Enhanced localization of amyloid β precursor protein in the rat hippocampus following long-term adrenalectomy

Using various antibodies to the amyloid ß precursor protein (APP) associated with Alzheimer's disease, we investigated changes in the distribution of APP in the hippocampus and neocortex of adrenalectomized (ADX) rats. In contrast to sham-operated controls, ADX rats euthanised after a survival period of 5 months showed striking APP reactivity in the CA1–CA4 fields and in the surviving cells in the dentate gyrus. Our results suggest the enhanced APP reactivity in hippocampal neurons may pertain to previous observations on the accumulation of APP fragments in the neocortex during ischemic or traumatic injury. Thus, long-term hormone deprivation would be another factor, which may influence the expression of APP in brain.     

突变型APP双荧光融合基因的构建和FRET对其表达的研究

目的探讨Alzheimer病的发病机制,研究淀粉样蛋白前体(APP)酶解过程,构建含有Swedish突变的双荧光真核表达系统。方法通过聚合酶链式反应(PCR)得到蓝色荧光蛋白(CFP)和黄色荧光蛋白碱基序列(YFP),生物合成含有Swedish突变的APP中间54个碱基片段(54bp),利用基因工程技术将CFP、54bp、YFP片段克隆至载体质粒pcDNA3.0中,得到重组质粒pcDNA3.0-CFP-54bp-YFP,将其转染至人神经母细胞瘤细胞(SH-SY5Y)中,利用激光共聚焦显微镜观察荧光表达,检测荧光共振能量转移(FRET)。结果酶切和基因序列分析证明重组质粒构建成功;对转染细胞的荧光和FRET检测发现融合基因能够准确表达荧光,同时可以观测到FRET现象。结论含有Swedish突变的荧光融合基因的构建为探索AD的发病机制、活体观察APP裂解奠定了基础。     

Caspase-3 activation in oligodendrocytes from the myelin-deficient rat

The myelin-deficient (MD) rat has a point mutation in its proteolipid protein (PLP) gene that causes severe dysmyelination and oligodendrocyte cell death. Using an in vitro model, we have shown that MD oligodendrocytes initially differentiate similarly to wild-type cells, expressing galactocerebroside, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin basic protein. However, at the time when PLP expression would normally begin, the MD oligodendrocytes die via an apoptotic pathway involving caspase activation. The active form of caspase-3 was detected, along with the cleavage products of poly-(ADP-ribose) polymerase (PARP) and spectrin, major targets of caspase-mediated proteolysis. A specific inhibitor of casapse-3, Ac-DEVD-CMK, reduced apoptosis in MD oligodendrocytes, but the rescued cells did not mature fully or express myelin-oligodendrocyte glycoprotein. These results suggest that mutant PLP affects not only cell death but also oligodendrocyte differentiation.     

The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects

The 4.2-kilodalton (kDa) polypeptide associated with the cerebral amyloid deposits of Alzheimer's disease (AD) derives from a much larger protein that is encoded by a gene on chromosome 21. In the present study, we have used antibodies raised against portions of the amyloid protein precursor (APP) to map its normal distribution and to gain further insights into the events that lead to amyloid deposition. Antibodies raised against several different portions of APP reacted with proteins having apparent molecular sizes of 65, 67, and 132 kDa on Western blots. In sections through the normal human brain, immunocytochemistry revealed punctate concentrations of the protein in pyramidal cells of the neocortex, particularly in associative regions, and intense staining in the CA1 pyramidal cells of the hippocampus. By electron microscopy, this punctate distribution coincided with dense concentrations of the protein in secondary lysosomes. In the hippocampus of several AD cases examined, abnormally dense immunostaining in enlarged intracellular domains accompanied a severe atrophy of the CA1 neurons. These data suggest that accumulations of APP in lysosomes of particular neurons may, in AD, lead to proteolytic events that form the insoluble 4.2-kDa amyloid peptide.     

