Endopeptidases 24.16 and 24.15 Are Responsible for the Degradation of Somatostatin, Neurotensin, and Other Neuropeptides by Cultivated Rat Cortical Astrocytes

Abstract: Several neuropeptides, including neurotensin, somatostatin, bradykinin, angiotensin II, substance P, and luteinizing hormone-releasing hormone but not vasopressin and oxytocin, were actively metabolized through proteolytic degradation by cultivated astrocytes obtained from rat cerebral cortex. Because phenanthroline was an effective degradation inhibitor, metalloproteases were responsible for neuropeptide fragmentation. Neurotensin was cleaved by astrocytes at the Pro¹⁰-Tyr¹¹ and Arg⁸- Arg⁹ bonds, whereas somatostatin was cleaved at the Phe⁶-Phe⁷ and Thr¹⁰-Phe¹¹ bonds. These cleavage sites have been found previously with endopeptidases 24.16 and 24.15 purified from rat brain. Addition of specific inhibitors of these proteases, the dipeptide Pro-He and N -[1-( RS )-carboxy-3-phenylpropyl]-Ala-Ala-Phe-4-aminobenzoate, significantly reduced the generation of the above neuropeptide fragments by astrocytes. The presence of endopeptidases 24.16 and 24.15 in homogenates of astrocytes could also be demonstrated by chromatographic separations of supernatant solubilized cell preparations. Proteolytic activity for neurotensin eluted after both gel and hydroxyapatite chromatography at the same positions as found for purified endopeptidase 24.16 or 24.15. In incubation experiments or in chromatographic separations no phosphoramidon-sensitive endopeptidase 24.11 (enkephalinase) or captopril-sensitive peptidyl dipeptidase A (angiotensin-converting enzyme) could be detected in cultivated astrocytes. Because astrocytes embrace the neuronal synapses where neuropeptides are released, we presume that the endopeptidases 24.16 and 24.15 on astrocytes are strategically located to contribute significantly to the inactivation of neurotensin, somatostatin, and other neuropeptides in the brain.     

Membrane Instability, Plasmalogen Content, and Alzheimer's Disease

Abstract: The normal stability of the cell membrane bilayer depends on its lipid composition being appropriate to the ambient (physiological) temperature, T_p. Membrane lipid composition may be altered by disease such that the bilayer is only stable at a new critical temperature, T^⋆, which may differ from T_p. In Alzheimer's disease (AD) temporal cortex, a defect of lipid composition has previously been identified, namely, a decrease in the ratio of plasmalogen to nonplasmalogen ethanolamine glycerophospholipids. Furthermore, for AD temporal cortex neural membranes, T^⋆≪ T_p, a finding confirmed in the present study in a larger series than previously, using a new method for obtaining T^⋆. This inequality between T^⋆ and T_p has been proposed as a putative contributory pathogenetic mechanism leading to membrane destabilisation in AD brain. The plasmalogen deficiency could account for the change in T^⋆ in AD, as shown by experiments where T^⋆ was measured for artificial lipid mixtures simulating brain membranes with varying plasmalogen/nonplasmalogen ratios. The critical temperature was found to be very sensitive to small alterations in plasmalogen content.     

Regional Membrane Phospholipid Alterations in Alzheimer's Disease

Regional levels of membrane phospholipids [phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC)] were measured in the brain of Alzheimer's disease (AD) and control subjects. The levels of PE-derived and PI-derived total fatty acids were significantly decreased in the hippocampus of AD subjects. Here significant decreases were found in PE-derived stearic, oleic and arachidonic and docosahexaenoic acids, and in PI-derived oleic and arachidonic acids. In the inferior parietal lobule of AD subjects, significant decreases were found only in PE and those decreases were contributed by stearic, oleic and arachidonic acids. In the superior and middle temporal gyri and cerebellum of AD subjects, no significant decreases were found in PC-, PE- and PI-derived fatty acids. The decrease of PE and PI, which are rich in oxidizable arachidonic and docosahexaenoic acids, but not of PC, which contains lesser amounts of these fatty acids, suggests a role for oxidative stress in the increased degradation of brain phospholipids in AD.     

