Eva Czirr

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.

Insensitivity to Aβ42-lowering nonsteroidal anti-inflammatory drugs and γ-secretase inhibitors is common among aggressive presenilin-1 mutations

Aβ42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of γ-secretase modulators that avoid side effects of pan-γ-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free γ-secretase assays indicating that they directly target the γ-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Aβ42-lowering NSAIDs. Of particular interest is the PS1-ΔExon9 mutation, which provokes a pathogenic increase in the Aβ42/Aβ40 ratio and dramatically reduces the cellular response to the Aβ42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr291–Ser319 including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-ΔExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Aβ42/Aβ40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Aβ42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to γ-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent γ-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Aβ42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at γ-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of γ-secretase modulators and inhibitors.

Presenilin‐1 but not amyloid precursor protein mutations present in mouse models of Alzheimer’s disease attenuate the response of cultured cells to γ‐secretase modulators regardless of their potency and structure

J. Neurochem. (2011) 116, 385–395.

α-secretase mediated conversion of the amyloid precursor protein derived membrane stub C99 to C83 limits Aβ generation

The Swedish mutation within the amyloid precursor protein (APP) causes early‐onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While β‐secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild‐type APP occurs mainly in endocytic compartments. However, we show that liberation of Aβ from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall β‐secretase cleaved soluble APP released from APPswe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased Aβ secretion. We point out that α‐secretase activity‐mediated conversion of C99 to C83 is the main cause of this Aβ reduction. Furthermore, we demonstrate that α‐secretase cleavage of C99 even contributes to the reduction of Aβ secretion of internalization deficient wild‐type APP. Therefore, inhibition of α‐secretase cleavage increased Aβ secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells.

Nonsteroidal Anti-Inflammatory Drugs and Ectodomain Shedding of the Amyloid Precursor Protein

Background: Epidemiological studies have suggested that long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced incidence of Alzheimer’s disease (AD). Several mechanisms have been proposed to explain these findings including increased shedding of the soluble ectodomain of the amyloid precursor protein (sAPP), which functions as a neurotrophic and neuroprotective factor in vitroand in vivo. Objective: To clarify whether NSAIDs consistently stimulate sAPP secretion. Methods: 293-EBNA cells with stable overexpression of an APP-alkaline phosphatase fusion protein (APP-AP), SH-SY5Y and PC12 cells or primary telencephalic chicken neurons were treated with ibuprofen or indomethacin. APP shedding was then determined by measuring AP activity in conditioned media, Western blot analysis with antibodies against total sAPP or specific for sAPP-α, or in a pulse-chase paradigm. Results: AP activity in conditioned media was not increased after NSAID treatment of 293-EBNA cells whereas it was elevated by phorbol ester. Surprisingly, ibuprofen or indomethacin treatment of SH-SY5Y and PC12 cells expressing endogenous APP did not cause changes in sAPP or sAPP-α secretion or downregulation of cellular APP. These findings were further corroborated in primary chicken neuronal cultures. Conclusions: Using various experimental settings, we were unable to confirm sAPP or sAPP-α stimulation with the NSAIDs ibuprofen and indomethacin in transfected and nontransfected cells of neuronal and nonneuronal origin. Importantly, these findings seem to rule out chronic sAPP stimulation as an alternative mechanism of NSAID action in AD.

BENEFICAL PROPERTIES OF A HUMAN PLASMA FRACTION FOR AGE-RELATED COGNITIVE DISORDERS

directly targeting CRH using a high affinity antibody.Methods:Using molecular cloning, vaccination, and hybridoma technology we have successfully generated and purified a monoclonal Anti CRH antibody for use as a potential therapeutic. This antibody is currently being tested as a therapeutic in both mutant APP and tau transgenic mice. Results:A one time intraperitoneal injection of 25 mg/kg of Anti-CRH antibody in mice is able to provide >80% suppression of the corticosterone response to acute restraint stress and is able to reverse the cushingoid phenotype seen in mice that overexpress CRHwithin the brain. This suppression of corticosterone from one dose remains present even four days past injection and possibly much longer. Conclusions:We have generated a high affinity (Kd<1.0E-12) monoclonal CRH antibody that is able to reduce the glucocorticoid response to acute restraint stress by greater than 80%while also maintaining basal glucocorticoid levels in mice. These studies have broad implication for stress-related disorders in addition to AD, and may herald a new class of central nervous system therapies that target neuropeptides using high affinity antibodies.