Early stages of probable Alzheimer disease are associated with changes in platelet amyloid precursor protein forms

Previous findings demonstrated an altered pattern of amyloid precursor protein (APP) forms in platelets of Alzheimer disease (AD) patients, compared both with healthy control subjects or patients with non-Alzheimer-type dementia. The present study aims to evaluate whether platelet APP form ratio (APPr) is altered in patients with early stage AD. We selected 40 patients with early stage AD and 40 age-matched healthy controls. Compared with controls (mean±SD=0.91±0.3), mean APPr was decreased in AD (mean±SD=0.46±0.26, p<0.0001). Sixteen very mild AD patients (clinical dementia rating=0.5), identified among the AD group, showed a significant decrease of APPr values (mean±SD=0.50±0.3, p<0.0001). These findings indicate that alteration of APP processing in platelets is an early event and suggest that this assay might be of diagnostic value in differentiating mild AD from normal ageing. Received: 20 November 2001 / Accepted in revised form: 16 June 2002     

Chronic elevation of amyloid precursor protein in the neocortex or hippocampus of marmosets with selective cholinergic lesions

Summary. In vitro studies have consistently demonstrated a link between cholinergic neurotransmission and amyloid precursor protein metabolism, although few studies have examined such a relationship in vivo and none have been conducted in primate species. The purpose of this study was to test the hypothesis that a reduction in cholinergic activity in neocortical and hippocampal areas consequent upon destruction of ascending cholinergic projections may lead to long-term changes in levels of amyloid precursor protein in these target areas in a primate species. The status of three synaptic proteins associated with neurotransmitter release, synaptophysin, syntaxin and SNAP-25, was also been examined. Selective immunolesions of the basal forebrain cholinergic projections led to increases in amyloid precursor protein-like immunoreactivity in hippocampus and cortex, measured 8 months postlesion. Furthermore, reductions in cortical and hippocampal SNAP-25, but not syntaxin or synaptophysin, immunoreactivity were observed. These results imply that the reduced cholinergic activity characteristic of Alzheimer's disease may contribute to the continuing emergence of neuropathology in addition to the well-known association with cognitive dysfunction. Received September 28, 2000; accepted January 31, 2001     

Frequency of mutations in the presenilin and amyloid precursor protein genes in early-onset Alzheimer disease in Spain

BACKGROUND: The relative contribution of mutations in the presenilin (PSEN) and amyloid precursor protein genes to autosomal dominant and other early-onset Alzheimer disease (AD) cases is not well established. OBJECTIVES: To clarify the respective contribution of the amyloid precursor protein and PSEN mutations to autosomal dominant AD and to determine its contribution to sporadic and familial nonautosomal dominant early-onset AD and familial late-onset AD in a referral-based Spanish population. SUBJECTS AND METHODS: Ninety-four patients with AD (60 with early-onset AD and 34 with late-onset AD) from 82 independent families were studied. According to the family history, patients were classified into the following groups: autosomal dominant, familial nonautosomal dominant, and sporadic. Mutational analysis of the coding regions of the amyloid precursor protein, presenilin 1, and presenilin 2 was performed in all patients. Apolipoprotein E was also genotyped. RESULTS: Of the 60 early-onset cases, 44 from 36 families had a positive family history (11 with autosomal dominant AD and 25 with familial nonautosomal dominant AD) and 16 were sporadic. The frequency of mutations was 54.6% (6/11) among the autosomal dominant group, 6.2% (1/16) among the sporadic group, and 4% (1/25) among the familial nonautosomal dominant AD group. Most PSEN mutations (92%) would have been detected using a cutoff age of 58 years. The apolipoprotein E epsilon4 allele frequency was increased among early-onset AD without PSEN mutations. CONCLUSIONS: More than half of the families with autosomal dominant early-onset AD can be explained by coding mutations in the PSEN genes. In the familial and sporadic early-onset groups mutations are rare. When family history is unavailable, an age of 58 years may be used as a cutoff point for genetic analysis. The increased apolipoprotein E epsilon4 allele in patients without PSEN mutations confirms that it is an important risk factor in the cause of non-PSEN early-onset AD.     

Measurement of alpha- and beta-secretase cleaved amyloid precursor protein in cerebrospinal fluid from Alzheimer patients

One of the major histopathological hallmarks of Alzheimer's disease (AD) is redundant senile plaques mainly composed of beta-amyloid (Abeta) aggregates. Alternative cleavage of the amyloid precursor protein (APP), occurring in both normal and AD subjects, results in the generation and secretion of soluble APP (sAPP) and Abeta. We examined the cerebrospinal fluid (CSF) for alpha- and beta-secretase cleaved sAPP (alpha-sAPP and beta-sAPP) in 81 sporadic AD patients, 19 patients with mild cognitive impairment, and 42 healthy controls by using newly developed sandwich enzyme-linked immunosorbent assay methods. We found that neither the level of CSF-alpha-sAPP nor CSF-beta-sAPP differed between sporadic AD patients and healthy controls. These findings further support the conclusion that there is no change in APP expression in sporadic AD. However, the level of CSF-beta-sAPP was significantly increased in patients with mild cognitive impairment compared to controls. We also investigated the relationship between the CSF level of alpha/beta-sAPP and Abeta(42) and the apoE epsilon 4 (apoE4) allele. Significantly lower levels of CSF-alpha-sAPP were found in AD patients possessing one or two apoE4 alleles than in those not possessing the apoE4 allele. Neither the levels of CSF-beta-sAPP nor CSF-Abeta(42) differed when comparing ApoE4 allele-positive with allele-negative individuals.     

