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.     

Cholesterol Potentiates β-Amyloid-Induced Toxicity in Human Neuroblastoma Cells: Involvement of Oxidative Stress

Alterations in brain cholesterol concentration and metabolism seem to be involved in Alzheimer’s disease (AD). In fact, several experimental studies have reported that modification of cholesterol content can influence the expression of the amyloid precursor protein (APP) and amyloid β peptide (Aβ) production. However, it remains to be determined if changes in neuronal cholesterol content may influence the toxicity of Aβ peptides and the mechanism involved. Aged mice, AD patients and neurons exposed to Aβ, show a significant increase in membrane-associated oxidative stress. Since Aβ is able to promote oxidative stress directly by catalytically producing H₂O₂ from cholesterol, the present work analyzed the effect of high cholesterol incorporated into human neuroblastoma cells in Aβ-mediated neurotoxicity and the role of reactive oxygen species (ROS) generation. Neuronal viability was studied also in the presence of 24S-hydroxycholesterol, the main cholesterol metabolite in brain, as well as the potential protective role of the lipophilic statin, lovastatin. Special issue article in honor of Dr. Ricardo Tapia.     

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.     

Upregulation of BACE1 and β-Amyloid Protein Mediated by Chronic Cerebral Hypoperfusion Contributes to Cognitive Impairment and Pathogenesis of Alzheimer’s Disease

Chronic cerebral hypoperfusion (CCH) increases the risk of Alzheimer disease (AD) through several biologically plausible pathways, but the relationship between CCH and the development of AD remains uncertain. To investigate expression of APP, BACE1 and Aβ in the hippocampus of BCCAO rats and study pathophysiological mechanism of AD from CCH. CCH rat model was established by chronic bilateral common carotid artery occlusion (BCCAO). Behavior was evaluated after BCCAO with Morris water maze and open-field task. Expression of Aβ was measured by enzyme linked immunosorbent assay (ELISA). β-Amyloid precursor protein cleavage enzyme 1 (BACE1) and β-amyloid precursor protein (APP) were tested by ELISA, Western blotting and RT-PCR. Cognitive impairment occurred with CCH by Morris water maze test and open-field task. The BACE1 and Aβ level in BCCAO rats was more increased than sham-operation control rats (P < 0.01) but APP had no difference(P > 0.05). The expression of BACE1 and Aβ has no inter-grouop difference in BCCAO rats (P > 0.05). The level of BACE1 and Aβ had positive correlation with cognitive impairment (P < 0.01) while no correlation was observed between APP and cognitive impairment. Chronic cerebral ischemia contributes to cognitive impairment and vascular pathogenesis of Alzheimer’s disease that chronic cerebral hypoperfusion increases BACE1 and Aβ level in brain.     

PMS777, a New Cholinesterase Inhibitor with Anti-Platelet Activated Factor Activity,Regulates Amyloid Precursor Protein Processing In Vitro

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically by progressive impairment of memory and cognition. Previous data have shown that beta-amyloid (Aβ) cascade plays a central role in AD pathophysiology and thus drugs regulate amyloid precursor protein (APP) metabolism may have therapeutic potential. Here the effects of PMS777, a new cholinesterase inhibitor with anti-platelet activated factor activity, on APP processing were investigated. Using SH-SY5Y^APP695 cells, it showed that PMS777 treatment caused significant decreased secretion of sAPPα into the conditioned media without affecting cellular holoAPP synthesis. When PC12 cells were incubated with PMS777, the same effect was observed. The data also indicated that 10 μM PMS777 incubation decreased the release of Aβ42 into the cell media as compared with vehicle group in SH-SY5Y^APP695 cells. Pretreatment of cells with M-receptor scopolamine antagonized the decreased secretion of sAPPα induced by PMS777, but N-receptor α-bungarotoxin pretreatment did not have such an effect. These results indicated that PMS777 could modulate APP processing in vitro and that decreasing Aβ generation might demonstrate its therapeutic potential in AD.     

