The proteome of the Hippocampal formation Cell types and sub fields of the hippocampal formation Regionally elevated expression in human hippocampal formation Regionally elevated expression in mouse hippocampal formation Regionally elevated expression in pig hippocampal formation Extended human hippocampal formation tissue section

The proteome of the Hippocampal formation

The hippocampal formation is a highly homologous structure across all mammals and, in primates, is located inside the temporal lobe, alongside the amygdala, medial to the inferior horn of the lateral ventricle (shown in figure 1). The hippocampus is pivotal in recollection of facts and formation of new (including spatial) memories. In fact, long-term potentiation (modifications in synaptic strength as a result of neural activity) in certain parts of the hippocampus has been suggested as a storage mechanism for memory. Information flows into and through the hippocampus through the following principal pathways: 1- the perforant pathway from the entorhinal cortex to granule cells of the dentate gyrus; 2- the mossy fiber pathway from the granule cells of the dentate gyrus to the pyramidal cells of the CA3 region; 3- the Schaffer collateral pathway from the CA3 to the CA1 region.

The transcriptome analysis shows that 83% (n=16392) of all human protein-coding genes (n=19670) are expressed in the human hippocampal formation. Human one-to-one orthologues were investigated in pig and mouse brain, suggesting that 11851 of all mouse one-to-one orthologues (n=15160) are expressed in the mouse hippocampal formation and that 12540 of all pig orthologues (n=14656) are expressed in the pig hippocampal formation.

Figure 1. Schematic drawing of the human brain, indicating the location of hippocampal formation from a coronal perspective.

Gene classification based on regional RNA expression in human, pig and mouse hippocampal formation defines 35 genes as hippocampal formation enriched in either of the three species, and 2 out of those genes show highest expression levels in hippocampal formation in all three species.

Cell types and sub fields of the hippocampal formation

The hippocampal formation is an inward folded continuation of the cerebral cortex. This brain region includes the entorhinal cortex, subiculum, hippocampus proper (cornu ammonis, CA1-3 and dentate gyrus. The main cell types of the hippocampal formation are glutatmatergic pyramidal neurons located in the cortical areas and CA regions, granule cell located in the dentate gyrus and interneurons populating different subfields of this brain structure. Pyramidal cells in the CA regions are densely packed in a single layer. The dendrites of these cells receiving input from other regions of the hippocampal formation are located on both sides of the pyramidal cell layer in the oriens layer (superficial) and radiatum layer. The dentate gyrus granule cells are also packed in a single cell layer. These cells have their dendritic trees located in the molecular layer. The axons of these neurons are called mossy fibers and innervate the CA3 pyramidal neurons. The dentate gyrus is one of the brain regions where in the fully developed brain still new neurons are formed (neurogenesis). These newborn neurons are formed on the interface between the polymorph layer and granular layer. More than 20 different populations of interneurons have been identified based on their location, molecular signature (calcium binding proteins, neuropeptides) and electrophysiological properties.

Protein profiles of hippocampus include several interesting patters, both in human and mouse, either related to certain cell types or selective synaptic fields, below are a few examples.

CCK
NECAB1
CALB2

TMEM132A
GRIA1
APLP1

Regionally elevated expression in human hippocampal formation

The transcriptome analysis shows that 83% (n=16392) of all human proteins (n=19670) are expressed in the hippocampal formation. 82 genes show an elevated expression level in the hippocampal formation compared to other regions of the brain.

8 regionally enriched genes
82 regionally elevated genes in total
34 of the hippocampal formation elevated genes are elevated in other tissues than the brain.
48 of the hippocampal formation elevated genes are elevated in the brain.

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

Specificity	Number of Human elevated genes
Region enriched	8
Group enriched	63
Region enhanced	11
Elevated	82

Elevated expression in hippocampal formation compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in hippocampal formation 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 hippocampal formation compared to the average of all regions), The number of genes in the individual category is shown in Table 1. In Table 2, the 8 genes with high level of regional specificity are listed.

Table 2. The 8 genes with the highest level of enriched expression in human hippocampal formation. "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
NEUROG3	Neurogenin 3	Intracellular	20
FIBCD1	Fibrinogen C domain containing 1	Intracellular	11
DMRT3	Doublesex and mab-3 related transcription factor 3	Intracellular	7
STAP1	Signal transducing adaptor family member 1	Intracellular	7
ASIP	Agouti signaling protein	Secreted	6
FNDC1	Fibronectin type III domain containing 1	Intracellular,Secreted	6
CCBE1	Collagen and calcium binding EGF domains 1	Intracellular,Secreted	4
TULP1	Tubby like protein 1	Intracellular	4

Hippocampus share many regionally elevated genes with other forebrain regions, such as cerebral cortex, amygdala and basal ganglia. Both neuronal and glial proteins are found among the regionally elevated targets.

ICAM5
MT1A
NRGN

Regionally elevated expression in mouse hippocampal formation

Although the volume of the hippocampus is much smaller in mice than in humans, the basic hippocampal architecture is common to both species. However, substantial species differences exist, which argue against the frequent portrayal of hippocampus as a phylogenetically primitive brain region.

The transcriptome analysis shows that 60% (n=11851) of all mouse one-to-one ortologues genes (n=15160) are expressed in the hippocampal formation. 146 genes show an elevated expression in hippocampal formation compared to other regions of the brain.

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

Specificity	Number of Mouse elevated genes
Region enriched	17
Group enriched	118
Region enhanced	11
Elevated	146

Figure 2. Schematic drawing of the mouse brain, indicating the location of hippocampal formation from a coronal perspective.

