The hypothalamus proteome Microanatomy Regionally elevated expression in human hypothalamus Regionally elevated expression in mouse hypothalamus Regionally elevated expression in pig hypothalamus List of proteins stained on human hypothalamus tissue section

The hypothalamus proteome

The hypothalamus is a part of the diencephalon, together with the thalamus, epithalamus, subthalamus and metathalamus. The hypothalamus develops from the embryonic diencephalon and is located at the bottom of the dienchepalon. In humans, it is an almond-like small structure. The hypothalamus is the center of the vegetative nervous system as well as a key regulator of the endocrine system.

The transcriptome analysis shows that 82% (n=16165) of all human protein-coding genes (n=19670) are expressed in the human hypothalamus. Human one-to-one orthologues were investigated in pig and mouse brain, suggesting that 11844 of all mouse one-to-one orthologues (n=15160) are expressed in the mouse hypothalamus and that 12526 of all pig orthologues (n=14656) are expressed in the pig hypothalamus.

Gene classification based on regional RNA expression in human, pig and mouse hypothalamus defines 37 genes as hypothalamus enriched in either of the three species, and 18 out of those genes show highest expression levels in hypothalamus in all three species.

Figure 1. Schematic drawing of the human brain, indicating the location of hypothalamus below the thalamus, at the bottom of diencephalon from a sagittal view.

Anatomically, the hypothalamus can be divided to anterior, tuberal and posterior parts. The anterior part involves the preoptic nucleus (thermoregulation), the medial preoptic nucleus (releases GnRH hormone), supraoptic nucleus (releases oxytocin - OXT and vasopressin - AVP), the paraventricular nucleus (releases the thyrotropin releasing hormone - TRH, corticotropin releasing factor - CRH, oxytocin, vasopressin and somatostatin hormones), the anterior hypothalamic nucleus (involved in thermoregulation) and the suprachismatic nucleus (involved in the regulation of the circadian rhythms). The tuberal hypothalamus consists of a medial and a lateral part. The medial part involves the dorsomedial- (regulation of blood pressure and heart rate), ventromedial- (regulation of satiety) and arcuate nucleus (regulation of food intake, among others). The lateral part of the tuberal hypothalamus is a complex hub of neuronal networks; it contains, among others, the orexin and MCH neurons that regulate food intake and the sleep/wake cycle. The lateral hypothalamus partially extends also to the anterior and posterior hypothalamus. The posterior hypothalamus consists of the mammillary nuclei, the lateral nucleus (that involved in the regulation of blood pressure) and the tuberomamillary nucleus (that involved in the regulation of arousal, energy balance, learning-memory processes and sleep).

GNRH1
TRH
PMCH

Microanatomy

Histologically, the hypothalamus could be divided to magnocellular and microcellular neurosecretory neurons and to non-secretory neurons. The magnocellular neurons releases oxytocin and vasopressin in the neurohypophysis. The axons of these magnocellular neurons form the suproopticohyphophyseal tract. The microcellular neurons secretes the ‘releasing’ and ‘release inhibiting’ hormones to the portal veins that will transport these hormones to the adenohyphophysis. The axons of the microcellular neurons form the tuberoinfundibular tract. The non-secretory neurons are involved in the regulation of multiple and complex vegetative functions. The tuberomamillary and mammillary nuclei are important parts of the limbic-system. The hypothalamus neurons express several neuropeptides and several hypothalamic neuronal populations and sub-populations can be identified by their neuropeptide markers. (e.g. orexin- and MCH-expressing neurons in the lateral hypothalamus; NPY- and POMC-expressing neurons in the arcuate nucleus etc.)

GAL
GAL
NUCB2

Galanin (GAL) is a physiologically active neuropeptide that is implicated in nociception, feeding and energy homeostasis, osmotic regulation and water balance. Galanin immunoreactivity can be observed in the tuberal hypothalamus. Nucleobindin 2 (NUCB2) is a calcium-binding protein and has a role in eating regulation in the hypothalamus NUCB2 immunoreactivity is observed in the hypothalamic paraventricular nucleus.

Regionally elevated expression in human hypothalamus

The transcriptome analysis shows that 82% (n=16165) of all human proteins (n=19670) are expressed in the hypothalamus. 80 genes show an elevated expression level in hypothalamus compared to other regions of the brain.

19 regionally enriched genes
80 regionally elevated genes in total
39 of the hypothalamus elevated genes are elevated in other tissues than the brain.
41 of the hypothalamus elevated genes are elevated in the brain.

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

Specificity	Number of Human elevated genes
Region enriched	19
Group enriched	49
Region enhanced	12
Elevated	80

Elevated expression in hypothalamus compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in hypothalamus 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 hypothalamus 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 19 enriched genes are listed.

