Intracellular cholesterol homeostasis and amyloid precursor protein processing

Many preclinical and clinical studies have implied a role for cholesterol in the pathogenesis of Alzheimer's disease (AD). In this review we will discuss the movement of intracellular cholesterol and how normal distribution, transport, and export of cholesterol are vital for regulation of the AD related protein, Aβ. We focus on cholesterol distribution in the plasma membrane, transport through the endosomal/lysosomal system, control of cholesterol intracellular signaling at the endoplasmic reticulum and Golgi, the HMG-CoA reductase pathway and finally export of cholesterol from the cell.     

Copper promotes the trafficking of the amyloid precursor protein

Accumulation of the amyloid β peptide in the cortical and hippocampal regions of the brain is a major pathological feature of Alzheimer disease. Amyloid β peptide is generated from the sequential protease cleavage of the amyloid precursor protein (APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution, including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis. Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper homeostasis and the role of copper in Alzheimer disease.     

The amyloid precursor protein: beyond amyloid

Mutations in PSEN1 and PSEN2 genes account for the majority of cases of early-onset familial Alzheimer disease. Since the first prediction of a genetic link between PSEN1 and PSEN2 with Alzheimer's disease, many research groups from both academia and pharmaceutical industry have sought to unravel how pathogenic mutations in PSEN cause presenile dementia. PSEN genes encode polytopic membrane proteins termed presenilins (PS1 and PS2), which function as the catalytic subunit of γ-secretase, an intramembrane protease that has a wide spectrum of type I membrane protein substrates. Sequential cleavage of amyloid precursor protein by BACE and γ-secretase releases highly fibrillogenic β-amyloid peptides, which accumulate in the brains of aged individuals and patients with Alzheimer's disease. Familial Alzheimer's disease-associated presenilin variants are thought to exert their pathogenic function by selectively elevating the levels of highly amyloidogenic Aβ42 peptides. In addition to Alzheimer's disease, several recent studies have linked PSEN1 to familiar frontotemporal dementia. Here, we review the biology of PS1, its role in γ-secretase activity, and discuss recent developments in the cell biology of PS1 with respect to Alzheimer's disease pathogenesis.     

Porcine prion protein amyloid

ABSTRACT

Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.     

Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

β-amyloid (Aβ) is the main constituent of senile plaques seen in Alzheimer's disease. Aβ is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases β- and β-secretase. In this study, we examined content and localization of β-secretase-cleaved APP (β-sAPP) in brain tissue sections from the frontal, temporal and occipital lobe. Strong granular β-sAPP staining was found throughout the gray matter of all three areas, while white matter staining was considerably weaker. β-sAPP was found to be localized in astrocytes and in axons. We found the β-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal β-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. β-sAPP was also found surrounding senile plaques and cerebral blood vessels. The results presented here show altered β-sAPP staining in the AD brain, suggestive of abnormal processing and transport of APP.     

The in vivo brain interactome of the amyloid precursor protein

Despite intense research efforts, the physiological function and molecular environment of the amyloid precursor protein has remained enigmatic. Here we describe the application of time-controlled transcardiac perfusion cross-linking, a method for the in vivo mapping of protein interactions in intact tissue, to study the interactome of the amyloid precursor protein (APP). To gain insights into the specificity of reported protein interactions the study was extended to the mammalian amyloid precursor-like proteins (APLP1 and APLP2). To rule out sampling bias as an explanation for differences in the individual datasets, a small scale quantitative iTRAQ (isobaric tags for relative and absolute quantitation)-based comparison of APP, APLP1, and APLP2 interactomes was carried out. An interactome map was derived that confirmed eight previously reported interactions of APP and revealed the identity of more than 30 additional proteins that reside in spatial proximity to APP in the brain. Subsequent validation studies confirmed a physiological interaction between APP and leucine-rich repeat and Ig domain-containing protein 1, demonstrated a strong influence of Ig domain-containing protein 1 on the proteolytic processing of APP, and consolidated similarities in the biology of APP and p75.     

Differential brain expression of the Alzheimer''s amyloid precursor protein.

