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.     

Application of Surface-Enhanced Raman Spectroscopy for Detection of Beta Amyloid Using Nanoshells

Currently, no methods exist for the definitive diagnosis of AD premortem. β-amyloid, the primary component of the senile plaques found in patients with this disease, is believed to play a role in its neurotoxicity. We are developing a nanoshell substrate, functionalized with sialic acid residues to mimic neuron cell surfaces, for the surface-enhanced Raman detection of β-amyloid. It is our hope that this sensing mechanism will be able to detect the toxic form of β-amyloid, with structural and concentration information, to aid in the diagnosis of AD and provide insight into the relationship between β-amyloid and disease progression. We have been successfully able to functionalize the nanoshells with the sialic acid residues to allow for the specific binding of β-amyloid to the substrate. We have also shown that a surface-enhanced Raman spectroscopy response using nanoshells is stable and concentration-dependent with detection into the picomolar range.     

Amyloid β-protein toxicity and oxidative stress in Alzheimer’s disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by loss of memory and progressive decline of cognitive abilities. Although the pathogenesis of this disease is not known and is still under intensive investigation, there are several hypotheses which address certain aspects of the disease. This review focuses on the oxidative-stress hypothesis of AD and on novel antioxidative approaches to an effective neuroprotection for the prevention and therapy of this neurodegenerative disorder. The toxicity of the AD-associated amyloid β-protein (Aβ), the induction of oxidative stress by Aβ in neurons, and potential sources of oxidative events in brain tissue are discussed. Received: 20 February 1997 / Accepted: 9 May 1997     

Homocysteine Thiolactone and Human Cholinesterases

1. The cholinergic system is important in cognition and behavior as well as in the function of the cerebral vasculature. 2. Hyperhomocysteinemia is a risk factor for development of both dementia and cerebrovascular disease. 3. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are serine hydrolase enzymes that catalyze the hydrolysis of the neurotransmitter acetylcholine, a key process in the regulation of the cholinergic system. 4. It has been hypothesized that the deleterious effects of elevated homocysteine may, in part, be due to its actions on cholinesterases. 5. To further test this hypothesis, homocysteine and a number of its metabolites and analogues were examined for effects on the activity of human cholinesterases. 6. Homocysteine itself did not have any measurable effect on the activity of these enzymes. 7. Homocysteine thiolactone, the cyclic metabolite of homocysteine, slowly and irreversibly inhibited the activity of human AChE. 8. Conversely, this metabolite and some of its analogues significantly enhanced the activity of human BuChE. 9. Structure–activity studies indicated that the unprotonated amino group of homocysteine thiolactone and related compounds represents the essential feature for activation of BuChE, whereas the thioester linkage appears to be responsible for the slow AChE inactivation. 10. It is concluded that hyperhomocysteinemia may exert its adverse effects, in part, through the metabolite of homocysteine, homocysteine thiolactone, which is capable of altering the activity of human cholinesterases, the most pronounced effect being BuChE activation.     

Amyloid beta: Functional protein or biological junk?

During a decade there was a dogma that Alzheimer’s amyloid beta (Aβ) is produced only upon the disease, and that this protein is neurotoxic for neurons and brain tissue. Current scientific evidence demonstrates that Aβ is an essential molecule in synaptic plasticity that underlines learning and memory. Therefore, it was hypothesized that the change of Aβ biology in Alzheimer’s disease (as well as in a number of other human pathologies, including cardiovascular disease, Niemann-Pick type C disease and Down syndrome) represents a physiological mechanism serving to compensate the impaired brain structure or function. This review summarizes experimental evidence on Aβ as a functional player in synaptic plasticity and neurochemical pathways.     

Dysregulation of Calcium Homeostasis in Alzheimer’s Disease

The accumulation of oligomeric species of β-amyloid protein in the brain is considered to be a key factor that causes Alzheimer’s disease (AD). However, despite many years of research, the mechanism of neurotoxicity in AD remains obscure. Recent evidence strongly supports the theory that Ca²⁺ dysregulation is involved in AD. Amyloid proteins have been found to induce Ca²⁺ influx into neurons, and studies on transgenic mice suggest that this Ca²⁺ influx may alter neuronal excitability. The identification of a risk factor gene for AD that may be involved in the regulation of Ca²⁺ homeostasis and recent findings which suggest that presenilins may be involved in the regulation of intracellular Ca²⁺ stores provide converging lines of evidence that support the idea that Ca²⁺ dysregulation is a key step in the pathogenesis of AD. Special issue article in Honor of Dr. Graham Johnston.     

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.     