Galanin-Like Immunoreactivity Is Increased in the Postmortem Cerebral Cortex from Patients with Alzheimer's Disease

Abstract: Galanin is a peptide that is associated with cholinergic neurons of the basal forebrain and, thus, of interest for the neuropathology of Alzheimer's disease. In the present study, human galanin-like immunoreactivity was measured in postmortem human cerebral cortical tissues by using a homologous radioimmunoassay. In an initial study, six cerebral cortical regions were evaluated from nine elderly controls, 13 neuropathologically verified Alzheimer's disease patients, and 19 elderly schizophrenics. A significant 65% increase in galanin was found in frontal cortex Brodmann area 8 of Alzheimer's disease patients compared with controls. In contrast, cerebral cortical tissues from elderly schizophrenics were not different from those from elderly controls in any region. In a second study, 10 cerebral cortical regions were evaluated from 50 neuropathologically verified Alzheimer's disease patients and nine elderly controls. Concentrations of galanin were increased significantly 26–61% in six of 10 cerebral cortical regions examined (Brodmann areas F8, F44, T20, T21, T36, and P22). Purification of brain extracts by size-exclusion Sephadex G-50 chromatography revealed that human galanin-like immunoreactivity eluted in two peaks of different molecular weights. These studies reveal increased concentrations of galanin in the cerebral cortex of Alzheimer's disease, similar to previous findings in basal forebrain tissue. Because galanin inhibits cholinergic neurotransmission, these findings may have important implications in the understanding of Alzheimer's disease neuropathology and associated cognitive deficits.     

阿尔茨海默症的诊断与治疗研究进展

阿尔茨海默症(AD)即老年痴呆症,是以老年斑和神经元纤维缠结为主要病理特征的神经退行性疾病.AD的发病机制是多种发病素、多种通路和分子机制的相互参与,例如信号异常、炎症和免疫系统、脂质转运、细胞内吞作用、细胞凋亡、氧化损伤和应激反应、tau 病理学、神经元和突触的损失、能量代谢等.目前没有一种 AD 治疗方法能从根本上停止其病理的退行性改变,但仍有多种治疗策略.我们从生物标志物、遗传、神经影像、药物治疗、β淀粉样蛋白免疫治疗方面,对阿尔茨海默症的治疗研究进展进行了简要综述.     

Tau蛋白在阿尔茨海默病中的作用

阿尔茨海默病(AD)是非常普遍的神经变性性疾病并且是老年人痴呆的主要原因。AD患者的症状特点包括进行性的认知障碍、记忆丧失和行为障碍,与大脑中的病理变化密切相关。AD现成为全球最严重的健康和社会经济问题。在AD患者脑中神经纤维网或神经营养障碍的过程中存在tau蛋白的异常。tau蛋白丧失其促微管组装的生物学功能,导致细胞骨架的破坏、丝状物形成和神经缠结,轴突运输损害,进而导致突触蛋白失去功能和神经退行性病变。其数量和结构的改变将会影响其功能而且会出现异常聚集。调节Tau蛋白的异常聚集的分子机制主要是一些翻译后修饰使其结构及构象发生变化。因此,异常磷酸化和截断的tau蛋白作为tau蛋白病理过程的关键机制而引起学者关注。本文描述了tau蛋白的结构和功能及其在AD中的主要病理变化,同时在本文中还涉及到磷酸化的tau蛋白是神经元对氧化应激的代偿反应这一观点。对tau蛋白进行更加全面的解读。     

DNA微阵列技术在阿尔茨海默病及其防治药物研究中的应用

在生物技术飞速发展的今天,DNA微阵列已成为功能基因组时代大规模、高通量乃至全基因组表达和功能研究的有力工具。阿尔茨海默病,因其发病机制复杂,迄今尚无定论,因此在临床上也缺乏有效的防治药物。简要综述DNA微阵列技术应用于阿尔茨海默病发病机制、早期诊断及防治药物等方面的研究进展。     

