Hypersensitivity to seizures in beta-amyloid precursor protein deficient mice

Secreted forms of the beta-amyloid precursor protein (beta-APP) have neuroprotective properties in vitro and may be involved in the containment of neuronal excitation. To test whether loss of secreted forms of beta-APP (sAPPs) may enhance excitotoxic responses, we injected mice homozygous for a targeted mutation of the beta-APP gene (beta-APPDelta/Delta) intraperitoneally with kainic acid. We found that in these mice, kainic acid induced seizures initiated earlier, and acute mortality was enhanced compared to isogenic wild-type mice independently from the callosal agenesis phenotype observed to occur at increased frequency in APP mutant mice. Expression of c-fos in cortex and cingulate gyrus was enhanced in beta-APPDelta/Delta mice, although the amount of structural damage and apoptosis in the hippocampal pyramidal cell layer and cortex was similar to that of controls. When cerebellar granule cell cultures and cortical neuronal cultures were challenged with glutamate receptor agonists, the rates of cell death and apoptosis of beta-APPDelta/Delta mice were indistinguishable from those of controls. Therefore, deficiency of sAPPs causes facilitation of seizure activity in the absence of enhanced cell death. Since enhanced seizures were observed also in mice homozygous for a deletion of the entire beta-APP gene, this phenotype results from a loss of APP rather than from a dominant effect of APPDelta.     

The metabolism of beta-amyloid converting enzyme and beta-amyloid precursor protein processing

Herein we investigated the processing of beta-secretase (BACE), implicated in Alzheimer's disease through processing of beta-amyloid precursor protein (betaAPP), into smaller metabolites. Four products of approximately 34, approximately 12, approximately 8, and approximately 5kDa were identified, none of which were generated autocatalytically. The approximately 34 and approximately 12kDa forms are held together by disulfide bridges. The approximately 34kDa form results from two cleavages: an N-terminal processing at RLPR(45) downward arrow by furin/PC5, and a C-terminal cleavage at SQDD(379) downward arrow by an unknown enzyme that also releases the C-terminal approximately 12kDa product. Microsequencing of the approximately 8 and approximately 5kDa fragments showed that they are the result of processing at VVFD(407) downward arrow and DMED(442) downward arrow, respectively. Mutagenesis of the identified cleavage sites revealed that the mutants D379A, D379L or D379E block the degradation of BACE into the approximately 12kDa product, confirming the importance of Asp(379). Notably, the D379E mutant results in higher betaAPP derived C99 levels. In contrast, D442A or D442E did not affect the production of the approximately 8 or approximately 5kDa products. The levels of the approximately 8 and approximately 5kDa products are significantly lower in the mutant D407A but less so D407E, likely due to the low efficacy of ER exit of the D407A mutant. Indeed, while co-expression of betaAPP with BACE results in enhanced production of Abeta(11-40), the D407A mutant produces mostly Abeta(40).     

Effect of estradiol on neuronal Swedish-mutated beta-amyloid precursor protein metabolism: reversal by astrocytic cells

Alzheimer's disease is the most frequent neurodegenerative disorder in the aged population and is characterized by the deposition of the 40/42-residue amyloid beta protein (Abeta), a proteolytic fragment of the beta-amyloid precursor protein (APP). Recently, it has been shown that physiological doses of estradiol reduce the generation of endogenous Abeta in primary cortical neurons. Here we investigate the influence of estrogen in amyloidogenesis and sAPPalpha secretion in the CNS. By means of primary cortical neurons overexpressing humanized APP(695) bearing the Swedish mutation (hAPP(695sw)), we analyzed APP maturation in the absence or in the presence of estrogen. We show that estrogen at a 2 microM concentration increases the release of the neuroprotective sAPPalpha fragment but does not reduce the release of Abeta in primary neurons overexpressing the Swedish-mutated form of APP. Furthermore, neurons cocultured with astrocytic cells or grown with astrocytes conditioned media do not exhibit the estrogen-induced increase in sAPPalpha secretion. Altogether, our data indicate that astrocytes interfere with estrogen in the regulation of sAPPalpha secretion, probably via secreted factor(s).     

