Amyloid precursor protein processing in sterol regulatory element-binding protein site 2 protease-deficient Chinese hamster ovary cells

Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.     

Giant adrenal myelolipomas: CT and MRI findings

A beta (beta/A4) is the major constituent of brain amyloid in Alzheimer's disease (AD), Down's syndrome (DS) and normal aged persons. This protein is presumably derived by normal proteolysis from a precursor protein (APP). In this study, C-terminal fragments of APP in a Tris/Triton soluble fraction were partially purified from DS brain by heparin-affinity and reverse phase chromatography, and analyzed by N-terminal amino acid sequencing after SDS polyacrylamide gel electrophoresis and Western blotting. We found at least six different C-terminal fragments including those with the entire A beta region. These results suggest that secretory processing of APP is heterogeneous and generates amyloidogenic C-terminal fragments.     

Molecular cloning of the cDNA for a human amyloid precursor protein homolog: evidence for a multigene family

Alzheimer's disease is a degenerative neurological disorder characterized by neural loss and brain lesions associated with plaques containing large amounts of the beta/A4 amyloid peptide. Molecular cloning of the cDNA for this peptide from human brain has shown it to be derived by proteolysis from a much larger precursor called the amyloid precursor protein (APP). The biological role of the precursor is unknown, but it has been shown to be transcribed in many human tissues in addition to brain. In the present report, we describe the molecular cloning from a human placental library of a full-length cDNA for a molecule closely related to APP. This novel molecule, which we have called amyloid precursor protein homolog (APPH), shares overall domain organization with APP. It is 763 amino acids in length and appears to encode a signal peptide, a large apparent extracellular domain including a Kunitz inhibitor domain, a transmembrane region, and a short cytoplasmic domain. Northern analysis indicates that it occurs in at least two molecular forms and is transcribed in human brain, heart, lung, liver, and kidney, in addition to placenta. On the basis of its extensive sequence similarity and conservation of domain structure, APPH is the nearest relative of APP yet identified in an emerging multigene family.     

Presenilin 1 regulates the processing of beta-amyloid precursor protein C-terminal fragments and the generation of amyloid beta-protein in endoplasmic reticulum and Golgi

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Abetatotal and Abeta42, Abeta42 was the major Abeta species in the ER fraction (Abeta42:Abetatotal ratio 0.5-1.0), whereas absolute levels of both Abeta42 and Abeta40 were higher in the Golgi fraction and the Abeta42:Abetatoal ratio was 0.05-0.16 there. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles.     

Protein kinase C activation increases release of secreted amyloid precursor protein without decreasing Abeta production in human primary neuron cultures

Overexpression and altered metabolism of amyloid precursor protein (APP) resulting in increased 4 kDa amyloid beta peptide (Abeta) production are believed to play a major role in Alzheimer's disease (AD). Therefore, reducing Abeta production in the brain is a possible therapy for AD. Because AD pathology is fairly restricted to the CNS of humans, we have established human cerebral primary neuron cultures to investigate the metabolism of APP. In many cell lines and rodent primary neuron cultures, phorbol ester activation of protein kinase C (PKC) increases the release of the secreted large N-terminal fragment of amyloid precursor protein (sAPP) and decreases Abeta release (; ; ). In contrast, we find that PKC activation in human primary neurons increases the rate of sAPP release and the production of APP C-terminal fragments and 4 kDa Abeta. Our results indicate species- and cell type-specific regulation of APP metabolism. Therefore, our results curtail the use of PKC activators in controlling human brain Abeta levels.     

Activation of protein kinase C inhibits cellular production of the amyloid beta-protein

The 39-43-amino acid amyloid beta-protein (A beta), which is progressively deposited in cerebral plaques and blood vessels in Alzheimer's disease (AD), is released by cultured human cells during normal metabolism. Here we show that agents which activate protein kinase C or otherwise enhance protein phosphorylation caused a substantial decrease in A beta production in vitro. Protein kinase C activation also markedly decreased A beta release from cells that express mutant forms of the beta-amyloid precursor protein genetically linked to familial AD. Inhibition of A beta secretion could also be effected by direct stimulation of m1 muscarinic acetylcholine receptors with carbachol. These results demonstrate that activation of the protein kinase C signal transduction pathways down-regulates the generation of the amyloidogenic A beta peptide. Pharmacologic agents that activate this system, including a variety of first messengers, could potentially slow the development or growth of some A beta plaques during the early stages of AD.     

