Abstract
To study the profile and regulation of apolipoprotein E (apoE) expression in the CNS, we generated mice in which apoE expression can be detected in vivo with unprecedented sensitivity and resolution. cDNA encoding enhanced green fluorescent protein (EGFP) with a stop codon was inserted by gene targeting into the apoE gene locus (EGFPapoE) immediately after the translation initiation site. Insertion of EGFP into one apoE allele provides a real-time location marker of apoE expression in vivo; the remaining allele is sufficient to maintain normal cellular physiology. In heterozygous EGFPapoE mice, EGFP was highly expressed in hepatocytes and peritoneal macrophages. EGFP was also expressed in brain astrocytes; however some astrocytes (∼25%) expressed no EGFP, suggesting that a subset of these cells does not express apoE. EGFP was expressed in <10% of microglia after kainic acid treatment, suggesting that microglia are not a major source of brain apoE. Although hippocampal neurons did not express EGFP under normal conditions, kainic acid treatment induced intense expression of EGFP in injured neurons, demonstrating apoE expression in neurons in response to excitotoxic injury. The neuronal expression was confirmed by in situ hybridization of mouse apoE mRNA and by anti-apoE immunostaining. Smooth muscle cells of large blood vessels and cells surrounding small vessels in the CNS also strongly expressed EGFP, as did cells in the choroid plexus. EGFPapoE reporter mice will be useful for studying the regulation of apoE expression in the CNS and might provide insights into the diverse mechanisms of apoE4-related neurodegeneration.
Introduction
The ε4 allele of the gene encoding apolipoprotein E (apoE) has been linked to late-onset familial and sporadic Alzheimer's disease (AD) and has a gene-dose effect on the risk and age of onset of the disease (Corder et al., 1993; Saunders et al., 1993; Roses, 1996; Tang et al., 1998; Romas et al., 2002). Individuals with two copies of the ε4 allele have a 50–90% chance of developing AD by the age of 85, compared with ∼45% for those with one allele (Corder et al., 1993; Farrer et al., 1997) and 20% for the general population (Corder et al., 1993). ApoE is found in amyloid plaques and neurofibrillary tangles (two neuropathological hallmarks of AD) and has been suggested to play important roles in the pathogenesis of these two lesions (Namba et al., 1991; Selkoe, 1991; Wisniewski and Frangione, 1992; Crowther, 1993; Strittmatter et al., 1993a; Roses, 1994; Holtzman et al., 2000; Irizarry et al., 2000; Tanzi and Bertram, 2001).
Initially, apoE was thought to be synthesized in the brain only by astrocytes, oligodendrocytes, and ependymal layer cells (Boyles et al., 1985; Poirier et al., 1991). Although not all studies support the notion (Page et al., 1998; Nishio et al., 2003), increasing evidence suggests that under diverse pathophysiological conditions, CNS neurons also express apoE, albeit at lower levels than astrocytes (Diedrich et al., 1991; Han et al., 1994; Bao et al., 1996; Beffert and Poirier, 1996; Metzger et al., 1996; Xu et al., 1998, 1999a,b). The cellular origin of apoE appears to influence its effects on AD pathology (Huang et al., 2004; Huang, 2006). Astrocyte-derived apoE3 and apoE4 have different effects on the production, deposition, and clearance of Aβ (LaDu et al., 1994; Bales et al., 1999; Holtzman et al., 2000; Irizarry et al., 2000; Ji et al., 2001; Vincent and Smith, 2001; Ye et al., 2005) and on cholesterol efflux (Fagan et al., 1999; Gong et al., 2002). Neuron-derived apoE3 and apoE4 differ in their susceptibility to proteolysis (Huang et al., 2001; Harris et al., 2003; Brecht et al., 2004; Chang et al., 2005) and in their effects on mitochondrial function (Chang et al., 2005), tau phosphorylation (Tesseur et al., 2000a,b; Huang et al., 2001; Harris et al., 2003, 2004a; Brecht et al., 2004), lysosomal leakage (Ji et al., 2002), neurodegeneration (Buttini et al., 1999; Buttini et al., 2002), androgen receptor deficiency (Raber et al., 2002), and cognitive decline (Raber et al., 1998, 2000, 2002). Therefore, a better understanding of the profile and regulation of apoE expression in the CNS is important for unraveling the mechanisms underlying apoE4-related neurodegeneration.
Regulation of apoE expression has been extensively studied in transfected cells (Reardon et al., 1986; Smith et al., 1988; García et al., 1996; Harris et al., 2004b) and in transgenic mice (Shachter et al., 1993; Simonet et al., 1993; Allan et al., 1995, 1997; Shih et al., 2000; Grehan et al., 2001a,b; Zheng et al., 2004) expressing apoE genomic or cDNA constructs. Although these systems have provided valuable information, practical considerations have limited systematic study of apoE expression. The high guanine and cytosine (GC) content of apoE coding sequences requires critical in situ hybridization conditions to limit nonspecific background signals while still providing acceptable sensitivity. Transfected cells are useful for mapping fine structures but have not yielded a fully accurate definition of any cell-specific regulation that reflects in vivo apoE expression. Transgenic models are hampered by variegated expression because of random integration of transgenes into the mouse genome. Enhancers and silencers of nearby endogenous genes can also interfere with transgene expression.
To avoid such limitations, we generated mice that express enhanced green fluorescent protein (EGFP) under control of the endogenous apoE gene promoter and enhancers (EGFPapoE), providing a real-time marker of in vivo apoE expression with unprecedented sensitivity and resolution. EGFP knock-in is a new approach to monitor gene expression in vivo (Aubert et al., 2003; Toyooka et al., 2003). Because it has no signal peptide, EGFP remains intracellular, and surrounding cells are not visible, avoiding confusion as to whether the immunostained apoE in certain cells is produced in situ or taken up through its receptors from the extracellular pool. Insertion of EGFP into one allele of the apoE gene provides a marker of apoE expression in vivo, and the remaining allele maintains normal lipid metabolism and cellular physiology and enables us to confirm the expression of apoE in various cells.
Materials and Methods
Reagents.
Minimum essential medium, Opti-MEM, and fetal bovine serum were obtained from Invitrogen (Rockville, MD). ECL was obtained from Amersham Biosciences (Arlington, IL). Polyclonal rabbit anti-mouse apoE antibody was kindly provided by Dr. Karl Weisgraber (Gladstone Institutes, San Francisco, CA). Kainic acid and monoclonal anti-α-actin were obtained from Sigma (St. Louis, MO). Monoclonal anti-neuron-specific nuclear protein (NeuN) and monoclonal anti-CD11b were from Chemicon (Temecula, CA). Rabbit anti-GFAP was from Dako (Carpinteria, CA). Fluorescein isothiocyanate- and Texas Red-coupled anti-rabbit and anti-mouse were from Vector Laboratories (Burlingame, CA).
Generation of EGFP knock-in mice.
The EGFP-targeting vector was electroporated into embryonic stem (ES) cells (129/Svjae) as described previously (Raffaï et al., 2001), and G418-resistant clones were selected and screened by PCR with primers covering Neo (forward) and 3′ flanking sequence of mouse Apoe (reverse). The DNA sequence recognized by the reverse primer was not included in the vector (Fig. 1A). Therefore, a 1.5 kb fragment could only be amplified from the DNA of the targeted ES cells. Of 200 clones screened, 21 were positive (Fig. 1B).
Generation and characterization of EGFP-targeted mouse ES cells. A, An EGFP cDNA with start and stop codons and a poly-A sequence (∼1 kb) was inserted into the basic gene-targeting vector used to generate apoE-Arg-61 knock-in mice at the Gladstone Institutes (Raffaï et al., 2001). Homologous recombination between the gene-targeting vector and the Apoe locus in ES cells introduces the EGFP cDNA. A Neo cassette was placed in intron 3. Targeted ES cell clones and mice were identified by PCR with primers and by digestion of genomic DNA with SacI and subsequent Southern blotting with an Apoe 3′ flanking sequence probe, which reveals an expanded 5.3 kb fragment; the wild-type fragment is 3.3 kb. B, PCR screening for ES cell clones with homologous recombination of EGFP revealed a 1.5 kb band in targeted (+/−) ES cells but no band in wild-type (WT) ES cells. C, Southern blotting revealed both 5.3 kb and 3.3 kb DNA fragments in targeted (+/−) ES cells but only a 3.3 kb fragment in wild-type ES cells.
The PCR-positive ES cell clones were also screened by Southern blotting. SacI-digested DNA was hybridized with a probe that detects fragments of 5.3 kb (targeted allele) and 3.3 kb (wild-type allele) in targeted ES cells (+/−) but only a 3.3 kb fragment in wild-type cells (Fig. 1A,C). Screening yielded 15 positive ES cell clones (Fig. 1C). Three were microinjected into C57BL/6 blastocysts in the Gladstone Blastocyst Core, yielding >50 chimeric mice harboring EGFP cDNA in the apoE locus. Six male chimeras (>90% brown fur) were crossed with C57BL/6 females to generate heterozygous EGFPapoE mice. Germline transmission resulted in heterozygous F1 EGFPapoE mice (confirmed by PCR and Southern blotting analyses; data not shown) that are 50% C57BL/6. Three additional crosses resulted in F4 mice that are >93% C57BL/6. Heterozygous EGFPapoE mice were then bred to generate homozygotes to maintain the line. For the current study, we used heterozygous EGFPapoE mice, generated by crossing homozygous EGFPapoE and wild-type C57BL/6 mice. Mice were weaned at 21 d of age, housed in a barrier facility at the Gladstone Animal Core with a 12 h light/dark cycle, and fed a chow diet containing 4.5% fat (Ralston Purina, St. Louis, MO).
Northern blotting and quantitative analysis of apoE mRNA.
Total RNA from brains of wild-type and heterozygous EGFPapoE mice was isolated with Triazol (Invitrogen). Total RNA (∼20 μg) was separated by electrophoresis in a 1% agarose gel containing 20% formaldehyde, transferred to Hybond membrane (Amersham Biosciences) and hybridized to a mouse apoE cDNA probe labeled with [32P]dCTP in Ultrahyb solution (Ambion, Austin, TX) at 60°C overnight. The blot was washed in high-stringency buffer (Ambion) at 68°C for 15 min (twice) and exposed to x-ray film for 2–6 h. The blot was then washed with a Strip-EZ buffer (Ambion), rehybridized to a mouse β-actin probe as an internal loading control, and exposed to x-ray film for 2–8 h. The bands of apoE and β-actin mRNAs were scanned, and the ratios of apoE to β-actin were calculated.
Preparation of mouse brain tissues.
Brains from wild-type or heterozygous EGFPapoE mice were collected after a 2 min transcardial perfusion with PBS. One hemibrain from each mouse was homogenized and analyzed for apoE by Western blotting (Huang et al., 2001). In brief, brain tissues were homogenized in ice-cold lysis buffer (50 mm Tris/HCl, pH 8.0, 150 mm NaCl, 4% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, and a mixture of protease and phosphatase inhibitors), placed in a TLA 100.3 rotor, and centrifuged at 35,000 rpm for 30 min at 4°C in an Optima TL ultracentrifuge (Beckman, Fullerton, CA).
Western blotting and quantitative analysis of apoE.
The supernatant (solubilized protein) was subjected to SDS-PAGE and analyzed by Western blotting with a rabbit polyclonal antibody against mouse apoE or a monoclonal antibody against α-actin. The bands of apoE and α-actin from individual mice were scanned, and their intensities were calculated (Huang et al., 2001).
Immunohistochemistry.
The other hemibrain from each mouse was fixed in 3% paraformaldehyde, sectioned, and stained with anti-mouse apoE, anti-NeuN (neuron marker), anti-GFAP (astrocyte marker), anti-α-actin (smooth muscle cell marker), and anti-CD11b (microglial marker) (Buttini et al., 1999; Huang et al., 2001). To block nonspecific reactions, all sections were incubated for 1 h in 10% normal serum from the species that produced the secondary antibodies (Jackson ImmunoResearch, West Grove, PA) in PBS or for 7 min in Superblock (Scytec, Logan, UT), followed by a 1 h incubation in PBS with primary antibodies. Sections were then washed three times in PBS and incubated for 1 h with the corresponding secondary antibodies coupled to Texas Red (Jackson ImmunoResearch). After three washes in PBS, the sections were mounted in VectaShield (Vector Laboratories) and examined for both green (EGFP) and red (other marker staining) channels with a Radiance 2000 laser-scanning confocal system (Bio-Rad, Hercules, CA) mounted on an Optiphot-2 microscope (Nikon, Tokyo, Japan). The images were processed with Photoshop (Adobe Systems, San Jose, CA).
Kainic acid injections.
Kainic acid crosses the blood–brain barrier and induces excitotoxic CNS injury, particularly in the hippocampus and neocortex (Spinler and Cziraky, 1994; Masliah et al., 1997). At 4–6 months of age, heterozygous EGFPapoE mice were injected intraperitoneally with kainic acid (Sigma) dissolved in saline (0.9%) at 25 mg/kg body weight in one dose, as described previously (Buttini et al., 1999). Within ∼15 min, all mice developed seizures. Seizure activity was assessed as described previously (Schauwecker and Steward, 1997). The groups did not differ in the time from injection to seizure onset or in the incidence, intensity, or duration of seizures (data not shown). Mice were killed 1 or 6 d after the injection of kainic acid.
In situ hybridization.
RNA probe complementary to nucleotides 492–783 of mouse apoE mRNA was labeled with [33P]UTP with an RNA transcription kit (Stratagene, La Jolla, CA). The labeled probe was purified through Micro Bio-Spin 30 chromatography columns (Bio-Rad). In situ hybridization was performed as described previously (Grehan et al., 2001b). Briefly, brain paraffin sections (7 μm) were incubated with 20 μg/ml proteinase K (Boehringer Mannheim, Indianapolis, IN) in 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, and 150 mm NaCl for 15 min at room temperature. Proteolytic activity was stopped by immersion for 10 min in 0.2% glycine in PBS. After fixation, acetylation, and dehydration, the sections were incubated for 14–18 h in a humidified chamber at 45°C with labeled probe in a buffer containing 50% formamide, 300 mm NaCl, 20 mm Tris, pH 8.0, 5 mm EDTA, 0.2% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 10% dextran sulfate, 250 μg/ml sperm DNA, and 0.1 mg/ml tRNA. After two washes at room temperature in 2× SSC and 1.0 mm EDTA for 10 min, the sections were immersed in 20 μg/ml ribonuclease (RNase) A (Sigma) in 500 mm NaCl and 10 mm Tris, pH 8.0, and 10 U/ml T1 RNase (Boehringer Mannheim) for 1 h at 37°C, washed at 60°C in six changes of 0.1× SSC with 1.0 mm EDTA for 4 h, rinsed twice for 10 min each in 0.5× SSC, and dehydrated. For dark-field and bright-field microscopy, the slides were dipped in NTB2 nuclear track emulsion (Eastman Kodak, Rochester, NY), incubated at 4°C for 2–5 d, and developed with D19 developer (Eastman Kodak). The sections were then stained with hematoxylin and eosin (Fisher Scientific, Tustin, CA). After dehydration in a graded series of ethanol (80, 95, and 100%), the slides were rinsed three times in xylene and overlaid with coverslips.
Statistical analysis.
Results are reported as mean ± SD. Differences were evaluated by a t test.
Results
Heterozygous EGFPapoE mice express both EGFP and apoE
As shown by Northern blotting, the average level of brain apoE mRNA in heterozygous EGFPapoE mice was 58% of that in wild-type mice (Fig. 2A), consistent with the expected inactivation of one apoE allele. The average level of apoE protein was 51% of wild-type, as shown by anti-apoE Western blotting (Fig. 2B). Similar Western blotting results were obtained from liver and peritoneal macrophages of heterozygous EGFPapoE mice (data not shown).
Heterozygous EGFPapoE mice express apoE at ∼50% of the level in wild-type mice. A, Northern blotting analysis of apoE and actin mRNA in brains of wild-type and heterozygous EGFPapoE mice at 5 months of age. B, Western blotting analysis of apoE and actin in brains of wild-type and heterozygous EGFPapoE mice at 5 months of age.
EGFP is expressed in hepatocytes and macrophages
EGFP was expressed at high levels in liver cells (Fig. 3A), the primary source of apoE in both humans and mice. Peritoneal macrophages also expressed EGFP, and the expression was enhanced by cholesterol loading (Fig. 3B), as reported previously (Basu et al., 1981). Thus, in heterozygous EGFPapoE mice, EGFP representing apoE was correctly expressed in hepatocytes and macrophages that normally express apoE.
Expression of EGFP in hepatocytes and peritoneal macrophages in a heterozygous EGFPapoE mouse at 2 months of age. A, Expression of EGFP in hepatocytes as determined by confocal microscopy. B, Peritoneal macrophages were cultured in vitro for 1 or 4 d and analyzed by confocal microscopy. Alternatively, after 4 d of culture, macrophages were incubated with acetylated LDL (AcLDL; 100 μg/ml) for 16 h and analyzed by confocal microscopy.
EGFP is expressed in many, but not all, CNS astrocytes
EGFP was also expressed in CNS astrocytes and was confirmed by anti-GFAP immunofluorescent staining (Fig. 4A–C). Strikingly, EGFP revealed the full cell volume of astrocytes with very fine resolution of cellular processes, whereas anti-GFAP staining highlighted only large processes. Thus, EGFP is better than immunostaining for characterizing cell identity and morphology. Interestingly, some GFAP-positive cells (26 ± 4%) did not express EGFP, suggesting that a subclass of astrocytes might not express apoE, at least under normal conditions.
Expression of EGFP in hippocampal astrocytes in heterozygous EGFPapoE mice at 4–6 months of age before (A–C) and 6 d after (D–F) kainic acid (KA) treatment (25 mg/kg body weight). Astrocytes were identified by anti-GFAP immunostaining and confocal microscopy. In the merged image, yellow indicates colocalization of EGFP (green) and GFAP (red).
To determine whether brain insults can induce EGFP-negative astrocytes to express EGFP, we treated heterozygous EGFPapoE mice with kainic acid, which can activate astrocytes, induce gliosis, and increase apoE expression in animal models (Sperk et al., 1983). Hippocampal astrocytes were activated, as indicated by much stronger GFAP staining and enlarged cell bodies and branches; however, 17 ± 3% of astrocytes still did not express EGFP (Fig. 4D–F).
EGFP is expressed in <10% of microglia after kainic acid treatment
EGFP was not expressed in CNS microglia as demonstrated by anti-CD11b, a microglial marker (Chen et al., 2005), immunofluorescent staining (Fig. 5A–C), suggesting that these cells do not express apoE under normal conditions. To determine whether brain insults can induce microglia to express EGFP, we treated heterozygous EGFPapoE mice with kainic acid, which can activate microglia and induce gliosis (Sperk et al., 1983). Hippocampal microglia were activated by kainic acid treatment, as indicated by much stronger CD11b staining and enlarged cell bodies and branches; however, only 6 ± 3% of microglia expressed EGFP (Fig. 5D–F).
Expression of EGFP in hippocampal microglia in heterozygous EGFPapoE mice at 4–6 months of age before (A–C) and 6 d after (D–F) kainic acid (KA) treatment (25 mg/kg body weight). Microglia were identified by anti-CD11b immunostaining and confocal microscopy. In the merged image, yellow (arrow) indicates colocalization of EGFP (green) and CD11b (red).
EGFP is expressed in hippocampal neurons in response to exitotoxic injury
EGFP was not expressed in hippocampal neurons in heterozygous EGFPapoE mice, as demonstrated by staining for NeuN (a neuronal marker) (Fig. 6A) and GFAP (Fig. 6B). Thus, hippocampal neurons do not express apoE under normal conditions. However, kainic acid treatment induced intense expression of EGFP in injured hippocampal neurons (Fig. 6C–G). Importantly, both EGFP and apoE were only present in injured neurons in the treated mice (Fig. 7A,B,D,E). Neuronal expression of apoE was confirmed by in situ hybridization of mouse apoE mRNA in heterozygous EGFPapoE mice treated with kainic acid (Fig. 7F). ApoE protein and mRNA were undetectable in hippocampal neurons in untreated mice (Fig. 7B,C), in which no neuronal injury was found, as determined by silver staining (Fig. 7A).
Hippocampal CA3 neurons express EGFP, representing apoE, in response to excitotoxic injury. Heterozygous 5-month-old EGFPapoE mice received peritoneal injections of kainic acid (KA; 25 mg/kg) (C–G), and the brains were collected 1 d (E–G) or 6 d (C, D) later. Untreated age-matched heterozygous EGFPapoE mice served as controls (A, B). Confocal images of immunostained brain sections were collected for EGFP (green) and anti-NeuN (red), a neuronal marker, or anti-GFAP (red), an astrocytic marker. Images in A–D and G are merged, and yellow indicates colocalization.
EGFP and apoE protein and mRNA are present only in injured hippocampal neurons in kainic acid-treated mice. Heterozygous 5-month-old EGFPapoE mice received peritoneal injections of kainic acid (KA; 25 mg/kg) (D–F), and the brains were collected 6 d later. Untreated age-matched heterozygous EGFPapoE mice served as controls (A–C). A, D, Gallyas silver staining of the hippocampal CA1 region. B, E, Merged confocal images of EGFP (green) and anti-apoE (red) in the CA1 region. C, F, In situ hybridization of mouse apoE mRNA in the CA1 region.
EGFP is expressed along CNS vessels and in the choroid plexus
EGFP was highly expressed along vessels in the CNS, as indicated by the close proximity or colocalization of EGFP with α-actin (Fig. 8A–C), a smooth muscle cell marker (Deaton et al., 2005). Interestingly, EGFP surrounded smooth muscle cells of small vessels in the hippocampus (Fig. 8A) and cortex (Fig. 8B) but colocalized with smooth muscle cells in large vessels in the cortex (Fig. 8C). In situ hybridization with a probe specific for mouse apoE demonstrated apoE mRNA in cells in or surrounding vessel walls (Fig. 8D). Anti-GFAP immunostaining indicated that the cells expressing EGFP along vessels were negative for GFAP, and thus were not astrocytes (Fig. 8E,F). EGFP was not expressed along veins in the CNS (data not shown). Cells of the choroid plexus also expressed high levels of EGFP (Fig. 9A) and contained apoE mRNA, as shown by in situ hybridization (Fig. 9B).
Expression of EGFP in smooth muscle cells in large blood vessels and cells surrounding small blood vessels in brains of heterozygous EGFPapoE mice. A–C, Merged confocal images of EGFP (green) and anti-α-actin (red), a smooth muscle cell marker, were collected from a 5-month-old heterozygous EGFPapoE mouse. A, Small blood vessels in the hippocampus. B, A small blood vessel in the cortex. C, A large blood vessel in the cortex. D, In situ hybridization shows mouse apoE mRNA along the wall of a large blood vessel in the cortex. E, F, Merged confocal images of EGFP (green) and anti-GFAP (red), an astrocyte marker, were collected from a 5-month-old heterozygous EGFPapoE mouse.
Expression of EGFP in choroid plexus cells of heterozygous EGFPapoE mice. A, Merged confocal image of EGFP (green) and anti-GFAP (red) from a 5-month-old EGFPapoE mouse. B, In situ hybridization revealed mouse apoE mRNA in the choroid plexus.
