Proteolytic Processing Of Amyloid Precursor Protein Research Articles

Apicidin attenuates memory deficits by reducing the Aβ load in APP/PS1 mice.

Amyloid beta (Aβ) is an important pathological feature of Alzheimer's disease (AD). A disintegrin and metalloproteinase 10 (ADAM10) can reduce the production of toxic Aβ by activating the nonamyloidogenic pathway of amyloid precursor protein (APP). We previously found that apicidin, which is a histone deacetylase (HDAC) inhibitor, can promote the expression of ADAM10 and reduce the production of Aβ in vitro. This study was designed to determine the potential of apicidin treatment to reverse learning and memory impairments in an AD mouse model and the possible correlation of these effects with ADAM10. Nine-month-old APP/PS1 mice and C57 mice received intraperitoneal injections of apicidin or vehicle for 2months. At 11 months of age, we evaluated the memory performance of mice with Morris water maze (MWM) and context fear conditioning tests. The Aβ levels were assessed in mouse brain using the immunohistochemical method and ELISA. The expression of corresponding protein involved in proteolytic processing of APP and the phosphorylation of tau were assessed by Western blotting. Apicidin reversed the deficits of spatial reference memory and contextual fear memory, attenuated the formation of Aβ-enriched plaques, and decreased the levels of soluble and insoluble Aβ40/42 in APP/PS1 mice. Moreover, apicidin significantly increased the expression of ADAM10, improved the level of sAPPα, and reduced the production of sAPPβ, but did not affect the levels of phosphorylated tau in APP/PS1 mice. Apicidin significantly improves the AD symptoms of APP/PS1 mice by regulating the expression of ADAM10, which may contribute to decreasing the levels of Aβ rather than decreasing the phosphorylation of tau.

Lipid Rafts and Amyloid Metabolism: Role in Pathogenesis of Alzheimer’s Disease

Brain lipids play an important role not only as ubiquitous structural membrane components providing the scaffolding and compartmentalisation outside and within the cells but also participating in various signalling processes either by facilitating them or by acting as signal molecules. Membrane lipids form highly specialised domains, called lipid rafts, which are more ordered structures than the rest of the membrane and are enriched in cholesterol and sphingolipids. These domains provide a platform for specific and targeted protein-lipid and protein-protein interactions and as such facilitate binding and/or enzymatic processes on the surface and within the membranes. These lipid-protein interactions are important for various signalling events and proper cell functioning. When normal structure and functions of lipid rafts is disturbed due to the changes in lipid metabolism, caused by various internal and environmental factors, it results in a cascade of pathological events. Among proteins whose metabolic pathways depend on the lipid raft structure and integrity is amyloid precursor protein (APP) – the protein highly implicated in the pathogenesis of Alzheimer’s disease (AD). Proteolytic processing of APP by a metalloproteinase called β-secretase (BACE1) and a multiprotein complex called γ-secretase results in production of the amyloid β peptide (Aβ) - one of the key molecules leading to development of AD. These events take place in the lipid rafts. Some lipid components of the rafts, including ganglioside GM1, facilitate Aβ aggregation and formation of its toxic oligomers. Understanding the mechanisms regulating lipid-protein interactions in the rafts might result in new therapeutic strategies and treatments. In this review we discuss the implications of lipids in APP processing and Aβ metabolism and possible therapeutic avenues derived from studying lipid raft structure and functions in normal and AD brain.

Phosphorylation Signaling in APP Processing in Alzheimer's Disease.

The abnormal accumulation of amyloid-β (Aβ) in the central nervous system is a hallmark of Alzheimer’s disease (AD). The regulation of the processing of the single- transmembrane amyloid precursor protein (APP) plays an important role in the generation of Aβ in the brain. The phosphorylation of APP and key enzymes involved in the proteolytic processing of APP has been demonstrated to be critical for modulating the generation of Aβ by either altering the subcellular localization of APP or changing the enzymatic activities of the secretases responsible for APP processing. In addition, the phosphorylation may also have an impact on the physiological function of these proteins. In this review, we summarize the kinases and signaling pathways that may participate in regulating the phosphorylation of APP and secretases and how this further affects the function and processing of APP and Aβ pathology. We also discuss the potential of approaches that modulate these phosphorylation-signaling pathways or kinases as interventions for AD pathology.

