Arg-61 apoE mice were generated as described and backcrossed with WT C57BL/6J mice for eight generations (8–10). Primary astrocyte cultures were dissociated from 3-day-old neonatal and 3-month-old adult mouse brains. Mice were housed and handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Neonatal astrocytes were cultured as described (10). Primary adult astrocyte cultures were prepared from the brains of 3-month-old WT, Arg-61 apoE, and apoE knock-out/WT heterozygous mice, according to published procedures with modifications (12). In brief, mouse brain cortex and hippocampus were dissected with a sterile razor blade and trypsinized for 20 min at 37 °C. Dissociated cells were filtered and suspended in fresh Dulbecco's modified Eagle's F-12 medium (Invitrogen) containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% Primocin (Invitrogen). The cell suspensions were plated and cultured in Dulbecco's modified Eagle's F-12 medium at 37 °C in a 5% CO₂ atmosphere. To confirm that the cells were astrocytes, cultures were stained with a monoclonal antibody against intermediate filament glial fibrillary acidic protein. All cells were glial fibrillary acidic protein-positive under our culture conditions (see supplemental Fig. 1). Astrocytes from neonatal and 3-month-old mouse brains were used in quantitative real-time PCR experiments. Astrocytes from 3-month-old mouse brains were used in functional studies, as the transporter system matures in adult astrocytes.

Astrocyte intracellular levels of apoE and OASIS (old astrocyte specifically induced substance) and the brain levels of OASIS were quantitated by immunoblotting. Primary astrocyte cultures were grown to 80% confluence in 6-well plates, harvested, and lysed. Brain tissues were homogenized in ice-cold lysis buffer (50 mmol/liter Tris-HCl, pH 8.0, 150 mmol/liter NaCl, 0.1% SDS, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and a mixture of protease and phosphatase inhibitors) and centrifuged at 30,000 rpm for 30 min at 4 °C in a TLA 100.3 rotor in an Optima TL ultracentrifuge (Beckman Instruments). Supernatants from both cell lysate and brain homogenates were collected, and 25 μg of protein from each sample were analyzed by SDS-PAGE gel electrophoresis. OASIS was detected with an antibody against recombinant mouse OASIS peptide (1:10,000, Strategic Diagnostics, Newark, DE). The sensitivity and specificity of anti-OASIS antibody was tested (see supplemental methods and supplemental Fig. 2). Mouse WT apoE and Arg-61 apoE were detected with an antibody against recombinant, full-length WT mouse apoE (1:10,000). Protein bands were detected by chemiluminescence with horseradish peroxidase-coupled anti-rabbit IgG secondary antibody. The blots were scanned, and protein levels were quantitated by densitometry (ImageJ, National Institutes of Health, Bethesda, MD).

Total RNA from primary astrocyte cultures (over 80% confluence in T-75 flask) was isolated with RNeasy mini columns (Qiagen, Valencia, CA). Reverse transcription reactions (Applied Biosystems, Foster City, CA) contained 300 ng of DNase-treated total RNA and random hexamer and oligo(dT) primers (2.5 μm each) (see supplemental Table 1). Diluted reactions (6 ng of cDNA) were analyzed with SYBR green PCR reagents in duplicate on an ABI Prism 7700 sequence detector (Applied Biosystems). OASIS, ATF4, and XBP-1 mRNA levels and the mRNA levels of downstream effectors were determined relative to standard curves made with serial dilutions of pooled cDNAs (30, 6, and 1.2 ng) from all samples. Values were normalized to mouse 18 S RNA.

Primary astrocytes were plated at a density of 5 × 10⁴ cells/ml and cultured for 48 h. Cell viability or apoptosis was detected by the Vybrant apoptosis assay kit 2 (Molecular Probes). Apoptotic cells and dead cells were stained with Alexa Fluor 488-conjugated annexin V or propidium iodide and separated by fluorescence emission at 530 and >575 nm, respectively. Cell distribution was detected and analyzed by flow cytometry.

Primary astrocytes were plated at 2500 cells/well in a 96-well plate and cultured for 48 h. Glucose uptake was detected with a fluorescent glucose derivative, 2-[N-(7-nitobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG, Molecular Probes) (13, 14). The concentration of 2-NBDG and the incubation time were optimized for measurement of glucose uptake in primary astrocyte cultures. Intracellular 2-NBDG was measured with a microplate reader with excitation at 475 nm and emission 545 nm (13). The intracellular 2-NBDG fluorescence intensities were normalized to total cellular protein levels to avoid the bias from the different cell numbers.

Primary astrocyte cultures (over 80% confluence in T-75 flask) were conditioned with serum-free Opti-MEM I medium for 48 h. The conditioned media from WT, Arg-61 apoE, and apoE knock-out/WT heterozygous mouse astrocytes were collected and filtered.

Neuro-2a cells (American Type Culture Collection, Manassas, VA) were maintained at 37 °C in a humidified 5% CO₂ incubator in MEM containing 10% fetal bovine serum supplemented with nonessential amino acids, penicillin, and streptomycin. Neuro-2a cells were plated in 24-well plates at 12,000 cells/well. To induce neurite outgrowth, serum-containing medium was replaced with serum-free Opti-MEM I medium containing 50% astrocyte-conditioned medium (ACM). Neuro-2a cells were cultured in Opti-MEM I/ACM for 72 h, fixed, and stained with Coomassie Blue (Bio-Rad) to enhance visualization of cell bodies and neurites. Neurite outgrowth was quantified as the percentage of cells with neurites longer than 1× the cell body diameter.

Quantitative data are expressed as mean ± S.E. Differences between two means were assessed with unpaired, two-tailed t tests. Differences among multiple means were evaluated by analysis of variance followed by a Tukey-Kramer post hoc test. For the age-dependent changes of OASIS levels, a two-way analysis of variance tested the effects of genotype and age.

Previously, using WT and Arg-61 apoE mice and human apoE3 and apoE4 knock-in mice, we demonstrated that domain interaction results in lower levels of Arg-61 apoE and apoE4 than of WT mouse apoE and apoE3 in the brain, as reflected by reduced secretion and not differences in transcription (10). To determine whether Arg-61 apoE accumulated in astrocytes or was degraded, cell lysates were examined. In lysates of primary astrocyte cultures, Arg-61 apoE levels were 49.6 ± 3.8% (p < 0.01) lower than WT levels, indicating that Arg-61 apoE was targeted for degradation (Fig. 1).

Next, we determined the levels of UPR components in primary astrocyte cultures by reverse transcription and real-time PCR. The levels of OASIS, an astrocyte-specific UPR transducer in the ATF6 (activating transcription factor) pathway (15), were 2.0 ± 0.2-fold higher in Arg-61 than in WT astrocytes, demonstrating transcriptional up-regulation (Fig. 2A). Expression of ATF4 and XBP-1 (X-box binding protein 1), components of two other common UPR pathways, PERK (PKR-like ER kinase) and IRE1 (inositol-requiring enzyme 1), were also up-regulated in Arg-61 astrocytes (1.3 ± 0.1- and 1.4 ± 0.2-fold higher than in WT astrocytes, respectively) (Fig. 2, B and C). Thus, domain interaction in Arg-61 apoE astrocytes elicited an ER stress response and activated all three UPR pathways.