Discussion
This study of EGFPapoE reporter mice provides insights into the profile and regulation of apoE expression in the CNS. Our findings demonstrate that neurons in the CNS produce apoE in response to injury. They also show that a subclass of astrocytes and most microglia do not express apoE, even after brain insults, and that many types of CNS cells in addition to astrocytes express apoE, including smooth muscle cells in larger blood vessels, cells surrounding small vessels, and cells of the choroid plexus. EGFPapoE reporter mice will be invaluable for studying the regulation of apoE expression in the CNS at different developmental stages or in response to various brain insults and will likely provide additional insights into the roles of apoE in neurobiology and the diverse mechanisms of apoE4-related neurodegeneration.
Our study lays to rest a long-standing controversy concerning the neuronal expression of apoE. Some previous studies showed that at least some neurons express apoE (Diedrich et al., 1991; Poirier et al., 1991; Han et al., 1994; Bao et al., 1996; Metzger et al., 1996; Xu et al., 1996, 1999a,b; Dupont-Wallois et al., 1997; Boschert et al., 1999; Ferreira et al., 2000; Dekroon and Armati, 2001; Hartman et al., 2001; Aoki et al., 2003; Harris et al., 2003); others suggested that they do not (Page et al., 1998; Nishio et al., 2003). More recent studies suggest that neurons might not normally express apoE but do so in response to brain injuries, such as excitotoxic or ischemic injury (Boschert et al., 1999; Aoki et al., 2003). ApoE is also expressed in primary cultured human and rat CNS neurons (Dekroon and Armati, 2001) and in many human neuronal cell lines, including SY-5Y, Kelly, and NT2 cells (Poirier et al., 1991; Dupont-Wallois et al., 1997; Ferreira et al., 2000; Hartman et al., 2001).
In previous studies, anti-apoE immunostaining and in situ hybridization were used to define apoE expression in neurons. Both methods have been criticized for technical or data interpretation limitations. For example, anti-apoE immunostaining can not differentiate whether the apoE in neurons is generated in those cells or taken up from the astrocyte-secreted pool. Likewise, in situ hybridization data are also questioned as false positive, because of the GC-rich nature of the apoE gene. In addition, in situ hybridization might also yield false-negative results because of the poor sensitivity of the technique in detecting low-abundance mRNA in cells. Our EGFPapoE reporter mice avoid those limitations. Thus, our findings conclusively demonstrate that neurons express apoE in response to excitotoxic injury and support the notion that understanding how apoE expression is regulated in neurons is important for unraveling the mechanisms of apoE4-related neurodegenerative disorders.
Surprisingly, ∼20% of hippocampal and cortical astrocytes did not produce apoE, even in response to brain insults. We speculate that apoE-positive and apoE-negative astrocytes play different roles in brain development, neuronal injury and repair, and even some disease processes. Therefore, it would be interesting to know whether aging or neurodegenerative disorders such as AD alter the ratio of those two types of astrocytes.
It has been reported that cultured rat primary microglia and murine microglial cell line express apoE in vitro (Xu et al., 2000; Naidu et al., 2002; Saura et al., 2003; Mori et al., 2004). ApoE protein has also been found in activated microglia in AD brains and in lesioned olfactory bulb of mice by immunocytochemistry (Uchihara et al., 1995; Nathan et al., 2001). We demonstrated in the EGFPapoE reporter mice that microglia do not express apoE under normal conditions, and <10% of microglia produce apoE in response to brain insults. Therefore, microglia are not a major source of brain apoE, at least in mice.
Smooth muscle cells of artery walls in peripheral tissues express apoE (Majack et al., 1988; Moore et al., 2004), and apoE has been detected in cells along vessels in the CNS by anti-apoE immunostaining (Boyles et al., 1985), but the source was unclear. We demonstrated that apoE is produced in smooth muscle cells in large vessels and in cells surrounding small vessels in the CNS. Clearly, those apoE-expressing cells surrounding small vessels are not astrocytes, because they are negative for anti-GFAP immunostaining. Although the cellular identity remains to be determined, apoE generated in these locations may be important in maintaining the integrity and normal function of the blood–brain barrier. In fact, apoE deficiency causes leakage of this structure (Fullerton et al., 2001; Methia et al., 2001). Furthermore, apoE has been found in lesions of cerebral amyloid angiopathy (CAA), and apoE4 is associated with increased severity of CAA in humans and amyloid protein precursor transgenic mice (Schmechel et al., 1993; Greenberg et al., 1995; Fryer et al., 2005).
