Non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease: old and new mechanisms of action

Alzheimer's disease (AD) is characterized by cerebral deposits of beta-amyloid (A beta) peptides and neurofibrillary tangles (NFT) which are surrounded by inflammatory cells. Epidemiological studies have shown that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. It has been postulated that some NSAIDs target pathological hallmarks of AD by interacting with several pathways, including the inhibition of cyclooxygenases (COX) and activation of the peroxisome proliferator-activated receptor gamma. A variety of experimental studies indicate that a subset of NSAIDs such as ibuprofen, flurbiprofen, indomethacin and sulindac also possess A beta-lowering properties in both AD transgenic mice and cell cultures of peripheral, glial and neuronal origin. While COX inhibition occurs at low concentrations in vitro (nM-low microm range), the A beta-lowering activity is observed at high concentrations (< or = 50 microm). Nonetheless, studies with flurbiprofen or ibuprofen in AD transgenic mice show that the effects on A beta levels or deposition are attained at plasma levels similar to those achieved in humans at therapeutic dosage. Still, it remains to be assessed whether adequate concentrations are reached in the brain. This is a crucial aspect that will allow defining the dose-window and the length of treatment in future clinical trials. Here, we will discuss how the combination of anti-amyloidogenic and anti-inflammatory activities of certain NSAIDs may produce a profile potentially relevant to their clinical use as disease-modifying agents for the treatment of AD.     

Presenilin clinical mutations can affect gamma-secretase activity by different mechanisms

Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the gamma-secretase complex and cleave many type I transmembrane proteins including beta-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause 'abnormal' gain or (partial) loss of function of gamma-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on gamma-secretase complex formation. A loss in APP and Notch substrate processing at epsilon and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the gamma site was affected in variable ways. PS1-Delta9 and PS1-L166P mutations caused a reduction in beta-amyloid peptide Abeta40 production whereas PS1-G384A mutant significantly increased Abeta42. Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Abeta than PS1. Finally, subtle differences in gamma-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect gamma-secretase structure or function in multiple ways.     

Behavioral phenotypes of amyloid-based genetically modified mouse models of Alzheimer's disease

Alzheimer's disease (AD) is the most common neurodegenerative affliction of the elderly, presenting with progressive memory loss and dementia and terminating with death. There have been significant advances in understanding the biology and subsequent diagnosis of AD; however, the furious pace of research has not yet translated into a disease-modifying treatment. While scientific inquiry in AD is largely centered on identifying biological players and pathological mechanisms, the day-to-day realities of AD patients and their caregivers revolve around their steady and heartbreaking cognitive decline. In the past decade, AD research has been fundamentally transformed by the development of genetically modified animal models of amyloid-driven neurodegeneration. These important in vivo models not only replicate some of the hallmark pathology of the disease, such as plaque-like amyloid accumulations and astrocytic inflammation, but also some of the cognitive impairments relevant to AD. In this article, we will provide a detailed review of the behavioral and cognitive deficits present in several transgenic mouse models of AD and discuss their functional changes in response to experimental treatments.     

gamma-Secretase complexes containing N- and C-terminal fragments of different presenilin origin retain normal gamma-secretase activity

The gamma-secretase complex processes substrate proteins within membranes and consists of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. PS harbours the enzymatic activity of the complex, and there are two mammalian PS homologues: PS1 and PS2. PS undergoes endoproteolysis, generating the N- and C-terminal fragments, NTF and CTF, which represent the active species of PS. To characterize the functional similarity between complexes of various PS composition, we analysed PS1, PS2, and chimeric PS composed of the NTF from PS1 and CTF from PS2, or vice versa, in assembly and function of the gamma-secretase complex. Chimeric PSs, like PS1 and PS2, undergo normal endoproteolysis when introduced into cells devoid of endogenous PS. Furthermore, PS2 CTF can, at least partially, restore processing in a truncated PS1, which cannot undergo endoproteolysis. All PS forms enable maturation of nicastrin and cleave full length Notch receptors, indicating that both PS1 and PS2 are present at the cell surface. Finally, when co-introduced as separate molecules, NTF and CTF of different PS origin reconstitute gamma-secretase activity. In conclusion, these data show that endoproteolysis, NTF-CTF interactions, and the assembly and activity of gamma-secretase complexes are very conserved between PS1 and PS2.     

