Sphingolipid storage impairs autophagic clearance of Alzheimer-associated proteins

Recent work from our laboratory demonstrates that the accumulation of sphingolipids (SLs) decreases the capacity of cells to clear potentially amyloidogenic fragments of the amyloid precursor protein (APP) during autophagy. APP is a type I membrane protein and could undergo sequential proteolytic processing by β- and γ-secretase resulting in the generation of the amyloid β-peptide (Aβ). Genetic, molecular and biochemical evidence indicates that the accumulation of toxic Aβ aggregates plays a critical role in the degeneration of neurons during the pathogenesis of Alzheimer disease (AD). Thus, SL storage could promote the accumulation of Ab in endosomal and lysosomal compartments and thereby induce characteristic cytopathological changes of AD.     

Cross-talk of membrane lipids and Alzheimer-related proteins

Alzheimer’s disease (AD) is neuropathologically characterized by the combined occurrence of extracellular β-amyloid plaques and intracellular neurofibrillary tangles in the brain. While plaques contain aggregated forms of the amyloid β-peptide (Aβ), tangles are formed by fibrillar forms of the microtubule associated protein tau. All mutations identified so far to cause familial forms of early onset AD (FAD) are localized close to or within the Aβ domain of the amyloid precursor protein (APP) or in the presenilin proteins that are essential components of a protease complex involved in the generation of Aβ. Mutations in the tau gene are not associated with FAD, but can cause other forms of dementia. The genetics of FAD together with biochemical and cell biological data, led to the formulation of the amyloid hypothesis, stating that accumulation and aggregation of Aβ is the primary event in the pathogenesis of AD, while tau might mediate its toxicity and neurodegeneration.The generation of Aβ involves sequential proteolytic cleavages of the amyloid precursor protein (APP) by enzymes called β-and γ-secretases. Notably, APP itself as well as the secretases are integral membrane proteins. Thus, it is very likely that membrane lipids are involved in the regulation of subcellular transport, activity, and metabolism of AD related proteins.Indeed, several studies indicate that membrane lipids, including cholesterol and sphingolipids (SLs) affect Aβ generation and aggregation. Interestingly, APP and other AD associated proteins, including β-and γ-secretases can, in turn, influence lipid metabolic pathways. Here, we review the close connection of cellular lipid metabolism and AD associated proteins and discuss potential mechanisms that could contribute to initiation and progression of AD.     

7-Deoxy-trans-dihydronarciclasine Reduces β-Amyloid and Ameliorates Memory Impairment in a Transgenic Model of Alzheimer’s Disease

The critical pathological feature of Alzheimer’s disease (AD) is the accumulation of β-amyloid (Aβ), the main constituent of amyloid plaques. β-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by β- and γ-secretase generating Aβ at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aβ at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aβ accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aβ production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aβ generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aβ, sAPPβ, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aβ and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.     

Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence.

A distinguishing feature of Alzheimer's disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4-amyloid peptide and the intracellular accumulation of potential amyloidogenic and non-amyloidogenic fragments. The relative amount of amyloid-containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. 'Humanizing' the rodent APP sequence by introducing three mutations in the beta A4-region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4-peptide to non-amyloidogenic p3-peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed-back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Flavonols and flavones as BACE-1 inhibitors: Structure–activity relationship in cell-free,cell-based and in silico studies reveal novel pharmacophore features

Generation and accumulation of the amyloid β peptide (Aβ) following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 (Beta-site APP Cleaving Enzyme-1, β-secretase) and γ-secretase is a main causal factor of Alzheimer's disease (AD). Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Aβ, is an attractive therapeutic approach for the treatment of AD. In this study, we discovered that natural flavonoids act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted Aβ in primary cortical neurons. In addition, we demonstrated the calculated docking poses of flavonoids to BACE-1 and revealed the interactions of flavonoids with the BACE-1 catalytic center. We firstly revealed novel pharmacophore features of flavonoids by using cell-free, cell-based and in silico docking studies. These results contribute to the development of new BACE-1 inhibitors for the treatment of AD.     

