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.     

Amyloid beta protein toxicity mediated by the formation of amyloid-beta protein precursor complexes

The amyloid-beta protein precursor, a type 1 transmembrane protein, gives rise to the amyloid beta-protein, a neurotoxic peptide postulated to be involved in the pathogenesis of Alzheimer's disease. Here, we show that soluble amyloid beta protein accelerates amyloid precursor protein complex formation, a process that contributes to neuronal cell death. The mechanism of cell death involves the recruitment of caspase-8 to the complex, followed by intracytoplasmic caspase cleavage of amyloid precursor protein. In vivo, the levels of soluble amyloid beta protein correlated with caspase-cleaved fragments of the amyloid precursor protein in brains of Alzheimer's disease subjects. These findings suggest that soluble amyloid beta protein-induced multimerization of the amyloid precursor protein may be another mechanism by which amyloid beta protein contributes to synapse loss and neuronal cell death seen in Alzheimer's disease.     

Role of amyloid precursor protein, amyloid-beta and gamma-secretase in cholesterol maintenance

Lipids play an important part as risk factors for Alzheimer's disease. This article summarizes the current understanding of the molecular mechanism by which amyloid-beta (Abeta) peptides regulate cholesterol and sphingomyelin metabolism, and how in return cholesterol and sphingomyelin regulate Abeta peptide production. An understanding of the physiological function of amyloid precursor protein processing and Abeta function is critical for the development of future therapeutic approaches, e.g. statin treatment.     

Long-term accumulation of amyloid-beta in axons following brain trauma without persistent upregulation of amyloid precursor protein genes

Brain trauma has been shown to be a risk factor for developing Alzheimer disease (AD), and AD-like plaques containing amyloid-beta (Abeta) peptides have been found in the brain shortly following trauma. Here, we evaluated the effects of brain trauma on the accumulation of Abeta and expression of amyloid precursor protein (APP) genes (APP695 and APP751/ 770) over 1 yr in a non-transgenic rodent model. Anesthetized male Sprague-Dawley rats were subjected to parasagittal fluid percussion brain injury of moderate severity (2.5-2.9 atm) or sham treatment and their brains were evaluated at 2, 4, 7, 14 days, and 1, 2, 6, 12 months following injury. Immunohistochemical analysis detected only weak Abeta staining by 2 wk following injury. However, by 1 month to 1 yr following injury, strong immunoreactivity for Abeta was found in damaged axons throughout the thalamus and white matter. Western blot analysis confirmed the accumulation of Abeta peptides in tissue from injured brains. Although in situ hybridization demonstrated an increased gene expression of APP751/770 surrounding the cortical lesion at 2 to 7 days following injury, this expression returned to baseline levels at all subsequent time points and no increase in the expression of APP695 was detected at any time point. These results demonstrate that long-termAbeta accumulation in damaged axons can be induced in a non-transgenic rodent model of brain trauma. Surprisingly, the extent of this Abeta production appeared to be dependent on the maturity of the injury, but uncoupled from the gene expression of APP. Together, these data suggest a mechanism that may contribute to long-term neurodegeneration following brain trauma.     

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.     

Cerebrolysin decreases amyloid-beta production by regulating amyloid protein precursor maturation in a transgenic model of Alzheimer's disease

Cerebrolysin is a peptide mixture with neurotrophic effects that might reduce the neurodegenerative pathology in Alzheimer's disease (AD). We have previously shown in an amyloid protein precursor (APP) transgenic (tg) mouse model of AD-like neuropathology that Cerebrolysin ameliorates behavioral deficits, is neuroprotective, and decreases amyloid burden; however, the mechanisms involved are not completely clear. Cerebrolysin might reduce amyloid deposition by regulating amyloid-beta (Abeta) degradation or by modulating APP expression, maturation, or processing. To investigate these possibilities, APP tg mice were treated for 6 months with Cerebrolysin and analyzed in the water maze, followed by RNA, immunoblot, and confocal microscopy analysis of full-length (FL) APP and its fragments, beta-secretase (BACE1), and Abeta-degrading enzymes [neprilysin (Nep) and insulin-degrading enzyme (IDE)]. Consistent with previous studies, Cerebrolysin ameliorated the performance deficits in the spatial learning portion of the water maze and reduced the synaptic pathology and amyloid burden in the brains of APP tg mice. These effects were associated with reduced levels of FL APP and APP C-terminal fragments, but levels of BACE1, Notch1, Nep, and IDE were unchanged. In contrast, levels of active cyclin-dependent kinase-5 (CDK5) and glycogen synthase kinase-3beta [GSK-3beta; but not stress-activated protein kinase-1 (SAPK1)], kinases that phosphorylate APP, were reduced. Furthermore, Cerebrolysin reduced the levels of phosphorylated APP and the accumulation of APP in the neuritic processes. Taken together, these results suggest that Cerebrolysin might reduce AD-like pathology in the APP tg mice by regulating APP maturation and transport to sites where Abeta protein is generated. This study clarifies the mechanisms through which Cerebrolysin might reduce Abeta production and deposition in AD and further supports the importance of this compound in the potential treatment of early AD.     

