Constitutive and Protein Kinase C-Regulated Secretory Cleavage of Alzheimer's β-Amyloid Precursor Protein: Different Control of Early and Late Events by the Proteasome

Abstract: The physiological processing of the β-amyloid precursor protein (βAPP) by a protease called α-secretase gives rise to APPα, a C-terminally truncated fragment of βAPP with known neurotrophic and cytoprotective properties. Several lines of evidence indicate that protein kinase C (PKC)-mediated events regulate this physiological pathway. We show here that the proteasome multicatalytic complex modulates the phorbol 12,13-dibutyrate-stimulated APPα secretion at several levels in human kidney 293 (HK293) cells. Two blocking agents of the proteasome, namely, Z -IE(Ot-Bu)A-leucinal and lactacystin, elicit a dual effect on PKC-regulated APPα secretion by metabolically labeled HK293 cells. Thus, short periods of preincubation (2–5 h) of the cells with the inhibitors trigger a drastic potentiation of APPα recovery, whereas long-term treatment of the cells (15–20 h) with the blocking agents leads to an overall decrease in the secretion of APPα. Such a dual effect was not observed on constitutive APPα secretion and intracellular formation generated by HK293 cells, which both only increase upon inhibitor treatments. Similar effects on the constitutive and PKC-regulated APPα secretion were observed with PC12 cells. Altogether, these data suggest distinct mechanisms underlying basal and PKC-regulated APPα production, indicating that this multicatalytic complex appears as a key contributor of the α-secretase pathway.     

Proteasome Contributes to the α-Secretase Pathway of Amyloid Precursor Protein in Human Cells

Abstract: A major histopathological hallmark in Alzheimer's disease consists of the extracellular deposition of the amyloid β-peptide (Aβ) that is proteolytically derived from the β-amyloid precursor protein (βAPP). An alternative, nonamyloidogenic cleavage, elicited by a protease called α-secretase, occurs inside the Aβ sequence and gives rise to APPα, a major secreted C-terminal-truncated form of βAPP. Here, we demonstrate that human embryonic kidney 293 (HK293) cells contain a chymotryptic-like activity that can be ascribed to the proteasome and that selective inhibitors of this enzyme reduce the phorbol 12,13-dibutyrate-sensitive APPα secretion by these cells. Furthermore, we establish that a specific proteasome blocker, lactacystin, also induces increased secretion of Aβ peptide in stably transfected HK293 cells overexpressing wild-type βAPP751. Altogether, this study represents the first identification of a proteolytic activity, namely, the proteasome, contributing likely through yet unknown intracellular relays, to the α-secretase pathway in human cells.     

α-Secretase-Derived Product of β-Amyloid Precursor Protein Is Decreased by Presenilin 1 Mutations Linked to Familial Alzheimer's Disease

Abstract: Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early-onset familial forms of Alzheimer's disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42-amino-acid-long species of β-amyloid peptide (Aβ). Here we show that overexpression of wild-type PS1 in HK293 cells increases Aβ40 secretion. By contrast, FAD-linked mutants of PS1 trigger increased secretion of both Aβ40 and Aβ42 but clearly favor the production of the latter species. We also demonstrate that overexpression of the wild-type PS1 augments the α-secretase-derived C-terminally truncated fragment of β-amyloid precursor protein (APPα) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APPα when compared with cells expressing wild-type PS1. This decrease was also observed when comparing double transfectants overexpressing wild-type β-amyloid precursor protein and either PS1 or its mutated congener M146V-PS1. Altogether, our data indicate that PS mutations linked to FAD not only trigger an increased ratio of Aβ42 over total Aβ secretion but concomitantly down-regulate the production of APPα.     

