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Mol Cell Proteomics. Jul 2009; 8(7): 1612–1622.
Published online Apr 7, 2009. doi:  10.1074/mcp.M800539-MCP200
PMCID: PMC2709189

Tissue Profiling of the Mammalian Central Nervous System Using Human Antibody-based Proteomics*

Abstract

A need exists for mapping the protein profiles in the human brain both during normal and disease conditions. Here we studied 800 antibodies generated toward human proteins as part of a Human Protein Atlas program and investigated their suitability for detailed analysis of various levels of a rat brain using immuno-based methods. In this way, the parallel, rather limited analysis of the human brain, restricted to four brain areas (cerebellum, cerebral cortex, hippocampus, and lateral subventricular zone), could be extended in the rat model to 25 selected areas of the brain. Approximately 100 antibodies (12%) revealed a distinct staining pattern and passed validation of specificity using Western blot analysis. These antibodies were applied to coronal sections of the rat brain at 0.7-mm intervals covering the entire brain. We have now produced detailed protein distribution profiles for these antibodies and acquired over 640 images that form the basis of a publicly available portal of an antibody-based Rodent Brain Protein Atlas database (www.proteinatlas.org/rodentbrain). Because of the systematic selection of target genes, the majority of antibodies included in this database are generated against proteins that have not been studied in the brain before. Furthermore optimized tissue processing and colchicine treatment allow a high quality, more extended annotation and detailed analysis of subcellular distributions and protein dynamics.

The brain is the most complex organ in the mammalian body. It processes sensory information from our external environment; produces behavior, emotions, and memories; and regulates the internal body homeostasis. To fulfill these diverse functions the brain harbors a myriad of neuronal networks processing information and connecting input and output systems. Because of the highly specialized functions, each neuron population is neurochemically specified expressing the necessary sets of proteins. Consequently a large number of genes are expressed in the mammalian brain. Based on microarray and in situ hybridization studies it is estimated that ~55–80% of all mouse genes are expressed in the brain (1, 2) (gene expression during developmental stages and pathological conditions not included). Interestingly 70% of these genes are expressed in different cell populations each covering less than 20% of the brain, indicating the complexity of the brain and the specialization of individual populations of neurons (1).

The success of humans as a species relies on our mental abilities, a result of brain development during evolution. The human brain is distinguished from other mammalian brains by its size; especially the neocortex involved in higher cognitive functions is greatly enlarged in humans. Despite this difference, the human brain has many similarities to brains of other mammalian species, and to some extent mammalian brains have a well preserved basic architecture (basic uniformity) (for reviews, see Refs. 3 and 4). Therefore, most human brain nuclei and connections have orthologs in other mammalian species ranging from great apes to rodents.

Genetic variation underpins interspecies variation in gene expression and assembly of proteins. The human and rat genomes encode similar numbers of genes of which the majority have persisted throughout evolution without deletion or duplication (5). It is evident that small changes in protein structure and altered expression levels of proteins influence brain development and form the basis of interspecies differences. However, most human genes have orthologs in rodents, and for most cell types in the brain their neurochemical specification has been preserved throughout evolution. Because of genomic homology and similarity in basic layout of the mammalian brain as well as the preservation of neurochemical specification of subsets of neurons throughout evolution, animal models have shown their value in medical neurosciences (6).

Advances in science are largely dependent on the processing of available information and the generation of new concepts and are driven by innovation and availability of new technologies. Recently mRNA-based techniques have emerged as an effective tool for genome wide analysis of expression levels in entire organs or disease-affected tissue. Results obtained from these studies are a source for identification of novel key molecules and have a predictive value to estimate changes in protein synthesis. There are several ongoing initiatives focusing on the expression profiles of the mammalian brain. The Allen Brain Atlas has produced detailed in situ hybridization profiles for over 20,000 genes in the mouse brain (1). The Gene Expression Nervous System Atlas (GENSAT) project uses enhanced green fluorescent protein reporter genes incorporated into bacterial artificial chromosome transgenic mice to visualize the expression profiles of the most important genes (7). This strategy can result in the identification of expressing cell types as the detailed morphology of enhanced green fluorescent protein-expressing cells is apparent. The Brain Maps project has a large collection of mammalian and non-mammalian brain maps using “classical” histochemical techniques but also includes a few protein distribution profiles visualized using immunohistochemistry (8).

