Secretion and accumulation of Abeta by brain vascular smooth muscle cells from AbetaPP-Swedish transgenic mice

Alzheimer amyloid-beta is deposited in the neuropil and in brain blood vessels in transgenic Tg2576 mice that overexpress human amyloid-beta precursor protein (AbetaPP) containing the Swedish mutation (AbetaPP-Swe). Because the AbetaPP transgene in Tg2576 mice is placed behind the PrP promoter, all amyloid-beta, including vascular amyloid, is considered to be of neuronal origin. We studied the expression of the transgenic AbetaPP in smooth muscle cells cultured from brain blood vessels from Tg2576 mice. We found that brain vascular smooth muscle cells overexpressed human AbetaPP-Swe approximately 4 times the physiological levels of mouse AbetaPP. The cultured cells secreted abundant Abeta1-40 and Abeta1-42 and formed intracellular Abeta-immunoreactive granules. The percentage of cells containing intracellular Abeta and the amount of intracellular Abeta were significantly higher in cultures obtained from 14-month-old than from 4-month-old mice, as tested on first or second passages. During cell senescence in culture, intracellular accumulation of Abeta and C-terminal fragments of AbetaPP increased in cells derived from both 4- and 14-month-old mice. Vascular muscle cells from Tg2576 mice appear to be a valuable model of the intracellular accumulation of Abeta. We suggest that vascular muscle cells may be involved in the production of cerebrovascular amyloid in Tg2576 mice.     

Apolipoprotein E alters metabolism of AbetaPP in cells engaged in beta-amyloidosis

Canine smooth muscle cells (SMCs), cultured from amyloid-affected brain blood vessels accumulate Alzheimer amyloid-beta peptide (Abeta) intracellularly, either spontaneously or after treatment with apolipoprotein E (apoE). ApoE is codeposited with Abeta, which suggests that apoE participates in Abeta accumulation. We tested the hypothesis that apoE-induced accumulation of Abeta in SMCs is caused by an increased production of amyloid-beta precursor protein (AbetaPP) and/or its altered metabolism. We found that 24 hours of treatment with apoE3 or apoE4 induced intracellular accumulation of Abeta-immunoreactive deposits in SMCs but did not influence AbetaPP production and processing. The treatment with apoE3 or E4 for 3 days resulted in the following: increased Abeta-accumulation; reduced levels of secreted Abeta; increased production and cellular retention of mature AbetaPP770; and reduced culture growth, cell proliferation, and viability. ApoE4, but not apoE3, increased cellular levels of mRNA AbetaPP 770 (the main form produced in SMCs) about ninefold. ApoE3 stimulated production and cellular retention of endogenous apoE. We hypothesize that Abeta accumulation is triggered by apoE, which may bind and immobilize soluble Abeta produced in SMCs. The newly formed Abeta deposits may further accelerate Abeta accumulation by altering metabolism of AbetaPP.     

C-terminal cleavage of the amyloid-beta protein precursor at Asp664: a switch associated with Alzheimer's disease

In addition to the proteolytic cleavages that give rise to amyloid-beta (Abeta), the amyloid-beta protein precursor (AbetaPP) is cleaved at Asp664 intracytoplasmically. This cleavage releases a cytotoxic peptide, APP-C31, removes AbetaPP-interaction motifs required for signaling and internalization, and is required for the generation of AD-like deficits in a mouse model of the disease. Although we and others had previously shown that Asp664 cleavage of AbetaPP is increased in AD brains, the distribution of the Asp664-cleaved forms of AbetaPP in non-diseased and AD brains at different ages had not been determined. Confirming previous reports, we found that Asp664-cleaved forms of AbetaPP were increased in neuronal cytoplasm and nuclei in early-stage AD brains but were absent in age-matched, non-diseased control brains and in late-stage AD brains. Remarkably, however, Asp664-cleaved AbetaPP was prominent in neuronal somata and in processes in entorhinal cortex and hippocampus of non-diseased human brains at ages <45 years. Our observations suggest that Asp664 cleavage of AbetaPP may be part of the normal proteolytic processing of AbetaPP in young (<45 years) human brain and that this cleavage is down-regulated with normal aging, but is aberrantly increased and altered in location in early AD.     