The Phosphatidyl Inositol 3 Kinase-Glycogen Synthase Kinase 3β Pathway Mediates Bilobalide-Induced Reduction in Amyloid β-Peptide

Bilobalide (BB), a sesquiterpenoid extract of Ginkgo biloba leaves, has been demonstrated to have neuroprotective effects. The neuroprotective mechanisms were suggested to be associated with modulation of intracellular signaling cascades such as the phosphatidyl inositol 3-kinase (PI3K) pathway. Since some members of intracellular signalling pathways such as PI3K have been demonstrated to be involved in amyloid precursor protein (APP) processing, the present study investigated whether BB has an influence on the β-secretase-mediated APP cleavage via PI3K-dependent pathway. Using HT22 cells and SAMP8 mice (a senescence-accelerated strain of mice), this study showed that BB treatment reduced generation of two β-secretase cleavage products of APP, the amyloid β-peptide (Aβ) and soluble APPβ (sAPPβ), via PI3K-dependent pathway. Additionally, glycogen synthase kinase 3β (GSK3β) signaling might be involved in BB-induced Aβ reduction as a downstream target of the activated PI3K pathway. BB showed no significant effects on β-site APP cleaving enzyme 1 (BACE-1) or γ-secretase but inhibited the β-secretase activity of another protease cathepsin B, suggesting that BB-induced Aβ reduction was probably mediated through modulation of cathepsin B rather than BACE-1. Similarly, inhibition of GSK3β did not affect BACE-1 activity but decreased cathepsin B activity, suggesting that the PI3K-GSK3β pathway was probably involved in BB-induced Aβ reduction. Increasing evidence suggests that decreasing Aβ production in the brain via modulation of APP metabolism should be beneficial for the prevention and treatment of Alzheimer’s disease (AD). BB may offer such an approach to combat AD.     

β-sitosterol inhibits high cholesterol-induced platelet β-amyloid release

Recently, increasing evidence has linked high cholesterol to the pathogenesis of Alzheimer’s disease (AD), suggesting that cholesterol may be a target for developing new compounds to prevent or treat AD. Plant sterols, a group of sterols enriched in plant oils, nuts, and avocados, have the structure very similar to that of cholesterol, and have been widely used to reduce blood cholesterol. Due to their cholesterol-lowering property, plant sterols such as β-sitosterol may also influence cholesterol-depending functions including its role in AD development. Using human platelets, a type of peripheral blood cells containing the most circulating amyloid precursor protein (APP), this study investigated the effect of β-sitosterol on high cholesterol-induced secretion of β amyloid protein (Aβ). It was found that β-sitosterol effectively inhibited high cholesterol-driven platelet Aβ release. In addition, β-sitosterol prevented high cholesterol-induced increase of activities of β- and γ-secretase, two APP cleaving enzymes to generate Aβ. Additional experiments showed that high cholesterol up-regulated lipid raft cholesterol. This effect of cholesterol could be suppressed by β-sitosterol. These findings suggest that β-sitosterol is able to inhibit high cholesterol-induced Aβ release probably through maintenance of membrane cholesterol homeostasis. Given that dietary plant sterols have the potential of penetrating the blood–brain barrier (BBB), these data suggest that plant sterols such as β-sitosterol may be useful in AD prevention.     

Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes

Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression. Special issue dedicated to Dr. Moussa Youdim.     

Phenotypic analysis of images of zebrafish treated with Alzheimer's γ-secretase inhibitors

Background  

Several γ-secretase inhibitors (GSI) are in clinical trials for the treatment of Alzheimer's disease (AD). This enzyme mediates the proteolytic cleavage of amyloid precursor protein (APP) to generate amyloid β protein, Aβ, the pathogenic protein in AD. The γ-secretase also cleaves Notch to generate Notch Intracellular domain (NICD), the signaling molecule that is implicated in tumorigenesis.     

Neuroinflammation in Lyme neuroborreliosis affects amyloid metabolism

Background  

The metabolism of amyloid precursor protein (APP) and β-amyloid (Aβ) is widely studied in Alzheimer's disease, where Aβ deposition and plaque development are essential components of the pathogenesis. However, the physiological role of amyloid in the adult nervous system remains largely unknown. We have previously found altered cerebral amyloid metabolism in other neuroinflammatory conditions. To further elucidate this, we investigated amyloid metabolism in patients with Lyme neuroborreliosis (LNB).     

