Caspase cleavage of the amyloid precursor protein modulates amyloid beta-protein toxicity

The amyloid beta-protein precursor (APP) is proteolytically cleaved to generate the amyloid beta-protein (Abeta), the principal constituent of senile plaques found in Alzheimer's disease (AD). In addition, Abeta in its oligomeric and fibrillar forms have been hypothesized to induce neuronal toxicity. We and others have previously shown that APP can be cleaved by caspases at the C-terminus to generate a potentially cytotoxic peptide termed C31. Furthermore, this cleavage event and caspase activation were increased in the brains of AD, but not control, cases. In this study, we show that in cultured cells, Abeta induces caspase cleavage of APP in the C-terminus and that the subsequent generation of C31 contributes to the apoptotic cell death associated with Abeta. Interestingly, both Abeta toxicity and C31 pathway are dependent on the presence of APP. Both APP-dependent Abeta toxicity and C31-induced apoptotic cell death involve apical or initiator caspases-8 and -9. Our results suggest that Abeta-mediated toxicity initiates a cascade of events that includes caspase activation and APP cleavage. These findings link C31 generation and its potential cell death activity to Abeta cytotoxicity, the leading mechanism proposed for neuronal death in AD.     

Regulation of amyloid beta-protein precursor by phosphorylation and protein interactions

Amyloid beta-protein precursor (APP), a type I membrane protein, is cleaved by primary alpha-or beta-secretase and secondary gamma-secretase. Cleavage of APP by beta- and gamma-secretases generates amyloid beta-protein, the main constituent of the cerebrovascular amyloid that accompanies Alzheimer disease. The generation and aggregation of amyloid beta-protein in the brain are believed to be a primary cause of Alzheimer disease pathogenesis, and indeed, early onset Alzheimer disease is genetically linked to APP and also to presenilins 1 and 2, which are components of gamma-secretase. Proteolytic cleavage of APP has been investigated as a candidate target for Alzheimer disease therapy, but the mechanisms regulating APP metabolism are still unclear. APP is a type I membrane protein with a short cytoplasmic region consisting of 47 amino acids. Recent research has elucidated the significance of the cytoplasmic region in the metabolism, trafficking, and physiological function of APP. The structure and function of the APP cytoplasmic domain can be modified by phosphorylation and through interaction with cytoplasmic proteins. This minireview summarizes a large body of recent information on the regulation of APP by phosphorylation and protein interaction, along with some of the physiological functions of APP. Recent findings regarding the regulation of APP processing contribute to the development of novel drugs and/or therapies for Alzheimer disease.     

Inhibition of familial cerebral amyloid angiopathy mutant amyloid beta-protein fibril assembly by myelin basic protein

Deposition of fibrillar amyloid beta-protein (Abeta) in the brain is a prominent pathological feature of Alzheimer disease and related disorders, including familial forms of cerebral amyloid angiopathy (CAA). Mutant forms of Abeta, including Dutch- and Iowa-type Abeta, which are responsible for familial CAA, deposit primarily as fibrillar amyloid along the cerebral vasculature and are either absent or present only as diffuse non-fibrillar plaques in the brain parenchyma. Despite the lack of parenchymal fibril formation in vivo, these CAA mutant Abeta peptides exhibit a markedly increased rate and extent of fibril formation in vitro compared with wild-type Abeta. Based on these conflicting observations, we sought to determine whether brain parenchymal factors that selectively interact with and modulate CAA mutant Abeta fibril assembly exist. Using a combination of immunoaffinity chromatography and mass spectrometry, we identified myelin basic protein (MBP) as a prominent brain parenchymal factor that preferentially binds to CAA mutant Abeta compared with wild-type Abeta. Surface plasmon resonance measurements confirmed that MBP bound more tightly to Dutch/Iowa CAA double mutant Abeta than to wild-type Abeta. Using a combination of biochemical and ultrastructural techniques, we found that MBP inhibited the fibril assembly of CAA mutant Abeta. Together, these findings suggest a possible role for MBP in regulating parenchymal fibrillar Abeta deposition in familial CAA.     