Isoform specific amyloid-beta protein precursor metabolism

Alzheimer's amyloid-beta protein precursor (AbetaPP) can occur in different isoforms, among them AbetaPP(751), which is the most abundant isoform in non-neuronal tissues, and AbetaPP(695), often referred to as the neuronal isoform. However, few isoform-specific roles have been addressed. In the work here described, AbetaPP isoforms, both endogenous and as cDNA fusions with green fluorescent protein (GFP), were used to permit isoform-specific monitoring in terms of intracellular processing and targeting. Differences were particularly marked in the turnover rates of the immature isoforms, with AbetaPP(751) having a faster turnover rate than AbetaPP(695) (0.8 h and 1.2 h respectively for endogenous proteins and 1.1 h and 2.3 h for transfected proteins). Hence, AbetaPP(751) matures faster. Additionally, AbetaPP(751) responded to both okadaic acid (OA) and phorbol 12-myristate 13-acetate (PMA), as determined by sAbetaPP production, with PMA inducing a more robust response. For the AbetaPP(695) isoform, however, although PMA produced a strong response, OA failed to elicit such an induction in sAbetaPP production, implicating isoform specificity in phosphorylation regulated events. In conclusion, it seems that the AbetaPP(695) isoform is processed/metabolized at a slower rate and responds differently to OA when compared to the AbetaPP(751) isoform. The relevance of isoform-specific processing in relation to Alzheimer's disease needs to be further investigated, given the predominance of the AbetaPP(695) isoform in neuronal tissues and isoform-specific alterations in expression levels associated with the pathology.     

Neurine, an acetylcholine autolysis product, elevates secreted amyloid-beta protein precursor and amyloid-beta peptide levels, and lowers neuronal cell viability in culture: a role in Alzheimer's disease?

Classical hallmarks of Alzheimer's disease (AD) are a synaptic loss, cholinergic neuron death, and abnormal protein deposition, particularly of toxic amyloid-beta peptide (Abeta) that is derived from amyloid-beta protein precursor (AbetaPP) by the action of beta- and gamma-secretases. The trigger(s) initiating the biochemical cascades that underpin these hallmarks have yet to be fully elucidated. The typical forebrain cholinergic cell demise associated with AD brain results in a loss of presynaptic cholinergic markers and acetylcholine (ACh). Neurine (vinyl-trimethyl-ammonium hydroxide) is a breakdown product of ACh, consequent to autolysis and is an organic poison found in cadavre brain. The time- and concentration-dependent actions of neurine were assessed in human neuroblastoma (NB, SK-N-SH) cells in culture by quantifying cell viability by lactate dehydrogenase (LDH) and MTS assay, and AbetaPP and Abeta levels by Western blot and ELISA. NB cells displayed evidence of toxicity to neurine at > or = 3 mg/ml, as demonstrated by elevated LDH levels in the culture media and a reduced cell viability shown by the MTS assay. Using subtoxic concentrations of neurine, elevations in AbetaPP and Abeta1-40 peptide levels were detected in conditioned media samples.     

AbetaPP-overexpression and proteasome inhibition increase alphaB-crystallin in cultured human muscle: relevance to inclusion-body myositis

Amyloid-beta precursor protein (AbetaPP) and its fragment amyloid-beta (Abeta) are increased in s-IBM muscle fibers and appear to play an important role in the pathogenic cascade. alphaB-Crystallin (alphaBC) was shown immunohistochemically to be accumulated in s-IBM muscle fibers, but the stressor(s) influencing alphaBC accumulation was not identified. We now demonstrate, using our experimental IBM model based on genetic overexpression of AbetaPP into cultured normal human muscle fibers, that: (1) AbetaPP overexpression increased alphaBC 3.7-fold (p=0.025); (2) additional inhibition of proteasome with epoxomicin increased alphaBC 7-fold (p=0.002); and (3) alphaBC physically associated with AbetaPP and Abeta oligomers. We also show that in biopsied s-IBM muscle fibers, alphaBC was similarly increased 3-fold (p=0.025) and physically associated with AbetaPP and Abeta oligomers. We propose that increased AbetaPP is a stressor increasing alphaBC expression in s-IBM muscle fibers. Determining the consequences of alphaBC association with Abeta oligomers could have clinical therapeutic relevance.     

An antibody to the beta-secretase cleavage site on amyloid-beta-protein precursor inhibits amyloid-beta production

Proteolytic cleavage of amyloid-beta-protein precursor (AbetaPP) by beta- and gamma-secretases results in production of the amyloid-beta peptide (Abeta) that accumulates in the brains of sufferers of Alzheimer's disease (AD). We have developed a monoclonal antibody, 2B12, which binds in the vicinity of the beta-secretase cleavage site on AbetaPP but does not bind within the Abeta region. We hypothesised that this antibody, directed against the substrate rather than the enzyme, could inhibit cleavage of AbetaPP by beta-secretase via steric hindrance and thus reduce downstream production of Abeta. The antibody would enter cells by binding to AbetaPP when it is at the cell surface and then be internalised with the protein. We subsequently demonstrated that, after addition of 2B12 to standard growth media, this antibody was indeed capable of inhibiting Abeta40 production in neuroblastoma and astrocytoma cells expressing native AbetaPP, as measured by an ELISA. This inhibition was both concentration- and time-dependent and was specific to 2B12. We were only able to inhibit approximately 50% of Abeta40 production suggesting that not all AbetaPP is trafficked to the cell surface. We propose that this antibody could be used as a novel, putative therapy for the treatment of AD.     