We previously described the possibilities of using antibodies raised against human proteins on rodent brain tissue (9). Here we show the first efforts to produce detailed proteome wide large scale tissue profiling maps of a mammalian brain using an antibody-based proteomics approach. In addition to the available, mentioned information on mRNA levels (Allen Brain Atlas), gene expression profiles (Gene Expression Nervous System Atlas), and detailed neuroanatomy (Brain Maps), antibody-based proteomics provide new information on cellular and subcellular distribution of gene products. This information will increase general knowledge and understanding of the organization and functioning of the brain. The study is based on antibodies generated as part of the Human Protein Atlas program aimed at exploring the protein expression patterns in normal and cancer tissues using tissue microarray-based immunohistochemistry and fluorescence-based confocal microscopy (10).

The Human Proteome Resource center aims to produce monospecific antibodies against every human gene. So far, the distribution profiles of 3,000 proteins in 48 human tissues, including four brain areas (cerebellum, cerebral cortex, the hippocampal formation, and lateral subventricular zone), and 20 cancers are available (Human Protein Atlas). The antibodies generated within the framework of this program are based on antigens selected as unique regions for each individual protein, called protein epitope signature tags (PrESTs)1 (11, 12). Over 5,000 antibodies have been generated and validated using Western blot analysis and protein arrays (13). The smaller size of the rat brain allows analysis of many brain areas and exposure of the antibodies to a very wide variety of proteins. Furthermore tissue can be processed under perfect conditions optimizing tissue antigenicity with flawless tissue morphology.

Here we describe the initial large scale mapping of 89 protein distribution profiles in 25 selected rat brain areas. By exposing systematically sampled rat brain tissue to our collection of monospecific antibodies a more detailed protein atlas of the mammalian brain was produced, expanding the four brain areas available in the human protein atlas to 25 brain areas (Fig. 1) involved in higher cognitive functions, sensation, emotion, maintenance of internal homeostasis, sleep, and motor and sexual behaviors. A database portal has been created to show selected images from the various regions of the brain.

Fig. 1.
Schematic overview of the 25 selected brain areas. Included are telencephalon (medial septum, lateral septum, horizontal/vertical diagonal band, prefrontal/cingulate/somatosensory/piriform/entorhinal cortex, ventral pallidum, stria terminalis, globus ...

EXPERIMENTAL PROCEDURES

All experiments on animals conformed to the European Communities Council Directive (86/609/EEC) and were approved by the local ethics committee (Stockholms Norra Djursförsöksetiska Nämnd; N396/06 N397/06). All performed experimental procedures have been described in detail previously (9).

Colchicine Treatment

Rats were anesthetized by an intraperitoneal injection of a mixture of Hypnorm and midazolam followed by an intracerebroventricular injection of the mitosis inhibitor colchicine (dissolved in 0.9% NaCl solution to a final concentration of 90 μg in 15 μl). Animals were sacrificed 24 h after colchicine injection.

Tissue Processing

Rats were deeply anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg) and were transcardially perfused with 300 ml of fixative composed of 4% paraformaldehyde and 0.2% picric acid in phosphate buffer that was preceded by a short prerinse (50 ml) with calcium-free Tyrode's solution. After dissection, brains were postfixed for 90 min at 4 °C and transferred to 0.01 m phosphate buffer containing 10% sucrose, 0.02% bacitracin, and 0.01% sodium azide for 48 h at 4 °C. After dehydration the brains were quickly frozen using CO2. Coronal sections (14 μm) were cut in a cryomicrotome and thaw mounted on gelatin-alum-coated slides with a sampling interval of 0.7 mm.

