Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

Effect of Ischemic Neuronal Insults on Amyloid Precursor Protein Processing

The nature of the association between ischemic stroke and Alzheimer’s disease (AD) at the cellular and molecular level is still unknown. We evaluated the effect of ischemic neuronal insults on the regulation of amyloid precursor protein (APP) processing. We used an in vitro model of cerebral ischemia (oxygen-glucose deprivation) to evaluate the effect of ischemic neuronal insults on the amyloidogenic and non-amyloidogenic pathways using human neuroblastoma cell line and primary cultured cells of transgenic mice which expressed human APP (Tg2576). Ischemic neuronal insults increased the production of Aβ in Tg2576 primary culture cells compared to controls. A disintegrin and metalloprotease 10 (ADAM 10) was markedly increased in early stage of ischemic insults, which was followed by decreased level of ADAM 10 expression in later stage. The protein and mRNA expression of β-site cleavage enzyme (BACE) and BACE activity was not significantly different between the group of ischemic insults and control. By contrast, the activity of γ-secretase was significantly increased after 4 h of ischemic insults, as compared to controls. The present study showed that the ischemic neuronal insults increased the production of Aβ by influencing APP metabolism, which may link the role of ischemic insults to the pathogenesis of AD.     

Mild cholesterol depletion reduces amyloid-β production by impairing APP trafficking to the cell surface

It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and γ-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Aβ₄₀ and Aβ₄₂ in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP–Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP–PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of γ-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts.     

Differential effects of γ-secretase and BACE1 inhibition on brain Aβ levels in vitro and in vivo

Alzheimer's disease (AD) is hypothesized to result from elevated brain levels of β-amyloid peptide (Aβ) which is the main component of plaques found in AD brains and which cause memory impairment in mice. Therefore, there has been a major focus on the development of inhibitors of the Aβ producing enzymes γ-secretase and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1). In this study, we investigated the Aβ-lowering effects of the BACE1 inhibitor LY2434074 in vitro and in vivo , comparing it to the well characterized γ-secretase inhibitor LY450139. We sampled interstitial fluid Aβ from awake APPswe/PS1dE9 AD mice by in vivo Aβ microdialysis. In addition, we measured levels of endogenous brain Aβ extracted from wildtype C57BL/6 mice. In our in vitro assays both compounds showed similar Aβ-lowering effects. However, while systemic administration of LY450139 resulted in transient reduction of Aβ in both in vivo models, we were unable to show any Aβ-lowering effect by systemic administration of the BACE1 inhibitor LY2434074 despite brain exposure exceeding the in vitro IC₅₀ value several fold. In contrast, significant reduction of 40–50% of interstitial fluid Aβ and wildtype cortical Aβ was observed when infusing LY2434074 directly into the brain by means of reverse microdialysis or by dosing the BACE1 inhibitor to p-glycoprotein (p-gp) mutant mice. The effects seen in p-gp mutant mice and subsequent data from our cell-based p-gp transport assay suggested that LY2434074 is a p-gp substrate. This may partly explain why BACE1 inhibition by LY2434074 has lower in vivo efficacy, with respect to decreased Aβ40 levels, compared with γ-secretase inhibition by LY450139.     

N-cadherin-based adhesion enhances Aβ release and decreases Aβ42/40 ratio

In neurons, Presenilin 1(PS1)/γ-secretase is located at the synapses, bound to N-cadherin. We have previously reported that N-cadherin-mediated cell–cell contact promotes cell-surface expression of PS1/γ-secretase. We postulated that N-cadherin-mediated trafficking of PS1 might impact synaptic PS1-amyloid precursor protein interactions and Aβ generation. In the present report, we evaluate the effect of N-cadherin-based contacts on Aβ production. We demonstrate that stable expression of N-cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Aβ in the medium. Moreover, N-cadherin expression decreased Aβ_42/40 ratio. The effect of N-cadherin expression on Aβ production was accompanied by the enhanced accessibility of PS1/γ-secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N-cadherin-mediated synaptic adhesion may modulate Aβ secretion as well as the Aβ_42/40 ratio via PS1/N-cadherin interactions.     

Etazolate, a neuroprotective drug linking GABA(A) receptor pharmacology to amyloid precursor protein processing

