The cerebellum-specific proteome Cell types specific for cerebellum Regionally elevated expression in human cerebellum Regionally elevated expression in mouse cerebellum Regionally elevated expression in pig cerebellum Extended human cerebellum data

The cerebellum-specific proteome

Cerebellum is involved in movments and coordination. The developmental origin of cerebellum is hindbrain, together with the brainstem (midbrain, pons and medulla oblangata). Cerebellum is attached to the Pons and all communication in and out of the cerebellum goes through Pons. Cerebellum is located underneath the cerebrum in the back of the head (see Figure 1).

The cerebellum has, similar to cerebrum, two hemispheres and a folded cortical surface, which in cerebellum has parallel groves. The cerebellar cortex includes three distinct layers; the molecular layer, Purkinje layer and granular layer. All three layers include a large number of specialized neurons, making the cerebellum the most neuronal dense region of the human brain. It is estimated to include 80% of all the neurons in the brain, but it only represents 10% of the brain volume.

The transcriptome analysis shows that 79% (n=15629) of all human protein-coding genes (n=19670) are expressed in the human cerebellum. Human one-to-one orthologues were investigated in pig and mouse brain, suggesting that 11380 of all mouse one-to-one orthologues (n=15160) are expressed in the mouse cerebellum and that 12200 of all pig orthologues (n=14656) are expressed in the pig cerebellum.

Figure 1. Schematic drawing of the human brain, indicating the location of cerebellum from a sagittal veiw.

Gene classification based on regional RNA expression in human, pig and mouse cerebellum defines 340 genes as cerebellum enriched in either of the three speces, and 95 out of those genes show highest expression levels in cerebellum in all three species.

Cell types specific for cerebellum

The Purkinje cells are one of the largest neurons in the human body, the cell body of Purkinje cells are located in the Purkinje layer while the dendritic tree of the Purkinje cells streches out in the molecular layer and the axon of Purkinje cells projects through the granular layer and further to deep cerebellar nuclei. Two examples of proteins specifically located to the Purkinje cells are MYH10, labeling the cell body and the stem of the dendritic tree, and HOMER3 which shows the extensive dendritic tree as well as the axonal projection. The Purkinje cell receives input from both the paralell fibers from the granular cell, projecting into the molecular layer, as well as stellate and basket cells located in the molecular layer. The synaptic inter-connections between basket cells and the initial segment of the Purkinje cell axon is especially clear by the protein detection of KIT.

MYH10
HOMER3
KIT

Example of a protein speclfically located to the cells of molecular layer is BHLHE22. Granular cells are highly abundant neurons in the cerebellum with round nucleus tightly packed, and with synaptic glomeruli formed by granular cells, golgi cells and mossy fibers (one of the major inputs to cerebellum from cerebral cortex and other regions). GABRB2 show synaptic positivity, including the synaptic glomeruli in granular layer. Bergmann glia are radial astrocytes specific for cerebellum located in the Purkinje layer with processes through the molecular layer all the way to the pial surface of the cerebellar cortex. Example of protein specifically detected in Bergmanglia is QKI.

BHLHE22
GABRB2
QKI

Above examples show the human cerebellum, and similar cell types and proteins can also be detected in the mouse cerebellum. For example, QKI show selective positivity in Bergmann glia, KCNIP4 show staining of the synaptic glomeruli in the granular layer and MX1 is located in the dendrictic processes of Purkinje cells.

QKI
KCNIP4
MX1

Regionally elevated expression in human cerebellum

Figure 2. Schematic drawing of the human brain, indicating the location of cerebellum from a coronal perspective.

The transcriptome analysis shows that 79% (n=15629) of all human proteins (n=19670) are expressed in the cerebellum, 13188 of all genes detected in the brain are also detected in cerebellum.

214 regionally enriched genes
373 regionally elevated genes in total
Shares the most group enriched genes with Pons & medulla
172 of the cerebellum elevated genes are elevated in other tissues than the brain.
201 of the cerebellum elevated genes are elevated in the brain.

