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 72% (n=14479) 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 166 genes as cerebellum enriched in either of the three species, and 49 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 72% (n=14479) of all human proteins (n=20090) are expressed in the cerebellum, 14479 of all genes detected in the brain are also detected in cerebellum.

  • 17 regionally enriched genes
  • 233 regionally elevated genes in total
  • Shares the most group enriched genes with Pons & medulla
  • 103 of the cerebellum elevated genes are elevated in other tissues than the brain.
  • 130 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 17
Group enriched 56
Region enhanced 160
Elevated 233

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 17 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
P3R3URF-PIK3R3 P3R3URF-PIK3R3 readthrough Intracellular 6
C5orf46 Chromosome 5 open reading frame 46 Secreted 5
EOMES Eomesodermin Intracellular 5
NAA11 N-alpha-acetyltransferase 11, NatA catalytic subunit Intracellular 5
RIMS1 Regulating synaptic membrane exocytosis 1 Intracellular 5
ZP2 Zona pellucida glycoprotein 2 Membrane,Secreted 5
AC004593.2 Chimerin 2 Intracellular 4
CLVS2 Clavesin 2 Intracellular 4
FGF3 Fibroblast growth factor 3 Secreted 4
GALNT5 Polypeptide N-acetylgalactosaminyltransferase 5 Intracellular 4
H4C3 H4 clustered histone 3 Intracellular 4
H4C5 H4 clustered histone 5 Intracellular 4

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.