Table 2. The 12 genes with the highest level of enriched expression in human hypothalamus. "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
NPVF	Neuropeptide VF precursor	Secreted	95
OXT	Oxytocin/neurophysin I prepropeptide	Secreted	59
HDC	Histidine decarboxylase	Intracellular	52
PMCH	Pro-melanin concentrating hormone	Secreted	27
SIM1	Single-minded family bHLH transcription factor 1	Intracellular	21
TMEM114	Transmembrane protein 114	Membrane	19
AVP	Arginine vasopressin	Secreted	18
GHRH	Growth hormone releasing hormone	Secreted	15
AGRP	Agouti related neuropeptide	Secreted	13
ELSPBP1	Epididymal sperm binding protein 1	Intracellular,Secreted	11
GAL	Galanin and GMAP prepropeptide	Secreted	8
SIX6	SIX homeobox 6	Intracellular	8

HCRT
AVP
OTP

AGRP
HDC

The (HCRT) gene produces two neuropeptides, orexin A and orexin B, and are implicated in the regulation of sleep and might also play a role in feeding behavior and homeostasis. Vasopressin (AVP) is synthesized in the supraoptic nucleus and paraventricular nucleus of the hypothalamus and is transported to the nerve endings in the neurohypophysis. When secreted into the bloodstream, vasopressin acts as an antidiuretic hormone in the kidneys. OTP is a transcription factor involved in the development of the hypothalamus. The agouti related neuropeptide (AGRP) has a role in feeding control and weight homeostasis. The HDC protein controls several processes such as gastric acid secretion and smooth muscle contraction.

Regionally elevated expression in mouse hypothalamus

The mouse hypothalamus is anatomically similar to the human hypothalamus, with similar topography of nuclei and similar neurochemical character (e.g. neuropeptide-expression) of the homologous nuclei.

The transcriptome analysis shows that 60% (n=11844) of all mouse one-to-one human orthologue genes (n=15160) are expressed in the hypothalamus and 99 genes show an elevated expression in hypothalamus compared to other regions of the brain.

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

Specificity	Number of Mouse elevated genes
Region enriched	22
Group enriched	43
Region enhanced	34
Elevated	99

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

Elevated expression in hypothalamus compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in hypothalamus 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 hypothalamus 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 22 enriched genes are listed.

Table 4: The 12 genes with the highest level of enriched expression in mouse hypothalamus. "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 tissue.

Gene	Description	Predicted location	RS-score
NKX2-4	NK2 homeobox 4	Intracellular	22
NR5A1	Nuclear receptor subfamily 5 group A member 1	Intracellular	20
QRFP	Pyroglutamylated RFamide peptide	Secreted	18
NPVF	Neuropeptide VF precursor	Secreted	17
AGRP	Agouti related neuropeptide	Secreted	16
FOXB2	Forkhead box B2	Intracellular	16
AC092442.1		Intracellular	15
FEZF1	FEZ family zinc finger 1	Intracellular	11
AVP	Arginine vasopressin	Secreted	10
GHRH	Growth hormone releasing hormone	Secreted	9
OTP	Orthopedia homeobox	Intracellular	7
SIM1	Single-minded family bHLH transcription factor 1	Intracellular	7

HCRT
AVP
OTP

Orexin (HCRT) immunostaining in the lateral hypothalamus. Vasopressin (AVP) peptide immunostaining in the paraventricular hypothalamic nucleus. Orthopedia homeobox protein (OTP) localization in the hypothalamic paraventricular nucleus.

3D imaging of mouse brain

This video demonstrates the 3D distribution of orexin immunostaining (HCRT) in the mouse lateral hypothalamus (iDISCO volume immunostaining). A descriptive movie including more details and an interview with Dr. Csaba Adori, Karolinska Institute, related Orexin and Narcolepsy is found here.

Regionally elevated expression in pig hypothalamus

The pig hypothalamus is anatomically similar to the human hypothalamus, with similar topography of nuclei and similar neurochemical character (e.g. neuropeptide-expression) of the homologous nuclei.

The transcriptome analysis shows that 64% (n=12526) of all pig one-to-one human orthologue genes (n=14656) are expressed in the hypothalamus. 44 genes show an elevated expression in hypothalamus compared to other regions of the brain.

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

Specificity	Number of Pig elevated genes
Region enriched	12
Group enriched	26
Region enhanced	6
Elevated	44

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

Elevated expression in hypothalamus compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in hypothalamus 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 hypothalamus 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 12 enriched genes are listed.

Table 6: The 12 genes with the highest level of enriched expression in pig hypothalamus. "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
HCRT	Hypocretin neuropeptide precursor	Secreted	57
NKX2-4	NK2 homeobox 4	Intracellular	32
PMCH	Pro-melanin concentrating hormone	Secreted	30
SIX6	SIX homeobox 6	Intracellular	12
CGA	Glycoprotein hormones, alpha polypeptide	Secreted	11
FEZF1	FEZ family zinc finger 1	Intracellular	9
GPR50	G protein-coupled receptor 50	Membrane	9
BSX	Brain specific homeobox	Intracellular	8
UCN3	Urocortin 3	Secreted	7
NPVF	Neuropeptide VF precursor	Secreted	4
OPN5	Opsin 5	Membrane	4
POMC	Proopiomelanocortin	Secreted	4

List of proteins stained on human hypothalamus tissue section

The standard setup in the Tissue Atlas, which profiles the human tissues, is based on Tissue Micro array technique, thus saving valuable tissue material and reagents. The TMA technique provides a wide tissue representation for protein profiling. Cerebral cortex, caudate nucleus, hippocampus and cerebellum are the four brain regions in the standard human brain profiling. In-depth analysis of the elevated genes in hypothalamus and literature search for hypothalamus-associated proteins served as a strategy when selecting which proteins to analyze in human hypothalamus samples.