老年痴呆与血浆同型半胱氨酸水平关系的临床研究

目的:探讨血浆同型半胱氨酸(homocysteine,Hcy)水平与老年痴呆的相关性。方法:应用全自动生化分析仪以循环酶法检测90例老年痴呆患者的血浆Hcy浓度,并与30例非痴呆患者作为同龄对照组血浆Hcy浓度进行比较;根据Hachinski缺血指数量表评分将老年痴呆分为阿尔茨海默病(Alzheimer’s disease,AD),混合性痴呆(mixed dementia,MD),血管性痴呆(vascular dementia,VD);根据简易精神状态检查量表(Mini-Mental State Examination,MMSE)评分划分痴呆患者严重程度,分为轻度、中度、重度。结果:AD、MD、VD组血浆Hcy水平均显著高于对照组(P<0.01);不同程度痴呆患者血浆Hcy水平有显著性差异(P<0.05),血浆Hcy浓度越高,认知功能损害的程度越重。结论:高Hcy血症可能是老年期痴呆发病的一个重要危险因素,且认知功能减退的程度与血浆同型半胱氨酸浓度呈正相关。     

Unifying features of systemic and cerebral amyloidosis

Amyloidosis is a generic term for a group of clinically and biochemically diverse diseases that are characterized by the deposition of an insoluble fibrillar protein in the extracellular space. Over 16 biochemically distinct amyloids are known. Despite this diversity, all amyloids have a particular ultrastructural and tinctorial appearance, a β-pleated sheet structure, and are codeposited with a group of amyloid-associated proteins. The most common amyloidosis is Alzheimer’s disease (AD), where Aβ is the main component of the amyloid. Recently it has been found that Aβ exists as a normal soluble protein (sAβ) in biological fluids. This links AD more closely to some of the systemic amyloidoses, where the amyloid precursor is found in the circulation normally. Numerous mutations have been found in the Aβ precursor (βPP) gene, associated with familial AD. Many mutations are also found in some of the hereditary systemic amyloidoses. For example, over 40 mutations in the transthyretin (TTR) gene are associated with amyloid. However, both Aβ and TTR related amyloid deposition can occur with no mutation. The pathogenesis of amyloid is complex, and appears to be associated with genetic and environmental risk factors that can be similar in the systemic and cerebral amyloidoses.     

Cerebral amyloid plaques in Alzheimer’s disease but not in scrapie-affected mice are closely associated with a local inflammatory process

Complement proteins of the classical pathway can be immunohistochemically identified in cerebral amyloid plaques in Alzheimer’s disease. Microglial cells in and around amyloid plaques express class II major histocompatibility (MHC) antigens and complement receptors CR3 and CR4. Negative immunostaining for immunoglobulins and for T-cell subsets in the brain parenchyma demonstrates a lack of evidence for the involvement of specific immune responses (such as an immune complex-mediated complement activation or a cell-mediated immune response) in cerebral amyloid deposits in Alzheimer’s disease. Cerebral amyloid plaques in scrapie-affected mice (slow-virus induced encephalopathy) do not contain complement factors C1q and C3c and are not clustered with microglial cells expressing MHC class II molecules or complement receptor CR3. The data presented suggest the induction of a reactive inflammatory process by β/A4 amyloid in the human brain, but not by scrapie-induced PrP amyloid in mice. Our findings do not support the hypothesis that the immune system is involved in the generation of amyloid plaques in Alzheimer’s disease. This study was partly supported by a grant from the Praeventiefonds, project 28–1945     

Ultrastructural localization of amyloid β/A4 protein precursor in the normal rat brain

The ultrastructural localization of amyloidβ/ A4 protein precursor (APP) was studied immunohistochemically in normal rat brains using antibodies against different portions of APP. In cerebral cortical neurons and Purkinje cells, APP reaction products were located in the cytoplasm and on cell surface membranes. Some Golgi apparatuses and rough endoplasmic reticulum also showed APP immunoreactivity on their membranes and some vesicles near the trans face of the Golgi apparatuses were stained. In the neuropil of the cerebral cortex and the cerebellar molecular layer, many cell processes, which surrounded synapses and were considered to be astrocytic, were APP-positive. Foot processes around capillaries and subpial astrocytic processes were also immuno-positive. At the ultrastructural level, APP-positive astrocytic processes were identified.     

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.     

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.     

冠心病患者血浆同型半胱氨酸和内皮功能的研究(英文)

目的:探讨冠心病患者血浆中同型半胱氨酸、内皮素、循环内皮细胞水平与冠状动脉粥样硬化性心脏病发生过程的关系。方法:对62例冠心病患者组和50例健康对照组分别采用高效液相色谱分析法和放射免疫分析法测定血浆中同型半胱氨酸水平和内皮素水平,同时测定血浆中循环内皮细胞的数量。结果:冠心病组同型半胱氨酸(21.27±5.73)μmol/L、内皮素(84.30±13.68)ng/L、循环内皮细胞(12.08±5.85)和cells/0.9μl明显高于对照组同型半胱氨酸(9.12±4.87)μmol/L、内皮素(47.65±12.71)ng/L、循环内皮细胞(2.35±1.02)cells/0.9μl,P均小于0.001。同型半胱氨酸与内皮素、循环内皮细胞呈正相关(r=0.601,P0.01;r=0.645,P0.01),且冠心病组血浆循环内皮细胞和内皮素呈正相关(r=0.850,P0.01)。结论:同型半胱氨酸及内皮素对冠状动脉粥样硬化性心脏病的发生发展有促进作用,对其检测有临床实用价值。     

