Enhanced amyloidogenic metabolism of the amyloid beta-protein precursor in the X11L-deficient mouse brain

X11L, a neuronal adaptor protein, associates with the cytoplasmic domain of APP and suppresses APP cellular metabolism. APP is the precursor of Abeta, whose metabolism is strongly implicated in Alzheimer disease pathogenesis. To examine the roles of X11L function in APP metabolism, including the generation of Abeta in the brain, we produced X11L-deficient mutant mice on the C57BL/6 background. The mutant mice did not exhibit histopathological alterations or compensatory changes in the expression of other X11 family proteins, X11 and X11L2. The expression level and distribution of APP in the brain of mutant mice were also identical to those in wild-type mice. However, in the hippocampus, where substantial levels of X11L and APP are expressed, the mutant mice exhibited a significant increase in the level of the C-terminal fragments of APP produced by cleavage with beta-secretase but not alpha-secretase. The levels of Abeta were increased in the hippocampus of aged mutant mice as compared with age-matched controls. These observations clearly indicate that X11L suppresses the amyloidogenic but not amyloidolytic processing of APP in regions of the brain such as the hippocampus, which express significant levels of X11L.     

The amyloidogenic pathway of amyloid precursor protein (APP) is independent of its cleavage by caspases

Amyloid beta-protein (A beta) is the main constituent of senile plaques in Alzheimer's disease and is derived by proteolysis from the amyloid precursor protein (APP). Generation and secretion of both A beta 40 and A beta 42 isoforms depend largely on internalization of APP and occurs mainly in the endocytic pathway. Evidence has also been presented (Gervais, F. G., Xu, D., Robertson, G. S., Vaillancourt, J. P., Zhu, Y., Huang, J., LeBlanc, A., Smith, D., Rigby, M., Shearman, M. S., Clarke, E. E., Zheng, H., Van der Ploeg, L. H. T., Ruffolo, S. C., Thornberry, N. A., Xanthoudakis, S., Zamboni, R. J., Roy, S., and Nicholson, D. W. (1999) Cell, 97, 395--406) that caspase cleavage of APP at its cytosolic tail affects its processing such that it is redirected to a more amyloidogenic pathway, resulting in enhanced A beta generation. However, caspase cleavage of APP also results in loss of its endocytosis signal (YENP), an event that would predict a decline in internalization and a concomitant decrease, not an increase, in A beta generation. In the present work, we examined whether caspase cleavage of APP is relevant to amyloidogenesis. We found that 1) caspase cleavage of APP results in reduced internalization and, accordingly, a decline in A beta secretion; 2) masking of the caspase site in APP did not affect A beta levels and, 3) caspase activation in cells by serum withdrawal did not increase A beta secretion. Thus, caspase cleavage of APP is unlikely to play a direct role in amyloidogenesis.     

Novel cadherin-related membrane proteins,Alcadeins, enhance the X11-like protein-mediated stabilization of amyloid beta-protein precursor metabolism

Previously we found that X11-like protein (X11L) associates with amyloid beta-protein precursor (APP). X11L stabilizes APP metabolism and suppresses the secretion of the amyloid beta-protein (Abeta) that are the pathogenic agents of Alzheimer's disease (AD). Here we found that Alcadein (Alc), a novel membrane protein family that contains cadherin motifs and originally reported as calsyntenins, also interacted with X11L. Alc was abundant in the brain and occurred in the same areas of the brain as X11L. X11L could simultaneously associate with APP and Alc, resulting in the formation of a tripartite complex in brain. The tripartite complex stabilized intracellular APP metabolism and enhanced the X11L-mediated suppression of Abeta secretion that is due to the retardation of intracellular APP maturation. X11L and Alc also formed another complex with C99, a carboxyl-terminal fragment of APP cleaved at the beta-site (CTFbeta). The formation of the Alc.X11L.C99 complex inhibited the interaction of C99 with presenilin, which strongly suppressed the gamma-cleavage of C99. In AD patient brains, Alc and APP were particularly colocalized in dystrophic neurites in senile plaques. Deficiencies in the X11L-mediated interaction between Alc and APP and/or CTFbeta enhanced the production of Abeta, which may be related to the development or progression of AD.     

