Effects of chronic alcohol consumption on expression levels of APP and Aβ-producing enzymes

Chronic alcohol consumption contributes to numerous diseases, including cancers, cardiovascular diseases, and liver cirrhosis. Epidemiological studies have shown that excessive alcohol consumption is a risk factor for dementia. Along this line, Alzheimer's disease (AD) is the most common form of dementia and is caused by the accumulation of amyloid-β (Aβ plaques in neurons. In this study, we hypothesized that chronic ethanol consumption is associated with pathological processing of APP in AD. To investigate the relationship between chronic alcohol consumption and Aβ production, brain samples from rats fed an alcohol liquid diet for 5 weeks were analyzed. We show that the expression levels of APP, BACE1, and immature nicastrin were increased in the cerebellum, hippocampus, and striatum of the alcohol-fed group compared to the control group. Total nicastrin and PS1 levels were induced in the hippocampus of alcohol-fed rats. These data suggest that the altered expression of APP and Aβ-producing enzymes possibly contributes to the chronic alcohol consumption-mediated pathogenesis of AD.     

慢性吸烟大鼠气道平滑肌大电导的钙激活钾通道和Kv1.5表达的变化

复制大鼠的慢性吸烟模型,采用气道反应性的测定、HE染色、免疫组织化学染色、原位杂交和免疫印迹实验等方法,观察吸烟对大鼠支气管平滑肌大电导的钙激活的钾通道(BKca)和电压依赖性延迟整流钾通道Kv1．5蛋白和mRNA表达的影响,以阐明吸烟引起的气道高反应性发病机制中钾通道表达变化的作用。结果显示：(1)慢性吸烟可降低大鼠大气道和小气道BKca和Kv1．5蛋白和mRNA表达;(2)大气道BKca的降低程度大于Kv1．5,小气道BKca和Kv1．5的降低程度无明显差异：(3)吸烟对全肺组织BKca和Kv1．5的蛋白表达无明显影响。上述结果提示,慢性吸烟可下调大鼠气道平滑肌钾通道BKca和Kv1．5的表达水平,是导致气道高反应的机制之一。     

NR2B-Containing NMDA Receptors Expression and Their Relationship to Apoptosis in Hippocampus of Alzheimer’s Disease-Like Rats

Although studies have shown that excitotoxicity mediated by N-methyl-D-aspartate receptors (NMDARs, NR) plays a prominent role in Alzheimer's disease (AD), the precise expression patterns of NMDARs and their relationship to apoptosis in AD have not been clearly established. In this study, we used Abeta (Aβ) 1-40 and AlCl(3) to establish AD rat model. The behavioral changes were detected by morris water maze and step-down test. The hippocampal amyloid deposition and pathological changes were determined by congo red and hematoxylin-eosin staining. Immunohistochemistry was used to detect expression of NR1, NR2A and NR2B, and TUNEL staining was used to detect apoptosis. Results showed that water maze testing escape latency of AD-like rats was prolonged significantly. Reaction time, basal number of errors, and number of errors of step-down test were increased significantly; latency period of step-down test was shortened significantly in AD-like rats. Amyloid substance deposition and obvious damage changes could be seen in hippocampus of AD-like rats. These results suggested that AD rat model could be successfully established by Aβ1-40 and AlCl(3). Results also showed that expression of NR1 and NR2B were significantly increased, but expression of NR2A had no significant change, in AD-like rat hippocampus. Meanwhile, apoptotic cells were significantly increased in AD-like rat hippocampus, especially in CA1 subfield and followed by dentate gyrus and CA3 subfield. These results implied that NR2B-, not NR2A-, containing NMDARs showed pathological high expression in AD-like rat hippocampus. This pathological high expression with apoptosis and selective vulnerability of hippocampus might be exist a specific relationship.     