Presenilin 2 familial Alzheimer's disease mutations result in partial loss of function and dramatic changes in Abeta 42/40 ratios

Gene knockout studies in mice suggest that presenilin 1 (PS1) is the major gamma-secretase and that it contributes disproportionately to amyloid beta (Abeta) peptide generation from beta-amyloid precursor protein (APP), whereas PS2 plays a more minor role. Based on this and other observations we hypothesized that familial Alzheimer's disease (FAD) mutations in PS2 would have a dramatic effect on function in order to have an observable effect on Abeta levels in the presence of normal PS1 alleles. Only four of the eight reported FAD mutations in PS2 have altered function in vitro suggesting that the other variants represent rare polymorphisms rather than disease-causing mutations. In support of our hypothesis, the four verified PS2 FAD mutations cause substantial changes in the Abeta 42/40 ratio, comparable with PS1 mutations that cause very-early-onset FAD. Most of the PS2 mutations also cause a significant decrease in Abeta 40, APP C-terminal fragment (CTF)gamma and Notch intracellular domain (NICD) production suggesting that they are partial loss of function mutations. PS2 M239V, its PS1 homolog M233V, and other FAD mutations within transmembrane (TM) 5 of PS1 differentially affect CTFgamma and NICD production suggesting that TM5 of PS are important for gamma-secretase cleavage of APP but not Notch.     

Apolipoprotein E and Alzheimer's disease: molecular mechanisms and therapeutic opportunities

Multiple genetic and environmental factors are likely to contribute to the development of Alzheimer's disease (AD). The most important known risk factor for AD is presence of the E4 isoform of apolipoprotein E (apoE). Epidemiological studies demonstrated that apoE4 carriers have a higher risk and develop the disease and an early onset. Moreover, apoE4 is the only molecule that has been associated with all the biochemical disturbances characteristic of the disease: amyloid-beta (Abeta) deposition, tangle formation, oxidative stress, lipid homeostasis deregulation, synaptic plasticity loss and cholinergic dysfunction. This large body of evidence suggest that apoE is a key player in the pathogenesis of AD. This short review examines the current facts and hypotheses of the association between apoE4 and AD, as well as the therapeutic possibilities that apoE might offer for the treatment of this disease.     

Analysis of transmembrane domain mutants is consistent with sequential cleavage of Notch by gamma-secretase

gamma-Secretase is a lipid-embedded, intramembrane-cleaving aspartyl protease that cleaves its substrates twice within their transmembrane domains (TMD): once near the cytosolic leaflet (at S3/epsilon) and again in the middle of the TMD (at S4/gamma). To address whether this unusual process occurs in two independent or interdependent steps, we investigated how mutations at the S3/epsilon site in Notch1-based substrates impact proteolysis. We demonstrate that such mutations greatly inhibit not only gamma-secretase-mediated cleavage at S3 but also at S4, independent of their impact on NICD stability. These results, together with our previous observations, suggest that hydrolysis at the center of the Notch transmembrane domain (S4/gamma) is dependent on the S3/epsilon cleavage. Notch (and perhaps all gamma-secretase substrates) may be cleaved by sequential proteolysis starting at S3.     

Generation of C-terminally truncated amyloid-beta peptides is dependent on gamma-secretase activity

Aberrant production of amyloid-beta peptides by processing of the beta-amyloid precursor protein leads to the formation of characteristic extracellular protein deposits which are thought to be the cause of Alzheimer's disease. Therefore, inhibiting the key enzymes responsible for amyloid-beta peptide generation, beta- and gamma-secretase may offer an opportunity to intervene with the progression of the disease. In human brain and cell culture systems a heterogeneous population of amyloid-beta peptides with various truncations is detected and at present, it is unclear how they are produced. We have used a combination of surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and a specific inhibitor of gamma-secretase to investigate whether the production of all amyloid-beta peptide species requires the action of gamma-secretase. Using this approach, we demonstrate that the production of all truncated amyloid-beta peptides except those released by the action of the nonamyloidogenic alpha-secretase enzyme or potentially beta-site betaAPP cleaving enzyme 2 depends on gamma-secretase activity. This indicates that none of these peptides are generated by a separate enzyme entity and a specific inhibitor of the gamma-secretase enzyme should havethe potential to block the generation of all amyloidogenicpeptides. Furthermore in the presence of gamma-secretase inhibitors, the observation of increased cleavage of the membrane-bound betaAPP C-terminal fragment C99 by alpha-secretase suggests that during its trafficking C99 encounters compartments in which alpha-secretase activity resides.     

