Biochemical and Histochemical Evidence of Nonspecific Binding of 7nAChR Antibodies to Mouse Brain Tissue
Alzheimer Research Laboratory, Department of Pharmacology and Therapeutics, University of South Florida, Tampa, Florida
Correspondence to: Marcia N. Gordon, University of South Florida, 12901 Bruce B. Downs Blvd., MDC Box 9, Tampa, FL 33612-4799. E-mail: mgordon{at}hsc.usf.edu
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Summary |
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(J Histochem Cytochem 52:13671375, 2004)
Key Words: transgenic lipopolysaccharide alpha 7 nicotinic receptor astrocytes immunohistochemistry Western blot
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Introduction |
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In human AD brain, nicotinic receptor populations have been extensively studied. Alpha 7 levels are generally decreased in AD, although there are variations in reports (reviewed in Perry et al. 2001). Declines in receptors may be due to cell loss or synaptic decline. Age-related changes in
7 expression in mice transgenic for a human mutated amyloid precursor protein (APP) have also been reported (Dineley et al. 2001
,2002
). Particular focus has been on
7 because of the potential interaction of the receptor with Aß peptides. Co-immunoprecipitation and receptor-binding assays indicate that Aß1-42 binds
7 with high affinity, but not
4ß2, in both human and rat tissue (Wang et al. 2000a
,b
). Studies conducted with wild-type
7 receptors indicate that Aß1-42 blocks the receptor (Liu et al. 2001
; Pettit et al. 2001
; Grassi et al. 2003
; Lee and Wang 2003
). Glial cells may also be involved in these interactions; recent reports have shown expression of
7 by reactive astrocytes in human AD brain, particularly in association with amyloid deposits (Wevers et al. 1999
; Graham et al. 2002
; Teaktong et al. 2003
).
In these experiments, we used standard protocols for immunohistochemical and Western blot analysis of 7 nAChR subunits in murine models of AD and neuroinflammation. Several commercially available antibodies were evaluated, and the results compared with genotyping and RNA analyses. Initially, four genotypes resulting from breeding transgenic mice carrying either mutant APP or mutant presenilin 1 (PS1) were examined for
7 expression. Various reports in the literature led us to expect decreases in levels of nicotinic receptors in amyloid-depositing mice, such as the APP and APP+PS1 transgenics. Although no decreases in neuronal staining were seen, a surprising finding was
7-immunopositive astroglia in apposition with compact plaques in APP and APP+PS1 mice, but in not PS1 or non-transgenics. To determine if amyloid was causing the astrocytic
7 expression, or if it was part of a more general inflammatory response, we injected lipopolysaccharide (LPS) intrahippocampally into APP and non-transgenic mice. Immunohistochemical analysis revealed many
7-positive astrocytes in the injected animals, leading us to believe that the expression was part of a general inflammatory response. In a final experiment using
7-null mice, the specificity of the antibodies was tested under both control and LPS-stimulated conditions. No discernible differences were seen between
7+/+ versus
7/ mice with any antibody used, regardless of the procedure. Genotyping and RNA analyses confirmed the disruption of the
7 allele and lack of
7 message in the knockouts. We therefore conclude that commercially available antibodies against
7 as used in the methods detailed here fail to specifically detect the subunits.
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Materials and Methods |
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Genotyping
APP and PS1 lines were analyzed as previously reported (Gordon et al. 2001). Mice from the
7-null mutation line were genotyped as follows: 2-mm diameter ear clips from the
7 heterozygous breedings were digested and the DNA extracted using Qiagen's DNeasy Kit (Valencia, CA) according to the manufacturer's instructions. Jackson Laboratories supplied the sequence of primers used to identify either the neo-cassette of the null mutation or the wild-type allele, for use with the polymerase chain reaction (PCR): forward, 5'-CCTGGTCCTGCTGTGTTAAACTGCTTC-3'; reverse WT (
7+), 5'-CTGCTGGGAAATCCTAGGCACACTTGAG-3'; reverse Neo (
7), 5'-GACAAGACCGGCTTCCATCC-3'. Thermocycling conditions were as follows: 95C for 4 min; 30 cycles of 95C for 15 sec, 60C for 60 sec, 72C for 60 sec; 72C for 7.5 min; store at 4C. PCR products were run on a 1% agarose gel, using ethidium bromide ultraviolet (UV) detection of bands at 440 bp (
7+) or 750 bp (
7).
Intrahippocampal Injections
APP mice aged 16 months, 7-null mice aged 11 months, and their non-transgenic littermates were used in these studies. Mice were anesthetized using isoflurane and immobilized in a stereotaxic apparatus. One-µl injections of either saline or 4 µg/µl LPS (Salmonella abortus equi, Sigma, St Louis, MO) were delivered over a 2-min period into the hippocampus (stereotaxic coordinates from bregma: 2.7 mm posterior; ±2.5 mm lateral; 3.0 mm ventral). This procedure had been previously demonstrated in our lab to induce a neuroinflammatory response without adversely affecting animal survival (DiCarlo et al. 2001
). All animal work was conducted under National Institutes of Health guidelines and approved by the University of South Florida's institutional animal care and use committee. Animals were singly housed for the seven-day posttreatment survival period under standard vivarium conditions.
Tissue Preparation
Mice were anesthetized with pentobarbital (200 mg/kg IP), then perfused transcardially with 25 ml of saline. The brain was removed, the right hemisphere dissected into regions, and the tissue stored at 80C for subsequent biochemical analyses. For immunohistochemistry, left hemispheres were transferred into a 4% paraformaldehyde solution for 24 hr, then processed through a cryprotection schedule of 10%, 20%, then 30% sucrose (24 hr in each solution). The tissue was sectioned horizontally on a sliding microtome at 25 µm. Sections were then stored in Dulbecco's phosphate-buffered saline (DPBS), pH 7.4, with sodium azide (100 mM) at 4C.
RNA Analysis
Mice obtained from the 7-heterozygous breedings were analyzed for mRNA using reverse transcription followed by PCR. The procedure was originally described in detail elsewhere (Dickey et al. 2003
). RNA was extracted from the injected hippocampus of
7+ and
7 mice, using Qiagen's RNeasy procedure. RNA concentration was determined with Molecular Probes RiboGreen RNA quantitation kit (Molecular Probes, Eugene, OR). Reverse transcription with mMLV (Invitrogen, Carlsbad, CA) was performed, and the resulting cDNA subjected to PCR. Primers used to identify
7 were directed toward portions of exons 910: forward, 5'-GTGGGCCTCTCAGTGGTCGT-3'; reverse, 5'-GTCCCCATCAGAGGGGTGTG-3'. Thermocycling conditions were as follows: 95C for 3 min; 45 cycles of 95C for 15 sec, 60C for 60 sec, 72C for 60 sec; 72C for 7.5 min; store at 4C. PCR products were run on a 1% agarose gel, using ethidium bromide UV detection of bands at 381 bp.
Histology
Immunohistochemical analysis of 7 nAChRs was performed using 25-µm free-floating sections spaced 300 µm apart through the hippocampus. Details of this procedure were originally described elsewhere (Gordon et al. 1997
). All steps were performed on a rotating platform at
40 rpm, room temperature, unless otherwise stated. Sections were first blocked for endogenous peroxidases (10% methanol, 3% hydrogen peroxide in DPBS) for 15 min, then washed three times for 5 min with DPBS. The tissue was then permeabilized in a solution of 100 mM lysine, 0.2% Triton X-100, and 4% normal goat or horse serum (Pel Freeze, Rogers, AK) in DPBS for 30 min. Sections were then incubated overnight in the appropriate primary antibody in DPBS and 4% serum, without shaking (Table 1). The following day, sections were incubated with shaking in primary antibody for 1 hr, washed three times for 5 min with DPBS, and then incubated in the appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) at a concentration of 0.5 µg/ml in DPBS and 4% serum, for 2 hr. Sections were then washed three times for 5 min with DPBS, then incubated for 1 hr in Vectastain Elite ABC solution (Vector Laboratories) using 8 drops each of components A and B per 100 ml of DPBS. Sections were then washed two times for 5 min with DPBS, followed by a single wash in Tris-buffered saline (TBS), pH 7.6, for 5 min. The tissue was then incubated with a solution of 0.5% nickelous ammonium sulfate hexahydrate and 0.05% diaminobenzidine in TBS for 5 min. Color development was achieved by the addition of 0.03% hydrogen peroxide, and incubation for an additional 5 min, followed by a single wash in TBS, then two washes in DPBS, each for 5 min. Controls for nonspecific binding of the secondary antibody were performed by excluding primary antibodies. Stained sections were mounted onto slides and air dried overnight. Slides were then processed through a dehydration schedule of 10 dips in water, followed by two washes for 3 min each in 25%, 50%, and 75% ethanol, then three washes for 5 min each in 95% ethanol, 100% ethanol, and Histo-Clear (National Diagnostics, Atlanta, GA). Slides were coverslipped with DPX (E.M. Sciences, Fort Washington, PA) and allowed to dry overnight.