Immunocytochemistry

Specific PrESTs of 100–150 amino acids for each target protein were designed using bioinformatics tools (11) and available information on the human genome (Ensembl database). PrESTs were carefully selected, transmembrane regions and signal peptides were avoided, and only amino acid sequences with low homology to other human proteins were used to avoid cross-reactivity. Selected PrESTs were recombinantly produced in Escherichia coli and used to immunize rabbits, and monospecific polyclonal antibodies were generated through immunoaffinity purification of the resulting antisera (14). Detailed immunohistochemistry procedures are described elsewhere (9). Briefly after a quick rinse in PBS, thaw mounted sections were incubated for 16–24 h with the rabbit monospecific antibodies at a standard dilution (1:1,000) followed by 30-min incubation with horseradish peroxidase-conjugated swine anti-rabbit IgG (P0217, Dako, Copenhagen, Denmark). Immunoreactivity was visualized using the tyramide signal amplification system (TSA-Plus, NEL741B001KT, PerkinElmer Life Sciences). All used Human Proteome Resource center antibodies presented in this study are listed in Table I. For double staining immunohistochemistry, single staining (as described above) with antibodies against PDZ-binding kinase (PBK) (1:1,000; HPA005753) and C6orf64 (1:1,000; HPA007959) was followed by an overnight incubation at 4 °C with a goat anti-doublecortin (DCx) (1:200; sc-8066, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Ki67 (1:1,000; RM-9106, Thermo Scientific, Waltham, MA), guinea pig anti-vasopressin (1:1,000 preadsorbed with 10−5 m oxytocin peptide; Ref. 15), and rabbit anti-oxytocin (1:500; Ref. 16). Immunoreactivity was visualized by incubation with appropriate Rhodamine Red X-conjugated donkey anti-rabbit, anti-guinea pig, or anti-goat antibody (1:200; 711-295-152, Jackson ImmunoResearch Laboratories, West Grove, PA). The combination of tyramide signal amplification with direct fluorescence can be used for double labeling experiments using primary antibodies raised in the same species against proteins expressed in different cells or cellular compartments (17). This method was used for double labeling experiments using rabbit antibodies targeted against Ki67 and PBK. All antibodies generated and validated within the Human Protein Atlas project and used in this report will be available for the scientific community and can be purchased via Prestige Antibodies (Sigma-Aldrich) or Atlas Antibodies (Stockholm, Sweden).

Table I
List of antibodies presented including references to published immunohistochemical data on brain tissue

Imaging

After processing, sections were examined using a Nikon Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan) equipped with epifluorescence with appropriate filter combinations. Photographs were taken using a Hamamatsu ORCA-ER C4742-80 digital camera (Hamamatsu, Hamamatsu City, Japan). Images were acquired using a 10× objective covering an area of 1.3 × 1.0 mm with a 1 pixel/μm resolution. Scale bars in Fig. 3 and in the micrographs available on line at www.proteinatlas.org/rodentbrain are 100 μm. Confocal laser scanning was performed on a Bio-Rad Radiance Plus confocal laser scanning microscope. Digital images were slightly modified to optimize image resolution, brightness, and contrast using Adobe Photoshop 10.01 software (Adobe Systems Inc., San Jose, CA) to best represent the immunocytochemistry observed in the microscope.

Fig. 3.
Immunofluorescence micrographs showing a spectrum of staining patterns of 25 validated antibodies in 25 selected, different brain areas. Antibody HPA001303 (NMT2) stains the soma of neurons in many regions, including medial septum (A). HPA003342 (CXADR) ...

Western Blot

Animals were quickly euthanized in CO2-rich atmosphere, and samples of the somatosensory cortex, dorsal hippocampal formation (HiFo), and hypothalamus were collected and snap frozen using dry ice. Samples were homogenized, diluted to a final protein concentration of 2 μg/μl, and denatured. Equal amounts of somatosensory cortex, HiFo, and hypothalamus homogenates were mixed, and proteins were resolved using SDS-polyacrylamide electrophoreses on 10% gels and transferred to PVDF membrane (Immobilon-FL, Millipore, Bedford, MA). The membranes were probed with the rabbit primary antibodies followed by incubation with IRDye-800-conjugated goat anti-rabbit IgG (Rockland Immunochemicals, Philadelphia, PA). Blotted proteins were detected and analyzed using the Odyssey infrared imaging system (Li-cor, Lincoln, NE). Antibodies that showed a band of expected size or slightly bigger (±20% or +10 kDa), presumably due to posttranslational modification, passed Western blot validation. Antibodies that passed validation were divided in three groups based on their specificity: 1) only a single band of the expected size, 2) also unpredicted weaker bands visible, or 3) also unpredicted strong bands visible (Fig. 2.

Fig. 2.
Western blot results of all 32 antibodies used in the study. Left column, protein ladder (green); middle column, Western blot result (red); right column, Western blot result (red) and theoretical band of predicted size ±20% or +10 kDa (yellow ...

RESULTS AND DISCUSSION

Protein Atlas of the Mammalian Brain

This investigation involves a pilot study of 800 antibodies selected from the Human Protein Atlas program by the criteria that the antigen used for generation of the affinity reagent showed at least 75% homology with their rat protein orthologs and that the antibodies did not fail Western blot validation on human cell lines (18). All 800 antibodies were applied to coronal sections of the rat brain including cortex, the HiFo, thalamus, and hypothalamus. Antibodies that showed clear and selective immunoreactivity were further validated for specificity using Western blot analysis of rat brain homogenates and bioinformatics as described previously (9). All antibodies that passed validation (12% of all 800 antibodies tested) were applied on coronal sections (14 μm thick, 0.7-mm interval) of a “normal” and a colchicine-treated rat brain. From these, 25 selected immunohistochemistry images are shown in Fig. 3. General staining patterns were analyzed, and protein expression was annotated. Similar to the Human Protein Atlas, staining of neuronal and non-neuronal cells was determined. Tissue optimization and the size of the rat brain, presenting an overview of many brain structures in one single section, allowed a detailed description of cellular and subcellular localization of proteins. Therefore neuronal immunoreactivity was further divided into somatic, nuclear, axonal, dendritic, or synaptic (nerve ending) immunoreactivity. Also nuclear staining in non-neuronal cells could be determined, and different types of glia cells could be identified including astrocytes, microglia, radial glia, and tanycytes. The regional distribution of proteins among the 25 selected brain areas was determined, and representative images covering 1.3/1.0 mm of a particular area and its surroundings were captured (Fig. 3). Analysis of the rat brain was, however, not limited to the 25 selected areas. Also other interesting areas with strong immunoreactivity were included (Fig. 4).

Fig. 4.
Distribution of immunoreactivity beyond the 25 selected regions. Antisera producing strong staining included HPA005551 (CFH) showing dendrite/radial glia-like structures in cortical areas and hippocampal formation but also glia cells in the subfornical ...

A comparison of 48 antibodies generated within the Human Protein Atlas program with corresponding commercial analogs revealed similar staining patterns in 92% of the antibody pairs (19). Also in the rodent brain HPA antibodies against calretinin (CALB2) and PBK revealed similar staining patterns with equal detail compared with commercial and “academic” antibodies against these proteins (data not shown). PBK is highly enriched in neural stem cells (20). Antibody HPA005753 raised against human PBK strongly labeled cells in the subventricular zone and the dentate gyrus of the HiFo, areas known to contain neural progenitors. Double immunohistochemistry with DCx, an early marker for neurons, and Ki67, a nuclear marker for proliferating cells, revealed limited colocalization of PBK with DCx (Fig. 5A). Double labeling experiments using rabbit antibodies against the nuclear marker Ki67 and cytosolic marker PBK revealed coexistence of both markers in neural progenitors (Fig. 5, B and andC).C). Our PBK antibody showed immunoreactivity of the soma and proximal processes (Fig. 5C), making it possible to investigate subcellular co-distribution of proteins expressed in these dividing cells.