The binding of resveratrol to monomer and fibril amyloid beta

As currently understood, Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that is driven by the aggregation of amyloid beta (Aβ) protein. It has been shown that resveratrol (RES) may attenuate amyloid β peptide-induced toxicity, promote Aβ clearance and reduce senile plaques. However, it remains to be determined whether RES could interact directly with Aβ. The aim of the present study was to examine the direct binding of RES to monomer and fibril Aβ. Using surface plasmon resonance (SPR) and proton nuclear magnetic resonance (¹H NMR), our results identified the direct binding of RES to Aβ. The ability of RES to bind to both fibril and monomer Aβ(1–40 and 1–42) was further analyzed by SPR. The binding response of RES to fAβ(1–42) was higher than that to monomer Aβ(1–42), whereas the binding response of RES to fAβ(1–40) was lower than that to monomer Aβ(1–40). The K_D of RES for fibril Aβ(1–40 or 1–42) was higher than that for the corresponding monomer Aβ. Compared to the control compound Congo red (CR), the binding responses of RES to monomer Aβ(1–42) and Aβ(1–40) were stronger, but binding to fibril Aβ(1–42) was weaker, and the K_Ds of RES with both monomer and fibril Aβ(1–40) and Aβ(1–42) were higher than that of CR. When Aβ(1–40 or 1–42) was co-incubated with RES (50 μM), the thioflavin T fluorescence of the mixture was weakened, and the number and length of amyloid fibrils were decreased. Furthermore, the results of staining in consecutive brain slices from AD patients showed that RES (10⁻⁴ M) could stain senile plaques. These results indicated that RES could bind directly to Aβ in different states, which may provide new insight into the protective properties of RES against AD.     

Amyloid formation by salmon calcitonin

It is demonstrated using three independent methods that salmon calcitonin can form amyloid fibrils in vitro. Large aggregates are shown to exhibit a blue-green birefringence in cross polarised light after staining with congo red. Individual fibrils were observed using electron microscopy. These fibrils are approx. 50–60 Å in diameter and up to 20 000 Å in length and are similar in appearance to those observed in Alzheimer's disease. Finally, X-ray diffraction studies of the large aggregates reveal the cross-β conformation characteristic of the monomers in the fibre.     

Heat Shock Proteins and Amateur Chaperones in Amyloid-Beta Accumulation and Clearance in Alzheimer’s Disease

The pathologic lesions of Alzheimer’s disease (AD) are characterized by accumulation of protein aggregates consisting of intracellular or extracellular misfolded proteins. The amyloid-β (Aβ) protein accumulates extracellularly in senile plaques and cerebral amyloid angiopathy, whereas the hyperphosphorylated tau protein accumulates intracellularly as neurofibrillary tangles. “Professional chaperones”, such as the heat shock protein family, have a function in the prevention of protein misfolding and subsequent aggregation. “Amateur” chaperones, such as apolipoproteins and heparan sulfate proteoglycans, bind amyloidogenic proteins and may affect their aggregation process. Professional and amateur chaperones not only colocalize with the pathological lesions of AD, but may also be involved in conformational changes of Aβ, and in the clearance of Aβ from the brain via phagocytosis or active transport across the blood–brain barrier. Thus, both professional and amateur chaperones may be involved in the aggregation, accumulation, persistence, and clearance of Aβ and tau and in other Aβ-associated reactions such as inflammation associated with AD lesions, and may, therefore, serve as potential targets for therapeutic intervention.     

Applications of electron paramagnetic resonance to studies of neurological disease

Electron paramagnetic resonance spectroscopy (EPR) has the potential to give much detail on the structure of the paramagnetic transition ion coordination sites, principally of Cu²⁺, in a number of proteins associated with central nervous system diseases. Since these sites have been implicated in misfolding/mis-oligomerisation events associated with neurotoxic molecular species and/or the catalysis of damaging redox reactions in neurodegeneration, an understanding of their structure is important to the development of therapeutic agents. Nevertheless EPR, by its nature an in vitro technique, has its limitations in the study of such complex biochemical systems involving self-associating proteins that are sensitive to their chemical environment. These limitations are at the instrumental and theoretical level, which must be understood and the EPR data interpreted in the light of other biophysical and biochemical studies if useful conclusions are to be drawn. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience, held in Melbourne on 11 July 2007.