Microvascular pathology and vascular basement membrane components in Alzheimer's disease

Several factors have highlighted the vasculature in Alzheimer's disease (AD): Cerebral amyloid angiopathy (CAA) is common, amyloid fibrils emanate from the vascular basement membrane (VBM), and similar forms of β-amyloid are found in vascular and parenchymal amyloid accumulations. The present article discusses the presence of microvascular pathology in AD. Microangiopathy, in addition to neurofibrillary tangles, senile plaques, and CAA, is a common pathologic hallmark of AD. VBM components are associated with amyloid plaques, and nonamyloidotic alterations of the VBM occur in brain regions susceptible to AD lesions. Also, intra-VBM perivascular cells (traditionally called pericytes), a subset of which share the immunophenotype of microglia and other mononuclear phagocytic system (MPS) cells, have been implicated in vascular alterations and cerebrovascular amyloid deposition. Perivascular and parenchymal MPS cells have access to several sources of the β-amyloid protein precursor, including platelets, circulating white cells, and neurons. MPS cells would thus be ideally situated to uptake and process the precursor, and deposit β-amyloid in a fashion analogous to that seen in other forms of systemic and cerebral amyloidoses.     

Spontaneous generation of infectious nucleating amyloids in the transmissible and nontransmissible cerebral amyloidoses

The unconventional viruses of the transmissible subacute spongiform encephalopathies (kuru-CJD-GSS-FFI-scrapie-BSE) are nucleants spontaneously generated from host precursor proteins altered to β-pleated sheet configuration that polymerize into insoluble infectious amyloid fibrils. Thede novo conversion to infectious amyloids is facilitated or accelerated by many different point mutations causing amino acid changes, a stop codon, or octapeptide inserts that increase the likelihood of spontaneous conversion to infectious configuration by many orders of magnitude. Similar nucleating induction of configurational change to amyloid probably occurs in other amyloidoses of brain and in systemic amyloidoses. Thus, all amyloids, particularly so-called fibrillar amyloid enhancing factors, may be considered to be infectious scrapie-like agents. These events probably occur extracellularly, thus we are attempting to reproduce them in vitro, even from synthetic polypeptides.     

Small molecule inhibitors of lysozyme amyloid aggregation

Protein amyloid aggregation is associated with a number of important human pathologies, but the precise mechanisms underlying the toxicity of amyloid aggregates are still incompletely understood. In this context, drugs capable of blocking or interfering with the aggregation of amyloidogenic proteins should be considered in strategies aimed at the development of novel therapeutic agents. Human lysozyme variants have been shown to form massive amyloid deposits in the livers and kidneys of individuals affected by hereditary systemic amyloidosis. Currently, there are no clinical treatments available to prevent or reverse formation of such amyloid deposits. We have recently described a number of di- and trisubstituted aromatic compounds that block the formation of soluble oligomers and amyloid fibrils of the β-amyloid peptide (Aβ) and protect hippocampal neurons in culture from Aβ-induced toxicity. Here, we show that some of those compounds inhibit the formation and disrupt preformed amyloid fibrils from both human and hen egg white lysozyme. These results suggest that these small molecule compounds may serve as prototypes for the development of drugs for the prevention or treatment of different types of amyloidoses.     

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.     

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.     

Delineating the Mechanism of Alzheimer’s Disease Aβ Peptide Neurotoxicity

The Alzheimer’s disease neurotoxic amyloid-β (Aβ) peptide is derived from the larger amyloid precursor protein (APP) and is the principal component of the senile plaques in Alzheimer’s disease (AD) brains. This mechanism by which Aβ mediates neurotoxicity or neuronal dysfunction is not fully resolved. This review will outline some of the key determinants that modulate Aβ’s activity and the cellular pathways and mechanisms involved.     

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.     

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.     

