KR102684933B1 - Use of GAT-3 for the diagnosis of attention deficit / hyperactivity disorder - Google Patents

Abstract

The present invention relates to the use of GAT-3 for the diagnosis of attention deficit hyperactivity disorder (ADHD), in the striatum of the brain that controls hyperactivity in Git1 gene-deficient hetero (+/-) mice. By confirming that the amount of GABA increases, it can be used as an attention deficit/hyperactivity disorder (ADHD) animal model, and GAT-3 (GABA transporter-3) is expressed in Git1 gene-deficient hetero (+/-) mice. By confirming that the protein expression level increased and that hyperactivity was improved by administering SNAP5114, an inhibitor of GAT-3 (GABA transporter-3), GAT-3 (GABA transporter-3) It is provided as a biomarker for diagnosing attention deficit/hyperactivity disorder (ADHD).

Description

Use of GAT-3 for the diagnosis of attention deficit / hyperactivity disorder}

The present invention relates to the use of GAT-3 for the diagnosis of attention deficit hyperactivity disorder (ADHD).

Attention deficit hyperactivity disorder (ADHD) is one of the common mental disorders that first appears in childhood, but it can also develop in adulthood and throughout adulthood. It has been reported that approximately 4.1% of children aged 9 to 17 years suffer from ADHD. Children with ADHD are unable to concentrate on a task, sit quietly, act impulsively, and are unable to complete activities. If left untreated, the child's injury rate increases and the disorder has long-term negative effects on the child's ability to make friends and perform at school and/or studies. Over time, children with ADHD have an increased risk of developing depression, low self-esteem, and other emotional problems.

In most cases, infants and adults with ADHD are treated with psychostimulants such as amphetamines, methylphenidate, and pemoline. Additionally, antidepressants such as desimpramine, which act by selectively blocking the reuptake of norepinephrine, are also effective in some cases. Additionally, new drugs such as atomoxetine, which block the reuptake of norepinephrine and serotonin, may also be effective in treating this disorder. Although psychostimulants and monoamine reuptake inhibitors control activity levels and attention, they are not effective in treating the comorbid or concomitant cognitive deficits associated with ADHD.

The cause of ADHD is genetic factors, biochemical factors that are seen as a reaction to lead levels and food additives in instant foods, the view that there is a problem in appropriately selecting the stimulation delivered to the brain, and environmental factors, that is, the relationship between parents and children. The parents' social status and the mother's smoking and alcohol abuse during pregnancy were also mentioned, but discussions about the cause are still divided. However, neurobiological factors are considered more important than psychosocial factors, and accordingly, research on drug treatment and biological factors for this disease is actively underway. In particular, it is suggested that it may be a non-uniform group of diseases caused by abnormalities in the interconnectivity of several brain regions responsible for high-level cognitive functions rather than abnormalities in the development of a single nervous system due to biological factors. Looking at structural and functional brain imaging studies on children with ADHD until recently, the pathophysiology of ADHD is generally associated with dysfunction of the fronto-striatal tract, and methylphenidate (MPH) ) is known to be associated with changes in the function of the dopamine system in this area. ADHD symptoms are related to the regulation of noradrenergic nerves, which control the neural circuits of the frontal lobe in addition to dopaminergic nerves. Studies have also reported that ADHD behavior is caused by an imbalance between the regulation of noradrenergic and dopaminergic neurons. Comprehensively, it is understood that this is due to a decrease in the influence of catecholamines, which control glutamatergic neurons and GABA (g-aminobutyric acid) neurons, on the postsynaptic region.

Korean Patent Publication No. 10-2005-0085538 (announced on August 29, 2005)

The purpose of the present invention is to provide a biomarker composition for diagnosing attention deficit hyperactivity disorder (ADHD).

The present invention provides a diagnostic biomarker composition for attention deficit hyperactivity disorder (ADHD) containing GAT-3 (GABA transporter-3) or the gene encoding it as an active ingredient.

In addition, the present invention provides a diagnostic kit for attention deficit hyperactivity disorder (ADHD) containing as an active ingredient an agent capable of detecting the expression level of the GAT-3 (GABA transporter-3) protein or the expression level of the gene encoding it. to provide.

In addition, the present invention includes the steps of measuring the expression level of GAT-3 (GABA transporter-3) protein or the expression level of the gene encoding it in a biological sample isolated from a subject suspected of having attention deficit hyperactivity disorder (ADHD); and comparing the expression level with a biological sample isolated from a normal person.

