Rivastigmine modifies the ?-secretase pathway and potentially early Alzheimer's disease.
ABSTRACT: Rivastigmine (or Exelon) is a cholinesterase inhibitor, currently used as a symptomatic treatment for mild-to-moderate Alzheimer's disease (AD). Amyloid-? peptide (A?) generated from its precursor protein (APP) by ?-secretase (or BACE1) and ?-secretase endoproteolysis. Alternative APP cleavage by ?-secretase (a family of membrane-bound metalloproteases- Adamalysins) precludes the generation of toxic A? and yields a neuroprotective and neurotrophic secreted sAPP? fragment. Several signal transduction pathways, including protein kinase C and MAP kinase, stimulate ?-secretase. We present data to suggest that rivastigmine, in addition to anticholinesterase activity, directs APP processing away from BACE1 and towards ?-secretases. We treated rat neuronal PC12 cells and primary human brain (PHB) cultures with rivastigmine and the ?-secretase inhibitor TAPI and assayed for levels of APP processing products and ?-secretases. We subsequently treated 3×Tg (transgenic) mice with rivastigmine and harvested hippocampi to assay for levels of APP processing products. We also assayed postmortem human control, AD, and AD brains from subjects treated with rivastigmine for levels of APP metabolites. Rivastigmine dose-dependently promoted ?-secretase activity by upregulating levels of ADAM-9, -10, and -17 ?-secretases in PHB cultures. Co-treatment with TAPI eliminated rivastigmine-induced sAPP? elevation. Rivastigmine treatment elevated levels of sAPP? in 3×Tg mice. Consistent with these results, we also found elevated sAPP? in postmortem brain samples from AD patients treated with rivastigmine. Rivastigmine can modify the levels of several shedding proteins and directs APP processing toward the non-amyloidogenic pathway. This novel property of rivastigmine can be therapeutically exploited for disease-modifying intervention that goes beyond symptomatic treatment for AD.
Project description:Amyloid-? (A?), the major component of neuritic plaques in Alzheimer's disease (AD), is derived from sequential proteolytic cleavage of amyloid protein precursor (APP) by secretases. In this study, we found that cystatin C (CysC), a natural cysteine protease inhibitor, is able to reduce A?40 secretion in human brain microvascular endothelial cells (HBMEC). The CysC-induced A?40 reduction was caused by degradation of ?-secretase BACE1 through the ubiquitin/proteasome pathway. In contrast, we found that CysC promoted secretion of soluble APP? indicating the activated non-amyloidogenic processing of APP in HBMEC. Further results revealed that ?-secretase ADAM10, which was transcriptionally upregulated in response to CysC, was required for the CysC-induced sAPP? secretion. Knockdown of SIRT1 abolished CysC-triggered ADAM10 upregulation and sAPP? production. Taken together, our results demonstrated that exogenously applied CysC can direct amyloidogenic APP processing to non-amyloidgenic pathway in brain endothelial cells, mediated by proteasomal degradation of BACE1 and SIRT1-mediated ADAM10 upregulation. Our study unveils previously unrecognized protective role of CysC in APP processing.
Project description:The ?-amyloid precursor protein (APP) plays a central role in the etiology of Alzheimer's disease (AD). However, its normal physiological functions are still unclear. APP is cleaved by various secretases whereby sequential processing by the ?- and ?-secretases produces the ?-amyloid peptide that is accumulating in plaques that typify AD. In addition, this produces secreted N-terminal sAPP? fragments and the APP intracellular domain (AICD). Alternative cleavage by ?-secretase results in slightly longer secreted sAPP? fragments and the identical AICD. Whereas the AICD has been connected with transcriptional regulation, sAPP? fragments have been suggested to have a neurotrophic and neuroprotective role . Moreover, expression of sAPP? in APP-deficient mice could rescue their deficits in learning, spatial memory, and long-term potentiation . Loss of the Drosophila APP-like (APPL) protein impairs associative olfactory memory formation and middle-term memory that can be rescued with a secreted APPL fragment . We now show that APPL is also essential for visual working memory. Interestingly, this short-term memory declines rapidly with age, and this is accompanied by enhanced processing of APPL in aged flies. Furthermore, reducing secretase-mediated proteolytic processing of APPL can prevent the age-related memory loss, whereas overexpression of the secretases aggravates the aging effect. Rescue experiments confirmed that this memory requires signaling of full-length APPL and that APPL negatively regulates the neuronal-adhesion molecule Fasciclin 2. Overexpression of APPL or one of its secreted N termini results in a dominant-negative interaction with the FASII receptor. Therefore, our results show that specific memory processes require distinct APPL products.