Hereditary cerebral hemorrhage with amyloidosis Dutch type (AbetaPP 693): decreased plasma amyloid-beta 42 concentration

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is a rare autosomal dominant disorder caused by an amyloid-beta precursor protein (AbetaPP) 693 mutation that clinically leads to recurrent hemorrhagic strokes and dementia. The disease is pathologically characterised by the deposition of Abeta in cerebral blood vessels and as plaques in the brain parenchyma. This study measured the Abeta40 and Abeta42 concentration in plasma of Dutch AbetaPP693 mutation carriers and controls. We found that the Abeta40 concentration was not different between AbetaPP693 mutation carriers and controls. However, the Abeta42 concentration was significantly decreased in the mutation carriers. No correlation exists between the APOE(epsilon)4 allele and the plasma of Abeta40 and Abeta42 levels in HCHWA-D patients. This finding contrasted with the increased concentrations found in Alzheimer's disease. Therefore it is suggested that the Dutch AbetaPP693 mutation located within the Abeta coding region of the AbetaPP gene has a different effect not only on clinical and pathological expression but also on Abeta processing.     

BACE1 and BACE2 in pathologic and normal human muscle

BACE1 and BACE2 are recently discovered enzymes participating in processing of amyloid beta precursor protein (AbetaPP). Their discovery is contributing importantly to understanding the mechanism of amyloid-beta generation, and hence the pathogenesis of Alzheimer's disease (AD). Sporadic inclusion-body myositis (s-IBM) and hereditary inclusion-body myopathy (h-IBM) are progressive muscle diseases in which overproduction of AbetaPP and accumulation of its presumably toxic proteolytic product amyloid-beta (Abeta) in abnormal muscle fibers appear to play an important upstream role in the pathogenic cascade. In normal human muscle AbetaPP was also shown to be present and presumably playing a role (a) at neuromuscular junctions and (b) during muscle development. To investigate whether BACE1 and BACE2 play a role in normal and diseased human muscle, we have now studied them by immunocytochemistry and immunoblotting in 35 human muscle biopsies, including: 5 s-IBM; 5 chromosome-9p1-linked quadriceps-sparing h-IBM; and 25 control muscle biopsies. In addition, expression of BACE1 and BACE2 was studied in normal cultured human muscle. Our studies demonstrate that BACE1 and BACE2 (a) are expressed in normal adult muscle at the postsynaptic domain of neuromuscular junctions, and in cultured human muscle; (b) are accumulated in the form of plaque-like inclusions in both s-IBM and h-IBM vacuolated muscle fibers; and (c) are immunoreactive in necrotizing muscle fibers. Accordingly, BACE1 and BACE2 participate in normal and abnormal processes of human muscle, suggesting that their functions are broader than previously thought.     

Presenilin structure in mechanisms leading to Alzheimer's disease

Molecular genetic studies of familial Alzheimer's disease by 1995 had clearly implicated three proteins as critical to Alzheimer's disease (AD), the amyloid-beta protein precursor (AbetaPP) and the two homologous presenilins, PS-1 and PS-2. To account for the roles of these proteins in AD, we had proposed that as an early and critical step in the mechanisms that lead to AD, the PS on the surface of a brain cell engages in a specific receptor-ligand intercellular interaction with AbetaPP on the surface of a neighboring cell. This cell-cell interaction is required to trigger off a cascade of processes that lead to the production of amyloid-beta (Abeta) from AbetaPP, leading to AD. At about this time, however, many established AD researchers had obtained data that appeared to disagree with our proposed mechanism. Their immediate objections to our proposal were based on their conclusions that 1) The PS proteins were exclusively intracellular, and were not expressed at the cell surface, and 2) The topography of the PS proteins in intracellular membranes exhibits either 6 or 8-TM spanning domains, not 7. Here we discuss the evidence for the 6-TM, 7-TM, 8-TM and other models of PS topography and offer possibilities for the differences in interpretation of the various sets of data. We review the experimental demonstration of the cell-surface expression and the 7-TM structure of PS, the functional consequences of this structure, and the findings that PS-1 and PS-2 are members of the superfamily of 7-TM heterotrimeric G-protein coupled receptors (GPCRs).     