Restraint Stress in Rats Alters Gene Transcription and Protein Translation in the Hippocampus

Stress is a relatively new and emerging risk factor for Alzheimer’s disease (AD). Severe stress can alter brain characteristics such as neuronal plasticity, due to changes in the metabolism of cytoskeletal proteins. In this study, male Wistar rats were exposed to restraint stress (RS) for 5 h daily for different time periods. At the end of the exposure periods, the amounts of β-actin, cofilin, amyloid precursor protein (APP) and mitogen-activated protein kinase 1 (MAPK-1) RNAs and proteins were investigated. The mRNA expressions of β-actin, cofilin and MAPK-1 followed U-shaped time course. Acute (3 days) and chronic (21 days) RS caused a fourfold and tenfold increases, respectively, in hippocampal β-actin mRNA expression. In the case of cofilin mRNA expression, elevations were detected in the hippocampus on days 3, 7 and 21. The APP mRNA level was increased on day 21. On protein level, chronic stress elevated the levels of β-actin, cofilin and APP in the hippocampus. These results suggest that stress causes the induction of some genes and proteins that are also elevated in AD selectively in the hippocampal region of the rat brain.     

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.     

Reduced Membrane Lipids in the Cortex of Alzheimer’s Disease Transgenic Mice

Accumulating evidence suggests that the conversion of Aβ peptides to soluble, neurotoxic polymers is the key event in the development of Alzheimer’s disease (AD). Moreover, interactions between Aβ peptides and neuronal membrane lipids likely play a vital role in developing the neurotoxicity associated with AD. The aim of this study is to assess whether lipid matrix of neuronal membranes is affected by the accumulation of Aβ peptides in double transgenic mouse model of AD expressing both mutant human β-amyloid precursor protein (APP) and presenilin 1 (PS1). We apply high pressure liquid chromatography with an evaporative light scattering detector to compare levels of cholesterol, galactocerebrosides, and phospholipid subclasses simultaneously in cortex samples between AD double transgenic mice at 4 months of age when Aβ production and amyloid plaque deposition is just beginning and at 9 months, when there is advanced Aβ levels and plaque deposition compared to age-matched wild-type (B6/SJL) mice. Both cholesterol (CL) and phospholipids (PL) are significantly lower in 9-month-old AD mice than the same age of B6/SJL mice. Among PL subclasses, phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) are selectively reduced in 9-month-old AD mice. The molar ratios of CL to PL in 9-month-old AD mice (1.19 ± 0.27) were significantly higher than those of 9-month-old B6/SJL mice (0.81 ± 0.08). In keeping with decreased levels of PL, there are also significant reductions of very long-chain n-3 fatty acids (docosahexaenoic acid) and n-6 fatty acid (arachidonic acid) in 9-month-old AD mice. On the other hand, ratios of total n-6 to total n-3 fatty acids were significantly higher in 9-month-old AD mice than in the same age of B6/SJL mice. Taken together, our present data support a role for the interactions of amyloid-β peptide and neuronal membranes in the subsequent development of AD. Special issue article in honor of Dr. George DeVries.     

Oligodendrocytes are a Novel Source of Amyloid Peptide Generation

Alzheimer’s disease is characterised by regional neuronal degeneration, synaptic loss, and the progressive deposition of the 4 kDa β-amyloid peptide (Aβ) in senile plaques and accumulation of tau protein as neurofibrillary tangles. Aβ derives from the larger precursor molecule, amyloid precursor protein (APP) by proteolytic processing via β- and γ-secretases. While APP expression is well documented in neurons and astrocytes, the case for oligodendrocytes is less clear. The latter cell type is reported to express different isoforms of APP, and we have confirmed this observation by immunocytochemistry in cultures of differentiated rat cortical oligodendrocytes. Moreover, by means of a sensitive electrochemiluminescent immunoassay employing Aβ C-terminal specific antibodies, mature oligodendrocytes are shown to secrete the 40 and 42 amino acid Aβ species (Aβ40 and Aβ42). Secretion of Aβ peptides was reduced by incubating oligodendrocytes with α- and β-secretase inhibitors, or a γ-secretase inhibitor. Disturbances of APP processing and/or synthesis in oligodendrocytes may account for some myelin disorders observed in Alzheimer’s disease and other senile dementias.     

