JPWO2006093138A1 - Method of determining tendency to fall into apoptosis and use thereof - Google Patents

Abstract

  Provide an effective means for diagnosing Alzheimer's disease. The amount of ATBF1 in the hippocampal pyramidal cells to be examined, which has been separated from the living body (corpse in the case of autopsy), is detected, and the detection result is used to determine whether or not the hippocampal pyramidal cells to be examined shift to neuronal cell death. In a preferred embodiment, the localization of a specific ATBF1 fragment produced by post-translational processing is detected to determine whether the test hippocampal pyramidal cells shift to neuronal cell death.

Description

  The present invention relates to methods for determining the propensity of certain cells to undergo apoptosis. More specifically, the present invention relates to a method for determining the tendency of hippocampal pyramidal cells to undergo apoptosis, a reagent used in the method, a diagnostic method using the method, and the like. The present invention is used for diagnosis of Alzheimer's disease, research on Alzheimer's disease, and the like.

One of the problems that must be considered all over the world beyond the boundaries of countries and regions is the problem of dementing diseases including Alzheimer's disease. The number of people with dementia, including Alzheimer's disease, has been increasing year by year with the aging of each country. Currently, the number is about 18 million in the world, and about 1.6 million in Japan. In the past, cerebral circulatory disorders (vascular dementia such as cerebral infarction and cerebral hemorrhage) were the main causes of dementia, but recent epidemiological surveys show that the proportion of Alzheimer's disease tends to increase. Currently, among the clearly recognized risk factors for Alzheimer's disease, as the only certain risk factor is aging, the proportion of Alzheimer's disease among dementia diseases increases year by year as the population ages. It seems that there is no doubt that it will continue to follow.
Japan has entered an aging society unprecedented in history, and it is estimated that 2-3% of the elderly will have Alzheimer's disease. It is estimated that the number of patients will increase from 450,000 to 550,000 in 2000, and will double to 700,000 to 800,000 in 2020, further increasing. We cannot escape from such problems spreading not only to Japan but to the world.

Currently, there is no other definitive diagnosis of Alzheimer's disease than neuropathological search. Therefore, in reality, it is extremely difficult to make a definite diagnosis in the true sense of life. However, in the clinical setting, diagnosis is made based on clinical symptoms and history of medical history, and as auxiliary tests, image diagnosis such as CT, MRI, PET/SPECT, electroencephalogram, and blood/cerebrospinal fluid biochemical tests are performed. ..
Until a few years ago, the pathological definitive diagnosis of Alzheimer's disease had not been emphasized because there was no cure. However, recent research, elucidation of the causative gene in familial Alzheimer's disease, has revealed the formation process of amyloid β protein, which is the basis of senile plaques, and the mechanism of phosphorylation of tau protein. It has been revealed that cholesterol metabolism and the like are greatly involved in the pathological condition of Alzheimer's disease because it is a risk factor. Based on these findings, new therapeutic methods are being developed, and pathological analysis and care of Alzheimer's disease are making great progress. Especially today, cholinesterase inhibitors have been actually used in clinical situations and achieved some results, and it has become possible to suppress the progress of symptoms to some extent in the early stage of disease onset. In addition, the development of vaccine therapy is currently stopped at the clinical trial stage, but it seems that it will be restarted in the near future and will make a great contribution to the prevention and treatment of Alzheimer's disease. In addition, with the recent remarkable technological advances, image diagnosis such as CT, MRI, and PET/SPECT has come to be focused on early diagnosis and prognostic diagnosis of Alzheimer's disease. In particular, the development of PET probe is making it possible to even visualize senile plaques.

The first step in treating a disease begins with an accurate diagnosis of the condition and the causative disease. Alzheimer's disease is no exception. Although not so common in Japan, brain tissue biopsy is performed in Europe and the United States when searching for causative diseases including Alzheimer's disease, various degenerative diseases, brain tumors, encephalitis, and brain infectious diseases, and determining treatment strategies. The importance of the pathological diagnosis by is increasing. The technology of brain tissue biopsy also uses CT and MRI as a guide, and stereobiopsy (localization biopsy) using a stereotaxic device is common, and it is possible to accurately biopsy a minimal area. In the case of Alzheimer's disease, according to Braak's staging, the parahippocampal gyrus (medial aspect of temporal lobe) is the first lesion in the hippocampus region (hippocampus, hippocampal limb, parahippocampal gyrus). The pathological definitive diagnosis can be made only after the lesion reaches (the part including Ammon's angle, Cornu Ammonis, CA1-CA4).
In Japan, pathological diagnosis by biopsy is not currently used for the definite diagnosis of Alzheimer's disease while living. For various reasons such as ethical issues and safety issues, the hippocampus or its vicinity is not actually targeted for biopsy. However, including the case where the hippocampus is the causative site of epileptic seizures, there are many examples of actually inserting electrodes into the hippocampus or unilateral hippocampal dissection. Therefore, pinpoint and accurate biopsy of the same site has no technical problem at present. Considering the diagnosis status of Alzheimer's disease by biopsy in Europe and America, there is no doubt that the need will gradually increase in the future.

Alzheimer's disease was first reported in 1906 by Alzheimer, a German psychiatrist and neuropathologist. A specific neuropathological change was reported in a 51-year-old woman who died of amnesia and disorientation as the initial symptoms, then depression, hallucinations, and significant dementia over the past four and a half years. One of the characteristics is that there are many patchy structures (senile plaque; SP) that are stained by silver staining in the interstitial area of nerve cells. Secondly, they are stained by silver staining in nerve cells. A large number of fibrous structures (neurofibrillary tangle; NFT) appear. In research to date, SP is composed of amyloid β protein aggregation and accumulation in extracellular tissue and associated tissue reaction (degenerated neurites and reactive glial cells), and the disease specificity in Alzheimer's disease is compared. It is said to be expensive. NFT is formed in nerve cells, and is a cytoskeletal protein tau (tau) that is abnormally phosphorylated and aggregated and accumulated. It is also known to appear in other diseases and its disease specificity is not so high. Has been done. Against this background, there are three main pathological findings that are currently used in definitive diagnosis. That is, (A) SP due to amyloid β protein deposition, (B) NFT formation due to abnormal aggregation of tau, and (C) nerve cell death due to simple atrophy. A definitive diagnosis is made by comprehensively judging how the three findings appear and progress. Complementing with the cell death in the above findings, there are two types of neuronal cell death morphologically. One is cell death associated with NFT formation, and the other is death that causes cells to atrophy and disappear. This process is called "simple atrophy". In Alzheimer's disease, nerve cells degenerate and fall out, that is, nerve cell death is the basis of the disease state. In particular, the hippocampal region (hippocampus, hippocampal limb, parahippocampal gyrus) is the most highly affected part of the Alzheimer's disease brain, and it is a well-known fact that it corresponds to a small amount of memory loss in the earliest stages.
The reports relevant to the present invention are listed below.

Miura et al. Cloning and characterization of an ATBF1 isoform that expresses in a neuronal differentiation-dependent manner.J. Biol. Chem. (1995) 270: 26840-26848 Kaspar et al. Myb-interacting protein, ATBF1, represses transcriptional activity of Myb oncoprotein. J. Biol. Chem. (1999) 274: 14422-14428 Ishii et al. ATBF1-A protein, but not ATBF1-B, is preferentially expressed in developing rat brain.J. Compartive Neurology (2003) 465: 57-71. Miura et al. Susceptibility to killer T cells of gastric cancer cells enhanced by mitomycin-C induction of ATBF1 and activation of p21 (Waf1-/Cip1) promoter. Microbiol. Immunol (2004) 48: 137-145 Sun X et al. Frequent somatic mutations of the transcription factor ATBF1 in human prostate cancer. Nat Genet. 2005 Apr;37(4):407-412.

By the way, in the actual neuropathological diagnosis, (A) SP due to amyloid β protein deposition, (B) NFT formation due to abnormal aggregation of tau, are microscopic observations after plating with silver and Galyas Braak. You can decide. In addition, (C) neuronal death due to simple atrophy can be judged by microscopic observation if it is possible to examine the size reduction of the entire brain, as in the brain of an autopsy case. However, assuming that a small amount of hippocampal brain tissue is biopsied, the determination of “neuronal cell death due to simple atrophy” is difficult because the atrophy cannot be clearly defined, and it is expected that the diagnosis will be difficult. If the pathological findings (A) and (B) described above are present at the same time, the diagnosis of Alzheimer's disease can be made. However, each is not a finding specific to Alzheimer's disease, and individually exists in other dementia diseases. Therefore, there is a demand for a simple "cell death determination method" for avoiding misdiagnosis with other diseases and determining (C) neuronal cell death due to simple atrophy with a small amount of brain tissue to ensure a definitive diagnosis. By the way.
The ability to accurately determine "neuronal death due to simple atrophy" in the hippocampal cranial nerve tissue that was biopsied in a small amount is extremely important in the future for the definite diagnosis of Alzheimer's disease and the determination of treatment policy. On the other hand, the judgment is required to be accurate, as well as quick and simple. Considering that the hippocampal region is an important region related to memory, it is impossible to obtain a large amount of cranial nerve tissue by biopsy. Therefore, accurate, simple, and reproducible determination of "neuronal cell death" is not expected until accurate diagnosis, and further the therapeutic effect by starting treatment as early as possible. Therefore, if a method capable of determining "neuronal cell death" in addition to being accurate and without imposing an excessive burden on the patient is developed, its contribution to Alzheimer's disease diagnosis is immeasurable. It is expected that it will be a great gospel for Alzheimer's disease patients, contributing to the analysis of the pathological condition of Alzheimer's disease, which is expected to develop further in the future, the development of therapeutic methods for stopping the progression of dementia.
In view of the above background, the present invention aims to provide an effective means for diagnosing Alzheimer's disease.

For the above purpose, the present inventors have paid attention to ATBF1 (AT motif binding factor 1). Two isoforms (ATBF1-A and ATBF1-B) are known to exist in ATBF1. ATBF1-A and ATBF1-B are formed by using different promoters and alternative splicing (see Non-Patent Document 1). ATBF1-A has a structure in which the protein N-terminal side is longer than ATBF1-B by 920 amino acids. As shown in Examples described later, the present inventors have established two types of antibodies (antibody name; NT440 and 1) that specifically recognize the N-terminal side of ATBF1-A protein (site corresponding to exon 3 of ATBF1 gene). -12), an antibody that specifically recognizes the central portion (the site corresponding to exon 10 of the ATBF1 gene (exon that was previously thought to be exon 8 and described as exon 9 in Non-Patent Document 5)) (Antibody name; D1-120), C-terminal side (site corresponding to exon 11 of the ATBF1 gene (exon that was previously considered to be exon 9 and described as exon 10 in Non-Patent Document 5)) The localization of ATBF1 in hippocampal pyramidal cells was searched using an antibody that specifically recognizes (antibody name: AT6) (see FIG. 1 for nuclear antibody recognition site, exon sequence, etc.). As a result, the following findings were obtained.
(1) The amount of ATBF1 was significantly increased in the hippocampal pyramidal cells of Alzheimer's disease (abbreviated as AD hereinafter) brain in which a high degree of brain atrophy was observed. That is, a high correlation was observed between the increase in ATBF1 amount and the neuronal cell death in AD. From this result, it was considered that the neuronal cell death could be judged by using the increase in ATBF1 amount as an index.
(2) ATBF1 protein undergoes processing that is divided into at least three parts, and is a part recognized by D1-120 in hippocampal pyramidal cells of AD brain ATBF1 (a part containing a region corresponding to exon 10 of the ATBF1 gene) Yes, it was located in the center of ATBF1-A) and localized in the nucleus (mainly in the nucleus), and its amount increased. That is, a high correlation was observed between the localization of the partial ATBF1 amount in the nucleus and the expression level thereof and the neuronal cell death in AD. From this result, it was considered that the detection of the partial ATBF1 (especially clarifying the localization mode) is effective for the determination of neuronal cell death.
(3) In the hippocampal pyramidal cells of AD brain, the partial ATBF1 recognized by AT6 (the portion containing the C-terminal region corresponding to exon 11 of the ATBF1 gene) is localized in the cytoplasm (localization mainly in the cytoplasm), The amount was also increasing. That is, a high correlation was observed between the amount of the partial ATBF1 localized in the cytoplasm and the expression level thereof and the neuronal cell death in AD. From this result, it was considered that the detection of the partial ATBF1 (especially to clarify the localization mode) is effective for the determination of neuronal cell death.
(4) In the hippocampal pyramidal cells of AD brain, the part ATBF1 (the part containing the N-terminal region corresponding to exon 3 of the ATBF1 gene) recognized by 1-12 is localized in the cytoplasm (localization mainly in the cytoplasm). ), the amount was increasing. That is, a high correlation was observed between the amount of the partial ATBF1 localized in the cytoplasm and the expression level thereof and the neuronal cell death in AD. From this result, it was considered that the detection of the partial ATBF1 (especially clarifying the localization mode) is effective for the determination of neuronal cell death.

Here, when an increase in ATBF1 amount, etc. is regarded as a tendency to shift to apoptosis, and a high degree of cerebral atrophy was observed in the brain tissue in which an increase in ATBF1 amount etc. was actually observed, it was considered that " It can be said that there is no substantial difference between the “determination” and the “determination of the tendency of nerve cells to undergo apoptosis”. Considering this, from the above findings, it is effective to detect the intracellular abundance of ATBF1 and/or the localization of a specific portion of ATBF1 as a means for determining the tendency of nerve cells to undergo apoptosis. I can say. In other words, it was considered that the detection could provide useful information for diagnosing Alzheimer's disease, which is the tendency of the hippocampal pyramidal cells of the control to fall into apoptosis.
The present invention has been completed based on the above achievements and findings, and provides the following configurations.
The present invention is a method for determining the tendency of hippocampal pyramidal cells to undergo apoptosis, which comprises the following steps.
(a) Preparing hippocampal pyramidal cells collected from the subject, and (b) detecting ATBF1 amount in the hippocampal pyramidal cells.
In one aspect of the present invention, in step (b), one or more selected from the group consisting of the following (1) to (4) is detected.
(1) Total amount of ATBF1 in the hippocampal pyramidal cells; (2) Nuclear abundance and/or cytoplasmic abundance of partial ATBF1 containing a region corresponding to exon 10 of the ATBF1 gene in the hippocampal pyramidal cells; 3) Nuclear abundance and/or cytoplasmic abundance of partial ATBF1 containing C-terminal region corresponding to exon 11 of ATBF1 gene in the hippocampal pyramidal cells; (4) N-terminal region corresponding to exon 3 of ATBF1 gene Abundance in the nucleus and/or cytoplasm in the hippocampal pyramidal cells of the partial ATBF1 containing
In a preferred aspect of the present invention, at least (2) is detected. In a further preferred aspect of the present invention, the above (2) to (4) are detected in step (b).
In the method of the present invention, immunohistochemical staining is preferably used as the detection method.
In another aspect, the present invention provides reagents and kits that can be used in the method of the present invention. The reagent of the present invention is an anti-ATBF1 antibody reagent for determining an apoptosis tendency. As anti-ATBF1, (1) an antibody recognizing a partial ATBF1 containing a region corresponding to exon 10 of the ATBF1 gene, (2) an antibody recognizing a partial ATBF1 containing a C-terminal region corresponding to exon 11 of the ATBF1 gene, or (3 ) An antibody that recognizes a partial ATBF (partial ATBF1-A) containing an N-terminal region corresponding to exon 3 of the ATBF1 gene can be used.
The kit of the present invention contains one or more antibodies selected from the group consisting of the following (1) to (3).
(1) an antibody recognizing a partial ATBF1 containing a region corresponding to exon 10 of the ATBF1 gene, (2) an antibody recognizing a partial ATBF1 containing a C-terminal region corresponding to exon 11 of the ATBF1 gene, and (3) an ATBF1 gene A group consisting of antibodies that recognize a partial ATBF (partial ATBF1-A) containing an N-terminal region corresponding to exon 3 of.
In a preferred embodiment, the kit of the invention additionally comprises ATBF1. When an antibody prepared by using an antigen that is a fusion protein with a tag or a carrier protein is used in the kit, the kit may further include a tag or a carrier protein.

