Implication of apoE isoforms in cholesterol metabolism by primary rat hippocampal neurons and astrocytes

Apolipoprotein E (apoE) has been genetically linked to late-onset Alzheimer's disease. From the three common alleles (epsilon2, epsilon3 and epsilon4), epsilon4 has been suggested to promote amyloid beta (Ass) plaque fibrillation, one hallmark of Alzheimer's disease. It has been demonstrated that altered lipid content of hippocampal plasma membrane coincides with the disease. In this study, we show for the first time that the apoE dependent cholesterol metabolism in hippocampal neurons is higher than that of hippocampal astrocytes. Further, apoE-bound cholesterol is highly incorporated in membranous compartments in hippocampal neurons, whereas hippocampal astrocytes show higher intracellular distribution. This is an effect that coincides with cell-type dependent difference of low density lipoprotein receptor (LDLR) family member expression. Hippocampal neurons express high levels of the LDLR related protein (LRP), whereas hippocampal astrocytes are highly positive for LDLR. We could also demonstrate an apoE isoform (apoE2, apoE3 and apoE4) dependent cholesterol uptake in both cells types. In hippocampal neurons, we could find a decreased apoE4-bound cholesterol uptake. In contrast, hippocampal astrocytes show decreased internalization of apoE2-bound cholesterol. In addition, lipidated apoE4 is little associated with neurites in hippocampal neurons in comparison to the other two isoforms. In contrary, hippocampal astrocytes show faint apoE2 immunocytostaining intensity. Data presented indicate that the role of apoE4 in cholesterol homeostasis and apolipoprotein cell association is more pronounced in hippocampal neurons, showing significant alterations compared to the other two isoforms, suggesting that hippocampal neurons are affected by apoE4 associated altered cholesterol metabolism compared to hippocampal astrocytes.     

Expression of beta-amyloid precursor protein in reactive astrocytes following neuronal damage

Although the beta-amyloid peptide is an established core component of neuritic plaques that accumulate in Alzheimer's disease, the mechanisms responsible for its deposition are not well understood. We now report that lesions of rat hippocampal neurons cause a time-dependent, long-lasting elevation of immunoreactivity for the beta-amyloid precursor protein (APP) in neighboring astrocytes, a cell type not normally containing the protein. The increase represents astroglial expression of the protein rather than a scavenging of APP released by damaged neurons. Immunoelectron microscopy confirmed that APP-containing cells are reactive astroglia, both surrounding capillaries and within the neuropil. These results demonstrate that neuronal damage stimulates APP expression in adult brain and suggest that reactive astrocytes may be a source of the beta-amyloid that forms neuropathological plaques in Alzheimer's disease.     

Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin

Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were greater than 95% astrocytes and less than 3% microglia. Under serum-free conditions, leupeptin induced a 1.4- to 2.0-fold increase, maximal by 48 hours, in cathepsin D protein quantified by a radioimmunoassay. Cathepsin D enzymatic activity, inhibitable by pepstatin, also increased. Northern blot analysis demonstrated that leupeptin also increased cathepsin D mRNA expression. Kinetic analysis indicated that maximal cathepsin D mRNA levels are detected 24 h after stimulation with leupeptin. Exposure of astrocytes under the same conditions to rat recombinant interferon-gamma, human recombinant tumor necrosis factor-alpha, human recombinant interleukin-1 beta, lipopolysaccharide, calcium ionophore, or a combination of these reagents did not increase the level of cathepsin D above controls. These results indicate that astrocytic cathepsin D mRNA and protein can be induced by selected materials. Furthermore, the effects attributed to leupeptin as a proteinase inhibitor may be modified by its ability to increase cathepsin D activity.     

Gene Expression Profiles of Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

Cholinergic neurons of the nucleus basalis (NB) are selectively vulnerable in Alzheimer's disease (AD), yet the molecular mechanisms associated with their dysfunction remain unknown. We used single cell RNA amplification and custom array technology to examine the expression of functional classes of mRNAs found in anterior NB neurons from normal aged and AD subjects. mRNAs encoding neurotrophin receptors, synaptic proteins, protein phosphatases, and amyloid-related proteins were evaluated. We found that trkB and trkC mRNAs were selectively down-regulated in NB neurons, whereas p75^NTR mRNA levels remained stable in end stage AD. TrkA mRNA was reduced by approximately 28%, but did not reach statistical significance. There was a down-regulation of synaptophysin, synaptotagmin, and protein phosphatases PP1 and PP1 mRNAs in AD. In contrast, we found a selective up-regulation of cathepsin D mRNA in NB neurons in AD brain. Thus, anterior NB neurons undergo selective alterations in gene expression in AD. These results may provide clues to the molecular pathogenesis of NB neuronal degeneration during AD.     

Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1-42)

Neuronal nicotinic acetylcholine receptors (nAChRs) are thought to be involved in the pathogenesis of Alzheimer's disease (AD). Interestingly, in the brains of patients with this disease, losses of several subtypes of nAChRs on neurons have been reported, while an increase in alpha7 nAChRs was recently detected in the astrocytes. However, little is presently known about the expressions of individual subunits of nAChR on rat astrocytes in primary culture or the possible influence of exposure to beta-amyloid peptide (Abeta), a neuropathological hallmark of AD, on this expression. Thus, in the present investigation the levels of individual nAChR subunits on primary rat astrocytes and the possible direct influence of Abetas on the receptors were examined by RT-PCR, Western blotting, monitoring intracellular free calcium and immunohistochemistry. The alpha4, alpha7, beta2 and beta3 subunits and related calcium channel responses were found in these cells, whereas neither alpha2 nor alpha3 could be detected. Elevation in the levels of alpha7, alpha4 and beta2 mRNAs and proteins were observed in astrocytes exposed to 0.1-100nM Abeta(1-42). In contrast, incubation with 1muM Abeta(1-42) or Abeta(35-25) did not affect these levels. We propose that the enhanced expression of alpha7, alpha4 and beta2 nAChRs by astrocytes stimulated directly by nanomolar concentrations of Abeta(1-42) might be related to ongoing defensive or compensative mechanisms.     

A novel member of the leucine-rich repeat superfamily induced in rat astrocytes by beta-amyloid.

beta-Amyloid (Abeta) deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer's disease (AD). Although the molecular mechanisms by which Abeta contributes to the progression of neuropathologic changes have not been established entirely, there is little doubt that the association of Abeta with astrocytes, the predominant cell type in brain, has significant influence on exacerbation of the disease. In an effort to identify key molecules involved in AD, we investigated Abeta-responsive genes using rat astrocytes. In this study, we identified a novel Abeta-induced rat gene, designated as Lib, encoding a type I transmembrane protein with an extracellular domain that contains fifteen leucine-rich repeats (LRRs). Human counterpart of rat Lib is located on chromosome 3q29 and human Lib mRNA found in particularly placenta. Lib mRNA levels in rat C6 astrocytoma cells can be increased by pro-inflammatory cytokines and the rat Lib-transfected cells express Lib protein on the cell surfaces. Lib appears to be a member of the LRR superfamily which is involved in cell-cell and/or -extracellular matrix interactions including adhesion or target recognition in neuroinflammatory states.     

Altered cathepsin D metabolism in PHEX antisense human osteoblast cells

X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX gene in osteoblast cells, leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism, but also by locally released osteoblastic mineralization inhibitory factor(s), referred to as minhibin. In our present study, we found that suppression of PHEX expression by PHEX antisense in human osteoblast cells caused an increase in cathepsin D expression at protein, but not mRNA, levels. This was associated with a decrease in cathepsin D degradation and an increased cathepsin D release into culture media. Our results also showed that lowering cathepsin D activity in antisense cell conditioned media abolished their inhibitory effect on osteoblast cell calcification, suggesting the involvement of cathepsin D in mediating the minhibin activity of the antisense cell conditioned media.     

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.     

Cholesterol metabolism, apolipoprotein E, adenosine triphosphate-binding cassette transporters, and Alzheimer's disease