Soluble aggregates of the amyloid-β peptide are trapped by serum albumin to enhance amyloid-β activation of endothelial cells

Background  

Self-assembly of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). As a result, synthetic molecules capable of inhibiting Aβ self-assembly could serve as therapeutic agents and endogenous molecules that modulate Aβ self-assembly may influence disease progression. However, increasing evidence implicating a principal pathogenic role for small soluble Aβ aggregates warns that inhibition at intermediate stages of Aβ self-assembly may prove detrimental. Here, we explore the inhibition of Aβ_1–40 self-assembly by serum albumin, the most abundant plasma protein, and the influence of this inhibition on Aβ_1–40 activation of endothelial cells for monocyte adhesion.     

Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid β protein

Background  

The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by α-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of β- and γ-secretases generates the amyloid β protein (Aβ). In this study, we investigated the effects of modulators of endocytosis on APP processing.     

β-Amyloid protein (Aβ) and human amylin regulation of apoptotic genes occurs through the amylin receptor

Deposition of amyloid-beta (Aβ) protein, a 39–43 amino acid peptide, in the brain is a major pathological feature of Alzheimer’s disease (AD). We have previously provided evidence that in primary cultures of rat basal forebrain and human fetal neurons (HFNs), neurotoxic effects of oligomeric Aβ are expressed through the amylin receptor. In this study, we utilized RT-PCR arrays to compare RNA expression levels of 84 markers for pro and anti- apoptotic signalling pathways following exposure of HFNs to either Aβ_1-42 (20 μM) or human amylin (2 μM). Oligomeric Aβ_1-42 or human amylin was applied to HFNs alone or after pre-treatment of cultures with the amylin receptor antagonist, AC253. Changes in RNA levels were then quantified and compared to each other in order to identify increases or decreases in gene expression of apoptotic markers. Applications of Aβ_1-42 or human amylin, but not the inactive inverse sequence Aβ_42-1 or rat amylin, resulted in a time-dependent marked increase in mediators of apoptosis including a 10- to 30-fold elevations in caspases 3, 6, 9, BID and XIAP levels. Amylin receptor antagonists, AC253 (10 μM) or AC187 (10 μM), significantly attenuated the induction of several pro-apoptotic mediators up-regulated following exposure to Aβ_1-42 or human amylin and increased the expression of several anti-apoptotic markers. These data allow us to identify key elements in the Aβ-induced apoptosis that are blocked by antagonism of the amylin receptor and further support the potential for amylin receptor blockade as a potential therapeutic avenue in AD.     

Feedback Regulation of SREBP and Aromatase in A β(25-35)-Supplemented Human Neuroblastoma Cells

1. The analogies between the processing of amyloid precursor protein (APP) and other transmembrane sterol regulatory element binding proteins (SREBPs) inspired us to conduct further studies on whether β-amyloid (Aβ) affects aromatase by interacting with APP and SREBP.2. In this study, cultured human neuroblastoma cells (SHSY-5Y) were incubated in experimental media (media without FBS, the main cholesterol source) in the presence or absence of Aβ (1 μM) for 24 h.3. Cellular extracts were subjected to immunoblot analysis using anti-APP, anti-aromatase and anti-SREBP-1. In these cell lines, we detected aromatase (55 kDa), SREBP cleavage product (68 kDa) and APP precursor (100–95 kDa) and cleavage product (60 kDa) by immunoblotting. Aromatase and SREBP levels were elevated in the cells incubated 24 h in experimental media and were attenuated in Aβ-supplemented experimental media.4. The disturbance of cholesterol homeostasis appears to be an important factor in the pathogenesis of Alzheimer's disease. These findings may have important implications for understanding the mechanisms of the aromatase enzyme gene in disease states such as Alzheimer’s.