Ontogeny of pineal protein kinase C activity

The developmental appearance of protein kinase C (PKC) activity in the rat pineal gland was investigated. Enzyme activity could be detected before birth in both cytosol and membrane fractions. A small peak in activity was seen between -2 and 4 days of age, coinciding with a temporary redistribution of activity to the membrane fraction (4% increasing to 17%). After 10 days of age both cytosol and membrane activity increased progressively to reach adult levels by 30 days. Inhibition of daily adrenergic stimulation to the gland by decentralizing or removing the superior cervical ganglia or exposing rats to constant light for 14 days did not reduce PKC activity. These results indicate that PKC activity is located in pinealocytes rather than in the presynaptic adrenergic terminals and that adrenergic stimulation is not necessary to maintain the high level of PKC activity in the pineal.     

Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase

We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.     

Constitutive activity of membrane-inserted protein kinase C

Incubation of purified protein kinase C (PKC) with phospholipid vesicles produced two populations of membrane-bound PKC: one population was dissociated by calcium chelation and the other was not. The second population appeared to be inserted into the membrane. The activity of membrane-inserted PKC was Ca2+-independent and was only modestly sensitive to phorbol esters. Insertion was caused by high calcium concentrations or by phorbol esters plus low calcium. These conditions correlated with those needed to activate PKC; insertion into the membrane may be a primary mechanism of PKC activation. PKC may be a long-term cell regulator which becomes inserted into the membrane upon appearance of the second messengers, calcium and diacylglycerol, and remains in an active membrane-bound state when the second messengers have been removed.     

Binding of gelsolin, a secretory protein, to amyloid beta-protein.

Soluble amyloid beta-protein (Abeta) is normally present in the cerebrospinal fluid (CSF) and plasma. However, it is fibrillized and deposited as plaques in the brains of patients with Alzheimer's disease. Cerebrospinal fluid (CSF) contains several circulating proteins (apolipoprotein E, apolipoprotein J, and transthyretin) that bind to Abeta. We report here that gelsolin, a secretory protein, also binds to Abeta in a concentration-dependent manner. Under similar conditions, other proteins such as G-actin, protein kinase C, polyglutamic acid, and gelatin did not bind to Abeta. Solid phase binding assays showed two Abeta binding sites on gelsolin that have dissociation constants (Kd) of 1.38 and 2.55 microM. Abeta was found to co-immunoprecipitate along with gelsolin from the plasma, suggesting that gelsolin-Abeta complex exists under physiological conditions. The gelsolin-Abeta complex was sodium dodecyl sulfate (SDS)stable in the absence of reducing agent, but was dissociated when the SDS stop solution contained dithiothreitol (reducing agent). This study suggests that the function of secretory gelsolin in the CSF and plasma is to bind and sequester Abeta.     

Stimulated platelets release amyloid beta-protein precursor

Human platelets can be stimulated by thrombin or ionomycin to secrete soluble truncated amyloid beta-protein precursor and particulate membrane fragments which contain C-terminal and N-terminal immunoreactive amyloid beta-protein precursor. This suggests a possible circulating source of beta-protein in serum which may play a role in the formation of amyloid deposits. The release of soluble amyloid beta-protein precursor could be involved in normal platelet physiology.     

Vitamin E inhibits protein kinase C activity

Vitamin E (dl-alpha-tocopherol) has been found to inhibit in vitro brain protein kinase c with a half inhibitory concentration of 450 microM. The known plasma concentrations of vitamin E are one order of magnitude lower than the protein kinase c half-inhibitory concentration but it is also known that, at the membrane level where the active protein kinase c is located, the lipophilic vitamin E is more concentrated (Burton, G.W., Joyce, A. and Ingold, K.U. and Locke, S. (1983) Arch. Biochem. Biophys. 221, 281-290). It appears that vitamin E, in addition to its antioxidant function, may play a role in regulating the activity of protein kinase c.     