Gamma-secretase: substrates and inhibitors

The amyloid β-protein (Aβ) deposited in Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a secreted proteolytic product of the amyloid β-protein precursor (APP). Generation of Aβ from the APP requires two sequential proteolytic events, β-secretase cleavage to generate the amino terminus, followed by γ-secretase cleavage to generate the carboxyl terminus. Because this process is a central event in the pathogenesis of AD, γ-secretase is believed to be an excellent therapeutic target. γ-Secretase activity has been demonstrated to be membrane-associated, with the cleavage site primarily determined by the location of the substrate with respect to the membrane. It has also been shown that this unusual proteolytic activity not only occurs for APP, but also for proteins involved in morphogenic processes or cell proliferation and differentiation such as Notch and ErbB4. Thus far, all γ-secretase substrates are involved in some form of nuclear signaling. These recent findings have important implications for the development of pharmacological interventions that target γ-secretase.     

The Polyphenol Piceid Destabilizes Preformed Amyloid Fibrils and Oligomers In Vitro: Hypothesis on Possible Molecular Mechanisms

Alzheimer’s disease (AD) is characterized by deposits of amyloid in various tissues. The neuronal cytotoxicity of Aβ peptides is attributed not only to various mechanisms but also to amyloid fibrils and soluble oligomeric intermediates. Consequently, finding molecules to prevent or reverse the oligomerization and fibrillization of Aβ could be of therapeutic value in the treatment of AD. We show that piceid, a polyphenol of the stilbene family, destabilized fibrils and oligomers to give back monomers that are not neurotoxic molecules. The mechanism of this destabilization could be a dynamic interaction between the polyphenol and the Aβ that could open the hydrophobic zipper and shift the reversible equilibrium “random coil⇔β-sheet” to the disordered structure.     

The role of cholesterol in pathogenesis of Alzheimer's disease: dual metabolic interaction between amyloid beta-protein and cholesterol

The implication that cholesterol plays an essential role in the pathogenesis of Alzheimer’s disease (AD) is based on the 1993 finding that the presence of apolipoprotein E (apoE) allele ε4 is a strong risk factor for developing AD. Since apoE is a regulator of lipid metabolism, it is reasonable to assume that lipids such as cholesterol are involved in the pathogenesis of AD. Recent epidemiological and biochemical studies have strengthened this assumption by demonstrating the association between cholesterol and AD, and by proving that the cellular cholesterol level regulates synthesis of amyloid β-protein (Aβ). Yet several studies have demonstrated that oligomeric Aβ affects the cellular cholesterol level, which in turn has a variety of effects on AD-related pathologies, including modulation of tau phosphorylation, synapse formation and maintenance of its function, and the neurodegenerative process. All these findings suggest that the involvement of cholesterol in the pathogenesis of AD is dualistic—it is involved in Aβ generation and in the amyloid cascade, leading to disruption of synaptic plasticity, promotion of tau phosphorylation, and eventual neurodegeneration. This review article describes recent findings that may lead to the development of a strategy for AD prevention by decreasing the cellular cholesterol level, and also focuses on the impact of Aβ on cholesterol metabolism in AD and mild cognitive impairment (MCI), which may result in promotion of the amyloid cascade at later stages of the AD process.     

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).     

Neprilysin Deficiency-Dependent Impairment of Cognitive Functions in a Mouse Model of Amyloidosis

Alzheimer’s disease, responsible for the vast majority of dementia cases in the elderly population, is caused by accumulation of toxic levels of amyloid β peptide (Aβ) in the brain. Neprilysin is a major enzyme responsible for the degradation of Aβ in vivo. We have previously shown that elevation of neprilysin levels in the brain delays the deposition of Aβ -plaques in a mouse model of amyloidosis and that lack of neprilysin leads to increased Aβ generation and to signs of incipient neurodegeneration in mouse brains. This study was designed to test whether low brain levels of neprilysin affect the amyloid pathology or perturb the learning and memory performance of mice. Double-mutated mice carrying a targeted depletion of one allele of Mme, the gene encoding neprilysin, and over-expressing human amyloid precursor protein (APP), exhibited a reinforced amyloid pathology in comparison with their APP transgenic littermates. Moreover, in contrast to their parental lines, these mice were impaired in the Morris water maze learning and memory paradigm and showed facilitated extinction in the conditioned taste aversion test. These data suggest that even a partial neprilysin deficiency, as is found during aging, exacerbates amyloid pathology and may impair cognitive functions. Special issue to Honor Dr. Akitane Mori.     

