Accumulation of amyloid precursor fragment in Alzheimer plaques.

Regenerative and degenerative neurites are components of classical senile plaques found in brain tissue of patients with Alzheimer's disease (AD). Amyloid beta/A4-protein derived from its precursor, amyloid beta/A4-protein precursor (APP/ABPP), constitutes the major portion of the amyloid core of senile plaques. A large N-terminal portion of APP (approximately Mr 100,000) is released from cells, leaving a minor C-terminal portion (approximately Mr 15,000) behind. A series of antisera against various sequences of APP were prepared and used to study the localization of each sequence in brain tissue. Plaque neurites stained as intensely as neuronal cell bodies with three antisera against the N-terminal portion of APP (N-terminal to a.a. 225), whereas five other antisera directed against the other C-terminal portions of APP (a.a. 284 to C-terminal) and antisera against the Kunitz-type protease inhibitor portion of APP stained plaque neurites less intensely than neuronal cell bodies in the hippocampus. These results suggest that a major part of the APP present in the neuritic component of senile plaques is a fragment representing the N-terminal one-third of the molecule.     

Increased S100 beta neurotrophic activity in Alzheimer's disease temporal lobe.

The confirming diagnosis of Alzheimer's disease includes an assessment of the concentration of neuritic plaques in the temporal lobe of the brain. The presence of abnormal levels of neurotrophic factors in Alzheimer's disease is one possible explanation for the increased concentration of aggregates of overgrown neurites in the neuritic plaques of Alzheimer's disease. The protein S100 beta, a neurotrophic factor produced by astroglia in the brain, induces neurite outgrowth in cerebral cortical neurons. The generation of specific S100 beta antibodies, the cloning of a full-length cDNA encoding the S100 beta mRNA, and the development of a neurite extension assay system for S100 beta allowed testing of the hypothesis that Alzheimer's disease S100 beta expression is elevated in brain temporal lobe where neuritic plaques are concentrated. The levels of S100 beta protein, mRNA, and specific neurotrophic activity were elevated 10-20-fold in extracts of temporal lobe from autopsy samples of Alzheimer's disease patients compared to those of aged control patients. The cells containing the increased S100 beta were reactive astrocytes; the neuritic plaques were surrounded by S100 beta-containing astrocytes. The elevated levels of S100 beta provides a link between the prominent reactive gliosis and neuritic plaque formation in this common disease of the elderly and raises the possibility that S100 beta contributes to Alzheimer's disease neuropathology.     

Perlecan domain V inhibits α2 integrin-mediated amyloid-β neurotoxicity

Amyloid-β (Aβ) peptide is a key component of amyloid plaques, one of the pathological features of Alzheimer's disease. Another feature is pronounced cell loss in the brain leading to an enlargement of the ventricular area and a decrease in brain weight and volume. Aβ plaque deposition and neuronal toxicity can be modeled by treating human cortical neuronal cultures with Aβ and showing robust Aβ deposition and neurotoxicity that is mediated by α2β1 and αvβ1 integrins. The current study expands on these findings by showing that the domain V of perlecan, a known α2 integrin ligand, inhibits Aβ neurotoxicity in an α2 integrin-dependent manner. Additionally, Aβ binds more efficiently to cells expressing activated α2 integrin. Finally the inhibition of Aβ neurotoxicity with domain V is synergistic with inhibitors of αv integrin and β1 integrin. We propose that domain V and potentially other α2 integrin ligands could be a new therapeutic approach for inhibiting the Aβ plaque deposition and neurotoxicity observed in Alzheimer's disease.     

Neuronal activity regulates the regional vulnerability to amyloid-β deposition

Amyloid-β (Aβ) plaque deposition in specific brain regions is a pathological hallmark of Alzheimer's disease. However, the mechanism underlying the regional vulnerability to Aβ deposition in Alzheimer's disease is unknown. Herein, we provide evidence that endogenous neuronal activity regulates the regional concentration of interstitial fluid (ISF) Aβ, which drives local Aβ aggregation. Using in vivo microdialysis, we show that ISF Aβ concentrations in several brain regions of APP transgenic mice before plaque deposition were commensurate with the degree of subsequent plaque deposition and with the concentration of lactate, a marker of neuronal activity. Furthermore, unilateral vibrissal stimulation increased ISF Aβ, and unilateral vibrissal deprivation decreased ISF Aβ and lactate, in contralateral barrel cortex. Long-term unilateral vibrissal deprivation decreased amyloid plaque formation and growth. Our results suggest a mechanism to account for the vulnerability of specific brain regions to Aβ deposition in Alzheimer's disease.     