Amyloid precursor protein (APP) regulates synaptic structure and function

The amyloid precursor protein (APP) plays a critical role in Alzheimer's disease (AD) pathogenesis. APP is proteolytically cleaved by β- and γ-secretases to generate the amyloid β-protein (Aβ), the core protein component of senile plaques in AD. It is also cleaved by α-secretase to release the large soluble APP (sAPP) luminal domain that has been shown to exhibit trophic properties. Increasing evidence points to the development of synaptic deficits and dendritic spine loss prior to deposition of amyloid in transgenic mouse models that overexpress APP and Aβ peptides. The consequence of loss of APP, however, is unsettled. In this study, we investigated whether APP itself plays a role in regulating synaptic structure and function using an APP knock-out (APP-/-) mouse model. We examined dendritic spines in primary cultures of hippocampal neurons and CA1 neurons of hippocampus from APP-/- mice. In the cultured neurons, there was a significant decrease (~35%) in spine density in neurons derived from APP-/- mice compared to littermate control neurons that were partially restored with sAPPα-conditioned medium. In APP-/- mice in vivo, spine numbers were also significantly reduced but by a smaller magnitude (~15%). Furthermore, apical dendritic length and dendritic arborization were markedly diminished in hippocampal neurons. These abnormalities in neuronal morphology were accompanied by reduction in long-term potentiation. Strikingly, all these changes in vivo were only seen in mice that were 12-15months in age but not in younger animals. We propose that APP, specifically sAPP, is necessary for the maintenance of dendritic integrity in the hippocampus in an age-associated manner. Finally, these age-related changes may contribute to AD pathology independent of Aβ-mediated synaptic toxicity.     

TrkA pathway activation induced by amyloid-beta (Abeta)

Amyloid-beta (Aβ), a cytotoxic fragment of Amyloid Precursor Protein (APP), has been implicated in the etiopathogenesis of Alzheimer's disease (AD). Since several neurotrophins signalling pathways may be activated in response to toxic insults, we investigated whether a similar response is triggered also by Aβ. After Aβ (25–35) peptide administration to cultured rat hippocampal neurons, the nerve growth factor (NGF) and its receptor (TrkA) mRNA expression is up-regulated. Moreover, we observe an increased cellular TrkA expression (4.5 fold) and NGF release in the culture medium (5-fold). Concomitantly, TrkA, Akt and glycogen synthase kinase 3β (Gsk3β) phosphorylation significantly increase. Interestingly, when cells were treated with Aβ (25–35) in the presence of blocking antibody against NGF, only a partial TrkA activation (2-fold) was observed. These results have been confirmed by using pathophysiological Aβ (1–42) oligomers. Our data provide the evidence that Aβ induces the TrkA pathway activation directly by itself and indirectly promoting NGF secretion.     

Cellular forms of the rat and human beta-amyloid precursor protein (BAPP)

Using synthetic peptide antisera generated against beta-amyloid precursor protein (BAPP) sequences we demonstrate that the newly synthesized BAPP appears as a 94 kDa peptide in rat brain RNA cell-free translates or polysome runoffs, while from similar human brain material we see two bands at 94 and 120 kDa. Immunoblots reveal likely post-translational modification of BAPP, and possibly an age-dependent, endogenous proteolysis of the protein in rat brain. On human brain immunoblots an unusual post-translational modification or possibly oligomerization of BAPP is seen, particularly in cortical regions.     

Alzheimer's disease: a dysfunction of the amyloid precursor protein(1)

In this review, we argue that at least one insult that causes Alzheimer's disease (AD) is disruption of the normal function of the amyloid precursor protein (APP). Familial Alzheimer's disease (FAD) mutations in APP cause a disease phenotype that is identical (with the exception that they cause an earlier onset of the disease) to that of 'sporadic' AD. This suggests that there are molecular pathways common to FAD and sporadic AD. In addition, all individuals with Down syndrome, who carry an extra copy of chromosome 21 and overexpress APP several-fold in the brain, develop the pathology of AD if they live past the age of 40. These data support the primacy of APP in the disease. Although APP is the source of the beta-amyloid (Abeta) that is deposited in amyloid plaques in AD brain, the primacy of APP in AD may not lie in the production of Abeta from this molecule. We suggest instead that APP normally functions in the brain as a cell surface signaling molecule, and that a disruption of this normal function of APP is at least one cause of the neurodegeneration and consequent dementia in AD. We hypothesize in addition that disruption of the normal signaling function of APP causes cell cycle abnormalities in the neuron, and that these abnormalities constitute one mechanism of neuronal death in AD. Data supporting these hypotheses have come from investigations of the molecular consequences of neuronal expression of FAD mutants of APP or overexpression of wild type APP, as well as from identification of binding proteins for the carboxyl-terminus (C-terminus) of APP.     