Hypoglycemia enhances the expression of mRNA encoding beta-amyloid precursor protein in rat primary cortical astroglial cells

Deposition of beta-amyloid (A beta) is a characteristic feature of the pathology of Alzheimer's disease (AD). Since glucose metabolism and the consequential ATP production are depressed in the temporal and parietal regions of the cortex in patients with AD, we designed the present study to investigate the possible role of hypometabolism in the pathogenesis of AD. We incubated rat primary cortical astroglial cells for 2 h to 4 days in a media deprived of 95% of its glucose and assessed the expression and alternative splicing of the mRNA that encoding beta-amyloid precursor protein (APP) using RT-PCR. Hypoglycemia caused a time-dependent increase in APP mRNA expression, which reaches a peak level of 173.2% of control expression (P < 0.05) at 24 h of hypoglycemia. Noteworthy, hypoglycemia favors the alternative splicing that includes the exon 7 segment, which encodes a Kunitz-type serine protease inhibitor domain. This study demonstrates that hypoglycemia increases APP mRNA expression in astroglial cells and processing of APP mRNA to a form that may encourage A beta deposits in AD. These data suggest that the observed hypometabolism in AD may contribute to its deposition of A beta in affected brain regions.     

The rat central nervous system expresses Alzheimer's amyloid precursor protein APP695, but not APP677 (L-APP form)

A novel splicing form of beta A4 amyloid precursor protein (APP) lacking exon 15, corresponding to 18 residues, was first reported in leukocytes and then in ubiquitous organs. To determine which APP molecules (APP695, APP751, or APP770) either with (N-APP) or without (L-APP; leukocyte-derived APP) exon 15 were expressed in various organs, we investigated the alternative splicing at exon 15 in the rat brain, kidney, heart, and testis by a PCR analysis of reverse-transcribed RNA and Southern blot analysis. Regarding APP695 without exons 7 and 8, L-APP was either seldom or never expressed in the brain, whereas both N- and L-APP were expressed in other organs. On the other hand, regarding APP751/770 containing exon 7, which codes for the Kunitz-type serine protease inhibitor domain, both N- and L-APP were expressed in all the organs examined, including the brain. These results suggest that a particular alternative regulation system related to exon 15 might be present in only APP695 of the brain and influence the proteolytic processing of APP.     

Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides

The Alzheimer amyloid precursor protein (APP) is cleaved by several proteases, the most studied, but still unidentified ones, are those involved in the release of a fragment of APP, the amyloidogenic beta-protein A beta. Proteolysis by gamma-secretase is the last processing step resulting in release of A beta. Cleavage occurs after residue 40 of A beta [A beta(1-40)], occasionally after residue 42 [A beta(1-42)]. Even slightly increased amounts of this A beta(1-42) might be sufficient to cause Alzheimer's disease (AD) (reviewed in ref. 1, 2). It is thus generally believed that inhibition of this enzyme could aid in prevention of AD. Unexpectedly we have identified in neurons the endoplasmic reticulum (ER) as the site for generation of A beta(1-42) and the trans-Golgi network (TGN) as the site for A beta(1-40) generation. It is interesting that intracellular generation of A beta seemed to be unique to neurons, because we found that nonneuronal cells produced significant amounts of A beta(1-40) and A beta(1-42) only at the cell surface. The specific production of the critical A beta isoform in the ER of neurons links this compartment with the generation of A beta and explains why primarily ER localized (mutant) proteins such as the presenilins could induce AD. We suggest that the earliest event taking place in AD might be the generation of A beta(1-42) in the ER.     