Glutamate receptor dysregulation in the hippocampus of transgenic mice carrying mutated human amyloid precursor protein

Alzheimer's disease transgenic mice overexpressing human amyloid precursor protein (hAPP) with the Swedish double mutation (hAPP(Sw)) develop age-related amyloid deposition and behavioral and electrophysiologic changes by an unknown mechanism. Analysis of glutamatergic receptor subtypes in 4- and 15-month-old heterozygous hAPP(Sw) transgenic mice revealed a selective increase in AMPA receptor binding in the hippocampus of 15-month-old transgenic mice, which have established cortical and hippocampal amyloid deposits. There were no significant alterations of GluR1, GluR2, and GluR4 protein expression by semiquantitative confocal analysis or GluR1 mRNA by in situ hybridization. There was no significant alteration in NMDA, in group I and II metabotropic glutamate and in muscarinic receptor binding, or in striatal dopamine and adenosine receptor binding in 15-month-old mice. These data suggest that mutant APP overexpression or age-related amyloid deposition produce a subtle specific alteration in hippocampal glutamate receptors with aging.     

Expressing mRNAs for presenilin-1 and amyloid precursor protein (APP-695) from same neuronal populations in rat hippocampus

Presenilin-1 gene (PS1, s182) encoding a protein that contains seven transmembrane domains was recently identified as a risk factor for Alzheimer’s disease (AD). Mutations on this gene may contribute to pathogenesis of AD. In this study, we used in situ hybridization histochemistry to detect the expression of PS1 in neurons in the hippocampal formation and cerebral cortex of the rat brain. Specifically, mRNAs of PS1 were predominantly found in pyramidal neurons in CA1, CA3, and granule cells in the hilar region of the dentate gyrus of the hippocampus and in the external and internal pyramidal layers of the cerebral cortex. The same neuronal populations express the amyloid protein precursor (APP-695). Transgenic mice expressing a mutated APP-695 produced increased levels of β-amyloid (Aβ) and exhibited behavioral deficits. These results lead us to suggest that when PS1 and APP-695 colocalize in neurons in the brain, PS1 may play a regulatory role in production of (Aβ) protein of AD.     

Abnormalities in the pattern of platelet amyloid precursor protein forms in patients with mild cognitive impairment and Alzheimer disease.

CONTEXT: Patients affected by sporadic Alzheimer disease (AD) show a significant alteration of amyloid precursor protein (APP) forms in platelets when compared with patients with dementia but without AD and age-matched controls. OBJECTIVE: To evaluate the ratio of platelet APP forms (APPr) in early-stage AD and mild cognitive impairment (MCI) and its potential as a biomarker for the early identification of AD. SETTING: Community population-based sample of patients admitted to 4 AD centers for investigation of cognitive disturbances. DESIGN AND METHODS: Thirty-five patients with mild AD (mAD), 21 patients with very mild AD (vmAD), 30 subjects with MCI, and 25 age-matched controls were included. The APPr was evaluated by Western blot analysis in platelet homogenate. RESULTS: Compared with controls (mean +/- SD, 0.93 +/- 0.3), the mean APPr was decreased in patients with mAD (0.44 +/- 0.24; P<.001) and patients with vmAD (0.49 +/- 0.3; P<.001). Regarding the MCI group, a significant decrease in APPr was found compared with controls (0.62 +/- 0.33; P<.001). Fixing a cutoff score of 0.6, sensitivity was 88.6% (31/35) for patients with mAD and 85.7% (18/21) for patients with vmAD, whereas specificity was 88% (22/25) for controls. Among patients with MCI, 18 (60%) of 30 individuals displayed APPr values below the cutoff. CONCLUSIONS: Alteration of platelet APP forms is an early event in AD, and the measurement of APPr may be useful for the identification of preclinical AD in patients with MCI.     

