Use of PLCXD3 as a treatment target for memory, cognition or learning disorder

Abstract

The present invention relates to the use of PLCXD3 as a therapeutic target for memory, cognitive, or learning disabilities. Specifically, the present invention relates to a method for providing information necessary for diagnosing memory, cognitive, or learning disabilities, comprising the steps of: (a) measuring the expression level of PLCXD3 in a biological sample isolated from an individual; and (b) comparing the PLCXD3 expression level from step (a) with the expression level of a normal control group, and determining that there is a memory, cognitive, or learning disability if the PLCXD3 expression level is lower than that of the normal control group. Furthermore, the present invention relates to an animal model having memory, cognitive, or learning disabilities in which the Plcxd3 gene is knocked out, a method for preparing the same, and a screening method for a therapeutic agent for memory, cognitive, or learning disabilities using the animal model in which the Plcxd3 gene is knocked out. Moreover, the present invention relates to a pharmaceutical composition for the prevention or treatment of memory, cognitive, or learning disabilities comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient. In addition, the present invention is characterized by confirming a decrease in working memory and an increase in amyloid plaques and brain inflammation when PLCXD3 expression is inhibited in an animal model of Alzheimer's disease, thereby identifying that PLCXD3 is a factor that plays an important role in the development of Alzheimer's disease, and at the same time identifying that a PLCXD3 expression promoter or an activator thereof can prevent or treat Alzheimer's disease.

Description

Technology Field

[0001] The present invention relates to the use of PLCXD3 as a therapeutic target for memory, cognitive, or learning disabilities. Background Technology

[0002] The entire world is rapidly entering an aging society, and accordingly, humans are seeking a high quality of life that allows them to live a long and healthy life free from disease. As the elderly population increases due to the extension of life expectancy resulting from scientific advancements, the incidence of degenerative brain diseases caused by the decline in brain function is skyrocketing.

[0003] Although the brain has various functions, memory, cognition, and learning are among its major functions; without these abilities, it is difficult to perform daily activities and poses a threat to survival.

[0004] Meanwhile, bipolar disorder is one of the most common mental disorders with an unknown mechanism. With a lifetime prevalence of 2–5%, it is a major mental illness that typically develops in late adolescence or early adulthood and affects a person throughout their life. As a representative mood disorder characterized by repeated elevations and depressions in mood, it is marked by the recurrent recurrence and chronicity of manic, depressive, and mixed episodes.

[0005] Family studies of patients with bipolar disorder have shown that, compared to the general population, the prevalence of bipolar disorder is four times higher and that of depression is two times higher. Twin studies of bipolar disorder have shown a 65-100% concordance rate in identical twins and a 10-30% concordance rate in fraternal twins. Synthesizing various epidemiological studies, bipolar disorder can be considered a highly heritable disease with a heritability of 60-80%.

[0006] Although many genetic studies have been conducted on bipolar disorder, it has been reported that it may occur in various chromosomal regions and among the various candidate genes presented, the results regarding mutual association were incomplete. Despite numerous studies aimed at identifying genes that regulate emotional states associated with bipolar disorder, the causative genes and basic mechanisms causing bipolar disorder have not yet been identified.

[0007] Meanwhile, phospholipase C (PLC) is an enzyme that hydrolyzes the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in response to external stimuli such as hormones, neurotransmitters, and neurotrophic factors. Additionally, six subtypes (β, γ, δ, ε, ζ, and η) of the PLC enzyme exist and have been identified in other human tissues, including the brain, heart, spleen, thymus, and hematopoietic cells. Most PLC subtypes (excluding PLC-β and PLC-γ) possess a C2 domain (core domain) that plays an essential role in producing IP3 by interacting with membrane-associated receptors in response to external stimuli. Furthermore, all PLC subtypes exhibit tissue-specific patterns in the localization of enzymes with unique functions for regulating cellular metabolism.

[0008] According to recent research, a class of PLC called PI-PLC (phosphoinositide-specific phospholipase C) has been identified. PI-PLC is referred to as the PLCXD enzyme, a subgroup of the PLC family that contains only the catalytic X-domain, which is the X-domain responsible for producing IP3. Additionally, three different isoforms of PLCXD (PLCXD1, PLCXD2, and PLCXD3) have been identified; each enzyme is thought to possess unique functions depending on tissue distribution and cellular location, and PLCXD enzymes have been found to function as active phosphodiesterases by influencing the increase of inositol phosphate.

[0009] Among these, PLCXD3 is known to be localized to the cytoplasm and perinuclear vesicles of HeLa cells, but there has been almost no research on specifically what functions PLCXD3 performs.

[0010] Accordingly, to investigate the role of PLCXD3 in brain function, the inventors prepared an animal model lacking the Plcxd3 gene (Knock-out, KO) using the CRISPER / Cas9 system method, and through comparative analysis between the animal model lacking Plcxd3 and a normal animal model, it was confirmed that the deficiency of the Plcxd3 gene leads to imbalances in motor ability, spatial memory ability, cognitive ability, learning memory, and social ability.

[0011] In addition, to investigate the role of Plc-xd3 in bipolar disorder-related behaviors by brain region in animal models lacking the Plcxd3 gene throughout the brain, the inventors prepared animal models with knock-down (KD) or over-expression (OE) of the amygdala and hippocampus-specific Plcxd3 gene. As a result of analyzing each prepared animal model, it was confirmed that when the amygdala-specific Plcxd3 gene expression was knock-down (KD), anxiety responses were reduced, short-term spatial memory and social memory were impaired, and fear formation did not occur normally compared to the normal control group, and when the amygdala-specific Plcxd3 gene was over-expressed (OE), short-term spatial memory and social memory were impaired compared to the normal control group. In addition, it was confirmed that when the expression of the Plcxd3 gene was specifically reduced (KD) or overexpressed (OE) in the hippocampal CA1, hyperactivity increased compared to normal control groups, while short-term spatial memory and social memory showed no difference.