Changes in caspase expression in Alzheimer's disease: comparison with development and aging

Changes in caspase-2 and -3 protein levels in Alzheimer's disease (AD) brains were assessed in comparison with their expression in development and aging. The protein levels of caspase-2 and -3 were significantly increased in AD brains, caspase-2 in the particulate fraction and caspase-3 in both the cytosolic and particulate fractions, compared with controls. Immunoblot analysis of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that caspase-3 levels were high from E19 to 2 weeks, while caspase-2 remained high from 4 to 96 weeks, indicating that the expression of caspase-2 and -3 is differentially regulated during development and aging. These results suggest that caspase-2 and -3 are upregulated in AD and that the changes in AD are different from those observed in senescent brains.     

Nicotine increases the expression of high affinity nerve growth factor receptors in both in vitro and in vivo

Impairment in nerve growth factor (NGF)-mediated support to basal forebrain cholinergic neurons may represent an initial insult to certain neural cells in Alzheimer's disease (AD). High affinity NGF receptor (TrkA) levels are decreased in AD brains as compared to age-matched control brains. One of the approaches suggested for the treatment of AD exploits the ability of small molecular substances to enhance the expression of endogenous growth factors and/or their receptors. The purpose of this study was to determine whether treatment with nicotine in both in vitro and in vivo settings would increase the neural expression of TrkA receptors. Using a differentiated PC12 neuronal-like system, chronic nicotine treatment increased cell surface TrkA receptor expression. Nicotine's action was blocked by co-treatment with either the non-competitive nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine or with the alpha7 nAChR-selective antagonist methyllycaconitine. Surprisingly, certain low doses of mecamylamine alone also increased TrkA receptor levels. Rats prepared with chronic indwelling intravenous catheters were continuously infused with nicotine to deliver a total dose of 12 mg/kg over 24 hr. This treatment resulted in a significant 44% increase in TrkA receptor expression in the hippocampus. As in the cell experiments, mecamylamine also increased hippocampal TrkA receptor expression. In fact, the ratio of the maximal mecamylamine response to the maximal nicotine response that was measured in vitro, i.e., 0.43 was remarkably similar to that for the in vivo experiment, i.e., 0.47. Since in our previous studies the increase in TrkA expression produced by nicotine was shown to be related to its cytoprotective actions, these results suggest that nicotine's neuroprotective actions might also be mediated through the drug's interaction with central alpha7 nAChRs and subsequent increase in TrkA receptor expression.     

Neuronal localization of the TNFalpha converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques

The tumor necrosis factor (TNF)-alpha converting enzyme (TACE) can cleave the cell-surface ectodomain of the amyloid-beta precursor protein (APP), thus decreasing the generation of amyloid-beta (Abeta) by cultured non-neuronal cells. While the amyloidogenic processing of APP in neurons is linked to the pathogenesis of Alzheimer's disease (AD), the expression of TACE in neurons has not yet been examined. Thus, we assessed TACE expression in a series of neuronal and non-neuronal cell types by Western blots. We found that TACE was present in neurons and was only faintly detectable in lysates of astrocytes, oligodendrocytes, and microglial cells. Immunohistochemical analysis was used to determine the cellular localization of TACE in the human brain, and its expression was detected in distinct neuronal populations, including pyramidal neurons of the cerebral cortex and granular cell layer neurons in the hippocampus. Very low levels of TACE were seen in the cerebellum, with Purkinje cells at the granular-molecular boundary staining faintly. Because TACE was localized predominantly in areas of the brain that are affected by amyloid plaques in AD, we examined its expression in a series of AD brains. We found that AD and control brains showed similar levels of TACE staining, as well as similar patterns of TACE expression. By double labeling for Abeta plaques and TACE, we found that TACE-positive neurons often colocalized with amyloid plaques in AD brains. These observations support a neuronal role for TACE and suggest a mechanism for its involvement in AD pathogenesis as an antagonist of Abeta formation.     

