Antisera to an amino-terminal peptide detect the amyloid protein precursor of Alzheimer's disease and recognize senile plaques

The cerebral amyloid deposited in Alzheimer's disease (AD) contains a 4.2 kDa beta amyloid polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). Three beta APP mRNAs encoding proteins of 695, 751, and 770 amino acids have previously been identified. In each of these, there is a single membrane-spanning domain close to the carboxyl-terminus of the beta APP, and the 42 amino acid beta AP sequence extends from within the membrane-spanning domain into the large extracellular region of the beta APP. We raised rabbit antisera to a peptide corresponding to amino acids 45-62 near the amino-terminus of the beta APP. We show that these antisera detect the beta APP by demonstrating that they (i) label a set of approximately 120 kDa membrane-associated proteins in human brain previously detected by antisera to the carboxyl-terminus of beta APP and (ii) label a set of approximately 120 kDa membrane-associated proteins that are selectively overexpressed in cells transfected with a full length beta APP expression construct. The beta APP45-62 antisera specifically stain senile plaques in AD brains. This finding, along with the previous demonstration that antisera to the carboxyl-terminus of the beta APP label senile plaques, indicates that both near amino-terminal and carboxyl-terminal domains of the beta APP are present in senile plaques and suggests that proteolytic processing of the full length beta APP molecule into insoluble amyloid fibrils occurs in a highly localized fashion at the sites of amyloid deposition in AD brains.     

Soluble derivatives of the beta amyloid protein precursor of Alzheimer's disease are labeled by antisera to the beta amyloid protein

The amyloid deposited in Alzheimer's disease (AD) is composed primarily of a 39-42 residue polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). In previous studies, we and others identified full-length, membrane-associated forms of the beta APP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble approximately 125 and approximately 105 kDa forms of the beta APP found in human cerebrospinal fluid are specifically labeled by several different antisera to the beta AP. This finding indicates that both soluble derivatives contain all or part of the beta AP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.     

Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices.

Recent studies indicated that the formation of a major constituent of Alzheimer's disease (AD) senile plaques, called beta A4-peptide, does not result from normal processing of its precursor, amyloid precursor protein (APP). Since proteolytic cleavage of APP inside its beta A4 sequence was found to be part of APP processing the formation of the beta A4-peptide seems to be prevented under normal conditions. We considered whether in AD one of the endogenous proteinase inhibitors might interfere with APP processing. After we had recently found that cultured human neuronal cells synthesize the most potent of the known human proteinase inhibitors, alpha-2-macroglobulin (alpha 2M), upon stimulation with the inflammatory mediator interleukin-6 (IL-6) we now investigated whether alpha 2M and IL-6 could be detected in AD brains. Here we report that AD cortical senile plaques display strong alpha 2M and IL-6 immunoreactivity while no such immunoreactivity was found in age-matched control brains. Strong perinuclear alpha 2M immunoreactivity in hippocampal CA1 neurons of Alzheimer's disease brains indicates that neuronal cells are the site of alpha 2M synthesis in AD brains. We did not detect elevated IL-6 or alpha 2M levels in the cerebrospinal fluid of AD patients. Our data indicate that a sequence of immunological events which seem to be restricted to the local cortical environment is part of AD pathology.     

Attenuated hippocampus-dependent learning and memory decline in transgenic TgAPPswe Fischer-344 rats

Alzheimer's disease (AD) is characterized by increased beta amyloid (Abeta) levels, extracellular Abeta deposits in senile plaques, neurofibrillary tangles, and neuronal loss. However, the physiological role of normal levels of Abeta and its parent protein, the amyloid precursor protein (APP) are unknown. Here we report that low-level transgenic (Tg) expression of the Swedish APP mutant gene (APPswe) in Fischer-344 rats results in attenuated age-dependent cognitive performance decline in 2 hippocampus-dependent learning and memory tasks compared with age-matched nontransgenic Fischer-344 controls. TgAPPswe rats exhibit mild increases in brain APP mRNA (56.8%), Abeta-42 (21%), and Abeta-40 (6.1%) peptide levels at 12 mo of age, with no extracellular Abeta deposits or senile plaques at 6, 12, and 18 mo of age, whereas 3- to 6-fold increases in Abeta levels are detected in plaque-positive human AD patients and transgenic mouse models. The data support the hypothesis that a threshold paradigm underlies Abeta-related pathology, below which APP expression may play a physiological role in specific hippocampus-dependent tasks, most likely related to its neurotrophic role.     

Molecular dissection of the interaction between amyloid precursor protein and its neuronal trafficking receptor SorLA/LR11

SorLA/LR11 is a sorting receptor that regulates the intracellular transport and processing of the amyloid precursor protein (APP) in neurons. SorLA/LR11-mediated binding results in sequestration of APP in the Golgi and in protection from processing into the amyloid-beta peptide (Abeta), the principal component of senile plaques in Alzheimer's disease (AD). To gain insight into the molecular mechanisms governing sorLA and APP interaction, we have dissected the respective protein interacting domains. Using a fluorescence resonance energy transfer (FRET) based assay of protein proximity, we identified binding sites in the extracellular regions of both proteins. Fine mapping by surface plasmon resonance analysis and analytical ultracentrifugation of recombinant APP and sorLA fragments further narrowed down the binding domains to the cluster of complement-type repeats in sorLA that forms a 1:1 stoichiometric complex with the carbohydrate-linked domain of APP. These data shed new light on the molecular determinants of neuronal APP trafficking and processing and on possible targets for intervention with senile plaque formation in patients with AD.     