[0012] In addition, it was confirmed that Plcxd3 gene deficiency in an Alzheimer's animal model (5xFAD) leads to a decrease in working memory and a significant increase in amyloid plaques and brain inflammatory lesions.

[0013] Accordingly, the inventors have completed the present invention by confirming that the Plcxd3 gene can be targeted as a novel target for the prevention, improvement, or treatment of memory impairment, cognitive impairment, brain developmental disorders, and neurodegenerative diseases. Prior art literature

[0014] 1. Korean Published Patent 10-2022-00241612. U.S. Published Patent US2020 / 0368239 The problem to be solved

[0015] Therefore, the object of the present invention is to provide a method for providing information necessary for diagnosing memory, cognitive, or learning disabilities, comprising: (a) a step of measuring the single expression level of PLCXD3 in a biological sample isolated from an individual; and (b) a step of comparing the PLCXD3 expression level of step (a) with the expression level of a normal control group and determining that there is a memory, cognitive, or learning disability if the PLCXD3 expression level is lower than that of the normal control group.

[0016] In addition, the present invention provides an animal model having memory, cognitive, or learning disabilities in which the Plcxd3 gene is knocked out, and a method for producing the same.

[0017] In addition, the present invention provides a method for screening therapeutic agents for memory, cognitive, or learning disorders using an animal model having memory, cognitive, or learning disorders in which the Plcxd3 gene is knocked out.

[0018] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of memory, cognitive, or learning disorders, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

[0019] In addition, the present invention provides a health functional food for the prevention or improvement of memory, cognitive, or learning disabilities, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

[0020] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neuroinflammation, tau-related diseases, or degenerative brain diseases, characterized by comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and having inhibition of the activity of microglia or astrocytes; and inhibition of amyloid plaques.

[0021] Furthermore, the present invention provides a health functional food for the prevention or improvement of neuroinflammation, tau-related diseases, or degenerative brain diseases, characterized by comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and having inhibition of the activity of microglia or astrocytes; and inhibition of amyloid plaques. means of solving the problem

[0022] To achieve the above objective, the present invention provides a method for providing information necessary for diagnosing a memory, cognitive, or learning disorder, comprising: (a) measuring the expression level of PLCXD3 in a biological sample isolated from an individual; and (b) comparing the PLCXD3 expression level of step (a) with the expression level of a normal control group, and determining that if the PLCXD3 expression level is lower than that of the normal control group, the individual has a memory, cognitive, or learning disorder.

[0023] In one embodiment of the present invention, the expression level may be the mRNA level of the Plcxd3 gene or the PLCXD3 protein expression level.

[0024] In one embodiment of the present invention, the Plcxd3 gene may be composed of the nucleotide sequence of SEQ ID NO. 1, and the PLCXD3 protein may be composed of the amino acid sequence of SEQ ID NO. 2.

[0025] In one embodiment of the present invention, the memory, cognitive, or learning disability may be a memory, cognitive, or learning disability caused by aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, CNS hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug intoxication, epilepsy, seizures, hippocampal sclerosis, headache, cerebral senility, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, neurological disease, cardiovascular disease, amnesia, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or attention deficit.

[0026] In addition, the present invention provides an animal model having memory, cognitive, or learning disabilities in which the Plcxd3 gene is knocked out.

[0027] In one embodiment of the present invention, the deficiency of the Plcxd3 gene may be induced by the CRISPR / Cas9 system.

[0028] In one embodiment of the present invention, the TCAACGAG sequence of the Plcxd3 gene may be targeted to induce a deficiency of the Plcxd3 gene.

[0029] In addition, the present invention provides a method for producing an animal model having memory, cognitive, or learning disabilities, comprising the step of inducing a knockout of the Plcxd3 gene.

[0030] In one embodiment of the present invention, the deficiency of the Plcxd3 gene may be induced by targeting the TCAACGAG sequence of the Plcxd3 gene with a CRISPR / Cas9 system.

[0031] In addition, the present invention provides a method for screening a therapeutic agent for memory, cognitive, or learning disabilities, comprising: (a) treating an animal model of the present invention with a candidate drug; and (b) comparing the animal model treated with the candidate drug in (a) with an untreated control group to determine whether there is an improvement in memory, cognitive, or learning disabilities.

[0032] In one embodiment of the present invention, determining whether there is an improvement in memory, cognition, or learning disability may be done by analyzing the Novel tank test, Alarm substance test, Open field test (OFT), Y-maze test, Novel object recognition test (NORT), Passive avoidance test (PAT), or Social interaction test (SAT).

[0033] In one embodiment of the present invention, the method may further include the step of determining the candidate drug as a therapeutic agent capable of improving, preventing, or treating memory, cognitive, or learning disorders if, in an animal model treated with the candidate drug, compared to an untreated control group, there is an increase in the number of dendritic spines in the cortex and hippocampus; an increase in the expression of β1 and β2, which are PLC subfamily members; an increase in the production of IP3; an increase in the expression of nrsn1, glun2b, and creb, which are learning or memory-related factors; or an increase in the expression of gng2, gpr183, and ephb2, which are upstream target factors of the PLC family.

[0034] In one embodiment of the present invention, the memory, cognitive, or learning disability may be memory, cognitive, or learning disability caused by aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, CNS hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug intoxication, epilepsy, seizures, hippocampal sclerosis, headache, cerebral senility, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, neurological disease, cardiovascular disease, amnesia, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or attention deficit.

[0035] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of memory, cognitive, or learning disorders, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

[0036] In one embodiment of the present invention, the Plcxd3 gene may be composed of the nucleotide sequence of SEQ ID NO. 1, and the PLCXD3 protein may be composed of the amino acid sequence of SEQ ID NO. 2.

