Down-regulation of the ATP-binding cassette transporter 2 (Abca2) reduces amyloid-β production by altering Nicastrin maturation and intracellular localization

Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-β (Aβ) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aβ generation, without affecting Notch cleavage.     

Hydrogen sulfide improves spatial memory impairment and decreases production of Aβ in APP/PS1 transgenic mice

Alzheimer’s disease (AD) is defined both by its progressive cognitive deterioration and hallmark increase in neuronal Aβ plaque formation. However, many of the underlying neurobiological facets of this disease are still being elucidated. Previous research has demonstrated that production of neuronal hydrogen sulfide (H₂S) is significantly decreased in patients with AD. Moreover, systemic plasma H₂S levels are negatively correlated with its severity. However, how a decrease in H₂S production might be correlated with either the etiology or pathophysiology of AD remains unknown. To better understand the role of H₂S in AD, we examined both levels of H₂S and the expression and activity H₂S-synthesizing enzyme (cystathionine beta synthase or CBS) in an APP/PS1 transgenic mouse line at 3, 6, 9 and 12 months. After intraperitoneal (i.p.) administration of an H₂S donor (NaHS) into APP/PS1 mice, application of exogenous H₂S resulted in improved spatial learning and memory acquisition in APP/PS1 mice. H₂S administration also led to significant decrease in extracellular levels of Aβ40 and Aβ42, the expression of BACE1 and PS1, and a significant increase of ADAM17 expression. Similarly, an increase in non-amyloidogenic C83 fragment generation and a decrease in amyloidogenic C99 fragment generation were also observed. Thus, NaHS application resulted in a shift from the plaque-forming beta pathway to the non-plaque forming alpha pathway of APP cleavage in 6 and 12 month APP/PS1 mice. These results indicate the importance of H₂S to AD severity and that administration of exogenous H₂S can promote a non-amyloidogenic processing of APP.     

Antagonist of peroxisome proliferator-activated receptor γ induces cerebellar amyloid-β levels and motor dysfunction in APP/PS1 transgenic mice

Recent evidences show that peroxisome proliferator-activated receptor γ (PPARγ) is involved in the modulation of the amyloid-β (Aβ) cascade causing Alzheimer’s disease (AD) and treatment with PPARγ agonists protects against AD pathology. However, the function of PPARγ steady-state activity in Aβ cascade and AD pathology remains unclear. In this study, an antagonist of PPARγ, GW9662, was injected into the fourth ventricle of APP/PS1 transgenic mice to inhibit PPARγ activity in cerebellum. The results show that inhibition of PPARγ significantly induced Aβ levels in cerebellum and caused cerebellar motor dysfunction in APP/PS1 transgenic mice. Moreover, GW9662 treatment markedly decreased the cerebellar levels of insulin-degrading enzyme (IDE), which is responsible for the cellular degradation of Aβ. Since cerebellum is spared from significant Aβ accumulation and neurotoxicity in AD patients and animal models, these findings suggest a crucial role of PPARγ steady-state activity in protection of cerebellum against AD pathology.     

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ_(1–40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3 × Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB.     

Depletion of CXCR2 inhibits γ-secretase activity and amyloid-β production in a murine model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to progressive cognitive decline. Recent studies from our group and others have suggested that certain G-protein coupled receptors (GPCRs) can influence the processing of the amyloid precursor protein (APP). Earlier, we demonstrated that stimulation of a chemokine receptor, CXCR2, results in enhanced γ-secretase activity and in increased amyloid-beta (Aβ) production. Taken together, results obtained from in vitro studies indicate that therapeutic targeting of CXCR2 might aid in lowering Aβ levels in the AD brain. To better understand the precise function and to predict the consequences of CXCR2 depletion in the AD brain, we have crossed CXCR2 knockout mice with mice expressing presenilin (PS1 M146L) and APPsw mutations (PSAPP). Our present study confirms that CXCR2 depletion results in reduction of Aβ with concurrent increases of γ-secretase substrates. At the mechanistic level, the effect of CXCR2 on γ-secretase was not found to occur via their direct interaction. Furthermore, we provide evidence that Aβ promotes endocytosis of CXCR2 via increasing levels of CXCR2 ligands. In conclusion, our current study confirms the regulatory role of CXCR2 in APP processing, and poses it as a potential target for developing novel therapeutics for intervention in AD.     

