A Comparative Study of β-Amyloid Peptides Aβ1-42 and Aβ25-35 Toxicity in Organotypic Hippocampal Slice Cultures

Accumulation of the neurotoxic amyloid β-peptide (Aβ) in the brain is a hallmark of Alzheimer’s disease (AD). Several synthetic Aβ peptides have been used to study the mechanisms of toxicity. Here, we sought to establish comparability between two commonly used Aβ peptides Aβ1-42 and Aβ25-35 on an in vitro model of Aβ toxicity. For this purpose we used organotypic slice cultures of rat hippocampus and observed that both Aβ peptides caused similar toxic effects regarding to propidium iodide uptake and caspase-3 activation. In addition, we also did not observe any effect of both peptides on Akt and PTEN phosphorylation; otherwise the phosphorylation of GSK-3β was increased. Although further studies are necessary for understanding mechanisms underlying Aβ peptide toxicity, our results provide strong evidence that Aβ1-42 and the Aβ25-35 peptides induce neural injury in a similar pattern and that Aβ25-35 is a convenient tool for the investigation of neurotoxic mechanisms involved in AD.     

Role of A β and the α 7 nicotinic acetylcholine receptor in regulating synaptic plasticity in Alzheimer's disease

Alzheimer's disease (AD) is caused by the accumulation of β-amyloid protein (Aβ) in the brain. The aggregation of β-amyloid protein to higher molecular weight fibrillar forms is also considered to be an important step in the pathogenesis of the disease. The memory problems associated with AD are likely to be caused by changes in synaptic plasticity. Recent studies suggest that Aβ binds to the α 7 nicotinic acetylcholine receptor (α 7 nAChR), which plays an important role in synaptic plasticity and memory. A loop domain localized towards the C-terminus of the extracellular region of the receptor has been identified as forming part of a putative Aβ-binding site. In cell culture experiments, the binding of Aβ to the α 7 nAChR has been found to cause an increase in the level of acetylcholinesterase, which is also increased around amyloid plaques in the AD brain. These studies indicate that the Aβ-binding site on the α 7 nAChR receptor is an important new target for therapeutic development in AD.     

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.     

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.     

Autoinsertion of soluble oligomers of Alzheimer's Aβ(1–42) peptide into cholesterol-containing membranes is accompanied by relocation of the sterol towards the bilayer surface

Background  

Soluble Alzheimer's Aβ oligomers autoinsert into neuronal cell membranes, contributing to the pathology of Alzheimer's Disease (AD), and elevated serum cholesterol is a risk factor for AD, but the reason is unknown. We investigated potential connections between these two observations at the membrane level by testing the hypothesis that Aβ(1–42) relocates membrane cholesterol.     

Aβ Toxicity in Alzheimer's Disease

Alzheimer's Disease (AD), the most common age-related neurodegenerative disorder, is characterized by progressive cognitive decline, synaptic loss, the formation of extracellular β-amyloid plaques and intracellular neurofibrillary tangles, and neuronal cell death. Despite the massive neuronal loss in the ‘late stage’ of disease, dendritic spine loss represents the best pathological correlate to the cognitive impairment in AD patients. The ‘amyloid hypothesis’ of AD recognizes the Aβ peptide as the principal player in the pathological process. Many lines of evidence point out to the neurotoxicity of Aβ, highlighting the correlation between soluble Aβ oligomer accumulation, rather than insoluble Aβ fibrils and disease progression. Pathological increase of Aβ in AD brains, resulting from an imbalance between its production, aggregation and clearance, might target mitochondrial function promoting a progressive synaptic impairment. The knowledge of the exact mechanisms by which Aβ peptide impairs neuronal function will help us to design new pharmacological tools for preventing AD neurodegeneration.     

DNA Immunization Against Amyloid beta 42 has High Potential as Safe Therapy for Alzheimer’s Disease as it Diminishes Antigen-Specific Th1 and Th17 Cell Proliferation

The pathogenesis of Alzheimer’s disease (AD) has been strongly associated with the accumulation of amyloid beta (Aβ) peptides in brain, and immunotherapy targeting Aβ provides potential for AD prevention. A clinical trial in which AD patients were immunized with Aβ42 peptide was stopped when 6% of participants showed meningoencephalitis, apparently due to an inflammatory Th1 immune response. Previously, we and other have shown that Aβ42 DNA vaccination via gene gun generates a Th2 cellular immune response, which was shown by analyses of the respective antibody isotype profiles. We also determined that in vitro T cell proliferation in response to Aβ42 peptide re-stimulation was absent in DNA Aβ42 trimer-immunized mice when compared to Aβ42 peptide-immunized mice. To further characterize this observation prospectively and longitudinally, we analyzed the immune response in wild-type mice after vaccination with Aβ42 trimer DNA and Aβ42 peptide with Quil A adjuvant. Wild-type mice were immunized with short-term (1–3× vaccinations) or long-term (6× vacinations) immunization strategies. Antibody titers and isotype profiles of the Aβ42 specific antibodies, as well as cytokine profiles and cell proliferation studies from this longitudinal study were determined. Sufficient antibody titers to effectively reduce Aβ42, but an absent T cell proliferative response and no IFNγ or IL-17 secretion after Aβ42 DNA trimer immunization minimizes the risk of inflammatory activities of the immune system towards the self antigen Aβ42 in brain. Therefore, Aβ42 DNA trimer immunization has a high probability to be effective and safe to treat patients with early AD.     