Gene silencing through methylation: an epigenetic intervention on Alzheimer disease

Alzheimer disease (AD) is among the few diseases that may display high homocysteine (HCY) and low B12 and folate in blood. This observation has raised the suspect that amyloid-beta overproduction and accumulation, which may be the cause of the disease, could be due to the loss of epigenetic control in the expression of the genes involved in AbetaPP (amyloid-beta protein precursor) processing. We have shown, in cell culture, that two of the genes responsible for amyloid-beta production are controlled by the methylation of their promoters. The process is strictly related to S-adenosylmethionine (SAM) metabolism. SAM is a natural compound, mainly produced by the liver, which has been found at very low concentrations in AD brains. A further support to this thesis came from the observation that in elderly DNA methylations are consistently lower than in young and mid aged people. We are actually experimenting in transgenic mice the possibility to prevent or to arrest amyloid-beta accumulation, through SAM administration, and therefore its significance and the use of this drug for the treatment of the disease.     

Purification and mass spectrometric analysis of the delta opioid receptor

A mouse delta opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B(max) of 10.5 pmol/mg protein. [3H]Bremazocine exhibited high affinity binding to the epitope-tagged delta opioid receptor with a K(D) of 1.4 nM. The agonists DADL, morphine, and DAMGO competitively inhibited bremazocine binding to the tagged delta receptor with K(I)'s of 0.9, 370, and 620 nM, respectively. Chronic treatment of cells expressing the epitope-tagged delta receptor with DADL resulted in downregulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The delta receptor was solubilized from HEK 293 cell membranes with n-dodecyl-beta-d-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of Sephacryl S300 gel filtration chromatography, wheat germ agglutinin (WGA)-agarose chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and SDS/PAGE permitted purification of the receptor. The purified delta opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 65 kDa. MALDI-TOF mass spectrometry was used to identify and characterize peptides derived from the delta opioid receptor following in-gel digestion with trypsin, and precursor-derived ms/ms confirmed the identity of peptides derived from enzymatic digestion of the delta opioid receptor.     

Three lipoprotein receptors and cholesterol in inclusion-body myositis muscle

BACKGROUND: An important aspect of inclusion-body myositis (IBM) vacuolated muscle fibers (VMF) is abnormal accumulation of amyloid-beta precursor protein (AbetaPP) epitopes and its product, amyloid-beta (Abeta), and of phosphorylated tau (p-tau) in the form of paired helical filaments. Lipoprotein receptors and cholesterol are known to play an important role in AbetaPP processing, Abeta production, and tau phosphorylation. METHODS: In 10 IBM and 22 control muscle biopsies the authors immunolocalized low-density lipoprotein receptor (LDLR), very low-density lipoprotein receptor (VLDLR), and low-density lipoprotein receptor-related protein (LRP), and colocalized them with Abeta, p-tau, APOE, and free cholesterol. RESULTS: In each biopsy, virtually all IBM VMF had strong LDLR-immunoreactive inclusions, which colocalized with Abeta, APOE, p-tau, and free cholesterol. VLDLR was increased mainly diffusely, but in approximately 50% of the VMF it was also accumulated in the form of inclusions colocalizing with Abeta, APOE, and free cholesterol, but not with p-tau. LRP inclusions were present in a few VMF. In all myopathies, a subset of regenerating and necrotizing muscle fibers had prominent diffuse accumulation of both LDLR and free cholesterol. At normal neuromuscular junctions (NMJ) postsynaptically, LDLR and VLDLR, but not LRP, were immunoreactive. CONCLUSIONS: 1) Abnormal accumulation of LDLR, VLDLR, LRP, and cholesterol within IBM vacuolated muscle fibers suggests novel roles for them in the IBM pathogenesis. 2) Expression of LDLR and VLDLR at normal NMJ suggests physiologic roles for them in transsynaptic signaling pathways, increased internalization of lipoproteins there, or both. 3) Increased LDLR and free cholesterol in some regenerating and necrotizing muscle fibers suggest a role for them in human muscle fiber growth and repair and necrotic death.     