Table 1: Number of genes within the different categories of regionally elevated expression, in human cerebellum

Specificity	Number of Human elevated genes
Region enriched	214
Group enriched	81
Region enhanced	78
Elevated	373

Elevated expression in cerebellum compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in cerebellum compared to all other regions), group enriched (at least four-fold higher mRNA levels in a group of 2-5 regions) and regionally enhanced (at least four-fold higher mRNA levels in cerebellum compared to the average of all regions), The number of genes in the individaul category is shown in Table 1. In Table 2, the 12 genes with the highest level of regional specificity among the 214 enriched genes are listed.

Table 2. The 12 genes with the highest level of enriched expression in human cerebellum. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
SPINK6	Serine peptidase inhibitor, Kazal type 6	Secreted	78
BARHL2	BarH like homeobox 2	Intracellular	42
CCDC155	Coiled-coil domain containing 155	Intracellular,Membrane	39
FGF3	Fibroblast growth factor 3	Secreted	38
GABRA6	Gamma-aminobutyric acid type A receptor alpha6 subunit	Intracellular,Membrane	36
FAT2	FAT atypical cadherin 2	Membrane	34
PRR35	Proline rich 35	Intracellular	27
CDH15	Cadherin 15	Membrane	24
CRTAM	Cytotoxic and regulatory T cell molecule	Membrane	24
ZIC5	Zic family member 5	Intracellular	24
CBLN3	Cerebellin 3 precursor	Intracellular,Secreted	23
SLC22A31	Solute carrier family 22 member 31	Membrane	23

Proteins specifically localized in human cerebellum

Proteins with elevated expression in cerebellum compared to the other brain regions were detected in the different cell types and layers of cerebellum. Protein specifically detected in cell of molecular layer (SCGN), the granular cells (PAX6) and the synaptic glomeruli of the granular layer (TMEM266) were all identified among the cerebellum elevated genes.

SCGN
PAX6
TMEM266

Several genes with cerebellum elevated expression were localized to Purkinje cells, for example ARHGEF33 and PPP1R17. Proteins detected in Bergmann glia were also found among the cerebellum elevated genes, for example DAO.

ARHGEF33
PPP1R17
DAO

Regionally elevated expression in mouse cerebellum

The mouse cerebellum is anatomically similar to the human cerebellum, with folded cortex and the three distinct layers of cells; granular layer, Purkinje layer and molecular layer, all containing different neuronal cell types. The transcriptome analysis shows that 58% (n=11380) of all mouse genes with one-to-one human orthologue (n=15160) are expressed in the cerebellum. 179 genes show an elevated expression in cerebellum compared to other regions of the brain.

Table 3: Number of genes within the different categories of regionally elevated expression, in mouse cerebellum

Specificity	Number of Mouse elevated genes
Region enriched	101
Group enriched	42
Region enhanced	36
Elevated	179

Figure 2.Schematic drawing of the mouse brain, indicating the location of cerebellum from a sagittal view.

Elevated expression in cerebellum compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in cerebellum compared to all other regions), group enriched (at least four-fold higher mRNA levels in a group of 2-5 regions) and regionally enhanced (at least four-fold higher mRNA levels in cerebellum compared to the average of all regions), The number of genes in the individual category is shown in Table 3. In Table 4, the 12 genes with the highest level of regional specificity among the 101 enriched genes are listed.

Table 4: The 12 genes with the highest level of enriched expression in mouse cerebellum. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
SYCP1	Synaptonemal complex protein 1	Intracellular	51
CRTAM	Cytotoxic and regulatory T cell molecule	Membrane	44
PCP2	Purkinje cell protein 2	Intracellular	43
GABRA6	Gamma-aminobutyric acid type A receptor alpha6 subunit	Intracellular,Membrane	40
CA8	Carbonic anhydrase 8	Intracellular	37
PKP3	Plakophilin 3	Intracellular	37
MYBPC3	Myosin binding protein C, cardiac	Intracellular	33
ARHGEF33	Rho guanine nucleotide exchange factor 33	Intracellular	32
BCL2L15	BCL2 like 15	Intracellular	32
PPP1R17	Protein phosphatase 1 regulatory subunit 17	Intracellular	32
HES3	Hes family bHLH transcription factor 3	Intracellular	30
CNPY1	Canopy FGF signaling regulator 1	Intracellular	29

One example of protein specifically localized to cerebellum is CBLN3 with a synaptic location most abundant in the molecular layer.

