Amyloid-β Protein Precursor Metabolism Research Articles

EDEM1 Regulates Amyloid Precursor Protein (APP) Metabolism and Amyloid-β Production

Endoplasmic reticulum (ER) degradation-enhancing α-mannosidase-like protein 1 (EDEM1) is a quality control factor directly involved in the endoplasmic reticulum-associated degradation (ERAD) process. It recognizes terminally misfolded proteins and directs them to retrotranslocation which is followed by proteasomal degradation in the cytosol. The amyloid-β precursor protein (APP) is synthesized and N-glycosylated in the ER and transported to the Golgi for maturation before being delivered to the cell surface. The amyloidogenic cleavage pathway of APP leads to production of amyloid-β (Aβ), deposited in the brains of Alzheimer’s disease (AD) patients. Here, using biochemical methods applied to human embryonic kidney, HEK293, and SH-SY5Y neuroblastoma cells, we show that EDEM1 is an important regulatory factor involved in APP metabolism. We find that APP cellular levels are significantly reduced after EDEM1 overproduction and are increased in cells with downregulated EDEM1. We also report on EDEM1-dependent transport of APP from the ER to the cytosol that leads to proteasomal degradation of APP. EDEM1 directly interacts with APP. Furthermore, overproduction of EDEM1 results in decreased Aβ40 and Aβ42 secretion. These findings indicate that EDEM1 is a novel regulator of APP metabolism through ERAD.

Amyloidogenic processing of amyloid β protein precursor (APP) is enhanced in the brains of alcadein α–deficient mice

Alzheimer's disease (AD) is a very common neurodegenerative disorder, chiefly caused by increased production of neurotoxic β-amyloid (Aβ) peptide generated from proteolytic cleavage of β-amyloid protein precursor (APP). Except for familial AD arising from mutations in the APP and presenilin (PSEN) genes, the molecular mechanisms regulating the amyloidogenic processing of APP are largely unclear. Alcadein α/calsyntenin1 (ALCα/CLSTN1) is a neuronal type I transmembrane protein that forms a complex with APP, mediated by the neuronal adaptor protein X11-like (X11L or MINT2). Formation of the ALCα-X11L-APP tripartite complex suppresses Aβ generation in vitro, and X11L-deficient mice exhibit enhanced amyloidogenic processing of endogenous APP. However, the role of ALCα in APP metabolism in vivo remains unclear. Here, by generating ALCα-deficient mice and using immunohistochemistry, immunoblotting, and co-immunoprecipitation analyses, we verified the role of ALCα in the suppression of amyloidogenic processing of endogenous APP in vivo We observed that ALCα deficiency attenuates the association of X11L with APP, significantly enhances amyloidogenic β-site cleavage of APP, especially in endosomes, and increases the generation of endogenous Aβ in the brain. Furthermore, we noted amyloid plaque formation in the brains of human APP-transgenic mice in an ALCα-deficient background. These results unveil a potential role of ALCα in protecting cerebral neurons from Aβ-dependent pathogenicity in AD.

Stabilization of intracellular trafficking and metabolism of amyloid β-protein precursor and Alcadein β by apolipoprotein E

Intracellular metabolism of amyloid β-protein precursor (APP) is important for the pathogenesis of Alzheimer’s disease (AD). Alcadeins (Alcα, Alcβ, and Alcγ) are neural membrane proteins similar to APP in their localization, metabolism, and cellular function. Isoform ε4 of apolipoprotein E (ApoE) is a major risk factor for AD. We found that ApoE expression attenuated intracellular trafficking of APP and Alcβ, resulting in metabolic stabilization of both proteins. By contrast, Alcα intracellular proteolysis was facilitated by ApoE expression, which was not due to an increase in the primary cleavage of Alcα. This difference may result from binding of ApoE to membrane proteins.

Cytoplasmic Fragment of Alcadein α Generated by Regulated Intramembrane Proteolysis Enhances Amyloid β-Protein Precursor (APP) Transport into the Late Secretory Pathway and Facilitates APP Cleavage

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo.