  According to the present invention, the hippocampal tissue collected from the brain can be used to easily determine the tendency of pyramidal cells (large nerve cells) important for diagnosis of Alzheimer's disease to undergo apoptosis, that is, nerve cell death. ..

FIG. 1 is a schematic diagram of NT440, 1-12, which recognizes the N-terminal part of ATBF1, D1-120, which recognizes the central part, and AT6 which recognizes the C-terminal, correspondence between each exon of ATBF1 gene and protein sequence, immunity. 1 shows the amino acid sequence of the polypeptide used in. Regarding NT440, 1-12, the difference between ATBF1 sequence in human and mouse was shown. FIG. 2 is a diagram schematically showing the relationship between ATBF1 and p21 and p53. The mechanism of cell cycle arrest by the use of mitomycin C in gastric cancer cells caused by DNA damage and the mechanism of ATBF1 involvement is outlined. FIG. 3 shows ATBF1 expression in P19 cells and search by flow cytometry. The upper row shows triple-stained images of DAPI (staining of nuclear DNA), ATBF1, and β-tubulin. FIG. 3a shows P19 cells that were not treated with retinoic acid (RA). Neither ATBF1 nor β-tubulin expression was observed. FIG. 3b shows P19 cells 24 hours after the action of retinoic acid (RA), which is negative for β-tubulin but has ATBF1 stained in the cytoplasm. FIG. 3c shows P19 cells in which the action of retinoic acid (RA) was stopped and the culture was continued for another 4 days. ATBF1 staining is concentrated in the nucleus, and β-tubulin-positive neurites extend from the cytoplasm to the periphery of the cell group. Figures 3d, 3e, and 3f are flow cytometric analyzes of P19 cells shown in Figures 3a, 3b, and 3c, respectively. Only in FIG. 3f is cell cycle arrest observed. Figure 4a shows the potential location of the nuclear retention signal in ATBF1 by computer analysis. The presence of sequences similar to the consensus sequence is predicted at two locations (starting at amino acids 277 and 2987, respectively). Figure 4b also shows the potential location of the nuclear export signal in ATBF1. The existence of the nuclear export signal is predicted in three places (starting from amino acids 1267, 2471, and 2504, respectively). The expression site of ATBF1 in P19 cells when fibronectin and poly-L-ornithine were applied to a culture dish after retinoic acid treatment and not applied is shown. FIG. 5a shows ATBF1 expression in P19 cells without application. The cells are in suspension and ATBF1 appears in the cytoplasm but does not translocate to the nucleus. FIG. 5b shows ATBF1 expression in P19 cells 3 hours after culturing after coating fibronectin and poly-L-ornithine on a culture dish. Translocation of ATBF1 to the nucleus is observed. FIG. 5c shows the state after 24 hours, and ATBF1 expression in the nucleus is clearly enhanced. FIG. 6 shows the effect of Leptomycin B on ATBF1 nuclear translocation in P19 cultured cells. FIG. 6a shows the expression state of ATBF1 in P19 cells when Leptomycin B was not allowed to act. Confirm the presence of ATBF1 in the nucleus. FIG. 6b shows the expression state of ATBF in P19 cells when Leptomysin B was allowed to act. It is clear that the nuclear concentration of ATBF1 increased. FIG. 7 shows the results of an experiment for investigating the effect of the action of two PI3K family protein antagonists (Wortmannin, caffeine) on the translocation of ATBF1 to the nucleus in P19 cells. FIG. 7a shows ATBF1 expression in the nucleus of P19 cells. FIG. 7b shows that ATBF1 is inhibited from translocating into the nucleus by the action of Wortmannin and ATBF1 exists in the cytoplasmic center. Figure 7c shows the effect of caffeine. It can be seen that the transfer of ATBF1 to the nucleus was more completely inhibited as compared to Wortmannin. FIG. 8 shows the findings of a confocal laser scanning microscope photograph in which the full-length ATBF1 cDNA was forcibly expressed in Neuro2A cells derived from a mouse neuroblastoma cell line. BrdU was added to the culture medium for 1 hour immediately before the cells were fixedly observed to label the DNA-synthesizing cells. BrdU-incorporated cells were detected with a green fluorescent secondary antibody, and at the same time, the HA tag added to the forced expression vector was detected with a red fluorescent secondary antibody. As shown by the arrow in FIG. 8a, the green color of BrdU-positive cells overlapped with the red color of the HA tag, and a group of cells that developed yellow color was detected. On the other hand, as shown by the arrow heads in Fig. 8b, all the HABF-tagged ATBF1-introduced cells show a red color, indicating that they do not overlap with the green color of BrdU-positive cells. The results of this experiment show that the forced expression of ATBF1 cDNA completely suppresses the cell cycle. These facts are summarized as a bar graph in Figure 8c. FIG. 8c shows the results of the introduction experiment of only the HA tag without ATBF1, showing that about 40% of BrdU-positive cells were double-positive with the HA tag and exhibited a yellow color, as shown by the shaded area. On the other hand, it has been shown that all of the HA-tagged gene-transfected cells containing ATBF1 cDNA are red and BrdU-negative. In addition to the results of these gene transfer experiments, the cell cycle of each cell group was assayed using FACScan. Compared with the cell cycle of the transgenic group containing only the HA tag shown in FIG. 8d, the cell cycle of the ATBF1 cDNA transgenic group shown in FIG. 8e is 10% in the M1 region, that is, in the G1/G0 phase cell group. From the above increase, it was clarified that the forced expression of ATBF1 arrests the cell cycle almost completely in G1/G0 phase when considering it together with the DNA transfer efficiency. FIG. 9 shows the difference in staining property by the antigen activation method performed in each of the cases of non-invasive cancer and invasive cancer of the urothelial carcinoma of the bladder. Autoclaving was performed using various buffers, and ATBF1 staining was performed. The black circles (●) indicate that the staining results mainly on the nucleus were obtained. White circles (◯) indicate that the results of staining mainly of cytoplasm were obtained. FIG. 10 shows the difference in staining property by the antigen activation method performed for each of non-invasive cancer and invasive cancer cases of urothelial cancer of the bladder. Microwave oven treatment was performed using various buffers, and ATBF1 staining was performed. The black circles (●) indicate that the staining results mainly on the nucleus were obtained. White circles (◯) indicate that the results of staining mainly of cytoplasm were obtained. FIG. 11 shows the difference in staining property by the antigen activation method performed in each of the cases of non-invasive cancer and invasive cancer of the urothelial cancer of the bladder. A pressure cooker treatment was performed using various buffers, and ATBF1 staining was performed. The black circles (●) indicate that the staining results mainly on the nucleus were obtained. White circles (◯) indicate that the results of staining mainly of cytoplasm were obtained. FIG. 12 shows a representative example of the difference in staining property depending on the selection of the antigen activation method performed in each of the cases of non-invasive cancer and invasive cancer of the urothelial cancer of the bladder. The upper row (Fig. 12a, c, e) shows non-invasive cancer, and the lower row (Fig. 12b, d, f) shows invasive cancer. The left side (Fig. 12a, b) shows a case in which 50 mM Tris-HCl buffer pH 10.0 was used and autoclaved. ATBF1 staining in the nucleus is observed in both non-invasive cancer (FIG. 12a) and invasive cancer (FIG. 12b). The middle (Fig. 12c, d) shows the case where microwave treatment was performed using DAKO TRS pH 6.0. In both the non-invasive cancer (FIG. 12c) and the invasive cancer (FIG. 12d), the cytoplasmic predominant cytoplasmic staining around the nucleus is recognized like the cells shown by the arrows. The right (Fig. 12e, f) shows the case where the pressure cooker treatment was performed using 10 mM citrate buffer pH 6.0. In non-invasive cancer (Fig. 12e), ATBF1 is localized in the nucleus, and in invasive cancer (Fig. 12f), ATBF1 is localized in the perinuclear cytoplasmic body, as shown by the arrow, and the difference in localization between the nucleus and the cytoplasmic body is highlighted. Is possible. 13 hippocampal region (hippocampus) in the human normal brain, an anatomical drawing of a frontal cross-sectional, Ammon's horn (C ornu A mmonis, abbreviated as CA) CA1-CA4 (Lorente de No hippocampal classification), dentate An outline of the position of the gyrus, hippocampus (hippocampus, front hippocampus), and parahippocampus (arrow) is shown. Furthermore, the directions of the outside (front side) of the brain and the inside (brain stem side) of the brain are shown. FIG. 14 shows immunostaining findings of ATBF1 (NT440 portion) obtained by using the brains of two female autopsy cases, 79 years old and 88 years old, which were judged to be normal without suffering from dementia. Findings on pyramidal cells that appear to be representative (Fig. 14a-l) and distribution of NT440-positive cells in the hippocampus (Fig. 14m) are illustrated. Solid arrows indicate positive images in pyramidal nuclei, dashed arrows indicate positive images in pyramidal cytoplasm, arrowheads indicate cells lacking staining in both nucleus and cytoplasm. Stainability in hippocampal pyramidal cells is mainly localized in the cytoplasm, but in some pyramidal cells, localization in the nucleus is also recognized, and cells lacking staining in both the nuclear cytoplasm are also present. FIG. 15 shows ATBF1 (NT440 part) obtained by using the brains of two female autopsy cases, 78 years old and 84 years old, who were clinically diagnosed with Alzheimer's disease and whose definitive diagnosis was obtained by autopsy. Immunostaining findings. Findings on pyramidal cells that appear to be representative (Fig. 15a-l) and distribution of NT440-positive cells in the hippocampus (Fig. 15m) are illustrated. The dashed arrow indicates a positive image in the pyramidal cytoplasm. Staining is localized mainly in the cytoplasm in almost all pyramidal cells. FIG. 16 is an immunostaining finding of ATBF1 (1-12 part) obtained by using the brains of two female autopsy cases, 79 years old and 88 years old, which were judged to be normal without suffering from dementia. .. Findings on pyramidal cells that appear to be representative (Fig. 16a-l) and distribution of 1-12 positive cells in the hippocampus (Fig. 16m) are illustrated. Solid arrows indicate positive images in pyramidal nuclei, dashed arrows indicate positive images in pyramidal cytoplasm, arrowheads indicate cells lacking staining in both nucleus and cytoplasm. 1-12 staining is mainly localized in the cytoplasm of hippocampal pyramidal cells, but some pyramidal cells are also localized in the nucleus, and cells lacking staining in both the nuclear cytoplasm are also present. FIG. 17 is an autopsy case clinically diagnosed with Alzheimer's disease and confirmed by pathological findings at autopsy. Both 78-year-old and 84-year-old ATBF1 (1-12 parts) obtained by using the brains of two women ) Immunostaining findings. Findings on the pyramidal cells that appear to be representative (Fig. 17a-l) and the distribution of 1-12 positive cells in the hippocampus (Fig. 17m) are illustrated. The dashed arrow indicates a positive image in the pyramidal cytoplasm, and the arrowhead indicates cells lacking staining in both the nucleus and cytoplasm. 1-12 staining is localized in the cytoplasm in almost all pyramidal cells. A very small number of cells lacking stainability are also present. FIG. 18 is an immunostaining finding of ATBF1 (D1-120 portion) obtained by using the brains of two female autopsy cases, 79 years old and 88 years old, which were judged to be normal without suffering from dementia disease. .. Findings on pyramidal cells that appear to be representative (Fig. 18a-l) and distribution of D1-120 positive cells in the hippocampus (Fig. 18m) are illustrated. Solid arrows indicate positive images in pyramidal nuclei, dashed arrows indicate positive images in pyramidal cytoplasm, arrowheads indicate cells lacking staining in both nucleus and cytoplasm. The dyeability of D1-120 in hippocampal pyramidal cells was small in both the nucleus and cytoplasm, and the tendency that the dyeability was concentrated in the nucleus or cytoplasm was unclear. In addition, some cells have a lack of staining in both nuclear cytoplasm. FIG. 19 shows ATBF1 (D1-120 part) obtained by using the brains of two female autopsy cases, 78 years old and 84 years old, who were clinically diagnosed with Alzheimer's disease and were confirmed to be confirmed by pathological findings at autopsy. ) Immunostaining findings. Findings on pyramidal cells that appear to be representative (Fig. 19a-l) and distribution of D1-120 positive cells in the hippocampus (Fig. 19m) are illustrated. Solid arrows indicate positive images in pyramidal nuclei, dashed arrows indicate positive images in pyramidal cytoplasm, and white arrows indicate cells lacking staining in the nucleus and having localized staining only in the cytoplasm. The dyeability of D1-120 in pyramidal cells is mainly nuclear, and the staining intensity is highly increased. There are a small number of cells in which the staining property is partially lost in the nucleus and the staining property is localized only in the cytoplasm, but these are scattered. FIG. 20 shows immunostaining findings of ATBF1 (AT6 portion) obtained by using the brains of two female autopsy cases, 79 years old and 88 years old, which were judged to be normal without suffering from dementia. Findings on pyramidal cells that appear to be representative (Fig. 20a-l) and the distribution of AT6-positive cells in the hippocampus (Fig. 20m) are illustrated. Solid arrows indicate positive images in pyramidal nuclei, dashed arrows indicate positive images in pyramidal cytoplasm, arrowheads indicate cells lacking staining in both nucleus and cytoplasm. AT6 staining in hippocampal pyramidal cells is small in both the nucleus and cytoplasm, and the tendency of staining to concentrate in the nucleus or cytoplasm is unclear. Some cells have a lack of staining in both nuclear cytoplasm. FIG. 21 shows ATBF1 (AT6 part) obtained by using the brains of two females, both at the age of 78 and at the age of 84, which were clinically diagnosed with Alzheimer's disease and confirmed by pathological findings at necropsy. Immunostaining findings. Findings on pyramidal cells that appear to be representative (Figure 21a-l) and distribution of AT6-positive cells in the hippocampus (Figure 21m) are illustrated. The dashed arrow indicates a positive image in the pyramidal cytoplasm. In hippocampal pyramidal cells, ATBF1 (AT6 portion) staining is highly increased, and the localization of staining is clearly cytoplasmic. FIG. 22 shows an outline of the site in the ATBF1-A protein pointed out by the antibody (FIG. 22A), and is a schematic view of the nuclear and cytoplasmic migration direction of each antibody pointing out ATBF1 and the tendency of increasing staining (FIG. 22B). ). The staining of ATBF1, that is, the amount of ATBF1 protein and its localization in the cell is illustrated by the shaded box, and how the intracellular localization of ATBF1 changes as normal pyramidal cells change to Alzheimer's disease. I showed you what to do. Fig. 23 shows a new view of the conventional pathological diagnosis method for Alzheimer's disease for the case used this time (Fig. 23A), and summarizes the features of Alzheimer's disease using this time Alzheimer's disease. This is illustrated together with the findings by the disease diagnosis method (Fig. 23B). Regarding ATBF1 findings, particular attention is paid to the staining intensity in typical hippocampal pyramidal cells. FIG. 24 shows an immunostaining of ATBF1 (D1-120 part) obtained by using a hippocampus of a 78-year-old female brain autopsy, which was clinically diagnosed with Alzheimer's disease and whose definitive diagnosis was obtained by autopsy. Comparison of findings (Fig. 24a, c) and findings of Galyas Braak (GB) staining (Fig. 24b, d). 24b and d are enlarged views of the square black-stained area of FIGS. 24a and 24c. The solid line arrow exemplifies a typical cone cell showing a strongly positive image of each staining (D1-120, GB), and the broken line arrow exemplifies a typical cone cell showing a weak positive image of GB staining. Fig. 25 shows immunostaining of ATBF1 (D1-120 part) obtained by using a brain hippocampus of an autopsy case 84 years old, which was clinically diagnosed with Alzheimer's disease and whose definitive diagnosis was obtained by pathological findings at autopsy. Comparison of findings (Fig. 25a, c) and Galyas Braak (GB) staining findings (Fig. 25b, d). 25b and d are enlarged views of the square black-stained area of FIGS. 25a and 25c. Solid arrows show representative pyramidal cells showing strong positive images of each staining (D1-120, GB), and broken line arrows show representative pyramidal cells showing weak positive images of GB staining. FIG. 26 is a schematic diagram of the use of different promoters shown in Non-Patent Documents 1 and 5, the mechanism of two kinds of ATBF1-A and B mRNA by alternative splicing, and the production of mutant protein by abnormal skipping of exon 10 in human malignant tumor. It is explanatory drawing (nonpatent literature 5). It is a figure which shows the arrangement|sequence of each exon (exons 2-11) of ATBF1. The underlined part is the exon region. The exon number is attached to the upper right of the sequence of each exon. The exon number is ATBF1-B specific untranslated region exon existing at the 5'-most upstream of ATBF1 gene, followed by ATBF1-A specific untranslated region exon 2, and the first ATBF1-A. The exon including the translation region is numbered 3, and the exons following this are numbered 4 to 11 in order. Continuation of FIG. 27. 28 continuation. Continuation of FIG. 29.

