Increase In apoE Secretion Research Articles

NGF Modulates Cholesterol Metabolism and Stimulates ApoE Secretion in Glial Cells Conferring Neuroprotection against Oxidative Stress.

Cholesterol plays a crucial role in the brain, where its metabolism is particularly regulated by astrocytic activity. Indeed, adult neurons suppress their own cholesterol biosynthesis and import this sterol through ApoE-rich particles secreted from astrocytes. Recent evidence suggests that nerve growth factor (NGF) may exert neurotrophic activity by influencing cell metabolism. Nevertheless, the effect of NGF on glial cholesterol homeostasis has still not been elucidated. Thus, the aim of this project is to assess whether NGF could influence cholesterol metabolism in glial cells. To reach this objective, the U373 astrocyte-derived cell line was used as an experimental model. Immunoblot and ELISA analysis showed that proteins and enzymes belonging to the cholesterol metabolism network were increased upon NGF treatment in glial cells. Furthermore, NGF significantly increased ApoE secretion and the amount of extracellular cholesterol in the culture medium. Co-culture and U373-conditioned medium experiments demonstrated that NGF treatment efficiently counteracted rotenone-mediated cytotoxicity in N1E-115 neuronal cells. Conversely, neuroprotection mediated by NGF treatment was suppressed when N1E-115 were co-cultured with ApoE-silenced U373 cells. Taken together, these data suggest that NGF controls cholesterol homeostasis in glial cells. More importantly, NGF exerts neuroprotection against oxidative stress, which is likely associated with the induction of glial ApoE secretion.

Dietary Lactoferrin Supplementation Prevents Memory Impairment and Reduces Amyloid-β Generation in J20 Mice.

Lactoferrin (LF) is present in senile plaques and neurofibrillary tangles in the brains of Alzheimer's disease (AD) patients and amyloid-β protein precursor transgenic (AβPP-Tg) mice. LF has anti-inflammatory and antioxidant functions, which exert neuroprotective effects against AD. However, its effects on memory impairment and AD pathogenesis have not been fully examined. In this study, we examined the effects of LF on memory impairment and AD pathogenesis in AβPP-Tg mice (J20 mice). Nine-month-old J20 mice were fed with control, 2% lactoferrin-containing (LF), and 0.5% pepsin-hydrolyzed lactoferrin-containing (LF-hyd) diets for 3 months. We found that both the LF and LF-hyd diets attenuated memory impairment in J20 mice and decreased brain Aβ40 and Aβ42 levels through the inhibition of amyloidogenic processing of AβPP, as it decreased β-site amyloid protein precursor cleaving enzyme 1 (BACE1) levels. Furthermore, we found for the first time that LF and LF-hyd treatments increased both ApoE secretion and ATP-binding cassette A1 (ABCA1) protein levels in the brains of J20 mice and in primary astrocyte cultures. Moreover, LF and LF-hyd promoted extracellular degradation of Aβ in primary astrocyte cultures. These findings indicate that the reduction in Aβ levels in the brains of mice fed with both the LF and LF-hyd diets may also be mediated by increased ApoE secretion and ABCA1 protein levels, which in turn leads to the enhanced degradation of Aβ in the brains of J20 mice. Our findings suggest that LF and LF-hyd can be used for the treatment and/or prevention of the development of AD.

5-HT3 Antagonist Ondansetron Increases apoE Secretion by Modulating the LXR-ABCA1 Pathway.

Apolipoprotein E (apoE) is linked to the risk for Alzheimer’s disease (AD) and thus has been suggested to be an important therapeutic target. In our drug screening effort, we identified Ondansetron (OS), an FDA-approved 5-HT3 antagonist, as an apoE-modulating drug. OS at low micromolar concentrations significantly increased apoE secretion from immortalized astrocytes and primary astrocytes derived from apoE3 and apoE4-targeted replacement mice without generating cellular toxicity. Other 5-HT3 antagonists also had similar effects as OS, though their effects were milder and required higher concentrations. Antagonists for other 5-HT receptors did not increase apoE secretion. OS also increased mRNA and protein levels of the ATB-binding cassette protein A1 (ABCA1), which is involved in lipidation and secretion of apoE. Accordingly, OS increased high molecular weight apoE. Moreover, the liver X receptor (LXR) and ABCA1 antagonists blocked the OS-induced increase of apoE secretion, indicating that the LXR-ABCA1 pathway is involved in the OS-mediated facilitation of apoE secretion from astrocytes. The effects of OS on apoE and ABCA1 were also observed in human astrocytes derived from induced pluripotent stem cells (iPSC) carrying the APOE ε3/ε3 and APOE ε4/ε4 genotypes. Oral administration of OS at clinically-relevant doses affected apoE levels in the liver, though the effects in the brain were not observed. Collectively, though further studies are needed to probe its effects in vivo, OS could be a potential therapeutic drug for AD by modulating poE metabolism through the LXR-ABCA1 pathway.

Class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

Despite the important role of apolipoprotein E (apoE) secretion from astrocytes in brain lipid metabolism and the strong association of apoE4, one of the human apoE isoforms, with sporadic and late onset forms of Alzheimer’s disease (AD) little is known about the regulation of astrocytic apoE. Utilizing annotated chemical libraries and a phenotypic screening strategy that measured apoE secretion from a human astrocytoma cell line, inhibition of pan class I histone deacetylases (HDACs) was identified as a mechanism to increase apoE secretion. Knocking down select HDAC family members alone or in combination revealed that inhibition of the class I HDAC family was responsible for enhancing apoE secretion. Knocking down LXRα and LXRβ genes revealed that the increase in astrocytic apoE in response to HDAC inhibition occurred via an LXR-independent pathway. Collectively, these data suggest that pan class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

Bioactive Compound Screen for Pharmacological Enhancers of Apolipoprotein E in Primary Human Astrocytes

Pharmacological screening in physiologically relevant brain cells is crucial for identifying neuroactive compounds that better translate into invivo biology and efficacious therapeutics. Pharmacological enhancement of apolipoprotein E (apoE), a cholesterol-transporting apolipoprotein, has been proposed as a promising therapeutic approach for Alzheimer's disease. Several nuclear receptor agonists were initially shown to increase brain apoE levels together with ATP-binding cassette transporter 1 (ABCA1), but their underlying mechanisms remain unclear. To gain an insight on brain apoE regulation, we performed an unbiased high-throughput screening of known drugs and bioactive compounds in cultured human primary astrocytes, the major apoE-producing cell type in thebrain. We have identified several small molecules that increase apoE secretion via previously unknownmechanisms, including those not co-inducing ABCA1. These newly identified compounds are active preferentially in human astrocytes but not in an astrocytoma cell line, furnishing new tools for investigating biological pathways underlying brain apoE production.

Retinoic Acid Isomers Facilitate Apolipoprotein E Production and Lipidation in Astrocytes through the Retinoid X Receptor/Retinoic Acid Receptor Pathway

Apolipoprotein E (apoE) is the major cholesterol transport protein in the brain. Among the three human APOE alleles (APOE2, APOE3, and APOE4), APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease (AD). The accumulation of amyloid-β (Aβ) is a central event in AD pathogenesis. Increasing evidence demonstrates that apoE isoforms differentially regulate AD-related pathways through both Aβ-dependent and -independent mechanisms; therefore, modulating apoE secretion, lipidation, and function might be an attractive approach for AD therapy. We performed a drug screen for compounds that modulate apoE production in immortalized astrocytes derived from apoE3-targeted replacement mice. Here, we report that retinoic acid (RA) isomers, including all-trans-RA, 9-cis-RA, and 13-cis-RA, significantly increase apoE secretion to ~4-fold of control through retinoid X receptor (RXR) and RA receptor. These effects on modulating apoE are comparable with the effects recently reported for the RXR agonist bexarotene. Furthermore, all of these compounds increased the expression of the cholesterol transporter ABCA1 and ABCG1 levels and decreased cellular uptake of Aβ in an apoE-dependent manner. Both bexarotene and 9-cis-RA promote the lipidation status of apoE, in which 9-cis-RA promotes a stronger effect and exhibits less cytotoxicity compared with bexarotene. Importantly, we showed that oral administration of bexarotene and 9-cis-RA significantly increases apoE, ABCA1, and ABCG1 levels in mouse brains. Taken together, our results demonstrate that RXR/RA receptor agonists, including several RA isomers, are effective modulators of apoE secretion and lipidation and may be explored as potential drugs for AD therapy.

Hormonal modulators of glial ABCA1 and apoE levels

Apolipoprotein E (apoE) is the major lipid carrier in the central nervous system. As apoE plays a major role in the pathogenesis of Alzheimer disease (AD) and also mediates repair pathways after several forms of acute brain injury, modulating the expression, secretion, or function of apoE may provide potential therapeutic approaches for several neurological disorders. Here we show that progesterone and a synthetic progestin, lynestrenol, significantly induce apoE secretion from human CCF-STTG1 astrocytoma cells, whereas estrogens and the progesterone metabolite allopregnanolone have negligible effects. Intriguingly, lynestrenol also increases expression of the cholesterol transporter ABCA1 in CCF-STTG1 astrocytoma cells, primary murine glia, and immortalized murine astrocytes that express human apoE3. The progesterone receptor inhibitor RU486 attenuates the effect of progestins on apoE expression in CCF-STTG1 astrocytoma cells but has no effect on ABCA1 expression in all glial cell models tested, suggesting that the progesterone receptor (PR) may participate in apoE but does not affect ABCA1 regulation. These results suggest that selective reproductive steroid hormones have the potential to influence glial lipid homeostasis through liver X receptor-dependent and progesterone receptor-dependent pathways.

Physiological role of hepatic NPC1L1 in human cholesterol and lipoprotein metabolism: New perspectives and open questions

The importance of the cholesterol transporter Niemann-Pick C1 like protein 1 (NPC1L1)in the small intestine for efficient absorption of cholesterol is well documented and its physiological importance is clear (1). Similarly, the drug ezetimibe (Zetia™), which blocks function of this transporter, reduces plasma cholesterol, primarily LDL, by dramatically reducing intestinal absorption of dietary and biliary cholesterol (2). There are controversies about the exact mechanism by which NPC1L1 functions: whether the protein is on the apical cell membrane or intracellular vesicles or both (3–5), and whether ezetimibe directly blocks NPC1L1 or interrupts its association with other proteins in the sterol absorption and transport pathway (6–8). Nevertheless, it is now accepted that this protein is the key player in sterol absorption, although other transporters may play ancillary roles (9, 10).

Human apoA-I increases macrophage foam cell derived PLTP activity without affecting the PLTP mass

Backgroundphospholipid transfer protein (PLTP) plays important roles in lipoprotein metabolism and atherosclerosis and is expressed by macrophages and macrophage foam cells (MFCs). The aim of the present study was to determine whether the major protein from HDL, apoA-I, affects PLTP derived from MFCs.Resultsas cell model we used human THP-1 monocytes incubated with acetylated LDL, to generate MFC. The addition of apoA-I to the cell media increased apoE secretion from the cells, in a concentration dependent fashion, without affecting cellular apoE levels. In contrast, apoA-I had no effect on PLTP synthesis and secretion, but strongly induced the PLTP activity in the media. ApoA-I also increased phospholipid transfer activity of PLTP isolated from human plasma. This effect was dependent on apoA-I concentration but independent on apoA-I lipidation status. ApoE, ApoA-II and apoA-IV, but not immunoglobulins or bovine serum albumin, also increased PLTP activity. We also report that apoA-I protects PLTP from heat inactivation.ConclusionapoA-I enhances the phospholipid transfer activity of PLTP secreted from macrophage foam cells without affecting the PLTP mass.

Decreased Atherosclerosis in Low-Density Lipoprotein Receptor Knockout Mice Transplanted With Abcg1 −/− Bone Marrow

Recent studies indicate that the ATP-binding cassette transporter ABCG1 can promote cholesterol efflux from macrophages to high-density lipoprotein. This study was designed to assess the in vivo role of macrophage ABCG1 in atherosclerosis. Bone marrow from Abcg1-/- mice was transplanted into irradiated Ldlr-/- recipients, and atherosclerosis was evaluated by aortic root assay after 7 or 11 weeks of feeding on a Western diet. After 7 weeks, there was no difference in lesion area in mice receiving either wild-type or Abcg1-/- bone marrow, whereas after 11 weeks, lesion area was moderately but significantly reduced in Abcg1-/- recipients. ABCG1-deficient peritoneal macrophages showed induction of several liver X receptor target genes, such as Abca1 and Srebp1c, and a dramatic increase in apolipoprotein E (apoE) protein both in cell media and lysates, without parallel change in apoE mRNA. Abca1 knockdown prevented the increase in apoE secretion but had minimal effects on apoE accumulation in cell lysates of Abcg1-/- macrophages. Plasma apoE levels were markedly increased in recipients of Abcg1-/- bone marrow. These studies reveal an inverse relationship between Abcg1 expression and apoE accumulation and secretion in macrophages. The reduced atherosclerosis in recipients of Abcg1-deficient bone marrow may be explained by induction of Abca1 and an associated increase in macrophage apoE secretion.

Reduced CSF PLTP activity in Alzheimer's disease and other neurologic diseases; PLTP induces ApoE secretion in primary human astrocytes in vitro

Phospholipid transfer protein (PLTP) plays a pivotal role in cellular lipid efflux and modulation of lipoprotein metabolism. PLTP is distributed widely in the central nervous system (CNS), is synthesized by glia and neurons, and is active in cerebrospinal fluid (CSF). The aims of this study were to test the hypothesis that patients with Alzheimer's disease (AD) have altered PLTP-mediated phospholipid transfer activity in CSF, and to examine the potential relationship between PLTP activity and apolipoprotein E (apoE) levels in CSF. We assessed PLTP activity and apoE concentration in CSF of patients with probable AD (n = 50), multiple sclerosis (MS; n = 9), other neurologic diseases (n = 21), and neurologically healthy controls (n = 40). PLTP activity in AD was reduced compared to that in controls (P < 0.001), with approximately half of the AD patients with PLTP activity values below all controls. Patients with MS had lower PLTP activity than AD patients (P < 0.001). PLTP activity was highly correlated with PLTP mass, as estimated by Western blot (r = 0.006; P < 0.01). CSF PLTP activity positively correlated with apoE concentration in AD (R = 0.435; P = 0.002) and controls (R = 0.456; P = 0.003). Anti-apoE immunoaffinity chromatography and Western blot analyses indicated that some CSF PLTP is associated with apoE-containing lipoproteins. Exogenous addition of recombinant PLTP to primary human astrocytes significantly increased apoE secretion to the conditioned medium. The findings of reduced PLTP activity in AD CSF, and the observation that PLTP can influence apoE secretion in astrocytes suggest a potential link between alterations in the brain lipid metabolism and AD pathogenesis.

Divergent Effects of Peroxisome Proliferator-activated Receptor γ Agonists and Tumor Necrosis Factor α on Adipocyte ApoE Expression

ApoE is expressed in multiple mammalian cell types in which it supports cellular differentiated function. In this report we demonstrate that apoE expression in adipocytes is regulated by factors involved in modulating systemic insulin sensitivity. Systemic treatment with pioglitazone increased systemic insulin sensitivity and increased apoE mRNA levels in adipose tissue by 2-3-fold. Treatment of cultured 3T3-L1 adipocytes with ciglitazone increased apoE mRNA levels by 2-4-fold in a dose-dependent manner and increased apoE secretion from cells. Conversely, treatment of adipocytes with tumor necrosis factor (TNF) alpha reduced apoE mRNA levels and apoE secretion by 60%. Neither insulin nor a peroxisome proliferator-activated receptor (PPAR) alpha agonist regulated adipocyte apoE gene expression. In addition, treatment of human monocyte-derived macrophages with ciglitazone did not regulate expression of apoE. Additional analyses using reporter genes indicated that the effect of TNFalpha and PPARgamma agonists on the apoE gene was mediated via distinct gene control elements. The TNFalpha effect was mediated by elements within the proximal promoter, whereas the PPARgamma effect was mediated by elements within a downstream enhancer. However, the addition of TNFalpha substantially reduced the absolute levels of apoE reporter gene response even in the presence of ciglitazone. These results indicate for the first time that adipose tissue expression of apoE is modulated by physiologic regulators of insulin sensitivity.

Progressive changes in regulation of apolipoproteins E and J in glial cultures during postnatal development and aging

Apolipoprotein (Apo) E and ApoJ are lipid- and cholesterol-carriers in the central nervous system and are implicated in age-related neurodegenerative diseases. The primary source of secreted ApoE and ApoJ (clusterin) in the brain is glia. Regulation of these apolipoproteins in mixed glial cultures from rat cerebral cortex differed most strongly between neonatal- and adult-derived glia. Basal secretion of ApoJ was two-fold greater in neonatal than adult glia. Responses to cytokines also differed by donor age. In adult glia, IL-6 increased ApoE secretion, but slightly decreased ApoJ. Both IL-1β and TNFα treatments increased ApoJ secretion from adult glia, with little effect on ApoE. In contrast to adult glia, neonatal ApoJ secretion did not respond to IL-1β, IL-6, or TNFα, and ApoE secretion from neonatal glia was slightly increased by IL-6. These differences may contribute to age-related neuroinflammatory processes, and are pertinent to the general use of neonatal-derived primary glia in models for neurodegenerative disease.

Apolipoprotein A-I-stimulated Apolipoprotein E Secretion from Human Macrophages Is Independent of Cholesterol Efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.

U373-MG response to interleukin-1beta-induced oxidative stress.

Oxidative stress has been involved in various neurological disorders and, in the central nervous system, astrocytes represent the cell type that contributes to neuroprotection via glutathione (GSH) metabolism, GSH-metabolizing enzymes like gamma-glutamyltransferase (GGT), and apoE secretion. In this study, using IL-1beta, a proinflammatory and prooxidant cytokine that is increased in numerous pathological situations, cells of astrocytoma cell line U373-MG were exposed to an oxidative stress, leading to c-Jun and c-Fos activation. IL-1beta decreased both GGT activity and intracellular GSH content and increased apoE secretion, initiating astroglial response to injury. We observed that antioxidants inhibit IL-1beta effects on c-Jun and c-Fos proteins, GGT activity and the GSH pool but not on apoE secretion. Our results allow us to conclude that neurological disorders associated with an IL-1beta-induced oxidative stress could be, at least experimentally, reversible in the presence of one antioxidant, N-acetylcysteine.

ApoE of the HepG2 Cell Surface Includes a Major Pool Associated with Chondroitin Sulfate Proteoglycans

We have investigated the association of apolipoprotein E (apoE) with the HepG2 cell surface (i.e. plasma membrane and extracellular matrix) using domain specific monoclonal antibodies against apoE. Growth in beta-D-xyloside decreased the incorporation of 35S into glycosaminoglycans by 31% and cell surface apoE by 45% with a concomitant increase in apoE secretion (4.3-fold), underlining the importance of glycosaminoglycan association of apoE. Heparinase (3-10 U/mL) or heparin (1 mg/mL) decreased apoE by 25 and 30.5%, respectively, which suggests that some apoE is associated with cell surface heparan sulfate proteoglycans. Chondroitinase ABC (1.5 U/mL) reduced cell surface apoE by 40%, indicating that a major pool of apoE is associated with chondroitin sulfate proteoglycans. Further enzymatic and displacement analysis suggested that cell surface apoE associates specifically with GAGs containing chondroitin-4-sulfates. 3H1, a monoclonal antibody that recognizes an epitope within the lipid-binding C-terminal domain of apoE, decreased binding of apoE to chondroitin sulfate proteoglycans in solid-phase assays by 77% and to heparan sulfate proteoglycans by 46%, suggesting that this region is of increased importance for binding to chondroitin sulfate proteoglycans. Previous studies with 3H1 demonstrated that apoE of the extracellular matrix is lipid-poor (Burgess, J. W., Gould, D. R., and Marcel, Y. L. (1998) J. Biol. Chem. 273, 5645-5654), but we show here that apoE on the remaining cell surface is lipid-associated. In summary, lipidated apoE associates with the HepG2 plasma membrane through interactions with chondroitin-4-sulfate containing GAGs and, to a lesser extent, HSPG.

Degradation of Macrophage ApoE in a Nonlysosomal Compartment: REGULATION BY STEROLS

Macrophage-derived apoE has been shown to play an important role in the susceptibility of the vessel wall to atherosclerosis. Previous studies have shown that macrophage sterol content modulates apoE synthesis and secretion, associated with a large transcriptional response of the apoE gene. The current studies were undertaken to evaluate the existence of additional post-transcriptional regulatory loci for the effect of sterols on apoE synthesis and secretion. Using a macrophage cell line transfected to constitutively express an apoE cDNA to facilitate detection of a post-transcriptional regulatory locus, we demonstrated that preincubations in 25-hydroxycholesterol and cholesterol lead to increased apoE secretion in pulse/chase experiments. Examination of cell lysates in these experiments showed that apoE not secreted by control cells was degraded and not detectable, suggesting that the preincubation in sterols increased secretion by decreasing degradation of newly synthesized apoE. The measurement of total protein and apoE degradation in cell fractions revealed an intermediate density fraction that degraded significant amounts of newly synthesized total protein and newly synthesized apoE. In this fraction, degradation of total protein and apoE was unaffected by chloroquine but was substantially reduced by N-acetyl-Leu-Leu-norleucinal plus N-acetyl-Leu-Leu-methioninal or by lactacystin, suggesting the involvement of proteasomes. Preincubation in sterol/oxysterol or acetylated low density lipoprotein did not modify total protein degradation by this fraction but inhibited apoE degradation. Similar results were obtained using intermediate density fractions isolated from human monocyte-derived macrophages. The results of our studies indicate that newly synthesized apoE in the macrophage can be degraded in an intermediate density nonlysosomal cellular compartment, which is sensitive to proteasomal inhibitors. Alteration of cellular lipid homeostasis by preincubation in sterol/oxysterol or acetylated low density lipoprotein inhibits apoE, but not total protein, degradation in this fraction. Inhibition of the degradation of apoE in this fraction likely contributes to the increased apoE secretion observed in sterol-enriched cells.

Altered apolipoprotein E secretion in cytokine treated human astrocyte cultures

Apolipoprotein E (ApoE), postulated to be a major lipid carrier protein in brain, is synthesized and secreted primarily by astrocytes and is involved in brain development and repair. We have analyzed its secretion in primary cultures of older (high passage) slowly dividing and younger (lower passage) rapidly dividing fetal human astrocytes exposed to various inflammatory and anti-inflammatory cytokines, alone and in combination. ApoE secretion was reduced in high passage astrocytes when compared to lower passage astrocytes. A further reduction in ApoE secretion in high passage cells was consistently produced by the combination of cytokines interleukin 1 (Il-1) alpha and beta and interferon (IFN-gamma) cytokines or by the basic fibroblast growth factor (basic-FGF) alone. Epidermal growth factor (EGF) increased ApoE secretion. The combination of these cytokine effects in chronically degenerating brain regions of Alzheimer's disease and other neurodegenerative diseases could reduce the amount of ApoE available for neuronal regeneration. EGF, or agents inducing EGF, could ameliorate these ApoE deficiencies.

Cell Surface Proteoglycans Modulate Net Synthesis and Secretion of Macrophage Apolipoprotein E

Using a macrophage cell line that constitutively expresses a human apolipoprotein E (apoE) cDNA, we have investigated the post-translational metabolism of endogenously produced apoE. Inhibition of lysosomal or cysteine proteases led to significant inhibition of apoE degradation but did not increase apoE secretion, indicating that cellular degradation is not limiting for apoE secretion in macrophages. Treatment of macrophages with inhibitors of proteoglycan synthesis (4-methylumbelliferyl-beta-D-xyloside) or sulfation (sodium chlorate) enhanced the release of apoE from cells and significantly attenuated the increase in secretion produced by incubation with phosphatidylcholine vesicles (PV). These observations suggested that a significant fraction of the apoE retained by cells (and released by incubation with PV) was associated with proteoglycans. Treatment of cells with exogenous heparinase led to a greater than 4-fold increase in apoE secretion and similarly attenuated the response to PV, suggesting that apoE was trapped in an extracellular proteoglycan matrix. This conclusion was confirmed in studies showing that PV could enhance the release of apoE from cells during an incubation at 4 degrees C, but this enhanced release was abolished in proteoglycan-depleted cells. Incubation with lactoferrin at 4 or 37 degrees C produced a similar decrement in cellular apoE, again indicating the existence of a cell surface pool of apoE. Pulse-chase studies showed that the apoE trapped in the proteoglycan matrix was susceptible to rapid cellular degradation such that net synthesis of apoE (secreted plus cell-associated) was increased significantly in proteoglycan-depleted cells compared with control cells as early as 45 min during a chase period.

Secretion of apolipoprotein E by an astrocytoma cell line.

Apolipoprotein (apo) E is a predominant protein in developing mammalian brain and in damaged peripheral nerve. Of particular interest is the observation that astrocytes in the central nervous system cease to produce apoE after nerve damage, whereas an increase in apoE production results after peripheral nerve injury. Differences in the response to injury with regard to the production of apoE may be related to dissimilarities in the abilities of the central and peripheral nervous systems to regenerate. As there are few data concerning the regulation of apoE gene expression in extrahepatic tissues, we employed a human astrocytoma cell line (CCF-STTG1) as a model to study apoE production in astrocytes. CCF-STTG1 cells secreted apoE constitutively in serum-free media. Cholesterol added to the media as cholesterol:phospholipid liposomes (2-100 micrograms/ml) or as human plasma LDL increased the amount of apoE secreted into the media, but had little or no effect on the relative abundance of apoE mRNA. By contrast, the commercially available triglyceride-phospholipid emulsion Intralipid added at dilutions of 1:50 to 1:500 caused a total inhibition of apoE secretion by the cells, but again, little change was noted in the relative abundance of apoE mRNA. Insulin (5 micrograms/ml) caused a 45-55% reduction in the amount of apoE secreted by the astrocytoma cells. Glucagon (5 micrograms/ml), on the other hand, did not increase apoE secretion, and apoE mRNA concentrations were not affected by either hormone treatment. ApoE was secreted from the astrocytoma cells associated with particles of plasma VLDL to IDL and HDL size. After feeding the cells with 20 micrograms/ml cholesterol as cholesterol:phospholipid liposomes, an increased proportion of apoE was secreted associated with the larger VLDL to IDL size particles, with a concomitant decrease in the proportion associated with the smaller HDL-size particles. When cells were incubated with 5 micrograms/ml insulin, most of the apoE was associated with the HDL-size particles. When cholesterol:phospholipid liposomes were added in the presence of insulin virtually all of the secreted apoE was found associated with the VLDL to IDL size particles. In summary, the regulation of apoE production in CCF-STTG1 cells in many respects resembles that of other cells, including hepatocytes. However, it is clear that there remain to be identified cell specific factors which regulate apoE production in astrocytes. The CCF-STTG1 cell line promises to provide a suitable model to investigate these questions.