Brain Serum Amyloid P Levels are Reduced in Individuals that Lack Dementia While Having Alzheimer’s Disease Neuropathology

The neuropathological signs of Alzheimer’s disease (AD) include beta amyloid plaques and neurofibrillary tangles. There is a significant population of individuals that have these key hallmarks but show no signs of cognitive impairment, termed non-demented with AD neuropathology (NDAN). The protective mechanism allowing these individuals to escape dementia is unknown. Serum amyloid P (SAP) is a serum protein associated with wound repair that is elevated in the brains of Alzheimer’s patients and binds to amyloid plaques. Using immunoblotting and immunohistochemistry, we evaluated SAP levels in postmortem samples of hippocampus and frontal cortex in age-matched controls, AD, and NDAN individuals. AD individuals had significantly increased SAP levels compared to normal controls, while NDAN samples had no significant difference in SAP levels compared to normal controls. Our results suggest that low levels of SAP in plaques marks the brains of individuals that escape dementia despite the presence of beta amyloid plaques and tangles.     

Up-regulation of calcineurin Abeta mRNA in the Alzheimer's disease brain: assessment by cDNA microarray

Recent advances in cDNA microarray technology have made it possible to analyze expression of more than 8000 genes. Using this technology, gene expression in the hippocampus containing neurofibrillary tangle-associated lesions from an Alzheimer's disease (AD) patient was compared with expression in the parietal cortex from the same patient that lacked these lesions. We also compared gene expression using a control brain. The top 20 named genes significantly up-regulated or down-regulated only in the AD brain were determined. The most up-regulated gene proved to be calcineurin Abeta mRNA (CAbeta). In situ hybridization histochemistry revealed that CAbeta was significantly up-regulated in pyramidal neurons of the hippocampus in the AD brain. RT-PCR analysis revealed that CAbeta was up-regulated in the hippocampus from two out of three AD brains while there were no changes in three control brains. Our study suggests that CAbeta may play a crucial role in the pathophysiological mechanisms in AD.     

Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer's disease

Deficits of cortical nicotinic acetylcholine receptors (nAChRs) have been observed in Alzheimer's disease (AD) by receptor binding assays. Little is known about the receptor subunit specificity influenced by AD, and it might be of importance for therapeutic strategies. In the present study, the protein levels of nAChR alpha3, alpha4, alpha7, and beta2 subunits were investigated using western blot analysis on postmortem brains of patients with AD and age-matched controls. The results showed that in human postmortem brain samples, bands with molecular masses of 52, 42, and 50 kDa were detected by anti-alpha4, anti-alpha7, and anti-beta2 antibodies, respectively. When anti-alpha3 antibody was used, one major band of 49 kDa and two minor bands of 70 and 38 kDa were detected. In AD patients, as compared with age-matched controls, the alpha4 subunit was reduced significantly by approximately 35 and 47% in the hippocampus and temporal cortex, respectively. A significant reduction of 25% in the alpha3 subunit was also observed in the hippocampus and a 29% reduction in the temporal cortex. For the alpha7 subunit, the protein level was reduced significantly by 36% in the hippocampus of AD patients, but no significant change was detected in the temporal cortex. In neither the hippocampus nor the temporal cortex was a significant difference observed in the beta2 subunit between AD patients and controls. These results reveal brain region-specific changes in the protein levels of the nAChR alpha3, alpha4, and alpha7 subunits in AD.     

Treatment of Alzheimer's Disease with Anti-Homocysteic Acid Antibody in 3xTg-AD Male Mice

Alzheimer''s disease (AD) is an age-associated progressive neurodegenerative disorder with dementia, the exact pathogenic mechanisms of which remain unknown. We previously reported that homocysteic acid (HA) may be one of the pathological biomarkers in the brain with AD and that the increased levels of HA may induce the accumulation of intraneuronal amyloid-beta (Aβ) peptides. In this study, we further investigated the pathological role of HA in a mouse model of AD. Four-month-old prepathological 3xTg-AD mice exhibited higher levels of HA in the hippocampus than did age-matched nontransgenic mice, suggesting that HA accumulation may precede both Aβ and tau pathologies. We then fed 3-month-old 3xTg-AD mice with vitamin B6-deficient food for 3 weeks to increase the HA levels in the brain. Concomitantly, mice received either saline or anti-HA antibody intraventricularly via a guide cannula every 3 days during the course of the B6-deficient diet. We found that mice that received anti-HA antibody significantly resisted cognitive impairment induced by vitamin B6 deficiency and that AD-related pathological changes in their brains was attenuated compared with the saline-injected control group. A similar neuroprotective effect was observed in 12-month-old 3xTg-AD mice that received anti-HA antibody injections while receiving the regular diet. We conclude that increased brain HA triggers memory impairment and that this condition deteriorates with amyloid and leads to subsequent neurodegeneration in mouse models of AD.     