(the term)
In the present specification, "detecting the amount of a certain substance (for example, the amount of ATBF1)" refers to grasping the existing amount of the detection target as an absolute amount or a relative amount. The relative amount here can be calculated, for example, by using the amount of the detection target found in the hippocampal pyramidal cells of autopsy cases that did not exhibit so-called dementia symptoms including Alzheimer's disease as the standard abundance. When determining the abundance in the nucleus and the abundance in the cytoplasm, the abundance in the nucleus can be determined as a relative amount based on the abundance in the cytoplasm. The abundance in the cytoplasm can be determined as the relative amount. The term "detection of the amount of a certain substance (for example, the amount of ATBF1)" refers to a concept that also includes checking whether or not a detection target (presence or absence) exists. Usually, the presence or absence of a detection target is checked at the same time as the abundance (if present). Strictly quantifying the detection target is not essential, and it is possible to determine the apoptotic tendency of the hippocampal pyramidal cells to be tested, for example, by comparing the detected amount in the pyramidal cells of the standard data searched at the same time. It is sufficient that the amount of the detection target can be measured to such an extent.

“ATBF1” refers to AT motif binding factor 1 (AT motif binding factor 1). ATBF1 is known to be a transcription factor that binds to the AT-rich domain of an AFP (alphafetoprotein) regulatory factor and down-regulates the expression of the AFP gene (see Non-Patent Document 1). As described above, "ATBF1" has two isoforms (ATBF1-A and ATBF1-B). The term "ATBF1" is used herein as a generic term for these two isoforms. Therefore, unless otherwise specified, the “ATBF1 amount” means the total amount of each isoform present. In the method of the present invention, in principle, the sum is set as the detection target. However, it does not prevent that only the amount of one of the isoforms is the detection target. In addition, when simply described as “ATBF1”, it means ATBF1 protein except when it is obvious that it has other meanings.
The structure of the ATBF1 gene is shown in FIG. 26 (see Non-Patent Document 1 and Non-Patent Document 5). The sequences of exons 2 to 11 of ATBF1 gene are shown in FIGS. 27 to 30. Exons 1 to 11 are present in the ATBF1 gene, and mRNAs of ATBF1-A and ATBF1-B are formed as a result of alternative splicing. Note that the regions shown as exons 10 and 11 in FIG. 26 are described as exons 9 and 10 in Non-Patent Document 5, respectively.
The amino acid sequences and nucleotide sequences corresponding to exon 3, exon 10 and exon 11 are shown by the following sequence numbers in the attached sequence listing.
Amino acid sequence of the region corresponding to exon 3 (SEQ ID NO: 11), base sequence of exon 3 (SEQ ID NO: 12), amino acid sequence of region corresponding to exon 10 (SEQ ID NO: 13), base sequence of exon 10 ( SEQ ID NO: 14), amino acid sequence of the region corresponding to exon 11 (SEQ ID NO: 15), base sequence of exon 11 (SEQ ID NO: 16).

(Apoptosis tendency determination method)
A first aspect of the present invention relates to a method for determining a tendency of hippocampal pyramidal cells to fall into apoptosis (also referred to herein as “apoptosis tendency”).
As described above, it can be considered that the “determination of apoptotic tendency” of hippocampal pyramidal cells is not substantially different from the “determination of cell death due to simple atrophy”. Therefore, the determination result of the apoptotic tendency can be used to diagnose Alzheimer's disease characterized by cell death due to simple atrophy. Thus, the method of the present invention provides useful information for diagnosis of Alzheimer's disease. It should be noted that the “determination of apoptotic tendency” does not determine the actual distinction between life and death of cells and the state of cerebral atrophy caused by cell death of many nerve cells.
In the determination of “propensity to fall into apoptosis (apoptosis tendency)”, the presence or absence of an apoptosis tendency is typically determined. It is also possible to determine whether or not the hippocampal pyramidal cells to be tested fall into apoptosis by setting the degree of apoptosis tendency to high or low. In the present specification, the determination result that “there is an apoptotic tendency” means that there is an extremely high possibility of falling into apoptosis, and it is considered that nerve cell death is recognized.

In one aspect of the apoptosis tendency determination method of the present invention, the following steps are performed.
(a) Preparing hippocampal pyramidal cells collected from the subject.
(b) Detecting the amount of ATBF1 in the hippocampal pyramidal cells.

(Step a)
In step a, hippocampal pyramidal cells collected from the subject are prepared. Not only humans, but also non-human primates such as monkeys and chimpanzees, rodents such as mice, rats and guinea pigs, birds such as chickens and quails, and the like can be tested. The information (judgment results) obtained when a non-human animal is used as a test object can be used for diagnosing Alzheimer's disease in the non-human animal, but rather it can be used to establish a diagnostic method for human Alzheimer's disease. It is useful because it can be used.
Particularly when collecting hippocampal pyramidal cells from a human body, a subject (patient) suspected of having Alzheimer's disease by another diagnostic method is usually a subject to be examined. Examples of other diagnostic methods here include clinical symptoms and medical history, CT examination, MRI examination, PET/SPECT examination, electroencephalogram examination, and diagnostic methods using biochemical examination. Usually, hippocampal pyramidal cells are collected from living brain tissue suspected of having Alzheimer's disease by one or more of these.

  Hippocampal pyramidal cells are collected from the brain and hippocampus regions of the subject. Specifically, in the case of a living body, a part of the brain hippocampal tissue of the subject is collected by biopsy (biopsy), and the collected tissue piece is used as a sample containing hippocampal pyramidal cells. In the case of an autopsy case (corpse), the whole or part of the hippocampal region collected at the time of autopsy can be used as a sample containing hippocampal pyramidal tissues. In this way, hippocampal pyramidal cells separated from a living body or a dead body are prepared. Here, "separated from a living body (or cadaver)" means that a part of the hippocampus of the living (or cadaver) brain tissue in which hippocampal pyramidal cells are present is excised, and the living body (or cadaver) derived from the hippocampus is derived. It is a completely isolated state. The hippocampal pyramidal cells are usually collected in a state of existing in a living body (or a cadaver), that is, in a state of being bound to various cells and interstitial connective tissue (that is, as a piece of tissue), and used in the method of the present invention. To be done.

(Step b)
In step b, the amount of ATBF1 in the hippocampal pyramidal cells (test hippocampal pyramidal cells) prepared in step a is detected. The propensity of the hippocampal pyramidal cells to be tested to undergo apoptosis is determined based on the resulting detected amount of ATBF1. Specifically, for example, when a large amount of ATBF1 is detected, it is determined that there is a tendency (or high) to fall into apoptosis, and cell death can be identified.
Here, "ATBF1" refers to AT motif binding factor 1. ATBF1 is known to be a transcription factor that binds to the AT-rich domain of an AFP (alphafetoprotein) regulatory factor and down-regulates the expression of the AFP gene (see Non-Patent Document 1). Two isoforms (ATBF1-A and ATBF1-B) are known to exist in ATBF1. ATBF1-A has a structure in which the protein N-terminal side is longer than ATBF1-B by 920 amino acids. The amino acid sequence of ATBF1-A (404 kDa) is shown in SEQ ID NO: 1. The nucleotide sequence (GenBank accession number: L32832) encoding ATBF1-A is shown in SEQ ID NO: 2. Similarly, the amino acid sequence of ATBF1-B (306 kDa) is shown in SEQ ID NO: 3, and the base sequence encoding ATBF1-B (GenBank accession number: L32833) is shown in SEQ ID NO: 4.

At least one of the following (1) to (4) is detected as the amount of ATBF1.
(1) Total amount of ATBF1 in pyramidal cells of hippocampus.
(2) Nuclear abundance and/or cytoplasmic abundance of partial ATBF1 containing a region corresponding to exon 10 of ATBF1 gene in hippocampal pyramidal cells.
(3) Nuclear abundance and/or cytoplasmic abundance of partial ATBF1 containing a C-terminal region corresponding to exon 11 of ATBF1 gene in hippocampal pyramidal cells.
(4) Nuclear abundance and/or cytoplasmic abundance of partial ATBF1 containing an N-terminal region corresponding to exon 3 of the ATBF1 gene in hippocampal pyramidal cells.

(1) Detection of Total ATBF1 Amount in Hippocampal Pyramidal Cells In the detection of total ATBF1 amount, both ATBF1-A and ATBF1-B are detection targets. However, ATBF1-A is mainly expressed in hippocampal pyramidal cells, and the expression level of ATBF1-B is negligible. Therefore, even if only the amount of ATBF1-A is detected, the same detection result as in the case of detecting both ATBF1-A and ATBF1-B is usually obtained.
Although the amount of ATBF1 is typically detected in the nucleus and cytoplasm of hippocampal pyramidal cells, ATBF1 may be detected only in the nucleus or cytoplasm. Alternatively, the amount of ATBF1 contained in the entire hippocampal pyramidal cell may be detected without distinguishing between the nucleus and the cytoplasm.
As shown in Examples described later, it was confirmed that ATBF1 was increased in hippocampal pyramidal cells of Alzheimer's disease brain. That is, it was found that an increase in the amount of ATBF1 is a suitable and important index for determining the apoptotic tendency. Therefore, for example, when a large amount of ATBF1 is detected in the hippocampal pyramidal cells to be tested, it can be determined that there is a tendency (or high) to fall into apoptosis (cell death is recognized).

(2) Detection of the nuclear abundance and/or cytoplasmic abundance of partial ATBF1 in hippocampal pyramidal cells containing the region corresponding to exon 10 of the ATBF1 gene In this detection, the region corresponding to exon 10 of the ATBF1 gene is included The amount of partial ATBF1 (hereinafter, also referred to as “first partial ATBF1”) in the hippocampal pyramidal cells in the nucleus and/or in the cytoplasm is to be detected. As shown in Examples below, in hippocampal pyramidal cells of Alzheimer's disease brain, (a) the first portion ATBF1 is increased, and (b) the first portion ATBF1 is localized mainly in the nucleus. Admitted. That is, it was found that (a) and (b) are suitable and important indexes for determining the apoptotic tendency. Therefore, for example, when a large amount of the first partial ATBF1 is detected in the test hippocampal pyramidal cells, it can be determined that the test hippocampal pyramidal cells tend to undergo apoptosis (or high) (cell death is recognized). When the determination is made using the expression level of the first partial ATBF1 in the hippocampal pyramidal cells as an index as described above, the nuclear abundance and the cytoplasmic abundance of the first partial ATBF1 are usually detected, but the apoptotic tendency In the (high) hippocampal pyramidal cells, the first part ATBF1 is localized mainly in the nucleus (that is, most of the first part ATBF1 is present in the nucleus), so even if only the amount in the nucleus is detected, It is possible to fully understand the increasing tendency of the first portion ATBF1. Therefore, instead of targeting the abundance in the nucleus and the abundance in the cytoplasm as detection targets, only the abundance in the nucleus may be the detection target.
On the other hand, when the determination is made using the localization mode of the first partial ATBF1 in hippocampal pyramidal cells as an index, the abundance in the nucleus and the abundance in the cytoplasm of the first partial ATBF1 are usually detected simultaneously. Then, the localization mode of the first partial ATBF1 is examined by comparing the detection results, and when the first partial ATBF1 is localized mainly in the nucleus, the hippocampal pyramidal cells to be examined tend to undergo apoptosis (or High). Thus, by comparing the amount detected in the nucleus with the amount detected in the cytoplasm, it becomes possible to clearly understand the subcellular localization mode of the first ATBF1. The intracellular localization of the first ATBF1 may be predicted and determined from the detection result of either the nuclear abundance or the cytoplasmic abundance of the first partial ATBF1. In this case, it is necessary to detect only the existing amount.