The expression of the Alzheimer amyloid protein precursor (AAPP) was examined in human, monkey, dog and rat brains. Two proteins, one identified as AAPP695 and the other as AAPP751, were immunoprecipitated from the in vitro translation of human, dog and rat brain polysomes. The AAPP751 to AAPP695 ratio was highest in human, intermediate in dog and lowest in rat brain polysomes. Human cerebral cortex contained higher levels of the AAPP751 mRNA than either dog or rat cortex. AAPP695 was detected in both cerebral cortex and cerebellum of all species examined. In contrast, AAPP751 was detected predominantly in the cortex of human, monkey and to a lesser extent dog brains while it was not detected in rat brain. These findings indicate that the amyloid precursors are differentially expressed in different mammalian brains and suggest that AAPP751 is mainly expressed in the brain regions involved in plaque formation.     

Copper refolding of prion protein

We have shown previously that normal mouse prion protein (MoPrP) binds copper ions during protein refolding and acquires antioxidant activity. In this report, we probe the structure of the copper refolded form of MoPrP to determine how copper binding alters the secondary and tertiary features of the protein. Circular dichroism showed that recombinant MoPrP prepared in the presence of copper (as Cu(++)) showed an increased signal in the 210-220 nm range of the spectrum. Changes in protein conformation were localised to the N-terminal region of MoPrP using a panel of antibodies to assess epitope accessibility. The copper refolded recombinant prion protein had reduced proteinase K (PK) sensitivity when compared to the non-copper liganded form. Reduced PK sensitivity was not due to aggregation however as high resolution electron microscopy showed a homogenous preparation with little aggregate when compared to the non-copper form. Finally, disruption of the single disulphide linkage in MoPrP significantly diminished the antioxidant activity of the copper refolded form suggesting that activity was not solely dependent on bound copper but also on a conformation enabled by the formation of the disulphide bond.     

Copper(II) inhibits in vitro conversion of prion protein into amyloid fibrils

In recent studies, the amyloid fibrils produced in vitro from recombinant prion protein encompassing residues 89-230 (rPrP 89-230) were shown to produce transmissible form of prion disease in transgenic mice (Legname et al., (2004) Science 305, 673-676). Long incubation time observed upon inoculation of the amyloid fibrils, however, suggests that the fibrils generated in vitro have low infectivity titers. These results emphasize the need to define optimal conditions for prion conversion in vitro, under which high levels of infectivity can be generated in a cell-free system. Because copper(II) has been implicated in normal and pathological functions of the prion protein, here we investigated the effect of Cu(2+) on cell-free conversion of recombinant PrP. Our results show that at pH 7.2 and at micromolar concentrations, Cu(2+) inhibited conversion of full-length recombinant PrP (rPrP 23-230) into amyloid fibrils. This effect was most pronounced for Cu(2+), and less so for Zn(2+), while Mn(2+) had no effect on the conversion. Cu(2+)-dependent inhibition of the amyloid formation was less effective at pH 6.0, at which rPrP 23-230 displays lower Cu(2+)-binding capacity. Using rPrP 89-230, we found that Cu(2+)-dependent inhibition occurred even in the absence of octarepeat region; however, it was less effective. Our further studies indicated that Cu(2+) inhibited conversion by stabilizing a nonamyloidogenic PK-resistant form of alpha-rPrP. Remarkably, Cu(2+) also had a profound effect on preformed amyloid fibrils. When added to the fibrils, Cu(2+) induced long-range coiling of individual fibrils and enhanced their PK-resistance. It, however, produced only minor changes in their secondary structures. In addition, Cu(2+) induced further aggregation of the amyloid fibrils into large clumps, presumably, through interfibrillar coordination of copper ions by octarepeats. Taken together, our studies suggest that the role of Cu(2+) in the pathogenesis of prion diseases is complex. Because Cu(2+) may inhibit prion replication, while at the same time stabilize disease-specific isoform against proteolytic clearance, the final outcome of copper-induced effect on progression of prion disease may not be straightforward.     