Cerebrovascular transforming growth factor-beta contributes to inflammation in the Alzheimer's disease brain

Inflammatory mechanisms are thought to contribute to lesion pathogenesis and neuronal cell death in Alzheimer's disease. Transforming growth factor-beta (TGF-beta) plays a central role in the response of the brain to injury, and is increased in the brain in Alzheimer's disease. In this study we determine whether expression of TGF-beta is abnormal in the microvasculature in Alzheimer's disease and whether TGF-beta affects vascular production of pro-inflammatory cytokines, interleukin (IL)-1 beta, and tumor necrosis factor (TNF)-alpha. Microvessels isolated from the cortices of Alzheimer's disease patients and age-matched controls are analyzed for microvessel-associated and released TGF-beta. Results from Western blot analysis and enzyme-linked immunosorbent assay indicate a higher level of TGF-beta in Alzheimer's disease vessels compared to controls. To determine whether TGF-beta affects vascular release of inflammatory factors, cultured brain endothelial cells are treated with TGF-beta and levels of IL-1 beta and TNF-alpha determined. Both enzyme-linked immunosorbent assay and Western blot analyses show that untreated endothelial cells express little IL-1 beta or TNF-alpha, but incubation with TGF-beta results in robust expression of these factors by brain endothelial cells. Our results suggest that vessel-derived TGF-beta contributes to inflammatory processes in the Alzheimer brain.     

An antiserum against amyloid β-protein precursor detects a unique peptide in Alzheimer brain

An antiserum was raised against an amino acid sequence predicted from the DNA sequence of amyloid β-protein precursor (ABPP), and it was then affinity-purified. This affinity-purified antibody (anti-GID) intensely stained neurons and dystrophic neurites in plaques of Alzheimer's disease (AD) patients, but marginally stained neurons of age-matched normal individuals. Anti-GID antibody detected a series of protein bands with a molecular weight centered at 100,000 and a second band at 55,000 on a blot of the human brain particulate fraction. It also stained a set of bands with a molecular weight around 95,000 and a doublet of M_r 16,000 in the soluble fraction. A band at M_r 35,000 was detected in the soluble fraction prepared from brain tissue of AD patients but not from control brain tissue. A strong immunostaining of AD sections with anti-GID and the presence of a M_r 35,000 band unique to AD might reflect an altered processing of ABPP in AD brains     

The molecular pathogenesis of astrogliosis in scrapie and Alzheimer''s disease

In slow infections caused by scrapie and other unconventional agents, and in Alzheimer's disease (AD), the formation of neuritic plaques and the increase in astrocytes and astrocyte-specific protein, glial fibrillary acidic protein (GFAP), are pathological changes common to both conditions. With the rationale that these parallels imply convergent pathogenetic mechanisms, we identified a gene whose expression increases in both. We now report the results of a more extensive analysis of this gene and show that by sequence analysis it is highly homologous and likely identical to GFAP. GFAP mRNA accumulates late in the course of scrapie in subpial and periventricular astrocytes and in cells in foci in the hippocampus. The increased abundance of GFAP mRNA is accompanied by an increase in the corresponding protein. GFAP mRNA is localized by in situ hybridization to the cell body and processes of astrocytes. In AD, the latter pattern predominates, consistent with induction of GFAP mRNA in the sites of synthesis in glial processes in the neuritic plaque.     