Trans fatty acids enhance amyloidogenic processing of the Alzheimer amyloid precursor protein (APP)

Hydrogenation of oils and diary products of ruminant animals leads to an increasing amount of trans fatty acids in the human diet. Trans fatty acids are incorporated in several lipids and accumulate in the membrane of cells. Here we systematically investigate whether the regulated intramembrane proteolysis of the amyloid precursor protein (APP) is affected by trans fatty acids compared to the cis conformation. Our experiments clearly show that trans fatty acids compared to cis fatty acids increase amyloidogenic and decrease nonamyloidogenic processing of APP, resulting in an increased production of amyloid beta (Aβ) peptides, main components of senile plaques, which are a characteristic neuropathological hallmark for Alzheimer's disease (AD). Moreover, our results show that oligomerization and aggregation of Aβ are increased by trans fatty acids. The mechanisms identified by this in vitro study suggest that the intake of trans fatty acids potentially increases the AD risk or causes an earlier onset of the disease.     

Increased amyloidogenic processing of transgenic human APP in X11-like deficient mouse brain

Background

X11-family proteins, including X11, X11-like (X11L) and X11-like 2 (X11L2), bind to the cytoplasmic domain of amyloid β-protein precursor (APP) and regulate APP metabolism. Both X11 and X11L are expressed specifically in brain, while X11L2 is expressed ubiquitously. X11L is predominantly expressed in excitatory neurons, in contrast to X11, which is strongly expressed in inhibitory neurons. In vivo gene-knockout studies targeting X11, X11L, or both, and studies of X11 or X11L transgenic mice have reported that X11-family proteins suppress the amyloidogenic processing of endogenous mouse APP and ectopic human APP with one exception: knockout of X11, X11L or X11L2 has been found to suppress amyloidogenic metabolism in transgenic mice overexpressing the human Swedish mutant APP (APPswe) and the mutant human PS1, which lacks exon 9 (PS1dE9). Therefore, the data on X11-family protein function in transgenic human APP metabolism in vivo are inconsistent.

Results

To confirm the interaction of X11L with human APP ectopically expressed in mouse brain, we examined the amyloidogenic metabolism of human APP in two lines of human APP transgenic mice generated to also lack X11L. In agreement with previous reports from our lab and others, we found that the amyloidogenic metabolism of human APP increased in the absence of X11L.

Conclusion

X11L appears to aid in the suppression of amyloidogenic processing of human APP in brain in vivo, as has been demonstrated by previous studies using several human APP transgenic lines with various genetic backgrounds. X11L appears to regulate human APP in a manner similar to that seen in endogenous mouse APP metabolism.

     

Homophilic interactions of the amyloid precursor protein (APP) ectodomain are regulated by the loop region and affect beta-secretase cleavage of APP

We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDS-labile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited cross-linking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of alpha-secretory APP increased, soluble beta-cleaved APP levels decreased. Because Abeta40 and Abeta42 decreased to similar levels as soluble beta-cleaved APP, we conclude either that beta-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.     

Regulation of FE65 nuclear translocation and function by amyloid beta-protein precursor in osmotically stressed cells

FE65, a neural adaptor protein, interacts with amyloid beta-protein precursor (APP) and is known to regulate amyloid beta generation from APP. FE65 also associates with nuclear proteins; however, its physiological function in the nucleus remains unclear. A fixed population of cytoplasmic FE65 is tethered to membranes by binding APP. This membrane-tethered FE65 is liberated from membranes by APP phosphorylation, which is facilitated by a stress-activated protein kinase in sorbitol-treated cells. Here we show that liberated FE65, which is distinct from "virgin" FE65 in the cytoplasm, translocates into the nucleus and accumulates in the nuclear matrix forming a patched structure. Targeting of FE65 into the nuclear matrix was suppressed by the APP intracellular domain fragment, which is generated by consecutive cleavages of APP. Thus, nuclear translocation of FE65 is under the regulation of APP. In the nucleus, FE65 induced gammaH2AX, which plays an important role in DNA repair as a cellular response by stress-damaged cells. These observations suggest that APP-regulated FE65 plays an important role in the early stress response of cells and that FE65 deregulated from APP induces apoptosis.     