It has been generally accepted that apoE in CAA is secreted by astrocytes (Fryer et al., 2003, 2005). ApoE4 might deliver more Aβ peptide than apoE3 from the brain parenchyma to vessel walls (Fryer et al., 2005). However, our observation that CNS cells in or around the vessel wall produce copious amounts of apoE raises the possibility that apoE4 generated in this location might retain more Aβ than apoE3, leading to more severe CAA in apoE4 carriers. In line with this hypothesis, CAA in AD brains occurs along the perivascular spaces of small blood vessels and between smooth muscle cells in large vessels (Weller et al., 1998). Interestingly, apoE was not expressed along veins in the CNS of the EGFPapoE mice, and CAA is seldom found along the veins in AD brains (Weller et al., 1998).
Finally, cells in the choroid plexus expressed high levels of apoE. The choroid plexus secretes CSF, expresses many receptors (e.g., apoE receptor-2), secretes numerous molecules (e.g., growth factors), transports nutrients from the blood to CSF, and clears brain metabolites (e.g., Aβ peptides) (Kim et al., 1996; Martel et al., 1997; Serot et al., 2003; Crossgrove et al., 2005; Moir and Tanzi, 2005). ApoE levels in the CSF have not been reported in mice, but in humans are 5–15% of those in plasma (Pitas et al., 1987; Fukumoto et al., 2003). Our results suggest that the choroid plexus is the major source of apoE in CSF. Because the choroid plexus clears brain Aβ peptides by transporting them from CSF to blood (Crossgrove et al., 2005) and because apoE isoforms interact differently with Aβ peptides (Strittmatter et al., 1993b; LaDu et al., 1994, 1995; Ma et al., 1994; Sanan et al., 1994; Wisniewski et al., 1994), apoE generated by the choroid plexus might affect Aβ clearance in an isoform-specific manner. In fact, apoE knock-out mice have higher Aβ levels in CSF than wild-type mice (DeMattos et al., 2004). Furthermore, the function of the choroid plexus declines with aging and in AD patients (Serot et al., 2003), which might lead to decreased clearance of Aβ peptides.
Clearly, apoE is expressed in different types of cells, in addition to astrocytes, in the CNS. We hypothesize that apoE from different cellular sources has distinct roles in both physiological and pathophysiological pathways, including the pathogenesis of AD (Huang et al., 2004; Huang, 2006). For example, apoE4 generated in injured neurons may be involved in mitochondrial dysfunction and neurofibrillary tangle formation (Huang et al., 2001; Harris et al., 2003; Brecht et al., 2004; Chang et al., 2005), apoE4 generated in astrocytes may be primarily responsible for amyloid plaque formation, apoE4 generated in or around blood vessels may be important for CAA formation, and apoE generated in the choroid plexus may participate in the clearance of Aβ peptides. This hypothesis is supported by the early observation that apoE generated locally in the liver is much more efficient than that generated in the periphery in mediating the hepatic clearance of remnant lipoproteins (Mahley and Ji, 1999; Raffaï et al., 2003).
Footnotes
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This work was supported in part by National Institutes of Heath Grants P01 AG022074 and R01 HL37063 and a postdoctoral fellowship from the John Douglas French Alzheimer's Foundation. We thank Dr. Karl Weisgraber for critical reading of this manuscript, Karina Fantillo for manuscript preparation, Stephen Ordway and Gary Howard for editorial assistance, John C. W. Carroll and John Hull for graphics, and Chris Goodfellow for photography.
- Correspondence should be addressed to Dr. Yadong Huang, Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158. Email: yhuang{at}gladstone.ucsf.edu