ADAM10 expression and promoter haplotype in Alzheimer's disease

Alzheimer's disease is confirmed at autopsy according to the accumulation of brain neuritic plaques and neurofibrillary tangles in the brain. Neuritic plaques contain amyloid-β (Aβ) and lower levels of Aβ correspond to an increase in ADAM10 α-secretase activity. ADAM10 α-secretase activity produces a soluble amyloid precursor protein (APP) alpha (sAPPα) product and negates the pathological production of Aβ. In this investigation, it was hypothesized that genetic variation with the ADAM10 promoter is associated with ADAM10 expression levels as well as cerebrospinal fluid sAPPα levels. Results from this investigation suggest that the ADAM10 rs514049-rs653765 C-A promoter haplotype is associated with: (1) higher CSF sAPPα levels in cognitively normal controls compared with Alzheimer's disease (AD) patients, (2) higher postmortem brain hippocampus, but not cerebellum, ADAM10 protein levels in subjects with low plaque scores compared with those with high plaque scores, and (3) higher promoter activity for promoter-only reporter constructs compared with promoter 3' untranslated region (3'UTR) constructs in the human neuroblastoma SHSY5Y cell line, but not in HepG2 or U118 cell lines. Taken together, these findings suggest that ADAM10 expression is modulated according to a promoter haplotype that is influenced in a brain region- and cell type-specific manner.     

Evidence that neurones accumulating amyloid can undergo lysis to form amyloid plaques in Alzheimer's disease

AIMS: Amyloid has recently been shown to accumulate intracellularly in the brains of patients with Alzheimer's disease (AD), yet amyloid plaques are generally thought to arise from gradual extracellular amyloid deposition. We have investigated the possibility of a link between these two apparently conflicting observations. METHODS AND RESULTS: Immunohistochemistry and digital image analysis was used to examine the detailed localization of beta-amyloid(42) (A beta 42), a major component of amyloid plaques, in the entorhinal cortex and hippocampus of AD brains. A beta 42 first selectively accumulates in the perikaryon of pyramidal cells as discrete, granules that appear to be cathepsin D-positive, suggesting that they may represent lysosomes or lysosome-derived structures. AD brain regions abundantly populated with pyramidal neurones exhibiting excessive A beta 42 accumulations also contained evidence of neuronal lysis. Lysis of these A beta 42-burdened neurones apparently resulted in a local, radial dispersion of their cytoplasmic contents, including A beta 42 and lysosomal enzymes, into the surrounding extracellular space. A nuclear remnant was found at the dense core of many amyloid plaques, strengthening the idea that each amyloid plaque represents the end product of a single neuronal cell lysis. The inverse relationship between the amyloid plaque density and pyramidal cell density in the AD brain regions also supports this possibility, as does the close correlation between plaque size and the size of local pyramidal cells. CONCLUSIONS: Our findings suggest that excessive intracellular accumulation of A beta 42-positive material in pyramidal cells can result in cell lysis, and that cell lysis is an important source of amyloid plaques and neuronal loss in AD brains.     

An integrative hypothesis concerning the pathogenesis and progression of Alzheimer''s disease

Observations, in Alzheimer's disease, in the pattern of nerve cell damage and loss, the pathology, microchemistry and immunology of senile plaques and neurofibrillary tangles and alterations in blood vessels are drawn together into a hypothesis that attempts to explain the pathogenesis and progression of the disorder. At the heart of this hypothesis lies a defect in blood brain barrier function and/or structure within the cerebral cortex and this defect may be the cause of the cerebral vessel amyloidosis common in many patients with Alzheimer's disease. Age-related alterations in blood brain barrier allow for damage to nerve terminals and limited formation of senile plaques within cerebral cortex; neurofibrillary tangles are formed within cortical and subcortical nerve cells which project to or near damaged vessels/senile plaques. Uptake of “neurotoxin” at affected terminals and retrograde transport to perikarya causes neurofibrillary tangles to be formed; their accumulation leads to perikaryal changes culminating in cell death and loss. Loss of cells in cortically projecting areas of subcortex such as nucleus basalis, locus caeruleus and dorsal raphe, which terminate on cerebral vessels, causes further blood brain barrier dysfunction, new plaque formation and continued cell loss in cortex and subcortex. Once started, such a process could be self-perpetuating and the initial site of damage could lie within the amygdala/hippocampus with putative pathogenic agent accessing the brain via the olfactory pathways.     

Astrocytes in Alzheimer''s disease gray matter express alpha 1-antichymotrypsin mRNA.

