Cerebellum Anatomical divisions Regionally elevated protein expression in human Regionally elevated protein expression in mouse Regionally elevated protein expression in pig Extended information

Cerebellum

Summary

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

The transcriptome analysis shows that 71% (n=14362) of all human protein-coding genes (n=20090) are expressed in the human cerebellum. Human one-to-one orthologues were investigated in pig and mouse brain, suggesting that 11817 of all mouse one-to-one orthologues (n=16320) are expressed in the mouse cerebellum and that 12560 of all pig orthologues (n=15829) are expressed in the pig cerebellum.

Gene classification based on regional RNA expression in human, pig and mouse cerebellum defines 251 genes as cerebellum enriched in either of the three species, and 64 out of those genes show highest expression levels in cerebellum in all three species.

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

Anatomical divisions

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 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 stretches 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 parallel 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 are clearly visualized by the protein detection of KIT.

MYH10
HOMER3
KIT

An example of a protein specifically located to the cells of the 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. An example of protein specifically detected in Bergman glia 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 dendritic processes of Purkinje cells.

QKI
KCNIP4
MX1

Regionally elevated protein expression in human

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

The transcriptome analysis shows that 71% (n=14362) of all human proteins (n=20090) are expressed in the cerebellum, 14362 of all genes detected in the brain are also detected in cerebellum.

129 regionally enriched genes
322 regionally elevated genes in total
Shares the most group enriched genes with Pons & medulla
162 of the cerebellum elevated genes are elevated in other tissues than the brain.
160 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	129
Group enriched	57
Region enhanced	136
Elevated	322

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 1. In Table 2, the 12 genes with the highest level of regional specificity among the 129 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
CCDC175	Coiled-coil domain containing 175	Intracellular	22
PRR35	Proline rich 35	Intracellular	20
PMIS2	PMIS2 transmembrane protein	Membrane	17
RHBG	Rh family B glycoprotein	Membrane	17
RIMS1	Regulating synaptic membrane exocytosis 1	Intracellular	16
CDH15	Cadherin 15	Membrane	15
EXPH5	Exophilin 5	Intracellular	15
ZP2	Zona pellucida glycoprotein 2	Membrane,Secreted	15
BARHL2	BarH like homeobox 2	Intracellular	14
GABRA6	Gamma-aminobutyric acid type A receptor subunit alpha6	Intracellular,Membrane	14
EFCAB9	EF-hand calcium binding domain 9	Intracellular	13
EOMES	Eomesodermin	Intracellular	13

Regionally elevated protein expression in mouse

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 59% (n=11817) of all mouse genes with one-to-one human orthologue (n=16320) are expressed in the cerebellum. 178 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	92
Group enriched	38
Region enhanced	48
Elevated	178

Figure 3.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 92 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	44
GABRA6	Gamma-aminobutyric acid type A receptor subunit alpha6	Intracellular,Membrane	40
CA8	Carbonic anhydrase 8	Intracellular	38
PKP3	Plakophilin 3	Intracellular	37
CPSF4L	Cleavage and polyadenylation specific factor 4 like	Intracellular	36
MYBPC3	Myosin binding protein C3	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
EMILIN3	Elastin microfibril interfacer 3	Secreted	30

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

CBLN3

Regionally elevated protein expression in pig

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 63% (n=12560) of all pig genes (n=15829) are expressed in the cerebellum. 155 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	77
Group enriched	24
Region enhanced	54
Elevated	155

Figure 4. 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 77 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	56
CBLN3	Cerebellin 3 precursor	Intracellular,Secreted	42
GABRA6	Gamma-aminobutyric acid type A receptor subunit alpha6	Intracellular,Membrane	37
PCP2	Purkinje cell protein 2	Intracellular	28
AVPR2	Arginine vasopressin receptor 2	Membrane	22
BARHL2	BarH like homeobox 2	Intracellular	20
COL27A1	Collagen type XXVII alpha 1 chain	Intracellular,Secreted	19
HENMT1	HEN methyltransferase 1	Intracellular	19
NEUROD1	Neuronal differentiation 1	Intracellular	19
CDH15	Cadherin 15	Membrane	17
GRIN2C	Glutamate ionotropic receptor NMDA type subunit 2C	Membrane	14
MAPK13	Mitogen-activated protein kinase 13	Intracellular	14

Extended information

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

Detailed annotation of the human cerebellum

A refined characterization of the protein location in the cerebellum of 903 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 providing 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.