β-Amyloid Precursor Protein Mutants Respond to γ-Secretase Modulators

Pathogenic generation of the 42-amino acid variant of the amyloid β-peptide (Aβ) by β- and γ-secretase cleavage of the β-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of Aβ₄₂ production by γ-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Aβ domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of γ-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the Aβ domain regarding their response to GSMs. We found that Aβ₄₂-increasing familial AD mutations of the γ-secretase cleavage site domain responded robustly to Aβ₄₂-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Aβ₄₂ produced. We thus expect that familial AD patients carrying mutations at the γ-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine-scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as other Aβ species (Aβ₄₁, Aβ₃₉) could also be lowered besides Aβ₄₂. Moreover, certain mutations simultaneously increased Aβ₄₂ and the shorter peptide Aβ₃₈, arguing that the proposed precursor-product relationship of these Aβ species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Aβ₄₂ generation (Gly-29, Gly-33) and potentially in GSM-binding (Lys-28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.     

Amyloid-β Peptide Exacerbates the Memory Deficit Caused by Amyloid Precursor Protein Loss-of-Function in Drosophila

The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD). APP can undergo two exclusive proteolytic pathways: cleavage by the α-secretase initiates the non-amyloidogenic pathway while cleavage by the β-secretase initiates the amyloidogenic pathway that leads, after a second cleavage by the γ-secretase, to amyloid-β (Aβ) peptides that can form toxic extracellular deposits, a hallmark of AD. The initial events leading to AD are still unknown. Importantly, aside from Aβ toxicity whose molecular mechanisms remain elusive, several studies have shown that APP plays a positive role in memory, raising the possibility that APP loss-of-function may participate to AD. We previously showed that APPL, the Drosophila APP ortholog, is required for associative memory in young flies. In the present report, we provide the first analysis of the amyloidogenic pathway’s influence on memory in the adult. We show that transient overexpression of the β-secretase in the mushroom bodies, the center for olfactory memory, did not alter memory. In sharp contrast, β-secretase overexpression affected memory when associated with APPL partial loss-of-function. Interestingly, similar results were observed with Drosophila Aβ peptide. Because Aβ overexpression impaired memory only when combined to APPL partial loss-of-function, the data suggest that Aβ affects memory through the APPL pathway. Thus, memory is altered by two connected mechanisms—APPL loss-of-function and amyloid peptide toxicity—revealing in Drosophila a functional interaction between APPL and amyloid peptide.     

AMPA Receptor Activation Promotes Non-Amyloidogenic Amyloid Precursor Protein Processing and Suppresses Neuronal Amyloid-β Production

Soluble oligomeric amyloid β peptide (Aβ) generated from processing of the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer''s Disease (AD) and through actions at glutamatergic synapses affects excitability and plasticity. The physiological control of APP processing is not fully understood but stimulation of synaptic NMDA receptors (NMDAR) can suppress Aβ levels through an ERK-dependent increase in α-secretase activity. AMPA-type glutamate receptors (AMPAR) couple to ERK phosphorylation independently of NMDAR activation raising the possibility that stimulation of AMPAR might similarly promote non-amyloidogenic APP processing. We have tested this hypothesis by investigating whether AMPAR directly regulate APP processing in cultured mouse cortical neurons, by analyzing APP C-terminal fragments (CTFs), soluble APP (sAPP), Aβ levels, and cleavage of an APP-GAL4 reporter protein. We report that direct stimulation of AMPAR increases non-amyloidogenic α-secretase-mediated APP processing and inhibits Aβ production. Processing was blocked by the matrix metalloproteinase inhibitor TAPI-1 but was only partially dependent on Ca²⁺ influx and ERK activity. AMPAR can therefore, be added to the repertoire of receptors that couple to non-amyloidogenic APP processing at glutamatergic synapses and thus pharmacological targeting of AMPAR could potentially influence the development and progression of Aβ pathology in AD.     

Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.     