Therapeutic targeting of nuclear receptors, liver X and retinoid X receptors, for Alzheimer's disease.

After 15years of research into Alzheimer's disease (AD) therapeutics, including billions of US dollars provided by federal agencies, pharmaceutical companies, and private foundations, there are still no meaningful therapies that can delay the onset or slow the progression of AD. An understanding of the proteolytic processing of amyloid precursor protein (APP) and the hypothesis that pathogenic mechanisms in familial and sporadic forms of AD are very similar led to the assumption that pharmacological inhibition of secretases or immunological approaches to clear amyloid depositions in the brain would have been the core to drug discovery strategies and successful therapies. However, there are other understudied approaches including targeting genes, gene networks, and metabolic pathways outside the proteolytic processing of APP. The advancement of newly developed sequencing technologies and mass spectrometry, as well as the availability of animal models expressing human apolipoprotein E isoforms, has been critical in rationalizing additional AD therapeutics. The purpose of this review is to present one of those approaches, based on the role of ligand-activated nuclear liver X and retinoid X receptors in the brain. This therapeutic approach was initially proposed utilizing in vitro models 15years ago and has since been examined in numerous studies using AD-like mouse models. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.

Full-length amyloid precursor protein regulates lipoprotein metabolism and amyloid-β clearance in human astrocytes

Mounting evidence suggests that alterations in cholesterol homeostasis are involved in Alzheimer's disease (AD) pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by proteolytic processing of APP have previously been implicated in the regulation of cholesterol metabolism. However, the physiological function of APP in regulating lipoprotein homeostasis in astrocytes, which are responsible for de novo cholesterol biosynthesis and regulation in the brain, remains unclear. To address this, here we used CRISPR/Cas9 genome editing to generate isogenic APP-knockout (KO) human induced pluripotent stem cells (hiPSCs) and differentiated them into human astrocytes. We found that APP-KO astrocytes have reduced cholesterol and elevated levels of sterol regulatory element-binding protein (SREBP) target gene transcripts and proteins, which were both downstream consequences of reduced lipoprotein endocytosis. To elucidate which APP fragments regulate cholesterol homeostasis and to examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series harboring the APP Swedish and APP V717F variants. Only astrocytes homozygous for the APP Swedish (APPSwe/Swe) mutation, which had reduced full-length APP (FL APP) due to increased β-secretase cleavage, recapitulated the APP-KO phenotypes. Astrocytic internalization of β-amyloid (Aβ), another ligand for low-density lipoprotein (LDL) receptors, was also impaired in APP-KO and APPSwe/Swe astrocytes. Finally, impairing cleavage of FL APP through β-secretase inhibition in APPSwe/Swe astrocytes reversed the LDL and Aβ endocytosis defects. In conclusion, FL APP is involved in the endocytosis of LDL receptor ligands and is required for proper cholesterol homeostasis and Aβ clearance in human astrocytes.

The physical approximation of APP and BACE-1: A key event in alzheimer's disease pathogenesis.

Alzheimer's disease (AD) is characterized by the accumulation of insoluble deposits of Amyloid β (Aβ) in brains. Aβ is derived by sequential cleavage of the amyloid precursor protein (APP) by β-site secretase enzyme (BACE-1) and γ-secretase. Proteolytic processing of APP by BACE-1 is the rate-limiting step in Aβ production, and this pathway is a prime target for AD drug development. Both APP and BACE-1 are membrane-spanning proteins, transported via secretory and endocytic pathways; and the physical interaction of APP and BACE-1 during trafficking is a key cell biological event initiating the amyloidogenic pathway. Here, we highlight recent research on intracellular trafficking/sorting of APP and BACE-1, and discuss how dysregulation of these pathways might lead to enhanced convergence of APP and BACE-1, and subsequent β-cleavage of APP. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 340-347, 2018.

Trafficking in Alzheimer's Disease: Modulation of APP Transport and Processing by the Transmembrane Proteins LRP1, SorLA, SorCS1c, Sortilin, and Calsyntenin.