Pharmacological modulation of the GABA_A receptor has gained increasing attention as a potential treatment for central processes affected in Alzheimer disease (AD), including neuronal survival and cognition. The proteolytic cleavage of the amyloid precursor protein (APP) through the α-secretase pathway decreases in AD, concurrent with cognitive impairment. This APP cleavage occurs within the β-amyloid peptide (Aβ) sequence, precluding formation of amyloidogenic peptides and leading to the release of the soluble N-terminal APP fragment (sAPPα) which is neurotrophic and procognitive. In this study, we show that at nanomolar-low micromolar concentrations, etazolate, a selective GABA_A receptor modulator, stimulates sAPPα production in rat cortical neurons and in guinea pig brains. Etazolate (20 nM–2 μM) dose-dependently protected rat cortical neurons against Aβ-induced toxicity. The neuroprotective effects of etazolate were fully blocked by GABA_A receptor antagonists indicating that this neuroprotection was due to GABA_A receptor signalling. Baclofen, a GABA_B receptor agonist failed to inhibit the Aβ-induced neuronal death. Furthermore, both pharmacological α-secretase pathway inhibition and sAPPα immunoneutralization approaches prevented etazolate neuroprotection against Aβ, indicating that etazolate exerts its neuroprotective effect via sAPPα induction. Our findings therefore indicate a relationship between GABA_A receptor signalling, the α-secretase pathway and neuroprotection, documenting a new therapeutic approach for AD treatment.     

An Etiological Role of Amyloidogenic Carboxyl-Terminal Fragments of the β-Amyloid Precursor Protein in Alzheimer's Disease

Abstract: Amyloid β protein (Aβ), 39–43 amino acids long, is the principal constituent of the extracellular amyloid deposits in brain that are characteristic of Alzheimer's disease (AD). Several lines of evidence indicate that Aβ may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of Aβ and the development of the disease. Thus, Aβ may not be the sole active fragment of β-amyloid precursor protein (βAPP) in the neurotoxicity associated with AD. Consequently, the possible effects of other cleaved products of βAPP need to be explored. The recent concentration on other potentially amyloidogenic products of βAPP has produced interesting candidates, the most promising of which are the amyloidogenic carboxyl-terminal (CT) fragments of βAPP. This review discusses a possible etiological role of CT fragments of βAPP in 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.     

Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

It has been suggested that cholesterol may modulate amyloid-β (Aβ) formation, a causative factor of Alzheimer’s disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (β-amyloid precursor protein (APP), β-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Aβ formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1^−/− cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, γ-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Aβ occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer’s disease and supports the role of lipid rafts in these processes.     

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β-protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro-blastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N-terminally truncated variant(s) Aβx-42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx-42, whereas extracellular levels of Aβ1–42 and Aβx-42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1 -induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx-42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx-42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific γ-secretase cleavage that occurs in the normal β-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β-secretase cleavage or (b) in the distinct sites where Aβx-42 and Aβ1–42 are generated.     

siRNA against presenilin 1 (PS1) down regulates amyloid β42 production in IMR-32 cells

Background  

One of the pathological hallmarks of Alzheimer's disease (AD) is the deposition of the ~4 kDa amyloid β protein (Aβ) within lesions known as senile plaques. Aβ is also deposited in the walls of cerebral blood vessels in many cases of AD. A substantial proportion of the Aβ that accumulates in the AD brain is deposited as Amyloid, which is highly insoluble, proteinaceous material with a β-pleated-sheet conformation and deposited extracellularly in the form of 5-10 nm wide straight fibrils. As γ-secretase catalyzes the final cleavage that releases the Aβ42 or 40 from amyloid β -protein precursor (APP), therefore, it is a potential therapeutic target for the treatment of AD. γ-Secretase cleavage is performed by a high molecular weight protein complex containing presenilins (PSs), nicastrin, Aph-1 and Pen-2. Previous studies have demonstrated that the presenilins (PS1 and PS2) are critical components of a large enzyme complex that performs γ-secretase cleavage.     

BACE-1 inhibition prevents the γ-secretase inhibitor evoked Aβ rise in human neuroblastoma SH-SY5Y cells

Background  

Accumulation of amyloid β-peptide (Aβ) in the plaques is one of the major pathological features in Alzheimer's disease (AD). Sequential cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE-1) and γ-secretase results in the formation of Aβ peptides. Preventing Aβ formation is believed to attenuate AD progression and BACE-1 and γ-secretase are thus attractive targets for AD drug development.     

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.     

Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

β-amyloid (Aβ) is the main constituent of senile plaques seen in Alzheimer's disease. Aβ is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases β- and β-secretase. In this study, we examined content and localization of β-secretase-cleaved APP (β-sAPP) in brain tissue sections from the frontal, temporal and occipital lobe. Strong granular β-sAPP staining was found throughout the gray matter of all three areas, while white matter staining was considerably weaker. β-sAPP was found to be localized in astrocytes and in axons. We found the β-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal β-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. β-sAPP was also found surrounding senile plaques and cerebral blood vessels. The results presented here show altered β-sAPP staining in the AD brain, suggestive of abnormal processing and transport of APP.     