Project description:<h4>Background</h4>Epidemiologic studies strongly suggest that the pathophysiology of late-onset Alzheimer disease (AD) versus early-onset AD has environmental rather than genetic causes, thus revealing potentially novel therapeutic targets to limit disease progression. Several studies supporting the "pathogen hypothesis" of AD demonstrate a strong association between pathogens and the production of ?-amyloid, the pathologic hallmark of AD. Although the mechanism of pathogen-induced neurodegeneration of AD remains unclear, astrocytes, a key player of the CNS innate immune response and producer/metabolizer of ?-amyloid, have been implicated. We hypothesized that Chlamydia pneumoniae infection of human astrocytes alters the expression of the amyloid precursor protein (APP)-processing secretases, ADAM10, BACE1, and PSEN1, to promote ?-amyloid formation. Utilizing immunofluorescent microscopy, molecular, and biochemical approaches, these studies explore the role of an intracellular respiratory pathogen, Chlamydia pneumoniae, as an environmental trigger for AD pathology. Human astrocytoma cells in vitro were infected with Chlamydia pneumoniae over the course of 6-72 h. The gene and protein expression, as well as the enzymatic activity of non-amyloidogenic (ADAM10), and pro-amyloidogenic (BACE1 and PSEN1) secretases were qualitatively and quantitatively assessed. In addition, the formation of toxic amyloid products as an outcome of pro-amyloidogenic APP processing was evaluated through various modalities.<h4>Results</h4>Chlamydia pneumoniae infection of human astrocytoma cells promoted the transcriptional upregulation of numerous genes implicated in host neuroinflammation, lipid homeostasis, microtubule function, and APP processing. Relative to that of uninfected astrocytes, BACE1 and PSEN1 protein levels were enhanced by nearly twofold at 48-72 h post-Chlamydia pneumoniae infection. The processing of APP in Chlamydia pneumoniae-infected astrocytes favors the pro-amyloidogenic pathway, as demonstrated by an increase in enzymatic activity of BACE1, while that of ADAM10 was decreased. Fluorescence intensity of ?-amyloid and ELISA-quantified levels of soluble-APP by products revealed temporally similar increases, confirming a BACE1/PSEN1-mediated processing of APP.<h4>Conclusions</h4>Our findings suggest that Chlamydia pneumoniae infection of human astrocytes promotes the pro-amyloidogenic pathway of APP processing through the upregulation of expression and activity of ?-secretase, upregulated expression of ?-secretase, and decreased activity of ?-secretase. These effects of astrocyte infection provide evidence for a direct link between Chlamydia pneumoniae and AD pathology.
Project description:A? peptides, the major components of Alzheimer's disease (AD) amyloid deposits, are released following sequential cleavages by secretases of its precursor named the amyloid precursor protein (APP). In addition to secretases, degradation pathways, in particular the endosomal/lysosomal and proteasomal systems have been reported to contribute to APP processing. However, the respective role of each of these pathways toward APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length wild type APP or the ?-secretase-derived C99 fragment (?-CTF) in which degradation pathways were selectively blocked using pharmacological drugs. APP metabolites, including carboxy-terminal fragments (CTFs), soluble APP (sAPP) and A? peptides were studied. In this report, we show that APP-CTFs produced from endogenous or overexpressed full-length APP are mainly processed by ?-secretase and the endosomal/lysosomal pathway, while in sharp contrast, overexpressed C99 is mainly degraded by the proteasome and to a lesser extent by ?-secretase.
