Thomas B. Ladd

Substrate-targeting γ-secretase modulators

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β deposition in Alzheimer’s disease. These data also demonstrate the existence and feasibility of ‘substrate targeting’ by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of ‘druggable’ targets.

Diverse compounds mimic Alzheimer disease–causing mutations by augmenting Aβ42 production

Increased Aβ42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Aβ42. Among the more potent Aβ42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2–selective NSAID. Many COX-2–selective NSAIDs tested raised Aβ42, including multiple COX-2–selective derivatives of two Aβ42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Aβ42. These compounds seem to target the γ-secretase complex, increasing γ-secretase–catalyzed production of Aβ42 in vitro. Short-term in vivo studies show that two Aβ42-raising compounds increase Aβ42 levels in the brains of mice. The elevations in Aβ42 by these compounds are comparable to the increases in Aβ42 induced by Alzheimer disease–causing mutations in the genes encoding amyloid β protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Aβ42 production in humans.

Overlapping profiles of Aβ peptides in the Alzheimer's disease and pathological aging brains

IntroductionA hallmark of Alzheimers disease (AD) is the presence of senile plaques composed of aggregated amyloid β (Aβ) peptides. Pathological aging (PA) is a postmortem classification that has been used to describe brains with plaque pathology similar in extent to AD, minimal cortical tau pathology, and no accompanying history of cognitive decline in the brain donor prior to death. PA may represent either a prodromal phase of AD, a benign form of Aβ accumulation, or inherent individual resistance to the toxic effects of Aβ accumulation. To attempt to distinguish between these possibilities we have systematically characterized Aβ peptides in a postmortem series of PA, AD and non-demented control (NDC) brains.MethodsAβ was sequentially extracted with tris buffered saline (TBS), radioimmunoprecipitation buffer (RIPA), 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA) from the pre-frontal cortex of 16 AD, eight PA, and six NDC patients. These extracts were analyzed by 1) a panel of Aβ sandwich ELISAs, 2) immunoprecipitation followed by mass spectrometry (IP/MS) and 3) western blotting. These studies enabled us to asses Aβ levels and solubility, peptide profiles and oligomeric assemblies.ResultsIn almost all extracts (TBS, RIPA, 2% SDS and 70% FA) the average levels of Aβ1-40, Aβ1-42, Aβ total, and Aβx-42 were greatest in AD. On average, levels were slightly lower in PA, and there was extensive overlap between Aβ levels in individual PA and AD cases. The profiles of Aβ peptides detected using IP/MS techniques also showed extensive similarity between the PA and AD brain extracts. In select AD brain extracts, we detected more amino-terminally truncated Aβ peptides compared to PA patients, but these peptides represented a minor portion of the Aβ observed. No consistent differences in the Aβ assemblies were observed by western blotting in the PA and AD groups.ConclusionsWe found extensive overlap with only subtle quantitative differences between Aβ levels, peptide profiles, solubility, and SDS-stable oligomeric assemblies in the PA and AD brains. These cross-sectional data indicate that Aβ accumulation in PA and AD is remarkably similar. Such data would be consistent with PA representing a prodromal stage of AD or a resistance to the toxic effects of Aβ.

Sortilin, SorCS1b, and SorLA Vps10p sorting receptors, are novel γ-secretase substrates