Ethanol-induced mitogen activated protein kinase activity mediated through protein kinase C.

The aim of this study was to determine the pathway(s) by which ethanol activates mitogen-activated protein kinase (MAPK) signaling and to determine the role of Ca2+ in the signaling process. MAPK signaling was determined by assessing MAPK activity, measuring phosphorylated extracellular signaling-regulated kinase (pp 44 ERK-1 and pp 42 ERK-2) expression and ERK activity by measuring ERK-2-dependent phosphorylation of a synthetic peptide as a MAPK substrate in rat vascular smooth muscle cells. Ethanol activated extracellular signal-regulated kinase expression (ERK 1 and 2) could be observed when vascular smooth muscle cells (VSMCs) were stimulated for 5 min or less, but was inhibited when cells are treated for 10 min or more with 1-16 mM of ethanol. Maximum ethanol-induced MAPK activity was observed within 5 min with 4 or 8 mM. Ethanol stimulated MAPK activity was blocked by the protein kinase C (PKC) inhibitor (GF109203X) and epidermal growth factor (EGF) receptor antagonist (PD153035) by 41 +/- 24 and 34 +/- 12.3%, respectively. The calcium channel blocker, diltiazem and the chelating agent, BAPTA, reduced the activation of MAPK activity by ethanol, significantly. The data demonstrate that ethanol-stimulated MAPK expression is mediated partially through both the EGF-receptor and PKC intermediates and that activation through the PKC intermediate is calcium-dependent.     

Regulation of protein kinase C activity by lipids

Protein kinase C is activated by the simultaneous presence of phospholipid, a diglyceride, and Ca2+. Under physiological conditions the activity of the enzyme is regulated by the availability of diglycerides, which are the products of phosphoinositide hydrolysis. The phospholipid-kinase interactions appear not to be of a highly specific nature. Phosphatidylserine (PS) is presumed to be the endogenous lipid that interacts with the kinase, but other acidic lipids can substitute. On the other hand, the kinase-diglyceride interactions are highly specific in nature, as would be expected of a physiological regulator. These interactions are stereo-specific and stoichiometric with respect to diglyceride. The specificity is directed toward the glycerol backbone and hydrophilic oxygen moieties of the diglyceride. The removal of one or more of the oxygen atoms or the addition of a single methyl group to the glycerol backbone virtually abolishes the activity of a putative diglyceride activator. The extreme specificity of the kinase toward the diglycerides, however, must be contrasted with the abilities of structurally diverse tumor promotors and irritants to activate the kinase. Specific small-molecule antagonists of protein kinase C have yet to be developed. The small-molecule antagonists that have been developed so far have been relatively nonspecific cationic lipids that appear to function by interfering with the interaction between the acidic phospholipids and Ca2+.     

Regulation of protein kinase C activity by gangliosides

The activity of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the presence of phosphatidylserine and its physiological regulator, diacylglycerol, could be suppressed by a mixture of brain gangliosides. Half-maximal inhibition was observed at 30 microM and was nearly complete at 100 microM. Inhibition was observed at all concentrations of Ca2+ between 10(-8) and 10(-4) M. Inhibition of protein kinase C activity could not be reversed by increasing the concentration of diacylglycerol or the substrate, histone. Inhibition was also observed when myelin basic protein or a synthetic myelin basic protein peptide was used as substrate. Among the individual gangliosides, the rank order of potency was GT1b greater than GD1a = GD1b greater than GM3 = GM1. Our results suggest that gangliosides may regulate the responsiveness of protein kinase C to diacylglycerol.     