Stable insertion of Alzheimer Abeta peptide into the ER membrane strongly correlates with its length

Alzheimer's disease is characterized by the deposition of amyloid beta-peptide (Abeta) plaques in the brain. Full-length amyloid-beta precursor protein (APP) is processed by alpha- and beta-secretases to yield soluble APP derivatives and membrane-bound C-terminal fragments, which are further processed by gamma-secretase to a non-amyloidogenic 3 kDa product or to Abeta fragments. As different Abeta fragments contain different parts of the APP transmembrane helix, one may speculate that they are retained more or less efficiently in the membrane. Here, we use the translocon-mediated insertion of different APP-derived polypeptide segments into the endoplasmic reticulum membrane to assess the propensities for membrane retention of Abeta fragments. Our results show a strong correlation between the length of an Abeta-derived segment and its ability to integrate into the microsomal membrane.     

The role of membrane trafficking in the processing of amyloid precursor protein and production of amyloid peptides in Alzheimer's disease

Alzheimer's disease (AD) is characterized by progressive accumulation of misfolded proteins, which form senile plaques and neurofibrillary tangles, and the release of inflammatory mediators by innate immune responses. β-Amyloid peptide (Aβ) is derived from sequential processing of the amyloid precursor protein (APP) by membrane-bound proteases, namely the β-secretase, BACE1, and γ-secretase. Membrane trafficking plays a key role in the regulation of APP processing as both APP and the processing secretases traffic along distinct pathways. Genome wide sequencing studies have identified several AD susceptibility genes which regulate membrane trafficking events. To understand the pathogenesis of AD it is critical that the cell biology of APP and Aβ production in neurons is well defined. This review discusses recent advances in unravelling the membrane trafficking events associated with the production of Aβ, and how AD susceptible alleles may perturb the sorting and transport of APP and BACE1. Mechanisms whereby inflammation may influence APP processing are also considered.     

Detection of soluble forms of the beta-amyloid precursor protein in human plasma

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.     

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.     

Molecular cloning, expression, and regulation of hippocampal amyloid precursor protein of senescence accelerated mouse (SAMP8).

Alzheimer's disease (AD) is associated with increased expression of amyloid precursor protein (APP) with a consequent deposition of amyloid beta peptide (Abeta) which forms characteristic senile plaques. We have noticed that the senescence accelerated mouse (SAMP8), a strain of mouse that exhibits age-dependent defects such as loss of memory and retention at an early age of 8-12 months, also produces increased amounts of APP and Abeta similar to those observed in Alzheimer's disease (AD). In order to investigate if this is due to mutations in APP similar to those observed in AD, and to develop molecular probes that regulate its expression, APP cDNA was cloned from the hippocampus of 8-month-old SAMP8 mouse. The nucleotide sequence is 99.7% homologous with that of mouse and rat, 88.7% with monkey, and 89.2% with human homologues. At the amino acid level, the homology was 99.2% and 97.6% with rodent and primate sequences, respectively. A single amino acid substitution of Alanine instead of Valine at position 300 was unique to SAMP8 mouse APP. However, no mutations similar to those reported in human familial AD were observed. When the cDNA was expressed in HeLa cells, glycosylated mature APP could be detected by immunoblotting technique. The expression could be regulated in a time- and concentration-dependent manner by using an antisense oligonucleotide specific to APP mRNA. Such regulation of APP expression may have a therapeutic application in vivo.     

Human Brain βA4 Amyloid Protein Precursor of Alzheimer''s Disease: Purification and Partial Characterization

The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4-kDa beta A4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100-110 kDa and 120-130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus-directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane-associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.     

Trafficking of Alzheimer's disease-related membrane proteins and its participation in disease pathogenesis

Alzheimer's disease (AD) is a common neurodegenerative disorder that causes senile dementia. The pathological characteristics are the appearance of neurofibrillary tangles comprising abnormally phosphorylated tau and senile plaques composed of amyloid beta-protein depositions. Amyloid beta-protein precursor (APP) and presenilin (PS) are known to be causative genes of familial AD. Recent analyses have documented that APP functions in the axonal transport of vesicles and PS regulates intracellular protein trafficking. Dystrophic neurites, in which APP and Alcadein accumulate in swollen axons, are also observed in AD brain. These pathological characteristics and the features of AD-related proteins suggest that AD is a disease of the vesicular transport system. Here we review recent progress of research on AD pathogenesis from the viewpoint of membrane trafficking.     

