KR102478215B1 - Use of DRG2 gene as dopamine release modulator - Google Patents

Abstract

The present invention relates to the use of the DRG2 gene as a dopamine release regulator, and more particularly, to a method for providing information for diagnosing dopamine dysfunctional diseases using the DRG2 gene; methods for screening candidates for preventing or treating dopamine dysfunction diseases; DRG2 gene knocked out (Knock-out) dopamine dysfunction disease animal model; And it relates to a method for screening a candidate substance for preventing or treating dopamine dysfunction disease using the animal model. The present invention first identified the use of the DRG2 gene as a dopamine release regulator from dopaminergic neurons in the brain as a new function. Therefore, by confirming DRG2 deficiency, abnormal expression or dysfunction, it is possible to diagnose various diseases (motor disorders, depression, emotional disorders, Parkinson's disease, etc.) related to dopaminergic action. In addition, in the case of animals in which the DRG2 gene is knocked out, it can be usefully used as an animal model for dopamine dysfunction disease.

Description

Use of DRG2 gene as dopamine release modulator}

The present invention relates to the use of the DRG2 gene as a dopamine release regulator, and more particularly, to a method for providing information for diagnosing dopamine dysfunctional diseases using the DRG2 gene; methods for screening candidates for preventing or treating dopamine dysfunction diseases; DRG2 gene knocked out (Knock-out) dopamine dysfunction disease animal model; And it relates to a method for screening a candidate substance for preventing or treating dopamine dysfunction disease using the animal model.

Dopamine is a neurotransmitter in the brain. Since this discovery was made in the 1950s, the function of dopamine in the brain has been intensively studied. To date, it has been established that dopamine is important in several aspects of brain function, including motor, cognitive, sensory, emotional and autonomic functions (eg, regulation of appetite, body temperature, sleep). Thus, modulation of dopaminergic function may be beneficial in the treatment of a wide range of disorders affecting brain function. In fact, drugs that act directly or indirectly at central dopamine receptors are commonly used in the treatment of neurological and psychiatric disorders such as Parkinson's disease and schizophrenia. However, currently available dopaminergic drugs can have serious side effects.

On the other hand, DRG (developmentally regulated GTP-binding protein) protein is one of the proteins belonging to GTPase that has G1 to G5 required for interaction with GTP (Leipe et al, 2002). Since most of these DRGs are recently accidentally discovered during the cloning process of genomic DNA or cDNA, their distinct functions are not well known. It was only mentioned about the possibility that DRG performs a key function in cells of various species (Li and Trueb, 2000), and through cDNA cloning, it was highly expressed in the early stage of mouse fetal brain differentiation, and as differentiation progressed, Decreased expression has been reported (Kumar et al., 1992). In addition, DRG is mainly present in the cytoplasm (Sazuka et al., 1992), and is present in almost all types of cells from bacteria to humans.

DRGs in eukaryotes can be classified into DRG1 and DRG2. DRG1 is known to exist at the 22q12-q13.1 region of human chromosome 22, and DRG2 is known to exist at the 17p11.2 region of human chromosome 17. (Li and Trueb., 2000). Among the two genes, DRG2 is a relatively recently discovered gene, and it is known that the DRG2 protein increases in the early stages of brain differentiation and then decreases as differentiation progresses. However, DRG2 was discovered relatively recently and its function has not yet been well elucidated.

Against this background, the present inventors investigated the expression pattern of DRG2 in adult mouse brain and the effect of DRG2 deficiency on mouse brain function using DRG2 knockout (KO) mice. As a result, DRG2 protein is mainly expressed in neurons, not glial cells, and DRG2 deficiency in DRG2 knockout (KO) mice does not affect dopamine neuron population or dopamine synthesis in the striatal region. The present invention was completed by identifying defects in dopamine release in striatal regions and motor function.

Korean Patent Registration No. 10-1303462 Korean Patent Publication No. 10-2015-0002935

Accordingly, an object of the present invention is to provide an information providing method for diagnosing dopamine dysfunctional diseases using the DRG2 gene.

Another object of the present invention is to provide a method for screening candidates for preventing or treating dopamine dysfunction diseases using the DRG2 gene.

Another object of the present invention is to provide a dopamine dysfunction disease animal model in which the DRG2 gene is knocked out.

Another object of the present invention is to provide a method for screening candidates for preventing or treating dopamine dysfunction diseases using the DRG2 gene knock-out animal model.

In order to achieve the object of the present invention as described above,

The present invention provides an information providing method for diagnosing dopamine dysfunction disease, comprising measuring the expression level of the DRG2 gene in a target sample to be analyzed and comparing it with a normal control group.

In one embodiment of the present invention, when the expression level of the DRG2 gene in the target sample is decreased compared to a normal control group, a dopamine dysfunction disease can be diagnosed.

In one embodiment of the present invention, the dopamine dysfunction disease is movement disorder, depression, emotional disorder, obsessive-compulsive disorder, autism, schizophrenia, attention deficit hyperactivity disorder, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia and Huntington's disease.

In addition, the present invention comprises the steps of a) contacting a test substance to be analyzed with brain-derived nerve cells; b) measuring the expression level of DRG2 in nerve cells in contact with the test substance; and c) providing a method for screening a candidate substance for preventing or treating dopamine dysfunction disease, comprising the step of selecting a test substance having an increased expression level of DRG2 compared to a control group not treated with the test substance.

In one embodiment of the present invention, the measurement of step b) is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, Western blot, Northern blot, It may be selected from the group consisting of enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoprecipitation assay, and immunohistochemical analysis.

In addition, the present invention provides a dopamine dysfunction disease animal model in which the DRG2 gene is knocked out.

In one embodiment of the present invention, the animal model can reduce dopamine release in the striatum of the brain by knocking out the DRG2 gene.

In one embodiment of the present invention, the animal may be selected from the group consisting of mouse, monkey, dog, cat, rabbit, rat, cow, sheep, pig and goat.

In one embodiment of the present invention, the dopamine dysfunction disease is movement disorder, depression, emotional disorder, obsessive-compulsive disorder, autism, schizophrenia, attention deficit hyperactivity disorder, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia and Huntington's disease.

In addition, the present invention comprises the steps of i) administering a test substance to the animal model of claim 6 as an experimental group; ii) measuring and comparing motor coordination between the experimental group in i) and a control animal model not treated with the test substance; And iii) selecting a test substance in which motor coordination is improved in the experimental group of i) compared to the control group.

The present invention first identified the use of the DRG2 gene as a dopamine release regulator from dopaminergic neurons in the brain as a new function. Therefore, by confirming DRG2 deficiency, abnormal expression or dysfunction, it is possible to diagnose various diseases (motor disorders, depression, emotional disorders, Parkinson's disease, etc.) related to dopaminergic action. In addition, in the case of animals in which the DRG2 gene is knocked out, it can be usefully used as an animal model for dopamine dysfunction disease.