Detection of the membrane-retained carboxy-terminal tail containing polypeptides of the amyloid precursor protein in tissue from Alzheimer's disease brain

A major hallmark of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques consisting primarily of amyloid beta peptide (A beta) which is derived from a larger beta-amyloid precursor protein (APP). APP is processed via secretory and endosomal/lysosomal pathways by a group of proteases called secretases. During the processing of APP, the carboxy-terminal tail fragment has been suggested to remain within the cell. To investigate the fate of this fragment, we generated an antibody specific for a nine amino acid residue, the sequence of which was derived from the carboxy-terminal putative cytoplasmic tail of APP. Computer analysis of the entire APP gene, searching for regions of greatest antigenicity, surface probability, hydrophilicity, and presence of beta turns, indicated that the cytoplasmic tail region is an immunodominant region of APP. The peptide coupled to keyhole limpet hemocyanin protein, produced a very high titer antibody (1:1 x 10(6)). To evaluate the specificity of the antibody, immunoprecipitation of in vitro transcribed and translated DNA encoding the carboxy-terminal amino acids of APP in wheat germ extract was carried out. A single immunoprecipitated band of the correct size was seen by SDS-PAGE. The antibody was also able to specifically detect the accumulation of the stable C-terminal tail containing fragments of APP in neurites of the amygdala and hippocampus regions of the human brain tissue from AD subjects, but did not react with age-matched control normal brain tissue. The localization of the C-terminal tail of APP within the brain tissue of AD patients underscores the likely importance of the C-terminus in the pathogenesis of AD.     

The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles

Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.     

Altered processing of Alzheimer amyloid precursor protein in response to neuronal degeneration

In the brains of individuals with Alzheimer disease, senile plaques containing aggregates of beta-amyloid peptide, derived from the beta-amyloid precursor protein (APP), are seen in association with degenerating nerve terminals. It is not known whether the degenerating nerve terminals cause the formation of these aggregates or whether beta-amyloid peptide in the aggregates causes nerve-terminal degeneration. In the present study of rat brain, degeneration either of local neurons or of nerve terminals caused decreased levels of a neuron-enriched isoform of APP, increased levels of a glia-enriched isoform of APP, and increased levels of potentially amyloidogenic, as well as nonamyloidogenic, COOH-terminal fragments of APP. Our results demonstrate that neuronal degeneration affects APP processing and suggest that it may contribute to amyloid formation in mammalian brain.     

The effects of perturbed energy metabolism on the processing of amyloid precursor protein in PC12 cells

The mismetabolism of amyloid precursor protein (APP), favouring the production of A beta, is considered to be central to the pathogenesis of Alzheimer's disease (AD). However it remains to be established whether the causative factor is the reported toxicity of A beta or reduced production of secretory derivatives of APP which may have trophic or neuroprotective properties. One possible contributory factor to an imbalance in APP metabolism is the impaired cellular energy availability described in AD. The aim of this study was to investigate processing of APP-like proteins following inhibition of oxidative energy metabolism in PC12 cells. Under these conditions, intracellular and secreted APP-like proteins were significantly reduced. Treatment of energy perturbed cells with the lysosomotropic agent chloroquine restored intracellular concentrations of APP-like proteins to the control range, while the secretion was completely restored by activation of protein kinase C. These findings raise the possibility that energy related metabolic stress may lead to altered metabolism of APP-like proteins favouring a potentially amyloidogenic pathway. Furthermore, the observation that activation of PKC is able to overcome this potentially pathogenic process has important implications for treatment of AD with the current generation of cholinomimetic drugs, suggesting that such drugs may slow disease progression as well as improve cognitive dysfunction.     

The chaperone BiP/GRP78 binds to amyloid precursor protein and decreases Abeta40 and Abeta42 secretion

Recent studies of cellular amyloid precursor protein (APP) metabolism demonstrate a beta-/gamma-secretase pathway resident to the endoplasmic reticulum (ER)/Golgi resulting in intracellular generation of soluble APP (APPsbeta) and Abeta42 peptide. Thus, these intracellular compartments may be key sites of amyloidogenic APP metabolism and Alzheimer's disease pathogenesis. We hypothesized that the ER chaperone immunoglobulin binding protein (BiP/GRP78) binds to and facilitates correct folding of nascent APP. Metabolic labeling and immunoprecipitation of transiently transfected human embryonic kidney 293 cells demonstrated co-precipitation of APP with GRP78, revealing their transient interaction in the ER. Maturation of cellular APP was impaired by this interaction. Furthermore, the levels of APPs, Abeta40, and Abeta42 recovered in conditioned medium were lower compared with cells transfected with APP alone. Co-expression with APP of GRP78 T37G, an ATPase mutant, almost completely blocked cellular APP maturation as well as recovery of APPs, Abeta40, and Abeta42 in conditioned medium. The inhibitory effects of GRP78 and GRP78 T37G on Abeta40 and Abeta42 secretion were magnified by co-expression with the Swedish mutation of APP (K670N/M671L). Collectively, these data suggest a transient and direct interaction of GRP78 with APP in the ER that modulates intracellular APP maturation and processing and may facilitate its correct folding.     