According to the present invention, it was confirmed that the amount of GABA increased in the striatum of the brain, which controls hyperactivity, in Git1 gene-deficient hetero (+/-) mice, resulting in attention deficit hyperactivity disorder (attention deficit/hyperactivity disorder). disorder, ADHD), it can be used as an animal model, and the expression level of GAT-3 (GABA transporter-3) protein was confirmed to be increased in Git1 gene-deficient hetero (+/-) mice. By confirming that hyperactivity was improved by administering SNAP5114, an inhibitor of ), it was confirmed that GAT-3 (GABA transporter-3) can diagnose attention deficit/hyperactivity disorder (ADHD). It can serve as a biomarker.

Figure 1 shows the results of evaluating changes in the amount of GABA in an ADHD (attention deficit / hyperactivity disorder) animal model using fluorescent immunohistochemistry (fIHC).
Figure 2 shows the results of evaluating changes in the amount of GABA in an ADHD (attention deficit / hyperactivity disorder) animal model using electrophysiological measurements (patch clamp recording).
Figure 3 shows the results of measuring the expression level of GAT-3 (GABA transporter-3) in an ADHD (attention deficit / hyperactivity disorder) animal model.
Figure 4 shows the results of evaluating changes in hyperactivity following the administration of a GAT-3 (GABA transporter-3) inhibitor in an ADHD (attention deficit / hyperactivity disorder) animal model.

The terms used in this specification are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention or precedent of a person working in the art, the emergence of new technology, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless clearly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

The numerical range includes the values defined in the range above. Any maximum numerical limit given throughout this specification includes all lower numerical limits as if the lower numerical limit were explicitly written out. Every minimum numerical limit given throughout this specification includes every higher numerical limit as if such higher numerical limit was explicitly written out. All numerical limits given throughout this specification will include all better numerical ranges within the broader numerical range, as if the narrower numerical limits were clearly written.

Hereinafter, the present invention will be described in more detail.

The present invention provides a diagnostic biomarker composition for attention deficit hyperactivity disorder (ADHD) containing GAT-3 (GABA transporter-3) or the gene encoding it as an active ingredient.

In addition, the present invention provides a diagnostic kit for attention deficit hyperactivity disorder (ADHD) containing as an active ingredient an agent capable of detecting the expression level of the GAT-3 (GABA transporter-3) protein or the expression level of the gene encoding it. to provide.

Agents that can measure the expression level of the protein are antibodies, peptides, aptamers, or compounds that specifically bind to the protein, and agents that can measure the expression level of the gene are primers that specifically bind to the gene or It's a probe.

The antibodies include polyclonal antibodies, monoclonal antibodies, recombinant antibodies and intact forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule, such as Fab, F(ab' ), F(ab')2, and Fv. Antibodies can be easily produced using techniques well known in the field to which the present invention pertains, and antibodies that have been manufactured and sold commercially can be used.

Additionally, the level of protein can be measured by immunoassay or immunostaining. The method may be implemented in the form of a microchip or automated microarray system capable of detecting the biomarker protein or fragment thereof in a sample.

The immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification.

The protein level can be measured through multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), and selected reaction monitoring (SRM). ) or may be measured using immune multiple reaction monitoring (iMRM). MRM is a method of determining the exact fragment of a substance, breaking it in a mass spectrometer, then selecting a specific ion among the broken ions once more and obtaining the number using a continuously connected detector.

The 'gene expression level' can be measured using an antisense oligonucleotide, primer pair, or probe that specifically binds to the mRNA of the gene, and the agent for measuring the expression of the mRNA is an antisense oligonucleotide specific to the gene. selected from the group consisting of nucleotides, primer pairs, probes, and combinations thereof. That is, detection of nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers that hybridize to the nucleic acid molecule encoding the gene or the complement of the nucleic acid molecule, but is not limited thereto. For example, detection of mRNA using primers can be performed by amplifying the gene sequence using an amplification method such as PCR and then confirming amplification using methods known in the art, such as RT-PCR, competitive RT-PCR, quantitative It can be measured by, but is not limited to, RT-PCR, RNase protection assay, Northern blot, or DNA chip.

The "probe" refers to a nucleic acid fragment, such as RNA or DNA, of as short as a few bases or as long as several hundred bases, capable of binding specifically to mRNA, and is labeled to confirm the presence or absence of a specific mRNA and the amount of expression. You can. Probes may be manufactured in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art.