Table 4: The 12 genes with the highest level of enriched expression in mouse hippocampal formation. "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 (Region Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
DSP	Desmoplakin	Intracellular	23
LCT	Lactase	Membrane	16
UBASH3A	Ubiquitin associated and SH3 domain containing A	Intracellular	13
SPINK8	Serine peptidase inhibitor, Kazal type 8 (putative)	Secreted	9
USP50	Ubiquitin specific peptidase 50	Intracellular	9
CRLF1	Cytokine receptor like factor 1	Intracellular,Secreted	7
OLFML2B	Olfactomedin like 2B	Intracellular,Secreted	6
ADAMTS14	ADAM metallopeptidase with thrombospondin type 1 motif 14	Secreted	5
FERMT1	Fermitin family member 1	Intracellular	5
KLK8	Kallikrein related peptidase 8	Intracellular,Secreted	5
TMEM171	Transmembrane protein 171	Membrane	5
CTXND1	Cortexin domain containing 1	Membrane	4

ITPKA
PCP4
ARFGEF1

Regionally elevated expression in pig hippocampal formation

The volume of the hippocampus lies between that of mice and humans; however, the basic hippocampal architecture is common to all 3 species.

The transcriptome analysis shows that 64% (n=12540) of all pig genes (n=14656) are expressed in the hippocampal formation. 98 genes show an elevated expression in hippocampal formation compared to other regions of the brain.

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

Specificity	Number of Pig elevated genes
Region enriched	11
Group enriched	82
Region enhanced	5
Elevated	98

Figure 3. Schematic drawing of the pig brain, indicating the location of hippocampal formation from a coronal perspective.

Elevated expression in hippocampal formation compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in hippocampal formation 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 hippocampal formation 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 11 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
NEUROG2	Neurogenin 2	Intracellular	18
TRIM10	Tripartite motif containing 10	Intracellular	18
MYO15A	Myosin XVA	Intracellular	15
AMHR2	Anti-Mullerian hormone receptor type 2	Intracellular,Membrane	10
SLC27A2	Solute carrier family 27 member 2	Membrane	6
MYLK3	Myosin light chain kinase 3	Membrane	5
NPY2R	Neuropeptide Y receptor Y2	Membrane	5
CCL1	C-C motif chemokine ligand 1	Secreted	4
FIBCD1	Fibrinogen C domain containing 1	Intracellular	4
KCNG2	Potassium voltage-gated channel modifier subfamily G member 2	Membrane	4
KRT27	Keratin 27	Intracellular	4

Extended human hippocampal formation tissue section

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

Table 7. The following 27 genes have been analyzed using extended hippocampal formation samples.

Gene	Gene description	Staining pattern
BHLHE22	Basic helix-loop-helix family member e22	Nuclear staining in a subset of neurons.
CHRNA5	Cholinergic receptor nicotinic alpha 5 subunit	Strong staining in astrocytes.
CREG2	Cellular repressor of E1A stimulated genes 2	Cytoplasmic positivity in synapses and neuronal projections.
DCC	DCC netrin 1 receptor	Strong projection positivity.
DCX	Doublecortin	Cytoplasmic positivity in neurons.
DNER	Delta/notch like EGF repeat containing	Positive in a few neurons.
FGF2	Fibroblast growth factor 2	Nuclear staining in glia.
GRIA3	Glutamate ionotropic receptor AMPA type subunit 3	Weak to moderate staining in neuropil.
GRIK4	Glutamate ionotropic receptor kainate type subunit 4	Strong projection and synapse staining.
GRIN1	Glutamate ionotropic receptor NMDA type subunit 1	Synaptic and cytoplasmic staining in neurons.
KCNG2	Potassium voltage-gated channel modifier subfamily G member 2	Distinct astrocyte end-feet staining in hippocampus.
MAP2	Microtubule associated protein 2	Strong cytoplasmic positivity in neuronal cells.
NEFL	Neurofilament light	Intense neuronal projection positivity.
NPTX1	Neuronal pentraxin 1	Strong cytoplasmic staining in neurons. Strong synaptic and neuronal projection staining.
NTRK2	Neurotrophic receptor tyrosine kinase 2	Strong cytoplasmic positivity in glial cells.
PDE1B	Phosphodiesterase 1B	Strong neuronal staining and moderate synaptic staining.
PRKCE	Protein kinase C epsilon	Synaptic positivity.
PRKCG	Protein kinase C gamma	Cytoplasmic positivity in neuronal cells.
PTPRN	Protein tyrosine phosphatase, receptor type N	Cytoplasmic staining in neuronal cells. Synaptic positivity.
PTPRZ1	Protein tyrosine phosphatase, receptor type Z1	Strong in neurons.
RTP5	Receptor transporter protein 5 (putative)	Cytoplasmic staining in neuronal cells.
RUNDC3A	RUN domain containing 3A	Strong extracellular staining in hippocampus.
SYNGAP1	Synaptic Ras GTPase activating protein 1	Synaptic and cytoplasmic positivity in neuronal cells.
TMEM255A	Transmembrane protein 255A	Synaptic staining.
TTR	Transthyretin	Moderate positivity in plasma in hippocampus.
VCAN	Versican	Moderate secreted positivity in extracellular space.
YJEFN3	YjeF N-terminal domain containing 3	Strong cytoplasmic staining in a rare subset of hippocampal neurons and moderate positivity in nerve fibers.
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