[0037] In one embodiment of the present invention, the memory, cognitive, or learning disability may be memory, cognitive, or learning disability caused by aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, CNS hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug intoxication, epilepsy, seizures, hippocampal sclerosis, headache, cerebral senility, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, neurological disease, cardiovascular disease, amnesia, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or attention deficit.

[0038] In addition, the present invention provides a health functional food for the prevention or improvement of memory, cognitive, or learning disabilities, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

[0039] The present invention also provides a pharmaceutical composition for the prevention or treatment of neuroinflammation, tau-related diseases, or degenerative brain diseases, characterized by comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and having inhibition of the activity of microglia or astrocytes; and inhibition of amyloid plaques.

[0040] Furthermore, the present invention provides a health functional food for the prevention or improvement of neuroinflammation, tau-related diseases, or degenerative brain diseases, characterized by comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and having inhibition of the activity of microglia or astrocytes; and inhibition of amyloid plaques. Effects of the invention

[0041] According to the present invention, by confirming the possibility of developing degenerative brain diseases, including memory impairment, cognitive impairment, and learning impairment, which result from motor, spatial, cognition, learning, memory, and social imbalances caused by PLCXD3 gene deficiency, it was discovered that the PLCXD3 gene and PLCXD3 protein can be used as novel targets for the prevention, improvement, or treatment of such brain diseases. Therefore, the onset of brain diseases can be diagnosed by measuring the expression or activity level of PLCXD3. Furthermore, the animal model lacking PLCXD3 prepared in the present invention can be usefully utilized for screening therapeutic agents for memory, cognitive, or learning disabilities and related degenerative brain diseases. Brief explanation of the drawing

[0042] Figure 1 shows a comparison of a zebrafish with Plcxd3 KO in one embodiment of the present invention with a normal zebrafish, (A) showing the results of in situ-hybridization analysis of the Plcxd3 gene expression pattern (top) and morphological changes (bottom) in the zebrafish brain 48 hours after fertilization, (B) showing a comparison of the normal Plcxd3 sequence in exon 2 and the sequence of a mutant with + nucleotide 29 bp inserted for the production of Plcxd3 KO zebrafish, (C) showing a photograph observing the phenotype of the Plcxd3 KO zebrafish compared with a normal zebrafish at 5 hpf (days after fertilization), and (D) and (E) showing the results of measuring the distance traveled in a novel tank test and a warning substance test. Figure 2 shows the analysis of PLCXD3 expression levels in normal mouse brain regions, (A) is the result of analyzing brain region-specific PLCXD3 expression levels in the brain of a 3-month-old normal mouse by real-time PCR, (B) and (C) are the results of confirming PLCXD3 protein expression levels in the brain of a 3-month-old normal mouse by immunohistochemistry, and (D) is a photograph showing the PLCXD3 protein expression levels observed in the hippocampus of a 3-month-old normal mouse. Figure 3 shows that PLCXD3 is mainly expressed in excitatory and inhibitory neurons of normal mice, (A) shows the results of immunofluorescence staining performed in normal mice using anti-PLXD3 and anti-GluA2 antibodies, (B) to (G) show the quantitative results of (A), (H) shows the results of immunofluorescence staining performed in normal mice using anti-PLXD3 and anti-GAD67 antibodies together, and (I) to (N) show the quantitative results of (H). FIG. 4 illustrates the process of producing Plcxd3 KO mice in an embodiment of the present invention, wherein (A) shows the CRISPER / Cas9 system for generating Plcxd3 KO mice, (B) shows the target sequence of Plcxd3 for producing a frameshift mutant (8 bp, top, blue deleted), and shows the confirmation of normal littermates, Plcxd3 heterozygotes, and KO mice via PCR, (C) shows a schematic diagram of the mating and generation process of Plcxd3 KO mice, (D) shows the target sequence of the Plcxd3 frameshift mutant (8 bp deleted, top, blue) and primer sequences for detecting the Plcxd3 gene (yellow) in normal littermates and Plcxd3 KO mice, (E) shows a schematic diagram of the process for confirming Plcxd3 KO mice using RT-PCR or real-time PCR, and (F) to (G) show Plcxd3 KO mice This shows the results of analyzing the expression of Plcxd3 in the hippocampi of the offspring of the same litter using RT-PCR and real-time PCR. Figure 5 shows the results of the behavioral, spatial, cognitive, learning, memory, and social abilities in Plcxd3 KO mice, including (A) open field test, (B) Y-maze test, (C) novel object recognition test, (D) passive avoidance test, and (E) social relationship test. Figure 6 confirms the decrease in the number of dendritic spines in the cortex and hippocampus of Plcxd3 KO mice, (A) shows the number of dendritic spines in the cortex apex slope (AO) and basal shift (BS) regions of the cortex and hippocampus of 3-month-old Plcxd3 KO mice and normal littermates, confirmed by Golgi staining, (B) to (C) are quantitative graphs of the results of (A), (D) shows the number of dendritic spines in the cortex apex slope (AO) and basal shift (BS) regions of the cortex and hippocampus of 7-month-old Plcxd3 KO mice and normal littermates, confirmed by Golgi staining, and (E) to (F) are quantitative graphs of the results of (D). Figure 7 shows the effects of Plcxd3 KO on the expression levels of PLC-related molecules, (A) shows the IP3 concentration in the hippocampus of 3-month-old Plcxd3 KO mice and normal littermates measured using ELISA, (B) shows the expression levels of PLC isoforms analyzed by real-time PCR, (C) to (D) show the expression levels of learning and memory-related factors nrsn1 and glun2b analyzed by real-time PCR, (E) shows the mRNA levels of PLCXD3-related upstream receptors gng2, gpr183, and ephb2 in 7-month-old Plcxd3 KO mice and normal littermates analyzed by real-time PCR, and (F) shows the results of analyzing the mRNA levels of the creb and nrsn1 genes related to learning and memory in the hippocampus of 3-month-old Plcxd3 KO mice and normal littermates using real-time PCR. Figure 8 shows the changes in anxiety / hyperactivity, short-term spatial cognitive function, sociality, and fear response when amygdala-specific Plcxd3 gene expression is reduced (KD) in normal animals (normal mice), with (A) showing the results of the cross maze test, (B) the Y-maze test, (C) the social relationship test, and (D) the fear conditioning response test. Figure 9 shows the changes in anxiety / hyperactivity, short-term spatial cognitive function, and sociality in normal animals when the amygdala-specific Plcxd3 gene overexpressed (OE), with (A) showing the results of the cross maze test, (B) showing the results of the Y-maze test, and (C) showing the results of the social relationship test. Figure 10 shows the changes in anxiety / hyperactivity, short-term spatial cognitive function, and sociality in normal animals when hippocampal CA1-specific Plcxd3 gene expression is reduced (KD), with (A) showing the results of the cross maze test, (B) showing the results of the Y-maze test, and (C) showing the results of the social relationship test. Figure 11 shows the changes in anxiety / hyperactivity, short-term spatial cognitive function, and sociality in normal animals when the hippocampal CA1-specific Plcxd3 gene overexpressed (OE), with (A) showing the results of the cross maze test, (B) showing the results of the Y-maze test, and (C) showing the results of the social relationship test. Figure 12 shows (A) that the hippocampal CA1-specific Plcxd3 or control AAV virus was successfully infected when hippocampal CA1-specific Plcxd3 gene expression was reduced (KD) in animals with Alzheimer's disease, and (B) the results of the passive avoidance test. Figure 13 shows (A) PLCXD3 gene expression, (B) amyloid plaque change, (C) APP expression, (D) BACE1 expression, and (E) DYRK1A expression change in hippocampal CA1-specific Plcxd3 gene expression reduction (KD) in animals with Alzheimer's disease. Figure 14 shows the changes in microglia activity (AB) and astrocyte activity (CD) in hippocampal CA1-specific Plcxd3 gene expression (KD) in animals with Alzheimer's disease. Specific details for implementing the invention

