Cellular Membrane Fluidity in Amyloid Precursor Protein Processing

The senile plaque is a pathologic hallmark of Alzheimer's disease (AD). Amyloid-β peptide (Aβ), the main constituent of senile plaques, is neurotoxic especially in its oligomeric form. Aβ is derived from the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases in the amyloidogenic pathway. Alternatively, APP can be cleaved by α-secretases within the Aβ domain to produce neurotrophic and neuroprotective α-secretase-cleaved soluble APP (sAPPα) in the nonamyloidogenic pathway. Since APP and α-, β-, and γ-secretases are membrane proteins, APP processing should be highly dependent on the membrane composition and the biophysical properties of cellular membrane. In this review, we discuss the role of the biophysical properties of cellular membrane in APP processing, especially the effects of phospholipases A₂ (PLA₂s), fatty acids, cholesterol, and Aβ on membrane fluidity in relation to their effects on APP processing.     

Membrane rafts in Alzheimer's disease beta-amyloid production

Alzheimer's disease (AD), the most common age-associated dementing disorder, is pathologically manifested by progressive cognitive dysfunction concomitant with the accumulation of senile plaques consisting of amyloid-β (Aβ) peptide aggregates in the brain of affected individuals. Aβ is derived from a type I transmembrane protein, amyloid precursor protein (APP), by the sequential proteolytic events mediated by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Multiple lines of evidence have implicated cholesterol and cholesterol-rich membrane microdomains, termed lipid rafts in the amyloidogenic processing of APP. In this review, we summarize the cell biology of APP, β- and γ-secretases and the data on their association with lipid rafts. Then, we will discuss potential raft targeting signals identified in the secretases and their importance on amyloidogenic processing of APP.     

Sphingolipids: Critical players in Alzheimer's disease

Alzheimer's disease is characterized by the progressive accumulation of extracellular deposits of the amyloid β-peptide (Aβ) and intraneuronal aggregates of the microtubule associated protein tau. Strong genetic, biochemical and cell biological evidence indicates critical roles of Aβ in the initiation of the pathogenic process, while tau might mediate its toxicity and neurodegeneration. Aβ is generated by proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases. Alternatively, APP can also be cleaved by α-secretase within the Aβ domain, thereby precluding subsequent production of Aβ. APP and the three secretases are integral membrane proteins and follow secretory and endocytic trafficking pathways. Thus, the membrane lipid composition could play important roles in trafficking and metabolism of Alzheimer's disease related proteins. Sphingolipids and especially complex gangliosides are abundant and characteristic components of neuronal membranes. Together with cholesterol, they confer unique characteristics to membrane domains, thereby regulating subcellular trafficking and signaling pathways. Thus, sphingolipids emerged to important modulators of biological processes including cell growth, differentiation, and senescence. Defects in sphingolipid catabolism are long known to cause severe lysosomal storage disorders, often characterized by neurological phenotypes. In recent studies it became evident that impaired sphingolipid metabolism could also be involved in Alzheimer's disease.     

Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies

Alzheimer's disease (AD) is the most common type of dementia in elderly people. Senile plaques, a pathologic hallmark of AD, are composed of amyloid β peptide (Aβ). Aβ aggregation produces toxic oligomers and fibrils, causing neuronal dysfunction and memory loss. Aβ is generated from two sequential proteolytic cleavages of a membrane protein, amyloid precursor protein (APP), by β- and γ-secretases. The transmembrane (TM) domain of APP, APPTM, is the substrate of γ-secretase for Aβ production. The interaction between APPTM and γ-secretase determines the production of different species of Aβ. Although numerous experimental and theoretical studies of APPTM structure exist, experimental 3D structure of APPTM has not been obtained at atomic resolution. Using the pETM41 vector, we successfully expressed an MBP-APPTM fusion protein. By combining Ni-NTA chromatography, TEV protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically-labeled APPTM for NMR studies. The reconstitution of APPTM into micelles yielded high quality 2D (15)N-(1)H HSQC spectra. This reliable method for APPTM expression and purification lays a good foundation for future structural studies of APPTM using NMR.     