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.     

Dysfunction of TGF-β1 signaling in Alzheimer's disease: perspectives for neuroprotection

Alzheimer’s disease (AD) is a neurodegenerative disorder that affects about 35 million people worldwide. Current drugs for AD only treat the symptoms and do not interfere with the underlying pathogenic mechanisms of the disease. AD is characterized by the presence of β-amyloid (Aβ) plaques, neurofibrillary tangles, and neuronal loss. Identification of the molecular determinants underlying Aβ-induced neurodegeneration is an essential step for the development of disease-modifying drugs. Recently, an impairment of the transforming growth factor-β1 (TGF-β1) signaling pathway has been demonstrated to be specific to the AD brain and, particularly, to the early phase of the disease. TGF-β1 is a neurotrophic factor responsible for the initiation and maintenance of neuronal differentiation and synaptic plasticity. The deficiency of TGF-β1 signaling is associated with Aβ pathology and neurofibrillary tangle formation in AD animal models. Reduced TGF-β1 signaling seems to contribute both to microglial activation and to ectopic cell-cycle re-activation in neurons, two events that contribute to neurodegeneration in the AD brain. The neuroprotective features of TGF-β1 indicate the advantage of rescuing TGF-β1 signaling as a means to slow down the neurodegenerative process in AD.     

Herpes simplex virus interferes with amyloid precursor protein processing

Background  

The early events underlying Alzheimer's disease (AD) remain uncertain, although environmental factors may be involved. Work in this laboratory has shown that the combination of herpes simplex virus type 1 (HSV1) in brain and carriage of the APOE-ε4 allele of the APOE gene strongly increases the risk of developing AD. The development of AD is thought to involve abnormal aggregation or deposition of a 39–43 amino acid protein - β amyloid (Aβ) - within the brain. This is cleaved from the much larger transmembranal protein 'amyloid precursor protein' (APP). Any agent able to interfere directly with Aβ or APP metabolism may therefore have the capacity to contribute towards AD. One recent report showed that certain HSV1 glycoprotein peptides may aggregate like Aβ; a second study described a role for APP in transport of virus in squid axons. However to date the effects of acute herpesvirus infection on metabolism of APP in human neuronal-type cells have not been investigated. In order to find if HSV1 directly affects APP and its degradation, we have examined this protein from human neuroblastoma cells (normal and transfected with APP 695) infected with the virus, using Western blotting.     

Amyloid-β Decreases Nitric Oxide Production in Cultured Retinal Neurons: A Possible Mechanism for Synaptic Dysfunction in Alzheimer’s Disease?

The neurotoxicity of the amyloid-β peptide (Aβ) appears to be, at least in part, related to pathological activation of glutamate receptors by Aβ aggregates. However, the downstream signaling pathways leading to neurodegeneration are still incompletely understood. Hyperactivation of nitric oxide synthase (NOS) and increased nitric oxide (NO) production have been implicated in excitotoxic neuronal damage caused by overactivation of glutamate receptors, and it has been suggested that increased NO levels might also play a role in neurotoxicity in Alzheimer’s disease. We have examined the effect of blockade of NO production on the neurotoxicity instigated by Aβ₄₂ and by elevated concentrations of glutamate in chick embryo retinal neurons in culture. Results showed that l-nitroarginine methyl ester, a potent inhibitor of all NOS isoforms, had no protective effect against neuronal death induced by either Aβ₄₂ (20 μM) or glutamate (1 mM). Surprisingly, at short incubation times both Aβ and glutamate decreased NO production in retinal neuronal cultures in the absence of neuronal death. Thus, excitotoxic insults induced by Aβ and glutamate cause inhibition rather than activation of NO synthase in retinal neurons, suggesting that cell death induced by Aβ or glutamate is not related to increased NO production. On the other hand, considering the role of NO in long term potentiation and synaptic plasticity, the decrease in NO levels instigated by Aβ and glutamate suggests a possible mechanism leading to synaptic failure in 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.     