Effects of Huanglian-Jie-Du-Tang and Its Modified Formula on the Modulation of Amyloid-β Precursor Protein Processing in Alzheimer's Disease Models

Huanglian-Jie-Du-Tang (HLJDT) is a famous traditional Chinese herbal formula that has been widely used clinically to treat cerebral ischemia. Recently, we found that berberine, a major alkaloid compound in HLJDT, reduced amyloid-β (Aβ) accumulation in an Alzheimer’s disease (AD) mouse model. In this study, we compared the effects of HLJDT, four single component herbs of HLJDT (Rhizoma coptidis (RC), Radix scutellariae (RS), Cortex phellodendri (CP) and Fructus gardenia (FG)) and the modified formula of HLJDT (HLJDT-M, which is free of RS) on the regulatory processing of amyloid-β precursor protein (APP) in an in vitro model of AD. Here we show that treatment with HLJDT-M and its components RC, CP, and the main compound berberine on N2a mouse neuroblastoma cells stably expressing human APP with the Swedish mutation (N2a-SwedAPP) significantly decreased the levels of full-length APP, phosphorylated APP at threonine 668, C-terminal fragments of APP, soluble APP (sAPP)-α and sAPPβ-Swedish and reduced the generation of Aβ peptide in the cell lysates of N2a-SwedAPP. HLJDT-M showed more significant APP- and Aβ- reducing effects than berberine, RC or CP treatment alone. In contrast, HLJDT, its component RS and the main active compound of RS, baicalein, strongly increased the levels of all the metabolic products of APP in the cell lysates. The extract from FG, however, did not influence APP modulation. Interestingly, regular treatment of TgCRND8 APP transgenic mice with baicalein exacerbated the amyloid plaque burden, APP metabolism and Aβ production. Taken together, these data provide convincing evidence that HLJDT and baicalein treatment can increase the amyloidogenic metabolism of APP which is at least partly responsible for the baicalein-mediated Aβ plaque increase in the brains of TgCRND8 mice. On the other hand, HLJDT-M significantly decreased all the APP metabolic products including Aβ. Further study of HLJDT-M for therapeutic use in treating AD is warranted.

Sphingosine kinases modulate the secretion of amyloid β precursor protein from SH-SY5Y neuroblastoma cells: the role of α-synuclein.

Sphingosine kinases (SphK 1&2) are involved in the regulation of cell survival, differentiation and neurotransmitter secretion. Current data suggest potential links between sphingolipid signalling, α-synuclein (ASN) and Alzheimer's disease (AD). Our aim was to investigate the possible role of SphKs and ASN in the regulation of the production and secretion of the amyloid β precursor protein (APP). We have previously shown that ASN intensified the secretion and toxicity of amyloid β (Aβ) to the point where it caused cell death. Our current results show that APP, the precursor protein for Aβ, is also influenced by ASN. The stable overexpression of wtASN in SH-SY5Y cells caused a three-fold, significant increase of the cellular APP level. This suggests that the influence of ASN on Aβ metabolism may actually occur at the level of APP protein rather than only through the changes of its cleavage into Aβ. To elucidate the mechanisms of APP modulation the cells were exposed to S1P and an SphK inhibitor (SKI). 72 h S1P treatment at 5 µM caused a nearly 50% reduction of the cellular APP signal. S1P also caused a tendency towards higher APP secretion, though the results were insignificant. The inhibition of SphKs decreased medium APP levels in a dose-dependent manner, reaching significance at 5 µM SKI with a correspondingly elevated intracellular level. Thus, it is reasonable to expect that in fact the influence of SphK activity on APP might be pro-secretory. This would also be in agreement with numerous articles on SphK-dependent secretion in the literature. The chronic nature of AD further suggests that subtle alterations in APP metabolism could have the potential to drive important changes in brain condition.

High Glucose Promotes Aβ Production by Inhibiting APP Degradation

Abnormal deposition of neuriticplaques is the uniqueneuropathological hallmark of Alzheimer’s disease (AD).Amyloid β protein (Aβ), the major component of plaques, is generated from sequential cleavage of amyloidβ precursor protein (APP) by β-secretase and γ-secretase complex. Patients with diabetes mellitus (DM), characterized by chronic hyperglycemia,have increased risk of AD development.However, the role of high blood glucose in APP processing and Aβ generation remains elusive. In this study, we investigated the effect of high glucose on APP metabolism and Aβ generation in cultured human cells. We found that high glucose treatment significantly increased APP protein level in both neuronal-like and non-neuronal cells, and promoted Aβ generation. Furthermore, we found that high glucose-induced increase of APP level was not due to enhancement of APP gene transcription but resulted from inhibition of APP protein degradation. Taken together, our data indicated that hyperglycemia could promote AD pathogenesis by inhibiting APP degradation and enhancing Aβ production. More importantly, the elevation of APP level and Aβ generation by high glucose was caused by reduction of APP turnover rate.Thus,our study provides a molecular mechanism of increased risk of developing AD in patients withDMand suggests thatglycemic control might be potentially beneficial for reducing the incidence of AD in diabetic patients and delaying the AD progression.