Retention in Endoplasmic Reticulum 1 (RER1) Modulates Amyloid-β (Aβ) Production by Altering Trafficking of γ-Secretase and Amyloid Precursor Protein (APP)

The presence of neuritic plaques containing aggregated amyloid-β (Aβ) peptides in the brain parenchyma is a pathological hallmark of Alzheimer disease (AD). Aβ is generated by sequential cleavage of the amyloid β precursor protein (APP) by β- and γ-secretase, respectively. As APP processing to Aβ requires transport through the secretory pathway, trafficking of the substrate and access to the secretases are key factors that can influence Aβ production (Thinakaran, G., and Koo, E. H. (2008) Amyloid precursor protein trafficking, processing, and function. J. Biol. Chem. 283, 29615–29619). Here, we report that retention in endoplasmic reticulum 1 (RER1) associates with γ-secretase in early secretory compartments and regulates the intracellular trafficking of γ-secretase. RER1 overexpression decreases both γ-secretase localization on the cell surface and Aβ secretion and conversely RER1 knockdown increases the level of cell surface γ-secretase and increases Aβ secretion. Furthermore, we find that increased RER1 levels decrease mature APP and increase immature APP, resulting in less surface accumulation of APP. These data show that RER1 influences the trafficking and localization of both γ-secretase and APP, thereby regulating the production and secretion of Aβ peptides.     

Peptides of Presenilin-1 Bind the Amyloid Precursor Protein Ectodomain and Offer a Novel and Specific Therapeutic Approach to Reduce ?-Amyloid in Alzheimer’s Disease

β-Amyloid (Aβ) accumulation in the brain is widely accepted to be critical to the development of Alzheimer’s disease (AD). Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP) and Presenilin (PS), as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH₂-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH₂-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.     

Octyl Gallate Markedly Promotes Anti-Amyloidogenic Processing of APP through Estrogen Receptor-Mediated ADAM10 Activation

Our previous studies showed that the green tea-derived polyphenolic compound (−)-epigallocatechin-3 gallate (EGCG) reduces amyloid-β (Aβ) production in both neuronal and mouse Alzheimer’s disease (AD) models in concert with activation of estrogen receptor-α/phosphatidylinositide 3-kinase/protein kinase B (ERα/PI3K/Akt) signaling and anti-amyloidogenic amyloid precursor protein (APP) α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10) processing. Since the gallate moiety in EGCG may correspond to the 7α position of estrogen, thereby facilitating ER binding, we extensively screened the effect of other gallate containing phenolic compounds on APP anti-amyloidogenic processing. Octyl gallate (OG; 10 µM), drastically decreased Aβ generation, in concert with increased APP α-proteolysis, in murine neuron-like cells transfected with human wild-type APP or “Swedish” mutant APP. OG markedly increased production of the neuroprotective amino-terminal APP cleavage product, soluble APP-α (sAPPα). In accord with our previous study, these cleavage events were associated with increased ADAM10 maturation and reduced by blockade of ERα/PI3k/Akt signaling. To validate these findings in vivo, we treated Aβ-overproducing Tg2576 mice with OG daily for one week by intracerebroventricular injection and found decreased Aβ levels associated with increased sAPPα. These data indicate that OG increases anti-amyloidogenic APP α-secretase processing by activation of ERα/PI3k/Akt signaling and ADAM10, suggesting that this compound may be an effective treatment for AD.     

γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP)

The relative increase in Aβ42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid β-protein (Aβ) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aβ peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aβ42/Aβ40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aβ42 generation by altering processivity but not ε-cleavage site utilization.     

Biogenesis of gamma-secretase early in the secretory pathway

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ₄₂, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.     

The Resveratrol Trimer Miyabenol C Inhibits β-Secretase Activity and β-Amyloid Generation

Accumulation and deposition of amyloid-β peptide (Aβ) in the brain is a primary cause of the pathogenesis of Alzheimer’s disease (AD). Aβ is generated from amyloid-β precursor protein (APP) through sequential cleavages first by β-secretase and then by γ-secretase. Inhibiting β-secretase activity is believed to be one of the most promising strategies for AD treatment. In the present study, we found that a resveratrol trimer, miyabenol C, isolated from stems and leaves of the small-leaf grape (Vitisthunbergii var. taiwaniana), can markedly reduce Aβ and sAPPβ levels in both cell cultures and the brain of AD model mice. Mechanistic studies revealed that miyabenol C affects neither protein levels of APP, the two major α-secretases ADAM10 and TACE, and the γ-secretase component Presenilin 1, nor γ-secretase-mediated Notch processing and TACE activity. In contrast, although miyabenol C has no effect on altering protein levels of the β-secretase BACE1, it can inhibit both in vitro and in vivo β-secretase activity. Together, our results indicate that miyabenol C is a prominent β-secretase inhibitor and lead compound for AD drug development.     

Independent Relationship between Amyloid Precursor Protein (APP) Dimerization and γ-Secretase Processivity

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.     