CBLN3

Regionally elevated expression in pig cerebellum

The pig cerebellum is anatomically similar to the human cerebellum, with folded cortex and the three distinct layers of cells; granular layer, Purkinje layer and molecular layer, all containing different neuronal cell types. The transcriptome analysis shows that 62% (n=12200) of all pig genes (n=14656) are expressed in the cerebellum. 142 genes show an elevated expression in cerebellum compared to other regions of the brain.

Table 5: Number of genes within the different categories of regionally elevated expression, in pig cerebellum

Specificity	Number of Pig elevated genes
Region enriched	91
Group enriched	29
Region enhanced	22
Elevated	142

Figure 3. Schematic drawing of the pig brain, indicating the location of cerebellum from a sagittal view. .

Elevated expression in cerebellum compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in cerebellum compared to all other regions), group enriched (at least four-fold higher mRNA levels in a group of 2-5 regions) and regionally enhanced (at least four-fold higher mRNA levels in cerebellum compared to the average of all regions), The number of genes in the individual category is shown in Table 5. In Table 6, the 12 genes with the highest level of regional specificity among the 91 enriched genes are listed.

Table 6: The 12 genes with the highest level of enriched expression in pig cerebellum. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
FAT2	FAT atypical cadherin 2	Membrane	66
CBLN3	Cerebellin 3 precursor	Intracellular,Secreted	47
GABRA6	Gamma-aminobutyric acid type A receptor alpha6 subunit	Intracellular,Membrane	44
IL16	Interleukin 16	Intracellular,Secreted	37
YIPF7	Yip1 domain family member 7	Intracellular,Membrane	29
COL27A1	Collagen type XXVII alpha 1 chain	Intracellular,Secreted	23
TGM6	Transglutaminase 6	Intracellular	22
BARHL2	BarH like homeobox 2	Intracellular	20
JUNB	JunB proto-oncogene, AP-1 transcription factor subunit	Intracellular	20
NEUROD1	Neuronal differentiation 1	Intracellular	20
AVPR2	Arginine vasopressin receptor 2	Membrane	18
PCP2	Purkinje cell protein 2	Intracellular	18

Extended human cerebellum data

Detailed annotation of the human cerebellum

A refined characterization of the protein location in the cerebellum of 911 genes was performed by taking 19 different cellular compartments into consideration in the annotation, unlike the standard annotation which includes cells of the three different layers (molecular, Purkinje and granular).

Large sections of human cerebellum

The standard setup in the Tissue Atlas, that profiles human tissues, is based on Tissue Micro array technique, saving valuable tissue material as well as reagents and still provide a good tissue representation for protein profiling. However, due to the complex nature of the brain, with different cell types and subfields, we occasionally include a larger tissue sample to better understand the protein location. In table 7, the selected targets used for profiling on extended tissue material is listed.

Table 7. The following 8 genes have been analyzed using extended cerebellum samples.

Gene	Gene description	Staining pattern
CAMKK2	Calcium/calmodulin dependent protein kinase kinase 2	Moderate cytoplasmic positivity in cells in dentate nucleus.
CPLX4	Complexin 4	Synaptic positivity in granular layer.
SMIM18	Small integral membrane protein 18	Mainly strong staining in a set of neurons and projections in cerebellum molecular layer.
SPTBN4	Spectrin beta, non-erythrocytic 4	Strong neuronal projection positivity close to Purkinje cell bodies. Neurites in molecular layer.
TRIM46	Tripartite motif containing 46	Strong cytoplasmic positivity in neuronal projections.
TTC21B	Tetratricopeptide repeat domain 21B	Stains neurons and synapses in Dentate nucleus.
VCAN	Versican	Strong membranous and cytoplasmic positivity in cells in dentate nucleus.
ZP2	Zona pellucida glycoprotein 2	Moderate cytoplasmic positivity in synapses.