Fig. 5.
Double labeling experiments confirm labeling of cell types known to express target proteins. HPA005753 is raised against a mitotic kinase (PBK) known to be expressed in neuronal progenitors. Double labeling with the early neuronal marker DCx and the nuclear ...

A further example is calretinin (CALB2), which is related to calbindin-D28k and belongs to the family of calcium-binding proteins. Calretinin is expressed throughout the brain in many neuron populations (21), and calretinin antibodies are often used to label neuronal subpopulations (22). In the cerebral cortex calretinin is known to be expressed in some GABAergic interneurons. Antibody HPA007306 raised against human calretinin labeled many structures in the rat brain known to contain this protein. Staining of brain sections from a mouse expressing GFP under the promoter of GABA-synthesizing enzyme glutamate decarboxylase 67 (GAD67gfp/+) showed immunoreactivity of this antibody in a population of GABAergic interneurons in the cerebral cortex (Fig. 5, D and andE)E) as reported before (23).

Because of the systematic approach, a large portion (75–80%) of all 800 tested antibodies are raised against proteins that have not been studied in the rat brain using immunohistochemistry (see Table I for references of published immunohistochemical data), and thus the cellular distribution of these proteins is unknown. However, for many of them data on mRNA expression are available. During the tissue profiling we came across several interesting antibodies that only show immunoreactivity in a small population of brain cells that were not associated with those cells previously. Antibody HPA007959 is raised against a 20-kDa protein transcribed from an uncharacterized gene located on chromosome 6 (C6orf64) that has homologs in rat and mouse. This antibody, with a supportive score 1 Western blot (Fig. 2), only showed immunoreactivity in the hypothalamic paraventricular and supraoptic nuclei, staining the neuronal cell bodies and their projections through the median eminence. These magnocellular neurons are primarily responsible for the secretion of the peptide hormones vasopressin or oxytocin (24). Double immunohistochemistry revealed coexistence of C6orf64 with oxytocin (Fig. 6, B and D) but not vasopressin (Fig. 6, A and C). C6orf64 has no known function and shares no conserved domains with other known proteins. However, our results suggest a role related to the involvement of oxytocin in control of milk ejection or in the many other functions of this nonapeptide.

Fig. 6.
HPA007959 (C6orf64), raised against a protein of unknown function located on chromosome 6, labeled neurons in the paraventricular (A and B) and supraoptic (C and D) nuclei and their projections. Co-labeling with vasopressin (AVP) (A and C) and oxytocin ...

A Rodent Brain Protein Atlas

A new database portal (www.proteinatlas.org/rodentbrain) for protein profiles in rat brain has been launched as part of this study. The portal is publicly available without password protection, and the first version of the atlas contains 89 antibodies and 642 images. The relative expression of each protein is shown in each part of the brain as determined with immunohistochemistry and manually annotated using a four-color code from strong, medium, weak, and no staining, Selected images are shown for each protein as well as validation using Western blots. A dictionary has been created to show the regions annotated in the rodent brain.