Accumulation of autophagosomes contributes to enhanced amyloidogenic APP processing under insulin-resistant conditions

Alzheimer disease (AD) is sometimes referred to as type III diabetes because of the shared risk factors for the two disorders. Insulin resistance, one of the major components of type II diabetes mellitus (T2DM), is a known risk factor for AD. Insulin resistance increases amyloid-β peptide (Aβ) generation, but the exact mechanism underlying the linkage of insulin resistance to increased Aβ generation in the brain is unknown. In this study, we investigated the effect of insulin resistance on amyloid β (A4) precursor protein (APP) processing in mice fed a high-fat diet (HFD), and diabetic db/db mice. We found that insulin resistance promotes Aβ generation in the brain via altered insulin signal transduction, increased BACE1/β-secretase and γ-secretase activities, and accumulation of autophagosomes. Using an in vitro model of insulin resistance, we found that defects in insulin signal transduction affect autophagic flux by inhibiting the mechanistic target of rapamycin (MTOR) pathway. The insulin resistance-induced autophagosome accumulation resulted in alteration of APP processing through enrichment of secretase proteins in autophagosomes. We speculate that the insulin resistance that underlies the pathogenesis of T2DM might alter APP processing through autophagy activation, which might be involved in the pathogenesis of AD. Therefore, we propose that insulin resistance-induced autophagosome accumulation becomes a potential linker between AD and T2DM.     

Allosteric Modulation of PS1/γ-Secretase Conformation Correlates with Amyloid β42/40 Ratio

Background

Presenilin 1(PS1) is the catalytic subunit of γ-secretase, the enzyme responsible for the Aβ C-terminal cleavage site, which results in the production of Aβ peptides of various lengths. Production of longer forms of the Aβ peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of γ-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin.

Methodology/Principal Findings

To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the γ-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either γ-secretase components (PS1, Pen2, Aph1) or γ-secretase substrate (amyloid precursor protein, APP) and are known to change Aβ₄₂ production are associated with a consistent conformational change in PS1.

Conclusions/Significance

These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Aβ_42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/γ-secretase may provide insight into molecular mechanism of Aβ₄₂ generation, which could be exploited therapeutically.     

The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species

The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase.     

Traditional Chinese Nootropic Medicine Radix Polygalae and Its Active Constituent Onjisaponin B Reduce β-Amyloid Production and Improve Cognitive Impairments

Decline of cognitive function is the hallmark of Alzheimer’s disease (AD), regardless of the pathological mechanism. Traditional Chinese medicine has been used to combat cognitive impairments and has been shown to improve learning and memory. Radix Polygalae (RAPO) is a typical and widely used herbal medicine. In this study, we aimed to follow the β-amyloid (Aβ) reduction activity to identify active constituent(s) of RAPO. We found that Onjisaponin B of RAPO functioned as RAPO to suppress Aβ production without direct inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase activities. Our mechanistic study showed that Onjisaponin B promoted the degradation of amyloid precursor protein (APP). Further, oral administration of Onjisaponin B ameliorated Aβ pathology and behavioral defects in APP/PS1 mice. Taken together, our results indicate that Onjisaponin B is effective against AD, providing a new therapeutic agent for further drug discovery.     

The Novel Membrane Protein TMEM59 Modulates Complex Glycosylation,Cell Surface Expression,and Secretion of the Amyloid Precursor Protein

Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases α- and β-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid β peptide (Aβ). At present, little is known about the cellular mechanisms that control APP shedding and Aβ generation. Here, we identified a novel protein, transmembrane protein 59 (TMEM59), as a new modulator of APP shedding. TMEM59 was found to be a ubiquitously expressed, Golgi-localized protein. TMEM59 transfection inhibited complex N- and O-glycosylation of APP in cultured cells. Additionally, TMEM59 induced APP retention in the Golgi and inhibited Aβ generation as well as APP cleavage by α- and β-secretase cleavage, which occur at the plasma membrane and in the endosomes, respectively. Moreover, TMEM59 inhibited the complex N-glycosylation of the prion protein, suggesting a more general modulation of Golgi glycosylation reactions. Importantly, TMEM59 did not affect the secretion of soluble proteins or the α-secretase like shedding of tumor necrosis factor α, demonstrating that TMEM59 did not disturb the general Golgi function. The phenotype of TMEM59 transfection on APP glycosylation and shedding was similar to the one observed in cells lacking conserved oligomeric Golgi (COG) proteins COG1 and COG2. Both proteins are required for normal localization and activity of Golgi glycosylation enzymes. In summary, this study shows that TMEM59 expression modulates complex N- and O-glycosylation and suggests that TMEM59 affects APP shedding by reducing access of APP to the cellular compartments, where it is normally cleaved by α- and β-secretase.     