The amyloid precursor protein (APP), one key player in Alzheimer's disease (AD), is extensively processed by different proteases. This leads to the generation of diverging fragments including the amyloid β (Aβ) peptide, which accumulates in brains of AD patients. Subcellular trafficking of APP is an important aspect for its proteolytic conversion, since the various secretases which cleave APP are located in different cellular compartments. As a consequence, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The mechanisms underlying intracellular APP transport are critical to understand AD pathogenesis and can serve as a target for future pharmacological interventions. In the recent years, a number of APP interacting proteins were identified which are implicated in sorting of APP, thereby influencing APP processing at different angles of the secretory or endocytic pathway. This review provides an update on the proteolytic processing of APP and the interplay of the transmembrane proteins low-density lipoprotein receptor-related protein 1, sortilin-receptor with A-type repeats, SorCS1c, sortilin, and calsyntenin. We discuss the specific interactions with APP, the capacity to modulate the intracellular itinerary and the proteolytic conversion of APP, a possible involvement in the clearance of Aβ, and the implications of these transmembrane proteins in AD and other neurodegenerative diseases.

Direct imaging of APP proteolysis in living cells.

Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α- and the β-secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α-secretase cleavage without perturbing the β-secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

Mutants of metal binding site M1 in APP E2 show metal specific differences in binding of heparin but not of sorLA.

The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimer's disease (AD) and appear to be essential for neuronal development and cell homeostasis. Proteolytic processing of APP and its physiological function depend on its interaction with heparin and are influenced by the binding of metal ions and sorLA. We created various mutations of metal binding site M1 residing within the extracellular E2 domain of APP. Using isothermal titration calorimetry and circular dichroism spectroscopy, we analyzed the binding of Cu(2+) and Zn(2+) to APP E2 and identified two mutations that are most suited for functional studies to dissect ion specific effects of metal binding. The H313A mutation abrogates only copper-based effects, whereas the H382A mutation weakens any metal binding at M1 of APP E2. Subsequently, we tested the effect of Cu(2+) and Zn(2+) on the binding of heparin and sorLA to APP E2 using a chromatographic technique and surface plasmon resonance. We show that Zn(2+) and to a larger degree also Cu(2+) enhance the binding of heparin to APP E2, consistent with an extracellular regulation of the function of APP by both metal ions. In contrast, neither ion seemed to affect the interaction between APP E2 and sorLA. This supports an intracellular interaction between the latter two partners that would not sense extracellular variations of metal ions upon synaptic activity.

Perturbations of the Straight Transmembrane α-Helical Structure of the Amyloid Precursor Protein Affect Its Processing by γ-Secretase

The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase of its C-terminal fragment produces amyloid-β (Aβ) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight α-helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the γ-secretase enzyme, as suggested by a series of Gly(700) mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of Aβ38 compared with Aβ40 peptides, which is reminiscent of the effect of γ-secretase modulators inhibitors.

F-box Only Protein 2 (Fbxo2) Regulates Amyloid Precursor Protein Levels and Processing

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

FKBP12 regulates the localization and processing of amyloid precursor protein in human cell lines

One of the pathological hallmarks of Alzheimer's disease is the presence of insoluble extracellular amyloid plaques. These plaques are mainly constituted of amyloid beta peptide (A beta), a proteolytic product of amyloid precursor protein (APP). APP processing also generates the APP intracellular domain (AICD). We have previously demonstrated that AICD interacts with FKBP12, a peptidyl-prolyl cis-trans isomerase (PPIase) ubiquitous in nerve systems. This interaction was interfered by FK506, a clinically used immunosuppressant that has recently been reported to be neuroprotective. To elucidate the roles of FKBP12 in the pathogenesis of Alzheimer's disease, the effect of FKBP12 overexpression on APP processing was evaluated. Our results revealed that APP processing was shifted towards the amyloidogenic pathway, accompanied by a change in the subcellular localization of APP, upon FKBP12 overexpression. This FKBP12-overexpression-induced effect was reverted by FK506. These findings support our hypothesis that FKBP12 may participate in the regulation of APP processing. FKBP12 overexpression may lead to the stabilization of a certain isomer (presumably the cis form) of the Thr668-Pro669 peptide bond in AICD, therefore change its affinity to flotillin-1 or other raft-associated proteins, and eventually change the localization pattern and cause a shift in the proteolytic processing of APP.

