectrin-like proteins could provide a mechanism by which brief increases in intracellular free calcium levels modify the structure of the submembraneous cytoskeleton and the distribution of cell surface receptors and alter cell shape.