APP processing induced by herpes simplex virus type 1 (HSV-1) yields several APP fragments in human and rat neuronal cells

Lifelong latent infections of the trigeminal ganglion by the neurotropic herpes simplex virus type 1 (HSV-1) are characterized by periodic reactivation. During these episodes, newly produced virions may also reach the central nervous system (CNS), causing productive but generally asymptomatic infections. Epidemiological and experimental findings suggest that HSV-1 might contribute to the pathogenesis of Alzheimer's disease (AD). This multifactorial neurodegenerative disorder is related to an overproduction of amyloid beta (Aβ) and other neurotoxic peptides, which occurs during amyloidogenic endoproteolytic processing of the transmembrane amyloid precursor protein (APP). The aim of our study was to identify the effects of productive HSV-1 infection on APP processing in neuronal cells. We found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons is followed by multiple cleavages of APP, which result in the intra- and/or extra-cellular accumulation of various neurotoxic species. These include: i) APP fragments (APP-Fs) of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aβ; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aβ(1-40) and Aβ(1-42). Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aβ oligomer. The multiple cleavages of APP that occur in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell β-secretase, γ-secretase, and caspase-3-like enzymes. These findings demonstrate that HSV-1 infection of neuronal cells can generate multiple APP fragments with well-documented neurotoxic potentials. It is tempting to speculate that intra- and extracellular accumulation of these species in the CNS resulting from repeated HSV-1 reactivation could, in the presence of other risk factors, play a co-factorial role in the development of AD.     

Early Presymptomatic Changes in the Proteome of Mitochondria-Associated Membrane in the APP/PS1 Mouse Model of Alzheimer’s Disease

Intracellular β-amyloid (Aβ) accumulation is an early event in Alzheimer’s disease (AD) progression. Recently, it has been uncovered that presenilins (PSs), the key components of the amyloid precursor protein (APP) processing and the β-amyloid producing γ-secretase complex, are highly enriched in a special sub-compartment of the endoplasmic reticulum (ER) functionally connected to mitochondria, called mitochondria-associated ER membrane (MAM). A current hypothesis of pathogenesis of Alzheimer’s diseases (AD) suggests that MAM is involved in the initial phase of AD. Since MAM supplies mitochondria with essential proteins, the increasing level of PSs and β-amyloid could lead to metabolic dysfunction because of the impairment of ER-mitochondrion crosstalk. To reveal the early molecular changes of this subcellular compartment in AD development MAM fraction was isolated from the cerebral cortex of 3 months old APP/PS1 mouse model of AD and age-matched C57BL/6 control mice, then mass spectrometry-based quantitative proteome analysis was performed. The enrichment and purity of MAM preparations were validated with EM, LC-MS/MS and protein enrichment analysis. Label-free LC-MS/MS was used to reveal the differences between the proteome of the transgenic and control mice. We obtained 77 increased and 49 decreased protein level changes in the range of ??6.365 to +?2.988, which have mitochondrial, ER or ribosomal localization according to Gene Ontology database. The highest degree of difference between the two groups was shown by the ATP-binding cassette G1 (Abcg1) which plays a crucial role in cholesterol metabolism and suppresses Aβ accumulation. Most of the other protein changes were associated with increased protein synthesis, endoplasmic-reticulum-associated protein degradation (ERAD), oxidative stress response, decreased mitochondrial protein transport and ATP production. The interaction network analysis revealed a strong relationship between the detected MAM protein changes and AD. Moreover, it explored several MAM proteins with hub position suggesting their importance in Aβ induced early MAM dysregulation. Our identified MAM protein changes precede the onset of dementia-like symptoms in the APP/PS1 model, suggesting their importance in the development of AD.     