Suppression of APP-containing vesicle trafficking and production of β-amyloid by AID/DHHC-12 protein

The metabolism of amyloid β-protein precursor (APP) is regulated by various cytoplasmic and/or membrane-associated proteins, some of which are involved in the regulation of intracellular membrane trafficking. We found that a protein containing Asp–His–His–Cys (DHHC) domain, alcadein and APP interacting DHHC protein (AID)/DHHC-12, strongly inhibited APP metabolism, including amyloid β-protein (Aβ) generation. In cells expressing AID/DHHC-12, APP was tethered in the Golgi, and APP-containing vesicles disappeared from the cytoplasm. Although DHHC domain-containing proteins are involved in protein palmitoylation, a AID/DHHC-12 mutant of which the enzyme activity was impaired by replacing the DHHC sequence with Ala–Ala–His–Ser (AAHS) made no detectable difference in the generation and trafficking of APP-containing vesicles in the cytoplasm or the metabolism of APP. Furthermore, the mutant AID/DHHC-12 significantly increased non-amyloidogenic α-cleavage of APP along with activation of a disintegrin and metalloproteinase 17, a major α-secretase, suggesting that protein palmitoylation involved in the regulation of α-secretase activity. AID/DHHC-12 can modify APP metabolism, including Aβ generation in multiple ways by regulating the generation and/or trafficking of APP-containing vesicles from the Golgi and their entry into the late secretary pathway in an enzymatic activity-independent manner, and the α-cleavage of APP in the enzymatic activity-dependent manner.     

Human Tau may Modify Glucocorticoids-Mediated Regulation of cAMP-dependent Kinase and Phosphorylated cAMP Response Element Binding Protein

Phosphorylation of the cAMP response element binding protein (CREB) by cAMP-dependent kinase (PKA) is critical to memory formation. However, activation of PKA can also increase tau phosphorylation, which may contribute to memory impairment. Therefore, the regulation of PKA may be part of the mechanism by which glucocorticoids (GCs) influence memory. Additionally, the cellular response to GCs may be affected by the presence of human tau. The goal of this paper was to study GCs-mediated regulation of PKA as well as CREB and tau phosphorylation in wild-type HEK293 cells and HEK293 cells stably expressing human tau441 (HEK293/tau441 cells). By using dexamethasone (DEX) as GCs, we found that DEX induced a tau-dependent selective decrease in the level of PKA RIIβ subunit protein. The observed decrease in RIIβ expression was not due to alterations of mRNA levels and was reversed by inhibiting the proteasome with lactacystin. Moreover, the decrease in RIIβ did not diminish the co-localization of the catalytic subunit of PKA with tau and might contribute to the DEX-induced increase in tau phosphorylation at Ser-214. DEX also induced a tau-dependent decrease in CREB phosphorylation that could not be reversed by activating PKA with forskolin. Taken together, these results show that human tau protein may alter the GCs-mediated regulation of PKA activity and CREB phosphorylation.     

Alterations of cerebral cortex and hippocampal proteasome subunit expression and function in a traumatic brain injury rat model

Following cellular stress or tissue injury, the proteasome plays a critical role in protein degradation and signal transduction. The present study examined the β-subunit expression of constitutive proteasomes (β1, β2, and β5), immunoproteasomes (β1i, β2i, and β5i) and the 11S proteasome activator, PA28α, in the rat CNS after traumatic brain injury (TBI). Concomitant measures assessed changes in proteasome activities. Quantitative real time PCR results indicated that β1 and β2 mRNA levels were not changed, while β5 mRNA levels were significantly decreased in injured CNS following TBI. However, β1i, β2i, β5i, and PA28α mRNA levels were significantly increased in the injured CNS. Western blotting studies found that β1, β2, β5, β2i, and β5i subunit protein levels remained unchanged in the injured CNS, but β1i and PA28α protein levels were significantly elevated in ipsilateral cerebral cortex and hippocampus. Proteasome activity assays found that peptidyl glutamyl peptide hydrolase-like and chymotrypsin-like activity were significantly reduced in the CNS after TBI, and that trypsin-like proteasome activity was increased in the injured cerebral cortex. Our results demonstrated that both proteasome composition and function in the CNS were affected by trauma. Treatments that preserve proteasome function following CNS injury may be beneficial as an approach to cerebral neuroprotection.     