Stimulation of the amyloid‐β precursor protein metabolism by cAMP

Besides playing a pathogenic role in Alzheimer disease, amyloid‐β (Aβ) peptides are normally produced in low amounts in the brain and several lines of evidence suggest that they can modulate synaptic plasticity and memory. As cyclic adenosine monophosphate (cAMP) is known to be involved in the same processes and the blockade of its degradation by phosphodiesterase‐4 inhibitors has consistently shown beneficial effects on cognition, we investigated the possible correlation between this second messenger and Aβ peptides in neuronal N2a cells overexpressing the amyloid‐β precursor protein (APP). We herein report that the elevation of endogenous cAMP by rolipram increased APP protein expression and both its amyloidogenic and non‐amyloidogenic processing. The effects of rolipram were reproduced by both the cAMP membrane‐permeant analogue 8Br‐cAMP and the forskolin‐induced activation of adenylyl cyclase, but were not affected by the PKA inhibitor H‐89.Our results demonstrate that, in neuronal cells, APP metabolism is physiologically modulated by cAMP and suggest that this might represent an additional mechanism through which the second messenger could influence memory functions. This work was supported by grants from the Alzheimer's Association and the Italian Ministry of University and Research (PRIN 2009M8FKBB_002).

Cyclic adenosine monophosphate as an endogenous modulator of the amyloid‐β precursor protein metabolism

Besides playing a pathogenic role in Alzheimer disease, amyloid-beta peptides are normally produced in low amounts in the brain, and several lines of evidence suggest that they can modulate synaptic plasticity and memory. As cyclic adenosine monophosphate (cAMP) is known to be involved in the same processes and the blockade of its degradation by phosphodiesterase 4 inhibitors has consistently shown beneficial effects on cognition, we investigated the possible correlation between this second messenger and Aβ peptides in neuronal N2a cells overexpressing the amyloid-β precursor protein (APP). We herein report that the elevation of endogenous cAMP by rolipram increased APP protein expression and both its amyloidogenic and nonamyloidogenic processing. The effects of rolipram were reproduced by both the cAMP membrane-permeant analog 8Br-cAMP and the forskolin-induced activation of adenylyl cyclase but were not affected by the PKA inhibitor H-89. Our results demonstrate that, in neuronal cells, APP metabolism is physiologically modulated by cAMP and suggest that this might represent an additional mechanism through which the second messenger could influence memory functions.

Open–closed motion of Mint2 regulates APP metabolism

The amyloid-β protein precursor (APP) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Knock-out and transgenic mouse studies of the adaptor protein Mint2 have revealed that it is a major player in regulating APP metabolism physiologically through the binding of its phosphotyrosine-binding (PTB) domain to the intracellular domain of APP. However, the molecular mechanism of APP dynamically binding to Mint2 remains elusive. Here, we report the structures of APP peptide-free and APP peptide-bound C-terminal Mint2 mutants at resolutions of 2.7 and 3.3 Å, respectively. Our structures reveal that APP peptide-free Mint2 exists in a closed state in which the ARM domain blocks the peptide-binding groove of the PTB domain. In sharp contrast, APP peptide-bound Mint2 exists in an open state in which the ARM domain drastically swings away from the bound peptide. Mutants that control the open-closed motion of Mint2 dynamically regulated APP metabolism both in vitro and in vivo. Our results uncover a novel open-closed mechanism of the PTB domain dynamically binding to its peptide substrate. Moreover, such a conformational switch may represent a general regulation mode of APP family members by Mint proteins, providing useful information for the treatment of AD.