The Amyloid Precursor Protein Maintains an Ideal&Alpha-Helical Conformation in the Lipid Bilayer

The Amyloid Precursor Protein (APP) is a type-I transmembrane glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase produces amyloid-β (A&β) peptides of different lengths, the deposition of which is an early indicator of Alzheimer's disease (AD). At present, there is no consensus on the conformation of the APP transmembrane (TM) domain at the biological membrane. Although structures have been determined by nuclear magnetic resonance (NMR) in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight helical conformation once embedded in a membrane bilayer. APP-TM is highly flexible however, and its secondary structure is strongly influenced by the surrounding lipid environment. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance (DEER) spectroscopy. These structural and dynamic features are probably critical in the proteolytic processing of APP by the γ-secretase enzyme, as suggested by mutants mimicking influencing the APP flexibility that shows a relevant increase in the production of A&β38 compared to physiological A&β40 peptides.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Metal Binding Dictates Conformation and Function of the Amyloid Precursor Protein (APP) E2 Domain

The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimer's disease and appear essential for neuronal development and cell homeostasis in mammals. Proteolytic processing of APP is influenced by metal ions, protein ligands and its oligomerization state. However, the structural basis and functional mechanism of APP regulation are hitherto largely unknown. Here we identified a metal-dependent molecular switch located within the E2 domain of APP containing four evolutionary highly conserved histidine residues. Three X-ray structures of the metal-bound molecule were solved at 2.6–2.0 Å resolution. Using protein crystallographic and biochemical methods, we characterized this novel high-affinity binding site within the E2 domain that binds competitively to copper and zinc at physiological concentrations. Metal-specific coordination spheres induce large conformational changes and enforce distinct structural states, most likely regulating the physiological function of APP and its processing in Alzheimer's disease.

Crystal Structure of the E2 Domain of Amyloid Precursor Protein-like Protein 1 in Complex with Sucrose Octasulfate

Missense mutations in the amyloid precursor protein (APP) gene can cause familial Alzheimer disease. It is thought that APP and APP-like proteins (APLPs) may play a role in adhesion and signal transduction because their ectodomains interact with components of the extracellular matrix. Heparin binding induces dimerization of APP and APLPs. To help explain how these proteins interact with heparin, we have determined the crystal structure of the E2 domain of APLP1 in complex with sucrose octasulfate (SOS). A total of three SOS molecules are bound to the E2 dimer. Two SOSs are bound inside a narrow intersubdomain groove, and the third SOS is bound near the two-fold axis of the protein. Mutational analyses show that most residues interacting with SOS also contribute to heparin binding, although in varying degrees; a deep pocket, defined by His-376, Lys-422, and Arg-429, and an interfacial site between Lys-314 and its symmetry mate are most important in the binding of the negatively charged polysaccharide. Comparison with a lower resolution APP structure shows that all key heparin binding residues are conserved and identically positioned, suggesting that APLP1 and APP may bind heparin similarly. In transfected HEK-293 cells, mutating residues responsible for heparin binding causes little change in the proteolysis of APP by the secretases. However, mutating a pair of conserved basic residues (equivalent to Arg-414 and Arg-415 of APLP1) immediately adjacent to the heparin binding site affects both the maturation and the processing of APP.

DrosophilaAmyloid Precursor Protein-Like Is Required for Long-Term Memory

The amyloid precursor protein (APP) plays an important role in Alzheimer's disease (AD), a progressive neurodegenerative pathology that first manifests as a decline of memory. While the main hypothesis for AD pathology centers on the proteolytic processing of APP, very little is known about the physiological function of the APP protein in the adult brain. Likewise, whether APP loss of function contributes to AD remains unclear. Drosophila has been used extensively as a model organism to study neuronal function and pathology. In addition, many of the molecular mechanisms underlying memory are thought to be conserved from flies to mammals, prompting us to study the function of APPL, the fly APP ortholog, during associative memory. It was previously shown that APPL expression is highly enriched in the mushroom bodies (MBs), a specialized brain structure involved in olfactory memory. We analyzed memory in flies in which APPL expression has been silenced specifically and transiently in the adult MBs. Our results show that in adult flies, APPL is not required for learning but is specifically involved in long-term memory, a long lasting memory whose formation requires de novo protein synthesis and is thought to require synaptic structural plasticity. These data support the hypothesis that disruption of normal APP function may contribute to early AD cognitive impairment.