Figure 1 shows multiple phenotypic features of DRG2 knockout (KO) mice. (A) and (B) are the results of measuring the expression levels of DRG2 in the cerebral cortex of each of DRG2 ^+/+ , DRG2 ^+/- and DRG2 ^-/- mice (1A: RT-PCR, 1B: Western blot). (C) is a photograph comparing body sizes of DRG2 ^+/+ , DRG2 ^+/- , and DRG2 ^-/- mice born from litters at 1 day (top) and 12 weeks (bottom). (D) shows survival curves over time of DRG2 ^+/+ , DRG2 ^+/- and DRG2 ^-/- mice (n = 12 for each group). (E) shows micro-CT scan images of DRG2 ^+/+ , DRG2 ^+/- , and DRG2 ^-/- mice at postnatal day 1 (top-whole body; mid-cranial lateral; bottom-top cranial surface; arrowheads-reduced). indicated mineralization; arrows - show reduction of the radius and ulna of the forelimbs and hindlimbs of DRG2 ^-/- mice). DRG2 ^-/- mice showed an enlarged anterior fontanelle (AF) and reduced premaxillary-maxillary (PM). Scale bar, 500 μm. (F) is a graph showing the body weight of DRG2 ^+/+ , DRG2 ^+/- , and DRG2 ^-/- mice (n = 12 for each group). One-way ANOVA: ^*** p <0.001. Error bars represent SEM.
Figure 2 is a profile of the expression pattern of DRG2 in the mouse brain. (A) is the result of Western blot analysis on the tissue distribution of DRG2 protein. (B) is a Western blot analysis result for DRG2 expression in mouse brain regions. (C) is a result of measuring changes in DRG2 protein expression according to age in four brain regions. DRG2 expression was normalized to β-actin. Each data point is presented as mean ± standard deviation (n = 6). (D) Results of in situ hybridization analysis of DRG2 expression in mouse brain. Sagittal sections of adult mouse brain were hybridized with the DRG2 antisense probe. Representative images of DRG2 mRNA (red dots) and nuclei counterstained with DAPI (blue) are shown. Boxed regions are shown at higher magnification in the lower panels. Scale bar 1 mm. (E) Results of immunohistochemical analysis using an anti-DRG2 primary antibody and an Alexa-488-labeled secondary antibody to confirm the level of DRG2 expression in sagittal and coronal sections of mouse brain. Nuclei were stained with DAPI. (a) Sagittal section of mouse brain. (bi) shows a higher magnification of specific regions within the sagittal image in (a) ((b) CTX, (c) THs, thalamus (d) PB, parabrachial nucleus (e) HY, hypothalamus (f) CB , (g) CTX, (h) HIP, (i) SN). Arrows in (f) indicate the Purkinje cell layer. Scale bar, 100 μm. (jl) shows a coronal section of a mouse brain in three different planes. A sagittal diagram of the brain showing the plane of the cross section was displayed within each image. AOB, accessory olfactory bulb; AUD, auditory areas; BMA, basomedial amygdala nuclear; CA1, cornu ammonis1; CA3, Cornu Ammonis3; DG, dentate gyrus; CB, cerebellum; CP, caudate putamen; CTX, cerebral cortex; ENT, entorhinal area; HIP, hippocampus; HY, hypothalamus; MB, mid brain; Mo, somatomotor area; NAc, nucleus accumbens; PB, parabrachial nucleus; Pir, piriform cortex; PTLP, posterior parietal association areas; RSP, retrosplenial area; SCN, suprachiasmatic nucleus; SN, substantia nigra; SNc, substantia nigra pars compacta; SNr, substantia pars reticulate; SS, somatosensory area; Str, striatum; SUB, subiculum; THs, thalamus; VIS, visual area; VTA, ventral tegmental area; WB, whole brain.
Figure 3 is a profile of the expression pattern of DRG2 in mouse neurons. (A) shows the results of immunohistochemical staining of the neuron marker Tuj1, the astrocyte marker GFAP, the microglia marker Iba1 and DRG2 in the substantia nigra (SN) and dentate gyrus (DG) neuron cell bodies of the hippocampus (HIP). Nuclei were stained with DAPI. Scale bar, 20 μm. (BE) is the result of in situ hybridization analysis of DRG2 expression in the mouse hippocampus. (B and C) Hippocampal sections of mouse brain were hybridized with antisense probes for vGlut2, GAD67 (grey dots), vGlut1, GAD65 (green dots) and DRG2 (red dots). Nuclei were stained with DAPI. Scale bar, 200 μm. (B, C) The boxed areas of the images are shown at higher magnification in the lower panels (D, E), respectively. Scale bar, 20 μm.
Figure 4a shows the results of a geotaxis test of DRG2 deficient mice. The left side is a series of photographs showing mice performing the attention test, and the right side is a graph measuring the delay time until completion of the attention test (mean ± standard deviation, maximum 60 seconds, n = 8 for each group). ^* p < 0.05, ^** p < 0.005.
Figure 4b shows the results of the rotarod test of DRG2 deficient mice. DRG2 ^+/+ and DRG2 ^-/- mice were compared at 6, 12, and 40 weeks of age. Female and male mice were compared at 6 weeks of age, and female mice were compared at 12 and 40 weeks of age. Data represent time to fall (mean ± standard deviation, maximum 300 s, n = 8 for each group). ^* p < 0.05, ^** p < 0.005, ^*** p < 0.001. Student's t-test.
Figure 4c shows the Y-maze test results of DRG2 deficient mice (percentage of alternating triplets, total number of passages and total distance). Both male and female DRG2 ^+/+ and DRG2 ^-/- mice were compared at 6 and 12 weeks of age. Data are presented as mean ± standard deviation (n = 8 for each group).
Figure 4d shows the results of the elevated plus maze (EPM) test of DRG2 deficient mice. Both male and female DRG2 ^+/+ and DRG2 ^-/- mice were compared at 6 and 40 weeks of age. Figures on the left are representative images showing typical examples of DRG2 ^+/+ and DRG2 ^-/- mice exploring in the EPM device. The graphs in the middle and on the right show the percentage of time spent in the open arm. Data are presented as mean ± standard deviation (n = 8 for each group). ^* p < 0.05.
FIG. 5a shows the results of double in situ hybridization analysis using antisense probes for DRG2 and tyrosine hydroxylase (TH) in the brains of DRG2 deficient mice. Coronal sections of adult mouse brains were hybridized with antisense probes for DRG2 (red) and TH (green). Nuclei were stained with DAPI. Top shows representative images of DRG2 and TH. Scale bar, 500 μm. Images in the middle and bottom are higher magnifications of the boxed regions of the VTA and SN in the top panel. Scale bar, 20 μm.
Figure 5b is the result of analyzing the DRG2 expression pattern of TH-positive and negative cells in the VTA region. Top is the result of in situ hybridization analysis of DRG2 and TH expression in the mouse VTA region. Scale bar, 20 μm. The middle image shows the boxed areas of (a) TH positive and (b) TH negative cells in the top panel at higher magnification. Scale bar, 5 μm. The lower graph shows the quantification of the number of DRG2 mRNA dots on TH-positive or TH-negative cells in the SN and VTA regions. Data are presented as mean ± standard deviation (n = 8 for each group). ^*** p < 0.0001.
In Figure 5c, the image on the left shows the results of immunohistochemical staining for TH neurons in Str, SN, NAc and VTA of 1-year-old DRG2 ^+/+ and DRG2 ^-/- mice (the dotted line indicates the area for quantification). shown, scale bar - 500 μm), the graph on the right shows the quantification of TH neurons in the image.
Figure 5d shows dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanilic acid (HVA), epinephrine in the striatum of DRG2 ^+/+ and DRG2 ^-/- mice. (EPI) and serotonin (5-HT) levels were measured and graphed. Data are presented as mean ± standard deviation (n = 8 for each group). ^** p < 0.005, ^*** p < 0.001.
5e is a result of fast-scan cyclic voltammetry (FSCV) analysis of dopamine release in striatal slices of DRG2 ^+/+ and DRG2 ^-/- mice. Dopamine release in coronal brain slices was evoked by a single electrical stimulation pulse and extracellular dopamine concentrations in the striatum were measured using FSCV. Data represent the area under the dopamine concentration curve (mean ± standard deviation, n = 3 for each group). ^** p < 0.001.
Figure 5f shows the effect of L-DOPA administration on the motor behavior of DRG2 ^-/- mice. DRG2−/− mice were injected ip L-DOPA (50 mg/kg) and tested on a rotating rod. Data represent the latencies to drop in the rotarod test (mean ± standard deviation, up to 300 s, n = 6 for each group). ^* p < 0.05, ^*** p < 0.001.
Figure 6 evaluates the expression pattern of DRG2 and the effect of DRG2 expression on brain weight in the mouse brain. (A) and (B) show the expression of DRG2 and cell-type specific markers (astrocytic marker GFAP; microglial marker Iba1; and neuron marker Tuj1) in the hippocampus (HIP) and cerebral cortex (CTX), respectively. Comparison was made by immunohistochemical analysis. Coronal sections of mouse brains were incubated with antibodies against DRG2, the neuron marker Tuj1, the astrocyte marker GFAP, and the microglia marker Iba1. Nuclei were stained with DAPI. Scale bar 20 μm. (C) confirms the expression level of DRG2 in neurons, astrocytes and microglia isolated from mouse embryonic brain through Western blot analysis. Equal amounts of total protein (10 μg of total protein for each sample) were loaded. (D) is a photograph comparing the brains of 3-month-old DRG2 ^+/+ , DRG2 ^+/- and DRG2 ^-/- mice. The graph on the left is the result of measuring the brain weight of each group (mean ± standard deviation, n = 6 for each group). (E) is a DAPI-stained photograph of the coronal plane of 3-month-old DRG2 ^+/+ and DRG2 ^-/- mice. The top two panels show images of the entire coronal section (scale bar, 1 mm), and the bottom two panels show the boxed area of the top two panels at higher magnification (scale bar, 500 μm).
7 is a result of confirming the intensity and distribution of tyrosine hydroxylase (TH)-positive neurons in the SN, VTA, NAc and Str regions of DRG2 ^+/+ and DRG2 ^-/- mice through immunohistochemical staining. Serial coronal sections of 24 month old DRG2 ^+/+ and DRG2 ^-/- mouse brains were incubated with antibodies to TH. (A) TH-positive neurons in the striatum (Str), substantia nigra (SN), and ventral tegmental area (VTA) were visualized by incubating with streptavidin-horseradish peroxidase complex ( Scale bar, 500 μm.). (C) TH-positive neurons of SN, Str, Nac core, and Nac shell were visualized by incubation with Alexa-488-labeled secondary antibody (scale bar, 100 μm). The bottom graph shows the intensity of TH-positive neurons in each brain region (mean ± standard deviation, n = 3 for each group).

The present inventors investigated the effect of DRG2 deficiency on mouse brain function using a DRG2 knockout (KO) animal model, and as a result, it was first identified that DRG2 functions as a dopamine release regulator from dopaminergic neurons.

In one aspect, the present invention provides an information providing method for diagnosing dopamine dysfunction disease, comprising measuring the expression level of the DRG2 gene in a target sample to be analyzed and comparing it with a normal control group.

As used herein, the term "dopamine" is a catecholamine-based organic compound, and refers to a hormone or neurotransmitter found in the central nervous system of various animals.

In the present invention, the term "dopamine dysfunction disease" is a disease that can be caused by abnormal dysfunction of dopamine, and includes movement disorders due to a decrease in dopamine secretion, depression, emotional disorders, obsessive-compulsive disorder, Alzheimer's disease, Parkinson's disease, and autism. It is a concept that includes diseases such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and mania, addiction, and impulse control disorders due to an abnormal increase in dopamine secretion.

In the information providing method of the present invention, when the expression level of the DRG2 gene in the target sample is decreased compared to the normal control group, it can be diagnosed as a dopamine dysfunction disease.

The dopamine dysfunction diseases may include movement disorders, depression, emotional disorders, obsessive-compulsive disorder, autism, schizophrenia, attention deficit hyperactivity disorder, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia and Huntington's disease, but However, as long as it is a disease related to dopamine dysfunction, the type is not particularly limited.

As another aspect, the present invention provides a step of contacting a test substance to be analyzed with brain-derived nerve cells; b) measuring the expression level of DRG2 in nerve cells in contact with the test substance; and c) providing a method for screening a candidate substance for preventing or treating dopaminergic dysfunction disease, comprising the step of selecting a test substance having an increased expression level of DRG2 compared to a control group not treated with the test substance.

In step a) of the present invention, the nerve cell (neuron) is a nerve cell derived from brain tissue, and preferably may be a nerve cell derived from substantia nigra (SN), hippocampus (HIP), and cortex (CTX) regions of the midbrain. .

Methods for measuring the expression level of DRG2 in step b) of the present invention include reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, Western blot, Northern blot , enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoprecipitation assay, and immunohistochemical analysis.

As another aspect, the present invention provides a dopamine dysfunction disease animal model in which the DRG2 gene is knocked out.

The DRG2 gene may have a polynucleotide sequence of SEQ ID NO: 1 or 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence homology thereto.

As used herein, the term "sequence homology" refers to nucleotide sequence identity in the target gene sequence, after aligning the sequences with respect to the donor and target gene polynucleotide sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity. It is defined as the percent identity of a nucleotide residue in a donor sequence compared to a residue. Alignment to determine percent nucleotide sequence homology can be achieved in a variety of ways within the skill of the art, for example, using commercially available computer software BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software. there is. Those skilled in the art can determine appropriate parameters for alignment of sequences, such as any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared.

As used herein, the term "disease model animal" refers to an animal having a disease very similar to a human disease. In the study of human disease, disease model animals have significance due to physiological or genetic similarities between humans and animals. In disease research, biomedical disease model animals provide materials for research on various causes, developmental processes, and diagnosis of diseases, and through disease model animal studies, disease-related genes can be identified and interactions between genes can be understood. In addition, through the actual efficacy and toxicity test of the developed new drug candidate, basic data can be obtained to determine the feasibility of commercialization.

As used herein, the term "knock-out" refers to a modification on a gene sequence that results in a decrease in the function of a target gene, preferably resulting in undetectable or meaningless expression of the target gene. In the present invention, knockout of the DRG2 gene means that the function of the DRG2 gene is substantially reduced so that expression cannot be detected or exists only at an insignificant level. A knockout transformant can be a transgenic animal having a heterozygous knockout of the DRG2 gene or a homozygous knockout of the DRG2 gene. Knockout includes, for example, exposure of the animal to substances that enhance target gene modification, introduction of enzymes that enhance recombination at the target gene site, or other methods that direct target gene modification after birth. Also included are conditional knockouts in which transformation can occur.

In the present invention, the term "animal" means any mammal other than human. The animals include animals of all ages including embryos, fetuses, neonates and adults. Animals for use in the present invention are available, for example, from commercial sources. These animals include laboratory animals or other animals, rabbits, rodents (eg mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (eg chickens, turkeys, ducks, geese), primates (eg chimpanzees, monkeys, rhesus monkeys).

In the present invention, the term "target gene" refers to a nucleic acid encoding an intracellular polypeptide, unless specifically specified otherwise. A "target sequence", unless specifically indicated otherwise, is a portion of a target gene, e.g., one or more exon sequences of a target gene, an intron sequence or regulatory sequence of a target gene, or an exon and intron sequence, an intron of a target gene. and regulatory sequences, exons and regulatory sequences, or combinations of exons, introns and regulatory sequences.

The animal model of the present invention reduces dopamine release in the striatum of the brain by knocking out the DRG2 gene, resulting in motor coordination disorder and anxious behavior, resulting in dopamine dysfunction It can be used as a disease animal model.

The dopamine dysfunction diseases may include movement disorders, depression, emotional disorders, obsessive-compulsive disorder, autism, schizophrenia, attention deficit hyperactivity disorder, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia and Huntington's disease, but However, if it is a disease related to dopamine dysfunction, the type is not particularly limited.

In another aspect, the present invention provides a test group comprising: i) administering a test substance to a dopamine dysfunction disease animal model in which the DRG2 gene is knocked out as an experimental group; ii) measuring and comparing motor coordination between the experimental group in i) and a control animal model not treated with the test substance; And iii) selecting a test substance in which motor coordination is improved in the experimental group of i) compared to the control group.

In step ii), in addition to measuring motor coordination between the experimental group in which the test substance was administered to the dopamine dysfunction disease animal model in which the DRG2 gene was knocked out, and the control animal model not treated with the test substance, additionally Depressive behavior can be further measured.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Example>

1. Materials and Methods

<1-1> Mouse specification management

DRG2 ^+/- -C57BL/6 (heterozygous mutant) and DRG2 ^-/- -C57BL/6 (homozygous mutant) mice and genotyping primer sets have been previously described (Mani, M.; Lee, UH; Yoon , NA; Kim, HJ; Ko, MS; Seol, W.; Joe, Y.; Chung, HT; Lee, BJ; Moon, CH; et al. Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling. Mol. Biol. Cell 2016, 27, 334-348.). After mating the heterozygous mutant mice, DRG2 ^+/+ -C57BL/6 (wild type) and DRG2 ^-/- -C57BL/6 (homozygous mutant) mice were identified through genotype analysis, and DRG2 ^-/- mice After backcrossing to a C57BL/6 WT background for 10 generations, they were maintained in the same animal facility at Korea Brain Research Institute (KBRI). Mice were housed in groups of 2-5 animals per cage and were allowed free access to food and water. It was turned on at 07:00 to maintain a 12-12 light/dark cycle, and humidity (about 55%) was maintained through a constant air flow under an ambient temperature of 20-22°C. Animal health was monitored regularly. All experimental designs were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of KBRI (IACUC-18-00018) (2018. 08. 07). Behavioral testing was performed during circadian light cycles in the laboratory. The rotarod test, the Y-maze test, and the elevated plus maze test (Elevated Plus Maze test) were performed using the same mice. On the day of testing, mice were habituated to the testing room and given 30 minutes to acclimate to their home cages.

<1-2> Rotarod Test

A rotarod test was performed to evaluate motor ability learning. Prior to the training session, mice were habituated to stay on the stationary drum for 3 min. The habit was repeated for 1 minute each day immediately prior to the session. Mice were tested on a rotating rod (Yusung Lab, Seoul, Korea) at a fixed speed of 20 rpm for 300 seconds, and mouse behavior was evaluated by testing three times per session for three consecutive days. A rest period of 180 s was allowed between each trial. The end of the test was the time when the mouse fell off the bar or when the mouse reached 300 seconds. Delay time was recorded for each trial.

<1-3> Y-maze test

For spatial learning, this experiment used a homemade Y-maze containing three identical arms (50 cm long, 16 cm wide, 32 cm high, set at 120° to each other) illuminated by a dim light. The visual details of the testing room were kept constant throughout the training session. The day before the start of the test, mice were habituated to free exploration of the maze for 5 minutes. The experimental animals were placed at the starting point and visited three open arms for 8 minutes. During the testing interval, each experimental animal was housed in its home cage. For quantitative analysis, the percentage of alternation triplets (entering three different zones sequentially) and the total number of entries were recorded online with a motion video analysis system (Ethovision 2.3, Noldus, Ltd., Wageningen, NL). , USA).

<1-4> Elevated Plus Maze (EPM) test

To measure anxiety-like behavior, a cross-type elevated maze test (Elevated Plus Maze test) was conducted. This device consists of two closed arms (length 50.8 cm, width 12 cm, height 40.6-cm) and two open arms (length 50.8 cm and width 12 cm) surrounded by side walls, and the height of the EPM is raised 72.4 cm from the ground. A common central platform (10 × 10 cm) is formed on it. Mice were habituated by placing them in the center of the maze and exploring for 10 minutes. Then, each mouse was individually placed on a central platform facing the open arm and allowed to freely explore the maze for 5 min. After each session, the device was washed with 10% ethanol. Mouse activity was recorded online using a motion video analysis system (Ethovision 2.3, Noldus, Ltd., Wageningen, NL, USA). The percentage of time spent in the open arm was used as an indicator of anxiety-like behavior.

<1-5> Negative Geotaxis Test

Negative geotaxis was assessed according to the protocol of a previous report. Briefly, mice were habituated by placing them on a grid for 5 min, and each neonate (P4 or older) was placed on a rough surface grid at an angle of approximately 35° with the mouse head facing down. ) was tested by placing. Pups were scored as follows: if they turned around or climbed up in less than 60 seconds, tried and failed to turn around in less than 60 seconds, failed to perform the test due to muscle weakness that made them barely able to stand or complete paralysis.

<1-6> In Situ Hybridization

Briefly, frozen sections were cut coronally between the hippocampal formation. Sections were thaw-mounted on Superfrost Plus Microscope Slides (# 12-550-15, ThermoFisher Scientific, Mass., USA). Sections were fixed in 4% paraformaldehyde for 10 minutes, dehydrated in increasing concentrations of ethanol for 5 minutes, and then air dried. Tissue sections were then pretreated for protease digestion for 10 minutes at room temperature. Probe hybridization and amplification were performed at 40°C using a HybEZ hybridization oven (Advanced Cell Diagnostics, Hayward, CA, USA). The probes used in this study were Mm-DRG2dml(nt) 2-306, Mm-Gad1-C2dml nt 62-3113, Mm-Gad2-C3dml nt 552-1506, Mm-Slc17a7/Vglut1-C3 nt 464-1415, These were three synthetic oligonucleotides complementary to nt 1986-2998 of Mm-Slc17a6/Vglut2-C2, and nt 483-1603 of Mm-TH-C2 (Advanced Cell Diagnostics, Hayward, CA, USA). The labeled probes were conjugated to Alexa Fluor 488, Atto 550 and Atto 647. Tissue sections were hybridized with the labeled probe mixture at 40° C. for 2 hours per slide. Unbound hybridization probes were removed by washing the sections 3 times with 1x wash buffer for 2 minutes at room temperature. For signal amplification, a process of reacting at 40°C for 30 minutes with Amplifier 1-FL, 15 minutes with Amplifier 2-FL, 30 minutes with Amplifier 3-FL, and 15 minutes with Amplifier 4 Alt B-FL was included. Thereafter, each amplifier solution was removed by washing with 1x washing buffer for 2 minutes at room temperature. The slides were observed, analyzed, and photographed using the TCS SP8 Dichroic/CS (Leica, Wetzlar, Germany), and the images were analyzed using the ImageJ program (NIH).

<1-7> Immunoblotting, immunostaining and quantification of TH-positive neurons

To collect mouse tissue, the mouse was deeply anesthetized with a 2.5% Avertin solution, and blood was removed by transcardial perfusion with an ice-cold PBS solution. Each organ tissue was rapidly dissected and flash-frozen in liquid nitrogen and stored at -80°C. Mouse tissue lysates were homogenized with RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) and T-PER buffer containing protease and phosphatase inhibitors (ThermoFisher Scientific, MA, USA). Tissue extracts were centrifuged twice at 14,000 rpm at 4° C. for 20 minutes to collect the supernatant. Equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Subsequently, the membrane was blocked with TBS containing 5% (w/v) nonfat milk (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature. Contains anti-DRG2 (anti mouse-DRG2 (Proteintech, Rosemont, IL, USA)) and anti-β-actin primary antibody (anti-β-actin (Cell Signaling, Danvers, MA, USA)) in 5% normal goat serum diluted in TBS-T (TBS-Tween 20; 0.05% Tween 20) and reacted with the membrane overnight at 4°C. The membrane was washed 4 times for 15 minutes each time at room temperature in TBST to remove unbound antibody. The secondary antibody conjugated with HRP was reacted for 1 hour at room temperature. After extensive washing with TBST, immunoreactivity was detected using the GE HealthCare Chemi image documentation system.

Immunocytochemistry was performed on samples obtained from mice deeply anesthetized with 2.5% Avertin solution, perfused by transcardial perfusion with cold PBS solution, and then harvested with 4% paraformaldehyde (PFA) in PBS. Dissected brains were maintained in 4% PFA in PBS overnight at 4°C, then fixed brains were maintained in 10% sucrose 1x PBS buffer containing 4% PFA at 4°C overnight. Thereafter, the fixed brains were sectioned using a cryostat (CM1850, Leica, Wetzlar, Germany) at a thickness of 50-100 μm before O.C.T. compound (Sakura Finetek, Torrance, CA, USA). Each brain slice was washed once with PBS, permeabilized with PBS containing 0.2% Triton X-100 for 1 hour at room temperature, and then blocked with PBS containing 2.5% normal goat serum for 1 hour at room temperature. . Primary antibodies (rabbit anti-DRG2 (NOVUS, Centennial, CO, USA), mouse anti-Tuj1 (Abcam, Cambridge, UK), mouse anti-GFAP (Abcam, Cambridge, UK), goat anti-Iba1 (Abcam, Cambridge , UK), and mouse anti-TH (Sigma-Aldrich, St. Louis, MO, USA)) were diluted in blocking buffer and applied to sections overnight at 4°C. Sections were then washed 4 times with PBS containing 0.1% Tween 20 for 15 minutes each time and conjugated with Alexa Fluor 488/594 (ThermoFisher Scientific, Waltham, MA, USA) for 2 hours at room temperature or overnight at 4°C. Staining with secondary antibody followed by DAPI staining in PBS for 15 min. Biotinylated primary antibody was used for TH neuron staining, followed by addition of an anti-biotin secondary antibody followed by incubation with streptavidin-horseradish peroxidase complex for 1 hour, followed by dialysis. Exposure to minobenzidine (diaminobenzidine). Sections were additionally washed three times with PBS containing 0.1% Tween 20 and washed twice with water. The sections were mounted on a VECTASHIELD (Vector Laboratories, Burlingame, CA, USA) and examined on a Nikon A1rsi/Ni-E inverted confocal microscope with a 60x, 1.4NA oil immersion objective at KBRI's Advanced Neural Imaging Center. was used to capture images of the cross section.

The number of TH positive cells was counted manually without genotype. When TH positive neurons are seen, starting with the first slide of substantia nigra (SN), ventral tegmental area (VTA), septal nucleus (NAc) and striatum (Str) sections of the midbrain, all TH neurons span the entire midbrain in both hemispheres. The substantia nigra (SN) and ventral tegmental area (VTA) were counted every 6 slides. The estimated total number of TH positive neurons in the brains of DRG2 ^−/− mice was calculated by adding the number of TH positive cells and expressed as % of TH positive neurons in the brains of DRG2 ^+/+ mice. TH staining intensity of striatum (Str) and septal nucleus (NAc) was measured by ImageJ software. Three mice from each group were analyzed at 24 months of age.

<1-8> Measurement of dopamine release using high-performance liquid chromatography (HPLC)

Dissected striatal (Str) tissue was frozen in liquid nitrogen and stored at -80 °C. Striatal (Str) tissue samples were homogenized in 0.3 N perchloric acid followed by sonication at 12V 7 times. Lysates were centrifuged at 18,000 x g for 10 minutes at 4°C. A sample of the supernatant (10 μL) was injected into an HPLC-ECD system configured with an UltiMate™ 3000 ECD-3000RS (ThermoFisher Scientific, Waltham, MA, USA). Hypersil™ BDS C18 column (3 μm) with Hypersil™ BDS guard column (28103-013001, ThermoFisher Scientific, Waltham, MA, USA) and UniGuard™ guard cartridge holder (852-00, ThermoFisher Scientific, Waltham, MA, USA) , 3 x 150 mm), chromatographic separation was done. The mobile phase was Dionex™ mobile phase (70-3829, ThermoFisher Scientific, Waltham, MA, USA). The column temperature was 35°C and the flow rate was 0.5 mL/min. ECD cell potentials were 250 mV and 400 mV. The peak of each neurotransmitter was identified by retention time compared to the time of the standard dopamine peak. Concentrations were calculated by comparing the peak area of the sample chromatogram to that of the standard dopamine chromatogram. Dionex™ Chromeleon™ CDS software (ThermoFisher Scientific, Waltham, MA, USA) was used for data collection and processing. All standard neurotransmitters including dopamine (H8502), DOPAC (850217), 3-MT (M4251), HVA (H1252), EPI (E4375) and 5-HT (H9523) were purchased from Sigma-Aldrich.

<1-9> Measurement of dopamine release from Str by slice fast scan cyclic voltammetry (FSCV)

Electrically evoked dopamine release in the striatum (Str) was measured by FSCV. Mice were anesthetized using isoflurane and sacrificed. Brains were rapidly removed and brain tissue preparations were prepared in cold, preoxygenated cells containing 119 mM NaCl, 26.2 mM NaHCO3, 11 mM glucose, 2.5 mM KCl, 2.5 mM CaCl2 and 1.3 mM MgSO4 in 1 mM phosphate buffer, pH 7.2-7.4. It began with the fabrication of vibratome sections from artificial cerebrospinal fluid (aCSF). Coronal brain slices (300 μm) were maintained in oxygenated artificial cerebrospinal fluid (aCSF) for at least 1 hour at room temperature. For recording, brain slices were transferred to a locally configured submersion recording chamber and perfused with oxygenated aCSF at 1 mL/min at 32°C. Carbon fiber microelectrodes were prepared as described above (Martel, P.; Leo, D.; Fulton, S.; Berard, M.; Trudeau, L.E. Role of Kv1 potassium channels in regulating dopamine release and presynaptic D2 receptor function. PLoS ONE 2011, 6, e20402.), the reference electrode was made of chlorinated silver wire. For dopamine recordings in the striatum (Str), the electrode potential was linearly scanned from -0.4 to 1.2 V versus Ag/AgCl at 400 V/s and then again to -0.4 V again. Scanning was repeated every 100 ms. Dopamine release was stimulated by 1-pulse stimulation (monophasic, 350 μA, 4-ms pulse width) from an adjacent bipolar stimulating electrode (Plastics One, Roanoke, VA, USA) located on the slice surface 100–200 μm away from the carbon fiber microelectrode. evoked every minute. A carbon fiber microelectrode was placed 100 μm below the slice surface. Cyclic voltammetry was recorded at the carbon fiber microelectrode every 100 ms by a ChemClamp voltage clamp amplifier (Dagan Corporation, Minneapolis, MN, USA). Voltammetry readings were collected and analyzed using LabVIEW-based (National Instruments, Austin, TX, USA) custom software (Demon Voltammetry).

<1-10> Application of L-DOPA

Mice received a single intraperitoneal injection of L-DOPA (50 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) (25 mg/kg) dissolved in PBS containing 0.25% ascorbic acid. 3 hours after L-DOPA administration, a rotarod test was performed.

<1-11> Separation of neurons, astrocytes and microglia

Primary cortical neuron cultures were prepared from 16-day-old embryonic mice as previously described (Araki, W., et al., Overexpression of presenilin-2 enhances apoptotic death of cultured cortical neurons. Ann N Y Acad Sci, 2000. 920: p.241-4./ Enokido, Y., et al., Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture. Brain Res, 1992. 599(2): p. 71.). Briefly, mouse embryos were subjected to decapitation, and brains were rapidly removed and placed in culture dishes containing HBSS (Thermo Scientific, Waltham, Mass.). The cortex was dissociated, transferred to a conical tube and washed twice in HBSS (Thermo Scientific). Cortical tissue was enzymatically digested with preheated papain (20 units/ml) (Worthington Biochemical Corporation) and DNase I (0.005%) at 37° C. for 30 min. Tissues were mechanically dissociated (ground) with 1000 μL and 200 μL pipette tips for complete tissue homogenization. Cortical cells were centrifuged at 800 rpm for 10 min at room temperature, and the obtained cells were supplemented with 2 mM glutamine (Thermo Scientific), N2 supplement (Thermo Scientific), B27 supplement (Thermo Scientific) and penicillin-streptomycin (Thermo Scientific). The neurobasal media to be plated were dispensed onto plates coated with poly-D-lysine (Sigma-Aldrich). The culture medium was replaced every 5 days and then every 3 days, and after culturing for 18 days, the cells were used in experiments.

Astrocytes and microglia cultures were prepared from the brain by the method of McCarthy and de Vellis (McCarthy, KD and J. de Vellis, Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol, 1980. 85( 3): p. 890-902.). Briefly, whole brains were homogenized on a 70-μm strainer. Cells were seeded into T75 culture flasks or 100 mm culture dishes. Cells were grown at 37 °C in a humidified atmosphere containing 5% CO ₂ . The culture medium was changed for the first 5 days and then every 2 days, and cells cultured for 14-21 days were used. The mixed glial cultures were shaken at 250 rpm for 4 hours to obtain secondary pure astrocyte cultures, and then the culture medium was discarded. After the astrocytes were dissociated using trypsin-EDTA (Life Technologies), centrifugation was performed at 2000 rpm for 30 minutes. Astrocytes obtained after centrifugation were seeded onto plates of DMEM (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and penicillin-streptomycin (Thermo Scientific). Pure microglia culture was performed using a mild trypsinization solution (155 mM NaCl, 2.9 mM Na2HPO4-7H2O, 1 mM KH2PO4, 0.2 mM EDTA, 0.5 mM CaCl2, 0.05% trypsin-EDTA (Ph 7.4)). Briefly, 5 ml of mild trypsinization solution was added to each 100 mm dish and the dish was incubated at 37°C for 30 to 60 minutes. The separated astrocyte layer was discarded, microglia were separated using trypsin-EDTA (Life Technologies), and then centrifuged at 2000 rpm for 30 minutes. Microglia obtained after centrifugation were seeded onto plates of DMEM (Life Technologies) supplemented with 10% heat-inactivated FBS (Thermo Scientific) and penicillin-streptomycin (Thermo Scientific).

<1-11> Statistical analysis

Analysis and quantification of data was performed with SigmaPlot 13.0 (Systat Software, Point Richmond, CA, USA), and data were presented as mean ± standard deviation. Results were analyzed by group or time using one-way ANOVA and considered significant when p<0.05. Significance is indicated as follows: * p < 0.05; **p < 0.01; ***p < 0.001; N.S. is not significant

2. Results

<2-1> Growth retardation and skeletal defects in DRG2 knockout (KO) mice

To determine the physiological function of DRG2, the phenotype of DRG2 ^-/- (homozygous mutant) mice compared to DRG2 ^+/+ (wild type (WT) control) and DRG2 ^+/- (heterozygous mutant) mice. Changes were observed first. The absence of DRG2 mRNA and protein in the brain cortex of DRG2 ^-/- mice was confirmed by RT-PCR (see Fig. 1A) and Western blotting (see Fig. 1B). DRG2 ^-/- mice appeared to be significantly smaller at birth (1 day old) than DRG2 ^+/+ and DRG2 ^+/- littermates, and this size difference persisted into adulthood (12 weeks of age) (see Figure 1C). ). Consistently, the body weight of DRG2 ^-/- mice was significantly smaller than that of wild-type (control) mice (see Fig. 1F). To determine whether the small body size of DRG2 ^-/- mice is related to skeletal changes, in this experiment we analyzed bone tissue from postnatal 1-day-old pups using micro-computed tomography (CT). As a result, long bones in the appendicular skeleton of DRG2 ^+/- mice were found to have apparently normal bone density, but DRG2 ^-/- mice were about 8 to 33% higher than those of wild-type (control) mice. appeared to be shorter. In addition, DRG2 ^-/- mice showed significantly reduced mineralization of the skull and increased non-ossified areas in the anterior fontanel (see Fig. 1E). However, although DRG2 ^-/- mice showed reduced skull ossification, there was no significant difference in total brain size, total brain weight, or brain structure (see Figs. 6D and 6E).

In addition, in this experiment, the survival rate after birth was further investigated to confirm the growth retardation phenomenon that causes the observed physiological changes. As a result, as shown in Figure 1D, in the case of DRG2 ^-/- mice, it was found that the survival rate was significantly reduced compared to the wild type (p <0.001), and the survival rate of DRG2 ^-/- mice after 1 year was the same as that of wild type mice. , whereas only 20% of the DRG2 ^+/- mice showed no significant change in survival (p > 0.05).

<2-2> Regional distribution of DRG2 protein in mouse brain

In this experiment, we characterized the detailed phenotypic expression pattern of mouse DRG2 to determine how the growth retardation induced by DRG2 deficiency affects brain function. The gene and protein expression of DRG2 in specific brain regions is unknown. Therefore, in this experiment, first, each organ tissue was stained using an antibody specific for the DRG2 protein, and brain-region specificity of the DRG2 protein was confirmed. As a result, DRG2 protein was strongly expressed in the brain, lung and spleen, whereas it was weakly expressed in the thymus, liver, testis, kidney and lung (see Fig. 2A). In the adult mouse brain, DRG2 protein is mainly expressed in the cortex (CTX), hippocampus (HIP), cerebellum (CB), and substantia nigra (SN), whereas less protein is expressed in the hypothalamus (HY) and striatum (Str). was detected (see Fig. 2B). The protein expression level of DRG2 did not show significant differences during postnatal development between brain regions with high expression (Fig. 2C).

In this experiment, in order to further determine the function of DRG2 in the brain, the expression pattern of DRG2 was confirmed in the adult mouse brain using in situ hybridization and immunohistochemistry. As a result, in situ hybridization revealed widespread expression of DRG2 across the mouse brain, but strong signals for DRG2 protein, including in the suprachiasmatic nucleus (SCN), were consistent by western blotting. detected (see Figure 2D). Immunohistochemical analysis showed that the expression pattern of DRG2 protein was similar to that of the DRG2 transcript, but the protein expression was different at the spatial level: accessory olfactory bulb, cortex (CTX), hippocampus (HIP), and midbrain. substantia nigra (SN), cerebellum (CB), and parabrachial nucleus showed high expression levels, whereas moderate expression was found in hypothalamus (HYP), septal nucleus (NAc), and thalamus (THs). Observed. As shown in the enlarged images, DRG2 protein was mainly detected as dots in the perinuclear region and in the Purkinje neuron cell body of the cerebellum. DRG2 protein was also highly expressed in layer VI of the cerebral cortex, cornu ammonis 3 (CA3) of the hippocampus (HIP), substantia nigra pars compacta (SNc) and substantia nigra pars reticulata (Fig. 2E see (a-i)). Next, DRG2 protein expression patterns were examined in coronal sections of mouse brains. DRG2 protein expression was found in the primary motor cortex (somatomotor area) and primary sensory cortex (somatosensory area), retrosplenial area, parietal association area, auditory area, visual area, and olfactory cortex (piriform). area) and dentate gyrus (DG), cornu ammonis 1 (CA1) and CA3 in the limbic system, and all cortical layers in the enolar area during hippocampal formation. DRG2 protein was also highly expressed in the basomedial nucleus of the amygdala (BMAa), thalamus, and midbrain (Fig. 2E (j-l)).

To further determine the cellular specificity of DRG2 expression in the mouse brain, this experiment compared the expression of DRG2 with that of cell-type specific markers (astrocytic marker GFAP; microglia marker Iba1; and neuronal marker Tuj1). Immunohistochemical analysis was performed for As a result, DRG2 was highly associated with Tuj1-positive neurons, but not GFAP-positive astrocytes or Iba1-positive microglia, in brain regions with strong expression, such as substantia nigra (SN), hippocampus (HIP) and cortex (CTX) of the midbrain. colocalized (see FIG. 3A and FIGS. 6A, 6B). To confirm this, neurons, astrocytes and microglia were isolated and the expression of DRG2 in these cells was analyzed using Western blot. As a result, DRG2 expression was detected from all types of cells, but the level of DRG2 expression in neurons (nerve cells) was higher than that in astrocytes and microglia (see Fig. 6C).

In addition, in this experiment, it was confirmed whether DRG2 was differentially expressed between excitatory and inhibitory neurons. In the hippocampus (HIP), DRG2 mRNA was mainly expressed in the dentate gyrus (DG) and Cornu Ammonis regions (CA1 and CA3) (see Figures 3B and 3C). In this experiment, double in situ hybridization was performed using antisense probes for excitatory neuronal markers such as vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2, respectively) and inhibitory neuronal markers (GAD65 and GAD67). ) was performed. As a result, VGLUT1 was mainly expressed in the cerebral cortex, hippocampus (HIP) and cerebellum (CB), whereas VGLUT2 was preferentially expressed in the thalamus (THs) and hypothalamus (HYP). Consistently, we found that DRG2 was expressed in the DG and CA1 regions of the hippocampus (HIP), which highly express VGLUT1, but no VGLUT2 was detected (see Fig. 3D). DRG2 co-localized with GAD65 and GAD67, although the expression levels of GAD65 and GAD67 were very low in the hippocampus (HIP) (see Fig. 3E). These results indicate that the double in situ hybridization was successful and that DRG2 co-localized with inhibitory neurons in the hippocampus (HIP) as well as excitatory neurons.

<2-3> DRG2 deficiency causes motor deficits and increases anxiety in mice

To clarify the effect of DRG2 deficiency on excitatory and inhibitory neurons in brain regions with high DRG2 expression, a series of behavioral tests were performed using DRG2 ^-/- mice. In this experiment, first, a negative geotaxis test was used to evaluate motor coordination and vestibular sensitivity. In this test, the latency required for a mouse to redirect to an overhead position on an inclined plane was recorded, and the time for a mouse that failed to redirect to an overhead position was recorded as 60 seconds. Although an initial delay in reorientation was observed in postnatal day 4 (P4) pups for wild-type mice, all wild-type (WT) mice readily repositioned their heads within 60 seconds during postnatal life. In contrast, pups of DRG2 ^-/- mice were unable to stand on an inclined plane until postnatal day 8 (P8) and did not complete the negative noticeability test, and were unable to complete the test by postnatal day 10 (P10). there was. Post Hoc Analysis showed significant differences between DRG2 ^-/- mice and wild-type control mice at P4 (p = 0.002), P6 (p = 0.002) and P8 (p = 0.015) (see Figure 4a). ).

In addition, a rotarod test was performed to investigate motor coordination and motor learning skills in various age groups. A fixed speed version of the rotarod task was applied to 6, 12 and 40 week old wildtype and DRG2 ^−/− mice. Compared to wild-type mice (300 seconds), 6-week-old female DRG2 ^−/− mice showed significantly reduced motor performance (243±22 seconds). 12- or 40-week-old female DRG2 ^-/- mice stayed much shorter (123±46 sec and 86±19 sec, respectively) than wild-type and 6-week-old DRG2 ^-/- mice (see FIG. 4B ). The reduced time in DRG2 ^−/− mice except for 40 week old DRG2 ^−/− mice observed in the rotarod test was improved by repeated testing 1 week later. These results suggest that DRG2 ^-/- mice have a defect in motor coordination but normal motor learning skills. Next, we evaluated whether DRG2 deficiency affects learning and memory behavior using the Y-maze test. As a result, the percentage of alternation triplets (when entering three different zones sequentially), the total number of passages, and the total distance at 12 weeks of age in male and female DRG2 ^-/- young (6 weeks old) and adult (12 weeks old) mice compared to wild type. side showed similar results (see Fig. 4c). These data suggest that DRG2 deficiency does not affect memory performance in mice.

In addition, the anxiety and fear-related behaviors of DRG2 ^-/- mice were evaluated using the Elevated Plus Maze (EPM) test. As a result, both male and female 12-week-old DRG2 ^-/- mice spent significantly less time in the open arm than wild-type mice of the same age (see Fig. 4d), suggesting that DRG2 deficiency causes anxiety-like behavior in mice. refers to increasing

<2-4> Abnormal behavior of DRG2-deficient mice is caused by changes in dopamine release

Dopaminergic neurons extend their axons into the striatum (Str) and secrete dopamine, which binds to dopamine receptors D1 and D2 expressed in the striatum (Str). Changes in the number of dopaminergic neurons and dopamine release in the striatum can affect striatal-related behaviors such as anxiety, motor coordination, gait movement, and sensitivity to reward. As changes in motor coordination and anxiety behavior were confirmed in DRG2 ^-/- mice, in this experiment, it was confirmed whether DRG2 deficiency causes deficiency in dopaminergic neurons. Accordingly, the present inventors applied double in situ hybridization using antisense probes for DRG2 and tyrosine hydroxylase (TH) to analyze the expression pattern of DRG2 in dopaminergic neurons (see Table 1 below). A high level of expression of DRG2 was detected in tyrosine hydroxylase (TH)-positive neurons of the ventral tegmental area (VTA) and substantia nigra (SN) of the midbrain (see FIG. 5a). Individual mRNA transcripts appeared as bright dots within cells, and the number of DRG2 mRNA dots was higher in TH-positive cells (SN and VTA regions, 3.2 ± 0.52 and 2.95 ± 0.64, respectively) than in TH-negative cells (SN and VTA regions, respectively). were significantly higher at 11.85 ± 1.26 and 13.10 ± 0.84, respectively (see Fig. 5b). Through the above results, it is possible to suggest a role of DRG2 in dopamine neurons, and it was confirmed that DRG2 deficiency can cause dopaminergic neuron dysfunction.

Apoptosis of dopamine-producing cells in the substantia nigra (SNc) and reduced striatal dopamine release can lead to dopamine neuron dysfunction. We therefore determined whether DRG2 deficiency induced changes in the population of tyrosine hydroxylase (TH)-positive neurons or in striatal dopamine release. Tyrosine hydroxylase (TH)-positive neurons were stained with an anti-TH antibody and detected using secondary antibodies conjugated with horseradish peroxidase complex or Alexa Fluor 488/594. First, as a result of quantifying tyrosine hydroxylase (TH) intensity and tyrosine hydroxylase (TH)-positive neurons, tyrosine hydroxylase in SN, VTA, NAc and Str regions between 24-month-old wild-type and DRG2 ^-/- mice No significant differences were found in the intensity and distribution of oxylase (TH)-positive neurons (see FIGS. 5C and 7A,B). In addition, DRG2 deficiency did not induce any changes in target regions of the nistriatal or mesolimbic pathways of tyrosine hydroxylase (TH)-positive neurons (see Fig. 7C). The above experimental results suggest that DRG2 deficiency does not affect dopamine neuron death.

Next, striatal monoamine levels involved in the regulation of motor behavior were analyzed. As a result, dopamine content (~29%, p = 0.0007), 3-methoxytyramine (3-MT) level (~27%, p = 0.0062) and epinephrine (EPI) level (~24%, p = 0.0062) in the striatum 0.0289) was confirmed to be significantly reduced in DRG2 ^-/- mice. However, the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and serotonin (5-hydroxytryptamine, 5-HT) showed no significant differences between wild-type and DRG2 ^-/- mice ( see Fig. 5d). Through the above experimental results, it was determined that DRG2 deficiency caused nigrostriatal dopaminergic dysfunction and reduced dopamine levels in the striatum.