Studying the pathogenesis of Alzheimer’s disease in a <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> model: Human APP overexpression in the brain of transgenic flies leads to deficit of the synaptic protein synaptotagmin

Alzheimer’s disease (AD) is a progressive neurodegenerative disease whose main pathomorphological sign is synapse degeneration in the cortex and hippocampus. Abnormal synaptogenesis precedes amyloidosis and neurodegeneration and correlates with memory impairment during the early clinical phase. Mutations in the amyloid precursor protein (APP) gene cause familial AD and enhance the secretion of amyloid-β protein (Aβ). However, it remains unclear in what way APP and Aβ- are involved in synaptic disorder in the absence of visible amyloid structures. In this study, the role of the human APP gene in synaptogenesis in transgenic lines of Drosophila melanogaster whose nerve cells express the human APP695 isoform, truncated APPs, and the presynaptic marker synaptotagmin containing the green fluorescent protein (GFP) sequence. The expression of APP and its truncated forms caused a decrease in the synaptotagmin content of antennal lobes (ALs) and mushroom bodies (MBs) of the D. melanogaster brain, as well as neurodegeneration that progressed with age. The results suggest that abnormal synaptogenesis and neurodegeneration occur in the Drosophila brain in the absence of β-. It is assumed that impaired cellular functions of APP and secretion of β- independently contribute to the pathogenesis of AD.     

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.     

Distinct amyloid precursor protein processing machineries of the olfactory system

Processing of amyloid precursor protein (APP) occurs through sequential cleavages first by β-secretase and then by the γ-secretase complex. However, abnormal processing of APP leads to excessive production of β-amyloid (Aβ) in the central nervous system (CNS), an event which is regarded as a primary cause of Alzheimer's disease (AD). In particular, gene mutations of the γ-secretase complex—which contains presenilin 1 or 2 as the catalytic core—could trigger marked Aβ accumulation.Olfactory dysfunction usually occurs before the onset of typical AD-related symptoms (eg, memory loss or muscle retardation), suggesting that the olfactory system may be one of the most vulnerable regions to AD. To date however, little is known about why the olfactory system is affected so early by AD prior to other regions. Thus, we examined the distribution of secretases and levels of APP processing in the olfactory system under either healthy or pathological conditions.Here, we show that the olfactory system has distinct APP processing machineries. In particular, we identified higher expressions levels and activity of γ-secretase in the olfactory epithelium (OE) than other regions of the brain. Moreover, APP c-terminal fragments (CTF) are markedly detected. During AD progression, we note increased expression of presenilin2 of γ-secretases in the OE, not in the OB, and show that neurotoxic Aβ*56 accumulates more quickly in the OE.Taken together, these results suggest that the olfactory system has distinct APP processing machineries under healthy and pathological conditions. This finding may provide a crucial understanding of the unique APP-processing mechanisms in the olfactory system, and further highlights the correlation between olfactory deficits and AD symptoms.     

Structural Determinants of the Alzheimer's Amyloid β-Peptide

Abstract: The hallmark event of Alzheimer's disease (AD) is the deposition of amyloid as insoluble fiber masses in extracellular neuritic plaques and around the walls of cerebral blood vessels. The main component of amyloid is a hydrophobic peptide, named amyloid β-peptide (βA4), which results from the processing of a much longer membrane amyloid precursor protein (APP). This review focuses on the structural features of βA4 and the factors that determine βA4 insolubilization. Theoretical and experimental studies of the primary structure of βA4 have shown that it is composed of a completely hydrophobic C-terminal domain, which adopts β-strand structure, and an N-terminal region, whose sequence permits different secondary structures. In fact, this region can exist as an α-helical or β-strand conformation depending on the environmental condition (pH and hydrophobicity surrounding the molecule). The effects of pH and hydrophobicity on βA4 structure may elucidate the mechanisms determining its aggregation and amyloid deposition in AD.     

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD.     