The serine protease inhibitor alpha 1-antichymotrypsin (ACT) has been shown to be tightly associated with the amyloid found in plaque cores and blood vessels in the brains of patients with Alzheimer's disease (AD). Although the ACT found in plaques could be derived from the high levels of ACT in serum, previous Northern analysis revealed that ACT mRNA is produced locally in AD gray matter at much higher levels than in control gray matter. To determine which brain cells express ACT mRNA, we conducted in situ hybridization with 35S-labeled cRNA probes on hippocampal sections from four AD and three control cases. To identify astrocytes unequivocally, some of the hybridized sections were immunostained for glial fibrillary acidic protein, which is astrocyte-specific. Our results showed numerous astrocytes that were intensely labeled by the probe for ACT mRNA throughout the subicular gray matter of the AD cases. In contrast, astrocytes in control gray matter were rarely labeled by the probe for ACT mRNA. Examination of plaque cores in the AD subiculum showed that some astrocytes intensely labeled by the probe for ACT mRNA were closely associated with virtually every plaque core. Our results also showed many astrocytes in both AD and control white matter that were intensely labeled by the probe for ACT mRNA, and a small fraction of the astrocytes in a juvenile cerebellar astrocytoma that we examined were found to produce high levels of ACT mRNA. In every area in which astrocytes expressing ACT mRNA were found, astrocytes producing no detectable ACT message were also present. Our findings indicate that astrocytes produce the increased ACT mRNA in AD gray matter observed by Northern analysis, but they also show that ACT mRNA expression by astrocytes is not unique to AD. The presence of astrocytes expressing ACT mRNA near, and extending processes towards, plaque cores strongly suggests that some if not all of the ACT associated with amyloid plaque cores is produced by astrocytes surrounding the cores.     

牛膝多肽促大鼠海马神经元突起的生长

目的 研究牛膝多肽(ABPP)对体外培养的大鼠海马神经元突起生长的促进作用及其对生长相关蛋白-43 (GAP-43)和神经丝蛋白(NF-H)mRNA和蛋白表达的影响. 方法 以体外原代培养的胎鼠海马神经元为研究模型,通过四甲基偶氮唑盐(MTT)检测ABPP对海马神经元细胞活性的影响,通过免疫荧光细胞化学法,采用Image-Pro Express软件测量不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药24h,对海马神经元神经突起生长的影响;通过实时荧光定量PCR法定量分析不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药6h,对海马神经元GAP-43和NF-H基因表达的影响;采用免疫印迹法观察不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药24h后,对海马神经元GAP-43 和NF-H蛋白表达的影响.同时应用胞外信号调节激酶(ERK)特异性拮抗剂PD98059与ABPP共培养海马神经元,分析ABPP对海马神经元的作用与ERK通路的关系. 结果 ABPP可有效地促进海马神经元突起的生长,加药后24h浓度为1.0mg/L,作用最强;实时荧光定量RT-PCR、免疫荧光细胞化学法和免疫印迹检测的结果表明,ABPP能增加体外培养的海马神经元GAP-43和NF-H的mRNA和蛋白表达,PD98059可抑制该过程. 结论 ABPP能促进体外培养的海马神经元神经突起的生长,其作用与上调GAP-43和NF-H 基因的表达相关,并可能与ERK通路有关.     

Microvessels from Alzheimer's disease brains kill neurons in vitro

Understanding the pathogenesis of Alzheimer's disease is of widespread interest because it is an increasingly prevalent disorder that is progressive, fatal, and currently untreatable. The dementia of Alzheimer's disease is caused by neuronal cell death. We demonstrate for the first time that blood vessels isolated from the brains of Alzheimer's disease patients can directly kill neurons in vitro. Either direct co-culture of Alzheimer's disease microvessels with neurons or incubation of cultured neurons with conditioned medium from microvessels results in neuronal cell death. In contrast, vessels from elderly nondemented donors are significantly (P<0.001) less lethal and brain vessels from younger donors are not neurotoxic. Neuronal killing by either direct co-culture with Alzheimer's disease microvessels or conditioned medium is dose- and time-dependent. Neuronal death can occur by either apoptotic or necrotic mechanisms. The microvessel factor is neurospecific, killing primary cortical neurons, cerebellar granule neurons, and differentiated PC-12 cells, but not non-neuronal cell types or undifferentiated PC-12 cells. Appearance of the neurotoxic factor is decreased by blocking microvessel protein synthesis with cycloheximide. The neurotoxic factor is soluble and likely a protein, because its activity is heat labile and trypsin sensitive. These findings implicate a novel mechanism of vascular-mediated neuronal cell death in Alzheimer's disease.