We have designed new non-peptidic potential inhibitors of gamma-secretase and examined their ability to prevent production of amyloid-beta 40 (Abeta40) and Abeta42 by human cells expressing wild-type and Swedish-mutant beta-amyloid precursor protein (betaAPP). Here we identify three such agents that markedly reduce recovery of both Abeta40 and Abeta42 produced by both cell lines, and increase that of C99 and C83, the carboxy-terminal fragments of betaAPP that are derived from beta-and alpha-secretase, respectively. Furthermore, we show that these inhibitors do not affect endoproteolysis of endogenous or overexpressed presenilins. These inhibitors are totally unable to affect the mDeltaEnotch-1 cleavage that leads to generation of the Notch intracellular domain (NICD). These represent the first non-peptidic inhibitors that are able to prevent gamma-secretase cleavage of betaAPP without affecting processing of mDeltaEnotch-1 or endoproteolysis of presenilins. The distinction between these two proteolytic events, which are both prevented by disruption of presenilin genes, indicates that although they are intimately linked with betaAPP and Notch maturation, presenilins are probably involved in the control of maturation processes upstream of enzymes that cleave gamma-secretase and Notch.

New protease inhibitors prevent gamma-secretase-mediated production of Abeta40/42 without affecting Notch cleavage.

The amyloid beta protein (Abeta) is derived from beta-amyloid precursor protein (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APPCTFbeta or C99), which is subsequently cleaved by gamma-secretase to produce Abeta. BACE (or BACE1), the major beta-secretase involved in cleaving APP, has been identified as a Type 1 membrane-associated aspartyl protease. In this study, we found that treatment with proteasome inhibitors resulted in an increase in APP C99 levels, suggesting that APP processing at the beta-secretase site may be affected by the ubiquitin-proteasome pathway. To investigate whether the degradation of BACE is mediated by the proteasome pathway, cells stably transfected with BACE were treated with lactacystin. We found that BACE protein degradation was inhibited by lactacystin in a time- and dose-dependent manner. Non-proteasome protease inhibitors had no effect on BACE degradation. BACE protein is ubiquitinated. Furthermore, lactacystin increased APP C99 production and Abeta generation. Our data demonstrate that the degradation of BACE proteins and APP processing are regulated by the ubiquitin-proteasome pathway.

Degradation of BACE by the ubiquitin-proteasome pathway.

The β-amyloid protein (Aβ) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by β-secretase generates a C-terminal fragment (APP-CTFβ), which is subsequently cleaved by γ-secretase to produce Aβ. The aim of this study was to examine the cleavage of APP-CTFβ by γ-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFβ sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFβ and Aβ were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFβ (hAPP-CTFβ) was converted to human Aβ (hAβ), which was rapidly secreted. The remaining 70% of the hAPP-CTFβ was degraded by an alternative pathway. The cleavage of hAPP-CTFβ to produce hAβ was inhibited by specific γ-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFβ and hAβ levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFβ is degraded by two distinct pathways, one involving γ-secretase, which produces Aβ, and a second major pathway involving direct cleavage of APP-CTFβ within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Aβ production in the AD brain.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.0014-2956.2001.02465.x

The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase.

Therapeutic approaches to Alzheimer's disease.

The present invention provides boronic acid compounds, boronic esters, and compositions thereof that can modulate apoptosis such as by inhibition of proteasome activity. The compounds and compositions can be used in methods of inducing apoptosis and treating diseases such as cancer and other disorders associated directly of indirectly with proteasome activity.

Proteasome inhibitors and methods of using the same

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of β-amyloid peptide (Aβ) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on Aβ formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of β-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress Aβ formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in Aβ formation.

Alzheimer's Disease:correlation of the suppression of β-Amyloid peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome

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Alzheimer's Disease:correlation of the suppression of β-Amyloid peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome