WO2004009842A2 - Methods for identifying genes related to malfunctions of the central nervous system - Google Patents

Abstract

The present invention relates to methods for identification of genes related to malfunctions of the central nervous system. In particular the present invention relates to identification of genes that are differentially expressed in hypothalamic subregions. Further aspects of the present invention relates to DNA sequences as well as recombinant polypeptides and uses thereof in prophylaxis, diagnosis and treatment of hypothalamic disorders.

Description

METHODS FOR IDENTIFYING GENES RELATED TO MALFUNCTIONS

OF THE CENTRAL NERVOUS SYSTEM

Technical field of the invention

The present invention concerns methods for identification of genes related to pathogenic conditions. In particular, the present invention relates to genes with a differential pattern of expression in specific areas of the brain. Such genes and/or their gene products potentially constitute novel drug targets for treatment of various human diseases as well as diagnostic targets.

Background of the invention

A number of human diseases are associated with alterations of gene expres- sion in confined areas of the brain.

In particular, a large number of somatic diseases are caused by hypothalamic dysfunctions or accompanied by altered hypothalamic function, which upon pharmacological correction will ameliorate symptoms or normalise the dysfunction. Comparative studies of the hypothalamus show that this region of the brain is anatomically, functionally, and physiologically very well conserved in vertebrates. Thus, several examples of dysfunctions originally observed in animal models have subsequently been shown to be analogous to similar human dysfunctions.

Well known examples of such dysfunctions in humans and animals are: hypothalamic hypogonadism and diabetes insipidus. Also, metabolic disorders such as obesity and accompanying diabetes mellitus and dyslipidaemia are frequently associated with abnormal function of hypothalamic neurons. Thus, several monogenetic diseases such as the metabolic syndromes associated with absent leptin synthesis (ob/ob mice), mutated leptin receptors (db/db mice, fa/fa rats), and the early onset obesity associated with melanocortin 4- receptor mutations are corrected by restoration of hypothalamic expression of the wild type gene. As a consequence, in most cases, data obtained using the hypothalamus from model animals excellently reflects human disorders involving dysfunctional hypothalamus and provides therapeutic targets for restoration of normal function.

As a central player of the limbic system, the hypothalamus is centrally placed as the overall conductor of such diverse functions as: reproduction and sexual behaviour, water and electrolyte homeostasis, energy homeostasis, blood glucose, emotions, mood, maternal behaviour, sleep and wakefulness, cir- cadian rhythms, memory, thermoregulation, blood pressure regulation, kid- ney function, endocrine system (thyroid, gonadal, adrenocortical, growth, mammary function, lactation), gastrointestinal function, and immune competence.

Drugs, compounds, gene therapies, and other therapeutic devices for amelio- rating, curing or modulating diseases with a hypothalamic component are suitable therapeutic tools for a number of diseases including:

Hypothermia, hyperthermia, obesity, dyslipidaemia, sarcopenia, anorexia nervosa, cancer cachexia, AIDS related wasting, bulimia nervosa, diabetes mellitus, hypoglycaemia, dehydration, polyuria, electrolyte disturbances (hy- ponatraemia, hypematraemia, hypokalaemia, hyperkalemia, hypocalcaemia, hypercalcaemia), diabetes insipidus, inappropriate syndrome of antidiuretic hormone (SIADH), autonomic dysfunction, arterial hypertension, arterial hypotension, (overhydration, water intoxication, sexual dysfunction, infertitility, precocious puberty, dysmenorrhea, oligomenorrhea, premature menopause, perimenopause and postmenopausal complications (including osteoporosis), hypogonadism, hyperprolactinaemia, galactorrhea, endogenous depression, stress, adrenocortical hyperfunction (Cushings disease), adrenocortical hypo- function (Addison disease), growth impairment, growth hormone insufficiency, growth hormone hypersecretion, hypothyroidism, hyperthyroidism, insomnia, Narcolepsy, somnolence, jet lag, Orcadian rhythm, control of mela- tonm mediated functions (tumour growth, reproductive function, jet lag, somnolence), inflammatory diseases, autoimmune inflammatory diseases, gas- troparesis, nausea, abdominal cramps, peptic ulcers, dyspepsia, diarrhoea, and obstipation

In mammals, energy homeostasis is regulated by neurons located in the hypothalamus The hypothalamus is organized as a collection of distinct autonomously active nuclei with discrete functions The hypothalamus governs several physiological variables regulated around an adjustable set point, including body composition and body temperature

The hypothalamus is a heterogeneously paired brain structure located below the thalamus on each side of the third ventricle The heterogeniety of the hypothalamus is well recognized, and is evident when microscopically examin- ing this structure in Nissl stained (Cresyl violet/Thionin) sections of the mammalian brain Based on Nissl stained material, groups or clusters of more or less densely packed neurons can be recognized More densely packed groups of neurons are classically termed "nuclei", whereas areas with more loosely packed neurons are termed "areas" or "zones" [Kπeg, 1932, Swanson, 1992] An example of a "nucleus" and an "area/zone" is given in Figure 1A

Figure 1A shows a Nissl stained (thionin) section through the rat hypothalamus corresponding to Plate 26 in the atlas by Swanson [Swanson, 1992] All nomenclature and abbreviations for hypothalamic and extrahypothalamic nuclei and areas used herein corresponds to the nomenclature used in the brain atlas by Swanson [Swanson, 1992]. Different nomenclatures are sometimes used in the literature in addition to the nomenclature suggested in Swanson.

In addition, the lateral hypothalamic area (LHA; Plate 22-33 [Swanson, 1992]) has been further subdivided according to Geeraedts and co-workers [Geeraedts et al., 1990a; Geeraedts et al., 1990b]. The hypothalamic paraventricular nucleus (PVH) is depicted in Figure 1A and from the figure (as well as from the Atlas) it can be seen that the PVH can be further sub- divided into so-called sub-nuclei; e.g. the dorsal parvicellular subnucleus (dpPVH), the posterior magnocellular subnucleus (pmlPVH) and the dorsal medial parvicellular subnucleus (mpdPVH).

On Figure 1A - below the PVH - the SBPV (=subparaventricular zone) is de- picted. This being an example of a "zone" (or "area" - that is a more loose collection of neurons). Hypothalamic "nuclei" and "areas" that are of importance in appetite and body-weight regulation include the following: the PVH, the hypothalamic arcuate nucleus (ARH; plate 26-30); the ventromedial hypothalamic nucleus (VMH, plate 26-30), the hypothalamic dorsomedial nucleus (DMH, plate 28-31 ), the lateral hypothalamic area (LHA, plate 22-33); the median eminence (Me, plate 26-30); the periventricular nucleus (PV, plate 19-31 ), the subparaventricular zone (SBPV).

Obesity is recognised as a disease, which requires medical attention and treatment. In humans, obesity is defined as a condition characterised by excess body fat mass relative to lean tissue mass, and this condition is generally reflected in a body mass index above 30 kg/m².

In addition to psychological problems caused by being severely obese, the condition is often characterised by accompanying metabolic disorders including insulin resistance and hypehnsulinaemia, which predispose to glucose intolerance. The medical condition characterized by visceral adiposity, insulin resistance, blood dyslipidaemia, and arterial hypertension is recognized as the metabolic syndrome and constitutes a severely elevated risk for development of type 2 diabetes and cardiovascular disease. Other secondary medical complications such as atherosclerosis, arterial hypertension, os- teoarthritis, certain cancers, as well as liver and gall bladder diseases sometimes accompany obesity.

Recently, laser capture microdissection has been introduced as a tool for the isolation of microscopically sized tissue samples. In most of the laser capture microdissection systems, a laser beam that is controlled by sophisticated software is used for circumcising the area of interest followed by isolation of the area. The isolation can be performed with non-contact methods where the isolated sample is catapulted or dropped from the slide into a centrifuge tube or cap, or with contact methods where the sample is fused with a membrane and isolated. The size of the area can be varied from single cells (for most systems) and up to several square millimiters containing thousands of cells.

Techniques for analysing gene expression include low throughput methods such as Northern blotting, multiplex PCR, real time PCR, and in situ hybridisation. More recently, various microarray based techniques have become available for high throughput analysis of gene expression. A common feature of these methods is that they only allow characterisation of genes that were previously described in the art.

Most of these methods for analysing gene expression require large amounts of RNA, a requirement that is almost impossible to fulfil when analysing the small amounts of tissue that can be obtained from distinct brain areas. Meth- ods generally employed to amplify cDNA comprise various polymerase chain reaction based (PCR) techniques. Another technique widely used for amplify- ing cDNA, T7-dhven linear amplification, has several drawbacks, the most important being that the gene fragments amplified are preferably derived only from the 3'-end of the mRNAs. T7 -driven amplification is based on the following principle: Isolated mRNA is reverse transcribed using a poly-dT primer with a T7 RNA polymerase promoter tag. Double-stranded cDNA is prepared and the tagged cDNA used as a template for transcription with T7 RNA polymerase. Another disadvantage of T7-driven amplification is that amplification of low amounts of RNA has to be performed in several rounds (e.g. [Ohyama et al., 2000; Scheidl et al., 2002]), making this procedure tedious and defective.

Genes differentially expressed in the brain of model animals under certain conditions have previously been described in a vast number of publications. Examples of methods used in such studies include in situ hybridisation, Northern blot, microarray based methods, etc.

A number of recent documents are addressing aspects of hypothalamus- specific genes:

Ziotopoulou et al. [Ziotopoulou et al., 2000] discloses differential expression of neuropeptides known to be implicated in weight regulation. These neu- ropeptides were examined in mice fed on different diets. RT-PCR analysis on RNA extracted from entire mice hypothalami were carried out in order to detect and quantify expression of NPY, AGRP, POMC, orexin, and SOCS-3.

Gautvik et al. [Gautvik et al., 1996] discloses identification of hypothalamus- specific transcripts in a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching for sequences expressed selectively in the hypothalamus. 94 clones from this library were selected for further analysis. Marathos-Flier et al., [Maratos-Flier et al,, 1997] discloses differential display analysis (DD-RTPCR) of hypothalamus tissue from normal and obese mice. In this study 10 entire hypothalami from each mouse type are excised and pooled in order to provide sufficient amounts of RNA for the analysis. In the introduction it is stressed that elucidation of the molecular mechanisms that contribute to hypothalamic dysfunction in obesity are difficult to carry out because hypothalamic tissue is scarce. It thus follows that is it very difficult to isolate differentially expressed genes from entire hypothalami.

Thus, it has so far not been possible and it has not even been attempted to specifically isolate anatomically or phenotypically distinct neurons in well- defined hypothalamic subregions. Isolation of entire hypothalami results in identification of genes that are differentially expressed in an area of the brain that is much larger than the desired target tissue or, in a worst-case scenario, that genes are erroneously concluded to be differentially expressed because the sampled area and/or the level of contamination from nearby areas may vary from sample to sample. This problem occurred in the example of CART (Cocaine and Amphetamine Regulated Transcript), which was originally identified as an transcript from striatum that was upregulated upon psychostimu- lation [Douglass et al., 1995]. It turned out however, that CART was most likely cloned because of an uneven dissection of samples [Vrang et al., 2002].

There is thus a need in the art for methods that enable identification of genes that are expressed in very confined areas of the hypothalamus, since many hypothalamic functions are governed by distinct autonomously active nuclei.

Summary of the invention

The present invention improves such approaches by making possible the identification of genes that are differentially expressed in well-defined groups of hypothalamic neurons under certain conditions. Such genes may or may not have been previously described. Such genes have most likely not previously been associated with hypothalamic malfunctions.

In the present invention, it is disclosed that genes differentially expressed in specific brain areas as a consequence of disease (or causing disease or propensity to develop disease) can be identified and/or cloned by combining brain sectioning and staining, laser capture microdissection, cDNA amplification by PCR, and subtraction cloning, and optionally microarray analysis. Such differentially expressed genes and/or their gene products represent potentially useful drug targets for use in treatment or prophylaxis of the particular hypothalamic malfunction.

Furthermore, the present invention demonstrates that genes expressed pref- erentially in distinct areas of the brain of model organisms can be identified and cloned. These genes exhibiting a spatially restricted expression represent good targets for specific modification of the function of the brain nucleus in which they are expressed and will thus constitute novel drug targets for drugs addressing the function(s) of distinct areas in the brain. These genes furthermore represent targets for diagnosis of conditions and diseases of plausible hypothalamic origin or with verified hypothalamic pathophysiology.

The present invention discloses methods for identification of genes involved in various hypothalamic diseases and/or malfunctions. These methods com- prise a combination of microdissection of tissue samples from brain cross sections, global reverse transcription and amplification by PCR, identification of differentially expressed cDNA by subtraction cloning, and optionally array analysis in order to identify genes that are differentially expressed in a hypothalamic subregion. Such genes and their gene products constitute potential drug targets and diagnostic targets. These drugs will ultimately lead to novel tools for treating obesity and accompanying metabolic disorders including type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, dyslipidaemia, and metabolic syndrome. Identification of differentially expressed genes likewise allows for the development of diagnostic tools that can be used for diagnosis as well as evaluation of propensity to develop cer- tain hypothalamus related diseases.

More specifically, the desired hypothalamic subregion (aggregate of functional equivalent neurons), which is also herein referred to as the first tissue sample, is isolated preferably using the laser capture technique (or other mi- croscope guided microdissection techniques). A second tissue sample is concurrently isolated using the same approach.

RNA is then extracted in parallel from the two isolated tissue samples and subsequently amplified using a global RT-PCR approach. Amplification by PCR is usually more efficient than T7-driven amplification. Although PCR amplification is not linear and may introduce misrepresentation of some cDNA species, this concern is of minor importance if samples to be compared are processed in parallel and thus, the representation of every single gene in the samples to be compared is skewed to the same degree. Subse- quently, subtraction cloning techniques are employed for identifying novel genes differentially expressed in the desired brain nuclei.

Differentially expressed genes may encode almost any gene product. Differentially expressed genes according to the present invention comprise: previ- ously unknown genes and splice variants as well as known genes not previously associated with a specific hypothalamic function. Differentially expressed genes that have been identified using the methods of the present invention also comprise genes that are specifically expressed in a subregion of the hypothalamus compared to other brain tissue types. Examples of such gene products include genes that constitute part of novel signalling pathways, novel parts of known pathways, enzymes, transcription factors, membrane proteins, organelle associated proteins, extracellular proteins, peptide hormones, etc. The products of these "novel" genes will be ob- vious targets for modification of the function of the specific brain nuclei in which they are expressed and are thus potential drug targets. Antisense nucleic acid molecules that are complementary to differentially expressed genes, or parts thereof, identified by the methods of the present invention can also be used for preparing medicaments for treatment of diseases char- acterised by hypothalamic pathophysiology. Other potential drug targets comprise regulatory sequences regulating expression of differentially expressed genes.

Description of the drawings

Figure 1 : Cross-sections of hypothalamus subregions from rats. Scale- bars^ 00 μm. A: Nissl-stained section illustrating the cytoarchitecture and the delineation of hypothalamic subregions. Dashed line shows the subdivisions of the PVH (=paraventricular nucleus of the hypothalamus). dpPVH=dorsal parvicellular subnucleus of the PVH; pmlPVH posterior mag- nocellular subnucleus; mpPVH medial parvicellular subnucleus of the PVH; SBPV=subparaventricular zone; 3V=third ventricle; AHN=anterior hypothalamic nucleus. B: Fluroscence microphotograph showing retrogradely labeled neurons in the PVH. Brightest neurons (whitest; thin arrows) are labelled with True Blue injected into the dorsal vagal complex; more faintly labelled neurons (fat arrows) contain the retrograde tracer Fluorogold (rats injected in- traperitoneally with Fluorogold). The two tracers are localized in different hypothalamic subregions. C: A hypothalamic subregion defined on the basis of neurotransmitter content. An antibody to oxytocin was used to label cells in the PVH containing this neuropeptide (dark neurons). D: A hypothalamic subregion defined on the basis of projection pattern. Cholera Toxin subunit B (ChB) was injected into the PVH and retrogradely labeled neurons in the DMH (neurons projecting to the PVN) are visualized using a peroxidase coupled ChB antibody and stained using diaminobenzidine as a chromogen.

Figure 2: Examples of laser microdissection of hypothalamic subregions defined on the basis of 12 μm thick Nissl stained brain sections. Scalebars=100 μm. A: Bilateral cutting of two PVH subregions - the posterior magnocellular subnucleus (pmlPVH) and the medial parvicellular subnucleus (mpPVH). The PVH boundaries are illustrated on the left side of the third ventricle (3V) by a dashed line. B: The right PVH from Figure 2A at a higher magnification. C: Two hypothalamic subregions cut from the dorsomedial hypothalamic nucleus (DMH) - the dorsal subnucleus (DMHd) and the compact subnucleus (DMHc).

Figure 3: Global amplification of RNA, cycle test for SMART cDNA synthesis. cDNA is amplified with a modified SMART kit and samples taken after 4, 6, 8, 10, 12, 14, 16, 18, and 20 cycles. These samples are run on a 1.2 % agarose gel. The optimal cycle number in this specific experiment is deduced to be 10 cycles of PCR where the visible products are sized approximately 0.3 to 10 kb. Top: number of cycles in second PCR. Left: marker sizes in kb.

Figure 4: Integrity check of cDNA. One μl of RNA purified from DMH was reverse-transcribed with Superscript (Invitrogen) and 35 cycles of PCR performed with various primers corresponding to the p619 (primer sets A to E), β-actin (primer set F), and GAPDH (primer set G) encoding cDNAs, respectively.

Figure 5: Reverse Northern blotting of clones derived from a DMH vs. CTX

(Cortex) screen. A. Radiolabeled cDNA amplified (SMART) from RNA iso- lated from Ctx (left) and DMH (right), respectively, was hybridised to a filter with immobilised cDNA (PCR product) from clones isolated in a DMH vs. Ctx cDNA subtraction screen. Position D12 contains cDNA from the TATA box binding protein (TBP) encoding cDNA and Position E12 contains cDNA from the β-actin cDNA. B. Scatter plot representation of signal Reverse Northern blotting using radiolabeled cDNA from DMH and CTX, respectively (left) and from CTX vs CTX, respectively (right). Background was subtracted from the filters and the signal from the beta-actin dot was used for normalization.

Figure 6: Flowchart visualizing the methods of the invention.

Figure 7: In situ hybridization using probe generated from clones in the DMH versus CTX screen. Slices from three different levels of Sprague-Dawley rat brains (PVN, DMH, and ARC) are used for in situ hybridization. The probes are generated by in vitro transcription of cloned cDNA from the DMH minus CTX subtraction experiment as indicated.

Figure 8: Regulation of necdin upon starvation of fatty Zucker (fa/fa) rats, fa/fa Zucker rats and wildtype rats (Fa/?) were freely fed or subjected to 48 hours fast (8 rats in each group). In situ hybridisation was performed and necdin expression in the arcuate nucleus quantified by densitometry of the exposed photographic film.

Detailed description of the invention

Diseases of hypothalamic origin or diseases characterised by hypothalamic pathophysiology have very diverse manifestations. The common theme of hypothalamic malfunctions is malfunction attributable to a pathophysiological condition in well defined neurones localised within a specific hypothalamic subregion. It is generally accepted that malfunctions in many, if not all, cases can be linked to an aberrant gene expression pattern. Genes that are differentially expressed and genes that are differentially expressed under certain conditions in a hypothalamic subregion and their gene products thus constitute desirable targets for treatment and prevention of various hypothalamic malfunctions. However, presently available methods have been unable to identify dysfunction associated with differentially expressed genes in specific subregions. Rather, the previously available methods have compared differential expression of genes in large heterogeneous regions covering several nuclei. There is therefore a need in the art for methods that enable identification of genes specifically related to hypothalamic malfunctions caused by dysfunction of specific neurons in clearly defined subregions, nuclei or subnuclei.

In particular, the present invention relates to a method of identifying differentially expressed nucleotide sequences in a tissue sample that corresponds to a hypothalamic subregion from a mammalian brain, said method comprising the following steps:

(i) isolating a subregion that is referred to as a first tissue sample, said sample being substantially free of cells from hypothalamic neighbouring tissues surrounding said hypothalamic subregion, (ii) isolating a second tissue sample,

(iii) isolating RNA from the first and the second tissue samples, (iv) synthesizing cDNA from the isolated RNA samples, (v) identifying cDNA sequences that are differentially expressed in the first tissue sample as compared with cDNA from the second tissue sample, and

(vi) optionally identifying a gene that corresponds to said cDNA sequence.

This method allows highly specific isolation of well-characterized groups of neurons or even single nerve cells. The present invention further relates to the following methods:

A method according to claim 1 , wherein at least one of the two tissue samples is taken from a DIO/DR rat. Said tissue samples are preferably isolated by laser capture microdissection.

A method wherein the brain tissue is stained prior to isolation of the first and/or the second tissue sample with a labelling agent selected from the group consisting of: immunostaining, expression of fluorescent proteins, Nissl staining (Cresyl violet, Thionin), haematoxylin-eosin, methylene blue, neutral red, any nuclear staining method, immunocytochemistry, endogenous expression of fluorescent proteins, application of neuronal tracers prior to sacrifice of the animal (such as cholera toxin B, horseradish peroxidase, fluorogold, true blue, fast blue, etc.). Staining allows identification of the hy- pothalamic subregion of interest.

A method that comprises a step wherein cDNA is amplified prior to identification of differentially expressed genes,

A method wherein differentially expressed genes are identified by differential display RT-PCR or cDNA subtraction. These methods allow identification of differentially expressed genes that were not necessarily previously associated with the hypothalamus or hypothalamic subregions.

A method wherein the differentially expressed genes are verified subsequent to their identification in order to confirm that the gene(s) in question is indeed differentially expressed. Methods for performing this verification include various PCR based methods, hybridization based methods, as well as micro array based methods. A method that comprises a step wherein the integrity of the isolated RNA or cDNA is verified prior to identification of differentially expressed genes.

A method that comprises a step wherein cDNA is amplified prior to identifica- tion of differentially expressed genes,

A method wherein cDNA amplification is carried out in a way that ensures preferential amplification of both the 3' and the 5' ends. When amplifying cDNA's, it has traditionally been difficult to obtain representation of the 5' ends. By using e.g. commercially available kits that have been designed to improve the representation of 5' ends, this problem can usually be circumvented.

A method where the mammalian brain is derived from a non-human animal, e.g. sheep, goat, rabbit, mouse, rat, horse, cow, dog, cat, primate, etc.

A method where a human counterpart gene to the differentially expressed gene is subsequently identified. Methods for identifying a human counterpart gene include various bioinformatics tools that the skilled man will be familiar with.

A method wherein brain tissue is stained prior to isolation of the first and/or the second tissue sample with a labelling agent selected from the group consisting of: immunostaining, expression of fluorescent proteins, Nissl staining (Cresyl violet, Thionin), haematoxylin-eosin, methylene blue, neutral red, any nuclear staining method, immunocytochemistry, endogenous expression of fluorescent proteins, application of neuronal tracers prior to sacrifice of the animal (such as cholera toxin B, horseradish peroxidase, fluorogold, true blue, fast blue, etc.). Staining allows identification of the hypothalamic subre- gion of interest. The present invention further relates to an isolated DNA sequence that is selected from the group consisting of SEQ ID NOs 1-11 , or a part thereof or an isolated DNA sequence that hybridises under stringent conditions with said sequence or is a degenerative of said sequence. DNA sequences ac- cording to the present invention may furthermore be used for designing a diagnostic kit for diagnosis of a hypothalamic condition. Said diagnostic kit may comprise e.g. specific primers, antibodies, probes, etc.

DNA sequences according to the present invention may be inserted into an expression vector. Said expression vector may be inserted into a host cell and said host cell may be cultivated in order to produce recombinant poly- peptides. The recombinant polypeptide may subsequently be used for designing drugs for treatment of hypothalamic diseases, and optionally for designing diagnostic kits for diagnosis of a hypothalamic condition.

The present invention further relates to antisense nucleic acid molecules that are complementary to DNA sequences according to the present invention. The antisense molecule may be in form as a DNA, PNA or LNA molecule. Antisense nucleic acids according to the present invention may be used for the preparation of a medicament for treatment of hypothalamic malfunctions.

The present invention also relates to methods of diagnosing a disease or malfunction of plausible hypothalamic origin or with known hypothalamic pathophysiology in an individual comprising determining the presence and/or amount or absense of a differentially expressed gene or gene product in a tissue or fluid sample from the individual, wherein said differentially expressed gene has been identified as being differentially expressed by any of the methods of the present invention. Methods of diagnosing a disease or malfunction of plausible hypothalamic origin or with known hypothalamic pathophysiology in an individual according to the present invention further comprise determining the presence and/or amount or absence of a marker in the upstream regulatory region of a differentially expressed gene, wherein said differentially expressed gene is identified by any of the methods according to the present invention.

Definitions:

Mammalian brain: According to the present invention, a mammalian brain may be derived from any mammal excluding humans. Examples of experimental animals from where the mammalian brain preferably may be derived include mice, rats, primates, rabbits, pigs, etc. The brain is situated at the anterior end of the spinal cord enclosed in the bony cranium and it comprises the forebrain, midbrain, and hindbrain.

Hypothalamic subregion: A hypothalamic subregion is herein defined as a microscopically identified group of nerve cells, or even a single nerve cell, located within the hypothalamus of a mammalian brain. The isolated hypothalamic subregion according to the invention is isolated in a manner that is so precise that only trace amounts (less than 20% by weight, preferably less than 5% by weight, more preferably less than 2% by weight and even more preferably less than 1 % by weight) of tissues from neighbouring areas are included in the isolated sample. A hypothalamic subregion typically comprises less than 10% of the entire hypothalamic volume, more preferably less than 5% and most preferably less than 3%. In addition to the neuronal cells, a hypothalamic subregion typically contains a number of non-neuronal cells such as glia cells and other cells.

Examples of a hypothalamic subregion according to the present invention include hypothalamic nuclei, hypothalamic subnuclei, single hypothalamus cells with a distinct appearance, hypothalamic areas, hypothalamic zones. As it is disclosed in the invention, a hypothalamic subregion according to the invention can only be identified and isolated in a sufficient precise manner using labelling methods combined with highly sophisticated microscope based equipment. A hypothalamic subregion can be identified in several different ways:

1 ) Identification based on appearence in a Nissl stain as a cytoarchitectoni- cally and morphologically distinct area.

2) Identification by using any nuclear staining method that is suitable for identifying hypothalamic subregions such as haematoxylin-eosin, methylene blue, neutral red.

3) Identification using fluorescent retrograde tracing methods to label groups of hypothalamic neurons projecting to one or more intra or extra hypothalamic sites injected with one or more retrograde tracers (e.g. True Blue, Fluorogold, Fast Blue, fluorescent microspheres; example in Figure 1 B).

4) Identification of cells or groups of cells containing e.g neurotransmitters; transporters; receptors; endogenous proteins, induced expression of a fluorescent protein or other cell specific protein (example in Figure 1c based on immunohistochemistry).

5) Identification of cells using immunohistochemical staining (using fluorescence or chromogen labeling methods) of retrogradely labeled neurons (using an antibody raised against any used retrograde neuronal tracer; e.g. the aforementioned as well as Cholera Toxin subunit B, Horseradish Peroxidase; example in Figure 1d). This is an example of how to identify a subregion on basis of function of the neurons rather than on basis on their morphology.

6) Identifying groups of neurons on basis of reporter driven gene expression. E.g. tissue-specific promoter driven expression of Green Flourescent protein or other fluorescent or otherwise microscopically recognizable proteins. Substantially free of cells from neighbouring tissues: This means that the tissue sample of interest (i.e. the first tissue sample) must correspond exactly to the tissue or even the single cell of interest. However, it is sometimes impossible to completely avoid inclusion of trace amounts of cells and tissues from areas surrounding the tissue sample of interest, i.e. neighbouring tissue areas. The amount of neighbouring tissue included in the sample of interest is therefore preferably less than 20%, more preferably less than 5%, even more preferably less than 2% and most preferably less than 1 % measured by weight of the tissue sample of interest.

First tissue sample: The first tissue sample is characterised as the tissue sample wherein differentially expressed genes of interest may be found by subtraction with a second tissue sample. A first tissue sample is taken from a cross section of a mammalian brain. A first tissue sample is an isolated hypo- thalamic subregion.

Second tissue sample: A second tissue sample is to be understood as the "reference tissue sample" in comparison with the first tissue sample. The second tissue sample can in principle comprise any brain tissue or single cell sample taken from within the same species or specimen from which the relevant hypothalamic subregion (first tissue sample) has been isolated, as long as the second tissue sample is not identical with the first tissue sample. Examples of second tissue samples include: cortex, thalamic nuclei, basal ganglia, hypothalamic subregions, mesencephalic, pontine and medullary nuclei, as well as cerebellar subregions. A second tissue sample can also be a sample taken from the same subregion as the first tissue sample but from another animal that has been treated differently (e.g. with pharmacological agents or fed on a different diet) or taken from a mammal with a different hypothalamus phenotype than the mammal from where the first tissue sample is taken. Tester and driver: Tester and driver samples correspond to cDNA from the first and second tissue samples respectively.

Isolating a hypothalamic subregion of interest: A preferred way of isolating a hypothalamic subregion of interest according to the present invention comprises the use of laser capture microdissection technologies to isolate tissue samples from brain cross sections. Other ways of isolating a microscopically identified tissue sample for use in the present invention include micromanipu- lated manual or automated dissection using principles different from the laser (e.g. Eppendorf MicroDissector where an extremely fine metal tip (Micro- Chisel) is oscillated at high levels of frequency and low amplitude and samples isolated by subsequent aspiration with a pipette).

Hybridising under stringent conditions: Hybridization under stringent condi- tions should in this context be understood as hybridisation under conditions that only allows closely related DNA sequences to hybridise. Hybridisation under stringent conditions may in the present invention be understood as hybridization under highly stringent conditions. Alternatively, hybridisation may also be carried out under moderately stringent conditions according to the present invention. Carrying out hybridization under highly stringent conditions will typically only allow specific hybridization between two sequences that have sequence homologies that are greater than 80% and preferably greater than 90%. Carrying out hybridization under moderately stringent conditions will typically allow specific hybridization between two sequences that have sequence homologies that are greater than 70%. How to carry out hybridization under moderate and highly stringent conditions has been described in greater detail in e.g. Sambrook et al. (2001 ) Molecular Cloning, CSHL PRESS.

Fragment: A fragment of a DNA sequence according to the present invention can be any fragment with a length of at least 30 bases. A fragment of an amino acid sequence according to the present invention can be any fragment with a length of at least 10 amino acids.

cDNA synthesis: cDNA molecules are preferably synthesized using methods that allow for subsequent amplification of the full-length cDNA molecules. Examples of kits and methods suitable for cDNA synthesis according to the present invention include but are not limited to: Capfinder and SMART II kits from Clontech, and FirstChoice™ RLM-RACE kit (with modifications) from Ambion.

DNA amplification: DNA molecules can be amplified by methods employing polymerase chain reaction (PCR) based techniques. PCR based techniques are described in greater detail in e.g. Sambrook et al. (2001 ) Molecular Cloning, CSHL PRESS. cDNA molecules are preferably amplified prior to subtrac- tion. Preferred methods or preferred kits for amplifying cDNA include but are not limited to: Capfinder and SMART II kits from Promega, Expand High Fidelity PCR System from Roche, any other PCR based kit employing proofreading thermostable DNA polymerases or mixtures of proofreading and non- proofreading polymerases.

Verify differentially expressed genes: Verification of differentially expressed genes according to the present invention can be carried out using a number of methods known in the art. Examples of such methods include but are not limited to: Northern blot, reverse Northern blot, real-time PCR, RT-PCR, and various microarray based methods, and Western blot/immunoblot based methods. Verification may take place by amplification using gene specific primers, hybridization (in situ/in vitro) using gene specific probes, immunoblot based methods using specific antibodies, or other methods available to the man skilled in the art (e.g. Sambrook et al. (2001 ) Molecular Cloning, CSHL PRESS). Expression vector: An expression vector according to the present invention can be any vector that has the ability to transcribe an inserted sequence into mRNA by means of either a constitutive or an inducible promoter. The transcribed mRNA may or may not be fused to an mRNA sequence enabling translation and it may or may not be fused to an mRNA sequence encoding a "tag sequence" that eases subsequent purification. Examples of expression vectors include but are not limited to pT7-7, pSKF101 , pSKF301 , pUR, pATH, pMAL-c2, pMAL-p2, pGEX1 , pGEX2T, pGEX3X, pESP-1 , pESP-2, pESP3, and CDM8.

Host cell: A host cell according to the present invention may be any cell that is transformable by an expression vector comprising an isolated DNA sequence according to the present invention and is able to express the encoded recombinant gene product. Examples of suitable host cells include, mammalian cells (such as CHO, COS, HeLa, C33A, Caski, HaCat, etc. cells), insect cells, yeast cells, bacteria (e.g. E. coli cells, Bifidobacterium Sp. cells, etc.)

The expression "antisense nucleic acid molecule" means any nucleic acid molecule in the form of a DNA, a RNA, a PNA ("peptide nucleic acid"), a LNA ("locked nucleic acid") or a phosphorothioate or derivatives, analogs or fragments thereof, including double-stranded siRNA (small interfering RNA) molecules, capable of down-regulating the expression of a particular protein encoded by a nucleic acid complementary of the antisense nucleic acid. The size of an antisense nucleic acid molecule may range from 15 bases to encompassing the entire coding sequence of the gene of interest.

The terms: "medicament and "drug" are used interchangably throughout the present invention. These terms cover any substance that posses a biological activity and may function as a therapeutical or profylactic compound in humans or animals, said compounds may furthermore comprise pharmaceuti- cally acceptable excipients that are selected in accordance with conventional pharmaceutical practise.

Diagnosis, according to the present invention, of a disease or malfunction of plausible or hypothalamic origin or with known hypothalamic pathophysiology might be carried out in several ways. Differential gene expression may e.g. be caused by mutations or other alterations in the DNA sequences that regulate transcription (e.g. promoters and/or enhancers) or it may be caused by mutations within the gene that cause the gene to be differentially expressed or alternatively the gene may be deleted, duplicated or in other ways altered. In such cases, diagnosis can be performed on basis of a DNA sample from an individual either by detecting and/or sequencing a marker or by cloning and sequencing a larger fragment of the genomic DNA.

The size of the marker or the length of the DNA strand that has to be identified may vary highly. In case of SNP polymorphism, as little as one specific nucleotide may be determined in order to diagnose specifically. In cases of larger genomic alterations, hundreds or maybe even thousands of bases may have to be determined. The diagnostic assay may also be designed on basis of detecting presence/absence/size/melting/temperature, etc. of PCR- fragments. However, in other cases, where there is either no genetic basis for the differential expression or the genetic basis for the altered expression is unknown, the gene products can be detected by either detecting the mRNA or protein products of the gene. This can be done by e.g. a sample of the cerebrospinal fluid or ultimately a brain biopsy.

Diagnosis can be performed by means of e.g. SNP-detection (single nucleotide polymorphism), or other PCR based methods by detection of the presence and/or amount, absence, and/or size of a PCR product. The PCR prod- uct may or may not be sequenced in order to perform the diagnosis. Diagnosis may furthermore be carried out on the level of DNA or mRNA using meth- ods based on hybridization, e.g. in situ hybridization, Southern blot, Northern blot, microarray based methods, etc. Finally, diagnosis may be performed by detection on the level of proteins using e.g. antibody based detection methods (Western blot, ELISA, protein microarray, etc.), assays detecting specific enzyme activities, radiolabelling based methods, etc.

The methods of the present invention are unique since they provide highly sophisticated ways of specifically isolating a hypothalamic subregion of interest substantially without including cells from neighbouring areas and regions in the isolated sample and thereby enabling identification of differentially expressed genes from this subregion that were not previously known to be associated with a specific hypothalamic disease or malfunction.

This is preferably accomplished through laser capture microdissection of brain sections in which a hypothalamic subregion is captured. RNA is then extracted from the isolated brain nucleus. The RNA is preferably amplified up to approximately 1 million-fold using e.g. global PCR amplification of full- length cDNA by PCR [Chenchik et al., 1998; Matz et al., 1999; Zhu et al., 2001 ; Zhumabayeva et al., 2001]. Preferably, cDNA is amplified by methods that ensure that both the 3' and the 5' ends of most of the cDNA species are amplified. Alternatively, amplification can be accomplished by the method of T7 based RNA amplification, a method that usually leads to preferential isolation of the 3'-end of genes.

In search for drug target genes, techniques for differential cloning and/or identification of genes are employed to isolate genes specifically expressed in the brain nucleus of interest. Various techniques such as RASH [Jiang et al., 2000], RDA [Hubank and Schatz, 1994], differential display PCR [Liang and Pardee, 1992] and, microarrays with oligonucleotides [Lockhart et al., 1996] or cDNAs [Schena et al., 1995] may be used to identify and/or validate genes differentially expressed. In order to initially identify genes that were not necessarily associated with a specific hypothalamic disease or condition, cDNA subtraction methods are used according to the present invention. Microarray based methods are preferably used subsequently in order to assay for the differential expression of already known genes or expressed se- quence tags (ESTs).

Differentially expressed genes may furthermore be used as diagnostic targets. It is possible to diagnose on basis of a DNA-containing sample in cases where the differential expression is caused by e.g. a mutated promoter and/or enhancer, or mutation(s) within the gene that cause the gene to be differentially expressed. Sources of genomic DNA include: urine, blood, sweat, saliva, tears, semen, bronchoalveolar lavage fluid, sputum, stick scra- bes (e.g. buccal swabs), hair, nails, dandruff, tissue samples, or other body fluid or tissues obtained from an individual. In other cases diagnosis where the RNA and/or protein corresponding to a specific gene is examined may be performed on basis of a sample of cerebrospinal fluid or ultimately a brain biopsy.

Identification of differentially expressed genes in the brain opens for new di- agnostic and therapeutic avenues. Differentially expressed genes are identified from first and second tissue samples. The two tissue samples can be different subregions from the same animal or identical subregions from phe- notypically different subjects of the same species. Both approaches to isolate and characterise differentially expressed genes are exemplified in the pre- sent text.

Using animal models to study a human disease constitutes a useful tool to learn about pathophysiology of the disease and test treatments before attempting human clinical trials. Several animal models of both monogenetic and polygenetic disorders mimicking human disease could be used as models of phenotypically different subjects: the spontaneous hypertensive (SHY) rat; the nonobese diabetic (NOD) mouse; the fatty (fa/fa) Zucker rat; the obese (ob/ob) mice; the lethal yellow (A^y) Agouti mouse; the obese Otsuka Tokushima Long Evans fatty (OTLEF) rat; Watanabe heritable hyperlipidae- mic rabbit.

Also, environmentally induced animal models of human disease could be employed: diet induced obesity, allergic autoimmune encephalitis, mycobac- terium cell wall induced arthritis; lesion induced osteoarthrosis; tumour induced cachexia; malnutrition induced musculoskeletal disorders etc. With the advent of genetically modified animals characterized by universal or tissue specific over-expression or lack of expression of single or multiple genes, it has become clear that the number of animal models mimicking specific human disease syndromes or single pathophysiological defects is virtually unlimited.

Using isolated RNA from both unaffected and affected animals, the present invention can be used to identify differentially expressed genes in specific cells of the central nervous system. The affected animals comprise the phenotype mimicking human disease whilst unaffected animals represent the phenotype of non-diseased humans. Upon sampling of differentially expressed genes in animal models of human disease, the identity of isolated cDNAs are characterised in genome databases and their pathophysiological relevance assessed using both bioinformatic tools, molecular approaches as well as common clinical judgement.

Once a novel gene is identified, the cDNA is full length cloned and the nature and function of the gene product assessed in a cellular expression system. The clinical and pharmacological relevance of identified differentially expressed genes are assessed in several ways. Firstly, using the humane genome database and available information about single nucleotide polymorphisms (SNPs) the possible linkage of certain point mutations in the identified genes and aggregation of human disease among bearers is investigated. As example, more than 50 alleles of the human melanocortin-4 receptor (MC-4R) exist, some of which are phenotypically related with early onset obesity whereas others have no overt phenotype. From both genetically modified mice having a life without the MC-4R, and from mice having the MC-4R function constantly antagonized by ectopically expressed Agouti protein (A^y mice), it is known that intact hypothalamic MC- 4R function is required for regulation of normal body weight, confirming that loss of function mutations of the human MC4-R yields an obese phenotype.

Secondly, the pathophysiological relevance of differentially expressed gene products is studied in human sufferers of specific diseases. Because patho- physiologically relevant gene products represent practically all types of pep- tides and proteins and direct or indirect products hereof, a broad spectrum of diagnostic tools should be employed to assess altered levels of the gene products. Thus, sputum, urine, stools, blood, plasma, cerebrospinal fluid as well as specific tissue biopsies constitute possible relevant samples from dis- ease-affected humans. In such samples, binding of specific tracers, presence of hormones, transmitters, transport proteins, enzymes, precursors and metabolites of enzymatically driven reactions, trace elements, transcription factors including co-repressors and co-activators can be studied to determine their validity as measures of disease (presence, severity, therapeutic re- sponse etc). Therefore, differentially expressed genes constitute both possible future drug targets as well as diagnostic markers of diseases useful for classification of diseases and their sensitivity to specific treatment modalities.

Validation of possible drug targets amenable for development of new chemi- cal compounds treating specific diseases involves several steps. The physiological relevance of differentially expressed gene products are assessed in permanently or transiently genetically modified animals constructed to over- express or eliminate expression of the gene product in question (site directed viral gene transfer or transgenic animals). In transgenic animals, altered gene expression might be universal (all somatic cells), site specific (driven by tis- sue dependent promoters), or time dependent (dependent on presence or absence of certain environmental cues/chemicals). Given that these studies further substantiate the pathophysiological relevance of the gene product, a range of molecular pharmacological experiments are initiated to ensure development of pharmacological tools capable of interfering with normal func- tions of the identified gene product.

Examples

To demonstrate the present invention, a number of novel genes preferentially expressed in the hypothalamic subregion consisting of the compact part of DMH (DMHc) were cloned and characterized. The DMH is known to be involved in the regulation of body weight in mammals and is a good target for the demonstration of this technique because a number of genes are known to be expressed specifically in this hypothalamic nucleus and thus, the success of this experiment can be readily assessed. Genes previously described to be differentially expressed in this subregion were identified based on "trial and error" experiments where specific genes suspected to be differentially expressed were confirmed or denied as being differentially expressed using techniques such as in situ hybridization, real-time PCR, and Northern blot- ting.

We refer to the flowchart in Fig. 6 for visualization of the methods used in the examples. The methods can briefly be described as follows:

The first tissue sample was DMHc and the second tissue sample was a cerebral cortex subregion. Both samples were excised and isolated from Nissl stained 12 micrometer thick frontal brain sections of normal Sprague- Dawley rats.

RNA was extracted from each area and amplified with a modified SMART kit (Clontech) protocol (figure 3). The presence of full-length cDNA was controlled by assaying for various fragments of the house keeping marker gene p619 (Sigma RNA inspector kit) before and/or after amplification (figure 4). Even the fragments situated in the 5'-most end of p619 were amplified efficiently using the present method.

It is thus demonstrated that cDNAs sized up to at least 14 kb can be generated from laser dissected brain nuclei. The cDNA is efficiently amplified from the minute amounts of RNA present in such samples.

One microgram of amplified cDNA from each area was used for cDNA subtraction [Jiang et al., 2000] using the cDNA from DMHc as the tester and the cDNA from the cerebral cortex subregion (CTX) as the driver. This method leads to the insertion of differentially expressed genes in a plasmid vector of choice and a number of clones encoding differentially expressed genes were thus isolated.

The clones isolated were subsequently subjected to Reverse Northern blotting. The inserts from approximately 90 clones were amplified by PCR and spotted on a nylon membrane. SMART amplified cDNA from DMHc and CTX, respectively, were labelled with ³²P and hybridised to the insert DNA on the membrane in two separate experiments (Figure 5). Compared with the β- actin standard, most of the clones obtained are labelled more with the amplified cDNA from DMHc than with the amplified cDNA from CTX, indicating that DMHc specific clones were indeed selectively captured by the present method. Other methods of confirming a differential pattern of expression include microarrays (preferred), so-called virtual Northern blotting where ampli- fied cDNA is run on a gel and tested with gene-specific probes, and real-time PCR.

Sequencing of 50 clones clearly demonstrated the specificity and efficiency of the methods of the present invention since a fraction of the 50 clones contains fragments of genes that were previously reported to be preferentially expressed in the DMHc. Examples of such "known" genes identified using the methods of the present invention are the galanin gene (found as an insert in 4 out of the 50 clones), and the angiotensinogen gene (found as an insert in 3 out of 50 clones). Furthermore, a number of "novel" genes that were not previously associated with hypothalamus were cloned as well. No expressed sequence tags (ESTs) or sequences encoding these genes were previously reported from rat and, perhaps more importantly, no homologous genes are present in public databases with mouse and human EST and annotated gene sequences. This clearly illustrates the potential of the present invention for the specific identification of novel genes with specific expression within a hypothalamic subregion.

The expression patterns of several novel genes isolated by cDNA subtraction between DMHc and CTX as described above were further examined by in situ hybridisation (figure 7). These genes were confirmed to be expressed in the DMH and, for some genes also PVN and ARC, whereas no or at least weaker expression was detected in CTX. Importantly, the level of expression of some of these genes is quite low, also in their confined areas of expres- sion, demonstrating the ability of this invention for the identification/cloning of sparsely expressed genes. Thus, the present invention provides a method for the isolation/identification of genes with expression confined to one or more hypothalamic subregions.

Furthermore, the present invention provides methods for the identification of genes involved in disease by the harvesting of identical hypothalamic subre- gions from two animals, one of these being a model animal for a disease. Subsequent amplification of cDNA and comparison of gene expression in the amplified cDNA from these hypothalamic subregions by subtraction or RNA profiling methods will yield a number of expressed gene tags that are obvious drug targets.

Example 1

Laser dissection microscopy

SuperFrost Plus slides (Menzel, Germany) were coated with pieces of Pen- Foil (PALM, Microlaser Technologies, Germany). Twelve micron sections of rat brain were cut on a cryostat and mounted on the coated slides. The slides were frozen at -20 degrees Celsius, transferred to -20 degrees ethanol (70% W/v) and fixed for 5 minutes. The slides were then frozen at -80 degrees Celsius, and counterstained in Thionein (on ice) using the following approximate time schedule: 70 % EtOH, 1 min.; 50 % EtOH, 1 min.; 0.1 % Thionein in 200 mM sodium acetate pH 4.0, 3.5 min.; 50% EtOH, 1 min.; 70% EtOH, 1 min.; 96% EtOH, 1 min.

The sections were air dried briefly and rapidly frozen at -80 °C. On the day of Laser Capture Microdissection, the slides were thawed and the PenFoil membrane gently released from the slide, turned up side down and re-glued with nail polish to a thin glass cover slip (0.17 mm thick).

The slides were microdissected on a P.A.L.M. laser microdissection system (PALM, Microlaser Technologies, Germany) where the central compact sub- nucleus of the DMH (DMHc, first tissue sample) and a cerebral cortex subregion (CTX, second tissue sample) were identified. The DMHc was identified as a densely packed group of neurons in Nissl stained sections (see e.g. Figure 1 and 2). The cortical sample was taken from the piriform cortex at the same rostrocaudal level as the DMHc sample. Samples were dissected with the dissection microscope using a computer assisted or manual microma- nipulator and the samples were transferred to an eppendorf tube using either manual or automated systems (laser blast, gravity or adherent films). Unilat- eral or bilateral samples of the specified hypothalamic subregions were gathered in sample tubes, one for each region, and kept at -80 C until further use.

Example 2

Isolation of RNA from laser dissected sections

200 μl TRI reagent (MRCgene, TR 118, U.S. patent 5,346,994) and 0.5 μl polyacryl carrier (MRCgene, PC 152) were added to each eppendorf tube containing the laser dissected material. The samples were then homogenized by vortexing for 1 min. Insoluble material was removed from the homoge- nates by centπfugation at 12,000 g for 10 minutes at 4 °C. The cleared su- pematants were transferred to fresh tubes and stored at room temperature (R.T.) for 5 min. Twenty μl of BCP (MRCgene, BP 151 ) was added to each tube and the mixtures were vortexed for 15 sees. The phases were separated by centrifugation (12,000 g for 15 min at 4 degrees C), the aqueous phases transferred to fresh tubes and precipitated with 100 μl isopropanol each.

The samples were stored at room temperature for 5-10 min and centrifuged at least 12,000 g for 8 min at 4-25 °C. The supernatants were removed and the pellet washed with 0.5 ml 75% ethanol, air dried, and dissolved in 4 μl RNase free water. For each sample, one microliter of the isolated RNA was used for fluorescence-based concentration measurement. (Ribogreen kit, Molecular Probes, R-11490).

Example 3

Assessment of RNA/cDNA quality

The quality of the cDNA synthesis and amplification was monitored by PCR as described in the RNA/cDNA Inspector Kit manual (Sigma, INSP-1 ), but the kit was modified in order to use primers specific for rat genes only. The primary feature of this procedure is the ability to test for the presence of intact cDNA sized up to 14 kb by the amplification of small fragments in the 15 kb long p619 cDNA using the following primers:

p619: Set A, pos. 12984 to 13892, SEQ ID NO 12: GGC AGT TGG AGC TGA ACA CA and SEQ ID NO 13: TGG AGG TCC AGA GGC TTC TT.

Set B, pos. 8290 to 8998, SEQ ID NO 14: GCT AAC CGC ACA GCC TTG TC and SEQ ID NO 15: GCC AGG TAG GCC AAT CCA GT.

Set C, pos. 5194 to 5802, SEQ ID NO 16: AGT GGC CGA TTG CAT CAC TA and SEQ ID NO 17: GAG TTC GGC TGC ATG TTG TT.

Set D, pos. 2280 to 2676, SEQ ID NO 18: ACA TAG TCT GGC ATG GAC TG and SEQ ID NO 19: CCG TTC TCG TAA TGG AGG TA.

Set E, pos. 1029 to 1329, SEQ ID NO 20: TTC ATC TGC TGA TCG GAG TC and SEQ ID NO 21 : TCC AGC TTC AAT GGT CTG TG. Beta-actin: Set F, pos 493 to 945, SEQ ID NO 22: TTG TGA TGG ACT CCG GAG AC and SEQ ID NO 23: CAC CAG ACA GCA CTG TGT TG.

GAPDH: Set G, pos 503 to 856, SEQ ID NO 24: TGC ATC CTG CAC CAC CAA CT and SEQ ID NO 25: CGC CTG CTT CAC CAC CTT C.

RNA isolated from DMHc and the CTX was reverse transcribed and subjected to PCR with the primers described above. The presence of PCR- products in all the lanes (figure 4) indicates that undegraded RNA was iso- lated from the micodissected hypothalamic subregions. . Importantly, similar results were obtained using the globally amplified cDNA as a template (data not shown), indicating that the SMART cDNA synthesis and amplification was successful.

Example 4

Synthesis and amplification of cDNA

cDNA was synthesized and amplified by using a modified SMART kit (Clon- tech PT3041 -1 ) as outlined below:

First strand cDNA synthesis

Three μl RNA sample were mixed with 1 μl cDNA synthesis (CDS) primer (10 pmol/μl) and 1 μl SMART II oligonucleotide (10 pmol/μl). The mixture was incubated at 70 °C for 2 min. The following was thereafter added to each tube: 2 μl 5X First-Strand Buffer (250 mM Tris-HCL pH 8.3, 375 mM KCI, 30 mM MgCI₂), 1 μl DTT (20 mM), 1 μl dNTP (10 mM), 0.5 μl RNAsin (Promega N2515, 20-40 u/μl), and 0.5 μl Powerscript reverse transcriptase. The tubes were incubated at 42 °C for 1 hr in an air incubator and placed on ice. Amplification of cDNA

The cDNA was amplified using the Expand High fidelity PCR system (Roche 1 732 641 ): To each tube was added: 35.4 μl H₂O, 1.6 μl 12.5 mM dNTP, and 3 μl cDNA-PCR primer. A cycle premix was made of: 34.9 μl H₂0, 10 μl 10X Expand high fidelity PCR buffer without MgCI₂, 3.6 μl 25 mM MgCI₂, 1 5 μl Enzyme mix. The cycle premix was added to the cDNA and the tube subjected to cycling following the program described below: 95 degrees 1 min. (1 cycle), 94 degrees 15 seconds, 65 degrees 30 seconds, 68 degrees 10 minutes (12 cycles). A second amplification was made by preparing mixtures A and B on ice. A: 35.4 μl H₂0, 1.6 μl 12.5 mM dNTP, 10 μl of the cDNA from the first amplification, 3 μl cDNA-PCR primer. B: 33.1 μl H₂0, 10 μl 10X Expand high fidelity PCR buffer without MgCI₂, 5.4 μl 25 mM MgCI₂, 1.5 μl En- zyme mix. A and B were mixed and the tube subjected to cycling following the program described above with at total cycle number of 18. Five μl samples were transferred to eppendorf tubes after 4, 6, 8, 10, 12, 14, 16, 18, and 20 cycles, respectively, and run on a 1.2% agarose gel. The optimal number of cycles was determined as described in the SMART manual (the optimal number of cycles is the number of cycles where the amount of PCR-products is not reaching saturation) and the remaining cDNA from the first amplification subjected to cycling in nine separate reactions as described above using this optimal cycle number.

The PCR products were pooled and purified using a Centricon YM50 column (Millipore 4224) as described by the manufacturer. Expected yield was approximately 20 μg. Example 5

Analysis of gene expression in the amplified cDNA

One μg of amplified cDNA from DMHc (tester) and CTX (driver) were used as input in a cDNA subtraction as described [Jiang et al., 2000] and a number of clones generated by ligating the subtracted cDNA to Xho I cut pBluescript II KS+ (Stratagene). The inserts of these clones as well as inserts encoding housekeeping gene cDNA were amplified by PCR followed by Reverse Northern blotting.

For Reverse Northern blotting, 10 μl of each PCR reaction was diluted with 90 μl of 0.22 M NaOH and incubated 15 minutes at 37 degrees Celsius. Then the DNA was applied to a Hybond N+ membrane in a dot-blot manifold in an 8X12 holes format. The membrane was soaked in neutralizing solution (0.5 M Tris-CI, 1.5 M NaCI pH 7.5) for 5 minutes and rinsed briefly in 2XSSC (0.3M NaCI, 0.03M Na-Citrate pH 7.0) followed by UV-crosslinking by exposure to 0,070 joules/cm₂. Then the filter was hybridised with 50 ng of the amplified cDNA (a mixture where half was cut with Bsp 1431 and the other half with EcoRII) from DMHc and CTX, respectively, and labelled with 32-P (Sigma, Random-primed labelling mix, R7522) using a Super HYB kit (Molecular Research Center, SK116) as described in the manual except that the last two washes were at 50 degrees Celsius to reduce cross hybridisation.

A Storm phosphor imager (AP Biotech) was used for detection and quantification of the signal. Example 6

Examples of genes isolated

Based on the reverse Northern blotting results with clones obtained by RASH, 50 clones were chosen for further characterization by sequencing.

Forty of these clones were unique, 2 previously unknown genes and 15 were represented in EST databases. A number of the previously characterized genes are known to be preferentially expressed in DMHc (and not in CTX). Thus, cDNAs corresponding to part of the galanin (4 of 50 clones) gene [Gundlach et al., 1990; Levin et al., 1987] as well as the angiotensinogen (3 of 50 clones) gene [Speth et al., 2001] were highly represented in the library. In situ hybridisation using clones representing previously unknown genes also revealed that mRNA corresponding to these clones is preferentially expressed in DMHc and not in CTX.

A number of the genes isolated in the DMHc minus CTX subtraction experiment were sequenced and the expression further analyzed by in situ hybridization.

In situ hybridization

Template preparation and in vitro transcription: Templates for transcription were prepared by PCR using the bacterial stocks directly as templates and the primers 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO 26) and 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO 27). The products were subsequently purified by the High Pure PCR purification kit (Roche, 1732676) and analyzed on agarose gels before use. cDNA probes were prepared as follows: 1x transcription buffer, 10mM DTT, RNase inhibitor (Promega, N2515, 0.5U/μl), CTP/ATP/GTP mix (1 mM ), S35- alpha-UTP or P33-alpha-UTP (Amersham Pharmacia), template (25 ng per 100 basepairs) and polymerase (T3 or T7, 40U) were mixed and incubated in a total volume of 25 μl for 2 hours at 37°C. Subsequently, DNA was digested by the addition of 1 μl RQ1 Dnase (Promega, M6101 ), 2 μl yeast tRNA and 1 μl RNase inhibitor (20-40 U/μl). The transcripts were purified using Micro- Biospin 30 columns (Qiagen) followed by precipitation in 2.0 M ammonium acetate and ethanol. The transcripts were diluted in a 1 :1 mixture of 100% deionized formamide and Tris (10 mM) EDTA (1 mM)-DTT (10 mM) buffer (pH 7.5). The specific activity of the generated transcripts was determined using a beta-counter.

Male Sprague Dawley rats (Charles River, Sweden) were sacrificed by decapitation and the brains were removed and frozen on dry ice. Twelve micron thick frontal sections were cut in a cryostat and mounted directly on SuperFrost™ Plus slides (Menzel, Germany). Dried slides were fixed for 5 min in 4% paraformaldehyde. The slides were next rinsed 2 x 5 min in phosphate buffered saline (PBS; pH 7.4) followed by a brief acetylation: 500μl acetic anhydride (100%) was added to 200ml 0.1 M triethanolamine and the slides immediately submerged for 2 min. Next, slides were passed through PBS twice (2 x 2 min) and finally through graded ethanol concentrations (30/60/80/96/99/99) and allowed to dry.

Immediately prior to hybridisation, the radioactively labeled probe was incubated for 3 min at 80°C (denaturation) and mixed with hybridization buffer. The hybridization buffer comprised 50% deionised formamide, 1X SALTS (300 mM NaCI , 10 mM Tris, 10 mM NaPO4 (pH 6.8), 5 mM EDTA, 0.02% Ficoll 400, 0.2% polyvinylpyrolidone (PVP-40, 40000 MW), 0.2% BSA Frac- tion V), 10% dextran sulphate, 1 μg/μl yeast tRNA and 9 mM DTT. Probe was added so that the final activity of the hybridization mix is approximately 15.000 cpm/μl. The hybridization mix was applied onto the sections (35 μl/section) and the sections were cover-slipped. Hybridization was performed overnight at 47°C and the slides were washed the next day in two stringency washes at 62 and 67°C.

The sections were washed for 1 hour at each temperature (lowest first) in a washing buffer comprising 50% formamide, 1 x SALTS and 10mM DTT. The sections were rinsed twice (2 x 2 min) in NTE buffer (0,5 M NaCI, 10 mM Tris-CI (pH 7,2), 1 mM EDTA) containing 10 mM DTT, and RNAse A treated (20 μg/ml; Boehringer-Mannheim) for 30 min. Next, the sections were rinsed twice for 5 min in NTE + 10mM DTT, 30 min in SSC (15mM NaCI, 1.5 mM trisodium citrate, pH= 7,0) comprising 1 mM DTT and finally dehydrated through a series of graded ethanol solutions containing 0.3 M ammonium acetate (30/60/80/90/99). After drying the hybridized sections were exposed to Kodak bio-max film.

Measurement of tissue-specific expression of differentially expressed genes by semiquantitative multiplex RT-PCR.

The procedure is based on methods described previously by Jensen et al. [Jensen et al., 1996].

Fresh tissue (hypothalamus, cerebellum, cerebrum, heart, liver, pancreas, fat, skeletal muscle, kidney, spleen, intestine, colon, and lung) is isolated from Sprague-Dawley rats and immediately submerged in RNAIater (Ambion, Texas, U.S.A.). Total RNA is extracted from the tissue using RNeasy spin columns (QIAGEN Inc., California, USA), following the manufacturer's instructions. First-strand cDNA is prepared using 1 μg total RNA, the Superscript RT kit, and random hexamer primers (GIBCO BRL, Gaithersburg, Maryland, USA), according to the manufacturer's instructions. The cDNA is diluted 1 :6 in distilled water, and PCR is carried out using 3 μL of the diluted cDNA and a PCR mix containing Biotaq DNA polymerase (2.5 U) and buffer (Bioline Ltd, London, UK ), 1.5 mM MgCI₂, dNTP mix (final concentrations of 60 μM of each dNTP, except dCTP, which is present at 30 μM [Sigma, St. Louis, U.S.A.]), and 1.25 μCi of [o-33P]-dCTP (2,000 Ci/mmol; Hartmann Analytic, Germany) in a 25 μL reaction volume, using a protocol provided by Promega.

Two primer sets (5 pmol of each primer) are included in each reaction, 1 set specific for the hypothalamic gene identified as described above, the second set specific for an internal standard. The internal standard could be any of a number of housekeeping genes including but not limited to elongation factor- 1 (EF-1cv), TATA box binding protein (TBP), beta-actin etc. The primers are chosen so the final products are in the range of approximately 150 to 300 bp. The PCR conditions are an initial incubation at 95°C for 2 minutes. This is followed by a number of cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 45 seconds. The number of cycles is chosen in the range where the limiting factor for the amount of product is the amount of input template cDNA, normally 18 to 25 cycles, the exact number depending on the expression of the gene examined and the efficiency of the PCR. The final PCR re- actions are mixed with 98% formamide denaturing loading buffer and separated on a 6% (wt/vol) polyacrylamide gel, containing 7 M urea. The gel is subsequently dried, exposed to a phosphorimager screen, and the resulting scan analyzed using ImageQuant (Amersham Biosciences, Sweden).

Bioinformatic analysis of isolated sequences

A number of bioinformatic tools are available on the Internet. Newly found clones are typically characterized using the programmes BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), which searches input sequence against the Genbank database (Expressed Sequence Tags, Genomic Databases, etc.), and BLAT (http://genome.ucsc.edu/cgi-bin/hqBlat), which searches input data against genomic databases of mouse, rat, or human DNA, and as output shows alignment with the genomic DNA sequence, with known mRNAs and/or ESTs, predicted genes etc.

Polypeptide sequences predicted from the sequences of ESTs and/or predicted genes overlapping the clones found in subtraction experiments can be analyzed using a plethora of programs. Most of these programs use evolutionary conserved domains for functional predictions. One example of such programs is the Panal program (http://mgd.ahc.umn.edu/panal/run_panal.html), which makes a simultaneous search on the input polypeptide sequence using the SMART, TIGRFAM, Prosite, BLOCKS, prints, and pfam conserved sequence signature databases for finding conserved sequence motifs that are indicative of polypeptide function.

Another approach, is used by the program ProtFun (http://www.cbs.dtu.dk/services/ProtFun/), which predicts protein function (using a neural network approach) from a number of functional attributes related to the linear sequence of amino acids. These attributes include features associated with simple aspects, such as the length, isoelectric point, polypeptide composition as well as features associated with more complex traits, such as posttranslational modifications and protein sorting [Jensen et al., 2002; Jensen et al., 2003].

The probability of a polypeptide for being secreted is of special interest when searching for new neuropeptides, and can be examined using the programs SignalP and TargetP. The SignalP (http://www.cbs.dtu.dk/services/SignalP- 2.0/, [Nielsen et al., 1997]) method incorporates a prediction of cleavage sites and a signal peptide/non-signal peptide prediction based on a combination of several artificial neural networks and hidden Markov models, whereas TargetP (http://www.cbs.dtu.dk/services/TargetP/, [Emanuelsson et al., 2000] predicts the subcellular location of eukaryotic protein sequences. The subcel- lular location assignment is based on the predicted presence of any of the N- terminal presequences chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP).

The following 9 clones were identified and analyzed as described above:

Clone P1 A11 (SEQ ID NO 1)

Sequencing of clone P1 A11 revealed the following sequence:

GAGGTTGTCCCCAACAGGACTGGGCACTTCCATGGCGACTCCCCACTG TCCGCGGGCTTCAGGGTGTCCTGTATAAACGTATACAGCGACTGTAGTT CCCTGCACCAGCCTCCCTGAGAAACTGCAGGGGGTGAGGCTCCTCCTG AGAGTGGAAGAGGACTTCCACGGAGGACTTGAGGCCCTCCAGGTC- TCGAG

BLAST and FASTA analyses (Wisconsin GCG package) of the cDNA inserted in clone P1 A11 revealed homology with a previously reported mouse gene. The mouse homologue (accession number Mm.141526) of this clone is present in the Unigene database (http://www.ncbi.nlm.nih.gov/UniGene/), and has a high degree of homology (87%) with a hypothetical human protein, FLJ20211 , accession number NP_060183.

The Mm.141526 mouse peptide has the following sequence:

1 magevtqalg gaqkigtltl lcdaktdgsf Ivhhflsfyl kanckvcfva Ivqsfshyni 61 vgqklgvslt aardrgqlvf leglkssvev Ifhsqdephp Iqflreagtg nlqslytfiq 121 dtlkpadsee spwkypvllv dnlsvllslg vgavavldfm qycratvcce Ikgnvvalvh 181 dtegatdegn dtllnglshq shlilraegl atgfckdvhg qlsilwrrps rstaqraqsl

241 tyqykiqdkn vsffakgmsp avl This sequence was used as input for the FunProt program. The output indicated that the protein is associated with the cell envelope and it might function as a growth factor.

In situ hybridization studies carried out as described above (Figure 7) showed that the P1A11 gene is expressed in hypothalamic subregions (DMHc and more), but also, in cortex and other brain areas, albeit at lower expression levels, indicating that the method described herein is suitable also for the identification/cloning of genes with minor expression differences.

Clone P1 B12 (SEQ ID NO 2)

Sequencing of clone P1 B12 revealed the following sequence:

TTGGCTGGAATAACCACCACTTGTATCATCGAAGAGTGGAGGAGGGTTT GGAGCTGCCCGGGTCCTATGCTTGGAACCGTGGACTCCATATCCCGAG TGGTAAGCCATGGCCGGACGTTCGCTCTACCCAAGGGTAGGCTTCGCT GAGTTCTGGGTACAAACACTACTGAAACAGCCGCTTCTCTTGCCTTGTC CTGCCCAAAGTTACCCGACCGTCCGACACGTCCTGGTCTCGAG

BLAST and FASTA analyses of the cDNA inserted in clone P1 B12 revealed homology with a previously reported rat sequence. The rat gene is contained in the unigene database (http://www.ncbi.nlm.nih.gov/HomoloGene/) (Rn. 7355) and has been noted to be homologous with a human putative trans- membrane protein, 54TM, accession number BC001299. This transmem- brane protein is a homologue to the yeast Golgi membrane protein Yifl p [Matern et al., 2000]. Clone P1 D4 (SEQ ID NO 3)

Sequencing of clone P1 D4 revealed the following sequence

TACAGCAGCACGTGCTACTGTGATCCAGTACTCAGGACATGATAGGGC CTAAGGTCATTATTACTATTTAAACTGGAGGTTTGTGTGGCTGAAGATAG GTTCACTGGGTTAAAAATGCTTGCTGCTCTTGCAGAAAGCCTCATGTTG GGTGGCTCACAACTACCTGGAACTCCAGCCCCAGGTCTCGAG

BLAST and BLAT, analysis of the cDNA inserted in clone P1 D4 revealed this cDNA as a splice variant of the gene encoding the Tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein eta (14-3-3, Accession number NM_013052, [Watanabe et al , 1991 ] The cloned cDNA fragment corresponds to a genomic sequence placed between exon 1 and exon 2 of the 14-3-3 eta encoding gene, leading to a splice variant with a coding potential for an N-terminally truncated 14-3-3 eta polypeptide

The 14-3-3 eta protein is an activator protein for tyrosin and tryptophan hy- droxylases (rate-limiting enzymes in catecholamine and serotonin biosynthe- sis) but is also a multifunctional regulatory protein in mechanisms in signal transduction and phosphorylation

14-3-3 proteins interact (and modulates the activity) with certain Regulators of G protein Signaling (RGS) proteins, including RGS7 RGS proteins facih- tate the conversion of G-protein bound GTP to GDP by accelerating the inac- tivation of GTP-bound Galpha. and Galpha_q subunits [Benzing et al , 2002] The physiological role of the splice variant is unknown, but interestingly, in situ hybridisation experiments specific for the splice variant and full-length 14-3-3 eta mRNA, respectively (figure 7), showed that the splice variant is specifically expressed in DMH and VMH and the arcuate nucleus, whereas the full-length 14-3-3 eta mRNA has a more uniform distribution, indicating a specific hypothalamic role of the splice variant.