BackgroundThe mammalian Vps10p sorting receptor family is a group of 5 type I membrane homologs (Sortilin, SorLA, and SorCS1-3). These receptors bind various cargo proteins via their luminal Vps10p domains and have been shown to mediate a variety of intracellular sorting and trafficking functions. These proteins are highly expressed in the brain. SorLA has been shown to be down regulated in Alzheimers disease brains, interact with ApoE, and modulate Aβ production. Sortilin has been shown to be part of proNGF mediated death signaling that results from a complex of Sortilin, p75NTR and proNGF. We have investigated and provide evidence for γ-secretase cleavage of this family of proteins.ResultsWe provide evidence that these receptors are substrates for presenilin dependent γ-secretase cleavage. γ-Secretase cleavage of these sorting receptors is inhibited by γ-secretase inhibitors and does not occur in PS1/PS2 knockout cells. Like most γ-secretase substrates, we find that ectodomain shedding precedes γ-secretase cleavage. The ectodomain cleavage is inhibited by a metalloprotease inhibitor and activated by PMA suggesting that it is mediated by an α-secretase like cleavage.ConclusionThese data indicate that the α- and γ-secretase cleavages of the mammalian Vps10p sorting receptors occur in a fashion analogous to other known γ-secretase substrates, and could possibly regulate the biological functions of these proteins.

Independent Generation of Aβ42 and Aβ38 Peptide Species by γ-Secretase

Proteolytic processing of the amyloid precursor protein by β- and γ-secretase generates the amyloid-β (Aβ) peptides, which are principal drug targets in Alzheimer disease therapeutics. γ-Secretase has imprecise cleavage specificity and generates themostabundant Aβ40 and Aβ42 species together with longer and shorter peptides such as Aβ38. Several mechanisms could explain the production of multiple Aβ peptides by γ-secretase, including sequential processing of longer into shorter Aβ peptides. A novel class of γ-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Aβ42 levels without a change in Aβ40 levels. A signature of GSMs is the concomitant increase in shorter Aβ peptides, such as Aβ38, leading to the suggestion that generation of Aβ42 and Aβ38 peptide species by γ-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Aβ42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Aβ38 production would be similarly affected indicating coupled generation of Aβ42 and Aβ38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Aβ38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Aβ peptide species. These data provide evidence that Aβ42 and Aβ38 species can be independently generated by γ-secretase and argue against a precursor-product relationship between these peptides.

A signal peptide peptidase (SPP) reporter activity assay based on the cleavage of type II membrane protein substrates provides further evidence for an inverted orientation of the SPP active site relative to presenilin.

Signal peptide peptidase (SPP) is an intramembrane-cleaving protease identified by its cleavage of several type II membrane signal peptides after signal peptidase cleavage. Here we describe a novel, quantitative, cell-based SPP reporter assay. This assay utilizes a substrate consisting of the NH2 terminus of the ATF6 transcription factor fused to a transmembrane domain susceptible to SPP cleavage in vitro. In cells, cleavage of the substrate releases ATF6 from the membrane. This cleavage can be monitored by detection of an epitope that is unmasked in the cleaved substrate or by luciferase activity induced by the cleaved ATF6 substrate binding to and activating an ATF6 luciferase reporter construct. Using this assay we show that (i) SPP is the first aspartyl intramembrane-cleaving protease whose activity increases proportionally to its overexpression and (ii) selectivity of various SPP and γ-secretase inhibitors can be rapidly evaluated. Because this assay was designed based on data suggesting that SPP has an orientation distinct from presenilin and cleaves type II membrane proteins, we determined whether the segment of SPP located between the two presumptive catalytic aspartates was in the lumen or cytoplasm. Using site-directed mutagenesis to insert an N-linked glycosylation site we show that a portion of this region is present in the lumen. These data provide strong evidence that although the SPP and presenilin active sites have some similarities, their presumptive catalytic domains are inverted. This assay should prove useful for additional functional studies of SPP as well as evaluation of SPP and γ-secretase inhibitors.