PURPOSE OF REVIEW: Recent evidence suggests that cholesterol metabolism participates in the pathogenesis of Alzheimer's disease. Apolipoprotein E is the main lipid carrier in the brain and the best-established risk factor for late-onset Alzheimer's disease. Intracellular cholesterol levels influence the generation of amyloid-beta peptides, the toxic species thought to be a primary cause of Alzheimer's disease. Finally, compounds that modulate cholesterol metabolism affect amyloid-beta generation. This review summarizes data linking apolipoprotein E and adenosine triphosphate-binding cassette transporters to aspects of cholesterol metabolism and Alzheimer's disease pathogenesis. RECENT FINDINGS: In vivo, the lipidation status of apolipoprotein E affects amyloid-beta burden in mice with Alzheimer's disease, which appears to caused by the modulation of amyloid-beta deposition or clearance rather than amyloid-beta production. State-of-the-art in-vivo assays reveal that amyloid-beta is cleared from the brain by multiple pathways. Members of the adenosine triphosphate-binding cassette superfamily of transporters regulate lipid homeostasis and apolipoprotein metabolism in the brain, and may affect Alzheimer's disease pathogenesis by modulating apolipoprotein E lipidation as well as intracellular sterol homeostasis. SUMMARY: Proteins involved in brain cholesterol metabolism may affect the pathogenesis of Alzheimer's disease. Compounds that modulate the expression of these proteins may be of therapeutic benefit in Alzheimer's disease.     

Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins

We have previously demonstrated that astrocytes synthesize and secrete apolipoprotein E in situ. In the present work, primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro. The astrocytes in culture contained immunoreactive apolipoprotein E in the area of the Golgi apparatus. Incubation of the astrocytes with [35S]methionine resulted in the secretion of labeled immunoprecipitable apolipoprotein E, which constituted 1-3% of the total secreted proteins. The apolipoprotein E secreted in culture and the apolipoprotein E in rat brain extracts differed from serum apolipoprotein E in two respects: both had a slightly higher apparent molecular weight (approx. 36,000) and more acidic isoforms than serum apolipoprotein E. Sialylation of the newly secreted apolipoprotein accounted for the difference in both the apparent molecular weight and isoelectric focusing pattern of newly secreted apolipoprotein E and plasma apolipoprotein E. The astrocytes possessed apolipoprotein B,E(LDL) receptors capable of binding and internalizing apolipoprotein E-containing lipoproteins. The uptake of lipoproteins by the cells led to a reduction in the number of cell surface receptors and to the intracellular accumulation of cholesteryl esters. Since apolipoprotein E is present within the brain, and since brain cells can express apolipoprotein B,E(LDL) receptors, apolipoprotein E-containing lipoproteins may function to redistribute lipid and regulate cholesterol homeostasis within the brain.     

The role of protein ubiquitination in neurodegenerative disease.

Ubiquitin immunocytochemistry with an antiserum which reacts with ubiquitin-protein conjugates demonstrates the presence of ubiquitinated proteins in filamentous inclusions found in neurones in the major human neurodegenerative diseases, i.e. Alzheimer's disease, diffuse Lewy body disease, motor neurone disease. Ubiquitin immunohistochemistry has revolutionized the neuropathological diagnosis of dementia showing that diffuse Lewy body disease is not, as previously supposed, a rare cause of dementia. The filamentous inclusions in neurones in the human neurodegenerative diseases can be divided into at least two types based on recent immunocytochemical studies. We have shown that a ubiquitin-carboxyl terminal hydrolase is present in Lewy bodies but not in neurofibrillary tangles in Alzheimer's disease. This observation is significant since it indicates that molecular pathological mechanisms in neurones in diffuse Lewy body disease are fundamentally different to Alzheimer's disease. Ubiquitin-protein conjugates are also found in vacuoles in areas of granulovacuolar degeneration in hippocampal neurones in Alzheimer's disease and in granulovacuoles in neurones of scrapie infected mouse brain. These locations suggest that ubiquitinated protein are present in the lysosome-related system of neurones. We have recently shown that ubiquitin-protein conjugates are indeed enriched some 12-fold in the lysosomes of normal fibroblasts and lymphocytes.     

Beta-amyloid peptides stimulate endozepine biosynthesis in cultured rat astrocytes

Accumulation of beta-amyloid peptide (Abeta), which is a landmark of Alzheimer's disease, may alter astrocyte functions before any visible symptoms of the disease occur. Here, we examined the effects of Abeta on biosynthesis and release of diazepam-binding inhibitor (DBI), a polypeptide primarily expressed by astroglial cells in the CNS. Quantitative RT-PCR and specific radioimmunoassay demonstrated that aggregated Abeta(25-35), at concentrations up to 10(-4) m, induced a dose-dependent increase in DBI mRNA expression and DBI-related peptide release from cultured rat astrocytes. These effects were totally suppressed when aggregation of Abeta(25-35) was prevented by Congo red. Measurement of the number of living cells revealed that Abeta(25-35) induced a trophic rather than a toxic effect on astrocytes. Administration of cycloheximide blocked Abeta(25-35)-induced increase of DBI gene expression and endozepine accumulation in astrocytes, indicating that protein synthesis is required for DBI gene expression. Altogether, the present data suggest that Abeta-induced activation of endozepine biosynthesis and release may contribute to astrocyte proliferation associated with Alzheimer's disease.     

