Cell type- and brain region-resolved mouse brain proteome

Abstract

Brain transcriptome and connectome maps are being generated, but an equivalent effort on the proteome is currently lacking. We performed high-resolution mass spectrometry-based proteomics for in-depth analysis of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type-specific proteins defined as tenfold more abundant than average expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.

Figure 1. Comparison of proteome and RNA-Seq data.

(a) Graphical illustration of the workflow for the cell type– and brain structure-resolved mouse brain proteome. DIV, days in vitro. (b) A bar chart showing number of proteins identified in adult mouse brain and each of the cultured cell type with FDR of 1% when analyzed as 6x SAX fractions. CGN, cerebellar granule neurons. (c) A total of 10,529 proteins were identified in all cell types and brain (∼84% of all identified proteins) and an average of ∼99% protein identifications were shared between at least two proteomes. (d) Single run analysis of the mouse brain, the different brain structures and the developing cerebellum; four biological replicates (triplicates for optic nerve and corpus callosum) were measured by single 4-h LC MS/MS runs.Numbers of identified proteins are indicated after matching between runs with fractionated brain and cell-type proteome runs in the MaxQuant environment. (e) RNA-Seq analysis of cultured cells and resulting density plots of gene expression levels. The density estimates of gene expression levels are shown for each cell type and for a combination over all cell types as indicated. The combined cell type was derived by extracting the largest expression value over all cell types. In all cell types, gene expression levels followed a binomial distribution (black). Geneexpression filtered for RPKM > 1 values are shown in blue. (f) Venn diagram of the number of expressed genes on the mRNA level and on the protein level.

Figure 2. Comparative analysis of cell proteomes.

(a) The matrix of 162 correlation plots revealed very high correlations between LFQ intensities in triplicates (Pearson correlation coefficient 0.94–0.98 between cell types). The color code follows the indicated values of correlation coefficient. (b) PCE. The proteome of all cell types and their differentiation states measured in triplicates segregated into major cell types based on component 1 and component 2, which account for 44.7% and 14.3% of variability, respectively. (c) Correlation plots of iBAQ intensities (proteome) versus RPKM values (transcriptome).The color follows the indicated values of correlation coefficient. (d) Fold expression of the indicated marker proteins in individual replicates is shown on a log₂ scale as points with mean in the specified cell type in comparison with other cell types. (e) Heat map of proteins differentially expressed across different cell types (n = 3 for each cell type). The top categories enriched for clusters are shown. Heat map of z-scored LFQ intensities of the significantly differentially expressed proteins after unsupervised hierarchical clustering. Proteins are divided into four clusters showing the top categorical annotations enriched after a Fisher’s exact test (P ═0.02).

Figure 3. Quantitative analysis of expressed genes.

(a) Analysis of specific GO annotation terms (indicated in red above the bar graph) is shown as the percent of the genes corresponding to the annotation term and the percent of the protein mass that was attributed to these annotations.The analysis was performed separately for all proteins identified in indicated cell type or for those proteins that were specific to the indicated cell type. (b) Cumulative protein mass from the highest to the lowest abundance proteins for the indicated cell type. The table liststotal number of proteins constituting different quantiles (Q1-Q4) and the percent of these proteins that showed cell type-specific expression. (c,d) Comparison of acutely isolated cells with cultured cells. (c) Cells were isolated using MACS microbeads coupled with antibodies to 04 for oligodendrocytes, PSA-NCAM for neuronal progenitors, CD11b for microglia and ACSA-2 for astrocytes. The heat map shows the Pearson correlation coefficientsbetween acutely isolated and cultured cell types. The color code indicates the values of the correlation coefficients. (d) Plot of GOCC enrichment of proteins > 10-fold enriched is shown. –log₁₀ P value is plotted against enrichment factor of the GOCC terms.

Figure 4. Abundant and enriched proteins in the mouse brain and its cell types.

(a) Scatter plot of log₂ fold expression versus log₂ LFQ intensity in the adult mouse brain in comparison to the mouse liver proteome. Among the top 40 most abundant and enriched proteins of the adult mouse brain are proteins of the myelin sheath (red), the cytoskeleton (blue) and synapses (green). (b) Scatter plot of log₂ fold expression versus log₂ LFQ intensity in the indicated cell type in comparison with other cell types with highlighting of known and previously unknown cell type-specific markers.