Selective interaction of amyloid precursor protein with different isoforms of neural cell adhesion molecule

Compare to the thoroughly studied beta-amyloid, the physiological function of amyloid precursor protein (APP) is not well understood. We now had identified neural cell adhesion molecule (NCAM)-140 as a potential interaction partner of APP. Our data indicated that NCAM-140, but not NCAM-180, binds to the conserved central extracellular domain of APP. We also found that the phosphorylation levels of ERK1 and ERK2 were increased when cells were co-transfected with NCAM-140 and APP indicate that the interaction between NCAM-140 and APP may involve the MAPK pathway. These findings demonstrated that NCAM-140 interacts with APP, potentially playing a role in neurite outgrowth and neural development.     

Characterization of the neurotrophic interaction between nerve growth factor and secreted alpha-amyloid precursor protein

The expression and secretion of amyloid precursor protein (beta APP) is increased in rat cerebral cortices that have been denervated by subcortical lesions of the nucleus basalis of Meynert. The physiological role of the secreted beta APP in response to this injury has not been established. We have previously shown that secreted beta APP produced by alpha-secretase activity (sAPP(alpha)) potentiates the neuritogenic activity of nerve growth factor (NGF) in vitro on naive PC12 cells. In this investigation, we have further characterized the neurotrophic interaction of NGF and sAPP(alpha) using differentiated PC12 cells and rat primary cortical neurons. NGF required the expression of beta APP to maintain a neuronal phenotype. Reduction of endogenous beta APP expression by introduction of antisense oligonucleotides in the presence of NGF resulted in loss of neurites from differentiated PC12 cells but no apparent cell death. Addition of exogenous sAPP(alpha) (60--200 pM) potentiated the protective activity of NGF in serum-deprived differentiated PC12 cells as determined by retention of neurites and cell viability. In addition, exogenous sAPP(alpha) increased neuron viability in both short-term (3 days) cortical neuron cultures grown in the absence of serum and in long-term (9 days) cultures grown with serum. Disruption of the insulin signaling pathway by reduction of IRS-1 expression inhibited the ability of sAPP(alpha) to potentiate neurotrophic activity. These observations suggest that sAPP(alpha) acts as an injury-induced neurotrophic factor that interacts with NGF to enhance neuronal viability using the insulin signaling pathway.     

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

Patients with the Lewy body variant (LBV) of Alzheimer's disease (AD) have ubiquitinated intraneuronal and neuritic accumulations of alpha-synuclein and show less neuron loss and tau pathology than other AD patients. Aged Tg2576 transgenic mice overexpressing human betaAPP695. KM670/671NL have limited neuron loss and tau pathology, but frequent ubiquitin- and alpha-synuclein-positive, tau-negative neurites resembling those seen in the LBV of AD.     

Involvement of amyloid beta precursor protein (AbetaPP) modulated copper homeostasis in Alzheimer's disease

AbetaPP is involved in Cu homeostasis in mouse and man. In vitro observations and in vivo data obtained from AbetaPP mouse models at least provide strong evidence that AbetaPP and Abeta overproduction enables intracellular Cu to be transported out of the cell. The increased Cu efflux seems to lead to a Cu deficiency and a subsequently reduced SOD-1 activity. Studies have shown that a disturbed metal-ion homeostasis with elevated serum Cu levels occurs in Alzheimer and Down's patients and lowered levels in post-mortem AD brain. We conclude that bioavailable Cu has beneficial and specific effects in Alzheimer's disease transgenic mice, and suggest that our observation can be regarded as a proof-of-concept for a prophylactic approach to overcome the observed CNS Cu deficiency in the brain of Alzheimer's disease patients.     

The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Aβ), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for β-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPβ, but not sAPPα. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that - in addition to its role in APP trafficking - LRP1 affects APP processing by competing for cleavage by BACE1.     

Mitochondrial abnormalities in neuroectodermal cells stably expressing human amyloid precursor protein (hAPP751)

Metabolic hypofunction is a common finding in a number of neurodegenerative diseases, including Alzheimer's disease (AD). The strong linkage between the amyloid precursor protein (APP) and AD led us to examine whether over-expression of this protein in CNS-type cells had an effect on mitochondria. We found abnormal morphology in mitochondria of the neuroectodermal progeny of P19 cells stably transfected with human APP751. In addition, the mitochondria of APP-transfected clones had a decreased mitochondrial membrane potential. These changes were independent of Abeta toxicity and distinct from complex I inhibition. Our results have important implications for the earliest events in the pathophysiology of AD and, by extrapolation, for intervention therapies.