Calorie restriction attenuates Alzheimer's disease type brain amyloidosis in Squirrel monkeys (Saimiri sciureus)

Recent studies from our laboratories and others suggest that calorie restriction (CR) may benefit Alzheimer's disease (AD) by preventing amyloid-beta (Abeta) neuropathology in the mouse models of AD. Moreover, we found that promotion of the NAD+-dependent SIRT1 mediated deacetylase activity, a key regulator in CR extension of life span, may be a mechanism by which CR influences AD-type neuropathology. In this study we continued to explore the role of CR in AD-type brain amyloidosis in Squirrel monkeys (Saimiri sciureus). Monkeys were maintained on the normal and CR diets throughout the entire lifespan until they died of natural causes. We found that 30% CR resulted in reduced contents of Abeta1-40 and Abeta1-42 peptides in the temporal cortex of Squirrel monkeys, relative to control (CON) fed monkeys. The decreased contents of cortical Abeta peptide inversely correlated with SIRT1 protein concentrations in the same brain region; no detectable change in total full-length amyloid-beta protein precursor (AbetaPP) level was found. Most interestingly, we found that 30% CR resulted in a select elevation of alpha- but not beta- or gamma- secretase activity which coincided with decreased ROCK1 protein content in the same brain region, relative to CON group. Collectively, the study suggests that investigation of the role of CR in non-human primates may provide a valuable approach for further clarifying the role of CR in AD.     

Correlation between platelet amyloid precursor protein isoform ratio and cognition in Alzheimer's disease

This study analyzed whether platelet amyloid beta-protein precursor (AbetaPP) isoform ratio correlates with cognition or cognitive decline in patients with Alzheimer's disease (AD). Platelet AbetaPP isoform ratio was measured, and cognitive assessment was performed using the Mini-Mental State Examination (MMSE) in 66 AD patients at baseline (T0) and in 29 of these patients in a one-year follow-up (T1). There was a significant correlation between the AbetaPP isoform ratios and MMSE scores in the 66 AD patients at T0. The T1 subjects were divided into two groups: 12 "no decliners" (MMSE score, T1-T0 > or = 0) and 17 "decliners" (MMSE score, T1-T0 < 0). The decliners group showed a significantly greater reduction of AbetaPP isoform ratio from T0 to T1 than the no decliners group. However, the decline of the ratio did not correlate with the decline of MMSE score. These findings indicate that AbetaPP isoform ratio correlates with cognition, and reduction in this ratio may be a marker for cognitive decline in AD patients.     

Purification and mass spectrometric analysis of the mu opioid receptor

A mouse mu opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B(max) of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged mu opioid receptor with a KD of 1.0 nM. The agonists [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and [D-Ala(2),D-Leu(5)]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged mu receptor with KI's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged mu receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The mu receptor was solubilized from HEK 293 cell membranes with n-dodecyl-beta-D-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)-agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified mu opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the mu opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the mu opioid receptor.     

Characterization of V642I-AbetaPP-induced cytotoxicity in primary neurons

Amyloid precursor protein (AbetaPP), a precursor of amyloid beta (Abeta) peptide, is one of the molecules involved in the pathogenesis of Alzheimer's disease (AD). Specific mutations in AbetaPP have been found in patients inheriting familial AD (FAD). These mutant AbetaPP proteins cause cell death in neuronal cell lines in vitro, but the molecular mechanism of cytotoxicity has not yet been clarified completely. We analyzed the cytotoxic mechanisms of the London-type AbetaPP mutant, V642I-AbetaPP, in primary cortical neurons utilizing an adenovirus-mediated gene transfer system. Expression of V642I-AbetaPP protein induced degeneration of the primary neurons. This cytotoxicity was blocked by pertussis toxin, a specific inhibitor for heterotrimeric G proteins, Go/i, and was suppressed by an inhibitor of caspase-3/7 and an antioxidant, glutathione ethyl ester. A specific inhibitor for NADPH oxidase, apocynin, but not a xanthine oxidase inhibitor or a nitric oxide inhibitor, blocked V642I-AbetaPP-induced cytotoxicity. Among mitogen-activated protein kinase (MAPK) family proteins, c-Jun N-terminal kinase (JNK) and p38MAPK, but not extracellular regulated kinase (ERK), were involved in this cytotoxic pathway. The V642I-AbetaPP-induced cytotoxicity was not suppressed by two secretase inhibitors, suggesting that Abeta does not play a major role in this cytotoxicity. Two neuroprotective factors, insulin-like growth factor I (IGF-I) and Humanin, protected these primary neurons from V642I-AbetaPP-induced cytotoxicity. Furthermore, interleukin-6 and -11 also attenuated this cytotoxicity. This study demonstrated that the signaling pathway activated by mutated AbetaPP in the primary neurons is the same as that by the other artificial insults such as antibody binding to AbetaPP and the artificial dimerization of cytoplasmic domain of AbetaPP. The potential of neurotrophic factors and cytokines in AD therapy is also indicated.