Decreased expression of hippocampal cholinergic neurostimulating peptide precursor protein mRNA in the hippocampus in Alzheimer disease

Hippocampal cholinergic neurostimulating peptide (HCNP) is involved in the phenotype development of the septo-hippocampal system. HCNP precursor protein (HCNP-pp) is known to interact with other molecules including phosphatidylethanolamine and Raf-1 kinase, and is also known as phosphatidylethanolamine-binding protein and raf kinase-inhibitory protein. To assess whether HCNP-pp is involved in the pathogenesis of Alzheimer disease (AD), the expression levels of its mRNA in the hippocampus of autopsy brains from patients with dementia (including AD and ischemic vascular dementia) were compared with those of non-demented control subjects. The in situ hybridization analysis revealed that the expression of HCNP-pp mRNA in patients with clinically late-onset AD was decreased in the hippocampal CA1 field, but not in the CA3 field or the dentate gyrus. The early-onset AD patients showed a wide range of expression levels in the hippocampal sub-regions. Northern blot analysis of HCNP-pp mRNA in brain tissue supported these observations. Since HCNP is known to stimulate the enzymatic activity of choline acetyltransferase in neurons, its low expression in the CAI field of AD patients may explain the downregulation of cholinergic neurons seen in these patients and may thus contribute to the pathogenic processes underlying AD.     

Notch 1 interacts with the amyloid precursor protein in a Numb-independent manner

We hypothesized that the physical interaction between the amyloid precursor protein (APP) and Notch 1 (N1) may be mediating the reported cross-talk between the respective signaling pathways. Immunoprecipitation of mouse N1 (mN1) or extracellular domain truncated mN1 (mN1-TM, mimics TACE-produced membrane-bound C-terminal fragment) specifically coprecipitated APP(751). Conversely, immunoprecipitation of APP(751) specifically coprecipitated mN1, furin-generated membrane-bound mN1 C-terminal fragment (f.mN1-TM), or mN1-TM. The London mutation of APP did not affect the APP(751)/mN1 interaction. Coexpression of APP(751) and mN1 did not affect APP processing or production of mN1 intracellular domain (mNICD). The APP(751)/mN1 interaction was Numb-independent, insofar as it was observed in HEK293 cells that lack detectable levels of Numb and was unaffected by the expression of exogenous Numb or deletion of the APP cytoplasmic domain, including the Numb-binding YENPTY sequence. This interaction was unaffected even when the N-terminal 647 amino acids of APP were replaced by a sequence of secreted alkaline phosphatase. These data combined with data showing interaction between mN1-TM and APP(751) suggest that their transmebrane domains and short sequences around them are sufficient for the interaction and that APP(751) and mN1 interact in cis. Our results imply novel functions of APP and/or N1 that derive from their interaction.     

Impaired Pavlovian cued fear conditioning in Tg2576 mice expressing a human mutant amyloid precursor protein gene

The processing of emotional and/or fear-related events is abnormal in patients with Alzheimer's disease. AD is accompanied by a number of neuropathological features, one of which is the deposition of amyloid plaques. The main aim of the present study was to examine the effects of a human amyloid precursor protein mutation on both the acquisition and expression of fear conditioning in Tg2576 mice. Sixteen-month-old, but not 4-month-old, transgenic mice showed aberrations in post-shock freezing during training. In a retention test carried out 24 h after training, Tg2576 mice showed comparable levels of conditioned fear elicited by contextual cues. However, freezing elicited by a tone conditioned stimulus was impaired in 16-month-old but not 4-month-old Tg2576 mice. The results are discussed with reference to the role of cue competition (overshadowing) in revealing fear conditioning deficits in Tg2576 mice.     

Roles of A beta and carboxyl terminal peptide fragments of amyloid precursor protein in Alzheimer disease

Several lines of evidence indicate that A beta may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of A beta and the development of the disease. Thus, A beta may not be the sole active fragment of beta-amyloid precursor protein (betaAPP) in the neurotoxicity assiciated with AD. We focused on the amyloidegenic carboxyl terminal fragments of betaAPP containing the full length of A beta (CT105). We synthesized a recombinant carboxyl-terminal 105 amino acid fragment of betaAPP and examined the effects of CT105 and A beta on cultured neurons, Ca++ uptake into rat brain microsomes, Na+-Ca++ exchange activity, ion channel forming activity in lipid bilayers and passive avoidance performance of mice. Our results suggest that the cytotoxic and channel inducing effects of CT105 are much more potent than that of A beta and toxic mechanisms of CT105 are different from those of A beta. Taken together, these lines of evidence postulate that CT is an alternative toxic element important in the generation of the symptoms common to AD.     