加兰他敏对阿尔茨海默病大鼠认知功能的影响

目的:研究加兰他敏对阿尔茨海默病大鼠认知功能的影响。方法:雄性SD大鼠35只,随机分为假手术组(10只)、链脲菌素(STZ)组(10只),加兰他敏组(15只)。侧脑室注射STZ制备阿尔茨海默病大鼠模型,水迷宫试验测定大鼠的学习记忆能力。加兰他敏3mg/kg,共6周。结果:术前三个组之间潜伏期及过平台次数无明显差异,术后第10天加兰他敏组潜伏期为48.64±8.41s,过平台次数为0.74±0.40,与STZ组比较,差异无统计学意义。治疗6周后加兰他敏组潜伏期为26.33±9.51s,过平台次数为3.33±1.51,与STZ组比较,差异有显著统计学意义。结论:加兰他敏对阿尔茨海默病大鼠的认知功能具有明显的改善作用。     

Membrane Phospholipid Alterations In Alzheimer's Disease: Deficiency of Ethanolamine Plasmalogens

The ethanolamine plasmalogens are decreased whereas serine glycerophospholipids are significantly increased in plasma membrane phospholipid in affected regions of brain in Alzheimer's disease. This may be due to stimulation of Ca²⁺-independent plasmalogen-selective phospholipase A₂, which was recently discovered in brain. This phospholipase A₂ differs from other Ca²⁺-independent phospholipases A₂ in response to ATP and various inhibitors. It may be responsible for excess release of arachidonic acid and accumulation of prostaglandins and lipid peroxides in AD. Accumulation of the above lipid metabolites due to abnormal receptor function and signal transduction may contribute to neurodegeneration in AD.     

The Involvement of Lipids in Alzheimer's Disease

It has been estimated that Alzheimer＇s disease （AD）, the most common form of dementia, will affect approximately 81 million individuals by 2040. To date, the actual cause and cascade of events in the progression of this disease have not been fully determined. Furthermore, there is currently no definitive blood test or simple diagnostic method for AD. Considerable efforts have been put into proteomic approaches to develop a diagnostic blood test, but to date these efforts have not been successful. More recently, there has been a stronger focus on lipidomic studies in the hope of increasing our understanding of the underlying mechanisms leading to AD and developing an AD blood test. It is well known that the strongest genetic risk factor for AD is the e4 variant of apolipoprotein E （APOE）. Evidence suggests that the ApoE protein, a major lipid transporter, plays a key role in the pathogenesis of AD, and its role in both normal and aberrant lipid metabolism warrants further extensive investigation. Here, we review ApoE-lipid interactions, as well as the roles that lipids may play in the pathogenesis of AD.     

Proteolytic Degradation of Alzheimer's Disease Amyloid β-Peptide by a Metalloproteinase from Microglia Cells

Abstract: The cerebral deposition of amyloid β-peptide (Aβ) is a histopathological characteristic of Alzheimer's disease. Because an impaired clearance of Aβ might be involved in the disease, we investigated the proteolytic degradation of synthetic Aβ (40-residue peptide) in cultures of glial cells and characterized a protease involved. Whereas rat astrocytes had a very low degradation capacity, cultivated rat microglia cells cleaved Aβ. Microglia activity was considerably enhanced by stimulation with lipopolysaccharide and to a lesser extent by phorbol esters. Most of the Aβ-degrading activity was released into the medium. By use of selective inhibitors the protease was characterized as a metalloprotease of ∼200 kDa that was different from neutral endopeptidase (a neuropeptide-degrading enzyme), matrix metalloproteases, or macrophage elastase. Its activity was efficiently reduced by four hydroxamic acid-based zinc-metalloprotease inhibitors that have been shown to inhibit membrane protein secretases (disintegrins). We conclude that activated microglia cells might impair amyloid plaque formation by release of a metalloprotease that degrades soluble Aβ before polymerization.     