  Preferably, the apoptotic tendency is determined using both of the above (a) and (b) as indicators. As a result, more accurate determination can be performed.

(3) Detection of abundance in the nucleus and/or cytoplasm of partial ATBF1 containing a C-terminal region corresponding to exon 11 of ATBF1 gene in hippocampal pyramidal cells In this detection, C corresponding to exon 11 of ATBF1 gene was detected. The nuclear abundance and/or cytoplasmic abundance of hippocampal pyramidal cells in the partial ATBF1 region including the terminal region (hereinafter, also referred to as “second partial ATBF1”) is the detection target. As shown in Examples described later, in hippocampal pyramidal cells of Alzheimer's disease brain, (c) the second partial ATBF1 is increased, and (d) the second partial ATBF1 is localized mainly in the cytoplasm. Was observed. That is, it was found that (c) and (d) are suitable indexes for determining the apoptotic tendency. Therefore, for example, when a large amount of the second partial ATBF1 is detected in hippocampal pyramidal cells, it can be determined that the tested hippocampal pyramidal cells tend to undergo apoptosis (or high) (cell death is recognized). When the determination is performed using the expression level of the second partial ATBF1 in hippocampal pyramidal cells as an index, the abundance in the nucleus and the abundance in the cytoplasm of the second partial ATBF1 is usually detected, but there is a tendency for apoptosis. In (high) hippocampal pyramidal cells, the second partial ATBF1 is localized mainly in the cytoplasm (that is, most of the second partial ATBF1 is present in the cytoplasm). It is possible to fully understand the increasing tendency of partial ATBF1. Therefore, instead of detecting the abundance in the nucleus and the abundance in the cytoplasm as detection targets, only the abundance in the cytoplasm may be detected.
On the other hand, when the determination is made using the localization mode of the second partial ATBF1 in hippocampal pyramidal cells as an index, the abundance in the nucleus and the abundance in the cytoplasm of the second partial ATBF1 are usually detected simultaneously. Then, the localization mode of the second partial ATBF1 was investigated by comparing the detection results, and when the second partial ATBF1 was localized mainly in the cytoplasm, the hippocampal pyramidal cells to be examined tended to undergo apoptosis (or High). Thus, by comparing the amount detected in the nucleus with the amount detected in the cytoplasm, it becomes possible to clearly understand the subcellular localization mode of the second partial ATBF1. The intracellular localization of the second partial ATBF1 may be predicted and determined from the detection result of either the nuclear abundance or the cytoplasmic abundance of the second partial ATBF1. In this case, it is necessary to detect only the existing amount.

  Preferably, the apoptotic tendency is determined using both of the above (c) and (d) as indicators. As a result, more accurate determination can be performed.

(4) Detection of the nuclear abundance and/or cytoplasmic abundance of partial ATBF1 containing the N-terminal region corresponding to exon 3 of the ATBF1 gene in the hippocampal pyramidal cells In this detection, N corresponding to exon 3 of the ATBF1 gene was detected. The nuclear abundance and/or cytoplasmic abundance of hippocampal pyramidal cells in the partial ATBF1 region including the terminal region (hereinafter, also referred to as “third partial ATBF1”) is to be detected. As shown in Examples described later, in hippocampal pyramidal cells of Alzheimer's disease brain, (e) the third portion ATBF1 is increased, and (f) the third portion ATBF1 is localized mainly in the cytoplasm. Was observed. That is, it has been found that (e) and (f) are suitable indices for determining the apoptotic tendency. Therefore, for example, when a large amount of the third partial ATBF1 is detected in hippocampal pyramidal cells, it can be determined that the hippocampal pyramidal cells tend to undergo apoptosis (or high) (cell death is recognized). Thus, when the determination is performed using the expression level of the third partial ATBF1 in hippocampal pyramidal cells as an index, the nuclear abundance and the cytoplasmic abundance of the third partial ATBF1 are usually detected, but there is an apoptotic tendency. In (high) hippocampal pyramidal cells, the third partial ATBF1 is localized mainly in the cytoplasm (that is, most of the third partial ATBF1 is present in the cytoplasm). It is possible to fully understand the increasing tendency of partial ATBF1. Therefore, instead of detecting the abundance in the nucleus and the abundance in the cytoplasm as detection targets, only the abundance in the cytoplasm may be detected.
On the other hand, when the determination is made using the localization mode of the third partial ATBF1 in hippocampal pyramidal cells as an index, the abundance in the nucleus and the abundance in the cytoplasm of the third partial ATBF1 are usually detected simultaneously. Then, by comparing the detection results, the localization mode of the third partial ATBF1 was investigated, and when the third partial ATBF1 was localized mainly in the cytoplasm, hippocampal pyramidal cells tended to undergo apoptosis (or high). To determine. Thus, by comparing the amount detected in the nucleus with the amount detected in the cytoplasm, it becomes possible to clearly understand the subcellular localization mode of the third partial ATBF1. The intracellular localization of the third partial ATBF1 may be predicted and determined from the detection result of either the nuclear abundance or the cytoplasmic abundance of the third partial ATBF1. In this case, it is necessary to detect only the existing amount.

  Preferably, the apoptotic tendency is judged using both of the above (e) and (f) as indicators. As a result, more accurate determination can be performed.

Preferably, two or more of the above (1) to (4) are detected, and the apoptotic tendency of the hippocampal pyramidal cells to be examined is determined in consideration of each detection result. If two or more detections are performed in this way, more detailed and accurate determination/evaluation can be performed.
It is preferable to include (2) detection of the first partial ATBF1 in the detection items. It can be said that the abundance or intracellular localization of the first partial ATBF1 detected in (2) is the most characteristic of the apoptotic tendency. Therefore, the detection of (2) is particularly important in determining the tendency of apoptosis.

  More preferred embodiments include the embodiments for detecting (2) and (3), the embodiments for detecting (2) and (4), and the embodiments for detecting (2) to (4). By increasing the number of detection items in this way, more accurate determination can be performed. Above all, it is most preferable to detect the above (2) to (4) and perform comprehensive evaluation using each detection result. This is because detailed information can be obtained and more highly accurate determination can be performed.

Here, exons 1 to 11 are present in the ATBF1 gene (see FIG. 26). Of these, exon 10 consists of the longest sequence and encodes all four homeodomains. As shown in the examples below, it was suggested that ATBF1 is cleaved into multiple parts in the post-translational process. It was also reported that abnormal skipping of exon 10 produces a mutant protein lacking the region corresponding to exon 10 (Non-Patent Document 5).
If ATBF1 is in a complete state in the cell (if it exists as a full-length protein), "a partial ATBF1 (first partial ATBF1) containing a region corresponding to exon 10 of the ATBF1 gene", "exon 11 of the ATBF1 gene""Partial ATBF1 (second partial ATBF1) containing the region corresponding to" and "Partial ATBF1 (third partial ATBF1) containing the region corresponding to exon 3 of the ATBF1 gene" exist as part of such ATBF1. To do. On the other hand, when ATBF1 is divided in the post-translational process, each partial ATBF1 exists as one (or a part thereof) of the partial ATBF1 generated by the division.
The first portion ATBF1 is recognized by the antibody D1-120 prepared by using a part of the central region of ATBF1-A protein (region other than the homeodomain of exon 10) as an antigen. Similarly, the second part ATBF1 is recognized by the antibody AT6 produced by using a part of the C-terminal region of the ATBF1-A protein as an antigen, and the third part ATBF1 is a part of the N-terminal region of the ATBF1-A protein as an antigen. It is recognized by the antibodies NT440 and 1-12 produced as. The method for producing these antibodies is described in detail in the section of Examples below.

The detection of the amount of ATBF1 or the amount of partial ATBF1 in each of the above steps is not limited to this, but is preferably performed by using an immunohistochemical staining method. According to the immunohistochemical staining method, the amount of ATBF1 and the like can be detected rapidly and with high sensitivity. Also, the operation is simple. Therefore, the burden on the subject (patient) due to the detection of the ATBF1 amount and the like is reduced.
In the immunohistochemical staining method, an antibody that specifically recognizes the detection target (anti-ATBF1 antibody, anti-first partial ATBF1 antibody, etc.) is used, and the amount of ATBF1 is detected using the binding property (binding amount) of the antibody as an index. It
In the immunohistochemical staining method, first, a test hippocampal pyramidal cell is first contacted with a specific antibody (for example, anti-ATBF1 antibody) to be detected. Then, the amount of the antibody bound to the whole cell, nucleus, and/or cytoplasm is measured. Then, from the measurement results, the abundance of the detection target in the whole hippocampal pyramidal cells, the nucleus, and/or the cytoplasm is calculated. Specifically, the method of the present invention can be carried out according to the immunohistochemical staining method shown below.

Immunohistochemical staining of the tissue containing the hippocampal pyramidal cells to be examined is generally performed by the following procedures (1) to (9). Regarding the immunohistochemical staining method of tissue, various literatures and books can be referred to (for example, “enzyme antibody method, 3rd revised edition”, Keiichi Watanabe, Kazuho Nakane, interdisciplinary project).
(1) Fixation/paraffin embedding Tissues collected from living organisms (corpses in the case of autopsy cases) are fixed with formalin or paraformaldehyde. After that, it is embedded in paraffin. Generally, it is dehydrated with alcohol, treated with xylene, and finally embedded in paraffin. The paraffin-embedded specimen is sliced to a desired thickness (for example, 3 to 5 μm) and spread on a glass slide. A frozen sample may be used instead of the paraffin-embedded sample.
(2) Deparaffinization Generally, it is treated sequentially with xylene, alcohol, and purified water.
(3) Pretreatment (antigen activation)
If necessary, enzyme treatment, heat treatment and/or pressure treatment or the like is performed for antigen activation.
(4) Removal of endogenous peroxidase When peroxidase is used as a labeling substance for staining, it is treated with hydrogen peroxide solution to remove the endogenous peroxidase activity.
(5) Inhibition of nonspecific reaction A section is treated with a bovine serum albumin solution (eg, 1% solution) for about several minutes to several tens of minutes to inhibit the nonspecific reaction. Note that this step may be omitted by performing the following primary antibody reaction using an antibody solution containing bovine serum albumin.
(5) Primary antibody reaction An antibody diluted to an appropriate concentration is dropped on a section on a slide glass and then reacted for several tens of minutes to several hours. After completion of the reaction, the plate is washed with an appropriate buffer such as phosphate buffer.
(6) Addition of labeling reagent Peroxidase is often used as a labeling substance. The secondary antibody bound with peroxidase is dropped on the section on the slide glass, and then reacted for several tens of minutes to several hours. After completion of the reaction, the plate is washed with an appropriate buffer such as phosphate buffer.
(7) Color reaction DAB (3,3'-diaminobenzidine) is dissolved in Tris buffer. Then, hydrogen peroxide solution is added. The color-developing solution thus prepared is allowed to penetrate the section for several minutes (for example, 5 minutes) to develop the color. After coloring, the section is thoroughly washed with tap water to remove DAB.
(8) Nuclear staining Nuclear staining is performed by reacting Mayer's hematoxylin for several seconds to several tens of seconds. Wash with running water and develop color (usually for a few minutes).
(9) Dehydration, clearing, encapsulation After dehydrating with alcohol, clearing treatment with xylene is performed, and finally it is sealed with synthetic resin, glycerin, rubber syrup, etc.

The antibody (detection antibody) used in the immunohistochemical staining method is not particularly limited in its type and origin as long as it has a specific binding property to a detection target. The detection antibody may be any of a polyclonal antibody, an oligoclonal antibody (a mixture of several kinds to several tens of kinds of antibodies), and a monoclonal antibody. As the polyclonal antibody or oligoclonal antibody, an IgG fraction derived from an antiserum obtained by immunizing an animal, or an affinity-purified antibody with an antigen can be used. The anti-ATBF1 antibody may be an antibody fragment such as Fab, Fab′, F(ab′) ₂ , scFv or dsFv antibody.
The D1-120 antibody shown in the Examples below can be used for the measurement of the total ATBF1 amount. This antibody specifically recognizes the D1-120 site (a region corresponding to exon 10 and a part of homeodomain 1 and a region immediately before it) which is a common part of ATBF1-A and ATBF1-B. Therefore, using this antibody, both ATBF1-A and ATBF1-B can be detected simultaneously. On the other hand, since this antibody specifically binds to the first partial ATBF1 produced by processing of ATBF1, the amount detected by this antibody reflects the amount of the first partial ATBF1. Therefore, by using this antibody, it becomes possible to grasp the amount of the first partial ATBF1 or its localization. Similarly, by using the antibody AT6 that recognizes the C-terminal region corresponding to exon 11 of the ATBF1 gene, it becomes possible to grasp the amount of the second partial ATBF1 containing the C-terminal region or its localization, and to determine the exon 3 of the ATBF1 gene. By using the antibody 1-12 that recognizes the N-terminal region corresponding to, it becomes possible to grasp the amount of the third partial ATBF1 containing the N-terminal region or its localization.

The anti-ATBF1 antibody and the like can be prepared using an immunological method, a phage display method, a ribosome display method and the like.
Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure. An antigen (for example, the D1-120 part of ATBF1 or a part thereof) is prepared, and an animal such as a rabbit is immunized with the prepared antigen. A non-human species (eg mouse) ATBF1 (or part thereof) can be used as an antigen. The antigen can be obtained by purifying a biological sample. Alternatively, an antigen obtained by using a gene recombination technique can be used. Recombinant human ATBF1 is prepared, for example, by introducing a gene encoding ATBF1 (which may be a part of the gene) into a suitable host using a vector and expressing it in the resulting recombinant cell. It
If it is expected that an effective immunity-inducing action cannot be expected due to its low molecular weight, it is preferable to use an antigen to which a carrier protein is bound. As the carrier protein, KLM (Keyhole Light Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin), etc. are used. A carbodiimide method, a glutaraldehyde method, a diazo condensation method, an MBS (maleimidobenzoyloxysuccinimide) method and the like can be used for binding the carrier protein. On the other hand, an antigen in which ATBF1 (or a part thereof) is expressed as a fusion protein with GST, β-galactosidase, maltose binding protein, or histidine (His) tag can be used. Such a fusion protein can be easily purified by a general-purpose method.