We next tested whether DRG2 deficiency affects presynaptic dopamine release using mouse brain slices containing substantia nigra (SN) and striatal (Str) regions of the midbrain. Brain slices were stimulated by bipolar stimulation electrodes, and dopamine release was measured using carbon fiber recording microelectrodes. Dopamine release in the brain slices of DRG2 ^-/- mice was measured to be significantly lower than that in brain slices of wild-type mice (see Fig. 5e). On the other hand, to determine whether reduced dopamine release causes altered motor coordination behavior in DRG2 -/ ^- mice, DRG2 ^-/- mice were treated with L-DOPA and a rotarod test was performed. Compared with no treatment or PBS treatment, administration of L-DOPA significantly increased motor coordination in DRG2 ^-/- mice in the rotarod test (see Fig. 5f). Taken together, the experimental results suggest that DRG2 deficiency reduces nigro-striatal dopamine release, and this reduction in dopamine release causes motor coordination and anxiety behavior.

Antisense probe used for double in situ hybridization Probe name catalog number where to buy DRG2 RNAscope®Probe- Mm-Drg2-O1 588531 Advanced Cell Diagnostics, Hayward, CA, USA Tyrosine hydroxylase (TH) RNAscope®Probe- Mm-tyrosine hydroxylase (Th)-C2 317621 Advanced Cell Diagnostics, Hayward, CA, USA

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

DRG2: Developmentally regulated GTP-binding protein 2
Str: Striatum
Ventral tegmental area (VTA)
SN: Substantia nigra
SNc: Substantia nigra pars compacta
SNr: Substantia pars reticulate
HIP: Hippocampu
CB: Cerebellum
CTX: cerebral cortex
SCN: suprachiasmatic nucleus
THs: Thalamus
PB: Parabrachial nucleus
HY or HYP: Hypothalamus
AOB: Accessory olfactory bulb
AUD: Auditory areas
BMA: Basomedial amygdala nuclear
CA1: Cornu ammonis1
CA3: Cornu ammonis3
DG: dentate gyrus
CP: Caudate putamen
ENT: Entorhinal area
MB: mid-brain
Mo: Momatomotor area
NAc: Nucleus accumbens
Pir: Piriform cortex
PTLP: Posterior parietal association areas
RSP: Retrosplenial area
SS: somatosensory area
SUB: Subiculum
VIS: visual area
WB: Whole brain
PD: Parkinson's disease
MT: microtubule
Micro-CT: Micro-computerized tomography
AF: Aanterior fontanel
PM: Premaxillary-maxillary
DOPAC: 3,4-dihydroxyphenylaceti
3-MT: 3-methoxytyramine
HVA: Homovanillic acid
EPI: Epinephrine
5-HT: Serotonin
TH: Tyrosine hydroxylase
DA: Dopamine
L-DOPA: l-3,4-dihydroxyphenylalanine
EN: Knockout

<110> Korea Brain Research Institute <120> Use of DRG2 gene as dopamine release modulator <130> NPDC-87628 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1095 <212> DNA <213> artificial sequence <220> <223> DRG2 polynucleotide sequence <400> 1 atggggatct tggagaaaat ctcggagatc gagaaggaga tcgctcgaac gcagaagaac 60 aaggccaccg agtaccacct gggcctgctg aaagcaaaac tggccaagta tcgggcccag 120 ctcctggaac catccaaatc cgcctcatcc aaaggagagg gctttgatgt catgaagtca 180 ggtgatgccc gtgtggcact gattggcttt ccctctgtgg gtaagtccac cttcttgagt 240 ctgatgacct ctacagccag cgaagctgcg tcctatgagt ttaccaccct gacgtgcatc 300 cctggggtca ttgaatacaa aggtgccaac atccagctct tggaccttcc tggaatcatt 360 gaaggagcag cacaaggccg aggccgtggc cggcaggtga ttgccgtagc acgcacagct 420 gatgtcgtcg tcatgatgct ggatgccacc aagggagatg tgcagaggtc cctgctggag 480 aaggagctgg agtctgtggg tattcgcctg aacaagcata agcccaacat ctatttcaag 540 cccaagaaag gtggtggcat ctcctttaac tcaacagtca cactgacaca atgctctgag 600 aagctggtgc agctcatcct acatgaatac aagatcttca acgccgaggt actcttccga 660 gaagactgct ctccagacga cttcatcgat gtgattgtgg gcaaccgggt gtatatgccc 720 tgcctctatg tgtataacaa aatcgaccag atctccatgg aagaagtaga ccgtttagct 780 cgaaagccca acagtgtagt tatcagttgc ggtatgaagt tgaacctcga ctacctgctg 840 gagatgctgt gggaggtacct ggccctcacc tgcatttaca ccaagaagag aggacagaga 900 ccggacttca cagacgccat cattctccgg aaaggggcct ctgttgaaca cgtgtgccac 960 cgcatccatc ggtcactggc cagccagttc aagtacgcct tggtgtgggg taccagcacc 1020 aagtacagcc cacagcgggt aggcctgacc cacactatgg agcatgagga cgtcatccag 1080 attgtgaaga agtag 1095

Claims (10)

As an information providing method for the diagnosis of dopamine dysfunction disease,
The method includes measuring the expression level of the DRG2 gene in a target sample to be analyzed and comparing it with a normal control group,
The dopamine dysfunction disease is characterized in that selected from the group consisting of movement disorders, depression, anxiety disorders and Parkinson's disease, information providing method for diagnosis of dopamine dysfunction disease. According to claim 1,
An information providing method for diagnosing dopamine dysfunctional disease, characterized in that diagnosing it as a dopamine dysfunctional disease when the expression level of the DRG2 gene in the target sample is reduced compared to a normal control group. delete As a method for screening candidates for preventing or treating dopamine dysfunction diseases,
The method comprises the steps of a) contacting a test substance to be analyzed with brain-derived nerve cells; b) measuring the expression level of DRG2 in nerve cells in contact with the test substance; and c) selecting a test substance having an increased expression level of DRG2 compared to a control group not treated with the test substance,
The dopamine dysfunction disease is a method for screening candidates for preventing or treating dopamine dysfunction disease, characterized in that selected from the group consisting of movement disorders, depression, anxiety disorders and Parkinson's disease. According to claim 4,
The measurement in step b) is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, Western blot, Northern blot, ELISA (enzyme linked immunosorbent assay), radiation Preventing or treating a dopamine dysfunction disease characterized by being selected from the group consisting of radioimmunoassay (RIA), radioimmunodiffusion, immunoprecipitation assay and immunohistochemical analysis Candidate screening method for As a non-human dopamine dysfunction disease animal model,
The animal model is a knock-out of the DRG2 gene,
The dopamine dysfunction disease is characterized in that selected from the group consisting of movement disorders, depression, anxiety disorders and Parkinson's disease, dopamine dysfunction disease animal model. According to claim 6,
The animal model is a dopamine dysfunction disease animal model, characterized in that the release of dopamine in the striatum of the brain is reduced by knocking out the DRG2 gene. According to claim 6,
The animal is a dopamine dysfunction disease animal model, characterized in that at least one species selected from the group consisting of mice, monkeys, dogs, cats, rabbits, rats, cows, sheep, pigs and goats. delete i) administering a test substance to the animal model of claim 6 as an experimental group;
ii) measuring and comparing motor coordination between the experimental group in i) and a control animal model not treated with the test substance; and
iii) a method for screening a candidate substance for preventing or treating dopamine dysfunction disease, comprising the step of selecting a test substance in which motor coordination is improved in the experimental group of i) compared to the control group.

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