RNA interference-mediated silencing of X11alpha and X11beta attenuates amyloid beta-protein levels via differential effects on beta-amyloid precursor protein processing

Processing of the beta-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by beta- and alpha-secretase to produce APP-C99 and APP-C83, which are further cleaved by gamma-secretase to produce amyloid beta-protein (Abeta) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11alpha (MINT1, encoded by gene APBA1) and X11beta (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11alpha and X11beta alters APP processing and Abeta production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11alpha and X11beta expression on APP processing and Abeta production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Abeta levels in both cell lines by inhibiting gamma-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Abeta levels, but apparently not via attenuation of gamma-secretase cleavage of APP. The notion of attenuating gamma-secretase cleavage of APP via the APP adaptor protein X11alpha is particularly attractive with regard to therapeutic potential given that side effects of gamma-secretase inhibition due to impaired proteolysis of other gamma-secretase substrates, e.g. Notch, might be avoided.     

gamma-Secretase cleavage and binding to FE65 regulate the nuclear translocation of the intracellular C-terminal domain (ICD) of the APP family of proteins

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) produces amyloid beta-protein (Abeta), the probable causative agent of Alzheimer's disease (AD), and is therefore an important target for therapeutic intervention. However, there is a burgeoning consensus that gamma-secretase, one of the proteases that generates Abeta, is also critical for the signal transduction of APP and a growing list of other receptors. APP is a member of a gene family that includes two amyloid precursor-like proteins, APLP1 and APLP2. Although APP and the APLPs undergo similar proteolytic processing, there is little information about the role of their gamma-secretase-generated intracellular domains (ICDs). Here, we show that APLP1 and 2 undergo presenilin-dependent RIP similar to APP, resulting in the release of a approximately 6 kDa ICD for each protein. Each of the ICDs are degraded by an insulin degrading enzyme-like activity, but they can be stabilized by members of the FE65 family and translocate to the nucleus. Given that modulation of APP processing is a therapeutic target and that the APLPs are processed in a manner similar to APP, any strategy aimed at altering APP proteolysis will have to take into account possible effects on signaling by APLP 1 and 2.     

Proteolytic processing of amyloid beta protein precursor (APP) by thrombin.

Search for proteases responsible for an altered processing of APP which generates intermediates containing beta/A4 peptide is preceding to understand the formation of beta amyloid deposits characteristic of Alzheimer's disease, since many studies reveal that APP is ordinarily processed so as not to generate beta amyloid. Here, we have examined the action of thrombin, a serine protease in the blood clotting, in APP processing. Thrombin cleaved the mouse recombinant APP695 in vitro, resulting in the accumulation of 28 kDa fragment. The immunoblot analysis showed that the fragment is derived from the carboxy-terminal side of the recombinant APP695. Further, amino acid sequencing exhibited that the fragment is generated by the cleavage at Arg 510-Ile 511 and therefore includes entire beta/A4 peptide. We consider that the 28 kDa fragment is a possible intermediate for beta/A4 peptide. Thus thrombin may be involved in the altered processing of APP.     

Phosphorylation of amyloid precursor protein (APP) at Tyr687 regulates APP processing by alpha- and gamma-secretase

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer’s disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by α-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by γ-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by α- and γ-secretases, determining the expression level of AICD.     

Protein kinase C regulation of intracellular and cell surface amyloid precursor protein (APP) cleavage in CHO695 cells

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP alpha-secretase cleavage has been shown to involve tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP(695) (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE.     

Synergistic effects of Munc18a and X11 proteins on amyloid precursor protein metabolism

X11 proteins have been shown to modulate metabolism of the amyloid precursor protein (APP) and to reduce the secretion of beta-amyloid peptides (Abeta) that are associated with Alzheimer's disease. Whereas X11alpha interacts with APP via its phosphotyrosine-binding domain, recent reports indicate that additional regulatory interactions involve the N terminus of X11. Here we report that the syntaxin-1a-binding protein Munc18a, which interacts with the Munc18a-interacting domain (MID) at the N terminus of X11, strongly regulates the actions of X11 on APP metabolism. When co-expressed with X11alpha, Munc18a potentiated the retention of APP and suppression of Abeta secretion by X11alpha. As a result, the constitutive release of Abeta40 was nearly abolished. Experiments using N terminus deletion mutants of X11alpha/beta and the MID-deficient X11gamma revealed that the majority of the regulatory effect by Munc18a occurred independent of a direct interaction of Munc18a with X11, although the presence of X11 was required. Munc18a expression induced a small increase in beta-secretase activity, whereas it also intensified the reduction in Abeta40 secretion by X11alpha. These data indicate that Munc18a in concert with X11 acts to suppress gamma-secretase processing. We conclude that Munc18a acts through direct and indirect interactions with X11 proteins and powerfully regulates APP metabolism and Abeta secretion.     

Focally elevated creatine detected in amyloid precursor protein (APP) transgenic mice and Alzheimer disease brain tissue

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the beta-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21-89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process.     