Changes in cathepsin gene expression and relative enzymatic activity during gilthead sea bream oogenesis

The aim of this study was to provide evidence on the modulation of lysosomal enzymes in terms of both gene expression and enzymatic activity during follicle maturation. For this purpose three lysosomal enzymes, cathepsins B, D, and L, were studied in relation to yolk formation and degradation, during the main phases of ovarian follicle growth in the pelagophil species, the sea bream Sparus aurata. Specific attention was focused on the gene expression quantification method, on the assay of enzymatic activities, and on the relationship between the proteolytic cleavage of yolk proteins (YPs), cathepsin gene expression and cathepsin activities. For the gene expression study, the cathepsins B-like and L-like mRNAs were isolated and partially or fully characterized, respectively; the sequences were used as design specific primers for the quantification of cathepsin gene expression by real-time PCR, in follicles at different stages of maturation. The enzymatic assays for cathepsins B, D, and L were optimized in terms of specificity, sensitivity and reliability, using specific substrates and inhibitors. In ovulated eggs, the lipovitellin I (LV I) was degraded and the changes in electrophoretic pattern were preceded by an increase in the activity of a cysteine proteinase, cathepsin L, and its mRNA. Cathepsin B did not appear to be involved in YP changes during the final maturation stage.     

A Novel Approach for Characterization of Cathepsin D Protease and Its Effect on Tau and ��-Amyloid Proteins

Cathepsin D is the lysosomal protease abundantly expressed in the brain. It plays an important role in the regulation of cellular apoptosis. In addition, cathepsin D has been shown to be involved in the pathogenesis of Alzheimer disease and autism. In this study, we developed a novel approach for the preparation of highly purified cathepsin D from the calf brain. This high grade purification is achieved by using DEAE-Sephacel Chromatography before the final step of applying to the Pepstatin-Sepharose 4B column. The properties of cathepsin D have also been studied. We show that cathepsin D cleaves both tau and β-amyloid precursor protein (APP). Both tau and APP are involved in the pathogenesis of Alzheimer's disease. Our findings strongly suggest a link between the lysosomal dysfunction of cathepsin D and the etiology of Alzheimer's disease. Our findings also indicate that cathepsin D could be a new approach to treating Alzheimer's disease.     

Identification of upregulated genes in scrapie-infected brain tissue

The pathogenesis of scrapie, and of neurodegenerative diseases in general, is still insufficiently understood and is therefore being intensely researched. There is abundant evidence that the activation of glial cells precedes neurodegeneration and may thus play an important role in disease development and progression. The identification of genes with altered expression patterns in the diseased brain may provide insight on the molecular level into the process which ultimately leads to neuronal loss. Differentially expressed genes in scrapie-infected brain tissue were enriched by the suppression subtractive hybridization technique, molecularly cloned, and further characterized. Northern blotting and nucleotide sequencing confirmed the identities of 19 upregulated genes, 11 of which were unknown to be affected by scrapie. A considerable number of these 19 genes, namely those encoding interferon-inducible protein 10 (IP-10), 2',5'-oligo(A) synthetase, Mx protein, IIGP protein, major histocompatibility complex classes I and II, complement, and beta(2)-microglobulin, were inducible by interferons (IFNs), suggesting that an IFN response is a possible mechanism of gene activation in scrapie. Among the newly found genes, that coding for 2',5'-oligo(A) synthetase is of special interest because it could contribute to the apoptotic loss of neuronal cells via RNase L activation. In addition, upregulation of the chemokine IP-10 and B-lymphocyte chemoattractant mRNAs was seen at relatively early stages of the disease and was sustained throughout disease development.     

Testosterone inhibits estrogen-induced mammary epithelial proliferation and suppresses estrogen receptor expression.