PEX11β induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development

Background  

Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although de novo biogenesis and divisions are likely important mechanisms, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as frogs where large embryonic yolk and fatty acid stores must be quickly metabolized, and resulting reactive oxygen species eliminated. Here we elucidate the role of Pex11β in regulating peroxisomal gene expression and number in Xenopus laevis embryogenesis.     

Effects of heparin and enoxaparin on APP processing and Aβ production in primary cortical neurons from Tg2576 mice

Background

Alzheimer''s disease (AD) is caused by accumulation of Aβ, which is produced through sequential cleavage of β-amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE1) and γ-secretase. Enoxaparin, a low molecular weight form of the glycosaminoglycan (GAG) heparin, has been reported to lower Aβ plaque deposition and improve cognitive function in AD transgenic mice.

Methodology/Principal Findings

We examined whether heparin and enoxaparin influence APP processing and inhibit Aβ production in primary cortical cell cultures. Heparin and enoxaparin were incubated with primary cortical cells derived from Tg2576 mice, and the level of APP and proteolytic products of APP (sAPPα, C99, C83 and Aβ) was measured by western blotting. Treatment of the cells with heparin or enoxaparin had no significant effect on the level of total APP. However, both GAGs decreased the level of C99 and C83, and inhibited sAPPα and Aβ secretion. Heparin also decreased the level of β-secretase (BACE1) and α-secretase (ADAM10). In contrast, heparin had no effect on the level of ADAM17.

Conclusions/Significance

The data indicate that heparin and enoxaparin decrease APP processing via both α- and β-secretase pathways. The possibility that GAGs may be beneficial for the treatment of AD needs further study.     

Zinc-induced dimerization of the amyloid-β metal-binding domain 1-16 is mediated by residues 11-14

Analysis of complex formation between amyloid-β fragments using surface plasmon resonance biosensing and electrospray mass spectrometry reveals that region 11-14 mediates zinc-induced dimerization of amyloid-β and may serve as a potential drug target for preventing development and progression of Alzheimer's disease.     

Intestine-specific expression of Apobec-1 rescues apolipoprotein B RNA editing and alters chylomicron production in Apobec1−/− mice

Intestinal apolipoprotein B (apoB) mRNA undergoes C-to-U editing, mediated by the catalytic deaminase apobec-1, which results in translation of apoB48. Apobec1^−/− mice produce only apoB100 and secrete larger chylomicron particles than those observed in wild-type (WT) mice. Here we show that transgenic rescue of intestinal apobec-1 expression (Apobec1^Int/O) restores C-to-U RNA editing of apoB mRNA in vivo, including the canonical site at position 6666 and also at approximately 20 other newly identified downstream sites present in WT mice. The small intestine of Apobec1^Int/O mice produces only apoB48, and the liver produces only apoB100. Serum chylomicron particles were smaller in Apobec1^Int/O mice compared with those from Apobec1^−/− mice, and the predominant fraction of serum apoB48 in Apobec1^Int/O mice migrated in lipoproteins smaller than chylomicrons, even when these mice were fed a high-fat diet. Because apoB48 arises exclusively from the intestine in Apobec1^Int/O mice and intestinal apoB48 synthesis and secretion rates were comparable to WT mice, we were able to infer the major sites of origin of serum apoB48 in WT mice. Our findings imply that less than 25% of serum apoB48 in WT mice arises from the intestine, with the majority originating from the liver.     

Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Aβ accumulation and memory deficits

Background

Preventing or reducing amyloid-beta (Aβ) accumulation in the brain is an important therapeutic strategy for Alzheimer’s disease (AD). Recent studies showed that the water channel aquaporin-4 (AQP4) mediates soluble Aβ clearance from the brain parenchyma along the paravascular pathway. However the direct evidence for roles of AQP4 in the pathophysiology of AD remains absent.

Results

Here, we reported that the deletion of AQP4 exacerbated cognitive deficits of 12-moth old APP/PS1 mice, with increases in Aβ accumulation, cerebral amyloid angiopathy and loss of synaptic protein and brain-derived neurotrophic factor in the hippocampus and cortex. Furthermore, AQP4 deficiency increased atrophy of astrocytes with significant decreases in interleukin-1 beta and nonsignficant decreases in interleukin-6 and tumor necrosis factor-alpha in hippocampal and cerebral samples.

Conclusions

These results suggest that AQP4 attenuates Aβ pathogenesis despite its potentially inflammatory side-effects, thus serving as a promising target for treating AD.