γ-Secretase Dependent Production of Intracellular Domains Is Reduced in Adult Compared to Embryonic Rat Brain Membranes

Background

γ-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid β-peptide (Aβ) and the APP intracellular domain. Aβ is widely believed to have a causative role in Alzheimer''s disease pathogenesis, and therefore modulation of γ-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of γ-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to γ-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs).

Principal Findings

Here, we investigated γ-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Aβ40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Aβ were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Aβ40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased.

Conclusions

In summary our data suggests that γ-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of γ-secretase inhibitors.     

Apolipoprotein A-I Deficiency Increases Cerebral Amyloid Angiopathy and Cognitive Deficits in APP/PS1ΔE9 Mice

A hallmark of Alzheimer disease (AD) is the deposition of amyloid β (Aβ) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases Aβ₄₀ aggregation and toxicity. Here we demonstrate that apoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of Aβ₄₂ and decreases Aβ₄₂ toxicity in primary brain cells. To determine the effects of apoA-I on AD phenotype in vivo, we crossed APP/PS1ΔE9 to apoA-I^KO mice. Using a Morris water maze, we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1ΔE9 mice. Further characterization of APP/PS1ΔE9/apoA-I^KO mice showed that apoA-I deficiency did not affect amyloid precursor protein processing, soluble Aβ oligomer levels, Aβ plaque load, or levels of insoluble Aβ in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble Aβ isolated from cerebral blood vessels. Our data show that in APP/PS1ΔE9/apoA-I^KO mice, insoluble Aβ₄₀ is increased more than 10-fold, and Aβ₄₂ is increased 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1ΔE9/apoA-I^KO mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated apoA-I significantly decreased Aβ toxicity against brain vascular smooth muscle cells. We conclude that lack of apoA-I aggravates the memory deficits in APP/PS1ΔE9 mice in parallel to significantly increased cerebral amyloid angiopathy.     

A TAG on to the neurogenic functions of APP

The proteolytic processing of amyloid β precursor protein (APP) has long been studied because of its association with the pathology of Alzheimer''s disease (AD). The ectodomain of APP is shed by α- or β-secretase cleavage. The remaining membrane bound stub can then undergo regulated intramembrane proteolysis (RIP) by γ-secretase. This cleavage can release amyloid β (Aβ) from the stub left by β-secretase cleavage but also releases the APP intracellular domain (AICD) after α- or β-secretase cleavage. The physiological functions of this proteolytic processing are not well understood. We compare the proteolytic processing of APP to the ligand-dependent RIP of Notch. In this review, we discuss recent evidence suggesting that TAG1 is a functional ligand for APP. The interaction between TAG1 and APP triggers γ-secretase-dependent release of AICD. TAG1, APP and Fe65 colocalise in the neurogenic ventricular zone and in fetal neural progenitor cells in vitro. Experiments in TAG1, APP and Fe65 null mice as well as TAG1 and APP double-null mice demonstrate that TAG1 induces a γ-secretase- and Fe65-dependent suppression of neurogenesis.Key words: Amyloid β precursor protein, APP, TAG1, AICD, Fe65, neurogenesis, Alzheimer''s disease     

Rheb GTPase Regulates β-Secretase Levels and Amyloid β Generation

The β-site amyloid precursor protein (APP)-cleaving enzyme 1 (β-secretase, BACE1) initiates amyloidogenic processing of APP to generate amyloid β (Aβ), which is a hallmark of Alzheimer disease (AD) pathology. Cerebral levels of BACE1 are elevated in individuals with AD, but the molecular mechanisms are not completely understood. We demonstrate that Rheb GTPase (Ras homolog enriched in brain), which induces mammalian target of rapamycin (mTOR) activity, is a physiological regulator of BACE1 stability and activity. Rheb overexpression depletes BACE1 protein levels and reduces Aβ generation, whereas the RNAi knockdown of endogenous Rheb promotes BACE1 accumulation, and this effect by Rheb is independent of its mTOR signaling. Moreover, GTP-bound Rheb interacts with BACE1 and degrades it through proteasomal and lysosomal pathways. Finally, we demonstrate that Rheb levels are down-regulated in the AD brain, which is consistent with an increased BACE1 expression. Altogether, our study defines Rheb as a novel physiological regulator of BACE1 levels and Aβ generation, and the Rheb-BACE1 circuitry may have a role in brain biology and disease.