γ-secretase processing of APLP1 leads to the production of a p3-like peptide that does not aggregate and is not toxic to neurons

The amyloid precursor-like protein-1 (APLP1) is a member of a protein family that includes the Alzheimer's disease-associated amyloid precursor protein (APP). While much is known about the proteolytic processing of APP, fewer details are available about APLP1. Using Chinese hamster ovarian cells stably transfected with human APLP1 and a novel juxtamembrane anti-APLP1 antibody, we demonstrate the detection of a secreted ∼ 3.5 kDa APLP1-derived peptide (ALP-1). The production of this peptide is abolished by inhibition of γ-secretase, but not β-secretase, suggesting that ALP-1 is analogous to the p3 fragment produced from APP. However, unlike p3 or Aβ, ALP-1 shows no obvious propensity for aggregation and is not toxic to neuronal cells. Moreover, using two distinct experimental paradigms, we demonstrate that neither cell-derived nor chemically synthesized ALP-1 influences the oligomerization or aggregation of Aβ.

Crystal Structure of the Human Fe65-PTB1 Domain

The neuronal adaptor protein Fe65 is involved in brain development, Alzheimer disease amyloid precursor protein (APP) signaling, and proteolytic processing of APP. It contains three protein-protein interaction domains, one WW domain, and a unique tandem array of phosphotyrosine-binding (PTB) domains. The N-terminal PTB domain (Fe65-PTB1) was shown to interact with a variety of proteins, including the low density lipoprotein receptor-related protein (LRP-1), the ApoEr2 receptor, and the histone acetyltransferase Tip60. We have determined the crystal structures of human Fe65-PTB1 in its apo- and in a phosphate-bound form at 2.2 and 2.7A resolution, respectively. The overall fold shows a PTB-typical pleckstrin homology domain superfold. Although Fe65-PTB1 has been classified on an evolutionary basis as a Dab-like PTB domain, it contains attributes of other PTB domain subfamilies. The phosphotyrosine-binding pocket resembles IRS-like PTB domains, and the bound phosphate occupies the binding site of the phosphotyrosine (Tyr(P)) within the canonical NPXpY recognition motif. In addition Fe65-PTB1 contains a loop insertion between helix alpha2 and strand beta2(alpha2/beta2 loop) similar to members of the Shc-like PTB domain subfamily. The structural comparison with the Dab1-PTB domain reveals a putative phospholipid-binding site opposite the peptide binding pocket. We suggest Fe65-PTB1 to interact with its target proteins involved in translocation and signaling of APP in a phosphorylation-dependent manner.

Involvement of myosin II‐B and protein kinase C in the processing of APP

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-β (Aβ) peptides cleaved from the amyloid precursor protein (APP). Although the proteolytic processing of APP has been implicated in the pathogenesis of AD, why and where this processing takes place in neurons remains unknown. Several studies support the hypothesis that APP functions as a vesicular receptor for cytoskeletal motor proteins. Our recent results strongly suggest that the trafficking of APP involves myosin II-B, important contributor to the cytoskeleton of neuronal cells (Massone et al., Biochem Biophys Res Commun. 2007 vol. 362 pp. 633–8). It is known that activation of protein kinase C (PKC) reduces secretion of Aβ favoring the non-amyloidogenic processing of APP. PKC phosphorylates myosin II-B, regulating the assembly of the motor protein and its shuttling between the cell cortex and the cytoplasm. These observations, together with our very recent results, are consistent with a model in which PKC-mediated phosphorylation of myosin II-B regulates shuttling of APP between the cell periphery and the perinuclear region, shifting APP away from the compartments where its amyloidogenic processing is favored or, alternatively, moving APP toward a non-amyloidogenic pathway. Work funded by Fondazione CARIGE (2007.0650-71) and PRIN (2006065711_002).

Inhibitors of Cathepsin B Improve Memory and Reduce β-Amyloid in Transgenic Alzheimer Disease Mice Expressing the Wild-type, but Not the Swedish Mutant, β-Secretase Site of the Amyloid Precursor Protein

Elucidation of Abeta-lowering agents that inhibit processing of the wild-type (WT) beta-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of beta-secretase activity in regulated secretory vesicles that produce beta-amyloid (Abeta). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT beta-secretase site but not the rare Swedish mutant beta-secretase site. In vivo treatment of London APP mice, expressing the WT beta-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Abeta40 and Abeta42, and reduced C-terminal beta-secretase fragment derived from APP by beta-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant beta-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Abeta in an AD animal model expressing the WT beta-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.