Immunohistochemical investigation of amyloid beta-protein (Abeta) in the brain of aged cats

To clarify the immunohistochemical features of amyloid deposits and cerebral amyloid angiopathy (CAA), the distribution of the amyloid beta-protein subtypes Abeta40, Abeta42, Abeta43 and Abeta precursor protein (APP) were examined in the brains of fourteen aged cats (7.5-21 year-old). Two types of plaques were detected. The first type was characterized by Ass positive antigenic material and detected in the cortical layers of the frontal and parietal lobes of all examined cats. The second type was characterized by diffuse positive immune staining representing diffuse plaques, which were detected only in the very aged cats (17-21 years old) and distributed throughout the cortical layers of the parietal lobes. Vascular amyloid and the amyloid deposits were strongly positive-stained with the antibody Abeta42. APP was exhibited in neurons and axons while the staining was stronger in the very aged cats (17-21 years old). Our findings suggest that the feline forms a spontaneous model for understanding the early changes of normal brain aging and the early stage of amyloid beta-protein deposition.     

Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13-16 A). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the PrlA4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent K(m) of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K(m) for proOmpA translocation.     

Seminal plasma proteins regulate the association of lipids and proteins within detergent-resistant membrane domains of bovine spermatozoa

Maturing spermatozoa acquire full fertilization competence by undergoing major changes in membrane fluidity and protein composition and localization. In epididymal spermatozoa, several proteins are associated with cholesterol- and sphingolipid-enriched detergent-resistant membrane (DRM) domains. These proteins dissociate from DRM in capacitated sperm cells, suggesting that DRM may play a role in the redistribution of integral and peripheral proteins in response to cholesterol removal. Since seminal plasma regulates sperm cell membrane fluidity, we hypothesized that seminal plasma factors could be involved in DRM disruption and redistribution of DRM-associated proteins. Our results indicate that: 1) the sperm-associated proteins, P25b and adenylate kinase 1, are linked to DRM of epididymal spermatozoa, but were exclusively associated with detergent-soluble material in ejaculated spermatozoa; 2) seminal plasma treatment of cauda epididymal spermatozoa significantly lowered the content of cholesterol and the ganglioside, GM1, in DRM; and 3), seminal plasma dissociates P25b from DRM in epididymal spermatozoa. We found that the seminal plasma protein, Niemann-Pick C2 protein, is involved in cholesterol and GM1 depletion within DRM, then leading to membrane redistribution of P25b that occurs in a very rapid and capacitation-independent manner. Together, these data suggest that DRM of ejaculated spermatozoa are reorganized by specific seminal plasma proteins, which induce lipid efflux as well as dissociation of DRM-anchored proteins. This process could be physiologically relevant in vivo to allow sperm survival and attachment within the female reproductive tract and to potentiate recognition, binding, and penetration of the oocyte.     

Intraneuronal amyloid precursor protein (APP) and appearance of extracellular beta-amyloid peptide (abeta) in the brain of aging kokanee salmon

Antibodies to human amyloid precursor protein (APP(695)) and beta-amyloid peptide (A beta(1-42)) were used to determine timing of amyloidosis in the brain of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages: immature (IM), maturing (MA), sexually mature (SM), and spawning (SP), representing a range of aging from somatically mature but sexually immature to spawning and somatic senescence. In IM fish, immunoreactive (ir) intracellular APP occurred in 18 of 23 brain regions. During sexual maturation and aging, the number of neurons expressing APP increased in 11 of these APP-ir regions. A beta-ir was absent in IM fish, present in seven regions in MA fish, moderately abundant in 15 regions in SM fish, and was most abundant in all brain regions of SP fish exhibiting A beta-ir. Intracellular APP-ir was observed in brain regions involved in sensory integration, olfaction, vision, stress responses, reproduction, and coordination. Intra- and extracellular A beta(1-42) immunoreactivity (A beta-ir) was present in all APP-ir regions except the nucleus lateralis tuberis (hypothalamus) and Purkinje cells (cerebellum). APP-ir and A beta deposition increase during aging. APP-ir is present in IM fish; A beta-ir usually appears first in MA or SM fish and increases in SM fish as does APP-ir. Extracellular A beta deposition dramatically increases between SM and SP stages (1-2 weeks) in all fish, indicating an extremely rapid and synchronized process. Rapid senescence observed in pacific salmon could make them a useful model to investigate timing of amyloidosis and neurodegeneration during brain aging.     

The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement.

FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.