Calpain inhibitor I decreases βA4 secretion from human embryonal kidney cells expressing β-amyloid precursor protein carrying the APP670/671 double mutation

doi:10.1016/0304-3940(95)12122-K

Neuroscience Letters

Volume 201, Issue 1, 1 December 1995, Pages 29-32

https://doi.org/10.1016/0304-3940(95)12122-K Get rights and content

Abstract

We have investigated the effects of the cell-penetrating cysteine protease inhibitors calpain inhibitor I (N-acetyl-Leu-Leu-norleucinal) and calpain inhibitor II (N-acetyl-Leu-Leu-methioninal) on the secretion of the β-amyloid peptide (βA4) using transiently transfected cells expressing β-amyloid precursor protein (APP) with the NL670/671 double mutation. Calpain inhibitor I markedly reduced the amounts of immunoprecipitable βA4 and p3 peptide released into the culture medium. Within the cells C-terminal APP fragments accumulated. Since βA4 secretion by cells expressing the 100 amino acid long APP C-terminus was also reduced by calpain inhibitor I, we conclude that this substance directly or indirectly interferes with the γ-secretase activity responsible for generating the βA4 and p3 C-termini.

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Citation Excerpt :
Nicastrin was independently isolated biochemically as a PSEN-associated protein and found to be essential for γ-secretase processing of both APP and Notch (Yu et al., 2000). A saturation screen in Caenorabditis elegans for PSEN modifiers identified these two proteins as well as Pen-2 (Francis et al., 2002). All four proteins (PSEN, nicastrin, Aph-1, and Pen-2) associate with one another and with an immobilized γ-secretase inhibitor (GSI) (Kimberly et al., 2003; Takasugi et al., 2003).
γ-Secretase is a protease complex responsible for cutting the transmembrane domain of the amyloid β-protein precursor (APP) to form the amyloid β-protein (Aβ), an aggregation-prone product that accumulates in the brain in Alzheimer’s disease. As evidence suggests that Aβ is critical to Alzheimer pathogenesis, γ-secretase is considered a key target for the development of disease-modifying therapeutics. The protease complex cuts many other substrates, and some of these proteolytic events are part of signaling pathways or other important cellular functions. Among these, proteolysis of the Notch receptor is essential for signaling that is involved in a number of cell-fate determinations. Many inhibitors of γ-secretase have been identified, but it is clear that drug candidates for Alzheimer’s disease should have minimal effects on the Notch signaling pathway, as serious safety issues have arisen with nonselective inhibitors. Two types of promising candidates that target this protease complex have emerged: the so-called “Notch-sparing” γ-secretase inhibitors, which block cleavage of APP selectively over that of Notch, and γ-secretase modulators, which shift the proportion of Aβ peptides produced in favor of shorter, less aggregation-prone species. The current status and prospects for these two general types of candidates will be discussed.
Adherence-dependent shifts in the patterns of β-amyloid peptides secreted by human mononuclear phagocytes
2008, Brain, Behavior, and Immunity
Cells of the mononuclear phagocyte system are closely associated with vascular and neuritic β-amyloid deposits in Alzheimer’s disease. Using one-dimensional and newly developed two-dimensional Aβ-SDS–PAGE Western immunoblot techniques (1D/2D-Aβ-WIB) we investigated the patterns of Aβ peptides released by primary non-adherent and adherence-activated human mononuclear phagocytes in vitro. An overall increase of total released Aβ peptides (Aβ_total) was observed in adherence-activated mononuclear phagocyte cultures. 2D-Aβ-WIB revealed that the proportion of Aβ_1–40 decreased significantly to 50.2 ± 5.4% (n = 10) of Aβ_total compared to 65.9 ± 5.6% (n = 7) in non-adherent cultures (p < 0.0001, t = 5.82). Aβ_1–42 accounted for only 3.0 ± 2.1% of Aβ_total and its proportion did not change significantly upon adherence (2.8 ± 0.5% of Aβ_total). In adherence-activated cultures we detected pronounced shifts in the fractional pattern of released Aβ peptides in favour of N-truncated species. The second most prominent Aβ peptide accounted for as much as 12.7 ± 3.0% of Aβ_total (2.0 ± 1.2% in non-adherent cultures; p < 0.0001, t = 9.00) and was identified as Aβ_2–40 by comigration with a synthetic peptide and by N-terminal-specific antibodies. A strong increase of a further Aβ immunoreactive spot migrating at pI 5.45 was observed. It accounted for 9.2 ± 1.7% of Aβ_total as compared to 1.0 ± 0.9% in non-adherent cultures (p < 0.0001, t = 11.61) and presumably represented a variant of Aβ_2–40 as determined by C-terminal Aβ₄₀-specific immunoprecipitation and N-terminal-specific immunodetection. Thus, mononuclear phagocytes might be one source of the N-truncated Aβ peptides regularly found in human plasma and are less likely to contribute substantially to plasma Aβ_1-42.
Platelet as a physiological model to investigate apoptotic mechanisms in Alzheimer β-amyloid peptide production
2008, Mechanisms of Ageing and Development
Although neuronal apoptosis in Alzheimer's disease is generally interpreted as the consequence of the toxicity of extracellular β-amyloid (Aβ) peptide aggregates, some experimental results provide evidence that the Aβ overproduction can be the result of a primary neuronal degeneration. As platelets are considered a good model where to study proteolytic processing of the amyloid precursor protein (APP), we exposed platelets to the proapoptotic agent ionomycin and analyzed Aβ40 and Aβ42 levels in the intracellular and extracellular compartments. The activation of apoptotic pathways in platelets has been verified by mitochondrial membrane depolarization, exposure of phosphatidylserine, protease activation and morphological changes. A significative increase in intraplatelet Aβ40, but not Aβ42, was observed after 10 min treatment with ionomycin. Thus, the activation of apoptotic pathways in platelets determines an altered processing of APP leading to elevated levels of intracellular Aβ40. The specific intracellular production of Aβ40 represents a potential threat to the cells since very high local Aβ40 concentration increases the risk of its aggregation and toxicity. As a result, Aβ40 might be dangerous even before it becomes secreted rendering neurons highly vulnerable.