Presenilin-directed inhibitors of gamma-secretase trigger caspase 3 activation in presenilin-expressing and presenilin-deficient cells

The amyloid beta peptide (Abeta) is generated by subsequent cleavages by beta- and gamma-secretases. Therefore, these two enzymes are putative therapeutic targets to prevent Abeta production, and hopefully to slow down or even stop the Alzheimer's disease (AD) neurodegenerative process. Several studies have revealed that gamma-secretase hydrolyses other important substrates besides beta-amyloid precursor protein (betaAPP) thus adding another level of complexity to designing fully AD-specific interfering drugs. Here we demonstrate that three distinct presenilin-directed gamma-secretase inhibitors as well as JLK compounds indirectly potentiate caspase 3 activity, the effector caspase of the apoptotic cascade. Thus, inhibitors were shown to drastically stimulate caspase 3 activity in wild-type mice blastocyst-derived and fibroblast cells. Interestingly, some of these inhibitors known to interact with presenilins also trigger caspase activation in presenilin-deficient cells. However, inhibitors do not affect recombinant caspase 3 activity, indicating that the effect on this enzyme was indirect. Furthermore, we established that caspase 3 activation was not due to an effect of gamma-secretase inhibitors on calpains, a family of proteolytic enzymes able to modulate caspase 3 activity. Altogether, our data demonstrate that presenilin-directed gamma-secretase inhibitors affect caspase 3 activity in a presenilin-independent manner. Therefore, as presenilin-dependent gamma-secretase activity is not specific for betaAPP and because its inhibitors clearly affect other vital cell functions, care should be taken in considering 'gamma-secretase' inhibitors as putative therapeutic tools to interfere with AD pathology.     

In vitro characterization of a gamma-secretase radiotracer in mammalian brain

Inhibition of gamma-secretase is a potential therapeutic target for Alzheimer's disease (AD). The present studies have characterized the in vitro properties of a radiolabeled small molecule gamma-secretase inhibitor, [3H]compound D (Yan et al., 2004, J. Neurosci.24, 2942-2952) in mammalian brain. [3H]Compound D was shown to bind with nanomolar affinity (Kd = 0.32-1.5 nM) to a single population of saturable sites in rat, rhesus and human brain cortex homogenates, the density of binding sites ranging from 4 to 7 nM across the species. Competition studies with a structurally diverse group of gamma-secretase inhibitors with a wide range of binding affinities showed that the binding affinities of these compounds correlated well with their ability to inhibit gamma-secretase in vitro. Autoradiographic studies showed that the specific binding of [3H]compound D was widely distributed throughout adult rat, rhesus and normal human brain. There did not appear to be any difference in distribution of [3H]compound D specific binding sites in AD cortex compared with control human cortex as measured using tissue section autoradiography, nor any correlation between gamma-secretase binding and plaque burden as measured immunohistochemically. [3H]compound D is a useful tool to probe the expression and pharmacology of gamma-secretase in mammalian brain.     

Conserved residues in juxtamembrane region of the extracellular domain of nicastrin are essential for gamma-secretase complex formation

The Alzheimer's disease-linked protein, presenilin, forms the active site of the gamma-secretase enzyme complex. However, three other proteins, nicastrin (NCT), PEN-2 and APH-1, are required for enzyme activity. This complex is responsible for cleaving the beta-amyloid precursor protein to produce amyloid beta and the intracellular domain (AICD). Although much research has focused on the regions of presenilin that are important for gamma-secretase function, less is known about NCT. To further our understanding of the role of NCT in gamma-secretase activity and complex formation, we have undertaken a systematic evaluation of conserved residues in the juxtamembrane region of the extracellular domain of NCT. Two mutants, S632A and W648A, greatly reduce gamma-secretase activity, as seen by a reduction in amyloid beta and AICD levels. Several lines of evidence suggest that these mutations result in reduced gamma-secretase activity because they affect the ability of NCT to stably associate with the other gamma-secretase components. Since NCT and APH-1 must first bind in order for presenilin and PEN-2 to stably join the complex, we propose that S632 and W648 are essential for a stable interaction with APH-1.     