The molecular genetics of Alzheimer's disease

The major pathological characteristic of Alzheimer's disease (AD) is the abnormal deposition of β-amyloid peptide (Aβ) in the brain. In some early onset cases, the disease develops because of mutations in the gene coding for β-amyloid precursor protein (βAPP). However, the majority of AD families in the early onset subgroup are linked to a locus on chromosome 14. The genetic analysis and age of onset correlates of both the βAPP gene and the chromosome 14 locus are discussed. We speculate on the mechanisms by which the βAPP mutations cause the disease and discuss recent advances in βAPP processing that may be relevant to the pathogenesis of the late-onset (common) form of the disease. In addition, we review the association of theAPOE locus with late-onset familial and nonfamilial disease. Further work is required to establish the effects of this locus on disease occurrence, age of onset, and progression. The molecular pathology of ApoE in relation to AD development and the identification of the chromosome 14 gene will greatly contribute to a general pathogenic model of AD, and will clarify the role of βAPP and its derivatives.     

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.     

Mitochondria,cholesterol and amyloid β peptide: a dangerous trio in Alzheimer disease

The molecular mechanisms of Alzheimer’s disease (AD) are not fully understood. Extensive evidence from experimental models has involved the overgeneration and accumulation of toxic amyloid β peptides (Aβ) in the onset and progression of the disease. The amyloidogenic processing of amyloid precursor protein into pathogenic Aβ fragments is thought to occur in specific domains of the plasma membrane and favored by cholesterol enrichment. Intracellular Aβ accumulation is known to induce oxidative stress, predominantly via mitochondria targeting of toxic Aβ. Recent evidence using mouse models of cholesterol loading has demonstrated that the specific mitochondrial cholesterol pool sensitizes neurons to Aβ-induced oxidant cell death and caspase-independent apoptosis due to selective mitochondrial GSH (mGSH) depletion induced by cholesterol-mediated perturbation of mitochondrial membrane dynamics. mGSH replenishment by permeable precursors such as glutathione ethyl ester protected against Aβ-mediated neurotoxicity and inflammation. Thus, these novel data expand the pathogenic role of cholesterol in AD indicating that in addition to fostering Aβ generation, mitochondrial cholesterol determines Aβ neurotoxicity via mGSH regulation.     

Cell based screening of inhibitors of transthyretin aggregation

The amyloidoses are the extracellular subset of a group of diseases in which in vivo protein misfolding leads to a pathologic gain of function, i.e., aggregation leading to protein deposition, with subsequent tissue damage. Wild-type and mutant transthyretins (TTR) are the etiologic agents in prototypic systemic amyloidoses. We describe a cell-based assay that measures the cytotoxicity of physiologic concentrations of the amyloidogenic Val30Met TTR variant (V30M TTR) using cells of the same lineage as the in vivo tissue target of amyloid deposition. We have utilized the assay to screen small molecules for their capacity to inhibit the TTR-induced cell damage. We compared the inhibitory activity of each compound with its ability to prevent TTR fibril formation in vitro. Our results emphasize the importance of screening compounds under physiologic conditions. Moreover, if a common conformational intermediate is responsible for cell death in all the amyloid diseases, the cell-based assay has the potential to aid in the discovery of compounds useful in the treatment of amyloidoses caused by other misfolded proteins as well as those caused by TTR.     

Increased bisecting and core-fucosylated <Emphasis Type="Italic">N</Emphasis>-glycans on mutant human amyloid precursor proteins

Alteration of glycoprotein glycans often changes various properties of the glycoprotein. To understand the significance of N-glycosylation in the pathogenesis of early-onset familial Alzheimer’s disease (AD) and in β-amyloid (Aβ) production, we examined whether the mutations in the amyloid precursor protein (APP) gene found in familial AD affect the N-glycans on APP. We purified the secreted forms of wild-type and mutant human APPs (both the Swedish type and the London type) produced by transfected C17 cells and determined the N-glycan structures of these three recombinant APPs. Although the major N-glycan species of the three APPs were similar, both mutant APPs contained higher contents of bisecting N-acetylglucosamine and core-fucose residues as compared to wild-type APP. These results demonstrate that familial AD mutations in the polypeptide backbone of APP can affect processing of the attached N-glycans; however, whether these changes in N-glycosylation affect Aβ production remains to be established. Keiko Akasaka-Manya and Hiroshi Manya contributed equally to this work.