Intracellular Trafficking of the Amyloid β-Protein Precursor (APP) Regulated by Novel Function of X11-Like

BackgroundAmyloid β (Aβ), a causative peptide of Alzheimer's disease, is generated by intracellular metabolism of amyloid β-protein precursor (APP). In general, mature APP (mAPP, N- and O-glycosylated form) is subject to successive cleavages by α- or β-, and γ-secretases in the late protein secretory pathway and/or at plasma membrane, while immature APP (imAPP, N-glycosylated form) locates in the early secretory pathway such as endoplasmic reticulum or cis-Golgi, in which imAPP is not subject to metabolic cleavages. X11-like (X11L) is a neural adaptor protein composed of a phosphotyrosine-binding (PTB) and two C-terminal PDZ domains. X11L suppresses amyloidogenic cleavage of mAPP by direct binding of X11L through its PTB domain, thereby generation of Aβ lowers. X11L expresses another function in the regulation of intracellular APP trafficking.MethodologyIn order to analyze novel function of X11L in intracellular trafficking of APP, we performed a functional dissection of X11L. Using cells expressing various domain-deleted X11L mutants, intracellular APP trafficking was examined along with analysis of APP metabolism including maturation (O-glycosylation), processing and localization of APP.ConclusionsX11L accumulates imAPP into the early secretory pathway by mediation of its C-terminal PDZ domains, without being bound to imAPP directly. With this novel function, X11L suppresses overall APP metabolism and results in further suppression of Aβ generation. Interestingly some of the accumulated imAPP in the early secretory pathway are likely to appear on plasma membrane by unidentified mechanism. Trafficking of imAPP to plasma membrane is observed in other X11 family proteins, X11 and X11L2, but not in other APP-binding partners such as FE65 and JIP1. It is herein clear that respective functional domains of X11L regulate APP metabolism at multiple steps in intracellular protein secretory pathways.

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

BackgroundX11-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.ResultsTo 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.ConclusionX11L 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.

Protein kinase C and rho activated coiled coil protein kinase 2 (ROCK2) modulate Alzheimer's APP metabolism and phosphorylation of the Vps10-domain protein, SorL1

BackgroundGeneration of the amyloid β (Aβ) peptide of Alzheimer's disease (AD) is differentially regulated through the intracellular trafficking of the amyloid β precursor protein (APP) within the secretory and endocytic pathways. Protein kinase C (PKC) and rho-activated coiled-coil kinases (ROCKs) are two "third messenger" signaling molecules that control the relative utilization of these two pathways. Several members of the Vps family of receptors (Vps35, SorL1, SorCS1) play important roles in post-trans-Golgi network (TGN) sorting and generation of APP derivatives, including Aβ at the TGN, endosome and the plasma membrane. We now report that Vps10-domain proteins are candidate substrates for PKC and/or ROCK2 and act as phospho-state-sensitive physiological effectors for post-TGN sorting of APP and its derivatives.ResultsAnalysis of the SorL1 cytoplasmic tail revealed multiple consensus sites for phosphorylation by protein kinases. SorL1 was subsequently identified as a phosphoprotein, based on sensitivity of its electrophoretic migration pattern to calf intestine alkaline phosphatase and on its reaction with anti-phospho-serine antibodies. Activation of PKC resulted in increased shedding of the ectodomains of both APP and SorL1, and this was paralleled by an apparent increase in the level of the phosphorylated form of SorL1. ROCK2, the neuronal isoform of another protein kinase, was found to form complexes with SorL1, and both ROCK2 inhibition and ROCK2 knockdown enhanced generation of both soluble APP and Aβ.ConclusionThese results highlight the potential importance of SorL1 in elucidating phospho-state sensitive mechanisms in the regulation of metabolism of APP and Aβ by PKC and ROCK2.

TMP21 Transmembrane Domain Regulates γ-Secretase Cleavage

TMP21 has been shown to be associated with the γ-secretase complex and can specifically regulate γ-cleavage without affecting ϵ-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of γ-secretase activity was investigated independent of ϵ-cleavage using an amyloid-β precursor proteinϵ (APPϵ) construct which lacks the amyloid intracellular domain domain. The APPϵ construct behaves similarly to the full-length precursor protein with respect to α- and β-cleavages and is able to undergo normal γ-processing. Co-expression of APPϵ and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Aβ-positive fragments, consistent with an inhibition of γ-secretase cleavage. The APPϵ system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the γ-secretase complex and could decrease γ-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Aβ production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased γ-cleavage activity.