Table 7. The following 41 genes have been analyzed using extended hypothalamus samples.

Gene	Gene description	Staining pattern
ADCYAP1	Adenylate cyclase activating polypeptide 1	Synaptic staining.
AGRP	Agouti related neuropeptide	Strong staining of synapses and neuronal projections
ARHGAP36	Rho GTPase activating protein 36	Strong in neuronal cells, synapses and projections.
AVP	Arginine vasopressin	Strong cytoplasmic staining in magnocellular neurosecretory neurons.
CALCR	Calcitonin receptor	Strong membranous staining in neurons.
CALY	Calcyon neuron specific vesicular protein	Cytoplasmic staining in neurons.
CARTPT	CART prepropeptide	Neuronal projections and synaptic positivity.
CCK	Cholecystokinin	Strong neuronal projection and extracellular staining.
CNGA3	Cyclic nucleotide gated channel alpha 3	Stains in neural projections and neuronal cell bodies.
CRABP1	Cellular retinoic acid binding protein 1	Strong in a subset of hypothalamic neurons.
DDX25	DEAD-box helicase 25	Strong staining processes and in subsets of astrocyte-like cells.
ECEL1	Endothelin converting enzyme like 1	Strong in neurons.
FEZF1	FEZ family zinc finger 1	Distinct positivity in processes and neuropil of hypothalamus.
GAL	Galanin and GMAP prepropeptide	Strong cytoplasmic positivity in neuronal cells and projections.
GATA2	GATA binding protein 2	Nuclear staining in a few neurons.
GNRH1	Gonadotropin releasing hormone 1	Strong cytoplasmic staining in a subset of hypothalamic neuronal cells.
GRIN2D	Glutamate ionotropic receptor NMDA type subunit 2D	Membranous and cytoplasmic staining in neurons. Projections and synapses are also positive.
HCRT	Hypocretin neuropeptide precursor	Strong staining in a subset of hypothalamic neurons.
HCRTR1	Hypocretin receptor 1	Positive projections and synapses.
HDC	Histidine decarboxylase	Strong staining in neurons of hypothalamus.
HMX2	H6 family homeobox 2	Strong staining in neurons and projections/synapses.
HTR7	5-hydroxytryptamine receptor 7	Cytoplasmic in a subset of neurons. Distinct staining in neuronal projections.
KISS1R	KISS1 receptor	Distinct neuronal projections and staining of neuronal cells.
LUZP2	Leucine zipper protein 2	Strong staining in subsets neuronal and glial projections.
MC3R	Melanocortin 3 receptor	Cytoplasmic positivity in a subset of hypothalamic neurons.
NPVF	Neuropeptide VF precursor	Moderate positivity mainly in glial cells in hypothalamus.
OTP	Orthopedia homeobox	Strong nuclear staining in subsets of neurons.
OXT	Oxytocin/neurophysin I prepropeptide	Strong cytoplasmic staining in magnocellular neurosecretory neurons.
PABPC1L2A	Poly(A) binding protein cytoplasmic 1 like 2A	Strong staining in processes and nerve fibers in hypothalamus.
PABPC1L2B	Poly(A) binding protein cytoplasmic 1 like 2B	Strong staining in processes and nerve fibers in hypothalamus.
PCSK2	Proprotein convertase subtilisin/kexin type 2	Cytoplasmic staining in a subset of glial cells.
PMCH	Pro-melanin concentrating hormone	Distinct cytoplasmic positivity in subsets of hypothalamic neurons and neural processes.
PPP1R17	Protein phosphatase 1 regulatory subunit 17	Cytoplasmic staining in subset of neurons and cell projections in hypothalamus.
PTPRN	Protein tyrosine phosphatase, receptor type N	Cytoplasmic staining in neuronal cells. Synaptic positivity.
QRFPR	Pyroglutamylated RFamide peptide receptor	Synaptic positivity.
RTP5	Receptor transporter protein 5 (putative)	Cytoplasmic staining in neuronal cells.
SCG2	Secretogranin II	Granular cytoplasmic staining in neurons. Positive axons.
SST	Somatostatin	Strong secreted and axonal and synapse staining in hypothalamus.
TPPP3	Tubulin polymerization promoting protein family member 3	Staining in in subsets of glial cells and ependymal cells in ventricle walls.
TRH	Thyrotropin releasing hormone	Strong positivity in subsets of hypothalamic neuronal cells and processes.
VGF	VGF nerve growth factor inducible	Strong positivity was observed in neuronal cells and neuronal processes. Glial cells were negative.
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