Project description:Autism spectrum disorder (ASD) and Fragile X syndrome (FXS) are developmental disorders. No validated blood-based biomarkers exist for either, which impedes bench-to-bedside approaches. Amyloid-? (A?) precursor protein (APP) and metabolites are usually associated with Alzheimer's disease (AD). APP cleavage by ?-secretase produces potentially neurotrophic secreted APP? (sAPP?) and the P3 peptide fragment. ?-site APP cleaving enzyme (BACE1) cleavage produces secreted APP? (sAPP?) and intact A?. Excess A? is potentially neurotoxic and can lead to atrophy of brain regions such as amygdala in AD. By contrast, amygdala is enlarged in ASD but not FXS. We previously reported elevated levels of sAPP? in ASD and FXS vs.We now report elevated plasma A? and total APP levels in FXS compared to both ASD and typically developing controls, and elevated levels of sAPP? in ASD and FXS vs.By contrast, plasma and brain sAPP? and A? were lower in ASD vs. controls but elevated in FXS plasma vs.We also detected age-dependent increase in an ?-secretase in ASD brains. We report a novel mechanistic difference in APP pathways between ASD (processing) and FXS (expression) leading to distinct APP metabolite profiles in these two disorders. These novel, distinctive biochemical differences between ASD and FXS pave the way for blood-based biomarkers for ASD and FXS.
Project description:Impaired glucose uptake in skeletal muscle is an important contributor to glucose intolerance in type 2 diabetes. The aspartate protease, beta-site APP-cleaving enzyme 1 (BACE1), a critical regulator of amyloid precursor protein (APP) processing, modulates in vivo glucose disposal and insulin sensitivity in mice. Insulin-independent pathways to stimulate glucose uptake and GLUT4 translocation may offer alternative therapeutic avenues for the treatment of diabetes. We therefore addressed whether BACE1 activity, via APP processing, in skeletal muscle modifies glucose uptake and oxidation independently of insulin.Skeletal muscle cell lines were used to investigate the effects of BACE1 and ?-secretase inhibition and BACE1 and APP overexpression on glucose uptake, GLUT4 cell surface translocation, glucose oxidation and cellular respiration.In the absence of insulin, reduction of BACE1 activity increased glucose uptake and oxidation, GLUT4myc cell surface translocation, and basal rate of oxygen consumption. In contrast, overexpressing BACE1 in C2C12 myotubes decreased glucose uptake, glucose oxidation and oxygen consumption rate. APP overexpression increased and ?-secretase inhibition decreased glucose uptake in C2C12 myotubes. The increase in glucose uptake elicited by BACE1 inhibition is dependent on phosphoinositide 3-kinase (PI3K) and mimicked by soluble APP? (sAPP?).Inhibition of muscle BACE1 activity increases insulin-independent, PI3K-dependent glucose uptake and cell surface translocation of GLUT4. As APP overexpression raises basal glucose uptake, and direct application of sAPP? increases PI3K-protein kinase B signalling and glucose uptake in myotubes, we suggest that ?-secretase-dependent shedding of sAPP? regulates insulin-independent glucose uptake in skeletal muscle.