Nerve and epidermal growth factors induce the release of the Alzheimer amyloid precursor from PC 12 cell cultures

Antisera against specific sites of the Alzheimer beta Amyloid protein precursor (beta APP) were used to study the effects of nerve and epidermal growth factors on the expression and processing of this protein in PC12 cell cultures. Two major beta APP proteins (140 and 105 kDa) both containing the Kunitz-protease inhibitor insert (KPI), were detected in cell extracts of naive PC12 cells. Treatment of these cultures with nerve growth factor (NGF) induced the release of two beta APP species 125 and 120 kDa, both of which contained the KPI domain and lacked the carboxy-terminal portion of the precursor. The released beta APP contained O-linked sugars. Only one of the released beta APP proteins bound to the lectin Concanavalin A indicating that they differ in their glycosylation. Epidermal growth factor (EGF) also induced the release of beta APP proteins into the culture medium with similar electrophoretic mobilities as those released by NGF.     

Evidence against the overexpression of APP in Down syndrome

Down syndrome (DS) is the most common genetic disorder with mental retardation and is caused by trisomy 21. By the age of 40 years, virtually all adults with DS have sufficient neuropathology for a diagnosis of Alzheimer's disease (AD), which is characterized by accumulation of amyloid-beta in senile plaques and formation of neurofibrillary tangles. Amyloid-beta derives from a longer precursor protein, APP, whose gene maps to chromosome 21. In DS, the early appearance of senile plaques is commonly associated with the presence of a third copy of the APP gene. Here we show DS brains and trisomic fibroblasts in which APP is not overexpressed, compared to euploid controls, challenging the notion that the widespread amyloid-beta deposits, consistently found in DS individuals, result from an extra copy of APP.     

Oligodendrocytes are a Novel Source of Amyloid Peptide Generation

Alzheimer’s disease is characterised by regional neuronal degeneration, synaptic loss, and the progressive deposition of the 4 kDa β-amyloid peptide (Aβ) in senile plaques and accumulation of tau protein as neurofibrillary tangles. Aβ derives from the larger precursor molecule, amyloid precursor protein (APP) by proteolytic processing via β- and γ-secretases. While APP expression is well documented in neurons and astrocytes, the case for oligodendrocytes is less clear. The latter cell type is reported to express different isoforms of APP, and we have confirmed this observation by immunocytochemistry in cultures of differentiated rat cortical oligodendrocytes. Moreover, by means of a sensitive electrochemiluminescent immunoassay employing Aβ C-terminal specific antibodies, mature oligodendrocytes are shown to secrete the 40 and 42 amino acid Aβ species (Aβ40 and Aβ42). Secretion of Aβ peptides was reduced by incubating oligodendrocytes with α- and β-secretase inhibitors, or a γ-secretase inhibitor. Disturbances of APP processing and/or synthesis in oligodendrocytes may account for some myelin disorders observed in Alzheimer’s disease and other senile dementias.     

Direct visualization of the gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus

Amyloid-beta, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase-mediated intramembranous cleavage. In addition to amyloid-beta, this cleavage produces a carboxyl-terminal intracellular fragment which has an unknown function. The carboxyl-terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl-terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP-CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP-CT-Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP-CT.     

Molecular biology of Alzheimer's amyloid—Dutch variant

Hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D) (or familial cerebral amyloid angiopathy) and familial Alzheimer's disease (FAD) share several properties. Both are autosomal dominant forms of cerebral amyloidosis characterized by beta-amyloid (A beta) deposition. In HCHWA-D the A beta is predominantly found in blood vessels and in early parenchymal plaques, whereas in AD parenchymal A beta deposits in the form of senile plaques and neurofibrillary tangles are a more prominent finding. Point mutations in the amyloid precursor protein (APP) have recently been described, in both conditions. A G to C transversion at codon 618 (extracellular portion of APP695), producing a single amino acid substitution of glutamine instead of glutamine acid, occurs in HCHWA-D; whereas mutations at codon 642 in the intramembrane region of APP695 (phenylalanine, isoleucine, or glycine instead of valine) are associated with early onset FAD. This suggests that the site of particular mutations in the APP gene and the type of amino acid substitution in the APP holoprotein are more important in determining clinicopathological phenotype and age at which A beta is deposited. Thus FAD and HCHWA-D can be regarded as two sides of the same coin.     

Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain

Transgenic mice overexpressing different forms of amyloid precursor protein (APP), i.e. wild type or clinical mutants, displayed an essentially comparable early phenotype in terms of behavior, differential glutamatergic responses, deficits in maintenance of long term potentiation, and premature death. The cognitive impairment, demonstrated in F1 hybrids of the different APP transgenic lines, was significantly different from nontransgenic littermates as early as 3 months of age. Biochemical analysis of secreted and membrane-bound APP, C-terminal "stubs," and Abeta(40) and Abeta(42) peptides in brain indicated that no single intermediate can be responsible for the complex of phenotypic dysfunctions. As expected, the Abeta(42) levels were most prominent in APP/London transgenic mice and correlated directly with the formation of amyloid plaques in older mice of this line. Plaques were associated with immunoreactivity for hyperphosphorylated tau, eventually signaling some form of tau pathology. In conclusion, the different APP transgenic mouse lines studied display cognitive deficits and phenotypic traits early in life that dissociated in time from the formation of amyloid plaques and will be good models for both early and late neuropathological and clinical aspects of Alzheimer's disease.