[0043] The present invention is characterized by providing the use of PLCXD3 (phospholipase C X-domain containing protein 3) as a therapeutic target for memory, cognitive, or learning disorders.

[0044] Currently, there is a complete lack of research regarding the relationship between PLCXD3 and brain disorders, including memory, cognitive, or learning disabilities.

[0045] While conducting research to discover new treatments for memory, cognitive, or learning disabilities and neurodegenerative diseases, the inventors prepared an animal model lacking the Plcxd3 gene and confirmed that memory, cognitive, or learning disabilities were induced in the Plcxd3-deficient animal model compared to normal animals, including hyperactivity, increased anxiety, reduced short-term / long-term memory, decreased cognitive function, learning disabilities, and impaired social behavior.

[0046] Accordingly, the inventors were able to determine that the Plcxd3 gene is associated with brain diseases including memory, cognitive, or learning disorders, and through the experiments of the following examples, they identified the role of PLCXD3 on brain function in zebrafish and mouse models lacking the Plcxd3 gene.

[0047] According to one embodiment of the present invention, when a novel tank test and an alarm substance test were performed on zebrafish lacking the Plcxd3 gene and normal zebrafish, it was found that the zebrafish lacking Plcxd3 exhibited a "hyperkinetic" pattern in all tests compared to normal zebrafish, and such hyperkinetic behavior can be considered to correspond to symptoms of bipolar disorder in humans.

[0048] In another embodiment of the present invention, mice lacking the Plcxd3 gene were prepared using a CRISPR / Cas9 system, and various brain function verification experiments were performed.

[0049] As a result, it was observed that mice lacking Plcxd3 spent less time in the central area of ​​the test space during open field tests compared to normal mice, confirming increased anxiety. Additionally, through the Y-maze, NORT, and PAT tests, mice lacking the Plcxd3 gene were found to have lower abilities in recognizing new objects and spatial awareness compared to normal mice, as well as lower learning ability and impaired social interaction behavior.

[0050] These results imply that PLCXD3 is involved in emotional behavior, spatial work, perception and learning, memory, and the regulation of social behavior.

[0051] In addition, in another embodiment of the present invention, the number of dendritic spines in the cerebral cortex and hippocampus of Plcxd3-deficient mice was analyzed, and it was found that the number of dendritic spines decreased in the absence of Plcxd3. These results imply that PLCXD3 acts on the formation of the number of dendritic spines associated with memory integration in the brain.

[0052] In addition, the inventors analyzed whether PLCXD3 affects the expression levels of genes related to learning, cognition, and memory. As a result, it was found that in Plcxd3-deficient mice, the mRNA levels of cytoplasmic vesicle component nrsn1, NMDA receptor subunit glun2b, and creb, which are factors related to learning and memory, were reduced. Furthermore, it was confirmed that the expression levels of upstream molecular target factors of the PLC family, such as gng2, gpr183, and ephb2, were all significantly reduced. In addition, it was confirmed that inhibiting PLCXD3 expression in animals with Alzheimer's disease resulted in a decrease in working memory and an increase in amyloid plaques and brain inflammation.

[0053] Through this, it was found that PLCXD3 plays an important role in brain functions, including memory, learning, cognition, and social behavior, as well as lesions in Alzheimer's disease.

[0054] Based on the above results, the inventors found that if the expression of PLCXD3 in the brain is reduced or suppressed, brain diseases including memory, cognitive, or learning disabilities may be induced, and that the onset of brain diseases including memory, cognitive, or learning disabilities can be predicted or diagnosed by measuring the expression level or activity of PLCXD3.

[0055] Therefore, the present invention can provide an animal model having memory, cognitive, or learning disabilities in which the Plcxd3 gene is knocked out.