Project description:Intramembrane proteolysis of transmembrane substrates by the presenilin-?-secretase complex is preceded and regulated by shedding of the substrate's ectodomain by ?- or ?-secretase. We asked whether ?- and ?-secretases interact to mediate efficient sequential processing of APP, generating the amyloid ? (A?) peptides that initiate Alzheimer's disease. We describe a hitherto unrecognized multiprotease complex containing active ?- and ?-secretases. BACE1 coimmunoprecipitated and cofractionated with ?-secretase in cultured cells and in mouse and human brain. An endogenous high molecular weight (HMW) complex (?5 MD) containing ?- and ?-secretases and holo-APP was catalytically active in vitro and generated a full array of A? peptides, with physiological A?42/40 ratios. The isolated complex responded properly to ?-secretase modulators. Alzheimer's-causing mutations in presenilin altered the A?42/40 peptide ratio generated by the HMW ?/?-secretase complex indistinguishably from that observed in whole cells. Thus, A? is generated from holo-APP by a BACE1-?-secretase complex that provides sequential, efficient RIP processing of full-length substrates to final products.
Project description:The competitive ectodomain shedding of amyloid-? precursor protein (APP) by ?-secretase and ?-secretase, and the subsequent regulated intramembrane proteolysis by ?-secretase are the key processes in amyloid-? peptides (A?) generation. Previous studies indicate that secretases form binary complex and the interactions between secretases take part in substrates processing. However, whether ?-, ?- and ?-secretase could form ternary complex remains to be explored. Here, we adopted bimolecular fluorescence complementation in combination with fluorescence resonance energy transfer (BiFC-FRET) to visualize the formation of triple secretase complex. We show that the interaction between ?-secretase ADAM10 and ?-secretase BACE1 could be monitored by BiFC assay and the binding of APP to ?-/?-secretase binary complex was revealed by BiFC-FRET. Further, we observed that ?-secretase interacts with ?-/?-secretase binary complex, providing evidence that ?-, ?- and ?-secretase might form a ternary complex. Thus our study extends the interplay among Alzheimer's disease (AD) related ?-/?-/?-secretase.
Project description:The BACE1 gene encodes the beta-site APP-cleaving enzyme 1 and has been associated with Alzheimer's disease (AD). BACE1 is the most important ?-secretase responsible for the generation of Alzheimer-associated amyloid ?-proteins (A?) and may play a role in the amyloidogenic process in AD. We hypothesized that BACE1 gene variants might influence BACE1 activity or other markers for APP metabolism in the cerebrospinal fluid (CSF) and thereby contribute to the development of AD. We genotyped a Swedish sample of 269 AD patients for the rs638405 single nucleotide polymorphism (SNP) in the BACE1 gene and correlated genotype data to a broad range of amyloid-related biomarkers in CSF, including BACE1 activity, levels of A?40, A?42, ?- and ?-cleaved soluble APP (?-sAPP and ?-sAPP), as well as markers for Alzheimer-type axonal degeneration, i.e., total-tau and phospho-tau181. Gene variants of BACE1 were neither associated with amyloid-related biomarkers, nor with markers for axonal degeneration in AD.
Project description:Deposition of amyloid ? protein (A?) to form neuritic plaques in the brain is the pathological hallmark of Alzheimer's disease (AD). A? is generated from sequential cleavages of the ?-amyloid precursor protein (APP) by the ?- and ?-secretases, and ?-site APP-cleaving enzyme 1 (BACE1) is the ?-secretase essential for A? generation. Previous studies have indicated that glycogen synthase kinase 3 (GSK3) may play a role in APP processing by modulating ?-secretase activity, thereby facilitating A? production. There are two highly conserved isoforms of GSK3: GSK3? and GSK3?. We now report that specific inhibition of GSK3?, but not GSK3?, reduced BACE1-mediated cleavage of APP and A? production by decreasing BACE1 gene transcription and expression. The regulation of BACE1 gene expression by GSK3? was dependent on NF-?B signaling. Inhibition of GSK3 signaling markedly reduced A? deposition and neuritic plaque formation, and rescued memory deficits in the double transgenic AD model mice. These data provide evidence for regulation of BACE1 expression and AD pathogenesis by GSK3? and that inhibition of GSK3 signaling can reduce A? neuropathology and alleviate memory deficits in AD model mice. Our study suggests that interventions that specifically target the ?-isoform of GSK3 may be a safe and effective approach for treating AD.