Brain S-Adenosylmethionine Levels Are Severely Decreased in Alzheimer's Disease

Abstract: S -Adenosylmethionine is an essential ubiquitous metabolite central to many biochemical pathways, including transmethylation and polyamine biosynthesis. Reduced CSF S -adenosylmethionine levels in Alzheimer's disease have been reported; however, no information is available regarding the status of S -adenosylmethionine or S -adenosylmethionine-dependent methylation in the brain of patients with this disorder. S -Adenosylmethionine concentrations were measured in postmortem brain of 11 patients with Alzheimer's disease. We found decreased levels of S -adenosylmethionine (−67 to −85%) and its demethylated product S -adenosylhomocysteine (−56 to −79%) in all brain areas examined (cerebral cortical subdivisions, hippocampus, and putamen) as compared with matched controls (n = 14). S -Adenosylmethionine and S -adenosylhomocysteine levels were normal in occipital cortex of patients with idiopathic Parkinson's disease (n = 10), suggesting that the decreased S -adenosylmethionine levels in Alzheimer's disease are not simply a consequence of a chronic, neurodegenerative condition. Reduced S -adenosylmethionine levels could be due to excessive utilization in polyamine biosynthesis. The severe reduction in levels of this essential biochemical substrate would be expected to compromise seriously metabolism and brain function in patients with Alzheimer's disease and may provide the basis for the observations of improved cognition in some Alzheimer's patients following S -adenosylmethionine therapy.     

Cortical Cytochrome Oxidase Activity Is Reduced in Alzheimer's Disease

Abstract: A defect in energy metabolism may play a role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. In the present study, we examined the activities of the enzymes that catalyze oxidative phosphorylation in frontal, temporal, parietal, and occipital cortex from Alzheimer's disease patients and age-matched controls. Complex I and complex II–III activities showed a small decrease in occipital cortex, but were unaffected in the other cortical areas. The most consistent change was a significant decrease of cytochrome oxidase (complex IV) activity of 25–30% in the four cortical regions examined. These results provide further evidence of a cytochrome oxidase defect in Alzheimer's disease postmortem brain tissue. A deficiency in this key energy-metabolizing enzyme could lead to a reduction in energy stores and thereby contribute to the neurodegenerative process.     

Alterations in immunological and neurological gene expression patterns in Alzheimer's disease tissues

Microarray technology was utilized to isolate disease-specific changes in gene expression by sampling across inferior parietal lobes of patients suffering from late onset AD or non-AD-associated dementia and non-demented controls. Primary focus was placed on understanding how inflammation plays a role in AD pathogenesis. Gene ontology analysis revealed that the most differentially expressed genes related to nervous system development and function and neurological disease followed by genes involved in inflammation and immunological signaling. Pathway analysis also implicated a role for chemokines and their receptors, specifically CXCR4 and CCR3, in AD. Immunohistological analysis revealed that these chemokine receptors are upregulated in AD patients. Western analysis demonstrated an increased activation of PKC, a downstream mediator of chemokine receptor signaling, in the majority of AD patients. A very specific cohort of genes related to amyloid beta accumulation and clearance were found to be significantly altered in AD. The most significantly downregulated gene in this data set was the endothelin converting enzyme 2 (ECE2), implicated in amyloid beta clearance. These data were subsequently confirmed by real-time PCR and Western blot analysis. Together, these findings open up new avenues of investigation and possible therapeutic strategies targeting inflammation and amyloid clearance in AD patients.     

阿尔采末病神经元凋亡发生机制的研究及四氢小檗碱的抗凋亡作用

本研究用免疫组织化学、细胞培养和膜片钳技术研究阿尔采末病 (AD)脑中的细胞凋亡 ,以及β 淀粉样肽 ( β AP)诱导离体培养海马神经元的凋亡及其离子机制。结果表明AD脑内存在细胞凋亡 ,β AP可以通过抑制神经元的电压依赖性K 和Na 通道促进神经元凋亡 ,一氧化氮与其有一定的协同作用。同时发现四氢小檗碱可通过降低细胞内Ca2 而减轻β AP引起的神经元凋亡。     

Transferrin and Iron in Normal, Alzheimer's Disease, and Parkinson's Disease Brain Regions