Trafficking regulation of proteins in Alzheimer’s disease

The β-amyloid (Aβ) peptide has been postulated to be a key determinant in the pathogenesis of Alzheimer’s disease (AD). Aβ is produced through sequential cleavage of the β-amyloid precursor protein (APP) by β- and γ-secretases. APP and relevant secretases are transmembrane proteins and traffic through the secretory pathway in a highly regulated fashion. Perturbation of their intracellular trafficking may affect dynamic interactions among these proteins, thus altering Aβ generation and accelerating disease pathogenesis. Herein, we review recent progress elucidating the regulation of intracellular trafficking of these essential protein components in AD.     

Synthesis of a potent photoreactive acidic γ-secretase modulator for target identification in cells

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimer´s disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Compounds named γ-secretase modulators (GSMs) can shift the substrate cleavage specificity of γ-secretase toward the production of non-amyloidogenic, shorter Aβ fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of γ-secretase), supporting a mode of action involving binding to γ-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in γ-secretase proteolytic specificity should pave the way for the development of improved drugs against AD.     

Generation of Alzheimer disease-associated amyloid β42/43 peptide by γ-secretase can be inhibited directly by modulation of membrane thickness

Pathogenic generation of amyloid β-peptide (Aβ) by sequential cleavage of β-amyloid precursor protein (APP) by β- and γ-secretases is widely believed to causally underlie Alzheimer disease (AD). β-Secretase initially cleaves APP thereby generating a membrane-bound APP C-terminal fragment, from which γ-secretase subsequently liberates 37-43-amino acid long Aβ species. Although the latter cleavages are intramembranous and although lipid alterations have been implicated in AD, little is known of how the γ-secretase-mediated release of the various Aβ species, in particular that of the pathogenic longer variants Aβ(42) and Aβ(43), is affected by the lipid environment. Using a cell-free system, we have directly and systematically investigated the activity of γ-secretase reconstituted in defined model membranes of different thicknesses. We found that bilayer thickness is a critical parameter affecting both total activity as well as cleavage specificity of γ-secretase. Whereas the generation of the pathogenic Aβ(42/43) species was markedly attenuated in thick membranes, that of the major and rather benign Aβ(40) species was enhanced. Moreover, the increased production of Aβ(42/43) by familial AD mutants of presenilin 1, the catalytic subunit of γ-secretase, could be substantially lowered in thick membranes. Our data demonstrate an effective modulation of γ-secretase activity by membrane thickness, which may provide an approach to lower the generation of the pathogenic Aβ(42/43) species.     

The role of membrane trafficking in the processing of amyloid precursor protein and production of amyloid peptides in Alzheimer's disease

Alzheimer's disease (AD) is characterized by progressive accumulation of misfolded proteins, which form senile plaques and neurofibrillary tangles, and the release of inflammatory mediators by innate immune responses. β-Amyloid peptide (Aβ) is derived from sequential processing of the amyloid precursor protein (APP) by membrane-bound proteases, namely the β-secretase, BACE1, and γ-secretase. Membrane trafficking plays a key role in the regulation of APP processing as both APP and the processing secretases traffic along distinct pathways. Genome wide sequencing studies have identified several AD susceptibility genes which regulate membrane trafficking events. To understand the pathogenesis of AD it is critical that the cell biology of APP and Aβ production in neurons is well defined. This review discusses recent advances in unravelling the membrane trafficking events associated with the production of Aβ, and how AD susceptible alleles may perturb the sorting and transport of APP and BACE1. Mechanisms whereby inflammation may influence APP processing are also considered.     

Presenilins: how much more than γ-secretase?!

AD (Alzheimer's disease) is a neurodegenerative disease characterized by a gradual loss of neurons and the accumulation of neurotoxic Aβ (amyloid β-peptide) and hyperphosphorylated tau. The discovery of mutations in three genes, PSEN1 (presenilin 1), PSEN2 (presenilin 2) and APP (amyloid precursor protein), in patients with FAD (familial AD) has made an important contribution towards an understanding of the disease aetiology; however, a complete molecular mechanism is still lacking. Both presenilins belong to the γ-secretase complex, and serve as the catalytic entity needed for the final cleavage of APP into Aβ. PSEN only functions within the γ-secretase complex through intra- and inter-molecular interactions with three other membrane components, including nicastrin, Aph-1 (anterior pharynx defective-1) and Pen-2 (PSEN enhancer-2). However, although the list of γ-secretase substrates is still expanding, other non-catalytic activities of presenilins are also increasing the complexity behind its molecular contribution towards AD. These γ-secretase-independent roles are so far mainly attributed to PSEN1, including the transport of membrane proteins, cell adhesion, ER (endoplasmic reticulum) Ca(2+) regulation and cell signalling. In the present minireview, we discuss the current understanding of the γ-secretase-independent roles of PSENs and their possible implications in respect of AD.     