The prion protein and neuronal zinc homeostasis

Although the prion protein (PrP) is known to be the causative agent of the neurodegenerative transmissible spongiform encephalopathies, its normal cellular function remains elusive. Octapeptide repeats in the N terminus of PrP bind metal ions and are required for the endocytosis of PrP upon exposure of cells to copper or zinc. As the concentration of zinc in the extracellular spaces of the brain is higher than that for copper, we put forward the hypothesis that PrP is involved in neuronal zinc homeostasis; PrP might be involved in transport of zinc into the cell or might act as a zinc sensor. In prion disease, when the protein undergoes a conformational change to the infectious form, this function of PrP in zinc homeostasis might be compromised.     

Glutamate-stimulated secretion of amyloid precursor protein from cortical rat brain slices

The aim of this study was to determine whether L-glutamate, a major excitatory transmitter in the cerebral cortex, modulates the proteolytic cleavage of the amyloid precursor protein (APP) in the brain through specific receptor activation. Native rat brain cerebral cortical slices were stimulated either with L-glutamate or various glutamate receptor agonists, and the soluble APP derivatives released into the incubation medium were assayed by Western blot analysis. Immunoprecipitation with specific antibodies revealed that in the medium only secretory forms of APP lacking intact C-terminus were present, whereas in the brain slices both C- and N-terminal intact APP products were detectable. L-glutamate induced the release of secretory APP from cortical slices in a concentration-dependent but biphasic manner, with the highest release at 50 μM L-glutamate and smaller effects at higher glutamate concentrations. To determine whether the effect of L-glutamate is mediated through distinct glutamate receptor subtypes, brain slices were incubated in the presence of various specific glutamate receptor agonists. Activation of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor with 50 nM (RS)-bromohomoibotenic acid resulted in a reduced release of secretory APP by 17%±3 (P<0.01, one tailed Student's t-test) compared to the incubation without any drug. Stimulation of the metabotropic glutamate receptor with 50 nM (2S,3S,4S)--(carboxycyclopropyl)-glycine (L-CCG-I) led to an enhanced release of secretory APP by 39%±3 (P<0.001), whereas activation of the N-methyl-D-aspartate (NMDA) receptor with 50 nM (1R,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1R,3R)-ACPD) did not significantly change the secretion of APP compared to the incubation without any drug. The data suggest that: (i) cortical glutamatergic neurotransmission is involved in APP metabolism; and (ii) the stimulation of APP cleavage in cerebral cortical brain slices is mainly mediated by the metabotropic but not the NMDA glutamate receptor subtype, whereas the AMPA receptor subtype seems to inhibit the secretory path of APP processing.     

Identification of prion amyloid filaments in scrapie-infected brain

Extracellular collections of abnormal filaments composed of prion proteins have been identified in the brains of scrapie-infected hamsters using immunoelectron microscopy. Some of the filaments were 1500 nm in length; generally, they exhibited a uniform diameter of 16 nm. Rarely, the filaments had a twisted appearance, raising the possibility that they are flattened cylinders or are composed of helically wound protofilaments. The prion filaments possess the same diameter and limited twisting as the shorter rod-shaped particles observed in purified preparations of prions. Both the filaments and rods are composed of PrP 27-30 molecules, as determined by immunoelectron microscopy using affinity-purified antibodies. The ultrastructural features of the prion filaments are similar to those reported for amyloid in many tissues including brain. These results provide the first evidence that prion proteins assemble into filaments within the brain and that these filaments accumulate in extracellular spaces to form amyloid plaques.     

Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain

Cholesterol is implicated to play a role in Alzheimer disease pathology. Therefore, the concentrations of cholesterol, its precursors, and its degradation products in brain homogenates of aging wild-type and beta-amyloid precursor protein transgenic mice carrying the Swedish mutation (APP23) were analyzed. Among the sterols measured, lanosterol is the first common intermediate of two different pathways, which use either desmosterol or lathosterol as the predominant precursors for de novo synthesis of brain cholesterol. In young mice, cholesterol is mainly synthesized via the desmosterol pathway, while in aged mice, lathosterol is the major precursor. 24S-hydroxycholesterol (cerebrosterol), which plays a key role in the removal of cholesterol from the brain, modestly increased during aging. No differences in the levels of cholesterol, its precursors, or its metabolites were found between wild-type and APP23 transgenic mice. Moreover, the levels of the exogenous plant sterols campesterol and sitosterol were significantly elevated in the brains of APP23 animals at age 12 and 18 months. This time point coincides with abundant plaque formation.     