[0056] The above-mentioned deficiency of the Plcxd3 gene can be performed by a method according to the CRISPR / Cas9 system, and in one embodiment of the present invention, the Plcxd3 gene was deficient by the CRISPR / Cas9 system by targeting the TCAACGAG sequence of the Plcxd3 gene.

[0057] In addition, the present invention may provide a method for producing an animal model having memory, cognitive, or learning disabilities, comprising a step of inducing a knockout of the Plcxd3 gene, and a specific method for production is described in the following examples.

[0058] In the present invention, animal models with the Plcxd3 gene knocked out were prepared using zebrafish and mice.

[0059] In the present invention, the term "knock out" refers to causing the loss of function of a target gene, meaning the elimination of gene expression by destroying or removing the target gene using genetic engineering methods. A method for inducing a knock out of a target gene may cause the loss of expression and function of the target gene by deleting a portion of the target gene's nucleotide sequence, substituting it with another nucleotide sequence, or introducing another nucleotide sequence.

[0060] When the above mouse is used as an animal model, the Plcxd3 gene to be deficient may be the Plcxd3 gene present in the mouse species, and in one embodiment of the present invention, the deficiency of the Plcxd3 gene in a mouse consisting of the nucleotide sequence of SEQ ID NO. 3 was induced. In addition, the amino acid sequence for the mouse PLCXD3 protein is shown in SEQ ID NO. 4.

[0061] In addition, the present invention may provide a method for providing information necessary for diagnosing memory, cognitive, or learning disabilities, comprising: (a) a step of measuring the expression level of PLCXD3 in a biological sample isolated from an individual; and (b) a step of comparing the PLCXD3 expression level of step (a) with the expression level of a normal control group and determining that there is a memory, cognitive, or learning disability if the PLCXD3 expression level is lower than that of the normal control group.

[0062] The expression level of the PLCXD3 above may be the mRNA level of the PLCXD3 gene or the expression level of the PLCXD3 protein, and the PLCXD3 gene may be composed of the nucleotide sequence of SEQ ID NO. 1 and the PLCXD3 protein may be composed of the amino acid sequence of SEQ ID NO. 2.

[0063] The method for measuring the expression level of PLCXD3 described above can be performed using a primer, probe, or antibody specific to the PLCXD3 gene or PLCXD3 protein, and can be selected from the group consisting of reverse transcription-polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), enzyme immunoassay (ELISA), immunohistochemistry, immunofluorescence staining, Western blotting, and flow cytometry, but is not limited thereto.

[0064] In addition, the present invention may provide a method for screening therapeutic agents for memory, cognitive, or learning disabilities using an animal model prepared according to the present invention, wherein the method comprises: (a) treating an animal model having memory, cognitive, or learning disabilities in which the Plcxd3 gene is knocked out with a candidate drug; and (b) comparing the animal model treated with the candidate drug in (a) with an untreated control group to determine whether there is an improvement in memory, cognitive, or learning disabilities.

[0065] Methods for verifying improvement in the above-mentioned memory, cognitive, or learning disabilities are not limited to the following and may be performed using the Novel Tank Test, Alarm Substance Test, Open Field Test (OFT), Y-Maze Test, Novel Object Recognition Test (NORT), Passive Avoidance Test (PAT), or Social Interaction Test (SAT).

[0066] In the above method, the criterion for determining a candidate drug as a treatment for memory, cognitive, or learning disorders is that if, in the various experimental results described above, memory, cognitive, or learning ability is improved or increased compared to a control group that was not treated with the candidate drug, the candidate drug can be determined as a treatment.

[0067] In addition, if, in an animal model treated with the above candidate drug, compared to an untreated control group, there is an increase in the number of dendritic spines in the cortex and hippocampus; an increase in the expression of β1 and β2, which are PLC subfamily members; an increase in the production of IP3; an increase in the expression of nrsn1, glun2b, and creb, which are learning or memory-related factors; or an increase in the expression of gng2, gpr183, and ephb2, which are upstream target factors of the PLC family, the above candidate drug can be considered as a therapeutic agent capable of improving, preventing, or treating memory, cognitive, or learning disorders.

[0068] In the present invention, memory, cognitive, or learning disabilities may include, but are not limited to, all brain diseases caused by a deficiency of PLCXD3, aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, CNS hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug intoxication, epilepsy, seizures, hippocampal sclerosis, headache, cerebral senility, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, neurological disease, cardiovascular disease, amnesia, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, memory, cognitive, or learning disabilities caused by cognitive deficit or attention deficit.

[0069] In addition, the above brain diseases may also include degenerative brain diseases. Degenerative brain diseases are progressive degenerative disorders associated with the central or peripheral nervous system characterized by damage to nerve structure and function, and are general neurodegenerative diseases that include brain shrinkage (atrophy) and neurodegeneration caused by the accumulation of amyloid beta, hyperphosphorylation of tau, and aggregation of α-synuclein, such as Alzheimer's disease and Parkinson's disease.

[0070] Furthermore, the present invention may provide a pharmaceutical composition for the prevention or treatment of memory, cognitive, or learning disorders, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

[0071] In one embodiment of the present invention, it was confirmed that memory, cognitive, or learning disorders were induced in an animal model in which the Plcxd3 gene was deficient and the expression and activity of Plcxd3 were suppressed.

[0072] Therefore, expression promoters capable of inducing the expression of the PLCXD3 gene or activators capable of promoting the activity of the PLCXD3 protein can produce effects of improving short-term and long-term memory, cognitive ability, or learning disabilities by preventing, improving, or treating memory, cognitive, or learning disabilities.