The "primer" is a short nucleic acid sequence that has a short free 3' hydroxyl group that can form base pairs with a complementary template and acts as a starting point for copying the template strand. says Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

In addition, the present invention includes the steps of measuring the expression level of GAT-3 (GABA transporter-3) protein or the expression level of the gene encoding it in a biological sample isolated from a subject suspected of having attention deficit hyperactivity disorder (ADHD); and comparing the expression level with a biological sample isolated from a normal person.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1. ADHD animal model preparation

Git1 gene deletion heterogeneous (+/-) mice were prepared as an attention deficit/hyperactivity disorder (ADHD) animal model. GIT1 gene deletion heterotype mice are obtained by crossing wild-type mice with GIT1 gene deletion knock-out type (KO type) mice. After creating a mating cage for the obtained heterotype mice, genotyping was performed to isolate the heterotype mice.

Open field tests were performed to evaluate behavioral patterns, and fluorescent immunostaining (Immunohistochemistry) and electrophysiological measurements (patch) were performed to confirm the characteristics of tonic GABA in the striatum, a representative brain region that controls hyperactivity. clamp recording) was performed.

Example 2. Assessment of GABA levels in ADHD animal models

1) Fluorescent Immunohistochemistry (fIHC)

In order to perform fluorescent immunohistochemistry (fIHC), perfusion was performed in mice. After primary perfusion with Phosphate-buffered saline (PBS), 0.05% glutaraldehyde was added to 4% PFA (Paraformaldehyde) for fixation. Afterwards, the brain was collected from the skull of the mouse, soaked in 4% PFA, and stored at 4°C for half a day. The fixed mouse brain was placed in a 30% sucrose solution and dried for 2 days. The dried brain was placed in OCT compound and stored in a mold at -80°C. After making the mold, the hippocampus and striatum were sectioned at a thickness of 30 μm using a cryotome. The cut slices were washed three times for 5 minutes with PBS and mixed with triton-X100 and normal goat serum for 1 hour. S100β and GABA were used as primary antibodies and reacted at 4°C for half a day. After antibody reaction, the cells were washed three times for 5 minutes with PBS and then reacted with a fluorescently conjugated secondary antibody at room temperature for 2 hours. After washing with PBS three times for 5 minutes, the brain tissue was stained on a glass slide using DAKO mounting solution.

As shown in Figure 1, it was confirmed that the amount of GABA in astrocytes of Git1 hetero type (+/-) mice increased. The left photo in Figure 1 is a fluorescent photo taken with a confocal microscope after fixing sections of the striatum region of Git1 wild type and hetero type mouse brains and fluorescent tissue immunostaining using specific antibodies. DAPI represents the nuclei, and s100b represents astrocytes. GABA is gamma amino butyric acid, which is a major inhibitory neurotransmitter in the central nervous system. The graph on the right of Figure 1 is a confocal micrograph analyzed in Image J. When the intensity of S100b was analyzed, no significance was found in the graph, but in the case of S100b positive GABA, that is, the intensity of GABA in astrocytes, in Git1 hetero type. appeared to increase significantly. Total GABA intensity and extra-astrocyte GABA levels were also found to increase overall in the Git1 hetero type.

2) Electrophysiological measurements (patch clamp recording)

Electrophysiological measurements (patch clamp recording) were performed by sectioning the mouse striatum at a thickness of 300 um. After bubbling with a mixed gas of 95% O _{2 and} 5% CO 2 , aCSF (unit: mM: 130 NaCl, 24 NaHCO ₃ , 1.25 NaH ₂ PO ₄ , 3.5 KCl, 1.5 CaCl ₂ , 1.5 MgCl ₂ , and 10 D(+)?glucose, pH 7.4). Measurement solution (containing 135 CsCl, 4 NaCl, 0.5 CaCl ₂ , 10 HEPES, 5 EGTA, 2 Mg-ATP, 0.5 Na ₂ -GTP, and 10 QX-314, adjusted to pH 7.2 with CsOH) was pipetted. The electrophysiological state was measured.

