The mouse brain proteome
Within mammalian evolution, the brain has undergone changes that resulted in increased capacities to process information and perform higher cognitive functions in primates and humans. These evolutionary processes mainly involve size expansion, not affecting the basic architecture of the brain and gene-expression in the various cell types that populate the brain. The driving force in evolution is genetic variability passed on from one generation to the other. Many of the mouse proteins have extensive homology with the human counterpart and this forms the basis of for using the mouse brain as a model for the corresponding human brain to explore the expression and distribution of proteins in the various regions and cells of the brain. The regional organization of brain anatomy separates the brain into regions, sub regions, nuclei and layers of specialized cells, enabling the specific function of each individual region. Transcriptomic data from the different regions facilitates classification of the regional expression variation within mouse brain.
The transcriptome analysis shows that 13214 of the human orthologues (n=15160) are expressed in the mouse brain, and 777 of these genes show a regionally elevated expression. 4510 human genes are missing mouse one-to-one orthologue and is therefore missing mouse expression data in the human protein atlas, expression data based on mouse genes are downloadable here.
Figure 1.Schematic drawing of the mouse brain, indicating the different regions and the anatomy from a sagittal view.
Table 1. The 10 regions of the brain and numbers of genes detected above cut off, indicating expression in that brain region, as well as number of genes classified as elevated in each region compared to the others based on transcript abundance in the individual regions (max NX of sub regions is used as representative). Same classification rules are used for the regional classification as the classification based on human tissue types
Table 2. The 12 genes with the highest level of regional enriched expression within the mouse brain and the regional distribution category. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.
In order to illustrate the relation of the different brain regions, a network plot was generated, displaying the number of genes shared between different regions. The majority of group enriched genes are shared between the forebrain regions. Cerebellum and olfactory bulb are the regions with largest number of regionally enriched genes. For more information and examples about the regionally elevated expression in the different regions, please visit the individual summary pages; olfactory bulb, cerebral cortex, hippocampal formation, amygdala, basal ganglia, hypothalamus, thalamus, midbrain, pons and medulla as well as cerebellum.
Figure 2. An interactive network plot of the regionally enriched and group enriched genes connected to their respective enriched region (black circles). Red nodes represent the number of regionally enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges.The network is limited to group enriched genes in combinations up to 4 regions, but the resulting lists show the compleate set of group enriched genes in the particular region.
Proteins localized in different regions of the mouse brain
In-depth analysis of the regionally elevated genes in the mouse brain, using antibody-based protein profiling, allowed us to understand the distribution of specific genes and their protein location. Proteins expressed by the different cells types of the brain can be identified using antibody-based protein localization as well as the regional variation of location when whole mouse brain sections are examined.
Other tissue types related to the brain
A brain region analysed but not included as one of the 10 major regions. Corpus callosum is the largest nerve tract in the brain, providing communication between left and right hemispheres. The neuronal fibers vary in density and the amount of myelination, reflecting their functionality. The structure is divided into subregions based on the target area it is connecting to. Studying brains of patients with severed corpus callosum, has brought answers on how each isolated hemisphere works.
Circumventricular organs in the brain include subcommissural organ, subfornical organ and median eminence, which are all part of in the protein profiling analysis of the mouse brain, but is currently missing RNA expression data.
Protein profiling in mouse brain
The much smaller mouse brain provides a more complete overview of many additional regions, such as thalamic, hypothalamic, and brainstem nuclei. This also enables annotation of cortical (layer 1-6) and hippocampal subfields (CA regions and dentate gyrus). Addition of brain regions with specialized functions increases the possibility of detecting protein expression and distribution of genes and proteins currently not detected in the human samples included in the human proteins atlas.
Figure 3. Example of a protein distribution in a single brain section of the mouse brain. A large 100 megapixel image with microscopic resolutions is generated. This image (a) reveals the regional protein distribution, in this case low density lipoprotein receptor-related protein associated protein 1 (LRPAP1). A more zoomed-in exploration of this image reveals protein levels in the 6 different cortical layers (b) with cellular resolution revealing information on the cellular and subcelluar distribution of proteins (c).
Mouse brain used for protein and mRNA expression analyses were collected and handled in accordance with Swedish laws and regulation, and all experiments were approved by the local ethical committee (Stockholms Norra Djurförsöksetiska Nämd N183/14). The animal experiments conformed to the European Communities Council Directive (86/609/EEC), and all efforts were made to minimize the suffering and the number of animals used. WT male (n = 2) and female (n = 2) C57BL/6J mice (2 month old) were obtained from Charles River Laboratories and maintained under standard conditions on a 12-hour day/night cycle, with water and food ad libitum. Mice were deeply anesthetized and transcardially perfused with 0.9% saline solution. Brains were quickly removed from the skull and dissected on a glass plate on ice. The entire brain was carefully dissected into 16 sub regions, and corpus callosum, pituitary gland and retina were also collected.
Due to the heterogeneous structure of the brain, with many nuclei and cell-types organized in complex networks, it is difficult to achieve a comprehensive overview. Analysis of more human brain samples, including smaller brain nuclei, is thus desirable in order to generate a more detailed map of protein distribution in the brain. Therefore, we here complemented the human brain atlas effort with a more comprehensive analysis of the mouse brain. Antibodies against proteins relevant for brain are selected based on homology, and evaluated for specificity before analysis on mouse brain sections. A series of mouse brain sections is explored for protein expression and distribution in a large number of brain regions. The complete workflow in the mouse brain atlas is described in the slideshow.
This protein atlas of the mouse brain is a collaborative project between the human protein atlas project and department of neuroscience at the Karolinska Institute and is supported by SciLifeLab strategic (SFO) and national infrastructure funding.
Relevant links and publications
Mulder J et al, 2007. Systematically generated antibodies against human gene products: high throughput screening on sections from the rat nervous system. Neuroscience.