[0073] In the present invention, "improvement of memory" and "improvement of cognitive ability" refer to the effect of maintaining cognitive ability by controlling harmful substances that damage brain cells, or improving reduced cognitive ability by controlling neurotransmitters in the brain, in response to memory decline, memory impairment, or cognitive decline that occurs when the brain atrophies and brain nerve cells are destroyed due to physical fatigue, sleep deprivation, excessive alcohol consumption, dementia, etc. The aforementioned memory refers to the ability to receive necessary information, store it in the brain, and retrieve and use it when needed, while cognitive ability refers to the ability to distinguish and perceive objects.

[0074] In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier. The composition including the pharmaceutically acceptable carrier may be in various formulations, oral or parenteral. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0075] Solid dosage forms for oral administration include tablets, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with one or more compounds. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0076] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, macrogol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc. may be used as bases for suppositories.

[0077] The above pharmaceutical composition may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid formulations, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories.

[0078] In addition, as described above, the pharmaceutical composition of the present invention can be administered to treat various brain diseases, including symptoms associated with memory, cognitive, or learning disorders.

[0079] The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including individual type and severity, age, gender, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0080] The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration. The typical dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0081] The administration route of the above pharmaceutical composition may be any general route capable of reaching the target tissue. Depending on the purpose, the composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or rectally, but is not limited thereto. Additionally, the composition may be administered by any device capable of delivering the active substance to target cells.

[0082] The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modulators for the prevention and treatment of memory, cognitive, or learning disabilities.

[0083] In addition, the present invention may provide a health functional food for the prevention or improvement of memory, cognitive, or learning disabilities, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

[0084] The health functional food of the present invention may further comprise a food science-acceptable carrier.

[0085] The above-mentioned health functional food may have a function that helps prevent, improve, or improve a disease intended for the present invention, and the food may include forms such as pills, powders, granules, infusions, tablets, capsules, or liquids, and there are no particular restrictions on the types of food to which the active ingredients of the present invention may be added, such as various beverages, chewing gum, tea, vitamin complexes, and health supplements.

[0086] In addition to the active ingredient of the present invention, other ingredients that do not interfere with the preventive, improving, or therapeutic effects may be added to the above-mentioned health functional food, and the types thereof are not particularly limited. For example, as with conventional foods, various herbal extracts, food-grade acceptable food additives, or natural carbohydrates may be contained as additional ingredients.

[0087] The term "health functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that have functional properties useful to the human body. Here, "functional properties" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Furthermore, unlike general pharmaceuticals, it has the advantage of being made from food ingredients, thus avoiding side effects that may occur with long-term use of pharmaceuticals, and can be highly portable.

[0088] There are no specific limitations on the types of health functional foods of the present invention, and specific examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all health functional foods in the conventional sense.

[0089] In addition, the present invention may provide a pharmaceutical composition for the prevention or treatment of neuroinflammation, tau-related diseases, or degenerative brain diseases, characterized by comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and having inhibition of the activity of microglia or astrocytes; and inhibition of amyloid plaques; and may provide a health functional food for the prevention or improvement of neuroinflammation, tau-related diseases, or degenerative brain diseases.

[0090] According to one embodiment of the present invention, when the expression of PLCXD3 was inhibited in a mouse model induced with Alzheimer's disease, it was confirmed that the activity of microglia or astrocytes increased and amyloid plaques increased. These results ultimately imply that PLCXD3, its expression promoter, or its activator can inhibit the activity of microglia or astrocytes and inhibit amyloid plaques, thereby enabling the prevention, improvement, or treatment of neuroinflammation, tau-related diseases, or degenerative brain diseases.

[0091] Microglial cells, which function as macrophages in the brain, are important effector cells that regulate immune responses within the central nervous system (CNS). Their activation plays a crucial role in maintaining CNS homeostasis by removing foreign substances caused by drugs or toxins and secreting nerve growth factors. However, exposure to harmful stressors—such as signals from damaged neurons, the accumulation of abnormally shaped proteins modified by external stimuli, or the invasion of pathogens—can lead to excessive microglia activity. This excessive increase in activity causes neuronal damage, potentially resulting in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and cerebral infarction. Furthermore, recent research has revealed that excessive inflammatory responses in microglial cells or astrocytes are a contributing factor to the development of neurodegenerative diseases.

[0092] Astrocytes are also known to play a crucial role in maintaining normal brain activity, specifically in the formation of synapses, regulation of synapse numbers, synaptic function, and the differentiation of neural stem cells into neurons. However, if these astrocytes become excessively reactive—that is, if they remain in a state of excessive activation—they can cause neuronal death and induce the death of neighboring neurons, thereby acting as a cause of neurodegenerative diseases. Therefore, inhibiting excessive astrocyte activation could also serve as a new treatment method for neurodegenerative diseases.

[0093] Furthermore, the neuroinflammatory response involves the activation of innate immune cells (microglia), the release of inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines, and macrophage infiltration, which induces neuronal cell death. Inflammatory activation of microglia and astrocytes is considered a crucial mechanism for pathological markers and the progression of neurodegenerative diseases. Strict regulation of microglia activity is essential for maintaining brain homeostasis and preventing infections and inflammatory diseases.

[0094] Furthermore, beta-amyloid (Aβ: amyloid beta) is derived from amyloid precursor proteins (APP) that are broken down by beta-secretase and gamma-secretase to produce Aβ; it is the main component of amyloid plaques found primarily in the brains of patients with brain diseases such as Alzheimer's disease, and its abnormal accumulation becomes a cause of various brain diseases.

[0095] Excessively accumulated beta-amyloid causes inflammatory reactions in the hippocampus, cerebral cortex, and surrounding cells, which can consequently damage nerve cells and impair even the neural networks that maintain normal brain function.