Coexistence of reactive plasticity and neurodegeneration in Alzheimer diseased brains

Alzheimer's disease (AD) is a pathological process characterized by neuron degeneration and, as recently suggested, brain plasticity. In this work, we compared the reactive plasticity in AD brains associated to O-glycosydically linked glycans, recognized by lectins from Amaranthus leucocarpus (ALL) and Macrobrachium rosenbergii (MRL), and the tau neuritic degeneration. The neuritic degenerative process was evaluated by the quantification of aggregated neuritic structures. Lesions were determined using antibodies against hyperphosphorylated-tau (AD2), amyloid-beta, and synaptophysin. In these conditions, we classified and quantified three pathological structures associated to the neuritic degenerative process: 1) Amyloid-beta deposits (AbetaDs), 2) Classic neuritic plaques (NPs), and 3) Dystrophic neurites clusters (DNCs) lacking amyloid-beta deposits. Reactive plasticity structures were constituted by meganeuritic clusters (MCs) and peri-neuronal sprouting in neurons of the CA4 region of the hippocampus, immunoreactive to synaptophysin (exclusively in AD brains) and GAP-43. Besides, MCs were associated to sialylated O-glycosydically linked glycans as determined by positive labeling with ALL and MRL. Considering that these lectins are specific for the synaptic sprouting process in AD, our results suggest the co-occurrence of of several areas of reactive plasticity and neuron degeneration in AD.     

Cigarette Smoke Inhibits Brain Mitochondrial Adaptations of Exercised Mice

Physical exercise and smoking are environmental factors that generally cause opposite health-promoting adaptations. Both physical exercise and smoking converge on mitochondrial adaptations in various tissues, including the pro-oxidant nervous system. Here, we analyzed the impact of cigarette smoking on exercise-induced brain mitochondrial adaptations in the hippocampus and pre-frontal cortex of adult mice. The animals were exposed to chronic cigarette smoke followed by 8 weeks of moderate-intensity physical exercise that increased mitochondrial activity in the hippocampus and pre-frontal cortex in the non-smoker mice. However, mice previously exposed to cigarette smoke did not present these exercise-induced mitochondrial adaptations. Our results suggest that smoking can inhibit some brain health-promoting changes induced by physical exercise.     

Altered expression of claudin family proteins in Alzheimer’s disease and vascular dementia brains

Claudins (Cls) are a multigene family of transmembrane proteins with different tissue distribution, which have an essential role in the formation and sealing capacity of tight junctions (TJs). At the level of the blood–brain barrier (BBB), TJs are the main molecular structures which separate the neuronal milieu from the circulatory space, by a restriction of the paracellular flow of water, ions and larger molecules into the brain. Different studies suggested recently significant BBB alterations in both vascular and degenerative dementia types. In a previous study we found in Alzheimer’s disease (AD) and vascular dementia (VaD) brains an altered expression of occludin, a molecular partner of Cls in the TJs structure. Therefore in this study, using an immunohistochemical approach, we investigated the expression of Cl family proteins (Cl‐2, Cl‐5 and Cl‐11) in frontal cortex of aged control, AD and VaD brains. To estimate the number of Cl‐expressing cells, we applied a random systematic sampling and the unbiased optical fractionator method. We found selected neurons, astrocytes, oligodendrocytes and endothelial cells expressing Cl‐2, Cl‐5 and Cl‐11 at detectable levels in all cases studied. We report a significant increase in ratio of neurons expressing Cl‐2, Cl‐5 and Cl‐11 in both AD and VaD as compared to aged controls. The ratio of astrocytes expressing Cl‐2 and Cl‐11 was significantly higher in AD and VaD as compared to aged controls. The ratio of oligodendrocytes expressing Cl‐11 was significantly higher in AD and the ratio of oligodendrocytes expressing Cl‐2 was significantly higher in VaD as compared to aged controls. Within the cerebral cortex, Cls were selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings suggest a new function of Cl family proteins which might be linked to response to cellular stress.     