     

Synergistic effects of electronegative-LDL- and palmitic-acid-triggered IL-1β production in macrophages via LOX-1- and voltage-gated-potassium-channel-dependent pathways

Electronegative LDL (LDL(-)) and free fatty acids (FFAs) are circulating risk factors for cardiovascular diseases (CVDs) and have been associated with inflammation. Interleukin-1 beta (IL-1β) represents a key cytokine in the development of CVD; however, the initial trigger of IL-1β in CVD remains to be explored. In this study, we investigated the combined effects of LDL(-) from the plasma of ST-segment elevation myocardial infarction (STEMI) patients or diet-induced hypercholesterolemic rabbits and bovine serum albumin bound palmitic acid (PA-BSA) on IL-1β production in macrophages. Macrophages derived from THP-1 cells or human peripheral blood mononuclear cells were independently treated with LDL(-), PA-BSA or cotreated with LDL(-) and PA-BSA. The results showed that nLDL and/or PA-BSA had no effect on IL-1β, and LDL(-) slightly increased IL-1β; however, cotreatment with LDL(-) and PA-BSA resulted in abundant secretion of IL-1β in macrophages. Rabbit LDL(-) induced the elevation of cellular pro-IL-1β and p-Iκ-Bα, but PA-BSA had no effect on pro-IL-1β or p-Iκ-Bα. In potassium-free buffer, LDL(-)-induced IL-1β reached a level similar to that induced by cotreatment with LDL(-) and PA-BSA. Moreover, LDL(-) and PA-BSA-induced IL-1β was inhibited in lectin-type oxidized LDL receptor-1 (LOX-1) knockdown cells and by blockers of voltage-gated potassium (Kv) channels. LDL(-) from diet-induced hypercholesterolemic rabbit had a similar effect as STEMI LDL(-) on IL-1β in macrophages. These results show that PA-BSA cooperates with LDL(-) to trigger IL-1β production in macrophages via a mechanism involving the LOX-1 and Kv channel pathways, which may play crucial roles in the regulation of inflammation in CVD.     

Desipramine administered chronically inhibits lipopolysaccharide-stimulated production of IL-1β in the brain and plasma of rats

Nowadays, it is assumed that therapeutic efficacy of antidepressants depends, at least partly, on their anti-inflammatory properties. The present study investigated for the first time the effect of 21-day oral administration of desipramine on the lipopolysaccharide (LPS)-stimulated IL-1β concentration in the olfactory bulb, hypothalamus, frontal cortex, hippocampus and plasma of rats, and on the LPS-induced IL-1β mRNA level in the olfactory bulb. Desipramine (15 mg/kg/day) reduced significantly the LPS (250 μg/kg i.p.)-induced IL-1β concentration in the olfactory bulb, hypothalamus and in plasma, and diminished the LPS effect on IL-1β mRNA in the olfactory bulb. Plasma concentration of desipramine was comparable to its therapeutic range. By using the α1/α2-adrenoceptor antagonist prazosin and the unspecific β-adrenoceptor antagonist propranolol given prior to LPS, we found that the effect of desipramine on LPS-induced IL-1β production was partially mediated by both adrenoceptors in the olfactory bulb and plasma, and that β-adrenoceptors contributed also to its effect on the stimulated IL-1β concentration in the hypothalamus. The effect of LPS on the cerebral IL-1β levels was, in part, mediated by β-adrenoceptors and, in a region-specific manner, by α1/α2-adrenoceptors. The findings provide evidence for central and peripheral anti-inflammatory activity of desipramine and confirm the impact of the noradrenergic system on IL-1β production induced by an immunostimulatory challenge.     

Effects of hypoxia and intracellular iron chelation on hypoxia-inducible factor-1α and -1β in the rat carotid body and glomus cells

The present investigation provides for the first time, unambiguous information on the occurrence of hypoxia-inducible factors (HIF-1 and HIF-1 proteins) in normoxia (Nx) and their interaction with hypoxia (Hx) and intracellular Fe²⁺ chelation in the rat carotid body (CB) glomus cells. HIF-1 bound to HIF-1 translocated into the nucleus is identified on the basis of immunohistochemistry and immunofluorescence. In Nx, a weak expression of HIF-1 was observed in CB glomus cells. However, exposure of CB and glomus cells to Hx (Po₂7 Torr) and Nx with ciclopirox olamine (CPX, 5 M) for 1 h showed a significant (P<0.001) increase in HIF-1 protein. The CBs and glomus cells exposed to Nx, Hx, and Nx with CPX showed a constant level of HIF-1 protein expression. HIF-1 subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. Hydroxylation of HIF-1 by prolyl hydroxylase for proteasomal degradation was dependent on iron, 2-oxoglutarate, and oxygen concentration. The intracellular iron that acts as a cofactor for prolyl hydroxylase activity belongs to the labile iron pool and can be easily chelated. Thus, chelation of intracellular labile iron by CPX in Nx significantly increased HIF-1 in CB glomus cells. Thus, the results are consistent with the hypothesis that HIF-1 which is present in the glomus cells translocates to the nucleus during exposure to Hx and to CPX in Nx.     