Figure 5. Brain region-resolved proteome

(a) PCA. The proteomes of major mouse brain regions (P60) were measured in quadruplicates (triplicates for optic nerve and corpus callosum) and segregated based on component 1 and component 2, which accounted for 34.8% and 24.5% of variability, respectively. (b) Heat map of proteins differentially expressed across the different brain regions. The heat map is based on the z-scored LFQ intensities of the significantly differentially expressed proteins after unsupervised hierarchical clustering. Proteins with more than fourfold expression differences are shown. (c) Scatter plot of log₂ fold expression versus log₂ LFQ intensity of the top 20 proteins in the indicated brain region in comparison with other brain region. The larger red circles indicate proteins chosen for the comparison with the corresponding transcripts analyzed by in situ hybridization in the Allen Brain Atlas project. Images are taken from the Allen Brain Atlas (http://mouse.brain-map.org). Image credit: Allen Institute for Brain Science. (d) Plot of GOCC enrichment of proteins > 10-fold enriched is shown. -log₁₀ P valueis plotted against enrichment factor of the GOCC terms.

Figure 6. Comparative pathway enrichment analysis identifies cell adhesion molecules enriched In oligodendrocytes and neurons.

(a) Annotation matrix of KEGG pathways enriched in different cell types shown as a heat map (red indicating KEGG pathways higher abundance and blue indicating lower abundance) after clustering of score differences from one-dimensional annotation testing (Online Methods). (b) Scatter plot for LFQ intensities of proteins corresponding to KEGG pathway cell adhesion molecules (CAM) in oligondendrocytes versus neurons. (c) Label-free quantification of individual triplicates is shown as points with mean ± s.e.m. for the IgLON family proteins in the different CNS cells types. (d) Scatter plot of log₂ fold expression versus log₂ z-normalized protein intensity of proteins corresponding to KEGG pathway cell adhesion molecules in brain versus liver.

Figure 7. Lsamp interacts with and is expressed on oligodendrocytes and neurons.

(a) Binding of Lsamp-Fc to neurons labeled with an antibody against βMI tubulin. Necl4-Fc served as a positive and Necl 1-Fc as a negative control.The Fc fragments were visualized with Cy3-conjugated anti-Fc antibodies. (b) Binding of Lsamp-Fc to oligodendrocytes labeled with an antibody against MBP. Necl 1-Fc served as a positive and Necl4-Fc as a negative control. (c) Lsamp-Fc did not bind to astrocytes labeled with an antibody against Gfap. (d) Immunoblot of secreted Fc fusion proteins containing the extracellular domains of the indicated proteins. (e) Immunofluorescence of mixed glial cultures shows staining of oligodendrocytes (O1; magenta), but not astrocytes (Gfap; green) with an antibody against Lsamp (red). (f) Top, Lsamp was present on a subpopulation of neurons (neurofilament 200 kDa) (top right). Arrow indicates toward an Lsamp-positive neuronal process, arrowhead indicates an Lsamp-negative process. Bottom, Lsamp was absent from microglia (Iba1), as shown by staining of a mixed glial culture. (g) Brain sections of wild-type mice were immunostained with antibodies against Lsamp (red) at P10, and Lsamp and MBP (green) at P30. Lsamp staining was enriched along the axonal tracts of the fimbria and anterior commissure (AC) at P10, but not at P30. Scale bars represent 10 μm.

Figure 8. Lsamp is a negative regulator of myelination in the fiber tracts of the fimbria-fornix.

(a) Electron microscopy images of the fimbria/fornix at P30 and P60 from controls (wild type, WT) and Lsamp knockout (KO) mice. Scale bar represents 1 |μm (b) Scatter plots of g-ratios of individual fibers of the fimbria/fornix at P20,P30 and P60 from control (black)and Lsamp KO mice (magenta). (c) The histogram shows the percentage of myelinated axons with respect to axon diameter at 0.3-μm intervals at P20, P30 and P60 for wild-type and Lsamp KO mice. There was a shift toward myelination of low-caliber axons in the mutant as compared with the control (chi-square test; from top to bottom: P ═ 4.5 x 10^-10, P ═ 2.8 x 10^-5, P ═ 0.36, ***P <0.0001).More than ∼250 axons for each genotype were counted (three animals per genotype) (d) Average g-ratio at P20, P30 and P60 for wild-type and Lsamp KO mice (Student's t test, P ═ 0.0105; n ═ 3 mice per genotype). Error bars represent s.d. (e) Percentage of myelinated and unmyelinated axons counted at P20, P30 and P60. More than 1,500 axons were counted for each time point (n ═ 5 mice for P20 and P30, n ═ 4 for P60) per genotype (bars show mean ± s.d.; Student's t-test; **P ═ 0.0055, ***P ═ 0006). (f,g) Coverslips were coated with 10 μg ml^-1 Fc-fusion proteins (IgLON family proteins and control), and oligodendrocyte precursor cells were plated and allowed to adhere and grow for 4 d. PLL and Necl1-Fc coating were used as positive controls. The purified supernatant of HEK 293T cells transfected with an empty vector (pcDNA) was used as negative control (bars show mean ± s.d.; ANOVA, P ⋖ 0.05, Dunnet post hoc test with pcDNA as control; n ═ 3 experiments; *P ═ 0.0087, **P ═ 0.0025). Scale bar represents 20 μm.