X11 Proteins Regulate the Translocation of Amyloid β-Protein Precursor (APP) into Detergent-resistant Membrane and Suppress the Amyloidogenic Cleavage of APP by β-Site-cleaving Enzyme in Brain

X11 and X11-like proteins (X11L) are neuronal adaptor proteins whose association to the cytoplasmic domain of amyloid beta-protein precursor (APP) suppresses the generation of amyloid beta-protein (Abeta) implicated in Alzheimer disease pathogenesis. The amyloidogenic, but not amyloidolytic, metabolism of APP was selectively increased in the brain of mutant mice lacking X11L (Sano, Y., Syuzo-Takabatake, A., Nakaya, T., Saito, Y., Tomita, S., Itohara, S., and Suzuki, T. (2006) J. Biol. Chem. 281, 37853-37860). To reveal the actual role of X11 proteins (X11s) in suppressing amyloidogenic cleavage of APP in vivo, we generated X11 and X11L double knock-out mice and analyzed the metabolism of APP. The mutant mice showed enhanced beta-site cleavage of APP along with increased accumulation of Abeta in brain and increased colocalization of APP with beta-site APP-cleaving enzyme (BACE). In the brains of mice deficient in both X11 and X11L, the apparent relative subcellular distributions of both mature APP and its beta-C-terminal fragment were shifted toward the detergent-resistant membrane (DRM) fraction, an organelle in which BACE is active and both X11s are not nearly found. These results indicate that X11s associate primarily with APP molecules that are outside of DRM, that the dissociation of APP-X11/X11L complexes leads to entry of APP into DRM, and that cleavage of uncomplexed APP by BACE within DRM is enhanced by X11s deficiency. Present results lead to an idea that the dysfunction of X11L in the interaction with APP may recruit more APP into DRM and increase the generation of Abeta even if BACE activity did not increase in brain.

P1-391: Intracellular dynamics of Alcadein family molecules and their metabolites in neuronal cells and tissues

Alcadein family proteins (Alcs) are evolutionarily conserved type I membrane proteins mainly expressed in neuronal tissues. Alcs associate with amyloid β-precursor protein (APP) through their cytoplasmic domains via an adaptor scaffolding protein X11-like (J. Biol. Chem. [2003] 278, 49448). Interestingly, despite their difference in the primary structures, Alcs and APP share common functional and biochemical features; Alc functions as a cargo receptor for kinesin-1 (EMBO J. [2007] 26, 1475), and Alcs undergo highly similar metabolism to those observed in APP. Alcs are subjected to be cleaved at primarily jaxtamembrane region by certain metalloproteinases and secondarily intramembrane region by γ-secretase to produce their ectodomain fragments (sAlc), small peptide fragments (β-Alc) and their intracellular domain fragments (AlcICD). This regulated intramembrane proteolysis (RIP) is very similar to APP-RIP, in which APP secretes sAPP, Aβ (or P3) and AICD. These functional and biochemical similarities suggest the possible availability of Alcs as an alternative biochemical marker for AD diagnosis. To analyze how Alcs behave in neuronal cells, we investigated the intracellular dynamics of Alcs and their metabolites. We expressed series of Alcadein family proteins and their deletion mutants mimicking the AlcICDs in neuroblastoma cells, and observed their intracellular dynamics utilizing several lines of biochemical and cell biological methodologies. Their expressions in mouse brain tissues were also examined. Several lines of evidence indicate that Alcs are transported to cell surface as effectively as APP, and Alcs are efficiently processed to generate AlcICDs that have potential to be localized in the nucleus. AlcICD is relatively stable in nucleus, and cytoplasmic AlcICD is susceptible to degradation by proteasome. In mouse brain, each Alc family members are differently expressed. The amount of AlcICDs in mouse brain increases as with its age, suggesting that Alc-RIP signal might function in maturating and/or aging neural cells. Alcs are efficiently processed as APP, providing additional evidence supporting the idea that Alcs would be an alternative biochemical marker reflecting alterations of APP metabolism in AD.