TARGETING THE EOTAXIN/CCR3 PATHWAY IN AGING-ASSOCIATED COGNITIVE DECLINE

after treatment. Student t-test was used to compare the different groups of animals. Animals were visually and histologically inspected for tumor formation. Results: Treatment of eSCs and mSCs by a less-invasive intravenous route in the MPTP PD rodent model provided augmentation in motor abilities at early (10 days post-treatment) and later (3 months post-treatment) time points. Also, this treatment was deemed to be safe from teratomas. Conclusions: Motor impairment observed in MPTP-induced PD mice ameliorates after the treatment with NPs, and this is more evident several days after the therapy. Therefore, peripheral administration of NPs could be a promising therapy to treat motor impairment associated to PD.

Presenilin-1 (PS1) and amyloid precursor protein (APP) mutations present in mouse models of Alzheimer's disease in their response to γ-secretase inhibitors and modulators

peptides mainly generate two isoform, Aß40 and Aß42 by enzymatic proteolysis of amyloid precursor protein (APP). In particular, the Aß42 is believed to be the major etiologic agent in pathogenesis of AD due to its higher fibrillation or oligomerization properties than that of Aß40. Recently we have established conformation dependent human antibody, B6, which binds to Aß42 fibril, but not to soluble form of Aß42, inhibiting Aß42 fibril formation. Concurrently, we have identified a mimotope of B6, B6-C15, using the PhD.C7C phage library. We chemically synthesized TAT-conjugated B6-C15 peptide, TATB6-C15. This synthetic peptide has inhibitory activity on Aß42 fibrillation. Furthermore, TAT-B6-C15 specifically binds to the oligomer form of Aß42, but not to freshly prepared monomer Aß42 nor its fibril form. In this study, we investigated the effect of this TAT-B6-C15 peptide on Aß40 assembly. Methods: Aß42 or Aß40 was incubated at 37 C in the presence or absence of TAT-B6-C15 peptide. Aß fibrillation was monitored by amyloid specific fluorescence dye, Thioflavin T. To identify the Aß conformers which specifically bound to TAT-B6-C15 peptide, we performed dot blot analysis. Aß conformers were periodically sampled after the onset of Aß40 or 42 fibrillation assay and hand-spotted onto nitrocellulose membrane, followed by incubation with detection probes such as TAT-B6-C15 or anti-Aß antibody. Results: The TAT-B6-C15 peptide exhibited inhibitory effect on Aß42, but not Aß40 fibrillation. Furthermore, the TAT-B6-C15 showed binding activity to the Aß42 prefibrillar oligomer, but not any Aß40 conformers. Conclusions: The mimotope peptide which identified as conformation dependent antibody epitope, specifically binds to prefibrillar oligomers of Aß42, inhibiting Aß42 but not Aß40 fibril formation.

P4-192: Mechanism of γ-secretase cleavage: Evidence for independent generation of Aβ42 and Aβ38 peptide species

changes in the precision of PS/ -secretase have any biological effects. Methods: We used a newly established luciferase reporter assay to evaluate the Notch signaling intensity. The improved construct contains four sequential RBP-J binding sites in the HES-1 promoter region (HES-Y). N-terminal capping antibodies which specifically recognize NICD-S or NICD-V were used to determine S3 cleavage precision. Results: We found that S3 cleavage mediated by PS/ -secretase has diversity, resulting in the production of two types of NICD (NICD-S and NICD-V) with apparently distinct ability to transmit Notch signaling. We also showed that the precision of S3 cleavage can be modulated by physiological factors, which affects the intensity of Notch signaling. Although many PS/ -secretase modifiers have been known to change the precision of -cleavage, it is unknown whether there are any methods which modify the S3 cleavage precision, thus affecting the intensity of Notch signaling. Using the Notch downstream assay and N-terminal capping antibodies which specifically recognize NICD-S or NICD-V, we searched for methods to modify S3 cleavage precision. Conclusions: Pathologically important A 42 upregulating process also exerts physiological function.