Identification of truncated C-terminal fragments of the Alzheimer's disease amyloid protein precursor derived from sequential proteolytic pathways.

Recent evidence supports the emerging hypothesis that the amyloid-ß precursor protein C-terminal fragments (APP-CTFs) and dysregulations in both their qualitative and quantitative productions may actively and directly contribute to the neuronal toxicity in early phases of Alzheimer's disease (AD). These new findings revealed the urgent needs and gaps in better understanding the metabolism and full spectrum of APP-CTFs. In this study, we characterized by mass spectrometry the full patterns of APP-CTFs in different cell types and in the brain of an AD APPPS1 mouse model. In these systems, we first discovered a series of 71-80 amino acids long N-terminally truncated APP-CTFs of unknown functions. We next demonstrated that these N-terminally truncated APP-CTFs are sequentially produced by the proteolytic processing of APP-C80, by an as yet unidentified protease. Finally, these N-terminally truncated APP-CTFs are likely protein substrates recognized and processed by the γ-secretase complex, leading to the production of N-terminally truncated Aß peptides. Together, our findings provide new insights into the metabolism of APP and offer potential new strategies to modulate the production of toxic Aß peptides in AD.

The metalloprotease ADAMTS4 generates N-truncated Aß4-x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer's disease.

Brain accumulation and aggregation of amyloid-ß (Aß) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aß peptides (mainly Aß1-40 and Aß1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aß peptides are truncated at the N-terminus, with Aß4-x peptides being particularly abundant. Aß4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aß peptide sequence, which facilitates Aß4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aß4-40 but unchanged levels of Aß1-x peptides. In the 5xFAD mouse model of amyloidosis, Aß4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4-/- knockout background, Aß4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aß species, but Aß4-40 peptides were absent in cultures derived from ADAMTS4-/- mice indicating that the enzyme was essential for Aß4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aß4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aß peptides.

Zinc and Copper Differentially Modulate Amyloid Precursor Protein Processing by γ-Secretase and Amyloid-ß Peptide Production.

Recent evidence suggests involvement of biometal homeostasis in the pathological mechanisms in Alzheimer's disease (AD). For example, increased intracellular copper or zinc has been linked to a reduction in secreted levels of the AD-causing amyloid-ß peptide (Aß). However, little is known about whether these biometals modulate the generation of Aß. In the present study we demonstrate in both cell-free and cell-based assays that zinc and copper regulate Aß production by distinct molecular mechanisms affecting the processing by γ-secretase of its Aß precursor protein substrate APP-C99. We found that Zn2+ induces APP-C99 dimerization, which prevents its cleavage by γ-secretase and Aß production, with an IC50 value of 15 µm Importantly, at this concentration, Zn2+ also drastically raised the production of the aggregation-prone Aß43 found in the senile plaques of AD brains and elevated the Aß43:Aß40 ratio, a promising biomarker for neurotoxicity and AD. We further demonstrate that the APP-C99 histidine residues His-6, His-13, and His-14 control the Zn2+-dependent APP-C99 dimerization and inhibition of Aß production, whereas the increased Aß43:Aß40 ratio is substrate dimerization-independent and involves the known Zn2+ binding lysine Lys-28 residue that orientates the APP-C99 transmembrane domain within the lipid bilayer. Unlike zinc, copper inhibited Aß production by directly targeting the subunits presenilin and nicastrin in the γ-secretase complex. Altogether, our data demonstrate that zinc and copper differentially modulate Aß production. They further suggest that dimerization of APP-C99 or the specific targeting of individual residues regulating the production of the long, toxic Aß species, may offer two therapeutic strategies for preventing AD.

Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Deregulation of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases (RTKs) has recently been demonstrated to predominately promote survival and chemoresistance of cancer cells. Intramembrane proteolysis mediated by presenilin/γ-secretase is known to regulate the homeostasis of some RTKs. In the present study, we demonstrate that AXL, but not TYRO3 or MERTK, is efficiently and sequentially cleaved by α- and γ-secretases in various types of cancer cell lines. Proteolytic processing of AXL redirected signaling toward a secretase-mediated pathway, away from the classic, well-known, ligand-dependent canonical RTK signaling pathway. The AXL intracellular domain cleavage product, but not full-length AXL, was further shown to translocate into the nucleus via a nuclear localization sequence that harbored a basic HRRKK motif. Of interest, we found that the γ-secretase-uncleavable AXL mutant caused an elevated chemoresistance in non-small-cell lung cancer cells. Altogether, our findings suggest that AXL can undergo sequential processing mediated by various proteases kept in a homeostatic balance. This newly discovered post-translational processing of AXL may provide an explanation for the diverse functions of AXL, especially in the context of drug resistance in cancer cells.-Lu, Y., Wan, J., Yang, Z., Lei, X., Niu, Q., Jiang, L., Passtoors, W. M., Zang, A., Fraering, P. C., Wu, F. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

The adipocyte differentiation protein APMAP is an endogenous suppressor of Aß production in the brain.

The deposition of amyloid-beta (Aß) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aß is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aß production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aß generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aß. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aß production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aß production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic system.

The lipidome associated with the γ-secretase complex is required for its integrity and activity.

γ-Secretase is a multi-subunit membrane protease complex that catalyses the final intramembrane cleavage of the ß-amyloid precursor protein (APP) during the neuronal production of amyloid-ß peptides (Aß), which are implicated as the causative agents of Alzheimer's disease (AD). In the present study, we report the reconstitution of a highly purified, active γ-secretase complex into proteoliposomes without exogenous lipids and provide the first direct evidence for the existence of a microenvironment of 53 molecular species from 11 major lipid classes specifically associated with the γ-secretase complex, including phosphatidylcholine and cholesterol. Importantly, we demonstrate that the pharmacological modulation of certain phospholipids abolishes both the integrity and the enzymatic activity of the intramembrane protease. Together, our findings highlight the importance of a specific lipid microenvironment for the structure and function of γ-secretase.

Perturbations of the straight transmembrane α-helical structure of the amyloid precursor protein affect its processing by γ-secretase.

The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase of its C-terminal fragment produces amyloid-ß (Aß) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight α-helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the γ-secretase enzyme, as suggested by a series of Gly(700) mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of Aß38 compared with Aß40 peptides, which is reminiscent of the effect of γ-secretase modulators inhibitors.

Inactivation of brain Cofilin-1 by age, Alzheimer's disease and γ-secretase.

Rapid remodeling of the actin cytoskeleton in the pre- and/or post-synaptic compartments is responsible for the regulation of neuronal plasticity,which is an important process for learning and memory. Cofilin1 plays an essential role in these processes and a dysregulation of its activity was associated with the cognitive decline observed during normal aging and Alzheimer's disease (AD). To understand the mechanism(s) regulating Cofilin1 activity we evaluated changes occurring with regard to Cofilin1 and its up-stream regulators Lim kinase-1 (LIMK1) and Slingshot phosphatase-1 (SSH1) in (i) human AD brain, (ii) 1-, 4-, and 10-months old APP/PS1 mice, (iii) wildtype 3-, 8-, 12-, 18- and 26-months old mice, as well as in cellular models including (iv) mouse primary cortical neurons (PCNs, cultured for 5, 10, 15 and 20 days in vitro) and (v) mouse embryonic fibroblasts (MEF). Interestingly,we found an increased Cofilin1 phosphorylation/inactivation with age and AD pathology, both in vivo and in vitro. These changes were associated with a major inactivation of SSH1. Interestingly, inhibition of ã-secretase activity with Compound-E (10 ìM) prevented Cofilin1 phosphorylation/inactivation through an increase of SSH1 activity in PCNs. Similarly, MEF cells double knock-out for ã-secretase catalytic subunits presenilin-1 and -2(MEFDKO) showed a strong decrease of both Cofilin1 and SSH1 phosphorylation,which were rescued by the over expression of human ã-secretase. Together, these results shed new light in understanding the molecular mechanisms promoting Cofilin1 dysregulation, both during aging and AD. They further have the potential to impact the development of therapies to safely treat AD.