Increased expression of the lysosomal cholesterol transporter NPC1 in Alzheimer's disease

The Niemann-Pick type C1 (NPC1) protein mediates the trafficking of cholesterol from lysosomes to other organelles. Mutations in the NPC1 gene lead to the retention of cholesterol and other lipids in the lysosomal compartment, and such defects are the basis of NPC disease. Several parallels exist between NPC disease and Alzheimer's disease (AD), including altered cholesterol homeostasis, changes in the lysosomal system, neurofibrillary tangles, and increased amyloid-beta generation. How the expression of NPC1 in the human brain is affected in AD has not been investigated so far. In the present study, we measured NPC1 mRNA and protein expression in three distinct regions of the human brain, and we revealed that NPC1 expression is upregulated at both mRNA and protein levels in the hippocampus and frontal cortex of AD patients compared to control individuals. In the cerebellum, a brain region that is relatively spared in AD, no difference in NPC1 expression was detected. Similarly, murine NPC1 mRNA levels were increased in the hippocampus of 12-month-old transgenic mice expressing a familial AD form of human amyloid-beta precursor protein (APP) and presenilin-1 (APP/PS1tg) compared to 12-month-old wild type mice, whereas no change in NPC1 was detected in mouse cerebellum. Immunohistochemical analysis of human hippocampus indicated that NPC1 expression was strongest in neurons. However, in vitro studies revealed that NPC1 expression was not induced by transfecting SK-N-SH neurons with human APP or by treating them with oligomeric amyloid-beta peptide. Total cholesterol levels were reduced in hippocampus from AD patients compared to control individuals, and it is therefore possible that the increased expression of NPC1 is linked to perturbed cholesterol homeostasis in AD.     

Amyloid precursor protein and proinflammatory changes are regulated in brain and adipose tissue in a murine model of high fat diet-induced obesity

Background

Middle age obesity is recognized as a risk factor for Alzheimer''s disease (AD) although a mechanistic linkage remains unclear. Based upon the fact that obese adipose tissue and AD brains are both areas of proinflammatory change, a possible common event is chronic inflammation. Since an autosomal dominant form of AD is associated with mutations in the gene coding for the ubiquitously expressed transmembrane protein, amyloid precursor protein (APP) and recent evidence demonstrates increased APP levels in adipose tissue during obesity it is feasible that APP serves some function in both disease conditions.

Methodology/Principal Findings

To determine whether diet-induced obesity produced proinflammatory changes and altered APP expression in brain versus adipose tissue, 6 week old C57BL6/J mice were maintained on a control or high fat diet for 22 weeks. Protein levels and cell-specific APP expression along with markers of inflammation and immune cell activation were compared between hippocampus, abdominal subcutaneous fat and visceral pericardial fat. APP stimulation-dependent changes in macrophage and adipocyte culture phenotype were examined for comparison to the in vivo changes.

Conclusions/Significance

Adipose tissue and brain from high fat diet fed animals demonstrated increased TNF-α and microglial and macrophage activation. Both brains and adipose tissue also had elevated APP levels localizing to neurons and macrophage/adipocytes, respectively. APP agonist antibody stimulation of macrophage cultures increased specific cytokine secretion with no obvious effects on adipocyte culture phenotype. These data support the hypothesis that high fat diet-dependent obesity results in concomitant pro-inflammatory changes in brain and adipose tissue that is characterized, in part, by increased levels of APP that may be contributing specifically to inflammatory changes that occur.