Protein Kinase C Activation Potentiates the Rapid Secretion of the Amyloid Precursor Protein from Rat Cortical Synaptosomes

Abstract : In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AβPP) by the α-secretase pathway within the βA4 domain to generate a soluble secreted N-terminal fragment (AβPP_s). AβPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform β1 and novel PKCε from cytosol to membrane fractions, but there was no alteration in the proportion of AβPP associated with the Tritonsoluble and -insoluble fractions. AβPP_s was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AβPP_s was only partially blocked by the PKC inhibitor GF109203X (2.5 μ M ), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AβPP_s secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the α-secretase activity leading to the secretion of AβPP_s can occur at the level of the presynaptic terminal.     

Generation and Regulation of β-Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "β-secretase" pathway, which generates β-amyloid (Aβ_1–40/42; ∼4 kDa), and the "α-secretase" pathway, which generates a smaller fragment, the "p3" peptide (Aβ_17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [³⁵S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp¹), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu¹¹) at the N terminus, rather than Aβ(Leu¹⁷) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPP_α release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.     

N-cadherin-based adhesion enhances Aβ release and decreases Aβ42/40 ratio

In neurons, Presenilin 1(PS1)/γ-secretase is located at the synapses, bound to N-cadherin. We have previously reported that N-cadherin-mediated cell–cell contact promotes cell-surface expression of PS1/γ-secretase. We postulated that N-cadherin-mediated trafficking of PS1 might impact synaptic PS1-amyloid precursor protein interactions and Aβ generation. In the present report, we evaluate the effect of N-cadherin-based contacts on Aβ production. We demonstrate that stable expression of N-cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Aβ in the medium. Moreover, N-cadherin expression decreased Aβ_42/40 ratio. The effect of N-cadherin expression on Aβ production was accompanied by the enhanced accessibility of PS1/γ-secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N-cadherin-mediated synaptic adhesion may modulate Aβ secretion as well as the Aβ_42/40 ratio via PS1/N-cadherin interactions.     

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester-Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP₇₅₁ to examine whether the α-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the α-secretase cleavage of APP₇₅₁. At 1 µ M , forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α-secretase cleavage of APP₇₅₁. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.     

Endosomal Sorting of Amyloid Precursor Protein-P-Selectin Chimeras Influences Secretase Processing

Amyloid β protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where α-secretase resides, or moves to acidic compartment(s) for β-secretase exposure. While the trans-Golgi network contains β-secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that β-secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in β-secretase processing of amyloid precursor protein, the wild-type and endosomal sorting mutant P-selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P-selectin, which is sorted from early to late endosomes, undergoes significantly less α-secretase cleavage, and more β-secretase cleavage, than amyloid precursor protein/P-selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P-selectin chimeras to α- and β-secretase activities, and suggest that, because delivery to late endocytic compartments favors β-secretase processing of amyloid precursor protein, there is likely limited β-secretase activity in early endosomes or at the cell surface. We propose that the trans-Golgi network may be involved in both secretory and endocytic generation of amyloid β protein.     

ATP-binding cassette transporter A7 regulates processing of amyloid precursor protein in vitro