Age and damage induced changes in amyloid protein precursor immunohistochemistry in the rat brain

Alzheimer's disease (AD) is characterized by the extensive deposition of the 42-amino-acid β-amyloid or A4 protein in neuritic plaques and neurofibrillary tangles within the brain. This protein is liberated from the much larger amyloid protein precursor (APP). Multiple species of APP have been proposed, including several forms that contain a 56 amino acid insert sequence analogous to the Kunitz protease inhibitors. Although expression of APP mRNA is reportedly altered in AD brain and various roles for APP have been proposed, the pathogenesis of amyloid deposition and AD remains unclear. AD is also characterized by specific memory impairments associated with decreased cholinergic activity. While aging rats do not develop mature amyloid pathology, behaviorally impaired aged rats demonstrate an analogous cholinergic decline. In this study, we examined behaviorally characterized aged rats and normal young controls for changes in APP immunohistochemistry by using anti-APP antibodies, which detect N- or C-terminal regions and which distinguish APP species with or without the Kunitz protease inhibitor domain. The results show specific age- and behavior-related changes in cortical APP immunoreactivity as well as limited numbers of APP immunoreactive deposits in the aged rats. Additionally, we found that lesions of the fimbria-fornix pathway, which in part mimic the memory impairments and loss of cholinergic activity seen in AD, result in the marked accumulation of APP immunoreactive material in the region of cholinergic fiber degeneration in the hippocampus. These findings are discussed in relation to the pathogenesis of AD in humans. © 1994 Wiley-Liss, Inc.     

Early-onset Alzheimer disease caused by a new mutation (V717L) in the amyloid precursor protein gene

CONTEXT: Alzheimer disease is the most common form of dementia. Mutations in the genes amyloid precursor protein (APP), presenilin 1(PS1) and presenilin 2(PS2) have been found in early-onset familial forms of Alzheimer disease OBJECTIVE: To determine the cause of dementia in a family with early-onset illness. DESIGN, SETTING, AND PARTICIPANTS: A family with a history of dementia was referred to the Indiana Alzheimer Disease Center, Indianapolis. All the research in this study was done in a university or university hospital. The proband and her 4 siblings took part in the study. The proband, who is still alive, showed symptoms of Alzheimer disease at 38 years of age. Genomic DNA was obtained from blood samples of 5 family members. The APPandPS1genes of the proband were screened for mutations by amplification followed by direct sequencing. RESULTS: Sequence of exon 17 of the APPgene revealed a single nucleotide (guanine to cytosine) substitution in 1 allele, resulting in an amino acid change at codon 717 (valine to leucine). Each of the proband's siblings were tested for this mutation by direct sequencing. Two of the 4 were found to have the mutation; one of whom was recently clinically diagnosed at the age of 36 years. CONCLUSIONS: A novel mutation in the APPgene (V717L) has been found in a family with a history of dementia, beginning in the mid to late 30s. The age of onset in this family is earlier than most of the other families with Alzheimer disease who also have APPmutations. Arch Neurol. 2000.     

Plaque-associated α-synuclein (NACP) pathology in aged transgenic mice expressing amyloid precursor protein

Patients with the Lewy body variant (LBV) of Alzheimer's disease (AD) have ubiquitinated intraneuronal and neuritic accumulations of α-synuclein and show less neuron loss and tau pathology than other AD patients. Aged Tg2576 transgenic mice overexpressing human βAPP695. KM670/671NL have limited neuron loss and tau pathology, but frequent ubiquitin- and α-synuclein-positive, tau-negative neurites resembling those seen in the LBV of AD.     

Caspase-3 activation in the hippocampus of rat pups is modulated by the effectiveness of escape behavior in response to footshock

We studied the time course of caspase-3 activity in the hippocampus of rat pups characterized by different efficacy of performance, i.e., number of escapes, induced by a brief footshock (40 V) on the 13th postnatal day (PND). The pups could escape footshock by crossing an open door (EF group). After crossing, the stimulation was stopped and resumed within 1 min until the total time of footshock reached 200 s. The data for the EF group were compared with the data of passive control group. The second control group included rat pups subjected to inescapable stimulation (IF group). Immediately after the stimulation, hippocampal caspase-3 activity was decreased in the EF group; this effect was not observed in the IF group. The lowest enzyme activity was found in the hippocampus of the animals with the largest number of escapes. By PND 17, the caspase-3 activity in the hippocampus of the “most successful” pups reached the control level and, in the IF group, the caspase-3 activity was suppressed. Thereafter (21–26 PND), the caspase-3 activity significantly increased in the EF group, but not in the IF group. The maximum caspase-3 levels was found in the hippocampus of the most unsuccessful rats (1–2 escapes).