As shown in Figure 2, the amount of tonic GABA measured in the striatum tends to decrease in the hetero type (+/-) and knock-out type (-/-) compared to the wild type (+/+). indicated. The upper left corner of Figure 2 shows the brain slice containing the striatum, which is the brain region where patch clamp recording was performed, and the location of the recording pipette. The upper right graph of Figure 2 shows traces of tonic GABA current measured in medium spiny neurons of the striatum of wild type, hetero type, and knock-out type. The gray bar indicates full activation current induced by processing GABA, and the red bar represents the time of processing bicuculine, an antagonist of GABA-A receptors. Base line current was blocked by bicuculine treatment, and full current and tonic current were measured as indicated by the light blue arrow. The bottom of Figure 2 is a graph that averages the amplitude and frequency of all recorded traces and analyzes the significance. The tonic GABA current amplitude (pA) at the bottom right is the largest in the wild type and significantly decreased in the hetero type, and the knock-out type showed the smallest value. The second graph, full activation current (pA), and the third graph, % of full activation, showed a similar trend to that of tonic GABA current.

Example 3. Evaluation of GAT-3 levels in ADHD animal models

In order to perform fluorescent immunohistochemistry (fIHC), perfusion was performed in mice. After primary perfusion with Phosphate-buffered saline (PBS), 0.05% glutaraldehyde was added to 4% PFA (Paraformaldehyde) for fixation. Afterwards, the brain was collected from the skull of the mouse, soaked in 4% PFA, and stored at 4°C for half a day. The fixed mouse brain was placed in a 30% sucrose solution and dried for 2 days. The dried brain was placed in OCT compound and stored in a mold at -80°C. After making the mold, the hippocampus and striatum were sectioned at a thickness of 30 μm using a cryotome. The cut slices were washed three times for 5 minutes with PBS and mixed with triton-X100 and normal goat serum for 1 hour. S100β and GAT-3 (GABA transporter-3) were used as primary antibodies and reacted at 4°C for half a day. After antibody reaction, the cells were washed three times for 5 minutes with PBS and then reacted with a fluorescently conjugated secondary antibody at room temperature for 2 hours. After washing with PBS three times for 5 minutes, the brain tissue was stained on a glass slide using DAKO mounting solution.

As shown in Figure 3, as a result of confirming the amount of GAT-3 (GABA transporter-3) protein expressed in astrocytes, the amount of GAT-3 protein was found in Git1 hetero type (+/-) compared to Wild type (+/+) mice. ) was found to be expressed at a high level. The above results show that in mice with attention deficit/hyperactivity disorder (ADHD), GAT-3 (GABA transporter-3), a transporter through which GABA enters astrocytes, is excessively expressed, leading to the inflow of GABA into astrocytes. This means that it increases.

As shown on the left of Figure 3, the expression level of GAT-3, which is expressed in large quantities in astrocytes, was found to be significantly increased in the Git1 hetero type. As shown on the right side of Figure 3, no significance was found in the bar graph analyzing the intensity of S100b, but in the case of S100b positive GAT-3, that is, the intensity of GAT3 in astrocytes was found to be significantly increased in the Git1 hetero type. Using IMARIS, one astrocyte was photographed at the single cell level and GAT-3 expression was analyzed. As a result of graphical analysis, GAT-3 expression in Git1 hetero type was found to be significantly increased compared to wild type.

Example 4. Changes in hyperactivity following treatment with GAT-3 inhibitors

Considering that the change in the amount of GABA associated with astrocytes in ADHD model mice is related to the transport protein that brings GABA into astrocytes, treatment with SNAP5114, an inhibitor of GAT-3 (GABA transporter-3), An experiment was conducted. An open field test was performed to evaluate behavioral improvements. Handling was carried out 5 days before the open field test to minimize the stress felt by the mouse on the experimenter. The mouse was placed in the center of an open field test cage (30 The measured video was analyzed for locomotor activity through ANYmaze.

The left side of Figure 4 tracks the mouse movement in an open field and shows the distance the mouse moved and the degree of activity. The right side of Figure 4 shows a graph showing the average value of the distance moved by each object used in the experiment. As shown in Figure 4, it was confirmed that Git1 hetero type (+/-) mice (HE) exhibit hyperactivity, indicating more movement compared to wild type (+/+) mice (WT). . When Git1 hetero type (+/-) mice were treated with SNAP5114 (HE SNAP), hyperactivity disorder was shown to be improved to the level of WT.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (5)

delete delete delete delete Measuring the expression level of GAT-3 (GABA transporter-3) protein or the expression level of the gene encoding it in a biological sample isolated from a subject suspected of having attention deficit hyperactivity disorder (ADHD);
Comparing the expression level with a biological sample isolated from a normal person; and
Attention deficit hyperactivity disorder (ADHD), which includes determining that the subject is likely to have ADHD if the expression level of the GAT-3 (GABA transporter-3) or the gene encoding it increases compared to a biological sample isolated from a normal person. How to provide information for the diagnosis of ADHD).