Activation loop phosphorylation-independent kinase activity of human protein kinase C zeta

Atypical protein kinase C zeta (PKCzeta) plays an important role in cell proliferation and survival. PKCzeta and its truncated form containing only the kinase domain, CATzeta, have been reported to be activated by the phosphorylation of threonine 410 in the activation loop. We expressed both the full length PKCzeta and CATzeta in a baculovirus/insect cell over-expression system and purified the proteins for biochemical characterization. Ion exchange chromatography of CATzeta revealed three species with different levels of phosphorylation at Thr-410 and allowed the isolation of the CATzeta protein devoid of phosphorylation at Thr-410. All three species of CATzeta were active and their activity was not correlated with phosphorylation at Thr-410, indicating that the kinase activity of CATzeta did not depend solely on activation loop phosphorylation. Tyrosine phosphorylation was detected in all three species of CATzeta and the full length PKCzeta. Homology structural modeling of PKCzeta revealed a conserved, predicted-to-be phosphorylated tyrosine residue, Tyr-428, in the close proximity of the RD motif of the catalytic loop and of Thr-410 in the activation loop. The structural analysis indicated that phospho-Tyr-428 would interact with two key, positively-charged residues to form a triad conformation similar to that formed by phospho-Thr-410. Based on these observations, it is possible that the Thr-410 phosphorylation-independent kinase activity of CATzeta is regulated by the phosphorylation of Tyr-428. This alternative mode of PKCzeta activation is supported by the observed stimulation of PKCzeta kinase activity upon phosphorylation at the equivalent site by Abl, and may be involved in resistance to drug-induced apoptosis.     

Protease nexin-2/amyloid beta-protein precursor in blood is a platelet-specific protein

The protease inhibitor, protease nexin-2 (PN-2), is the secreted form of the amyloid beta-protein precursor (APP) which contains the Kunitz protease inhibitor domain. PN-2/APP is an abundant platelet alpha-granule protein which is secreted upon platelet activation. PN-2/APP mRNA is present in cultured endothelial cells and the protein has been detected in plasma. In the present studies we quantitated PN-2/APP in platelets, plasma and several different cell types of the vasculature to identify the repository of the protein in the circulatory system. We report that PN-2/APP is predominantly a platelet protein in the vascular compartment. Lysates of unstimulated umbilical vein endothelial cells, granulocytes or monocytes contained little PN-2/APP based on sensitive functional protease binding and immunoblotting assays. Quantitative immunoblotting studies demonstrated that normal citrated-plasma contains less than or equal to 60 pM PN-2/APP. In contrast, platelets can contribute up to 30 nM PN-2/APP, indicating that they are the major source of the protein in blood.     

Accumulation and aggregation of amyloid beta-protein in late endosomes of Niemann-pick type C cells

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta-protein (Abeta) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.     

Action of linoleic acid on phospholipase C activity at the rectal level of the African locust under the control of protein kinase C

Linoleic acid (18:2n-6), linolenic acid and eicosatrienoic acid stimulated fluid reabsorption in locust rectum. Only 18:2n-6 was able to enhance phospholipase C activity, inositol_(1,4,5) trisphosphate production and to increase cytosolic free Ca²⁺ concentrations in epithelial cells via the opening of L-type Ca²⁺ channels. These effects resemble those exerted by neuroparsin, an antidiuretic neuronal hormone extracted from the storage lobes of the locust corpora cardiaca. As for neuroparsin, the effects of 18:2n-6 were abolished after pre-treatment with the protein kinase C inhibitor, polymyxin B. The results were consistent with a regulation of neuroparsin-sensitive phospholipase C activity by 18:2n-6 under control of protein kinase C, possibly by increasing membrane fluidity. Cyclooxygenase inhibitors attenuated the effects of 18:2n-6. This demonstrated that the results should be produced via the metabolites of 18:2n-6 HODEs rather than the PUFA itself.     

GM1 ganglioside-mediated accumulation of amyloid beta-protein on cell membranes

The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta is the key step in the development of Alzheimer's disease. Liposomal studies proposed that Abeta specifically recognizes a cholesterol-dependent cluster of monosialoganglioside GM1 and a conformationally altered form of Abeta promotes the aggregation of the protein. In this study, the accumulation of Abeta on living cells was investigated for the first time. The interaction of fluorescein-labeled Abeta (FL-Abeta) with rat pheochromocytoma PC12 cells was visualized using confocal laser microscopy. FL-Abeta was found to colocalize with GM1-rich domains on cell membranes and to accumulate in a concentration- and time-dependent manner, leading to cytotoxicity. Cholesterol depletion significantly reduced Abeta accumulation. These observations corroborate the GM1-mediated Abeta accumulation model.     