  The present inventors have found that in fetal and adult rat brain tissues, undifferentiated embryonal carcinoma cell lines (P19 cells) and two types of neuroblastoma cell lines (NB-1, GOTO) in actual tissues by Western blotting. The size of ATBF1 protein was assayed in. The antibody used at that time was D1-120 which detects the central part of the ATBF1-A (404 kDa) protein. As a result of Western blotting using these antibodies, the size of the protein recognized by the antibody (that is, the ATBF1 protein) is 404 kDa depending on the tumor cells, 210 kDa, which is about half the size, and many smaller sizes. there were. However, when the antibody against the site corresponding to D1-120 is used for the determination of the apoptotic tendency in hippocampal pyramidal cells in the practice of the present invention, it is considered unnecessary to consider this difference in protein size. However, in order to obtain the same results as those shown in the below-mentioned examples when using recombinant ATBF1, the ATBF1-A amino acid sequence D1 of the ATBF1 gene (which may be a part of the gene) is used. It is considered necessary to select the gene portion encoding the -120 portion (site corresponding to exon 10) and use an antibody prepared using an antigen prepared by introducing it into a suitable host using a vector. As shown by the results of experiments using NT440, 1-12, and AT6, the detection results (localization mode, etc.) when using an anti-ATBF1 antibody that recognizes a site greatly separated from the D1-120 site are D1- The result is completely different from the detection result when 120 is used. However, in the vicinity thereof, it is possible that the partial ATBF1 (first partial ATBF1) detected by D1-120 can be similarly detected using an antibody that recognizes a site other than the D1-120 site. Whether a certain antibody has the same specificity as D1-120 can be verified by a preliminary experiment using P19 cells or the like, a search experiment for localization of ATBF1 using hippocampus of Alzheimer's disease or control brain, etc. .. As a result, if it is determined that the antibody has the same specificity as D1-120, the antibody can be used for the same purpose as D1-120.

  If necessary, immunization is repeated, and when the antibody titer rises sufficiently, blood is collected and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified. On the other hand, a monoclonal antibody can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer rises sufficiently. Next, the obtained antibody-producing cells are fused with myeloma cells to obtain hybridomas. Then, after hybridizing this hybridoma, the clone which produces the antibody which has high specificity with respect to a target protein is selected. The desired antibody can be obtained by purifying the culture solution of the selected clone. On the other hand, the desired antibody can also be obtained by proliferating the hybridomas to a desired number or more, transplanting the hybridomas into the abdominal cavity of an animal (eg, mouse), proliferating in the ascites and purifying the ascites. Affinity chromatography using protein G, protein A or the like is preferably used for the purification of the culture medium or the ascites. Affinity chromatography in which the antigen is immobilized can also be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods may be used alone or in any combination.

  Various modifications can be made to the obtained antibody, provided that the specific binding property to the detection target is retained. Such modified antibodies may be used in the present invention.

  When a labeled antibody is used as the anti-ATBF1 antibody or the like, it is possible to directly detect the amount of bound antibody using the labeled amount as an index. Therefore, the method is simpler. On the other hand, in addition to the need to prepare an anti-ATBF1 antibody having a labeling substance bound thereto, there is a problem that the detection sensitivity is generally low. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody bound with a labeling substance or a method using a polymer bound with a secondary antibody and a labeling substance. The secondary antibody here is an antibody having a specific binding property to the anti-ATBF1 antibody or the like, and when the anti-ATBF1 antibody or the like is prepared as a rabbit antibody, the anti-rabbit IgG antibody is used. Labeled secondary antibodies that can be used against antibodies of various species such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Ltd. and Cosmo Bio Co., Ltd.), depending on the anti-ATBF1 antibody used in the present invention. Appropriate ones can be appropriately selected and used.

  The labeling substance may be any of peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin, and radioactive substances. What is selected is preferably used. In particular, the method of reacting avidin peroxidase using biotin as a labeling substance enables more sensitive detection.

A second aspect of the present invention provides a reagent (reagent for determining an apoptosis tendency) and a kit (kit for determining an apoptosis tendency) for carrying out the method of the present invention.
One embodiment of the reagent of the present invention is the anti-ATBF1 antibody (antibody for detection) used when carrying out the method of the present invention by the immunological method as described above. The antibody here may be any of a polyclonal antibody, an oligoclonal antibody (a mixture of several kinds to several tens kinds of antibodies), and a monoclonal antibody. As the polyclonal antibody or oligoclonal antibody, an IgG fraction derived from an antiserum obtained by immunizing an animal, or an affinity-purified antibody with an antigen can be used. The anti-ATBF1 antibody may be an antibody fragment such as Fab, Fab′, F(ab′) ₂ , scFv or dsFv antibody. As described above, the antibody may be labeled as desired.
As an antibody for a detection antibody, (1) an antibody that recognizes a partial ATBF1 containing a region corresponding to exon 10 of the ATBF1 gene, and (2) an antibody that recognizes a partial ATBF1 containing a C-terminal region corresponding to exon 11 of the ATBF1 gene. (3) An antibody that recognizes a partial ATBF (partial ATBF1-A) containing an N-terminal region corresponding to exon 3 of the ATBF1 gene can be preferably used.
The antibody (1) can detect the partial ATBF1 containing the region corresponding to exon 10, that is, the first partial ATBF1. The antibody (2) can detect the partial ATBF1 containing the C-terminal region corresponding to exon 11, that is, the second partial ATBF1. The antibody (3) can detect the partial ATBF (partial ATBF1-A) containing the N-terminal region corresponding to exon 3, that is, the third partial ATBF1.
Specific examples of the antibody (1), the antibody (2), and the antibody (3) include the followings used in Examples described later.
(1) antibody: D1-120, (2) antibody: AT6, (3) antibody: 1-12, NT440 (NT440-1, NT440-2, NT440-3 mixture)
Details of the characteristics of these antibodies, such as the recognition site and the correspondence with the exon of the ATBF1 gene, are shown in FIG.

The kit of the present invention contains a reagent having a specific binding property to ATBF1. A preferred example of the reagent is an anti-ATBF1 antibody, but is not limited to this. By using the kit of the present invention, the method of the present invention can be carried out more easily and in a shorter time.
As a preferred embodiment, an immunological measurement (detection) kit containing an anti-ATBF1 antibody is provided. In the case of the kit for the method for directly detecting the binding amount of the anti-ATBF1 antibody, a labeled anti-ATBF1 antibody is used. On the other hand, in the case of a kit for an indirect detection method, an unlabeled anti-ATBF1 antibody is used. In this case, a secondary antibody labeled with a labeling substance (labeled secondary antibody) may be included in the kit. When a kit for a detection method using a polymer in which a secondary antibody and a labeling substance are bound is used, the polymer may be included in the kit.

On the other hand, the kit may further include ATBF1 (antigen). Typically, the kit contains ATBF1 that is substantially the same as or equivalent to the one used as the antigen when producing the anti-ATBF1 antibody used in the kit. Therefore, it does not have to be the full-length ATBF1. It may also be recombinant ATBF1. ATBF1 is used to confirm that the stainability obtained using the kit is based on the specific binding between anti-ATBF1 antibody and ATBF1. Specifically, first, anti-ATBF1 antibody is treated with ATBF1. Immunostaining is performed using the treated anti-ATBF1 antibody. The stained image obtained is compared with the stained image obtained using untreated anti-ATBF1 antibody. If strong staining is observed in the latter stained image, it can be confirmed that the staining is based on the specific binding between the anti-ATBF1 antibody and ATBF1.
On the other hand, when an anti-ATBF1 antibody prepared by using a fusion protein with a tag or a carrier protein (hereinafter referred to as a tag) as an antigen is used in the kit, the tag used may be further included in the kit. .. The tag or the like is necessary when there is a possibility that the anti-ATBF1 antibody constituting the kit contains a reactive antibody in the tag or the like used in the production process. By using the tag or the like as described below, it can be confirmed that the staining property obtained by using the kit is based on the specific binding between the anti-ATBF1 antibody and the ATBF1 antibody. First, anti-ATBF1 antibody is treated with this tag or the like. The sample is immunostained with the treated anti-ATBF1 antibody. The stained image obtained is compared with the stained image obtained using untreated anti-ATBF1 antibody. If there is no difference in staining between the two, it can be confirmed that the staining in the latter stained image is based on the specific binding between the anti-ATBF1 antibody and ATBF1.
The kit of the present invention contains one or more reagents necessary for carrying out immunostaining such as antigen-antibody reaction and staining (for example, formalin and paraffin for tissue fixation/embedding, BSA for inhibiting non-specific binding). , DAB and other color-forming reagents, hematoxylin solution for nuclear staining, instruments, etc. may be further included. Further, usually, the kit of the present invention is accompanied by an instruction manual.
Hereinafter, the present invention will be described in more detail with reference to examples (including experimental examples).

1. Examination of the relationship between ATBF1 and cell cycle control system The present inventors have studied the molecular interaction by the yeast two-hybrid method, which is a molecular biology method. We have discovered a new fact that ATBF1 binds not only to DNA but also to the cytoskeletal protein GFAP (glial fibrillary acidic protein) present in the cytoplasm. Although it has been naturally expected and observed that DNA-binding proteins exist in the nucleus in physiological conditions, ATBF1 is also expected to be a nuclear protein, but it not only functions in the nucleus but also in the cytoplasm and cytoskeletal protein. It means that the possibility exists in combination with. This was the starting point for finding the translocation of ATBF1 into the cytoplasm. Moreover, in another experimental system using gastric cancer cultured cells as previously reported (Non-Patent Document 4), the present inventors have found that ATBF1 binds to p53 protein in the nucleus, activates p21 promoter, and suppresses cell cycle. It has been found to work (see Figure 2). Therefore, we focused on the cell cycle control function of ATBF1, and decided to investigate the trend of ATBF1 in various cancer cells. As a result, in some cancer cells, ATBF1 (using D1-120 that detects the central part of ATBF1-A) as a nuclear protein is localized in the nucleus. In the cancer cells that have become infected, ATBF1 is observed to be exported from the nucleus to the cytoplasm, and the present inventors have noticed the importance of the new fact that there is an example in which the staining property of ATBF1 in the nucleus is extremely reduced. .. Furthermore, the inventors have found that these phenomena can be applied to normal nerve cells other than cancer cells through research on P19, an undifferentiated embryonal cancer cell line differentiated into nerve cells. That is, when the staining of ATBF1 in the nucleus of a cancer cell is extremely reduced, the fact that the cancer cell acquires a high degree of proliferative ability, the expression of ATBF1 in P19 cells is not observed, and while ATBF1 is localized in the cytoplasm, Contrary to the fact that proliferative activity is maintained, it is a phenomenon in which nerve cells, which are normal cells, transition to apoptosis when ATBF1 staining in the nucleus of brain neurons is extremely enhanced. Utilizing this phenomenon, the discoverers have established a method for determining neuronal cell death, which is important for diagnosing Alzheimer's disease. As shown below, basic experiments such as examination of staining conditions including antigen activation were performed using various cancer tissues rather than brain hippocampal tissues, but the conditions that were useful in cancer tissues were also in brain hippocampal tissues. I have confirmed that it can be used in exactly the same way.

2. Preparation of anti-ATBF1 antibody (see Figure 1)
2-1. Preparation of antigen (for antibody D1-120)
The 41 amino acid residues of mouse ATBF1 (Ido et al., (1996). Gene, 168, 227-231) (2114 to 2154: LQTLPAQLPPQLGPVEPLPADLAQLYQHQLNPTLLQQQNKR: SEQ ID NO: 5 and antigen portion at the time of producing antibody D1-120) are glutathione S. Recombinant peptide fused to transferase (GST) was used as the antigen. The 41 amino acid residues described above are completely identical to the amino acid residues (2170 to 2147) of human ATBF1.
Specifically, the antigen was prepared by the following procedures (1) and (2). Details of antigen preparation and antibody preparation are described in a previous report (J. Compartive Neurology (2003) 465: 57-71: Non-Patent Document 3).
(1) The target amino acid portion was excised from mouse cDNA as described above, and recombined (subcloned) into the vector pGEX-KT for producing GST fusion protein.
(2) The gene was introduced into Escherichia coli AD202, and the protein expressed in AD202 was purified by a conventional method using Sepharose-glutathione beaded agarose (Sigma) (for example, "First recombinant protein purification handbook"). Published 1999, see Amersham Pharmacia Biotech, Inc.).

2-2. Immunization and antibody separation/purification 2-1. The anti-ATBF1 antibody (D1-120) was obtained by the following procedures (1) to (4) using the antigen (ATBF1-GST fusion protein) prepared in (1).
(1) An antigen (1 mg/ml) melted in PBS (pH 7.5) was mixed with Titer Max Gold (CytRx) in the same amount to prepare an emulsion for immunization.
(2) 2 ml of emulsion was subcutaneously injected to the back of a rabbit to immunize (5 times on days 0, 14, 28, 49 and 70).
(3) At the time when 91 days had elapsed, blood was sacrificed and blood serum was separated.
(4) An anti-ATBF1 antibody was obtained after purifying the antigen-antibody column from the serum by preparing an antigen column with the antigen used for immunization (see, for example, “The First Antibody Purification Handbook”, 2000, Amersham Pharmacia Biotech Co., Ltd.). ).

2-3. Production of the other three types of antibodies (see Figure 1)
First, each antigen was prepared by the following procedure.
(1) In case of antibody NT440 (polyclonal antibody)
Three types of amino acid residues of human ATBF1-A (see Non-Patent Document 1) (4 to 15: CDSPVVSGKDNG: SEQ ID NO: 6, 429 to 445: C KSSEGKDSGAAEGEKQE: SEQ ID NO: 7, 500 to 516: C PSELDEELEDRPHEEPG: The synthetic peptide of SEQ ID NO: 8) was mixed and [ C underlined at the N-terminal of the synthetic peptides of SEQ ID NO: 7 and 8 was added to the original human amino acid sequence to ensure stability of the synthetic peptide. Used as a peptide antigen.
(2) In the case of antibody 1-12 (monoclonal antibody)
Amino acid residues of the human ATBF1-A (143~155: C IVESL S 14 8 QLTQGGG: SEQ ID NO: 9) using a synthetic peptide, with the addition of C (underlined) at the N-terminus, 148 th serine (underlined And a peptide obtained by phosphorylating 148 on the right shoulder) was used as an antigen. Therefore, this antibody is designed to recognize only when serine at position 148 of ATBF1-A is phosphorylated.
(3) In case of antibody AT6 (polyclonal antibody)
Amino acid residues common to human ATBF1-A and B (3405 to 3549: PGASPSPDKDPAKESPKPEEQKNTPREVSPLLPKLPEEPEAESKSADSLYDPFIVPKVQYKLVCRKCQAGFSDEEAARSHLKSLCFFGQSVNLQEMVLHVPTGGGGGGSGGGGGGGGGGGGGGSYHCLACESALCGEEASQ)

  An antibody (polyclonal or monoclonal) was produced using each of the prepared antigens and purified. Preparation of polyclonal antibody, purification was carried out in the same manner as the method described in the section of D1-120 antibody, preparation of monoclonal antibody, purification was carried out using various literature and methods similar to the literature (for example, "enzyme antibody method, Revised 3rd edition", edited by Keiichi Watanabe and Kazuho Nakane, interdisciplinary project).