Pivotal role of RanBP9 in integrin-dependent focal adhesion signaling and assembly

Accumulation of the amyloid β (Aβ) peptide derived from the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease (AD). We previously reported that the scaffolding protein RanBP9 is markedly increased in AD brains and promotes Aβ generation by scaffolding APP/BACE1/LRP complexes together and accelerating APP endocytosis. Because APP, LRP, and RanBP9 all physically interact with β-integrins, we investigated whether RanBP9 alters integrin-dependent cell adhesion and focal adhesion signaling. Here, we show that RanBP9 overexpression dramatically disrupts integrin-dependent cell attachment and spreading in NIH3T3 and hippocampus-derived HT22 cells, concomitant with strongly decreased Pyk2/paxillin signaling and talin/vinculin localization in focal adhesion complexes. Conversely, RanBP9 knockdown robustly promotes cell attachment, spreading, and focal adhesion signaling and assembly. Cell surface biotinylation and endocytosis assays reveal that RanBP9 overexpression and RanBP9 siRNA potently reduces and increases surface β1-integrin and LRP by accelerating and inhibiting their endocytosis, respectively. Primary hippocampal neurons derived from RanBP9-transgenic mice also demonstrate severely reduced levels of surface β1-integrin, LRP, and APP, as well as neurite arborization. Therefore, these data indicate that RanBP9 simultaneously inhibits cell-adhesive processes and enhances Aβ generation by accelerating APP, LRP, and β1-integrin endocytosis.     

Alzheimer’s disease and Notch signaling

Cleavage of the amyloid precursor protein (APP) by γ-secretase generates a neurotoxic amyloid β-peptide (Aβ) that is thought to be associated with the neurodegeneration observed in Alzheimer’s disease (AD) patients. Presenilin is the catalytic member of the γ-secretase proteolytic complex and mutations in presenilins are the major cause of early-onset familial Alzheimer’s disease. In addition to APP, γ-secretase substrates include Notch1 homologues, Notch ligands Delta and Jagged, and additional type I membrane proteins, raising concerns about mechanism-based toxicities that might arise as a consequence of inhibiting γ-secretase. Notch signaling is involved in tumorigenesis as well as in determining the fates of neural and nonneural cells during development and in adults. Alterations in proteolysis of the Notch by γ-secretase could be involved in the pathogenesis of AD. Inconsistently, several recent observations have indicated that enhanced Notch signaling and expression could be instrumental in neurodegeneration in AD. Therefore, detailed and precise study of Notch signaling in AD is important for elucidating diverse mechanisms of pathogenesis and potentially for treating and preventing Alzheimer’s disease.     

Lysophosphatidic acid induces increased BACE1 expression and Aβ formation

The abnormal production and accumulation of β-amyloid peptide (Aβ), which is produced from amyloid precursor protein (APP) by the sequential actions of β-secretase and γ-secretase, are thought to be the initial causative events in the development of Alzheimer's disease (AD). Accumulating evidence suggests that vascular factors play an important role in the pathogenesis of AD. Specifically, studies have suggested that one vascular factor in particular, oxidized low density lipoprotein (oxLDL), may play an important role in regulating Aβ formation in AD. However, the mechanism by which oxLDL modulates Aβ formation remains elusive. In this study, we report several new findings that provide biochemical evidence suggesting that the cardiovascular risk factor oxLDL may contribute to Alzheimer's disease by increasing Aβ production. First, we found that lysophosphatidic acid (LPA), the most bioactive component of oxLDL induces increased production of Aβ. Second, our data strongly indicate that LPA induces increased Aβ production via upregulating β-secretase expression. Third, our data strongly support the notion that different isoforms of protein kinase C (PKC) may play different roles in regulating APP processing. Specifically, most PKC members, such as PKCα, PKCβ, and PKCε, are implicated in regulating α-secretase-mediated APP processing; however, PKCδ, a member of the novel PKC subfamily, is involved in LPA-induced upregulation of β-secretase expression and Aβ production. These findings may contribute to a better understanding of the mechanisms by which the cardiovascular risk factor oxLDL is involved in Alzheimer's disease.