Clone P1 D6 (SEQ ID NO 4)

Sequencing of clone P1 D6 revealed the following sequence:

CTCGAGACCTGGCAGAGTCACACAGGGTTTGAGTCTGCAGTCTGTTTTC TGTCCCCTCAGGGTGGGGTGTTACTGATCTTCCTGATGTGCGTGGTGC CACCAGCCAGCTCTGAGGTCTCCCAGCTACTCACTGCTCTTGGTATCCT GATTTAAAGATGGTTTTTGTTGCTATAGAATTCTTTTTATGATAGCCCTTG GTGGAGGCGCTCTGTAGGGCTGGCACCTGGTCTCGAG

No homologous ESTs or known genes were revealed by BLAST and FASTA analysis of the sequence shown above, but BLAT analysis combined with rtPCR with primers predicted to amplify P1 D6 fragments and 3'-RACE revealed the following open reading frame containing cDNA sequence (SEQ ID NO 10):

CCCATGTCAAAAAGCCAGGCATGGCTGCAAAGGCCTGCAGCCCAGCAC TGGTGGGCAAAGGCAGGTGATCCCAAGAGCCCGTTGAGCTGAAATGTT GAGCTCTTGGTTTGATGAGAGACACTGTCTCAAGGCAATAAGGTGGGA GTGACAGAAGAGGACACACACATGGTCACACATGCACACATGCATATGC ACACTCTCAAGCACACACATGTGCACACACACATGCCCATCCTCCCCAA GGCCAAAAGAGAAAAGAAAGGCTGCCAAAAGTGGGCCACAGCAGGCCA GTGATGGGCTGCCGACCTGCTTTCTGGCCAGGTCCTCACTGACCAGTC TCACCAGAGCCCTGGTCAGGGCCACTGATAGACGCCGGGTCTGACTGC TCATAAGCCTGTCTGTTCCCAGGCTTGCATGAGAGATTCCTGCAATAGC ATGGACCTGAAGAAGGAGGTGGAGCTCCTCCAGCACCTGCAGCTCAGC CCGCCCGTGTTGGGACTTCAGAAGGCTGTACTGAACATCCTGAGAGTG TCCCTGTCCTGGCTGGAGGAGACAGAGCAGCTGCTCGGGGACCTCAAC ATCGAGCTGTCAGATTCCGACAAAGGGTTCTCTCTGTGTCTGATATACC TTCTGGAACATTACAAGAAAATTATGATCCAGTCCGAGGAACTTCAGGC CCAGGTGAATGCTTCTCTGGAAACCCAGCAGTCTCTGCAGGAGGAGAA CCTGGCTGAAAAGGAGAAGCTGACAGAGAAGCTAGAACAAGAAGAGAA GCTGAAAGCCAGAATTCAGCAGCTGACAGAAGAGAAGGCGGCTCTGGA AGAGAGCGTCGCAGAGGAGAAAAACAAATTGCAGGGGGACCTGGAGAT GACACAAGCTCGGGTCCATGAGCTGGAGAACGACTTGGCTTGTCAAAA GGAGGTCTTGGAGAGCAGTGTGACTCAAGAGAAAAGGAAAATGCGGGA AGTGCTGGAGGCAGAAAGGAGGAAGGCACAAGACCTGGAGAATCAGCT GACCCAGCAGAAGGAGATCTCAGAGAGTAACACGTACGAGAAACTCAA GATGAGAGACACTCTGGAGAAGGAGAAGAGGAGGATACAGGACCTGGA GAATCGCCTGACCAAGCAAAGAGAGCAGTTACAGGGCAAGGAAAAACC TGCTGATTGTGTAGTCACTACACAGAAACACAGCAAGCGCAACAAGGTT GTCAGTATAAACCTATAATTCCAGCCACTCAGGAGGCTGAGGTCATGCA AGACTAGCCTGGGCTGCAGAGTGAGCTCAGGGTCAGCTTAGAAACCTG GTAAAGCCCTGTCTCGGAATTAAAAAGCAAAGAGACCTGAAGATAAAAA CCCAGAGATGTGTCGCTTGCCTAGCATGTGCAAGGCGCTGGGTTGGAA GTCCACAGCTAAATGTCAGGCAAGCTCACAGAGGCAAAGCCCTTACCA GGTGCCAGCCCTACAGAGCGCCTCCACCAAGGGCTATCATAAAAAGAA TTCTATAGCAACAAAAACCATCYTTAAATCAGGATACCAAGAGCAGTGAG TAGCTGGGAGACCTCAGAGCTGGCTGGTGGCACCACGCACATCAGGAA GATCAGTAACACCCCACCCTGAGGGGACAGAAAACAGACTGCAGAGCA AACCCTGTGTGACTCTGCCAGGGATTCTGTACCCAATCTCAAACCCCAG GAAACCAAACCGAAAATGAAGACGTTAAAAGCTTCCGCCAGACGGAGG GAACAGTGGGCAGATGGAACCACTAGACTGGGCAGGAAATACTTTGAA ACACAGGTCAGAGAACTGGTTAATCTCTACACCGTCTGAGGAATCCCAA TAGCTCACTGAAAAGACAGGCCAATGGCCCAACCAAAGCACTCACCAG GCACCTGACAGACTGGCTTGTTTTATTTTATGAGGCCAGGGAATGGAGC CCAGGGCCTCTCACACAGCGGCCAGGTGCTTTCCCACTGAGTTATATC CAAAGTTTTTATTTGTTTGTTTTATTTTATTTTTGAAATGGAGTCTCATTAA TTTGTCCAAGTTGACCTCGAACTCACTATGTAACCCAGGCTTGTTTCAGC TCAGCCATCCTCCTGCCTCTCAACCTTTCAAGTAGCTGGAATCATAGAC CAGCCCCACACCTGACCTGAATACACTTTTTTTCTCTCTTCAAAAAAAA

Which has a coding potential for the following polypeptide (SEQ ID NO 11 ):

MRDSCNSMDLKKEVELLQHLQLSPPVLGLQKAVLNILRVSLSWLEETEQLL GDLNIELSDSDKGFSLCLIYLLEHYKKIMIQSEELQAQVNASLETQQSLQEEN LAEKEKLTEKLEQEEKLKARIQQLTEEKAALEESVAEEKNKLQGDLEMTQA RVHELENDLACQKEVLESSVTQEKRKMREVLEAERRKAQDLENQLTQQKE ISESNTYEKLKMRDTLEKEKRRIQDLENRLTKQREQLQGKEKPADCVVTTQ KHSKRNKWSINL

The polypeptide sequence contains a leucine zipper motif and might therefore encode a transcription factor.

In situ hybridisation (Figure 7, panel P1 D6) revealed that the P1 D6 mRNA is expressed in only the ependymal lining of the third ventricle, showing a role in hypothalamic function.

Clone P1 E3 (SEQ ID NO 5)

Sequencing of clone P1 E3 revealed the following sequence:

CTCGAGACCTGGCCAGGGGGCGGGTGGAGACCTGTGGAGGAGACAGT TGAATCGCTTAAAGGAGGTGAAGGCCTTTGTCACTCAGGACATTCAACT GTACCACAACCTGGTGATGAAGCACCTCCCTGGTCTCGAG

A mouse homologue of this gene has previously been identified (Unigene Mm.34046) and the protein has recently been characterized and named Se- lenoprotein M (SelM) because it contains a selenocysteine amino acid residue [Korotkov et al., 2002]. SelM harbors characteristics of neuropeptides such as presence of a peptidase cleavage site (RPDWN) and a signal peptide. The protein seems to be localized in the endoplasmic reticulum and the Golgi system.

In situ hybridization (Figure 7, panel P1 E3) shows signal in the PVN, the retrochiasmatic area, in the VMH and in the compact part of the DMH. Also the Arcuate nucleus contains this transcript. Thus, the intrinsic characteristics of the SelM peptide as well as the subcellular localization and the hypothalamic expression makes SelM a potential candidate for regulation of appetite and/or metabolism.

Clone P1 E6 (SEQ ID NO 6)

Sequencing of clone P1 E6 revealed the following sequence:

CTCGAGACCAGGGGTCCCCTGGTTTGCACTTGAATTCGTAGTCCTCCCA CACTGCATCAGGTGCACTGGCATTGGGGTTCACTGTGCCCTGCAGGAT CACCTCAGTCCGTTCTTTGGTGATACTTCCGAAGGCCCCATAGGTGTTG ACAATCCTGAGTGGGTTGAAGGAGGTGTTCATGGCCTGCTTGGAGCTTA GGAGG TTGATAACCA CGGGTACGCT GAGCCAGGTC TCGAG

A mouse homologue of this gene has previously been identified (Unigene Mm.12787) and a leucine zipper motif indicates that the protein might be a transcription factor.

In situ hybridization (Figure 7, panel P1 E6) shows that this protein is specifically expressed in the DMH, the PVN, and the VMH. This transcript is virtually absent in the rest of the brain. Clone P1 G1 (SEQ ID NO 7)

Sequencing of clone P1 G1 revealed the following sequence:

CTCGAGAGATCCCAGCACCGCCGGCCCCTGCCCAGCTGGTGCAGAAG GCGCACGAGCTCATGTGGTACGTGTTGGCCAAGGACCAGAAGAGGATG GTCCTCTGGTTTCCAGACATGGTGAAAGAGGTCATGGGCAGCTACAAG AAGTGGTGCAGAAGCATCCTCAGGCGCACCAGCGTCATCCTCGCCAGA GTTTTCGGGCTGCACCTGAGGCTGACCAATCTCCACACC ATGGAGTTCGCCCTGGTCAAAGCACTCAGCCCAGAAGAGCTGGACAGA GTCGCGCTGAACAACCGTATGCCCATGACAGGTCTCCTGCTCATGATCT CTCGRTCGACGGTATC

Clone P1 G1 contains part of the Necdin cDNA (Unigene Mm. 7089). Necdin is one of several genes deleted in the Prader-Willi syndrome, a syndrome characterized by mental retardation, decreased muscle tone, short stature, emotional lability, and an insatiable appetite, which can lead to life- threatening obesity.

Necdin null mice have partial early postnatal lethality [Gerard et al., 1999], have reduced numbers of oxytocin- (-29%, p=0.009) and luteinizing hormone-releasing hormone-producing hypothalamic (LHRH, -25%, p=0.016) neurons and display behavioural and cognitive disruptions that bear some resemblance to characteristics observed in humans with PWS although they show no sign of obesity at age 18 months [Muscatelli et al., 2000].

The Necdin polypeptide has been shown to have a plethora of functions and interaction partners and has a proposed role in cell cycle, apoptosis, and signal transduction. In situ hybridization signal from necdin is seen in PVN, Ret- rochiasmatic area and in the DMHc (compact part) as well as in the VMH and the arcuate nucleus. As seen from figure 7, panel P1 G1 , expression in cor- tex is low Other areas expressing Necdin includes the hippocampus (CA1 , CA2 and CA3), thalamus, amygdala and piπform cortex

The distribution in rat brain is similar to the distribution previously reported in mouse brain [Uetsuki et al , 1996] Importantly, the expression of necdin is regulated upon fasting in fatty Zucker (fa/fa) rats (figure 8), indicating a role of necdin in metabolic regulation

Clone P1 G5 (SEQ ID NO 8)

Sequencing of clone P1 G5 revealed the following sequence

CTCGAGACCTGGGCAATCAAATGGGAGAAACATTTCCCTGCCACTGCA GAATTGTATTCTGGAAAGAGAAAGAGAAGGAAAGAGAGAAGTACTTCTG GTTGGTGTGTAAGTGGATGTGGTTAAGGAGAGACACACCCGCAGATTCT CCGAGCAGCCTCTCTTCGTCTGGAACCCTGTGTTTAGAATTTGTAATGT GGGGGTTGGGGATTTAGCTCAGTGGTAGAGCGCTTGCCTAGCAAA TGCAAGGCCCTGGTCTCGAG

In situ hybridization studies indicate that this gene is highly expressed in hypothalamus (not shown)

Clone P1 G9 (SEQ ID NO 9)

Sequencing of clone P1 G9 revealed the following sequence

CTCGAGACCTGGTGAAGACTGGGTAGGGTCTGTGGCCATCTGACTCAG ACTAGCTTCAGGAACTCTTACAGTGTGTTCTGTTCCACGGCCTCCACGA AGAGTTCATAGTCCCCACAGTAGTGGTCCCCGTTGACAATCTGAGGTGG GGTGGCTTTGGGGTTGCCGGCCAAGGTGCGCATCTCATCTCGAAGGGC GTTGTCCTGGTCTCGAG BLAST and FASTA analyses of the cDNA inserted in clone P1 G9 revealed that the sequence of this cDNA has been previously reported and is present in the Unigene database (Rn. 18564). The translated cDNA has a low degree of homology (-40%) to SH3B_Mouse SH3 domain-binding glutamic acid rich protein, which shows a significant similarity to Glutaredoxin 1 (GRX1 ) of Es- cherichia coli and is predicted to belong to the thioredoxin superfamily. In situ hybridization studies showed hypothalamic expression of this gene (data not shown).

In conclusion, the identification of these clones demonstrates that the methods of the present invention can be used for identifying genes with a differential pattern of gene expression in confined areas of the hypothalamus. These genes include genes known in the art as well as known genes that were not previously associated with the hypothalamus as well as previously unknown genes.