Selective ectodomain phosphorylation and regulated cleavage of beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta APP) is a highly conserved integral membrane protein expressed in most mammalian tissues and found at highest levels in the nervous system. Cerebral deposition of the amyloid beta-peptide (A beta), derived by proteolysis of beta APP, is an early and invariant feature of Alzheimer's disease. Protein phosphorylation by protein kinase C (PKC) has been found to regulate the metabolism of beta APP into nonamyloidogenic and amyloidogenic derivatives, but both the mechanism of these effects and the nature of beta APP phosphorylation are unknown. When labeled in vivo with [32P]orthophosphate, beta APP was phosphorylated only on serine residues in the N-terminal half of the extracellular domain, resulting in the secretion of phosphorylated soluble beta APP. PKC-mediated stimulation of beta APP secretion and concurrent inhibition of A beta release did not involve enhanced phosphorylation of beta APP and proceeded in the absence of cytoplasmic or extracellular phosphorylation of the precursor. The region of beta APP required for this indirect regulation by PKC was largely restricted to a 64 amino acid stretch around the secretory cleavage site. Moreover, in a truncated molecule designed to release soluble beta APP without the need for proteolytic cleavage, secretion was no longer regulated by PKC. Our data indicate that PKC-mediated pathways play a pivotal role in the control of beta APP metabolism and amyloid formation. However, in contrast to current postulates, this regulation is independent of beta APP phosphorylation and instead involves phosphorylation of other substrates that alter beta APP processing, such as beta APP-cleaving proteases.     

Calnuc binds to Alzheimer's beta-amyloid precursor protein and affects its biogenesis

Calnuc, a Golgi calcium binding protein, plays a key role in the constitution of calcium storage. Abnormal calcium homeostasis has been linked to Alzheimer's disease (AD). Excessive production and/or accumulation of beta-amyloid (Abeta) peptides that are proteolytically derived from the beta-amyloid precursor protein (APP) have been linked to the pathogenesis of AD. APP has also been indicated to play multiple physiological functions. In this study, we demonstrate that calnuc interacts with APP through direct binding to the carboxyl-terminal region of APP, possibly in a calcium-sensitive manner. Immunofluorescence study revealed that the two proteins co-localize in the Golgi in both cultured cells and mouse brains. Over-expression of calnuc in neuroblastoma cells significantly reduces the level of endogenous APP. Conversely, down-regulation of calnuc by siRNA increases cellular levels of APP. Additionally, we show that over-expression of calnuc down-regulates the APP mRNA level and inhibits APP biosynthesis, which in turn results in a parallel reduction of APP proteolytic metabolites, sAPP, CTFs and Abeta. Furthermore, we found that the level of calnuc was significantly decreased in the brain of AD patients as compared with that of age-matched non-AD controls. Our results suggest a novel function of calnuc in modulating the levels of APP and its proteolytic metabolites, which may further affect the patho/physiological functions of APP including AD pathogenesis.     

Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate

Using a monoclonal antibody against the entire C-terminal end of human APP₆₉₅ (643–695 sequence) and a monoclonal antibody directed against human β[1–40] amyloid peptide (βA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of βA. According to the nature of N- and C-terminal amino acids we identified endogenous β-, γ-, ε-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the β-amyloid sequence at same sites as those observed in human βA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study βA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human βA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.     

Mitogenic heparin-binding lectin-like protein from cloned thymic myoid cells

A mitogenic heparin-binding (reactive) lectin-like protein (HBP) was purified from the extract of a cloned rat thymic myoid cell R615B2 by a one-step procedure of affinity chromatography on a heparin--Sepharose CL-6B column. Four distinct peptide bands with molecular weights of 10,000, 13,000, 13,700, and 14,600 were detected on SDS-polyacrylamide gel electrophoresis. This protein is mitogenic at concentrations of as low as 1.1-70.0 ng/ml for peanut lectin-nonagglutinated thymocytes and splenocytes from euthymic mice and rats but not for splenocytes from nude mice. These results indicate that thymic myoid cell-derived HBP is an important signal for one particular step in T-cell differentiation.     

Inhibition of trypsin by the beta-amyloid protein precursor. A comparative study between transfected cells, human brain and cerebrospinal fluid.

Soluble beta-amyloid protein precursors (beta-APPs) were studied in human brain and cerebrospinal fluid (CSF) after partial purification by ion exchange chromatography. Proteins were analysed in immunoblotting experiments using a monoclonal antibody directed against the N-terminal segment of the beta-APP 770, and by reverse enzymography. In the human brain and CSF, a protein which comigrates with the beta-APP 770 expressed by transfected CHO cells was able to inhibit trypsin.     