Lipopolysaccharide-induced cytokine production in peripheral blood mononuclear cells: intracellular localization of tumor necrosis factor α and interleukin 1β detected with a three-color immunofluorescence technique

 Lipopolysaccharide (LPS) can induce monocytes to produce various cytokines such as tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In the present study, the kinetics of both intracellular and extracellular accumulation of TNFα and IL-1β in LPS stimulated mononuclear cell (MNC) cultures has been determined. A three-color-immunofluorescence technique was used to detect intracellular accumulation of cytokines. Intracellular accumulation of TNFα in monocytes starts shortly after initiation of the culture; i.e., TNFα is detectable after 1 h, reaching a peak level after 3–4 hours with 50–65% of monocytes staining positive. In parallel with its increased intracellular presence, TNFα was also found in the culture supernatant. The intracellular accumulation of IL-1β in monocytes became detectable after 2 h of culture in the presence of LPS. After 4 h, a plateau was reached, with 90% of the monocytes being positive. In parallel, but with a little delay, IL-1β could be detected in the culture supernatant. TNFα and IL-1β can be produced simultaneously in the same monocytes as was shown by a three-color-immunofluorescence technique. It is concluded that TNFα and IL-1β are good parameters for the early measurement of monocyte activation and that both the intracellular accumulation in monocytes and the amount of secreted cytokines can be used for such a purpose. The intracellular accumulation in monocytes can be measured by the three-color-immunofluorescence technique described. Accepted: 27 August 1996     

Caveolin-1β promotes the production of active human microsomal glutathione S-transferase in induced intracellular vesicles in Spodoptera frugiperda 21 insect cells

The heterologous expression in Spodoptera frugiperda 21 (Sf21) insect cells of the β isoform of canine caveolin-1 (caveolin-1β), using a baculovirus-based vector, resulted in intracellular vesicles enriched in caveolin-1β. We investigated whether these vesicles could act as membrane reservoirs, and promote the production of an active membrane protein (MP) when co-expressed with caveolin-1β. We chose hMGST1 (human microsomal glutathione S-transferase 1) as the co-expressed MP. It belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral MPs, and, as a phase II detoxification enzyme, it catalyzes glutathione conjugation of lipophilic drugs present in the lipid membranes. In addition to its pharmaceutical interest, its GST activity can be conveniently measured. The expression of both MPs were followed by Western blots and membrane fractionation on density gradient, and their cell localization by immunolabeling and transmission electron microscopy. We showed that caveolin-1β kept its capacity to induce intracellular vesicles in the host when co-expressed with hMGST1, and that hMGST1 is in part addressed to these vesicles. Remarkably, a fourfold increase in the amount of active hMGST1 was found in the most enriched membrane fraction, along with an increase of its specific activity by 60% when it was co-expressed with caveolin-1β. Thus, heterologously expressed caveolin-1β was able to induce cytoplasmic vesicles in which a co-expressed exogenous MP is diverted and sequestered, providing a favorable environment for this cargo.     

TGF-β-mediated sustained ERK1/2 activity promotes the inhibition of intracellular growth of Mycobacterium avium in epithelioid cells surrogates

Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.     

The distribution of α1-antichymotrypsin and amyloid production in the brain in Alzheimer’s disease

In this immunohistopathological study α₁-antichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endothelial cells and the cytoplasm of astroglial cells in brains from patients with Alzheimer’s disease. Amyloid precursors stained with methenamine silver were arrayed mainly along the membranes, and amyloid fibrils, which stained densely with anti-α₁-antichymotrypsin, were in direct contact with the fibrous structures connecting with the membranes of vascular feet or astrocytic processes. From the above findings, α₁-antichymotrypsin seems to play a role in the production of amyloid fibrils in Alzheimer’s disease.