Identification of new Presenilin-1 phosphosites: implication for γ-secretase activity and Aß production.

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-beta (Aß) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ-secretase is responsible for the intramembrane proteolysis of the amyloid-ß precursor protein (APP), which leads to the production of the toxic Aß peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ-secretase activity, to reduce Aß42 production. Because phosphorylation of proteins is a post-translational modification known to modulate the activity of many different enzymes, we used electrospray (LC-MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ-secretase. We identified 11 new single or double phosphosites in two well-defined domains of Presenilin-1 (PS1), the catalytic subunit of the γ-secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ-secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ-secretase activity and the production of the Alzheimer's Aß peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aß plaque formation in AD. In this study, we identified 11 new phosphosites in Presenilin-1 (PS1), the catalytic subunit of the Alzheimer's γ-secretase complex. By combining a mutagenesis approach with cell-based and cell-free γ-secretase assays, we demonstrate that the new phosphosites do not modulate the maturation and activity of γ-secretase. Individual PS1 phosphosites shall thus not be considered therapeutic targets for reducing cerebral Aß plaque formation in Alzheimer's Disease. Aß, amyloid beta.

Novel therapeutic strategy for neurodegeneration by blocking Aß seeding mediated aggregation in models of Alzheimer's disease.

Aß accumulation plays a central role in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that the process of Aß nucleated polymerization is essential for Aß fibril formation, pathology spreading and toxicity. Therefore, targeting this process represents an effective therapeutic strategy to slow or block disease progression. To discover compounds that might interfere with the Aß seeding capacity, toxicity and pathology spreading, we screened a focused library of FDA-approved drugs in vitro using a seeding polymerization assay and identified small molecule inhibitors that specifically interfered with Aß seeding-mediated fibril growth and toxicity. Mitoxantrone, bithionol and hexachlorophene were found to be the strongest inhibitors of fibril growth and protected primary cortical neuronal cultures against Aß-induced toxicity. Next, we assessed the effects of these three inhibitors in vivo in the mThy1-APPtg mouse model of AD (8-month-old mice). We found that mitoxantrone and bithionol, but not hexachlorophene, stabilized diffuse amyloid plaques, reduced the levels of Aß42 oligomers and ameliorated synapse loss, neuronal damage and astrogliosis. Together, our findings suggest that targeting fibril growth and Aß seeding capacity constitutes a viable and effective strategy for protecting against neurodegeneration and disease progression in AD.

Production of active glycosylation-deficient γ-secretase complex for crystallization studies.

Alzheimer's disease (AD)-associated γ-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although γ-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. γ-Secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only γ-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of γ-secretase as a functional complex. We purified glycosylation-deficient γ-secretase from this recombinant cell line (CL1-9) and fully glycosylated γ-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient γ-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of γ-secretase and to structure-based drug discovery for AD.

The role of γ-secretase activating protein (GSAP) and imatinib in the regulation of γ-secretase activity and amyloid-ß generation.

γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-ß production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-ß production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-ß levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-ß generation. Likewise, purified recombinant GSAP had no effect on amyloid-ß generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-ß levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-ß levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-ß generation are uncertain.

Substrate-targeting gamma-secretase modulators.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-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 gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta 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 Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta 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.

Label-free imaging of cerebral ß-amyloidosis with extended-focus optical coherence microscopy.