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Western Blotting
Mice obtained from the 7-heterozygous breedings were analyzed for protein using SDS-PAGE followed by Western blot. Cerebral cortex previously stored at 80C was homogenized in 10 mM HEPES buffer, pH 7.4, containing a protease inhibitor cocktail (Roche, Indianapolis, IN). Crude protein concentrations were determined by the Bradford method using Bio-Rad Protein Assay Dye Reagent (Hercules, CA). Samples were denatured with Bio-Rad Laemmli sample buffer by boiling for 5 min. Ten µg of protein was loaded per well, and proteins were separated using 10% polyacrylamide Bio-Rad Ready Gels. Bio-Rad Precision-Plus Protein All Blue molecular weight standards were run for band identification. The separated proteins were transferred to Immobilon-P polyvinylidine fluoride membranes and immunoblotted (Millipore, Bedford, MA). Membranes were first blocked with 5% non-fat dry milk in borate-buffered saline, pH 8.5, and 0.05% Tween-20 (BST) for 1 hr on a rocking platform at room temperature. Membranes were then incubated with primary antibody in 2.5% non-fat dry milk in BST for one hour (Table 1). After washing three times for 5 min with BST, blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:10,000 dilution in 2.5% milk-BST for 30 min. Finally, membranes were triple washed in BST. Bands were identified using Western Blotting Luminol Reagent (Santa Cruz Biotechnology) for chemiluminescent detection and subsequent film exposure for 0.55 min. The presence of
7 protein was verified by comparing the protein bands to the molecular weight standard markers. The expected molecular weight of the
7 subunit was 56 kDa.
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Results |
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Discussion |
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Previous reports have shown the utilization of monoclonal antibodies from clones 306 and 319 for immunohistochemical analysis of rat brain (Del Toro et al. 1994). The results reported by Del Toro indicated ubiquitous neuronal expression, similar to that seen in our studies. Both the Del Toro procedure and ours used paraformaldehyde fixation of the brain prior to sucrose cryoprotection. Both procedures used 25-µm free-floating sections, an avidin-biotin-peroxidase protocol, and diaminobenzidine tetrahydrochloride color development. The immunohistochemical protocols used in our experiments were developed to yield significant positive stain while minimizing background. Various concentrations and combinations of primary (1:1001:10,000) and secondary (1:10001:10,000) antibodies were tested in an effort to identify conditions that stained the
7+/+ mice but not the
7-null mice, without success. Tissue perfusion (saline versus paraformaldehyde) and post-fixation times (224 hr) were also evaluated separately in non-transgenic
7+ mice. No differences in the immunolabeling patterns were seen with any of these variations. It is conceivable that the antigenic determinant(s) was modified by the paraformaldehyde treatment (Montero, 2003
). Another possibility is that the levels of
7 may be so low, or the amount of antibody needed to yield a signal so high, that cross-reacting protein binding masked any
7-positive cells. Other researchers who have worked with the
7-null mice did not use immunohistochemistry to demonstrate the absence of this receptor, but rather showed the absence of
-bungarotoxin binding in unfixed tissue (Orr-Urtreger et al. 1997
; Franceschini et al. 2002
; Wang et al. 2003
). Using our paraformaldehyde-treated tissue, we failed to detect specific labeling using fluorophore-conjugated
-bungarotoxin (data not shown).