��-Secretase Inhibitor Potency Is Decreased by Aberrant ��-Cleavage Location of the ��Swedish Mutant�� Amyloid Precursor Protein

The amyloid-β (Aβ) peptide, widely known as the causative molecule of Alzheimer disease (AD), is generated by the sequential cleavage of amyloid precursor protein (APP) by the aspartyl proteases BACE1/β-secretase and presenilin/γ-secretase. Inhibition of BACE1, therefore, is a promising strategy for preventing the progression of AD. However, β-secretase inhibitors (BSIs) exhibit unexpectedly low potency in cells expressing “Swedish mutant” APP (APPswe) and in the transgenic mouse Tg2576, an AD model overexpressing APPswe. The Swedish mutation dramatically accelerates β-cleavage of APP and hence the generation of Aβ; this acceleration has been assumed to underlie the poor inhibitory activity of BSI against APPswe processing. Here, we studied the mechanism by which the Swedish mutation causes this BSI potency decrease. Surprisingly, decreased BSI potency was not observed in an in vitro assay using purified BACE1 and substrates, indicating that the accelerated β-cleavage resulting from the Swedish mutation is not its underlying cause. By focusing on differences between the cell-based and in vitro assays, we have demonstrated here that the potency decrease is caused by the aberrant subcellular localization of APPswe processing and not by accelerated β-cleavage or the accumulation of the C-terminal fragment of β-cleaved APP. Because most patients with sporadic AD express wild type APP, our findings suggest that the wild type mouse is superior to the Tg2576 mouse as a model for determining the effective dose of BSI for AD patients. This work provides novel insights into the potency decrease of BSI and valuable suggestions for its development as a disease-modifying agent.     

γ-Secretase complexes containing caspase-cleaved presenilin-1 increase intracellular Aβ(42) /Aβ(40) ratio

Markers for caspase activation and apoptosis have been shown in brains of Alzheimer's disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid β-peptide (Aβ) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aβ(40) and Aβ(42) production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aβ(42) accompanied by increased intracellular Aβ(42) /Aβ(40) ratio without changing the Aβ secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aβ pattern with increased Aβ(42) /Aβ(40) ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aβ(42) /Aβ(40) ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents.     

Collapsin response mediator protein-2 hyperphosphorylation is an early event in Alzheimer's disease progression

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that can regulate microtubule assembly in neurons. This function of CRMP2 is regulated by phosphorylation by glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (Cdk5). Here, using novel phosphospecific antibodies, we demonstrate that phosphorylation of CRMP2 at Ser522 (Cdk5-mediated) is increased in Alzheimer's disease (AD) brain, while CRMP2 expression and phosphorylation of the closely related isoform CRMP4 are not altered. In addition, CRMP2 phosphorylation at the Cdk5 and GSK3 sites is increased in cortex and hippocampus of the triple transgenic mouse [presenilin-1 (PS1)(M146V)KI; Thy1.2-amyloid precursor protein (APP)(swe); Thy1.2tau(P301L)] that develops AD-like plaques and tangles, as well as the double (PS1(M146V)KI; Thy1.2-APP(swe)) transgenic mouse. The hyperphosphorylation is similar in magnitude to that in human AD and is evident by 2 months of age, ahead of plaque or tangle formation. Meanwhile, there is no change in CRMP2 phosphorylation in two other transgenic mouse lines that display elevated amyloid beta peptide levels (Tg2576 and APP/amyloid beta-binding alcohol dehydrogenase). Similarly, CRMP2 phosphorylation is normal in hippocampus and cortex of Tau(P301L) mice that develop tangles but not plaques. These observations implicate hyperphosphorylation of CRMP2 as an early event in the development of AD and suggest that it can be induced by a severe APP over-expression and/or processing defect.     

Lovastatin inhibits amyloid precursor protein (APP) beta-cleavage through reduction of APP distribution in Lubrol WX extractable low density lipid rafts

Previous studies have described that statins (inhibitors of cholesterol and isoprenoid biosynthesis) inhibit the output of amyloid-β (Aβ) in the animal model and thus decrease risk of Alzheimer's disease. However, their action mechanism(s) in Aβ precursor protein (APP) processing and Aβ generation is not fully understood. In this study, we report that lovastatin treatment reduced Aβ output in cultured hippocampal neurons as a result of reduced APP levels and β-secretase activities in low density Lubrol WX (non-ionic detergent) extractable lipid rafts (LDLR). Rather than altering cholesterol levels in lipid raft fractions and thus disrupting lipid raft structure, lovastatin decreased Aβ generation through down-regulating geranylgeranyl-pyrophosphate dependent endocytosis pathway. The inhibition of APP endocytosis by treatment with lovastatin and reduction of APP levels in LDLR fractions by treatment with phenylarsine oxide (a general endocytosis inhibitor) support the involvement of APP endocytosis in APP distribution in LDLR fractions and subsequent APP β-cleavage. Moreover, lovastatin-mediated down-regulation of endocytosis regulators, such as early endosomal antigen 1, dynamin-1, and phosphatidylinositol 3-kinase activity, indicates that lovastatin modulates APP endocytosis possibly through its pleiotropic effects on endocytic regulators. Collectively, these data report that lovastatin mediates inhibition of LDLR distribution and β-cleavage of APP in a geranylgeranyl-pyrophosphate and endocytosis-dependent manner.