P4-042: Alcadein, a presenilin/ γ-secretase substrate is a novel cargo receptor for kinesin-I

Alcadein (Alc) are type I membrane protein like amyloid β–protein precursor (APP), and the metabolism and trafficking of both proteins are regulated by their cytoplasmic domain through interaction with cytoplasmic adaptor proteins. We previously reported that APP and Alc form complex via cytoplasmic interaction with X11–like protein (X11L) in neuron, and both proteins are subject to coordinated metabolism when X11L is released from the complex. However, the process of complex formation remained unclear in cells. Here we found that APP and Alcα, a member of Alc family proteins, are subjected to an independent vesicular transport at different speeds with kinesin I motor. Furthermore, we found that JIP1b, a member of JNK–interacting proteins, plays an important role in the selection of cargos containing APP or Alca by kinesin I. Molecular mechanisms of intracellular trafficking and metabolism of APP and Alc will be discussed.

Role of 14-3-3γ in FE65-dependent Gene Transactivation Mediated by the Amyloid β-Protein Precursor Cytoplasmic Fragment

The amyloid beta-protein precursor intracellular domain fragment (AICD) is generated from amyloid beta-protein precursor by consecutive cleavages. AICD is thought to activate FE65-dependent gene expression, but the molecular mechanism remains under consideration. We found that dimeric 14-3-3gamma bound both AICD and FE65 simultaneously, and this binding facilitated FE65-dependent gene transactivation by enhancing the association of AICD with FE65. 14-3-3gamma bound to the 667VTPEER672 motif of AICD and, most interestingly, the phosphorylation of AICD at Thr-668 in this motif inhibited the interaction with 14-3-3gamma and blocked gene transactivation. 14-3-3gamma required a sequence between the WW domain and the first phosphotyrosine interaction domain of FE65 for association with FE65. Deletion of this region blocked 14-3-3gamma binding to FE65 and suppressed AICD-mediated FE65-dependent gene transactivation, although the deletion mutant FE65 was still able to bind Tip60, a histone acetyltransferase that forms a complex with FE65 in the nucleus. Taken together, these data demonstrate that 14-3-3gamma facilitates FE65-dependent gene transactivation by forming a complex containing AICD and FE65, and phosphorylation of AICD down-regulates FE65-dependent gene transactivation through the dissociation of 14-3-3gamma and/or FE65 from AICD. Our findings suggest that multiple interactions of AICD with FE65 and 14-3-3gamma modulate FE65-dependent gene transactivation.

Coordinated metabolism of Alcadein and amyloid beta-protein precursor regulates FE65-dependent gene transactivation.

The Alcadeins (Alcs)/calsyntenins and the amyloid beta-protein precursor (APP) associate with each other in the brain by binding via their cytoplasmic domains to X11L (the X11-like protein). We previously reported that the formation of this APP-X11L-Alc tripartite complex suppresses the metabolic cleavages of APP. We show here that the metabolism of the Alcs markedly resembles that of APP. The Alcs are subjected to a primary cleavage event that releases their extracellular domain. Alcs then undergo a secondary presenilin-dependent gamma-cleavage that leads to the secretion of the amyloid beta-protein-like peptide and the liberation of an intracellular domain fragment (AlcICD). However, when Alc is in the tripartite complex, it escapes from these cleavages, as does APP. We also found that AlcICD suppressed the FE65-dependent gene transactivation activity of the APP intracellular domain fragment, probably because AlcICD competes with the APP intracellular domain fragment for binding to FE65. We propose that the Alcs and APP are coordinately metabolized in neurons and that their cleaved cytoplasmic fragments are reciprocally involved in the regulation of FE65-dependent gene transactivation. Any imbalance in the metabolism of Alcs and APP may influence the FE65-dependent gene transactivation, which together with increased secretion of amyloid beta-protein may contribute to neural disorders.

Accumulation of amyloid β-protein precursor (APP) in Purkinje cells and increase of amino-terminal fragments of APP in cerebrum and cerebellum of aged rat brain

In aged rat brain, amyloid β-protein precursor (APP) is accumulated in dendrites and cell bodies of Purkinje cells as full-length or truncated APP, because dendrites and cell bodies are positively stained by antibodies against both the amino- and car☐y-termini of APP. Western blot analysis of homogenates of brains of aged and young rats showed no apparent differences except for an increase in amino-terminal fragments in cerebrum and cerebellum of aged rat. These results indicate that the expression, transport or metabolism of APP in specific regions of brains may be affected by the aging process.