A domain at the C-terminus of PS1 is required for presenilinase and gamma-secretase activities

The structural requirements for presenilin (PS) to produce active presenilinase and gamma-secretase enzymes are poorly understood. Here we investigate the role the cytoplasmic C-terminal region of PS1 plays in PS1 activity. Deletion or addition of residues at the PS C-terminus has been reported to inhibit presenilinase endoproteolysis of PS and alter gamma-secretase activity. In this study, we use a sensitive assay in PS1/2KO MEFs to define a domain at the extreme C-terminus of PS1 that is essential for both presenilinase and gamma-secretase activities. Progressive deletion of the C-terminus demonstrated that removal of nine residues produces a PS1 molecule (458ST) that lacks both presenilinase processing and gamma-secretase cleavage of Notch and APP substrates. In contrast, removal of four or five residues had no effect (462ST, 463ST), while intermediate truncations partially inhibited PS1 activity. The 458ST mutant was unable to replace endogenous wtPS1 in HEK293 cells. Although 458ST was able to form a gamma-secretase complex, this complex was not matured, illustrated by mutant PS1 instability, lack of endoproteolysis, and little production of mature Nicastrin. These data indicate that the C-terminal end of PS1 is essential for Nicastrin trafficking and modification as well as the replacement of endogenous PS1 by PS1 transgenes.     

APPepsilon, the epsilon-secretase-derived N-terminal product of the beta-amyloid precursor protein, behaves as a type I protein and undergoes alpha-, beta-, and gamma-secretase cleavages

beta-Amyloid peptide accumulates in the brain of patients affected by sporadic or familial forms of Alzheimer's disease. It derives from the proteolytic attacks of the beta-amyloid precursor protein (betaAPP) by beta- and gamma-secretase activities. An additional epsilon cleavage taking place a few residues C-terminal to the gamma-site has been reported, leading to the formation of an intracellular fragment referred to as APP intracellular domain C50. This epsilon cleavage received particular attention because it resembles the S3 Notch cleavage generating Notch intracellular domain. Indeed, APP intracellular domain, like its Notch counterpart, appears to mediate important physiological functions. gamma and epsilon cleavages on betaAPP appear spatio-temporally linked but pharmacologically distinct and discriminable by mutagenesis approaches. As these cleavages could be seen as either deleterious (gamma-site) or beneficial (epsilon-site), it appears of most interest to set up models aimed at studying these activities separately, particularly to design specific and bioavailable inhibitors. On the other hand, it is important to respect the topology of the substrates in order to examine physiologically relevant cleavages. Here we describe the obtention of cells overexpressing APPepsilon, the epsilon-secretase-derived N-terminal fragment of betaAPP. Interestingly, this N-terminal fragment of betaAPP was shown by biochemical and immunohistochemical approaches to behave as a genuine membrane-bound protein. APPepsilon undergoes constitutive and protein kinase C-regulated alpha-secretase cleavages. Furthermore, APPepsilon is targeted by the beta-secretase beta-site APP-cleaving enzyme and is subsequently cleaved by gamma-secretase. The resulting beta-amyloid peptide production is fully prevented by various gamma-secretase inhibitors. Altogether, our study shows that APPepsilon is a relevant betaAPP derivative to study gamma-secretase activities and to design specific inhibitors without facing any rate-limiting effect of epsilon-secretase-derived cleavage.     

Molecular mechanisms for Alzheimer's disease: implications for neuroimaging and therapeutics