Age-dependent degradation of amyloid precursor protein in the post-mortem mouse brain cortex

We have examined the degradation of amyloid precursor protein (APP) in the brain cortex of adult (24±2) and old (58±2) mice at different post-mortem time intervals (0, 1.5, 3, 6, 12 and 24 h). The brain cortex extract was prepared and processed for immunoblotting using antibodies against N-terminal 47–62 amino acids (Asp29) and central 301–316 amino acids containing Kunitz protease inhibitor (KPI) domain (Asp45) of APP. Asp29 (N-terminal) recognizes two bands of 140 and 112 kDa. The amount of 140 kDa is relatively higher in adult than old. The level of 112 kDa is 1.6 times lower in adult than old. It shows no remarkable change with varying post-mortem time. On the other hand, Asp45 (KPI) detects two bands of 110 and 116 kDa. While 116 kDa disappears rapidly after death of the animal, 110 kDa shows no remarkable change with different post-mortem periods. Further incubation of the disrupted tissue at 4 °C for 24 h and immunoblot analysis with Asp29 (N-terminal) shows 112 kDa in both ages but 58.5 kDa in adult and 70 kDa in old only. Analysis with Asp45 (KPI) shows only 54 kDa which increases after 3 h in adult but decreases significantly after 1.5 h and becomes undetectable at 24 h in old. Thus the present findings indicate that APP is degraded in a precise pattern and it depends on cellular intactness, post-mortem period and age of the animal.     

Specific biarsenical labeling of cell surface proteins allows fluorescent- and biotin-tagging of amyloid precursor protein and prion proteins

Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins by using prion protein (PrP) and amyloid precursor protein (APP) as models. In prion-infected cells, FlAsH-labeled tetracysteine-tagged PrP converted from the normal isoform (PrPsen) to the disease-associated isoform (PrPres), suggesting minimal steric effects of the tag. Pulse-chase analysis of PrP and APP by fluorescent gel imaging demonstrated the utility of IDEAL labeling in investigating protein metabolism by identifying an as-yet-unrecognized C-terminal fragment (C3) of PrPsen and by characterizing the kinetics of PrPres and APP metabolism. C3 generation and N-terminal truncation of PrPres were inhibited by the anti-prion compound E64, a cysteine protease inhibitor. Surprisingly, E64 did not inhibit the synthesis of new PrPres, providing insight into the mechanism by which E64 reduces steady-state PrPres levels in prion-infected cells. To expand the versatility of tetracysteine tagging, we created new Alexa Fluor- and biotin-conjugated tetracysteine-binding molecules that were applied to imaging PrP endocytosis and ultrastructural localization. IDEAL-labeling extends the use of biarsenical derivatives to extracellular proteins and beyond microscopic imaging.     

The biological role of the Alzheimer amyloid precursor protein in epithelial cells

Proteolytic processing of the Alzheimer amyloid precursor protein (APP) results in the generation of at least two distinct classes of biologically relevant peptides: (1) the amyloid beta peptides which are believed to be involved in the pathogenesis of Alzheimer's disease and (2) the soluble N-terminal ectodomain (sAPP) which exhibits a protective but as yet ill-defined effect on neurons and epithelial cells. In this report we present an overview on the functions of sAPP as an epithelial growth factor. This function involves specific binding of sAPP to membrane rafts and results in signal transduction and various physiological effects in epithelial cells as different as keratinocytes and thyrocytes. At nanomolar concentrations sAPP induces a two to fourfold increase in the rate of cell proliferation and cell migration. Specific inhibition of APP expression by antisense techniques results in decreased sAPP release and in reduced proliferative and motogenic activities. Proliferation and migration are known to be part of complex processes such as wound healing which, therefore, might be facilitated by the growth factor function of sAPP.