Lysine 624 of the Amyloid Precursor Protein (APP) Is a Critical Determinant of Amyloid β Peptide Length SUPPORT FOR A SEQUENTIAL MODEL OF γ-SECRETASE INTRAMEMBRANE PROTEOLYSIS AND REGULATION BY THE AMYLOID β PRECURSOR PROTEIN (APP) JUXTAMEMBRANE REGION

Background: γ-Secretase modulators (GSMs) bind APP near lysine 624. Results: Mutation of lysine 624 shifts cleavage toward smaller Aβ with no effect on ϵ cleavage. Conclusion: The amino acid at 624 in substrates affects the final γ-secretase cut. Significance: γ-Secretase cleavage likely begins at ϵ and proceeds up the transmembrane until Aβ is released, and GSMs may modulate this process through lysine 624. γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimers disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ϵ, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1–42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1–40 to 1–33 without significant changes to ϵ cleavage. These results further support a model where ϵ cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid β peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the APP juxtamembrane region

Background: γ-Secretase modulators (GSMs) bind APP near lysine 624. Results: Mutation of lysine 624 shifts cleavage toward smaller Aβ with no effect on ϵ cleavage. Conclusion: The amino acid at 624 in substrates affects the final γ-secretase cut. Significance: γ-Secretase cleavage likely begins at ϵ and proceeds up the transmembrane until Aβ is released, and GSMs may modulate this process through lysine 624. γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimers disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ϵ, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1–42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1–40 to 1–33 without significant changes to ϵ cleavage. These results further support a model where ϵ cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal peptide peptidase

Signal peptide peptidase (SPP) is an intramembrane cleaving protease (I‐CLiP) identified by its cleavage of several type II membrane signal peptides. To date, only human SPP has been directly shown to have proteolytic activity. Here we demonstrate that the most closely related human homologue of SPP, signal peptide peptidase like 3 (SPPL3), cleaves a SPP substrate, but a more distantly related homologue, signal peptide peptidase like 2b (SPPL2b), does not. These data provide strong evidence that the SPP and SPPL3 have conserved active sites and suggest that the active sites SPPL2b is distinct. We have also synthesized a cDNA designed to express the single SPP gene present in Plasmodium falciparum and cloned this into a mammalian expression vector. When the malaria SPP protein is expressed in mammalian cells it cleaves a SPP substrate. Notably, several human SPP inhibitors block the proteolytic activity of malarial SPP (mSPP). Studies from several model organisms that express multiple SPP homologs demonstrate that the silencing of a single SPP homologue is lethal. Based on these data, we hypothesize that mSPP is a potential a novel therapeutic target for malaria.—Nyborg, A. C., Ladd, T. B., Jansen, K., Kukar, T., Golde, T. E. Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal peptide peptidase. FASEB J. 20, 1671–1679 (2006)

Steroids as γ-secretase modulators

Aggregation and accumulation of Aβ42 play an initiating role in Alzheimers disease (AD); thus, selective lowering of Aβ42 by γ‐secretase modulators (GSMs) remains a promising approach to AD therapy. Based on evidence suggesting that steroids may influence Aβ production, we screened 170 steroids at 10 μM for effects on Aβ42 secreted from human APP‐overexpressing Chinese hamster ovary cells. Many acidic steroids lowered Aβ42, whereas many nonacidic steroids actually raised Aβ42. Studies on the more potent compounds showed that Aβ42‐lowering steroids were bonafide GSMs and Aβ42‐raising steroids were inverse GSMs. The most potent steroid GSM identified was 5β‐cholanic acid (EC50=5.7 μM; its endogenous analog lithocholic acid was virtually equipotent), and the most potent inverse GSM identified was 4‐androsten‐3‐one‐17β‐carboxylic acid ethyl ester (EC50=6.25 μM). In addition, we found that both estrogen and progesterone are weak inverse GSMs with further complex effects on APP processing. These data suggest that certain endogenous steroids may have the potential to act as GSMs and add to the evidence that cholesterol, cholesterol metabolites, and other steroids may play a role in modulating Aβ production and thus risk for AD. They also indicate that acidic steroids might serve as potential therapeutic leads for drug optimization/development.—Jung, J. I., Ladd, T. B., Kukar, T., Price, A. R., Moore, B. D., Koo, E. H., Golde, T. E., Felsenstein, K. M., Steroids as γ‐secretase modulators. FASEB J. 27, 3775–3785 (2013). www.fasebj.org