Alzheimer's disease is a progressive neurodegenerative disorder characterised by the gradual onset of dementia. The pathological hallmarks of the disease are beta-amyloid (Abeta) plaques, neurofibrillary tangles, synaptic loss and reactive gliosis. The current therapeutic effort is directed towards developing drugs that reduce Abeta burden or toxicity by inhibiting secretase cleavage, Abeta aggregation, Abeta toxicity, Abeta metal interactions or by promoting Abeta clearance. A number of clinical trials are currently in progress based on these different therapeutic strategies and they should indicate which, if any, of these approaches will be efficacious. Current diagnosis of Alzheimer's disease is made by clinical, neuropsychologic and neuroimaging assessments. Routine structural neuroimaging evaluation with computed tomography and magnetic resonance imaging is based on non-specific features such as atrophy, a late feature in the progression of the disease, hence the crucial importance of developing new approaches for early and specific recognition at the prodromal stages of Alzheimer's disease. Functional neuroimaging techniques such as functional magnetic resonance imaging, magnetic resonance spectroscopy, positron emission tomography and single photon emission computed tomography, possibly in conjunction with other related Abeta biomarkers in plasma and CSF, could prove to be valuable in the differential diagnosis of Alzheimer's disease, as well as in assessing prognosis. With the advent of new therapeutic strategies there is increasing interest in the development of magnetic resonance imaging contrast agents and positron emission tomography and single photon emission computed tomography radioligands that will permit the assessment of Abeta burden in vivo.     

Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) demonstrate excessive p53-dependent apoptosis and neuronal loss

Gamma-secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid beta protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other gamma-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.     

Presenilin function and gamma-secretase activity

Alzheimer's disease (AD) is the most common form of dementia and is characterized pathologically by the accumulation of beta-amyloid (Abeta) plaques and neurofibrillary tangles in the brain. Genetic studies of AD first highlighted the importance of the presenilins (PS). Subsequent functional studies have demonstrated that PS form the catalytic subunit of the gamma-secretase complex that produces the Abeta peptide, confirming the central role of PS in AD biology. Here, we review the studies that have characterized PS function in the gamma-secretase complex in Caenorhabditis elegans, mice and in in vitro cell culture systems, including studies of PS structure, PS interactions with substrates and other gamma-secretase complex members, and the evidence supporting the hypothesis that PS are aspartyl proteases that are active in intramembranous proteolysis. A thorough knowledge of the mechanism of PS cleavage in the context of the gamma-secretase complex will further our understanding of the molecular mechanisms that cause AD, and may allow the development of therapeutics that can alter Abeta production and modify the risk for AD.     

Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca(2+) homeostasis. We could confirm previous observations that endoplasmic reticulum Ca(2+) stores contain less calcium in cells with reduced APP gamma-secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca(2+) on mitochondria and of mitochondria on Ca(2+) homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca(2+) homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca(2+) storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.     

Amyloid precursor protein and Notch intracellular domains are generated after transport of their precursors to the cell surface

Alzheimer's disease is characterized by brain deposition of extracellular amyloid beta-peptide (Abeta)-containing plaques. The cellular site of gamma-secretase activity, which releases Abeta and the corresponding amyloid precursor protein intracellular domain (AICD), remains controversial. Proposed cleavage sites range from the endoplasmic reticulum (ER), the Golgi apparatus, and the cell surface to endosomal compartments. We now used C99-green fluorescent protein (GFP), a fluorescent reporter substrate for gamma-secretase activity and monitored AICD production in living cells. C99-GFP is efficiently cleaved by gamma-secretase, and AICD-GFP is released into the cytosol. Inhibiting gamma-secretase results in accumulation of C99-GFP in early endosomes. By blocking selective transport steps along the secretory pathway, we demonstrate that gamma-secretase does not cleave its substrates in the ER, the Golgi/trans-Golgi network, or in secretory vesicles. In contrast, inhibition of endocytosis did not inhibit cleavage of C99-GFP. Similar results were obtained for another gamma-secretase substrate, NotchDeltaE. Our results suggest that intracellular domains are generated by gamma-secretase at the plasma membrane and/or early endosomes.     

Immunological aspects of microglia: relevance to Alzheimer's disease

Alzheimer's disease (AD) is a progressive dementing neurologic illness, and the most frequent cause of dementia in the elderly. Neuritic plaques are one of the main neuropathological findings in AD, and the major protein component is the β-amyloid protein (Aβ). Another striking feature of neuritic plaques is the presence of activated microglia, cytokines, and complement components, suggestive of “inflammatory foci” within AD brain. In this review, we will examine the mechanisms by which microglia become activated in AD, emphasizing the role in the Aβ protein and proinflammatory cytokines. As well, pathways for suppression of microglial activation by immunosuppressive cytokines will be described. Inflammation mediated by activated microglia is an important component of AD pathophysiology, and strategies to control this response could provide new therapeutic approaches for the treatment of AD.