Abstract: Oxidant-mediated damage is suspected to be involved in the pathogenesis of several neurodegenerative disorders. Iron promotes conversion of hydrogen peroxide to hydroxyl radical and, thus, may contribute to oxidant stress. We measured iron and its transport protein transferrin in caudate, putamen, globus pallidus, substantia nigra, and frontal cortex of subjects with Alzheimer's disease (n = 14) and Parkinson's disease (n = 14), and in younger adult (n = 8) and elderly (n = 8) normal controls. Although there were no differences between control groups with regard to concentrations of iron and transferrin, iron was significantly increased ( p < 0.05) in Alzheimer's disease globus pallidus and frontal cortex and Parkinson's disease globus pallidus, and transferrin was significantly increased in Alzheimer's disease frontal cortex, compared with elderly controls. The transferrin/iron ratio, a measure of iron mobilization capacity, was decreased in globus pallidus and caudate in both disorders. Regional transferrin and iron concentrations were generally more highly correlated (Pearson's correlation coefficient) in elderly controls than in Alzheimer's and Parkinson's disease. The altered relationship between iron and transferrin provides further evidence that a disturbance in iron metabolism may be involved in both disorders.     

Proteomic Profiling and Neurodegeneration in Alzheimer's Disease

Quantitative proteome analysis of Alzheimer's disease (AD) brains was performed using 2-D gels to identify disease specific changes in protein expression. The task of characterizing the proteome and its components is now practically achievable because of the development and integration of four important tools: protein, EST, and complete genome sequence databases, mass spectrometry, matching software for protein sequences and protein separation technology. Mass spectrometry (MS) instrumentation has undergone a tremendous change over the past decade, culminating in the development of highly sensitive, robust instruments that can reliably analyze biomolecules, particularly proteins and peptides; we identified 35 proteins from over 100 protein spots on a 2-D gel. Using this current technology, protein-expression profiling, which is actually a specialized form of mining, is an important principal application of proteomics. The information obtained has tremendous potential as a means of determining the pathogenesis, and detecting disease markers and potential targets for drug therapy in AD.     

The Cognitive Psychopharmacology of Alzheimer's Disease: Focus on Cholinergic Systems

The primary pathology in Alzheimer's disease (DAT) occurs in the basal forebrain cholinergic system (BFCS), which provides the major cholinergic innervation to the neocortex, hippocampus and amygdala. Consistent with the 'cholinergic hypothesis' of dementia in DAT, the most effective treatments so far developed for DAT are drugs which act to boost the functions of the BFCS. These include the centrally acting cholinesterase inhibitor tacrine, and the cholinergic agonist nicotine, acute administration of which leads to an improvement in attentional functions, in line with recent animal studies of the role of the BFCS in cognition. We conclude that future research should include the development of more potent, longer-lasting, less toxic cholinergic agents, which appear to be the best candidates for alleviating the cognitive symptomatology of DAT. Such drugs may also be useful in the treatment of a number of other cognitive disorders, including Lewy body dementia, attention deficit/hyperactivity disorder, and schizophrenia.     

Altered Protein Tyrosine Phosphorylation in Alzheimer''s Disease

The activity of protein tyrosine kinase was determined in extracts from Alzheimer's disease brains and age- and postmortem time-matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p less than 0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine-positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's disease.     

Impaired proteasome function in Alzheimer's disease

Inhibition of proteasome activity is sufficient to induce neuron degeneration and death; however, altered proteasome activity in a neurodegenerative disorder has not been demonstrated. In the present study, we analyzed proteasome activity in short-postmortem-interval autopsied brains from 16 Alzheimer's disease (AD) and nine age- and sex-matched controls. A significant decrease in proteasome activity was observed in the hippocampus and parahippocampal gyrus (48%), superior and middle temporal gyri (38%), and inferior parietal lobule (28%) of AD patients compared with controls. In contrast, no significant decrease in proteasome activity was observed in either the occipital lobe or the cerebellum. The loss of proteasome activity was not associated with a decrease in proteasome expression, suggesting that the proteasome may become inhibited in AD by a posttranslational modification. Together, these data indicate a possible role for proteasome inhibition in the neurodegeneration associated with AD.