[0096] In addition, tau protein is a microtubule-associated protein with a molecular weight of 50,000 to 70,000 Da that is involved in the entanglement of nerve fibers. Tau protein is one of the microtubule-associated proteins present in the axons of normal neurons. When these tau proteins accumulate excessively, microtubules collapse, leading to the disintegration of the normal neuronal network. The cause of tau protein accumulation is known to be the excessive attachment of phosphorus to the tau protein, forming phosphorylated masses; furthermore, the accumulation of these tau proteins and the aggregation of neurons are known to be causes of various neurodegenerative diseases.

[0097] Therefore, PLCXD3, its expression promoter, or its activator having inhibition of microglia or astrocyte activity; and inhibition of amyloid plaque activity can be used as a substance capable of preventing, improving, or treating neuroinflammation, tau-related diseases, or degenerative brain diseases.

[0098] Furthermore, the contents of the pharmaceutical composition and health functional food according to the present invention are as described above.

[0100] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0102] <Preparation and Experimental Methods>

[0103] Preparation of Plcxd3 Knock-Out Zebrafish

[0104] To prepare sgRNA for zebrafish Plcxd3, a 20 bp target nucleotide sequence within exon 2 was selected using the CRISPR SCAN website (http: / / www.crisprscan.org / ). DNA templates for the sgRNA were prepared using fill-in PCR and DNA purification methods according to the manufacturer's protocol. In vitro transcription of the sgRNA was performed using the MaxiScript T7 kit (Ambion), and the synthesized RNA was precipitated with ammonium acetate. To prepare Plcxd3 mutations in the zebrafish genome, the CRISPR / Cas9 system was microinjected into zebrafish embryos at the single-cell stage (World Precision Instrument, USA). The microinjection mixture was prepared with 100 ng / μg sgRNA and 300 ng / μg Cas9 mRNA along with phenol red.

[0106] Preparation of Plcxd3 knock-out mice

[0107] To prepare Plcxd3-deficient mice, a single guide RNA (sgRNA) targeting the exon 2 region was designed using the MEGAshortscript™ kit (Thermo Fisher Scientific, Waltham, MA, USA). The sequence information used to design the gRNAs is as follows: crRNA1: 5'-TCTGGCTGCTCCGGGCCTAC-3', crRNA2: 5'-TTCAGTGCCAAAGTCAACGA-3', and crRNA3: 5'-GGGTCTGTCGTGTTTGCCCA-3'. To microinject Cas9 protein and sgRNA into fertilized eggs, male C57BL6 / J mice were reared individually for one week prior to mating, and female C57BL6 / J mice were administered 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin (HCG) for superovulation. C57BL6 / J male and female mice were mated after HCG injection, and fertilized embryos were obtained from mated C57BL6 / J female mice. sgRNA (40 ng / μl) and Cas9 protein (40 ng / μl) were injected into the cytoplasm of fertilized eggs at the single-cell stage via microinjection using a continuous flow injection mode. Surviving two-cell embryos were implanted into the fallopian tubes of quasi-pregnant females. F0 (founder mice) were analyzed from the offspring. F0 mice were crossed with normal C57BL6 / J mice, and F1 mice were crossed to obtain F2 homozygous mutants. To maintain a stable inbreeding background, female mice were backcrossed with parental inbreeding lines approximately every 10 generations. For genotyping analysis, forward primers were 5'-GTCCCATGATTCCTTCAGCT-3' and reverse primers were 5'-TTTCTCTGAGTCCACTTGCCA-3'.Deficiency (KO) mutants were screened by T7 endonuclease I analysis of PCR products, and several insertion or deletion mutations were identified, including a +1 bp insertion, a -8 bp deletion, and two -10 bp deletion mutations. Finally, -8 bp deletion mutant (Plcxd3 KO) mice were used in the experiment.

[0109] Immunohistochemistry (DAB staining)

[0110] Experiments were conducted under the approval of the Animal Care Committee of the Korea Brain Research Institute (IACUC-21-00059) for all animal experimental methods. Three-month-old normal mice were reared in a pathogen-free facility at 22±2°C, 50±5% humidity, and a 12-hour light-dark cycle, provided with food and water at will. The mice were perfused and fixed in PBS and 4% paraformaldehyde. To obtain brain sections for immunohistochemical analysis, brain tissue stored in 4% paraformaldehyde at 4°C for 24 hours was immersed in 30% sucrose in PBS for 72 hours and sectioned to a thickness of 30 µm using a cryostat microtome (Leica CM1850, Wetzlar, Germany). Subsequently, the sections were incubated in PBS with 30% H2O2 and incubated overnight at 4°C with rabbit-anti-PLCXD3 antibody. After washing the slices with PBS buffer, they were incubated with biotylated secondary antibodies at room temperature for 2 hours. After washing again with PBS buffer, ABC solution (Vector Laboratories, Burlingame, CA, USA) was added and incubated at room temperature for 1 hour. Subsequently, the samples were developed for 42 seconds using DAB solution (Vector Laboratories, Burlingame, CA, USA). Afterward, the samples were mounted on glass slides using Permount solution (Vector Labs, Burlingame, CA), images of the immunostained tissue were obtained (Pannoramic scan, 3dHistec, Budapest, Hungary), and the images were analyzed (ImageJ, National Institutes of Health, Bethesda, MD, USA).

[0112] Immunofluorescence staining (IF staining)

[0113] Plcxd3-deficient mice and litter-born normal mice were reared in a pathogen-free facility at 22±2°C, 50±5% humidity, and a 12-hour light-dark cycle, provided with random food and water. After behavioral tests, the mice were perfused and fixed in PBS and 4% paraformaldehyde, respectively. To obtain brain sections for immunofluorescence staining, brain tissue stored in 4% paraformaldehyde at 4°C for 24 hours was immersed in PBS containing 30% sucrose for 72 hours, and then sectioned to a thickness of 30 µm using a cryostat microtome (Leica CM1850, Wetzlar, Germany). The brain sections were blocked with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) at room temperature for 2 hours, followed by incubation with target antibodies overnight at 4°C. Details of the antibodies used are listed in the table below. Subsequently, the slices were washed with PBST buffer and incubated with a secondary antibody conjugated to Alexa 555 or Alexa 488 at room temperature for 2 hours. After mounting on glass slides using Vectashield mounting solution containing DAPI (Vector Labs, Burlingame, CA), fluorescence microscopy images of the immunostained tissue were obtained (DMi8, Leica Microsystems, Wetzlar, Germany) and analyzed (ImageJ, National Institutes of Health, Bethesda, Maryland, USA).