An ER stress response should result in cleavage of OASIS, generating an N-terminal fragment that is translocated to the nucleus, activating its target genes (16). Western blot analysis with an anti-OASIS antibody showed that OASIS was expressed as an ∼80-kDa protein with minimal cleavage in WT astrocytes. However, in Arg-61 apoE astrocytes, a 55-kDa band was more prominent, demonstrating increased cleavage (Fig. 2D). Arg-61 astrocytes also expressed higher levels of OASIS protein, indicating that domain interaction induced ER stress and up-regulated UPR pathways. Total OASIS protein expression was 1.7 ± 0.2-fold higher in Arg-61 apoE astrocytes, which had higher levels of both full-length protein (Fig. 2E) and the 55-kDa fragment (Fig. 2F) (2.5 ± 0.1 versus WT 2.0 ± 0.1 and 1.9 ± 0.2 versus WT 1.2 ± 0.3 arbitrary units, respectively; both p < 0.05).

The cleaved cytosolic N-terminal fragment of OASIS is a transcription factor that, together with ATF4 and spliced XBP-1, increases the expression of genes encoding effectors of UPR pathways, including ER chaperones, such as BiP (immunoglobulin heavy chain binding protein) and GRP 94 (glucose-regulated protein 94), and proteins that catalyze protein folding, such as protein disulfide isomerases (PDIs) (17–19). BiP, GRP94, and PDI mRNA levels were 1.3 ± 0.1-, 1.4 ± 0.1-, and 1.3 ± 0.1-fold higher, respectively, in Arg-61 apoE astrocytes than WT; p < 0.05) (Fig. 3, A–C). mRNA levels of GADD34 (growth arrest and DNA damage-inducible gene 34), which may protect against the destructive attenuation of translation mediated by PERK pathway activation (20), were also higher in Arg-61 apoE astrocytes (1.5 ± 0.2-fold, p < 0.05) (Fig. 3D).

ER-associated degradation is one of the common response mechanisms. Homocysteine-inducible ER protein (Herp), an ER-resident membrane protein, was hypothesized to function for ER-associated degradation (21, 22). The mRNA level of Herp was 1.4 ± 0.2-fold higher in Arg-61 apoE astrocytes (p < 0.05) (Fig. 3E). CHOP (C/EBP homologous protein), a member of the C/EBP family of bZIP transcription factors, is expressed at high levels in response to ER stress and is believed to be a downstream effector in all three common UPR pathways (23, 24). CHOP expression levels were 1.7 ± 0.2-fold higher (p < 0.05) in Arg-61 apoE astrocytes (Fig. 3F).

Previously, we showed that brain levels of Arg-61 apoE are lower than those of WT in E18 mouse embryos and remain lower up to 2 years of age (10). We, therefore, determined whether ER stress induced by domain interaction also existed in adult astrocytes. Indeed, mRNA levels of OASIS (Fig. 4A), ATF4 (Fig. 4C), and XBP-1 (Fig. 4D) were higher in cultures of astrocytes from adult (3-month-old) Arg-61 apoE mice (2.4 ± 0.6-, 2.2 ± 0.5-, and 3.1 ± 1.0-fold higher, respectively, than WT; p < 0.05).

A two-way analysis of variance test was used to determine whether there was an age effect on OASIS mRNA levels between the WT and Arg-61 apoE astrocytes from neonatal and adult brains. In WT adult astrocytes, OASIS mRNA levels were increased ∼25% (Fig. 4B). However, age had a major effect on OASIS expression in Arg-61 apoE astrocytes. The mRNA levels of OASIS were markedly elevated with ∼70% more than Arg-61 apoE neonatal astrocytes (p < 0.01, Fig. 4B), indicating that age-dependent expression of abnormally folded Arg-61 apoE exacerbates ER stress and UPR.

To rule out the possibility that the ER stress induced by domain interaction is due to the lower apoE protein levels, we examined astrocytes from 3-month-old heterozygous Apoe^+/− mice, with 50% lower apoE levels. mRNA levels of OASIS, ATF4, and XBP-1 were not elevated (83 ± 10%, 92 ± 11%, and 72 ± 15% of WT levels, respectively, p > 0.05) (Fig. 4). Thus, simply reducing the apoE protein level is not sufficient to induce ER stress in primary astrocyte cultures, indicating a specific effect of Arg-61 apoE or domain interaction.