The Swedish dilemma - the almost exclusive use of APPswe-based mouse models impedes adequate evaluation of alternative β-secretases

Alzheimer's disease (AD) is the most common form of dementia, however incurable so far. It is widely accepted that aggregated amyloid β (Aβ) peptides play a crucial role for the pathogenesis of AD, as they cause neurotoxicity and deposit as so-called Aβ plaques in AD patient brains. Aβ peptides derive from the amyloid precursor protein (APP) upon consecutive cleavage at the β- and γ-secretase site. Hence, mutations in the APP gene are often associated with autosomal dominant inherited AD. Almost thirty years ago, two mutations at the β-secretase site were observed in two Swedish families (termed Swedish APP (APPswe) mutations), which led to early-onset AD. Consequently, APPswe was established in almost every common AD mouse model, as it contributes to early Aβ plaque formation and cognitive impairments. Analyzing these APPswe-based mouse models, the aspartyl protease BACE1 has been evolving as the prominent β-secretase responsible for Aβ release in AD and as the most important therapeutic target for AD treatment. However, with respect to β-secretase processing, the very rare occurring APPswe variant substantially differs from wild-type APP. BACE1 dominates APPswe processing resulting in the release of Aβ1-x, whereas N-terminally truncated Aβ forms are scarcely generated. However, these N-terminally truncated Aβ species such as Aβ2-x, Aβ3-x and Aβ4-x are elevated in AD patient brains and exhibit an increased potential to aggregate compared to Aβ1-x peptides. Proteases such as meprin β, cathepsin B and ADAMTS4 were identified as alternative β-secretases being capable of generating these N-terminally truncated Aβ species from wild-type APP. However, neither meprin β nor cathepsin B are capable of generating N-terminally truncated Aβ peptides from APPswe. Hence, the role of BACE1 for the Aβ formation during AD might be overrepresented through the excessive use of APPswe mouse models. In this review we critically discuss the consideration of BACE1 as the most promising therapeutic target. Shifting the focus of AD research towards alternative β secretases might unveil promising alternatives to BACE1 inhibitors constantly failing in clinical trials due to ineffectiveness and harmful side effects.     

Opposite effects of P2X7 and P2Y2 nucleotide receptors on α-secretase-dependent APP processing in Neuro-2a cells

The amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases to release amyloid-β peptide (Aβ), the main component found in senile plaques of Alzheimer's disease (AD) patient brains. Alternatively, APP can be cleaved within the Aβ sequence by α-secretase, thus precluding the generation of Aβ. We have demonstrated that activation of the P2X7 receptor leads to a reduction of α-secretase activity in Neuro-2a cells. Moreover, the P2X7 ligand 2'(3')-O-(4-benzoylbenzoyl) ATP (BzATP) can also activate a different P2 receptor in these cells. This receptor, whose pharmacology resembles that of the P2Y(2) receptor, has an opposite effect, leading to increases in α-secretase activity. Our study suggests that P2X7R and P2Y(2)R could be novel therapeutic targets in AD.     

The role of G protein-coupled receptors in the pathology of Alzheimer's disease

G protein-coupled receptors (GPCRs) are involved in numerous key neurotransmitter systems in the brain that are disrupted in Alzheimer's disease (AD). GPCRs also directly influence the amyloid cascade through modulation of the α-, β- and γ-secretases, proteolysis of the amyloid precursor protein (APP), and regulation of amyloid-β degradation. Additionally, amyloid-β has been shown to perturb GPCR function. Emerging insights into the mechanistic link between GPCRs and AD highlight the potential of this class of receptors as a therapeutic target for AD.     

MEK inhibitors suppress β-amyloid production by altering the level of a β-C-terminal fragment of amyloid precursor protein in neuronal cells

β-Amyloid peptide (Aβ) is generated via sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases. Cell-based screening experiments disclosed that the MEK (MAP kinase kinase) inhibitors, U0126 and PD184352, suppress Aβ secretion from human neuronal SH-SY5Y cells expressing Swedish mutant APP. These inhibitors did not affect the cellular levels of APP but significantly reduced those of the APP β-C-terminal fragment (β-CTF). Additionally, β-CTF levels were markedly reduced by these inhibitors in cells expressing the fragment in a γ-secretase-independent and proteasome-dependent manner. Our results suggest that MEK inhibitors reduce Aβ generation via secretase-independent alteration of β-CTF levels.     