We demonstrate label-free imaging of cerebral ß-amyloidosis ex vivo and in a living mouse model of Alzheimer's disease using extended-focus Fourier domain optical coherence microscopy (xfOCM). xfOCM provides 3D, high-resolution images of individual ß-amyloid plaques in the brain parenchyma and vasculature and requires no staining of the alzheimeric sample under investigation. xfOCM also opens the possibility to perform minimally invasive studies of ß-amyloid pathology in vivo, without the use of labeling methods, which potentially confound experimental findings.

Highly efficient production of the Alzheimer's γ-secretase integral membrane protease complex by a multi-gene stable integration approach.

Inefficient production of membrane-embedded multi-protein complexes by conventional methods has largely prevented the generation of high-resolution structural information and the performance of high-throughput drug discovery screens for this class of proteins. Not exempt from this rule is γ-secretase, an intramembrane-cleaving protease complex regulating a multitude of signaling pathways and biological processes by influencing gene transcription. γ-Secretase is also implicated in the pathogenesis of Alzheimer's disease and several types of cancer. As an additional challenge, the reconstitution of the protease complex in its active form requires an intricate assembly and maturation process, including a highly regulated endoproteolytic processing of its catalytic component. In this article we report the application of a transposon-mediated multigene stable integration technology to produce active γ-secretase in mammalian cells in amounts adequate for crystallization studies and drug screening. Our strategy is expected to help elucidate the molecular mechanisms of intramembrane proteolysis. It is further expected to be widely used for the production of other multi-protein complexes for applications in structural biology and drug development.

A delay in vesicle endocytosis by a C-terminal fragment of N-cadherin enhances Aß synaptotoxicity.

Synaptotoxic Aß oligomers are thought to play a major role in the early pathology of Alzheimer´s disease (AD). However, the molecular mechanisms involved in Aß-induced synaptic dysfunction and synapse damage remain largely unclear. Previously, Aß synaptotoxicity has been reported to be enhanced by increased levels of a C-terminal fragment of the synaptic adhesion molecule N-cadherin that is generated by proteolytic shedding of the extracellular domains [1]. To address the molecular mechanisms involved in this process, we have now studied the functional synaptic changes induced by C-terminal fragments (CTF1) of synaptic adhesion proteins. We used synaptophysin-pHluorin (SypHy) fluorescence imaging to monitor synaptic vesicle exo- and endocytosis in cultures of mouse cortical neurons. We increased the levels of C-terminal fragments of synaptic adhesion proteins by pharmacologically inhibiting γ-secretase, which further degrades CTF1 fragments. We found that this intervention caused a delay in synaptic vesicle endocytosis. A similar effect was induced by overexpression of N-cadherin CTF1, but not by overexpression of Neurexin3ß CTF1. Based on these observations, we further studied whether directly modulating synaptic vesicle endocytosis enhances Aß synaptotoxicity. We pharmacologically induced a delayed synaptic vesicle endocytosis by a low concentration of the endocytosis inhibitor dynasore. This treatment enhanced synaptoxicity of Aß oligomers as indicated by a reduced frequency of miniature postsynaptic currents. In conclusion, we propose that delayed endocytosis results in prolonged exposure of synaptic vesicle membranes to the extracellular space, thus enabling enhanced vesicle membrane binding of Aß oligomers. This might in turn promote the endocytic uptake of toxic Aß oligomers and might thus play an important role in intracellular Aß-mediated synaptotoxicity in AD.

Generation of monoclonal antibody fragments binding the native γ-secretase complex for use in structural studies.

A detailed understanding of γ-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimer's disease. γ-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.

Processing of the synaptic cell adhesion molecule neurexin-3beta by Alzheimer disease alpha- and gamma-secretases.