Colchicine-treated Brain

Many proteins are rapidly transported from the cell body into the axon and nerve terminals and are thus difficult to detect in the cell soma. The mitosis inhibitor colchicine arrests this centrifugal transport and will cause protein accumulation in the soma. In addition, colchicine mimics the effect of nerve injury, and treatment with colchicine may identify proteins possibly involved in neuroplasticity. All validated antibodies were therefore also applied to sections of a colchicine-treated brain. Antibodies raised against amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 13 (ALS2CR13; HPA003229) and synaptosome-associated protein, 25 kDa (SNAP25; HPA001830) only showed immunoreactivity of nerve terminals in brain tissue of untreated rats. Under normal conditions ALS2CR13 strongly stained nerve endings in layer Ib of the piriform cortex (Fig. 7 A). In colchicine-treated brains ALS2CR13 was mostly located in the proximal dendrites (Fig. 7B) originating from cells located in layer II. In the somatosensory cortex SNAP25 was located in synapses (Fig. 7C). Blockade of axonal transport resulted in a reduced staining of nerve terminals, and the soma of SNAP25-expressing cell became visible (Fig. 7D). Based on a single immunohistochemical staining it is difficult to discriminate between post- or presynaptic localization of proteins. Arresting centrifugal transport of proteins could give an indication of protein distribution. In colchicine-treated brain sections ALS2CR13 showed a translocation to dendritic pronounced structures, whereas SNAP25 revealed a more axonal and somatic staining pattern after blocking protein transport. This could indicate a presynaptic distribution of SNAP25 and a postsynaptic localization of ALS2CR13. Furthermore by using colchicine we were able to identify proteins that are not expressed under normal conditions in the adult brain. For example, activating transcription factor 3 (ATF3) could not be detected in normal brain tissue using immunohistochemistry and Western blot. However, in brain tissue of colchicine-treated rats, a number of stained nuclei could be identified (data not shown); this is in line with earlier findings showing up-regulation of ATF3 after nerve injury (25).

Fig. 7.
Colchicine treatment prevents centrifugal transport, allowing analysis of protein dynamics and identification of cells producing these proteins. Antibody HPA003229 (ALS2CR13) reveals a synaptic staining in layer Ib of the piriform cortex (A). In brain ...

Concluding Remarks and Future Perspectives

Although many aspects of antibody generation can be controlled and, in fact, were controlled in this project, the actual immunization process is not well characterized. Immunization with the same protein fragment or even different bleeds from the same animal often results in variation in antibody titer affecting sensitivity and specificity of the produced antisera. Therefore validation of specificity has to be implemented even if antibodies have been affinity-purified using the antigen as affinity ligand. Sensitivity of the antibodies will determine the threshold of detection. Structures with high concentrations of target proteins can be visualized, and the quality of the antibodies will determine the level of detail that can be obtained. Therefore absence of staining can be a sensitivity issue, and negative results have to be interpreted with caution. This may be one reason for discrepancies between immunohistochemistry and results obtained with mRNA-based techniques. However, also the mRNA-based techniques may have sensitivity and specificity problems. Furthermore translation is regulated, and levels of mRNA do not always resemble actual protein concentrations (26, 27).

Within the Human Protein Atlas program, antibody generation for 16,000 genes has been initiated, and it has been estimated that a first draft of the human proteome can be achieved by 2015 (28). We show here that many of these antibodies can be used to explore the rat brain to potentially generate a brain atlas based on immunohistochemistry as a complement to the expression-based atlas programs (see above).

Antibody-based high throughput profiling of human cancers has already resulted in the identification of new biomarkers for different types of cancers (29, 30). The Human Brain Proteome, an initiative of the Human Proteome Organization (HUPO), aims to perform suitable quantitative proteomics on body fluids and brain tissue to analyze the brain proteome of human as well as mouse models with healthy, neurodiseased, and aged status with a focus on Alzheimer and Parkinson diseases (31). With the increasing number of antibodies within the human proteome resource program and the additional information on the brain distribution of a large set of proteins, high throughput antibody-based proteomics can be utilized to identify new diagnostic or therapeutic targets in neuropsychiatric and neurodegenerative diseases using human post-mortem tissue or rodent animal models.

Footnotes

* This work was supported by the Knut and Alice Wallenberg Foundation and Wallenberg Consortium North, Swedish Research Council Grant 04X-2887, and the Marianne and Marcus Wallenberg Foundation.

The abbreviations used are:

PrEST
protein epitope signature tag
CNS
central nervous system
DCx
doublecortin
GABA
γ-aminobutyric acid
GAD
glutamate decarboxylase
GFP
green fluorescent protein
HiFo
hippocampal formation
PBK
PDZ-binding kinase
PDZ
postsynaptic density-95/discs large/zona occludens-1
HPA
Human Protein Atlas.

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