3. Relationship between ATBF1 expression and cell cycle in cultured cancer cells Mouse undifferentiated embryonal carcininoma cell line P19 cultured cells were used to conduct an experiment to differentiate the cultured cells of undifferentiated cancer into nerve cells, and ATBF1 in the process A study was conducted focusing on the relationship between expression (using D1-120 that detects the central part of ATBF1-A) and cell cycle control.

3-1. ATBF1 expression at the start of P19 culture, cell cycle
At the start of culture of P19 cancer cells, cells in an undifferentiated and proliferating state with no particular stimulation of differentiation were collected and subjected to immunohistochemical staining (ATBF1 staining, D1-120) according to the procedure shown below. The cells were cultured on a chamber slide, fixed with 4% paraformaldehyde adjusted with PBS, and washed after fixation. Anti-ATBF1 antibody, D1-120 was 5μg / ml, the primary antibody solution dissolved in 0.05M Tris buffer (pH 7.6, 1% bovine serum albumin solution, containing sodium azide) was added dropwise to the section, The reaction was carried out at 37°C for about 30 minutes (primary antibody reaction). After thorough washing, the secondary antibody Alexa Fluor 594-conjugated goat anti-rabbit IgG (as shown in the following examples, when double staining with another monoclonal antibody is performed, Alexa Fluor 488-conjugated goat anti-rabbit IgG Mouse IgG was also used at the same time, both of which were made by Molecular Probes) and allowed to react with the sample at room temperature for about 1 hour (secondary antibody reaction). After thorough washing, encapsulation is performed. Observation was performed with a fluorescence microscope (AX70; manufactured by Olympus) or a confocal laser microscope (LSM5; manufactured by ZWISS). As a result of staining, ATBF1 was not expressed in both nucleus and cytoplasm (see FIG. 3a), and the cells showed a proliferative state by a search using flow cytometry, and cells in the cell cycle G1 phase as well as cells in the S phase, G2 However, a high proportion of cells in M phase were mixed (see FIG. 3d).

3-2. P19, ATBF1 expression 24 hours after retinoic acid treatment, cell cycle 24 hours after administration of retinoic acid, a drug that promotes neural differentiation in cultured cells, ATBF1 staining by the method described in 3-1 above, and further flow cytometry I did a search. Morphologically, the cells still maintained the undifferentiated growth state, but the expression of ATBF1 in the cytoplasm was observed (see FIG. 3b). In the search using flow cytometry, the cells still showed a proliferative state, and the proportions of S-phase cells, G2, M-phase cells as well as G1 phase cells were still high (see FIG. 3e).

3-3. Discontinuation of retinoic acid, expression of ATBF1 after 4 days, cell cycle retinoic acid was interrupted, and after further culturing for 4 days, ATBF1 staining was performed by the method described in 3-1 above, and further flow cytometric search was performed. Morphologically, the cultured cells changed into a group of cells differentiated into nerve cells having protrusions. Then, ATBF1 was translocated from the cytoplasm to the nucleus, and the expression mainly in the nucleus was changed (see FIG. 3c). In the search using flow cytometry, the proportion of S-phase cells, G2 cells, and M-phase cells was extremely reduced, and most of them were converted into G1 phase cell population (see FIG. 3f). This means a clear cell cycle arrest, and G1 arrest, in other words, growth suppression was observed. The results of this experiment show that ATBF1 actually translocates from the cytoplasm to the nucleus as expected from the presence of two putative Nuclear localization signals (see FIG. 4a) starting from the amino acid sequence numbers: 277 and 2987. Not only was it proved to be a protein exhibiting the above, but it also shows that cell cycle arrest occurs due to ATBF1 translocation to the nucleus.

4. Examination of regulatory mechanism of translocation of ATBF1 (using D1-120 that detects the central part of ATBF1-A) from the cytoplasm to the nucleus and export from the nucleus to the cytoplasm in P19 cells 4-1. Examination of the promotion mechanism of ATBF1 nuclear import
In P19 cells, when retinoic acid or its inducing chemical substance is allowed to act, ATBF1 mRNA transcription amount and intracellular protein amount increase. However, as long as the P19 cells are cultured in a state of being suspended in the culture medium, ATBF1 remains in the cytoplasm, and nuclear translocation is not observed. Here, we investigated to find out how ATBF1 expression changes by setting conditions such that cells adhere to culture dishes. The localization of ATBF1 in the cells was observed after applying fibronectin, laminin, gelatin, poly-L-ornithine, and poly-L-lysine, which have characteristics similar to the extracellular environment in vivo, to the culture dish. The above 3-1. ATBF1 staining was performed by the same method as described in 1. When fibronectin or the like is not applied to the culture dish, the cells are in a suspended state and ATBF1 appears in the cytoplasm but does not translocate to the nucleus (see FIG. 5a), whereas fibronectin and poly-L-ornithine appear in the culture dish. When the cells were applied to the cells to make them attachable, ATBF1 translocation to the nucleus was observed in a considerable number of cells within 3 hours after the culture (see FIG. 5b), and at 24 hours, almost all the cells had ATBF1 nuclei. Was observed (see Figure 5c). Even when laminin or gelatin was applied to the culture dish, P19 cells were able to adhere well to the culture dish surface, and even under these conditions, ATBF1 translocation to the nucleus was observed. This means that the translocation of ATBF1 from the cytoplasm to the nucleus is regulated by the influence of factors such as fibronectin (or adhesion stimulus itself) that promotes the adhesion of cells to culture dishes. The change from the floating state to the attached state may involve the receptors on the cell surface, and it was thought that the intracellular localization of ATBF1 was changed by transmitting information according to the extracellular environment into the cell. ..

4-2. Examination of ATBF1 export mechanism from nucleus to cytoplasm Recently, it has been reported that CRM1 (Export in 1 or chromosome region maintenance 1) regulates transport of various factors from nucleus to cytoplasm. In ATBF1, the presence of CRM1 target sequence, ie, nuclear export signal, is supposed at three positions (starting from 1267th, 2471th, and 2504th amino acid sequences, respectively) (see FIG. 4b). .. Therefore, it was expected that the action of the antibiotic Leptomycin B, which is an inhibitor of CRM1, would inhibit ATBF1 export from the nucleus to the cytoplasm. This time, using P19 cells, an experiment was conducted in which retinoic acid was allowed to act on the culture dish coated with the above-mentioned fibronectin and poly-L-ornithine, and the antibiotic Leptomycin B was further added, and ATBF1 was prepared by the method described in 3-1 above. The trend of ATBF1 was observed by performing staining (using D1-120 that detects the central portion of ATBF1-A). As a result, the nuclear concentration of ATBF1 in P19 cells was clearly increased by the action of Leptomysin B as compared with the expression level when Leptomycin B was not acted (see FIG. 6a) (see FIG. 6b). At the same time, the number of cells that fell into apoptosis increased obviously. Further, when the culture was further added for 1 day, most of the cells died on the next day. The results of this experiment indicate that the export of ATBF1 from the nucleus is regulated by CRM1 and that the state in which ATBF1 concentration increases in the nucleus promotes the apoptosis of P19 cells that have shown differentiation into neurons. Shows. The result of this experiment is that the state in which the nuclear concentration of ATBF1 (site detectable by D1-120) in hippocampal pyramidal cells of human brain is extremely increased is the state in which the transition to apoptosis is promoted, that is, It seems to give important implications for understanding what leads to the prediction of "cell death".

4-3. Examination of regulatory mechanism of translocation from cytoplasm to nucleus It is known that amino acid phosphorylation is involved in translocation of protein to nucleus/cytoplasm as a fact that was previously examined regarding various factors related to cell cycle. In particular, it has been found that phosphorylation and dephosphorylation of nuclear retention signals are important for nuclear translocation. Among the enzymes that catalyze phosphorylation, PI3K (phosphatidylinositol 3-kinase) family proteins have been well studied, and recently, like ATBF1, N-CoR (The nuclear receptor co-repressor), which functions by translocating into the nucleus and cytoplasm, functions. It was reported that PI3K is involved in phosphorylation of serine 401 at the time of localization from the nucleus to the cytoplasm (Hermanson O, et al, N-CoR controls differentiation of neural stem cells into astrocytes.Nature 419, 934-939, 2002). Based on the fact that ATBF1 expression and N-CoR expression in rat fetal brain neurons are complementary, we conclude that ATBF1 is transferred from the cytoplasm to the nucleus, contrary to the case where N-CoR is exported from the nucleus to the cytoplasm. We predicted that PI3K phosphorylation of the nuclear retention signal might be involved in the translocation. Therefore, in the same manner as described above, using P19 cells, an experiment was conducted in which two retinoic acids were allowed to act on a fibronectin-coated culture dish, and then two PI3K antagonists (Wortmannin, caffeine) were acted on, and the method described in 3-1 above. The ATBF1 trend was observed by performing ATBF1 staining (using D1-120) at. As a result, ATBF1 in P19 cells when no drug was added showed translocation to the nucleus (see FIG. 7a), whereas ATBF1 production itself was not affected in P19 cells when a drug was added. However, there was a tendency that the translocation of the protein into the nucleus was blocked (the effect of caffeine was stronger than that of Wortmannin) (see FIGS. 7b and 7c). At that time, the cells maintained a proliferative state with scattered mitotic figures. Cytoplasmic ATBF1 was observed to be assembled in a ring-like form in the cytoplasmic part around the nucleus (predicted in the endoplasmic reticulum), which is a phenomenon in which ATBF1 translocation from the cytoplasmic (endoplasmic reticulum) to the nucleus depends on PI3K. That is, the cells can maintain the proliferative state in the absence of ATBF1 translocation to the nucleus.

5. Study on the relationship between forced expression of ATBF1 and cell cycle 3. From these results, it was revealed that ATBF1 transits from the cytoplasm to the nucleus and then to the cytoplasm to switch the cell cycle timing and the proliferation state. In order to clarify whether these events occurred mainly due to ATBF1 or the result of the action of some other factor, the present inventors used an ATBF1 single expression vector to analyze mouse neuroblasts. A forced expression experiment was carried out using a neuron-derived cultured cell line Neuro2A cells. After the ATBF1 expression vector was introduced into Neuro2A to forcibly express ATBF1, ATBF1 staining (using D1-120 that detects the central part of ATBF1-A) was performed by the method described in 3-1 above. While observing the trend of ATBF1, we searched the cell cycle by flow cytometry. At the same time, BrdU (5-bromodeoxyuridine) was added to the culture medium to examine whether thymidine incorporation for cell DNA replication was carried out. As a result, it was clarified that BrdU was not taken up by the cells that were found to accumulate in the nucleus by forcedly expressing ATBF1 (see FIG. 8b), and the proportion of cells that arrested the cell cycle increased in the whole cultured cells. (See Figure 8c). These experimental results imply that nuclear expression of ATBF1 is a direct cause of cell DNA replication arrest and cell cycle arrest.
On the other hand, BrdU (5-bromodeoxyuridine) was added to the culture solution at the same time as the gene transfer of the ATBF1 expression vector to label cells during DNA replication. As a result, it was clarified that BrdU uptake was not recognized in all cells in which ATBF1 was forcibly expressed, and the fact that the cell cycle was arrested mainly due to ATBF1 was clarified.

6. Examination of optimal ATBF1 detection conditions (antigen activation conditions) when using anti-ATBF1 antibody To accurately detect ATBF1 from normal pathological specimens surgically collected and embedded in paraffin after fixation with formalin It is important to determine the antigen activation reaction conditions. Proper activation of antigen in both the nucleus and cytoplasm is particularly important for searching ATBF1 nucleocytoplasmic translocation. Of the bladder cancers surgically collected and diagnosed this time, papillary non-invasive bladder urothelial carcinoma in the epithelium (abbreviated as non-invasive carcinoma) and subepithelial invasive bladder urothelial carcinoma (invasive carcinoma) The following studies were conducted to find an optimal antigen activation method. The antibody used is D1-120. First, after fixing with formalin, a paraffin section was prepared and described in 7. ATBF1 staining was performed by the same procedure as in. However, as the antigen activation method, as shown below, a combination of 3 types of heat treatment and 9 types of activation liquid (a total of 27 types of combination) and 3 types of enzyme treatment were used, and their dyeability was examined. Three types of heat treatment: (1) autoclave, 121°C, 15 minutes, (2) pressure cooker, 4 minutes, (3) microwave oven, temperature setting not to boil, 15 minutes. 9 types of antigen stimulating solution: (1) DAKO Target Retrieval Solution PH 6.0, (2) DAKO Target Retrieval Solution High pH, pH 10.0, (3) 10 mM citrate buffer pH 6.0, (4) 10 mM NaOH citrate buffer pH7.0, (5) TE buffer (1 mM EDTA + 10 mM Tris-HCl buffer) pH9.0, (6) 50 mM Tris-HCl buffer pH10.0, (7) 20 mM Tris/0.65 mM EDTA/0.0005% Tween 20 , (8) 1 mM EDTA solution, pH 8.0, (9) 5% Urea solution. Three types of enzyme treatments: (1) trypsin, (2) pepsin, and (3) proteinase K.

  The staining results were summarized. Depending on the heat treatment method and the type of buffer, the staining intensity in non-invasive cancer and invasive cancer, and the staining pattern of nuclei and cytoplasm in cells changed. Although the 27 kinds of stainability were all different, the major tendency was that autoclave treatment tended to make the nucleus stainability remarkable, and cytoplasmic staining could not be obtained depending on the selection of buffer (see FIG. 9). On the contrary, in the microwave treatment, it became difficult to give the stainability of the nucleus, and the stainability of the cytoplasm tended to be conspicuous (see FIG. 10). In the pressure cooker treatment, the temperature was set between the autoclave and the microwave oven, and there was a tendency that the stainability of the nucleus and the cytoplasm could be simultaneously obtained (see FIG. 11). In addition, no staining was obtained with either enzyme treatment for both non-invasive and invasive cancers. Depending on the antigen retrieval method and the type of buffer, ATBF1 is localized in both the nucleus and cytoplasm in non-invasive cancers (see FIGS. 12a and c), and in both the nucleus and cytoplasm in invasive cancers (see FIGS. 12b and d). I was very confused at the evaluation. Although the objective evaluation seemed impossible at first glance, the evaluation of the tendency throughout the 27 kinds of staining was possible as shown in (1) and (2) below. In other words, (1) ATBF1 (D1-120 part) is a protein that can exist in both the nucleus and cytoplasm, but not in the membrane or plasma components. (2) In the non-invasive cancer and the invasive cancer used here, ATBF1 is present in both the nucleus and cytoplasm. However, there is a difference in the protein ratio of subcellular localization of nucleus/cytoplasm, and it can be judged that non-invasive cancer is mainly nuclear and invasive cancer is mainly cytoplasmic. Based on these two assessments, the correspondence for surgically excised and formalin-fixed specimens can be determined. That is, after the ATBF1 staining, it is important to realize not only the presence or absence of ATBF1 expression in cancer cells but also the relative comparison of the amount of ATBF1 in the nucleus and cytoplasm for the purpose of searching the migration of nucleocytoplasm. .. For that purpose, it was judged that it was optimal to use 10 mM citrate buffer (pH 6.0) and heat-treat for 4 minutes (110° C.) in a pressure cooker (see FIGS. 12e and f). Furthermore, heat treatment using microwave (95°C) or autoclave (121°C) is unsuitable for ATBF1 staining because it cannot clearly express the detection of ATBF1 localization migrating in the nucleus and cytoplasm. It was considered. This time, the only antibody used in this good antigen retrieval condition search experiment was D1-120, but the conditions used for D1-120 also when staining with other antibodies (NT440, 1-12, AT6) was performed. By selecting the same antigen stimulating conditions as in 1., it was possible to satisfactorily stain the difference in the nuclear and cytoplasmic localization of ATBF1 in cells (including normal and cancer cells). Therefore, it was determined that the best use of all four anti-ATBF1 antibodies was to use 10 mM citrate buffer (pH 6.0) and heat-treat for 4 minutes (110°C) in a pressure cooker.