Neurexins (NRXNs) are synaptic cell adhesion molecules having essential roles in the assembly and maturation of synapses into fully functional units. Immunocytochemical and electrophysiological studies have shown that specific binding across the synaptic cleft of the ectodomains of presynaptic NRXNs and postsynaptic neuroligins have the potential to bidirectionally coordinate and trigger synapse formation. Moreover, in vivo studies as well as genome-wide association studies pointed out implication of NRXNs in the pathogenesis of cognitive disorders including autism spectrum disorders and different types of addictions including opioid and alcohol dependences, suggesting an important role in synaptic function. Despite extensive investigations, the mechanisms by which NRXNs modulate the properties of synapses remain largely unknown. We report here that α- and γ-secretases can sequentially process NRXN3ß, leading to the formation of two final products, an ∼80-kDa N-terminal extracellular domain of Neurexin-3ß (sNRXN3ß) and an ∼12-kDa C-terminal intracellular NRXN3ß domain (NRXN3ß-ICD), both of them being potentially implicated in the regulation of NRXNs and neuroligins functions at the synapses or in yet unidentified signal transduction pathways. We further report that this processing is altered by several PS1 mutations in the catalytic subunit of the γ-secretase that cause early-onset familial Alzheimer disease.

Mercury is a direct and potent γ-secretase inhibitor affecting Notch processing and development in Drosophila.

Prenatal exposure to mercury causes neurodevelopmental disorders and neurological pathologies in infants, such as microcephaly and mental retardation. Despite critical importance, the molecular interactions leading to mercury toxicity are yet to be elucidated. We first used a cell-free assay to investigate mercury effects on purified γ-secretase activity. Next, we treated adult Drosophila melanogaster with mercury and collected control and mercury-treated embryos, which were subjected to mild hypotonic protein extraction, or immunostained to reveal nervous system morphology. Embryos left to develop into adults were examined for wing phenotypes. Relative to control metals, we found that mercury strongly inhibits in vitro γ-secretase processing of both amyloid-ß precursor protein (APP) and Notch. Mercury inhibited APP and Notch cleavage in a dose-dependent manner, with IC(50) values of 50-125 nM, and is therefore comparable in potency to benchmark γ-secretase inhibitors. Immunoblot analysis of embryonic protein extracts showed that mercury inhibits Notch cleavage by γ-secretase in vivo. This is accompanied by severe neurodevelopmental abnormalities in embryos and adult wing-notching phenotypes. Our findings provide first evidence that mercury is a direct and potent γ-secretase inhibitor and suggest that inhibition of γ-secretase and disruption of the Notch developmental pathway potentially contribute to mercury-induced toxicity in the nervous system.

Novel gamma-secretase inhibitors uncover a common nucleotide-binding site in JAK3, SIRT2, and PS1.

Gamma-secretase is an intramembrane-cleaving protease responsible for the final proteolytic event in the production of the amyloid-beta peptides (Abeta) implicated in Alzheimer's disease (AD). Inhibition of gamma-secretase activity is thus an attractive therapeutic strategy to slow down the pathogenesis of AD. Drugs often target more than one biomolecule because of conserved 3-dimensional structures in prospective protein binding sites. We have capitalized on this phenomenon of nature to identify new gamma-secretase inhibitors. Here we show that 2-hydroxy naphthyl derivatives, a previously identified subclass of NAD(+) analog inhibitors of sirtuin 2 (SIRT2), are direct gamma-secretase inhibitors. Subsequent structure-activity relationship studies further showed that 2-hydroxy-1-naphthaldehyde is the minimal pharmacophore for gamma-secretase inhibition. In evaluating target protein determinants of inhibition, we identified a common GXG signature nucleotide-binding site (NBS) shared by the gamma-secretase subunit presenilin-1 C-terminal fragment (PS1-CTF), SIRT2, and Janus kinase 3 (JAK3). Because a detailed 3-dimensional structure of gamma-secretase is beyond our knowledge, we took advantage of the known crystal structure of human JAK3 to model the NBS of the PS1-CTF, which includes the catalytic residue D385. Our results suggest that the flexible PS1-CTF (381)LGLG(384) loop comprises a substrate-docking site capable of recognizing specifically different gamma-secretase substrates.