The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of extracellular senile plaques composed of amyloid beta-peptide (A beta). Whereas most cases of AD occur sporadically, about 10% of AD cases are inherited as a fully penetrant autosomal dominant trait. Mutations in the recently cloned Presenilin genes (PS-1 and PS-2) are by far the most common cause of early onset familial AD. MATERIALS AND METHODS: Cellular expression of endogenous and overexpressed PS proteins was analyzed by immunocytochemistry and metabolic labeling followed by immunoprecipitation. In vivo phosphorylation sites of PS proteins were analyzed by extensive mutagenesis. RESULTS: PS-1 as well as PS-2 proteins were localized predominantly within the endoplasmic reticulum (ER). However, small amounts of the PS proteins were detected within the Golgi compartment, where they colocalize with the beta-amyloid precursor protein (beta APP). The PS-2 protein was found to be highly phosphorylated, whereas very little phosphorylation was observed for PS-1. The selective phosphorylation of PS-2 occurs exclusively on serine residues. In vivo phosphorylation of PS-2 was mapped to serine residues 7, 9, and 19 within an acidic stretch at the N terminus, which is absent in PS-1. casein kinase (CK)-1 and CK-2 were shown to phosphorylate the N terminus of PS-2 in vitro. CONCLUSIONS: The majority of PS proteins were detected in the ER where little if any proteolytic processing of beta APP was reported. ER retention of PS proteins might occur by intramolecular aggregation. Small amounts of PS proteins were also detected in the Golgi where they colocalized with beta APP. This might suggest that potential interactions between PS proteins and beta APP could occur within the Golgi. Selective phosphorylation of PS-2 proteins within the acidic domain missing in PS-1 indicates differences in the biological functions and regulation of the two highly homologous proteins.     

Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called beta-amyloid (Abeta) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Abeta that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-beta-cyclodextrin, we show that the formation of Abeta is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Abeta formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Abeta formation to occur and imply a link between cholesterol, Abeta, and Alzheimer's disease.     

Beta-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer's therapy

Inhibition of cerebral amyloid beta-protein deposition seems to be an important target for Alzheimer's disease therapy. Amyloidogenesis could be inhibited by short synthetic peptides designed as beta-sheet breakers. Here we demonstrate a 5-residue peptide that inhibits amyloid beta-protein fibrillogenesis, disassembles preformed fibrils in vitro and prevents neuronal death induced by fibrils in cell culture. In addition, the beta-sheet breaker peptide significantly reduces amyloid beta-protein deposition in vivo and completely blocks the formation of amyloid fibrils in a rat brain model of amyloidosis. These findings may provide the basis for a new therapeutic approach to prevent amyloidosis in Alzheimer's disease.     

C-terminal fragment of amyloid precursor protein inhibits calcium uptake into rat brain microsomes by Mg2+-Ca2+ ATPase

Numerous lines of evidence suggest that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (A beta). However, the possible role of other cleaved products of APP is less clear. In this study, the effects of a recombinant carboxy terminal 105 amino acid (CT105) fragment of APP on the calcium uptake by endoplasmic reticulum Mg2+-Ca2+ ATPase, the major mechanism for sequestering calcium in this organelle, were investigated. We found that CT 105 is a potent inhibitor of Mg2+-Ca2+ ATPase of endoplasmic reticulum, whereas A beta shows no effect. These results demonstrate that CT 105 inhibits the ability of brain microsomes to sequester calcium and suggest that this inhibitory effect of CT 105 may contribute to disruption of intracellular calcium concentration, possibly being involved in inducing the neural toxicity characteristic of AD.