Abnormal and deficient processing of beta-amyloid precursor protein in familial Alzheimer's disease lymphoblastoid cells

Western blot analysis showed abnormal processing of beta-amyloid precursor protein (APP) in lymphoblastoid cell lines (LCLs) of familial Alzheimer's disease (FAD). Antibody raised against central APP751 revealed that media of early and late-onset FAD LCLs had highly increased amounts of a 120 kD long-lived. SDS-stable, heat-labile complex of the Kunitz protease inhibitor domain of secreted APP and a approximately 70 kD FAD-specific, yet unidentified serine protease. Antibody against the beta A4-cytoplasmic domain showed a slower APP processing and increased amounts of 16 kD C-terminal preamyloid in lysates of early and late-onset FAD LCLs, first indicating a deficient intra-beta A4 proteolysis in FAD as a possible cause of abundant amyloid deposits in AD brain.     

Isolation of baculovirus-derived secreted and full-length beta-amyloid precursor protein

We have expressed two forms of the Alzheimer's beta-amyloid precursor protein (beta APP), the 695-amino acid form (695 beta APP), and the 751-amino acid form (751 beta APP) in a baculovirus system. Both forms were expressed as full-length precursor, and were subsequently processed in vivo to release extracellular secreted proteins. The secreted forms were cleaved from the full-length beta APP in a manner analogous to the cleavage of beta APP during constitutive secretion in mammalian cells (Weidemann, A., K?nig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., Beyreuther, K. (1989) Cell 57, 115-126; Oltersdorf, T., Ward, P. J., Henriksson, T., Beattie, E. C., Neve, R., Lieberburg, I., and Fritz, L. J. (1990) J. Biol. Chem. 265, 4492-4497). High levels of expression of 20-50 mg/liter were achieved. Both full-length and secreted forms of the beta-amyloid precursor proteins were purified using a combination of ion-exchange and immunoaffinity chromatography using a monoclonal antibody directed against beta APP. The 751 beta APP-derived full-length and secreted forms, which contain the Kunitz protease inhibitor domain, were shown to be as active in the inhibition of trypsin as is mammalian-derived secreted beta APP. The availability of purified full-length beta APP from the baculovirus system will be valuable for biochemical and cell biological analyses that may elucidate the mechanism of the inappropriate processing that leads to beta-amyloid formation in Alzheimer's disease.     

Amyloid-like properties of a synthetic peptide corresponding to the carboxy terminus of beta-amyloid protein precursor.

A synthetic peptide whose sequence corresponds to the 20 carboxy-terminal amino acids of beta-amyloid protein precursor (APP) was found to form fibrils in vitro. These fibrils showed birefringence in polarized light when stained with Congo red, fluoresced when bound with thioflavin S, were resistant to proteases, and had a cross-beta conformation. By contrast, peptides with other sequences from the intracellular domain of APP and a peptide corresponding to this entire domain did not exhibit the full range of beta-amyloid properties. These results suggest that a fragment from the C-terminus of the beta-amyloid protein precursor could bind to intraneuronal paired helical filaments and account for some of its amyloid-like properties.     

The beta-amyloid precursor protein is not processed by the regulated secretory pathway.

The beta-amyloid peptide is derived from a larger membrane bound protein and accumulates as amyloid in Alzheimer's diseased brains. beta-amyloid precursor protein (beta APP) proteolytically processed during constitutive secretion cannot be a source of deposited amyloid because this processing results in cleavage within the amyloidogenic peptide. To see if other secretory pathways could be responsible for generating potentially amyloidogenic molecules we tested the possibility that beta APP is targeted to the regulated secretory pathway. Stable AtT20 cell lines expressing exogenous human beta APP were genetically engineered. These cells were labeled with [35S]-methionine, and chased in the presence or absence of secretagogue. The beta APP both inside the cells and released from the cells was analyzed by immunoprecipitation and gel analysis. Quantitation of autoradiograms showed that virtually all of the synthesized beta APP was secreted by the constitutive pathway, and that no detectable (less than 1%) beta APP was targeted to the regulated secretory pathway.     

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.     