A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts
2001, Analytical Biochemistry
Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media. CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection. The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample. Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors. This will provide a pattern that might reflect more closely the real CP levels in intact cells. The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions.
The Protease Inhibitor, MG132, Blocks Maturation of the Amyloid Precursor Protein Swedish Mutant Preventing Cleavage by β-Secretase
2001, Journal of Biological Chemistry
Citation Excerpt :
This suggests that treatment of cells with high concentrations of peptide aldehydes may cause a more general impairment of the secretory pathway. This impairment blocks transport and processing of APPSw through the secretory pathway, thus explaining the decrease in Aβ peptide secretion observed by others (12-14). In previous studies, peptide aldehydes such as MG132 were thought to inhibit Aβ and p3 peptide secretion by blocking γ-secretase cleavage of the COOH-terminal fragment of APP (12, 13, 17, 18).
Amyloid (Aβ) peptides found aggregated into plaques in Alzheimer's disease are derived from the sequential cleavage of the amyloid precursor protein (APP) first by β- and then by γ-secretases. Peptide aldehydes, which inhibit cysteine proteases and proteasomes, reportedly block Aβ peptide secretion by interfering with γ-secretase cleavage. Using a novel, specific, and sensitive enzyme-linked immunosorbent assay for the β-secretase-cleaved fragment of the Swedish mutant of APP (APPSw), we determined that the peptide aldehyde, MG132, prevented β-secretase cleavage. This block in β-secretase cleavage was not observed withclasto-lactacystin β-lactone and thus, cannot be attributed to proteasomal inhibition. MG132 inhibition of β-secretase cleavage was compared with the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF inhibition of β-secretase cleavage was immediate and did not affect α-secretase cleavage. With MG132, inhibition was delayed and it decreased secretion of α-cleaved APPSw as well. Furthermore, MG132 treatment impaired maturation of full-length APPSw. Both inhibited intracellular formation of the β-cleaved product. These results suggest that peptide aldehydes such as MG132 have multiple effects on the maturation and processing of APP. We conclude that the MG132-induced decrease in β-secretase cleavage of APPSw is due to a block in maturation. This is sufficient to explain the previously reported peptide aldehyde-induced decrease in Aβ peptide secretion.
Factors influencing the processing and function of the amyloid β precursor protein - A potential therapeutic target in Alzheimer's disease?
2000, Pharmacology and Therapeutics
Citation Excerpt :
While the γ-secretase enzyme has not been identified yet, its actions, which may be associated with multiple proteases, are determined by the location of the AβPP within the plasma membrane (Murphy et al., 1999). Furthermore, the γ-secretases, which generate Aβ40 and Aβ42 may be distinct enzymes, with the γ-40 secretase being a cysteine protease and γ-42 being a serine protease (Klafki et al., 1995; Citron et al., 1996; FigueiredoPereira et al., 1999), with the generation of Aβ40 and Aβ42 occurring within different compartments of the cell. It has been proposed that Aβ40 is generated in the TGN, while Aβ42 is produced both within the ER and the TGN with the Aβ generated in the TGN being localised in two distinct pools (Greenfield et al., 1999).
The amyloid β precursor protein (AβPP), which plays a pivotal role in Alzheimer's disease (AD), can exist as either a membrane-bound or soluble protein. The former is cleaved at the level of the plasma membrane to generate the soluble form of the protein (AβPP_s). An alternative pathway exists, however, for the cleavage of AβPP to generate a 40–42 amino acid peptide termed amyloid β (Aβ), either within the lysosomal or the endoplasmic reticulum/Golgi compartments of the cell. In AD, there is an increase in the ratio of the 42 amino acid form of the Aβ peptide (Aβ₄₂) to Aβ₄₀. The Aβ₄₂ form is the more amyloidogenic form and has an increased potential to form the insoluble amyloid deposits characteristic of AD pathology. Studies on the familial form of the disease, with mutations in AβPP or in the presenilin proteins, have confirmed an increase in Aβ₄₂ generation associated with the early stages of the disease. This review will examine the factors that influence AβPP processing, how they may act to modulate the biological effects of AβPP_s and Aβ, and if they provide a viable target for therapeutic intervention to modify the rate of progression of the disease.

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¹: We thank D. Ristig for technical assistance and S. Frentzel for helpful discussions.

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Calpain inhibitor I decreases βA4 secretion from human embryonal kidney cells expressing β-amyloid precursor protein carrying the APP670/671 double mutation

Abstract

Degradation of secretory immunoglobulin M in B lymphocytes occurs in a postendoplasmic reticulum compartment and is mediated by a cysteine protease

J. Biol. Chem.

Generation of β-amyloid in the secretory pathway in neuronal and nonneuronal cells

High efficiency transformation of mammalian cells by plasmid DNA

Mol. Cell. Biol.

Mutation of the β-amyloid precursor protein in familial Alzheimer's disease increases β-protein production

Nature

Comparison of the effect of calpain inhibitors on two extralysosomal proteinases: the multicatalytic proteinase complex and m-calpain

J. Neurochem.

Targeting of cell-surface β-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments

Nature

Amyloid β-peptide is produced by cultured cells during normal metabolism

Nature

β-amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms

J. Biol. Chem.

Inhibition of β-amyloid formation identifies proteolytic precursors and subcellular site of catabolism

Neuron