A protective role of the low density lipoprotein receptor-related protein against amyloid beta-protein toxicity

In order to delineate the neuroprotective role of the low density lipoprotein receptor-related protein (LRP) against amyloid beta-protein toxicity, studies were performed in C6 cells challenged with amyloid beta-protein in the presence or absence of activated alpha(2)-macroglobulin. Toxicity was assessed via two cell viability assays. We found that this endocytic receptor conferred protection against amyloid beta-protein toxicity in the presence of activated alpha(2)-macroglobulin and its down-regulation via inhibition by receptor-associated protein or transfection of cells with presenilin 1, increased susceptibility to amyloid beta-protein toxicity. Increased surface LRP immunoreactivity in response to amyloid beta-protein challenge was associated with increased translocation of LRP from the endoplasmic reticulum to the surface, rather than from increased mRNA or protein expression. Furthermore, this translocation of LRP to the surface was mediated by a calcium/calmodulin protein kinase II-dependent signaling pathway. These studies provide evidence for a protective role of LRP against amyloid beta-protein toxicity and may explain the aggressive nature of presenilin-1 mutation in familial Alzheimer's disease.     

Fibrillar amyloid beta-protein binds protease nexin-2/amyloid beta-protein precursor: stimulation of its inhibition of coagulation factor XIa

Cerebrovascular deposition of fibrillar 39-42 amino acid amyloid beta-protein (Abeta), a condition known as cerebral amyloid angiopathy (CAA), is a key pathological feature of Alzheimer's disease and related disorders including hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Severe cases of CAA, particularly in HCHWA-D, lead to recurrent and often fatal hemorrhagic strokes. Although the reasons for this pathological consequence remain unclear, alterations in proteolytic hemostasis mechanisms have been implicated. For example, the Abeta parent molecule protease nexin-2/amyloid beta-protein precursor (PN-2/AbetaPP), which is elevated in HCHWA-D cerebral vessels with Abeta deposits, is a potent inhibitor of coagulation factor XIa (FXIa). Here we show that fibrillar HCHWA-D Abeta binds PN-2/AbetaPP, but not its isolated Kunitz-type proteinase inhibitor (KPI) domain, in a saturable, dose-dependent manner with a K(d) of approximately 28 nM. Neither PN-2/AbetaPP nor its KPI domain bound to nonfibrillar HCHWA-D Abeta. The fibrillar Abeta binding domain on PN-2/AbetaPP was localized to residues 18-119. PN-2/AbetaPP that bound to fibrillar HCHWA-D Abeta immobilized either in plastic wells or on the surface of cultured cerebrovascular smooth muscle cells was active in inhibiting FXIa. Quantitative kinetic measurements revealed that fibrillar HCHWA-D Abeta caused a >5-fold enhancement of FXIa inhibition by PN-2/AbetaPP. Similar stimulatory effects on FXIa inhibition by PN-2/AbetaPP were also observed with fibrillar wild-type Abeta. However, fibrillar Abeta had no effect on the inhibition of trypsin by PN-2/AbetaPP. These findings suggest that fibrillar Abeta deposits in cerebral vessels can effectively localize and enhance the anticoagulant functions of PN-2/AbetaPP, thereby contributing to a microenvironment conducive to hemorrhaging.     

Endoplasmic reticulum stress-inducible protein, Herp, enhances presenilin-mediated generation of amyloid beta-protein.

Presenilin (PS) is essential for the gamma-cleavage required for the generation of the C terminus of amyloid beta-protein (Abeta). However, the mechanism underlying PS-mediated gamma-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of gamma-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of Abeta generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated Abeta generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in Abeta accumulation, including the formation of senile plaques and vascular Abeta deposits.