7. Staining property of Alzheimer's disease brain tissue with anti-ATBF1 antibody The present inventors used ATBF1 in hippocampal pyramidal cells (using These antibodies were compared and examined in terms of intracellular abundance and localization of NT440, 1-12, D1-120 and AT6). The brains of two females, 78 years old and 84 years old, were clinically diagnosed with Alzheimer's disease and confirmed by pathological findings at necropsy. As a control, the brains of 2 females, both of the 79-year-old and 88-year-old autopsy cases, which are considered to be normal and do not suffer from dementia disease, were used.

7-1. Preparation and Staining of Specimen Whole brain tissue collected at autopsy was fixed with 4% paraformaldehyde. After the fixative had penetrated, the region including hippocampus, parahippocampal gyrus, and dentate gyrus was cut out, embedded in paraffin, sliced to about 3 μm, and extended on a slide glass. After deparaffinization, a citrate buffer solution (pH 6.0) was used for heat treatment for 4 minutes (110° C.) in a pressure cooker (see Example 5 for the reason why the antigen activation method was selected). Then, it was treated with hydrogen peroxide solution to remove the endogenous peroxidase. Next, 0.05M Tris buffer (pH 7.6, 1% bovine serum albumin solution, sodium azide) containing 4 kinds of anti-ATBF1 antibodies (NT440, 1-12, D1-120, AT6) at 5 μg/ml. A primary antibody solution dissolved in (containing) was dropped on the section and reacted at room temperature for about 1 hour (primary antibody reaction). After thorough washing, a secondary antibody (DAKO Enivision, Labeled polymer, HRP [Code No. K1491] Anti-mouse and Anti rabbit) was allowed to act on the sample and reacted at room temperature for about 1 hour (secondary antibody reaction). .. After thorough washing, DAB was dissolved in Tris buffer and a coloring solution containing hydrogen peroxide was permeated into the sample for 5 minutes to develop color (coloring reaction). After the color development, the sample was thoroughly washed with tap water to remove DAB. Then, after dyeing with Meyer's hematoxylin for about 15 seconds, it was washed with running water for 8 minutes for color development. Finally, through alcohol series and xylene series, clearing and encapsulation were performed. By staining human brain tissue by the above procedure, the localization in the nucleus and cytoplasm of pyramidal cells could be clarified with respect to the ATBF1 site that can be detected by four kinds of antibodies.

  The human hippocampus is located posterior to the corpus callosum and has a structure in which it is rolled up on the inner surface of the cerebral hemisphere (see FIG. 13). Strictly speaking, the hippocampus refers to the Cornu Ammonis (abbreviated as CA) and is divided into areas CA1-CA4 (Lorente de No hippocampus classification). This Ammon's angle, dentate gyrus and hippocampus (pre-hippocampus, hippocampus) are collectively called the hippocampal body. Neurons of the hippocampus (Ammon's angle) include large neurons called cone cells and small neurons called non-pyramid cells because of their large and cone-like appearance. Non-pyramidal cells (small nerve cells) are not easy to distinguish from glial cells due to their size, and lack certainty in determining nerve cell death. In comparison, cone cells (large nerve cells) are easy to find in a small amount of tissue and can be reliably distinguished from cells other than nerve cells such as glia, microglia and oligodendroglia that are present in the brain. , The inventors focused their observations on this cone cell. In the following, the localization of ATBF1 in pyramidal cells using each antibody is outlined. In particular, CA3-CA4 in which large pyramidal cells are present and subsegments of the dentate gyrus (see FIG. 13) are observed. This is the finding obtained. Therefore, if biopsy of the human hippocampus may be performed in the future, pinpointed stereotactic brain biopsy of the pyramidal cell layer of the subzone CA3-CA4 is required.

7-2. Stainability with NT440 First, using NT440 (see Fig. 1) capable of detecting the N-terminal side of ATBF1-A (see Fig. 1), the staining property in the majority of hippocampal pyramidal cells in the control normal brain was localized in the cytoplasm. Although shown, localization in the nucleus was also observed in some pyramidal cells (see FIG. 14). In contrast, NT440 staining in the hippocampus of Alzheimer's disease was localized mainly in the cytoplasm in almost all pyramidal cells (see FIG. 15). This change in localization can be said to be one of the features of NT440 staining in Alzheimer's disease. However, since the percentage of pyramidal cells whose staining property is localized in the nucleus is very small even in the normal hippocampus, it was judged that it is an auxiliary feature when diagnosing Alzheimer's disease from a very small amount of material.

7-3.1 Staining property Next, the N-terminal of ATBF1-A is detected in the same manner as NT440, but among them, it can be detected only when the 148th serine is phosphorylated 1-12 (Fig. (See 1), its staining was localized in the cytoplasm in the majority of hippocampal pyramidal cells in the control normal brain, but in some pyramidal cells it was also localized in the nucleus ( See FIG. 16.). In contrast, in the hippocampus with Alzheimer's disease, localization was mainly cytoplasmic in almost all pyramidal cells (see FIG. 17). Similar to NT440, this change in localization was considered to be a characteristic of 1-12 staining in Alzheimer's disease. Another distinguishing feature of NT440 is that the staining intensity of 1-12 is clearly higher in Alzheimer's disease hippocampus compared to normal brain. However, since the determination of the staining intensity usually requires comparison with other samples, it is difficult to determine whether the staining intensity is strong or weak when a normal brain or a brain with Alzheimer's disease is searched alone. Furthermore, regarding the change in localization from the nucleus to the cytoplasm, as in the case of NT440, the staining rate by 1-12 is very low even in the normal brain hippocampus because the percentage of pyramidal cells localized in the nucleus is very low. Although it was effective as an index for diagnosing Alzheimer's disease, it was judged that it remains a supplementary feature when diagnosing Alzheimer's disease from a very small amount of material.

7-4. Stainability with D1-120 Next, using D1-120 (see FIG. 1) capable of detecting the protein central part in the common part of ATBF1-A and B, D1-120 in hippocampal pyramidal cells was detected in normal brain tissue. The stainability was low in both the nucleus and cytoplasm, and the tendency of the stain to concentrate in the nucleus or cytoplasm was unclear (see FIG. 18). On the other hand, in hippocampal pyramidal cells with Alzheimer's disease, the amount of ATBF1 was highly increased, and the staining of D1-120 was clearly nuclear-dominant (see FIG. 19). This high degree of nuclear D1-120 staining is a phenomenon that can hardly be seen in normal brain tissue, whereas the concentration of expression in this nucleus is extremely high in cone cells of Alzheimer's disease. Therefore, it was judged that it could be the most prominent and most important feature worthy of diagnosing Alzheimer's disease from a very small amount of hippocampal material.

7-5. Stainability by AT6 Finally, using AT6 (see Fig. 1), which can detect the C-terminal of the protein at the common part of ATBF1-A and B, shows that in normal brain tissue, AT6 staining in hippocampal pyramidal cells is nuclear. The amount of cytoplasm was small, and the tendency of staining to concentrate in the nucleus or cytoplasm was unclear (see FIG. 20). In comparison, in hippocampal pyramidal cells with Alzheimer's disease, the amount of ATBF1 was highly increased, and AT6 staining was clearly cytoplasmic (see FIG. 21). This highly cytosolic AT6 staining is a phenomenon that can be found only in a small part in normal brain tissue (it is necessary to be careful in this respect. In other words, although it is a part, this phenomenon is normal. However, the probability of this concentration of cytoplasmic expression in Alzheimer's disease pyramidal cells is not as high as that of D1-120. It is quite high, so it is worth diagnosing Alzheimer's disease from a very small amount of hippocampal material. Therefore, it was judged that it could be the second most important feature that has value after D1-120.

  From the above examination of staining, the characteristics of ATBF1 staining in Alzheimer's pyramidal cells are (1) concentration of D1-120 in the nucleus (most important) and (2) localization of AT6 in the cytoplasm. Local concentration (second most important), (3) localization of 1-12 in the cytoplasm and enhancement of staining intensity (auxiliary), (4) localization of NT440 in the cytoplasm (auxiliary) 4 Can be summarized as points. In addition, an increase in the amount of ATBF1 as a whole cell is also one of the characteristics observed in the pyramidal cells of Alzheimer's disease. FIG. 22 illustrates ATBF1 staining, that is, the amount of ATBF1 protein and its intracellular localization by encircling the shaded boxes. How normal pyramidal cells become Alzheimer's disease It showed that it would change.

  Conventional neuropathological diagnosis of Alzheimer's disease is based on (A) Senile plaque SP (silvering method) due to amyloid β protein deposition, (B) Neurofibrillary tangle formation due to abnormal aggregation of tau (Galyas) (3) Braak staining) and (C) Neuronal cell death due to simple atrophy should be clarified. Therefore, the findings obtained by the conventional pathological diagnosis method for Alzheimer's disease in the case used this time are newly taken and are shown in Fig. 23 together with the findings by the Alzheimer disease diagnosis method using ATBF1 this time, and the difference and usefulness of the findings. The sex was compared and considered. According to the conventional classical observation method, in the hippocampal region of Alzheimer's disease brain, compared with the hippocampal region of normal brain, (a) a clear decrease in volume, (b) ordering of large pyramidal cells in the pyramidal cell layer The disappearance of parallel arrays, (c) scattered senile plaques (SP), and (d) scattered neurofibrillary tangles (NFT) in the nerve cells were observed. Therefore, the above four findings can be summarized as representative findings (Fig. 23). On the other hand, in the case of ATBF1 staining, (1) concentration of D1-120 localization in the nucleus (with enhancement of staining intensity), (2) concentration of localization of AT6 in cytoplasm (with enhancement of staining intensity) ), (3) Concentration of localization of 1-12 in cytoplasm (with enhancement of staining intensity), and (4) Concentration of localization of NT440 in cytoplasm.

  Now, among the diagnostic methods using ATBF1, D1-120 is also the site where various basic experiments are performed most closely, and it is easy to theoretically clarify the interpretation of its localization. Therefore, the localization of D1-120, which can be judged to have the highest diagnostic value, to the nucleus of pyramidal cells was confirmed by Galyas Braak (GB) staining, ie, NFT, in the hippocampal pyramidal cell layer of the same patient with Alzheimer's disease. An attempt was made to compare with scattered (see Figures 24 and 25). In FIG. 24, the D1-120 staining and the GB staining of the site (boxed) centering on the Ammon's angle CA3, 4 of the 78-year-old female Alzheimer's disease hippocampus were compared. The number of D1-120-positive pyramidal cells in the nucleus was 27, and it was easy to determine positive as most cells indicated by solid arrows. In contrast, 12 cells with NFT in the cells by GB staining showed clear NFT (solid arrow in the figure), and 12 cells with weak staining but suspected of having NFT (arrow in the figure). , 24 in total. Next, in FIG. 25, the D1-120 staining and the GB staining of the site (boxed) centering on the Ammon's angle CA3 of Alzheimer's disease of 84-year-old female were compared. The number of D1-120-positive pyramidal cells in the nucleus is 62, the number of cells in which NFT is present in the cells by GB staining is 22 in which NFT is clear, and the cell with weak staining, which is suspected of the presence of NFT 33 The total was 55. The nucleus-positive image of D1-120 is easier to judge because the stainability is clearer than the judgment of the presence of NFT by GB staining, and even if cells that are thinly stained by GB staining and difficult to judge are included. The number of pyramidal cells in which the D1-120 portion of ATBF1 was localized in the nucleus tended to be pointed out more than the total number of pyramidal cells in which NFT was observed in the cells by GB staining. The fact that the staining of D1-120 in the nucleus suggests a transition to apoptosis is inferred from the results of basic experiments, and this fact indicates that the ATBF1 portion that can be detected by the antibody D1-120 does not search for cells in which the localization is in the nucleus. , It is possible to simultaneously judge a cell group that will undergo neuronal cell death in the future due to NFT deposition and a cell group that is likely to undergo neuronal cell death in the future even if NFT deposition is small or absent. Seemed to indicate.

  Since the entire hippocampal region can be observed in autopsy cases, (A) Senile plaque SP due to amyloid β protein deposition (Silver method) [Conventional method (c) Same as scattered senile plaques (SP)], (B) Abnormal tau Neurofibrillary tangles due to aggregation NFT formation (Galyas Braak staining) scattered [conventional method (d) coincides with scattered neurofibrillary tangles (NFT) in nerve cells] (a) of the apparent hippocampal volume From the decrease, (b) the disappearance of the ordered parallel arrangement of large pyramidal cells in the pyramidal cell layer, (C) the neuronal cell death due to simple atrophy can be easily assumed. However, it is more sensitive than the scattered neurofibrillary tangle (NFT) in nerve cells due to (d) GB staining, especially in cases such as biopsy cases where it is not possible to clarify the volume decrease and cell array disorder with a small amount of sample. , Using the anti-ATBF1 antibody, which may include neurons that will die in the future even when NFT is not yet deposited (1) Concentration of D1-120 in the nucleus (1) It can be said that it is effective in diagnosing Alzheimer's disease with the increase in staining intensity. In addition, (2) AT6 cytoplasmic localization, (3) 1-12 cytoplasmic localization (with enhanced staining intensity), and (4) NT440 cytoplasmic localization. Provides useful information in determining Alzheimer's disease, albeit with secondary findings.