Example 7

Subtraction experiment with subtraction of identical areas from different rat strains

The DIO (Diet Induced Obesity) /DR (Diet Resistant) rat model of obesity has been established as two outbred Sprague-Dawley rat strains exhibiting dif- ferent sensitivity to a high energy diet. The DIO rats become obese when fed a high energy diet as compared to chow-fed controls, whereas the DR rats gain no more weight when fed the high energy diet than the controls and thus are resistant to the high energy diet [Levin and Dunn-Meynell, 2002; Levin et al., 1997; Levin and Keesey, 1998]. This rat model has a number of traits in common with human obesity such as the polygenetic inheritance and the status of a number of metabolic parameters. A number of hypothalamic nuclei are implicated in the regulation of appetite and energy expenditure, and the DMH has been implied as a key player in the setting and maintenance of the body weight set point, (see [Bemardis and Bellinger, 1998] for a review). Clearly, the present invention can be used to identify genes in the DMH and other hypothalamic nuclei with a differentially regulated expression in DIO vs DR rats.

The difference in gene expression in the hypothalamic nuclei of the DIO and DR rats may be constitutive and/or induced by a number of conditions, and therefore, the search for differentially expressed genes may be performed on animals subjected to a number of different feeding paradigms. These feeding paradigms could include but are not limited to:

• Chow fed animals • High energy fed animals before obesity develops

• High energy fed animals after obesity has developed,

• Overfed animals (force-fed or fed highly palatable diet) regaining normal weight.

One example suited to identify genes differentially expressed a priori in DR and DIO rats is described below. The genes identified in this experiment are differentially expressed in DR and DIO rats fed on chow diet and may represent genes whose differential expression confers different sensitivity to high energy diets. The feeding paradigms described above constitute examples of physiological parameters that can be explored in the DR/DIO or other animal model settings.

DIO and DR rats fed a standard chow are decapitated at 10 weeks of age, the brains quickly removed and frozen in 2-methyl butane cooled with dry ice. Then, laser dissection, RNA isolation, cDNA preparation and amplification is performed as described above. Subtraction is subsequently performed in two separate reactions, using the DIO and DR cDNA as tester and driver, respectively in the first reaction and using the DR and DIO cDNA as tester and driver, respectively in the second reaction. Subsequently, a number of clones are isolated. The genes are identified by sequencing and the expression dif- ferences validated by Reverse Northern blotting and in situ hybridizations as described above.

Example 8

Cloning of full-length cDNAs by RACE (rapid amplification of cDNA ends)

Background art

The deduction of structure-function relationship as well as a number of experimental designs for the analysis of the mode of action of a differentially regulated gene cloned as described above requires the knowledge and/or cloning of full-length cDNA. A number of options exist for the isolation of full- length cDNA when the sequence of a segment of the cDNA is well known. One example is the screening of full-length cDNA libraries, but the preparation and screening of such libraries is tedious and time-consuming, and am- plification of the gene by RACE will in most cases be more straightforward and rapid. The RACE technology requires the setup of two independent reactions, a 5'-RACE, where the 5'-end of the cDNA is amplified by PCR and cloned, and a 3'-RACE, where the 3'-end of the cDNA is amplified and cloned.

Gene specific primers (GSPs): The sequence information obtained from the subtraction cloning is used to design gene-specific primers that are designed to have a length of 23-28 nucleotides, a GC content of 50-70%, and a T_m >= 65 °C, preferably >70°C to facilitate the use of a touchdown PCR program where a high initial annealing temperature during the first rounds of the PCR is gradually lowered in the subsequent cycles. This leads to a better proportion between specific products and side-products. The results of the in situ hybridization experiments are used to determine the direction of the primers for use in 5'-RACE (GSP1 ) and 3'-RACE (GSP2), respectively.

The methods used for the RACE cloning of cDNA are adapted from the SMART™ RACE cDNA amplification kit (Clontech).

δ'-RACE

cDNA synthesis:

One microgram total RNA is mixed with 10 pmol 5'-CDS primer (5' - T25N-1N - 3' (N-ι=A,G, or C, N=A,G,C, or T) ; SEQ ID NO 28) and 10 pmol SMART II A oligonucleotide; SEQ ID NO 29: (5'- AAGCAGTGGTATCAACGCAGAGTACGCGGG-3' in a total volume of 5 μl. The tube is incubated at 70 °C for 2 minutes and placed on ice for 2 min. whereafter 2 μl first-strand buffer (250 mM Tris-HCI (pH 8.3), 375 mM KCI, 30 mM MgCI₂), 1 μl 20 mM DTT, 1 μl 10 mM dNTP, and 1 μl Powerscript reverse transcriptase is added and the tube is incubated at 42°C for 90 min- utes. The cDNA is diluted with 100 μl of 10 mM Tricine-KOH (pH 8.5), 1 mM EDTA and heated to 72°C for 7 minutes.

PCR:

cDNA (2.5 μl) is mixed with 5 μl of universal primer mix (0.4 μM of 5'- CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT; SEQ ID NO 30) and 2 μM of 5'-CTAATACGACTCACTATAGGGC-3'; SEQ ID NO 31 ), 1 μl of gene specific primer (10 μM), 34.5 μl water, 5 μl 10X Advantage 2 PCR buffer, 1 μl 10 mM dNTP mix (10 mM), and 1 μl 50 X advantage 2 po- lymerase mix. The tube is subjected cycling with the following program: 5 cycles of 94°C for 5 sec, 72 °C for 3 min. 5 cycles of 94°C for 5 sec, 70°C for 10 sec, and 72°C for 3 min. 25 cycles of 94°C for 5 sec, 68°C for 10 sec, and 72°C for 3 min.

The conditions of the PCRs (number of cycles, annealing temperature, extension time) are individually optimized for each different cDNA to assure that the highest possible amount of specific fragment is obtained.

3'-RACE

cDNA synthesis:

One microgram total RNA is mixed with 10 pmol 3'-CDS primer A(5'- AAGCAGTGGTATCAACGCAGAGTAC(T)₃oN.ιN-3' where N =A,C,G, or T, and N-i = A, G, or C; SEQ ID NO 32) in a total volume of 5 μl. The tube is incubated at 70 °C for 2 minutes and placed on ice for 2 min. whereafter 2 μl first-strand buffer (250 mM Tris-HCI (pH 8.3), 375 mM KCI, 30 mM MgCI₂), 1 μl 20 mM DTT, 1 μl 10 mM dNTP, and 1 μl Powerscript reverse transcriptase is added and the tubes are incubated at 42°C for 90 minutes. The cDNA is diluted with 100 μl of 10 mM Tricine-KOH (pH 8.5), 1 mM EDTA and heated to 72°C for 7 minutes.

PCR

cDNA (2.5 μl) is mixed with 5 μl of universal primer mix (0.4 μM of 5'- CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT; SEQ ID NO 33 and 2 μM of 5'-CTAATACGACTCACTATAGGGC-3'; SEQ ID NO 34), 1 μl of gene specific primer (10 μM), 34.5 μl water, 5 μl 10X Advantage 2 PCR buffer, 1 μl 10 mM dNTP mix (10 mM), and 1 μl 50 X advantage 2 polymerase mix. The tube is subjected cycling with the following program: 5 cycles of 94°C for 5 sec, 72 °C for 3 min. 5 cycles of 94°C for 5 sec, 70°C for 10 sec, and 72°C for 3 min. 25 cycles of 94°C for 5 sec, 68°C for 10 sec, and 72°C for 3 min.

General considerations

It may be necessary to optimize the conditions of the PCRs (number of cycles, annealing temperature, extension time, buffer composition, and polymerase) for each different cDNA to assure that the highest possible amount of specific fragment is obtained. The products of the PCR reactions are ana- lyzed on agarose gels, cloned into TA-cloning type vectors and characterized by sequencing.

The present invention is not limited by the teaching of the examples, but extends to the scope of the accompanying claims and equivalents thereby.

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Claims

Claims

1. A method of identifying differentially expressed nucleotide sequences in a tissue sample that corresponds to a hypothalamic subregion from a mammalian brain, said method comprising the following steps:

(vii) isolating a subregion that is referred to as a first tissue sample, said sample being substantially free of cells from hypothalamic neighbouring tissues surrounding said hypothalamic subregion, (viii) isolating a second tissue sample, (ix) isolating RNA from the first and the second tissue samples,

(x) synthesizing cDNA from the isolated RNA samples, (xi) identifying cDNA sequences that are differentially expressed in the first tissue sample as compared with cDNA from the second tissue sample, and (xii) optionally identifying a gene that corresponds to said cDNA sequence.

2. A method according to claim 1 , wherein at least one of the two tissue samples is taken from a DIO/DR rat.

3. A method according to claim 1 or 2 wherein at least one of the tissue samples is isolated by laser capture microdissection.

4. A method according to any of claims 1 -3 wherein differentially expressed genes are identified by differential display RT-PCR.

5. A method according to any of claims 1-4 wherein differentially expressed genes are identified by cDNA subtraction.

6. A method according to any of claims 1-5 wherein the differentially expressed genes are verified as being differentially expressed subsequent to their identification.

7. A method according to any of claims 1 -6 that furthermore comprises a step wherein the integrity of the isolated RNA or cDNA is verified prior to identification of differentially expressed genes.

8. A method according to any of claims 1-7 that furthermore comprises a step wherein cDNA is amplified prior to identification of differentially expressed genes.

9. A method according to claim 8 wherein cDNA amplification is carried out in a way that ensures preferential amplification of both the 3' and the 5' ends.

10. A method of claims 1 -9 that further comprises the step of verifying genes that are differentially expressed.

1 1. A method according to any of claims 1-10 where the mammalian brain is derived from a non-human animal.

12. A method according to claim 11 where a human counterpart gene to the differentially expressed gene is identified.

13. A method according to any of claims 1-12 wherein brain tissue is stained prior to isolation of the first and/or the second tissue sample with a labelling agent selected from the group consisting of: immunostaining, expression of fluorescent proteins, Nissl staining (Cresyl violet, Thionin), haema- toxylin-eosin, methylene blue, neutral red, any nuclear staining method, immunocytochemistry, endogenous expression of fluorescent proteins, application of neuronal tracers prior to sacrifice of the animal (such as cholera toxin B, horseradish peroxidase, fluorogold, true blue, or fast blue).

14. An isolated DNA sequence that is selected from the group consisting of SEQ ID NOs 1 -11 , or a part thereof.

15. An isolated DNA sequence that hybridises under stringent conditions with or is a degenerative of a sequence according to claim 14.

16. Use of a DNA sequence according to claim 14 or 15 for designing a diagnostic kit for diagnosis of a condition characterized by abnormal gene expression in the hypothalamus.

17. An expression vector comprising an isolated DNA sequence according to claim 14 or 15.

18. A host cell comprising an expression vector of claim 17.

19. A method of cultivating the host cell of claim 18 in order to produce recombinant polypeptides.

20. A recombinant polypeptide obtained by the cultivation method of claim 19.

21. Use of the recombinant polypeptide according to claim 20 for designing drugs for treatment of hypothalamic diseases.

22. Use of a recombinant polypeptide according to claim 20 for designing a diagnostic kit for diagnosis of a hypothalamic condition.

23. An antisense nucleic acid molecule that is complementary to a DNA sequence of claim 14.

24. An antisense nucleic acid of claim 23 that is an antisense DNA molecule.

25. An antisense nucleic acid of claim 23 that is an antisense PNA molecule.

26. An antisense nucleic acid of claim 23 that is an antisense LNA molecule.

27. Use of at least one antisense nucleic acid according to claims 23-26 for preparation of a medicament for treatment of hypothalamic malfunctions.

28. A method of diagnosing a disease or malfunction of plausible or hypothalamic origin or with known hypothalamic pathophysiology in an individual comprising determining the presence and/or amount or absense of a differentially expressed gene or gene product in a tissue or fluid sample from the individual, wherein said differentially expressed gene has been identified as being differentially expressed by any of the methods of claims 1 -13.

29. A method of diagnosing a disease or malfunction of plausible or hypothalamic origin or with known hypothalamic pathophysiology in an individual comprising determining presence and/or amount or absence of a marker in the upstream regulatory region of a differentially expressed gene, said differentially expressed gene being identified by any of the methods of claims 1 -13.