ATP-binding cassette transporter A7 (ABCA7) is expressed in the brain and, like its closest homolog ABCA1, belongs to the ABCA subfamily of full-length ABC transporters. ABCA1 promotes cellular cholesterol efflux to lipid-free apolipoprotein acceptors and also inhibits the production of neurotoxic β-amyloid (Aβ) peptides in vitro . The potential functions of ABCA7 in the brain are unknown. This study investigated the ability of ABCA7 to regulate cholesterol efflux to extracellular apolipoprotein acceptors and to modulate Aβ production. The transient expression of ABCA7 in human embryonic kidney cells significantly stimulated cholesterol efflux (fourfold) to apolipoprotein E (apoE) discoidal lipid complexes but not to lipid-free apoE or apoA-I. ABCA7 also significantly inhibited Aβ secretion from Chinese hamster ovary cells stably expressing human amyloid precursor protein (APP) or APP containing the Swedish K670M671→N670L671 mutations when compared with mock-transfected cells. Studies with fluorogenic substrates indicated that ABCA7 had no impact on α-, β-, or γ-secretase activities. Live cell imaging of Chinese hamster ovary cells expressing APP-GFP indicated an apparent retention of APP in a perinuclear location in ABCA7 co-transfected cells. These studies indicate that ABCA7 has the capacity to stimulate cellular cholesterol efflux to apoE discs and regulate APP processing resulting in an inhibition of Aβ production.     

Metabotropic Glutamate Receptor Subtype mGluR1α Stimulates the Secretion of the Amyloid β-Protein Precursor Ectodomain

Abstract: To examine the effects of glutamatergic neurotransmission on amyloid processing, we stably expressed the metabotropic glutamate receptor subtype 1α (mGluR1α) in HEK 293 cells. Both glutamate and the selective metabotropic agonist 1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) rapidly increased phosphatidylinositol (PI) turnover four- to fivefold compared with control cells that were transfected with the expression vector alone. Increased PI turnover was effectively blocked by the metabotropic antagonist α-methyl-4-carbophenylglycine (MCPG), indicating that heterologous expression of mGluR1α resulted in efficient coupling of the receptors to G protein and phospholipase C activation. Stimulation of mGluR1α with glutamate, quisqualate, or ACPD rapidly increased secretion of the APP ectodomain (APPs); these effects were blocked by MCPG. The metabotropic receptors were coupled to APP processing by protein kinases and by phospholipase A₂ (PLA₂), and melittin, a peptide that stimulates PLA₂, potently increased APPs secretion. These data indicate that mGluR1α can be involved in the regulation of APP processing. Together with previous findings that muscarinic and serotonergic receptor subtypes can increase the secretion of the APP ectodomain, these observations support the concept that proteolytic processing of APP is under the control of several major neurotransmitters.     

Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression

The RIC-3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC-3 (hRIC-3) protein enhances expression of α7 receptors and abolishes expression of α4β2 receptors. In vitro translation of hRIC-3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC-3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for α7 expression enhancement but not for α4β2 inhibition. A combination of increased α7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in α7 membrane expression induced by hRIC-3. Antibodies against hRIC-3 showed its expression in SH-SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC-3 in rat brain localized, in general, in places where α7 nAChRs were found.     

Regulation of α2A-Adrenergic Receptor Expression by Epinephrine in Cultured Astroglia from Rat Brain

Abstract: Epinephrine (Epi) mediates various physiological effects via α_2A-adrenergic receptors (α_2A-ARs). Studies in mice with a point mutation in the gene for α_2A-AR have shown that these receptors are responsible for the centrally mediated depressor effects of α₂-AR agonists. These studies underscore the importance of understanding the basic cellular mechanisms involved in the expression of α_2A-ARs, of which little is known. We use astroglia cultured from the hypothalamus and brainstem of adult Sprague-Dawley rats as a model system in which to study factors that regulate α_2A-AR expression. These cells contain α₂-ARs, which are predominately of the α_2A-AR subtype. Our studies have shown that Epi causes a dose- and time-dependent decrease in steady-state levels of α_2A-AR mRNA and number of α_2A-ARs, effects that are mediated via α₁- and β-adrenergic receptors (α₁-ARs and β-ARs). These effects of Epi on α_2A-AR mRNA and α_2A-AR number are mimicked by activation of protein kinase C or increases in cellular cyclic AMP, which are intracellular messengers activated by α₁-ARs and β-ARs, respectively. Taken together, these results indicate that expression of α_2A-ARs is regulated in a heterologous manner by Epi, via α₁-AR- and β-AR-mediated intracellular pathways.