Proteomic identification of specifically carbonylated brain proteins in APP(NLh)/APP(NLh) × PS-1(P264L)/PS-1(P264L) human double mutant knock-in mice model of Alzheimer disease as a function of age

Alzheimer disease (AD) is the most common type of dementia and is characterized pathologically by the presence of neurofibrillary tangles (NFTs), senile plaques (SPs), and loss of synapses. The main component of SP is amyloid-beta peptide (Aβ), a 39 to 43 amino acid peptide, generated by the proteolytic cleavage of amyloid precursor protein (APP) by the action of beta- and gamma-secretases. The presenilins (PS) are components of the γ-secretase, which contains the protease active center. Mutations in PS enhance the production of the Aβ42 peptide. To date, more than 160 mutations in PS1 have been identified. Many PS mutations increase the production of the β-secretase-mediated C-terminal (CT) 99 amino acid-long fragment (CT99), which is subsequently cleaved by γ-secretase to yield Aβ peptides. Aβ has been proposed to induce oxidative stress and neurotoxicity. Previous studies from our laboratory and others showed an age-dependent increase in oxidative stress markers, loss of lipid asymmetry, and Aβ production and amyloid deposition in the brain of APP/PS1 mice. In the present study, we used APP (NLh)/APP(NLh) × PS-1(P246L)/PS-1(P246L) human double mutant knock-in APP/PS-1 mice to identify specific targets of brain protein carbonylation in an age-dependent manner. We found a number of proteins that are oxidatively modified in APP/PS1 mice compared to age-matched controls. The relevance of the identified proteins to the progression and pathogenesis of AD is discussed.     

Proteases and lipoprotein receptors in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of senile dementia, and is a complex disorder. The pathological hallmarks of AD were discovered by Dr. Alois Alzheimer in 1907, and include deposits of amyloid or senile plaques and neurofibrillar tangles. Plaques are composed of a peptide, termed the Abeta peptide, that is derived by proteolytic processing of the amyloid precursor protein (APP), while neurofibrillar tangles result from a hyperphosphorylation of the tau protein. Mechanisms associated with the formation of plaques and neurofibrillar tangles and their respective contributions to the disease process have been intensely investigated. Proteolytic processing of APP that results in the generation of the Abeta peptide is now well understood and is influenced by several proteins. Recent evidence suggests that the Abeta levels are carefully regulated, and several proteases play an important role in removing the Abeta peptide. Finally, it is becoming apparent that several members of the LDL receptor family play important roles in the brain, and may modulate the course of AD.     

Convergence of Amyloid-β and Tau Pathologies on Mitochondria In Vivo

The histopathological characteristics of Alzheimer’s disease (AD) are amyloid-β (Aβ) containing plaques and neurofibrillary tangles (NFTs) as well as neuronal and synaptic loss. Until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. There is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several APP and tau transgenic mouse models. Recently, we examined mitochondrial function in a novel triple transgenic mouse model (pR5/APP/PS2)—^tripleAD mice—that combines both pathologic features of the disease in brain. Using comparative, quantitative proteomics (iTRAQ) and mass spectroscopy, we found a massive deregulation of 24 proteins, of which one third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Remarkably, deregulation of complex I was related to tau, whereas deregulation of complex IV was Aβ dependent, both at the protein and activity levels. The ^tripleAD mice showed synergistic effects of Aβ and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. At 12 months, the strongest defects on OXPHOS, synthesis of ATP and reactive oxygen species, were exhibited in the ^tripleAD mice, again emphasizing synergistic, age-associated effects of Aβ and tau in impairing mitochondria. This review highlights the convergence of Aβ and tau on mitochondria and establishes a molecular link in AD pathology in vivo.     

Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations

The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the ratio of the aggregation-prone 42-residue form (Aβ42) to the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, γ-secretase produces a range of Aβ peptides, the result of initial cutting at the ε site to form Aβ48 or Aβ49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aβ42:Aβ40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides.