Biomedical subjects

M J LaDu

Publications and source records attributed to M J LaDu.

15 recordsLinked to original sources

PubMed2000

SDS-stable complex formation between native apolipoprotein E3 and beta-amyloid peptides.

Extracellular senile plaques composed predominantly of fibrillar amyloid-beta (Abeta) are a major neuropathological feature of Alzheimer's disease (AD). Genetic evidence and in vivo studies suggest that apolipoprotein E (apoE) may contribute to amyloid clearance and/or deposition. In vitro studies demonstrate that native apoE2 and E3 form an SDS-stable complex with Abeta(1-40), while apoE4 forms little such complex. Our current work extends these observations by presenting evidence that apoE3 also binds to Abeta(1-42) and with less avidity to modified species of the peptide found in senile plaque cores. These modified peptides include a form that originates at residue 3-Glu as pyroglutamyl and another with isomerization at the 1-Asp and 7-Asp positions. In addition, we used binding reactions between apoE3 and various Abeta fragments, as well as binding reactions with apoE3 and Abeta(1-40) plus Abeta fragments as competitors, to identify the domain(s) of Abeta involved in the formation of an SDS-stable complex with apoE3. Residues 13-28 of Abeta appear to be necessary, while complex formation is further enhanced by the presence of residues at the C-terminus of the peptide. These results contribute to our understanding of the biochemical basis for the SDS-stable apoE3/Abeta complex and support the hypothesis that Abeta can be transported in vivo complexed with apoE. This complex may then be cleared from the interstitial space by apoE receptors in the brain or become part of an extracellular amyloid deposit.

Amino Acid Substitution↗

PubMed2000

Apolipoprotein E receptors mediate the effects of beta-amyloid on astrocyte cultures.

We have previously shown that beta-amyloid (Abeta) induces astrocyte activation in vitro and that this reaction is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. However, the effects of Abeta on endogenous apoE and apoJ levels and the potential role of apoE receptors in astrocyte activation have not been addressed. Three activating stimuli (lipopolysaccharide, dibutyryl cAMP, and aged Abeta 1-42) were used to induce activation of rat astrocyte cultures, as assessed by changes in morphology and an increase in interleukin-1beta. However, only Abeta also induced approximately 50% reduction in the amount of released apoE and apoJ and an 8-fold increase in the levels of cell-associated apoE and apoJ. Experiments using two concentrations of receptor-associated protein, an inhibitor of apoE receptors with a differential affinity for the low density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), suggest that LRP mediates Abeta-induced astrocyte activation, whereas LDLR mediates the Abeta-induced changes in apoE levels. Receptor-associated protein had no effect on apoJ levels or on activation by either dibutyryl cAMP or lipopolysaccharide. These data suggest that apoE receptors translate the presence of extracellular Abeta into cellular responses, both initiating and modulating the inflammatory response induced by Abeta.

Amyloid beta-Peptides↗

PubMed1999

Unique lipoproteins secreted by primary astrocytes from wild type, apoE (-/-), and human apoE transgenic mice.

Composition of central nervous system lipoproteins affects the metabolism of lipoprotein constituents within the brain. The epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease via an unknown mechanism(s). As glia are the primary central nervous system cell type that synthesize apoE, we characterized lipoproteins secreted by astrocytes from wild type (WT), apoE (-/-), and apoE transgenic mice expressing human apoE3 or apoE4 in a mouse apoE (-/-) background. Nondenaturing size exclusion chromatography demonstrates that WT, apoE3, and apoE4 astrocytes secrete particles the size of plasma high density lipoprotein (HDL) composed of phospholipid, free cholesterol, and protein, primarily apoE and apoJ. However, the lipid:apoE ratio of particles containing human apoE is significantly lower than WT. ApoE localizes across HDL-like particle sizes. ApoJ localizes to the smallest HDL-like particles. ApoE (-/-) astrocytes secrete little phospholipid or free cholesterol despite comparable apoJ expression, suggesting that apoE is required for normal secretion of astrocyte lipoproteins. Further, particles were not detected in apoE (-/-) samples by electron microscopy. Nondenaturing immunoprecipitation experiments indicate that apoE and apoJ reside predominantly on distinct particles. These studies suggest that apoE expression influences the unique structure of astrocyte lipoproteins, a process further modified by apoE species.

Animals↗

PubMed1998

Glial fibrillary acidic protein-apolipoprotein E (apoE) transgenic mice: astrocyte-specific expression and differing biological effects of astrocyte-secreted apoE3 and apoE4 lipoproteins.

The epsilon4 allele of apolipoprotein E (apoE) is associated with increased risk for Alzheimer's disease (AD) and poor outcome after brain injury. In the CNS, apoE is expressed by glia, predominantly astrocytes. To define the potential biological functions of different human apoE isoforms produced within the brain, transgenic mice were generated in which human apoE3 and apoE4 expression is under control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. These animals were then bred back to apoE knock-out mice. Human apoE protein is found within astrocytes and the neuropil throughout development and into the adult period, as assessed by immunocytochemistry and immunoblot analysis in several GFAP-apoE3 and E4 lines. Cultured astrocytes from these mice secrete apoE3 and apoE4 in lipoproteins that are high-density lipoprotein-like in size. When primary hippocampal neurons are grown in the presence of astrocyte monolayers derived from these transgenic mice, there is significantly greater neurite outgrowth from neurons grown in the presence of apoE3-secreting astrocytes compared with apoE4-secreting or apoE knock-out astrocytes. These effects are not dependent on direct astrocyte-neuron contact and appear to require the low-density lipoprotein receptor-related protein. These data suggest that astrocyte-secreted, apoE3-containing lipoproteins have different biological effects than apoE4-containing lipoproteins. In addition to providing information regarding the role of astrocyte-secreted apoE lipoproteins in the normal brain, these animals will also be useful in models of both AD and CNS injury.

Animals↗

PubMed1998

Isoform-specific effect of apolipoprotein E on cell survival and beta-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures.

Although the genetic link between the epsilon4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established, the isoform-specific activity of apoE underlying this correlation remains unclear. To determine whether apoE influences the neurotoxic actions of beta-amyloid (Abeta), we examined the effect of native preparations of apoE3 and E4 on Abeta-induced toxicity in primary cultures of rat hippocampal pyramidal neurons. The source of apoE was conditioned medium from HEK-293 cells stably transfected with human apoE3 or E4 cDNA. ApoE4 (10 microg/ml) alone was toxic to the cultures, whereas apoE3 had no effect. ApoE3 treatment prevented the toxicity induced by 10 microM Abeta(1-40) or Abeta(25-35). The apoE3 protective effect appears to be specific to Abeta-induced toxicity, because apoE3 did not protect against the cytotoxicity produced by NMDA or staurosporine, nor did apoE3 affect the increase in intracellular calcium induced by either NMDA or KCl. ApoE3 had no effect on the toxicity produced by Abeta in the presence of receptor-associated protein, an inhibitor of apoE receptors, particularly the LDL-receptor-related protein. Interaction with apoE receptors may not mediate the toxic actions of apoE4, because receptor-associated protein did not affect apoE4-induced neurotoxicity. Consistent with our previous biochemical experiments, analysis of the culture medium revealed that SDS-stable apoE3:Abeta complex is present in greater abundance than apoE4:Abeta complex. Thus, the protection from Abeta-induced neurotoxicity afforded by apoE3 treatment may result from clearance of the peptide by apoE3:Abeta complex formation and uptake by apoE receptors.

Amyloid beta-Peptides↗

PubMed1998

Nascent astrocyte particles differ from lipoproteins in CSF.

Little is known about lipid transport and metabolism in the brain. As a further step toward understanding the origin and function of CNS lipoproteins, we have characterized by size and density fractionation lipoprotein particles from human CSF and primary cultures of rat astrocytes. The fractions were analyzed for esterified and free cholesterol, triglyceride, phospholipid, albumin, and apolipoproteins (apo) E, AI, AII, and J. As determined by lipid and apolipoprotein profiles, gel electrophoresis, and electron microscopy, nascent astrocyte particles contain little core lipid, are primarily discoidal in shape, and contain apoE and apoJ. In contrast, CSF lipoproteins are the size and density of plasma high-density lipoprotein, contain the core lipid, esterified cholesterol, and are spherical. CSF lipoproteins were heterogeneous in apolipoprotein content with apoE, the most abundant apolipoprotein, localized to the largest particles, apoAI and apoAII localized to progressively smaller particles, and apoJ distributed relatively evenly across particle size. There was substantial loss of protein from both CSF and astrocyte particles after density centrifugation compared with gel-filtration chromatography. The differences between lipoproteins secreted by astrocytes and present in CSF suggest that in addition to delivery of their constituents to cells, lipoprotein particles secreted within the brain by astrocytes may have the potential to participate in cholesterol clearance, developing a core of esterified cholesterol before reaching the CSF. Study of the functional properties of both astrocyte-secreted and CSF lipoproteins isolated by techniques that preserve native particle structure may also provide insight into the function of apoE in the pathophysiology of specific neurological diseases such as Alzheimer's disease.

Animals↗

PubMed1998

Apolipoprotein E attenuates beta-amyloid-induced astrocyte activation.

A common feature of Alzheimer's disease pathology is an abundance of activated glia, indicative of an inflammatory reaction in the brain. The relationship between glial activation and neurodegeneration is not known, although several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology. As beta-amyloid (A beta) is one of several stimuli that can activate glia, it is important to determine how A beta-induced glial activation is influenced by other proteins present in the plaque, such as apolipoprotein E (apoE). We examined the effect of native preparations of apoE on activation of rat cortical astrocyte cultures by A beta1-42. The apoE source was conditioned medium from human embryonic kidney 293 cells stably transfected with human apoE3 or apoE4 cDNA. By morphological criteria, apoE inhibited A beta-induced astrocyte activation in three experimental paradigms: apoE pretreatment blocked subsequent A beta-induced activation, A beta aged in the presence of apoE did not activate astrocytes, and apoE addition to activated astrocytes transiently reversed the activated phenotype. No apoE isoform selectivity was observed. The effect of apoE appears to be specific to A beta, as apoE did not attenuate cyclic AMP-induced astrocyte activation. These data suggest that apoE may modulate the ability of A beta to induce inflammatory responses in the brain.

Amyloid beta-Peptides↗

PubMed1997

Association of human, rat, and rabbit apolipoprotein E with beta-amyloid.

In humans, apolipoprotein E (apoE) has three major isoforms, E2 (Cys112, Cys158), E3 (Cys112, Arg158), and E4 (Arg112, Arg158). While epsilon4 is a genetic risk factor for Alzheimer's disease (AD), epsilon2 may protect against late-onset AD. Using native preparations of apoE from conditioned tissue culture media or plasma lipoproteins, we have previously shown that when equivalent amounts of apoE3 or E4 were incubated with beta-amyloid (A beta), apoE3 formed 20 times as much SDS-stable complex with the peptide as apoE4. This preferential binding of A beta to apoE3 was abolished when apoE was purified by a process which includes delipidation and denaturation. Here we expand these observations to include A beta binding to lipoprotein-associated and purified apoE2. Lipoproteins isolated from the plasma of individuals homozygous for either epsilon2 or epsilon3 were incubated with A beta(1-40). SDS-stable complex formation was analyzed by a non-reducing gel shift assay, followed by immunoblotting with either A beta or apoE antibodies. ApoE2:A beta complex formation was comparable to apoE3:A beta in both native and purified preparations of apoE. In addition, lipoprotein-associated rat apoE (Arg112, Arg158), like human apoE4, did not form complex with A beta, while lipoprotein-associated rabbit apoE (Cys112, Arg158) did bind the peptide. These binding studies provide one possible explanation for protective effects of both apoE2 and E3 against the development of Alzheimer's disease.

Alzheimer Disease↗

PubMed1997

Effect of apolipoprotein E on neurite outgrowth and beta-amyloid-induced toxicity in developing rat primary hippocampal cultures.

The correlation between the epsilon 4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established. However, the role of apoE in normal as well as pathological brain processes remains unclear. We evaluated the effect of apoE treatment on development and beta-amyloid (A beta)-induced toxicity using primary cultures of developing rat hippocampal neurons. The source of apoE was conditioned media from HEK cells stably transfected with human apoE3 or apoE4 cDNA, a preparation where apoE is lipid-associated. Morphological and biochemical changes in the cultures were assessed at 1 and 3 days following low- and high-density plating with either apoE3 or E4 with or without A beta. Both apoE isoforms were neurotrophic, as measured by increased neurite length. Aged A beta(1-42), a peptide preparation exhibiting extensive fibril and aggregate formation, is toxic to these cultures. Addition of apoE3 and E4 significantly and comparably attenuated the A beta-induced reduction in both neurite length and cell viability. The level of protection against this toxicity was proportional to the neurotrophic actions of the two apoE isoforms. Thus, apoE acts as a potent growth factor in both the absence and the presence of A beta, supporting a potentially important role for apoE in neurobiology.

Amyloid beta-Peptides↗

PubMed1996

Neurons of the human frontal cortex display apolipoprotein E immunoreactivity: implications for Alzheimer's disease.

Apolipoprotein E (apoE) is a plasma protein that regulates lipid transport and cholesterol homeostasis. In humans, apoE occurs as 3 major isoforms (apoE2, E3, and E4). Genetic evidence demonstrates an overrepresentation of the apoE epsilon 4 allele in Alzheimer's disease (AD). While apoE immunoreactivity (IR) is associated with the amyloid plaques and neurofibrillary tangles of AD, few studies have characterized the localization of apoE in normal human brains. We examined the distribution of apoE in the cerebral cortex of normal aged individuals and compared the results to clinically diagnosed and pathologically confirmed AD cases. In addition, we characterized the apoE IR in brains from high plaque non-demented (HPND) cases. We observed consistent and widespread apoE staining in cortical neurons from normal and HPND individuals. This finding was confirmed by double immunostaining which colocalized apoE with microtubule-associated protein-2, as well as low density lipoprotein receptor-related protein, an apoE receptor found on neurons. In contrast, AD brains displayed apoE IR in plaques and neurofibrillary tangles with little neuronal staining. These data clearly establish the presence of apoE in normal neurons, supporting an intracellular role for apoE. Moreover, the results suggest that this function of apoE is disrupted in AD, where apoE staining of neurons was drastically reduced.

Aged↗

PubMed1995

Purification of apolipoprotein E attenuates isoform-specific binding to beta-amyloid.

Apolipoprotein E (apoE), particularly the e4 allele, is genetically linked to the incidence of Alzheimer's disease. In vitro, apoE has been shown to bind beta-amyloid (A beta), an amyloidogenic peptide that aggregates to form the primary component of senile plaques. In previous work, we demonstrated that apoE3 from tissue culture medium binds to A beta with greater avidity than apoE4 (LaDu, M. J., Falduto, M. T., Manelli, A. M., Reardon, C. A., Getz, G. S., and Frail, D. E. (1994) J. Biol. Chem. 269, 23403-23406). This is in contrast to data using purified apoE isoforms as substrate for A beta (Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L.-M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8098-8102). Here we resolve this apparent discrepancy by demonstrating that the preferential binding of A beta to apoE3 is attenuated and even abolished with purification, a process that includes delipidation and denaturation. We compared the A beta binding capacity of unpurified apoE isoforms from both tissue culture medium and intact human very low density lipoproteins with that of apoE purified from these two sources. The interaction of human A beta-(1-40)-peptide and apoE was analyzed by nonreducing SDS-polyacrylamide gel electrophoresis followed by Western immunoblotting for either A beta or apoE immunoreactivity. While the level of the apoE3.A beta complex was approximately 20-fold greater compared with the apoE4.A beta complex in unpurified conditioned medium, apoE3 and apoE4 purified from this medium bound to A beta with comparable avidity. Moreover, using endogenous apoE on very low density lipoproteins from plasma of apoE3/3 and apoE4/4 homozygotes, apoE3 was again a better substrate for A beta than apoE4. However, apoE purified from these plasma lipoproteins exhibited little isoform specificity in binding to A beta. These results suggest that native preparations of apoE may be a more physiologically relevant substrate for A beta binding than purified apoE and further underscore the importance of subtle differences in apoE conformation to its biological activity.

Alzheimer Disease↗

PubMed1994

Isoform-specific binding of apolipoprotein E to beta-amyloid.

Apolipoprotein E (apoE), particularly the e4 allele, is genetically linked to the incidence of Alzheimer's disease. ApoE is present in the extracellular senile plaques and intracellular neurofibrillary tangles associated with Alzheimer's disease. In vitro, apoE has been shown to bind beta-amyloid (A beta), an amyloidogenic proteolytic product of amyloid precursor protein. To analyze the interaction of A beta and apoE, we used Western immunoblotting of human A beta-(1-40)-peptide incubated with conditioned medium from HEK-293 cells transfected with either human apoE3 or apoE4 (products of the e3 and e4 alleles, respectively) cDNA. Nonreducing SDS-polyacrylamide gel electrophoresis revealed the presence of an approximately 45-kDa complex with both A beta and apoE immunoreactivity. The level of the apoE3.A beta complex was approximately 20-fold greater than that of the apoE4.A beta complex. This apoE isoform-specific binding pattern was maintained from pH 5.0 to 9.0, from 2 min to 24 h of peptide incubation, and at concentrations of apoE from 5 to 100 micrograms/ml and of A beta from 10 microM to 1 mM. The higher level of apoE3 binding to A beta is in contrast to previously published data using purified apoE (Strittmatter, W. J., Weisgraber, K.H., Huang, D. Y., Dong, L.-M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A.D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8098-8102). Factors responsible for the isoform-specific interactions between apoE and A beta will require further study before the apparent discrepancy between these data can be reconciled.

Amyloid beta-Peptides↗

PubMed1994

Expression of lipoprotein lipase during differentiation of cultured L6 muscle cells.

The presence of lipoprotein lipase (LPL) in L6 muscle cells is equivocal. Analysis of a 21-day time course indicates that these cells express both LPL activity and mRNA. Lipase activity peaked at 4 days after plating and decreased to a nadir at day 21 after plating. Characterization of lipase activity at 4 and 19 days after plating, corresponding to myoblasts and myotubes, respectively, indicated that most of the enzyme activity had the properties of LPL, including an alkaline pH optimum, a serum requirement, and inhibition by NaCl. LPL mRNA expression peaked at 7 days after plating and fell slightly (24%) at day 21. The primary LPL mRNA species in these cells is 3.7 kb in length. Lipase activity and LPL mRNA were highly correlated during the nine course (r = +0.82), suggesting transcriptional regulation of the enzyme. These data clearly demonstrate that L6 cells express LPL during differentiation.

Animals↗

PubMed1991

Characterization of serum-stimulated lipoprotein lipase from bovine heart.

1. A triglyceride (TG) lipase is present in whole homogenate and tissue extracts of beef myocardium with characteristics of lipoprotein lipase (LPL); i.e., activity is stimulated by serum, inhibited by NaCl and protamine sulfate, the protein binds to heparin-Sepharose, and the enzyme has an alkaline pH optimum. 2. This TG lipase, eluted from heparin-Sepharose at 0.9-1.0 M NaCl, has an apparent mol. wt of 64 K daltons. Its primary mRNA is 3.7 kb. 3. Expression of LPL mRNA and enzyme activities are in the ratio of approximately 20:8:1 for hearts of mouse, rat and beef, respectively and correlate with r = +0.99.

Animals↗

PubMedDate not supplied

Apolipoprotein E and apolipoprotein E receptors modulate A beta-induced glial neuroinflammatory responses.

Large numbers of activated glia are a common pathological feature of many neurodegenerative disorders, including Alzheimer's disease (AD). Several different stimuli, including lipopolysaccharide (LPS), dibutyryl (db)cAMP, and aged amyloid-beta 1-42 (A beta), can induce glial activation in vitro, as measured by morphological changes and the production of pro-inflammatory cytokines and oxidative stress molecules. Only A beta-induced activation is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. In addition, only A beta also induces an increase in the amount of endogenous apoE, the primary apolipoprotein expressed by astrocytes in the brain. The functional significance of the increase in apoE appears to be to limit the inflammatory response. Indeed, compared to wild type mice, glial cells cultured from apoE knockout mice exhibit an enhanced production of several pro-inflammatory markers in response to treatment with A beta and other activating stimuli. The mechanism for both the A beta-induced glial activation and the increase in apoE appears to involve apoE receptors, a variety of which are expressed by both neurons and glia. Experiments using receptor associated protein (RAP), an inhibitor of apoE receptors with a differential affinity for the low-density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), revealed that LRP mediates A beta-induced glial activation, while LDLR mediates the A beta-induced changes in apoE levels. In summary, both an apoE receptor agonist (apoE) and an antagonist (RAP) inhibit A beta-induced glial cell activation. Thus, apoE receptors appear to translate the presence of extracellular A beta into cellular responses, both initiating glial cell activation and limiting its scope by inducing apoE, an anti-inflammatory agent.

Amyloid beta-Peptides↗