Protective Effect of Isorhynchophylline Against β-Amyloid-Induced Neurotoxicity in PC12 Cells

Beta-amyloid peptide (Aβ), a major protein component of senile plaques, has been considered as a critical cause in the pathogenesis of Alzheimer’s disease (AD). Modulation of the Aβ-induced neurotoxicity has emerged as a possible therapeutic approach to ameliorate the onset and progression of AD. The present study aimed to evaluate the protective effect of isorhynchophylline, an oxindole alkaloid isolated from a Chinese herb Uncaria rhynchophylla, on Aβ-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The results showed that pretreatment with isorhynchophylline significantly elevated cell viability, decreased the levels of intracellular reactive oxygen species and malondialdehyde, increased the level of glutathione, and stabilized mitochondrial membrane potential in Aβ_25-35-treated PC12 cells. In addition, isorhynchophylline significantly suppressed the formation of DNA fragmentation and the activity of caspase-3 and moderated the ratio of Bcl-2/Bax. These results indicate that isorhynchophylline exerts a neuroprotective effect against Aβ_25-35-induced neurotoxicity in PC12 cells, at least in part, via inhibiting oxidative stress and suppressing the mitochondrial pathway of cellular apoptosis.     

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.     

Amyloid beta–heme peroxidase promoted protein nitrotyrosination: relevance to widespread protein nitration in Alzheimer’s disease

Amyloid beta (Aβ) peptide accumulation has been demonstrated to play a central role in Alzheimer’s disease (AD). Substantial evidence indicates that protein nitrotyrosination contributes to Aβ-dependent neurotoxicity; however, the molecular mechanism is unknown. Recent research has shown that Aβ complexes with heme to form Aβ–heme, and increases the pseudo-peroxidase activity of heme. We found that Aβ–heme uses H₂O₂ and NO₂ ⁻ to cause nitration of enolase and synaptic proteins more effectively than heme. Thus, the increased peroxidase activity of Aβ–heme may be the molecular link between excess Aβ and the widespread protein nitration in AD. Interestingly, the site of enolase nitration that was catalyzed by Aβ–heme is different from that induced by heme. Moreover, the secondary structural perturbations of Aβ–heme-treated and heme-treated enolase are also different. These observations suggest that Aβ–heme targets specific amino acid sequences in enolase. Furthermore, our data show that Aβ–heme peroxidase activity is independent of the aggregation state of Aβ, suggesting an important role of soluble Aβ in addition to Aβ aggregates and oligomers in AD pathogenesis.     

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.     

Quantitative Analysis of β-Amyloid Peptides Expressed in Human Cerebrospinal Fluid by an Improved Method of Antibody-Assisted Time-of-Flight Mass Spectrometry

Establishment of diagnostic measures for early stage Alzheimer’s disease (AD) and mild cognitive impairment (MCI) is of crucial importance. Using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS), antibody-assisted MS of cerebrospinal fluid (CSF) has enabled quantitative analysis of the ratio of β-amyloid (Aβ) peptides, Aβ1-42/Aβ1-40, which has a diagnostic value for AD/MCI. To apply the MS analysis to a far wider range of CSF samples, we have established a method to analyze Aβ peptides expressed in 100 μl CSF samples quantitatively. Pretreatment of CSF samples by limit-filtration to condense peptides, and modified washing procedure using urea as denaturant, Aβ peptides of interest can be assessed with higher sensitivity by five to tenfolds to the original method. This improvement enables quantitative analysis of Aβ species from a residual amount of CSF samples, which will be occasionally obtained in case of lumbar anesthesia prior to operation and spinal tap performed for routine diagnostic purposes. Prevalence of the new procedure as laboratory test, especially among the elderly consulting for neurological clinic, will enhance the number of subjects diagnosed at early stage of AD/MCI.     

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.     

BDNF-, IGF-1- and GDNF-Secreting Human Neural Progenitor Cells Rescue Amyloid β-Induced Toxicity in Cultured Rat Septal Neurons

Alzheimer’s disease (AD) is characterized by the depositions of amyloid-β (Aβ) proteins, resulting in a reduction of choline acetyltransferase (ChAT) activity of AD brain in the early stages of the disease. Several growth factors, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF)-1 and glial cell-derived neurotrophic factor (GDNF) are known to protect neuronal cell death in several neurodegenerative both in vitro and in vivo models. In this study, septal neurons were prepared from septal nucleus of embryonic (day 16-17) rat brain and treated with monomeric, oligomeric or fibrillar Aβ_1-42 peptide. Oligomeric Aβ_1-42, (10 μM) was the most potent at sublethal dose. Septal neuron cultures treated with BDNF, IGF-1 or GDNF or co-cultured with genetically modified human neural progenitor cells (hNPCs) secreting these neurotrophic factors (but not allowing contact between the two cell types), were protected from oligomeric Aβ_1-42 peptide-induced cell death, and these trophic factors enhanced cholinergic functions by increasing ChAT expression level. These results indicate the potential of employing transplanted hNPCs for treatment of AD.