Soluble beta-amyloid precursor protein is related to disease progression in amyotrophic lateral sclerosis

Background

Biomarkers of disease progression in amyotrophic lateral sclerosis (ALS) could support the identification of beneficial drugs in clinical trials. We aimed to test whether soluble fragments of beta-amyloid precursor protein (sAPPα and sAPPß) correlated with clinical subtypes of ALS and were of prognostic value.

Methodology/Principal Findings

In a cross-sectional study including patients with ALS (N = 68) with clinical follow-up data over 6 months, Parkinson''s disease (PD, N = 20), and age-matched controls (N = 40), cerebrospinal fluid (CSF) levels of sAPPα a, sAPPß and neurofilaments (NfH^SMI35) were measured by multiplex assay, Progranulin by ELISA. CSF sAPPα and sAPPß levels were lower in ALS with a rapidly-progressive disease course (p = 0.03, and p = 0.02) and with longer disease duration (p = 0.01 and p = 0.01, respectively). CSF NfH^SMI35 was elevated in ALS compared to PD and controls, with highest concentrations found in patients with rapid disease progression (p<0.01). High CSF NfH^SMI3 was linked to low CSF sAPPα and sAPPß (p = 0.001, and p = 0.007, respectively). The ratios CSF NfH^SMI35/CSF sAPPα,-ß were elevated in patients with fast progression of disease (p = 0.002 each). CSF Progranulin decreased with ongoing disease (p = 0.04).

Conclusions

This study provides new CSF candidate markers associated with progression of disease in ALS. The data suggest that a deficiency of cellular neuroprotective mechanisms (decrease of sAPP) is linked to progressive neuro-axonal damage (increase of NfH^SMI35) and to progression of disease.     

Serum is required for release of Alzheimer's amyloid precursor protein in neuroblastoma cells

The beta-amyloid peptide, the major component of the senile plaques that characterize Alzheimer's disease, is generated from a set of alternatively spliced beta-amyloid precursor proteins (APPs), which are proteolytically cleaved by the action of a set of enzymes referred to generically as secretases. The major processing pathway involves the proteolytic cleavage of APP by alpha-secretase and results in the release of soluble non-amyloidogenic full-length amino terminal fragments (sAPP), which appear to be involved in neurotrophic events. A reduced production of these neuroprotective sAPP would contribute, together with deposition of the beta-amyloid peptide, to the neurodegenerative processes that lead to the cellular death in Alzheimer's disease. In the present work, we describe a dramatic reduction of sAPP content in medium conditioned by neuronal cells grown under low-serum conditions, when compared with the levels released in the presence of 10% serum. The inhibitory effect on sAPP release appears to be quite specific since that reduction occurs without major changes in cell proliferation, expression of APP-mRNA or intracellular APP levels. Under low-serum conditions, cells showed a more differentiated morphology and no apoptotic signs were observed. Since the alpha-secretase has been described as a membrane anchored protein, our results suggest that the serum contains an essential factor(s) involved in the alpha-secretase activity.     

A comparison of three heparin-binding serine proteinase inhibitors.

The purpose of this study was to compare three heparin-binding plasma proteinase inhibitors in order to identify common and unique features of heparin binding and heparin-enhanced proteinase inhibition. Experiments with antithrombin, heparin cofactor, and protein C inhibitor were performed under identical conditions in order to facilitate comparisons. Synthetic peptides corresponding to the putative heparin binding regions of antithrombin, heparin cofactor, and protein C inhibitor bound to heparin directly and interfered in heparin-enhanced proteinase inhibition assays. All three inhibitors obeyed a ternary complex mechanism for heparin-enhanced thrombin inhibition, and the optimum heparin concentration was related to the apparent heparin affinity of the inhibitor. The maximum inhibition rate and rate enhancement due to heparin appeared to be unique properties of each inhibitor. In assays with heparin oligosaccharides of known size, only the antithrombin-thrombin reaction exhibited a sharp threshold for rate enhancement at 14-16 saccharide units. Acceleration of antithrombin inhibition of factor Xa, heparin cofactor inhibition of thrombin, and protein C inhibitor inhibition of thrombin, activated protein C, and factor Xa did not require a minimum saccharide size. The differences in heparin size dependence and rate enhancement of proteinase inhibition by these inhibitors might reflect differences in the importance of the ternary complex mechanism and other mechanisms, alterations in inhibitor reactivity, and orientation effects in heparin-enhanced proteinase inhibition.