  The high concentration of ATBF1 sites in the nucleus that can be detected by D1-120 is completely consistent with what the inventors had expected through basic experiments and should be positioned as the most important and striking feature of Alzheimer's disease pyramidal cells. Was completed. Antibodies that recognize different regions of ATBF1 other than D-120 (1-12, NT440, AT-6) were also found to show independent staining properties, and the processing and phosphorylation status of ATBF1-treated proteins was revealed. It seemed possible to know. Therefore, it could be judged that the utility value is sufficient although it is an auxiliary in the practical aspect of Alzheimer's disease diagnosis. To further complement the theoretical aspect, there are 1-12 and NT440 that recognize the amino acid sequence corresponding to exon 3, and the 1-12 antibody recognizes that 148th serine is phosphorylated, and NT440 is Recognizes with or without phosphorylation. It was found that the nuclear retention signal was present in the amino acid sequence corresponding to exon 3 but the nuclear export signal was not present (see FIG. 1 and FIG. 4), and this part was separated from other regions. In this case, the specific state exists only in the nucleus and not in the cytoplasm, and the 1-12 antibody and the NT440 antibody can detect this specific state. AT-6 recognizes the amino acid sequence corresponding to exon 11. Since neither the nuclear retention signal nor the nuclear export signal is structurally present in this amino acid sequence (see FIG. 1 and FIG. 4 ), when it is separated from other regions, it actively activates in the nucleus. It becomes a fragment that cannot move and is expected to accumulate mainly in the cytoplasm. This state can be specifically detected by AT-6. In other words, if a disease state in which the overall structure of ATBF1 is altered due to processing of ATBF1 is expected, consider the findings of 1-12, NT440, AT-6 in addition to D1-120. Is the preferred translation. In conclusion, it is considered that the certainty of the diagnosis will be increased by focusing on the findings of D1-120 and taking a method of assisting the diagnosis by integrating the findings of other antibodies. The simultaneous use of these antibodies (preferably simultaneous use of four antibodies) including identification of cell types is an effective method especially when handling a small sample such as a biopsy instead of an autopsy case as in this observation. Is expected to be.

  According to the histopathological observations up to now, it is said that the dying cone cells show a characteristic image when they disappear from the brain. However, it is difficult and reproducible for general observers. Moreover, the dead cells that have disappeared cannot be seen in reality, and although it is expressed as "neurocyte death due to atrophy", it is possible to morphologically grasp the cell death itself in a small amount of biopsy tissue. Has some difficulties. However, considering that ATBF1 that can be detected by D1-120 is concentrated in the nucleus as a tendency to shift to apoptosis, and in consideration of the fact that a high degree of brain atrophy is observed in such brain tissue, It can be judged that the meaning of determining "neuronal cell death" is not different from that of determining "nerve cells tend to undergo apoptosis". According to this idea, the amount of ATBF1 in the nucleus of the nerve cell (the site that can be detected by D1-120) is related to the judgment of “the tendency of the nerve cell to undergo apoptosis”, that is, the amount of ATBF1 in the nucleus of the nerve cell ( It is synonymous with that the site that can be detected with D1-120) is related to the judgment of "neuronal cell death", and the diagnostic method using this ATBF1 is effective for the diagnosis of "neuronal cell death due to atrophy" and the diagnosis of Alzheimer's disease. It is clear that

8. Construction of kit for determining apoptotic tendency (for determining Alzheimer's disease diagnosis) 8-1. Preparation of Reagents The following reagents were combined into a kit. Here, an example of construction of a kit containing D1-120 as an anti-ATBF1 antibody is shown.
Reagent A: Anti-ATBF1 antibody stock solution
B reagent: ATBF1 antigen solution
C reagent: GST antigen solution
Reagent A is the same as 2. It can be obtained by adjusting the anti-ATBF1 antibody prepared in (2) to 250 μg/ml. The B reagent is obtained by adjusting the recombinant peptide in which 41 amino acid residues of mouse ATBF1 (2114 to 2154: LQTLPAQLPPQLGPVEPLPADLAQLYQHQLNPTLLQQQNKR: SEQ ID NO: 5) of mouse ATBF1 adjusted at the time of preparing the D1-120 antibody to GST were adjusted to 2 mg/ml. The C reagent is obtained by adjusting GST (for example, Sigma) to 2 mg/ml.

8-2. Method of using the kit An example of the method of using the kit (immunohistological staining with DAB color development) is shown below.
(1) The brain hippocampal tissue to be examined, which was resected at the time of biopsy or necropsy by brain surgery, is fixed with 10% formalin and embedded in paraffin by the same procedure as in a normal pathological examination. Paraformaldehyde fixation may be used instead of formalin. There is no particular difference in the staining property of ATBF1 between formalin and paraformaldehyde fixation. The basis is to use the brain tissue material fixed immediately after collection.
(2) The tissue after embedding in paraffin is cut, sliced and placed on a slide glass. As the slide glass, for example, Superfrost, MAS coated, S-9441 (trade name, made by Matsunami) is used. The slide glass has excellent durability against heat treatment by a pressure cooker.
(3) The section is treated with a usual deparaffinization series used for ordinary pathological specimen preparation, and finally alcohol is replaced with purified water.
(4) Between the steps (3), add 2 liters of citrate buffer solution to the pressure cooker (the one used for general cooking can be used) and boil over high heat. A commercially available citrate buffer solution (for example, instant citrate buffer solution [20 times concentrated solution RM-102C], pH 6.0, manufactured by Mitsubishi Kagaku Yatron) can be used. In principle, do not reuse the buffer solution that has been heat-treated once.
(5) Put the section in a metal sample basket, remove the lid of the pressure cooker, and place it sideways in the buffer solution so that the surface on which the section is placed faces upward.
(6) After covering with a lid, heat with high heat until steam noise begins, then turn to low heat and continue heating for 4 minutes. Stop heating and let stand for 1 minute.
(7) Cool the entire pot with running water (tap water). After about 40 minutes, remove the lid of the pressure cooker, remove the cage from the cooker, and replace with purified water. Subsequently, in order to block the endogenous peroxidase, treatment with hydrogen peroxide solution is performed. Next, the solution is replaced with purified water, the hydrogen peroxide solution is removed, and then the sample is soaked in 20 mM Tris buffer. After that, the medium is replaced with 0.05 M Tris buffer pH 7.6.
(8) Prepare 0.05 M Tris buffer pH 7.6 containing 1% bovine serum albumin solution containing sodium azide, and dilute reagent A 50 times with this (antibody solution). The antibody solution is added to the section, and the mixture is allowed to react at room temperature for 1 hour under humidified conditions (in a moist chamber) (primary antibody reaction). Approximately 40 μl of antibody solution is used for one reaction for normal samples, and 80 μl for relatively large samples. Place the tip of the pipette on the front side of the slide, and let the antibody solution fit well to the section, taking care not to touch the section.
(9) Wash the sections with 0.05 M Tris buffer pH 7.6 (5 minutes, 2 times in total). After the cleaning process, wipe off excess cleaning liquid.
(10) HRP-labeled secondary antibody (DAKO Enivision, Labeled polymer, HRP (Code No. K1491) Anti-mouse and Anti-rabbit) is added to the section and reacted for 1 hour at room temperature (secondary antibody reaction).
(11) Wash as in (9).
(12) Two tablets of DAB Chromogen (DAKO, Code. S3000) are dissolved in 40 ml of 0.05M Tris buffer pH 7.6. After adding 30 μl of hydrogen peroxide solution to this, permeate the section (5 minutes) and develop color.
(13) Wash and remove the DAB liquid with running water (tap water).
(14) Stain the section with Mayer's hematoxylin for about 15 seconds.
(15) Place the sections in running water for 8 minutes (color wash).
(16) Pass through alcohol series and xylene series and clear and encapsulate as in the usual pathological specimen preparation.
(17) Microscopic observation is performed using the specimen obtained as a result of the above.

8-2-1. Confirmation of ATBF1(D1-120) specific staining 1
To confirm that the color development by DAB (dark brown) is the result of ATBF1-specific immunostaining, the following procedure is performed.
(1) A reagent (ATBF1 antibody stock solution) 10 μl, B reagent (ATBF1, D1-120 antigen) 1 μl, PBS 10 μl, and 5% BSA 4 μl are mixed in a microtube.
(2) Incubate the entire tube (total volume 25 μl) at 37°C for 2 hours.
(3) After completion of the reaction, 25 μl of a buffer solution (0.05 M Tris buffer solution pH 7.6 containing 1% bovine serum albumin solution containing sodium azide) used for antibody dilution is added. A total of 50 μl of the thus obtained solution is used as a reagent for the primary antibody reaction, and immunohistochemical staining is performed by the above method. As a result, if staining with DAB is not observed, it can be confirmed that ATBF1-specific immunostaining is performed.

8-2-2. Confirmation of ATBF1(D1-120) specific staining 2
Since it was prepared using GST fusion antigen, the anti-ATBF1 antibody (reagent A) may have mixed reactivity with GST. In order to confirm that the color development by DAB is not the result of the reactivity of the anti-ATBF1 antibody with GST, the following operation is performed.
(1) Mix A reagent (ATBF1, D1-120 antibody stock solution) 10 μl, C reagent (GST) 1 μl, PBS 10 μl, and 5% BSA 4 μl in a microtube.
(2) Incubate the entire tube (25 μl) at 37°C for 2 hours.
(3) After completion of the reaction, 25 μl of a buffer solution (0.05 M Tris buffer solution pH 7.6 containing 1% bovine serum albumin solution containing sodium azide) used for antibody dilution is added. A total of 50 μl of the thus obtained solution is used as a reagent for the primary antibody reaction, and immunohistochemical staining is performed by the above method. As a result, if DAB staining is observed, it can be confirmed that ATBF1-specific immunostaining is performed.

Although the kit constructed by using D1-120 as a detection antibody has been described above, a kit may be prepared by further combining an anti-ATBF1 antibody that recognizes a site different from the ATBF1 site recognized by the D1-120 antibody. As a specific example, a kit having the following configuration can be used.
A reagent: Anti-ATBF1 antibody stock solution (NT440, 1-12, D1-120, AT6 4 types of ATBF1 part)
B reagent: ATBF1 antigen solution (NT440 [mixture of 3 kinds of peptides], 1-12, D1-120, 4 kinds of AT6,)
C reagent: GST antigen solution Since this kit can obtain staining results with four types of antibodies, it is possible to determine the apoptosis tendency with higher reliability.

According to the present invention, the apoptotic tendency of the test hippocampal pyramidal cells can be determined. Thus, the present invention provides useful information for diagnosis of Alzheimer's disease. Conventionally, it was virtually impossible to diagnose Alzheimer's disease by utilizing a smaller hippocampal brain tissue, such as a stereotaxic biopsy. It is also effective for the target.
By using the present invention, for example, the progress of Alzheimer's disease using various model animals such as rat, mouse, and monkey, and the therapeutic effect of new therapeutic means are frequently monitored at a very small biopsy level. It will be possible to do so, and it will be useful for the development of a treatment method for this disease, which will become a major social problem in the future. The present invention can also be applied to autopsy cases. In addition, by utilizing the present invention as an adjunct to conventional diagnostic methods for Alzheimer's disease, it is possible to further clarify the transition of neuronal cells to apoptosis and increase the reliability of definitive diagnosis of Alzheimer's disease. Is.
The present invention can be applied to early diagnosis of Alzheimer's disease using an extremely small amount of biopsy material, if stereotactic brain biopsy of the hippocampal tissue of the brain becomes technically and ethically possible in the future. Furthermore, from the background that the PET probe that recognizes senile plaques has already been developed, the development of this method may become an important starting point for enabling neuron death to be captured more image-wise and easily. I think there is.

The present invention is not limited to the description of the embodiments and examples of the invention. Various modifications are also included in the present invention within the scope that can be easily conceived by those skilled in the art without departing from the scope of the claims.
The contents of papers, published patent publications, patent publications, and the like, which are specified in this specification, are all incorporated by reference.

Claims (9)

A method of determining the propensity of hippocampal pyramidal cells to undergo apoptosis, comprising the following steps:
(a) preparing hippocampal pyramidal cells collected from the subject, and
(b) Detecting the amount of ATBF1 in the hippocampal pyramidal cells. The determination method according to claim 1, wherein in the step (b), one or more selected from the group consisting of the following (1) to (4) is detected:
(1) the total amount of ATBF1 in the hippocampal pyramidal cells,
(2) The amount of partial ATBF1 containing a region corresponding to exon 10 of the ATBF1 gene in the hippocampal pyramidal cells in the nucleus and/or in the cytoplasm,
(3) Nuclear abundance and/or cytoplasmic abundance in the hippocampal pyramidal cells of a partial ATBF1 containing a C-terminal region corresponding to exon 11 of the ATBF1 gene,
(4) Nuclear abundance and/or cytoplasmic abundance of the partial ATBF1 containing an N-terminal region corresponding to exon 3 of the ATBF1 gene in the hippocampal pyramidal cells. The determination method according to claim 1, wherein the following (2) to (4) are detected in the step (b):
(2) The amount of partial ATBF1 containing a region corresponding to exon 10 of the ATBF1 gene in the hippocampal pyramidal cells in the nucleus and/or in the cytoplasm,
(3) Nuclear abundance and/or cytoplasmic abundance in the hippocampal pyramidal cells of a partial ATBF1 containing a C-terminal region corresponding to exon 11 of the ATBF1 gene,
(4) Nuclear abundance and/or cytoplasmic abundance of the partial ATBF1 containing an N-terminal region corresponding to exon 3 of the ATBF1 gene in the hippocampal pyramidal cells.   The determination method according to claim 1, wherein the detection is performed by using an immunohistochemical staining method.   A reagent for determining apoptosis tendency, which comprises an anti-ATBF1 antibody. The apoptotic tendency determination reagent according to claim 5, wherein the anti-ATBF1 antibody is an antibody selected from the group consisting of (1) to (3) below:
(1) an antibody that recognizes a partial ATBF1 containing a region corresponding to exon 10 of the ATBF1 gene,
(2) an antibody that recognizes a partial ATBF1 containing a C-terminal region corresponding to exon 11 of the ATBF1 gene,
(3) An antibody that recognizes a partial ATBF1 containing an N-terminal region corresponding to exon 3 of the ATBF1 gene. An apoptotic tendency determination kit comprising one or more antibodies selected from the group consisting of (1) to (3) below:
(1) an antibody that recognizes a partial ATBF1 containing a region corresponding to exon 10 of the ATBF1 gene,
(2) an antibody that recognizes a partial ATBF1 containing a C-terminal region corresponding to exon 11 of the ATBF1 gene,
(3) An antibody that recognizes a partial ATBF1 containing an N-terminal region corresponding to exon 3 of the ATBF1 gene.   The apoptotic tendency determination kit according to claim 7, further comprising ATBF1.   The apoptosis tendency determination kit according to claim 7 or 8, wherein the antibody is produced using an antigen that is a fusion protein with a tag or a carrier protein, and further comprises the tag or the carrier protein. ..