This study investigated the effect of sex steroids and tamoxifen on primate mammary epithelial proliferation and steroid receptor gene expression. Ovariectomized rhesus monkeys were treated with placebo, 17beta estradiol (E2) alone or in combination with progesterone (E2/P) or testosterone (E2/T), or tamoxifen for 3 days. E2 alone increased mammary epithelial proliferation by approximately sixfold (P:<0.0001) and increased mammary epithelial estrogen receptor (ERalpha) mRNA expression by approximately 50% (P:<0.0001; ERbeta mRNA was not detected in the primate mammary gland). Progesterone did not alter E2's proliferative effects, but testosterone reduced E2-induced proliferation by approximately 40% (P:<0.002) and entirely abolished E2-induced augmentation of ERalpha expression. Tamoxifen had a significant agonist effect in the ovariectomized monkey, producing a approximately threefold increase in mammary epithelial proliferation (P:<0.01), but tamoxifen also reduced ERalpha expression below placebo level. Androgen receptor (AR) mRNA was detected in mammary epithelium by in situ hybridization. AR mRNA levels were not altered by E2 alone but were significantly reduced by E2/T and tamoxifen treatment. Because combined E2/T and tamoxifen had similar effects on mammary epithelium, we investigated the regulation of known sex steroid-responsive mRNAs in the primate mammary epithelium. E2 alone had no effect on apolipoprotein D (ApoD) or IGF binding protein 5 (IGFBP5) expression, but E2/T and tamoxifen treatment groups both demonstrated identical alterations in these mRNAs (ApoD was decreased and IGFBP5 was increased). These observations showing androgen-induced down-regulation of mammary epithelial proliferation and ER expression suggest that combined estrogen/androgen hormone replacement therapy might reduce the risk of breast cancer associated with estrogen replacement. In addition, these novel findings on tamoxifen's androgen-like effects on primate mammary epithelial sex steroid receptor expression suggest that tamoxifen's protective action on mammary gland may involve androgenic effects.     

Abeta deposition and related pathology in an APP x PS1 transgenic mouse model of Alzheimer's disease

A transgenic mouse bearing mutant transgenes linked to familial forms of Alzheimer's disease (AD) for the amyloid precursor protein and presenilin-1 (TASTPM) showed Abeta plaque deposition and age-related histological changes in associated brain pathology. The Abeta present was of multiple forms, including species with a C-terminus at position 40 or 42, as well as an N-terminus at position 1 or truncated in a pyro-3-glutamate form. Endogenous rodent Abeta was also present in the deposits. Laser capture microdissection extracts showed that multimeric forms of Abeta were present in both plaque and tissue surrounding plaques. Associated with the Abeta deposits was evidence of an inflammatory response characterised by the presence of astrocytes. Also present in close association with the deposits was phosphorylated tau and cathepsin D immunolabelling. The incidence of astrocytes and of phosphorylated tau and cathepsin D load showed that both of these potential disease markers increased in parallel to the age of the mice and with Abeta deposition. Immunohistochemical labelling of neurons in the cortex and hippocampus of TASTPM mice suggested that the areas of Abeta deposition were associated with the loss of neurons. TASTPM mice, therefore, exhibit a number of the pathological characteristics of disease progression in AD and may provide a means for assessment of novel therapeutic agents directed towards modifying or halting disease progression.     

Modulation of apolipoprotein D and apolipoprotein E mRNA expression by growth arrest and identification of key elements in the promoter.

Apolipoprotein D (apoD) and apolipoprotein E (apoE) are co-expressed in many tissues, and, in certain neuropathological situations, their expression appears to be under coordinate regulation. We have previously shown that apoD gene expression in cultured human fibroblasts is up-regulated when the cells undergo growth arrest. Here, we demonstrate that, starting around day 2 of growth arrest, both apoD and apoE mRNA levels increase between 1.5- and 27-fold in other cell types, including mouse primary fibroblasts and fibroblast-like and human astrocytoma cell lines. To understand the regulatory mechanisms of apoD expression, we have used apoD promoter-luciferase reporter constructs to compare gene expression in growing cells and in cells that have undergone growth arrest. Analysis of gene expression in cells transfected with constructs with deletions and mutations in the apoD promoter and constructs with artificial promoters demonstrated that the region between nucleotides -174 and -4 is fully responsible for the basal gene expression, whereas the region from -558 to -179 is implicated in the induction of apoD expression following growth arrest. Within this region, an alternating purine-pyrimidine stretch and a pair of serum-responsive elements (SRE) were found to be major determinants of growth arrest-induced apoD gene expression. Evidence is also presented that SREs in the apoE promoter may contribute to the up-regulation of apoE gene expression following growth arrest.