The immunolabeling of reactive astrocytes in amyloid-depositing mice by mAb 319 was an interesting finding of these studies. Subsequent studies with LPS injection in 7 knockout mice revealed this labeling to be nonspecific. A recent report by Teaktong et al. (2003)
showed immunolabeled
7+ astrocytes in human AD brain. Cholinergic signaling by rat astrocytes has been demonstrated (Sharma and Vijayaraghavan, 2001
). Activation of these cells produced calcium flux that was blocked by
-bungarotoxin, indicating the presence of
7 nAChRs. The potential for some of the astrocyte
7 labeling to be specific should not be ruled out. Thus, in designing experiments to evaluate
7 expression, an alternative approach would be the use of
-bungarotoxin instead of anti-
7 antibodies. However, we were unable to develop adequate
-bungarotoxin labeling in formaldehyde-fixed sections.
Several reports have demonstrated Western blot analysis of 7 in rodent brain extracts using monoclonal antibodies from clones 306 and 319 (Schoepfer et al. 1990
; Del Toro et al. 1994
; Orr-Urtreger et al. 1997
; Dineley et al. 2001
; Fabian-Fine et al. 2001
). These reports have listed the apparent molecular weight of the
7 subunit as ranging from 48 to 72 kDa, although the calculated molecular weight is 56 kDa. Some of the investigators used standard homogenization procedures, SDS-PAGE separation, transfer, and subsequent immunoblotting, similar to the procedure described here. Other reports affinity purified the
7 nAChRs with cobratoxin or bungarotoxin prior to SDS-PAGE, resulting in a primary band at
56 kDa (Del Toro et al. 1994
; Orr-Urtreger et al. 1997
). The immunoblotting protocols used in our laboratory were developed to minimize nonspecific bands and background. Various concentrations and combinations of primary and secondary antibodies, ranging from 1:50 to 1:10,000, were tested in an effort to come up with conditions that differentiated between the
7+ and the
7-null mice, without success. Separately, various homogenization buffers (10 mM HEPES ± 12% Triton X-100, 50150 mM PBS ± 12% Triton X-100, 50 mM TBS ± 12% Triton X-100), vendors of secondary antibodies (Vector, Santa Cruz), blocking solutions (BST + 2.55% milk, tris buffered saline plus 0.05% tween-20 + 2.55% milk), and subcellular fractionation preparations (crude, membrane, and solubilized fractions) were tried, but failed to reveal differences in the staining pattern between
7+ and
7-null mice. Other researchers who have performed Western blot analysis of
7-null tissue did not use commercially available antibodies, and also affinity purified the
7 subunit before analysis (Orr-Urtreger et al. 1997
; Franceschini et al. 2002
; Wang et al. 2003
). Again, an alternative approach in designing experiments to evaluate
7 expression would be the use of
-bungarotoxin affinity purification. However, the large amounts of tissue needed for affinity purification complicate the procedure and preclude analysis of small brain regions (such as the hippocampus) in individual rodents.
There have been reports of a partial duplication of the human 7 nAChR gene, which has four novel N terminus exons and conserved exons 510 (Gault et al. 1998
; Villiger et al. 2002
). Such duplication has not been reported in rodents, but cannot be completely ruled out. If there were such duplication, it might account for the
7 protein detection in the absence of
7 gene expression. Still, one would expect at least a quantitative difference in the amounts of stained material. Alternatively spliced
7 mRNAs have recently been shown in mice (Saragoza et al. 2003
). The resultant mRNA includes a novel exon 9b. Additionally, another splice variant with novel exon 4b has been reported in rat (Severance et al. 2002
). The Saragoza variant could potentially interfere with our RNA analysis, in as much as the primers are designed to prime to exons 910. In contrast, the Severance variant does contain exons 910 and should be eliminated in the knockout mouse. However, alternatively spliced mRNAs would have no effect on the genotyping results, because exons 810 are interrupted in the knockout. Moreover, the complete absence of
7 mRNA in the null mice would require some mutation in any duplicated
7 gene, which would disrupt the annealing of the primer pairs. We find such circumstances unlikely to account for the results we have obtained here.
In conclusion, careful examination of protocols is necessary when drawing conclusions from immunodetection studies of 7 nAChRs. Localization of the
7 subunit with immunohistochemistry must be interpreted with caution. Confirmation with
-bungarotoxin-binding experiments is recommended, as well as RNA analysis, where applicable.
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Acknowledgments |
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Footnotes |
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