[0115] Reverse Transcription PCR

[0116] The levels of Plcxd3 mRNA expressed in the hippocampus of normal mice and Plcxd3-deficient mice were analyzed by RT-PCR. cDNA was obtained by reverse transcription using total RNA extracted with QIAzol Lysis Reagent (Qiagen, Cat No. 79306), and 30-cycle RT-PCR was performed using Prime Taq Premix (GeNetBio). The sequences of the primers used are shown in the table below. The amplification products (amplicons) were separated by electrophoresis on a 2% agarose gel supplemented with EcoDye (1:5000, Biofact, Daejeon, Korea), and the gel images were analyzed using the software Fusion Capt Advance (Vilber Lourmat, Eberhardzell, Germany).

[0118] Real-time PCR

[0119] The mRNA levels of Plcxd3 in the hippocampus of normal and Plcxd3-deficient mice were analyzed by real-time PCR. Specifically, cDNA synthesized with the Superscript cDNA Premix Kit II (GeNetBio) was analyzed by 50-cycle real-time PCR using the Fast SYBR Green Master Mix (Thermo Fisher Scientific, CA, USA) and the QuantStudio™ 5 system (Thermo Fisher Scientific). GAPDH Ct values ​​were used as normalization values. Changes in Plcxd3 mRNA levels in Plcxd3-deficient mice were analyzed based on normal mice. Additionally, the primer sequences used in the above experiment are listed in the table below.

[0121] IP 3 Assess

[0122] The effect of PLCXD3 on IP3 expression levels in the cortex and hippocampus was analyzed in normal and PLCXD3 mice. For this purpose, the hippocampus was dissected and homogenized in PBS buffer, then left on ice for 1 hour. Subsequently, the supernatant was collected after centrifugation at 10,000 rpm at 4°C for 15 minutes. Total protein concentration was measured using a BCA protein assay, and IP3 expression levels were measured using an ELISA kit (Cat no. CSB-E13410m, CUSABIO, Huston, TX, USA).

[0124] New tank test

[0125] Zebrafish are known to reside at the bottom of the tank and exhibit anxious behavior when exposed to unfamiliar environments. To investigate behavioral differences between Plcxd3-deficient mutants and normal specimens, each zebrafish was placed in a new tank (24 cm × 15 cm × 15 cm), filled to a water level of 12 cm, and maintained at a temperature of 28°C. The behavior of the zebrafish was recorded for 30 minutes using a video camera (Sony, HDRCX190, Japan). Image tracking of the video recordings was performed using EthoVision XT 7.0 motion tracking software (Noldus).

[0127] Alarm substance test

[0128] It is known that when zebrafish are harmed by predators, warning substances are released from their skin to transmit danger signals to other fish. Zebrafish exhibit behaviors such as panic and freezing when exposed to these warning substances. Accordingly, an experiment was conducted to determine whether these behavioral changes occur in Plcxd3-deficient mutants. The warning substance was prepared from zebrafish skin extract. The tank was prepared under the same conditions as the one used in the previously described novel tank test (24 cm × 15 cm × 15 cm). The water level in the tank was filled to 12 cm, and the temperature was maintained at 28°C. Movements were recorded for 30 minutes using a video camera (Sony, HDRCX190, Japan), and image tracking was performed by dividing the tank height into three sections using EthoVision XT 7.0 motion tracking software (Noldus).

[0130] Open field test (OFT)

[0131] The open field consisted of a brightly lit enclosure (40 × 40 cm) featuring a white plastic floor and transparent Plexiglas walls (Kinder Scientific, Poway, CA, USA). On the day of the test, mice were allowed to explore the enclosure for 5 minutes, and their behavioral performance was tracked and analyzed using video software from SMART video tracking software (SMART 3.0, Panlab, Llobregat, Spain). To analyze exploratory behavior in the open field, the distance traveled and the exploration time within the center or periphery of the enclosure were analyzed as outcome measures. After evaluating the activity of the open field, the equipment was cleaned with 70% ethanol to remove residual odors.

[0133] Y-maze test

[0134] To investigate the function of Plcxd3 in short-term and spatial memory impairment in Plcxd3-deficient mice, a Y-maze test was performed. The Y-maze consisted of three arms (35 cm x 7 cm x 15 cm) meeting at a 120° angle, and a single mouse was allowed to freely navigate the maze for 5 minutes during each test. Voluntary turns were recorded using a video camera for 5 minutes and then manually counted. The percentage of turns was calculated using the following formula.

[0135] [Alternation percentage(%) = (number of alternations / number of alternation triads) × 100].

[0137] New Object Recognition Test (NORT)

[0138] To determine the effects of Plcxd3 on long-term and cognitive memory, NOR tests were conducted. The device used for the NOR test was an open box with a side length of 40 cm and a height of 25 cm, and the NOR test consisted of two phases: training and testing. The training and testing phases were performed at 24-hour intervals. In the training phase, two identical objects were placed inside the box, and mice were instructed to explore the box for 5 minutes. In the testing phase, mice were returned to a box containing one familiar object and one new object for 5 minutes. During the testing phase, the positions of the two objects in the box were balanced, and after the trial, the box and objects were thoroughly cleaned with 70% ethanol to remove odors. Exploration time was calculated manually by reviewing video recordings of each test. Behavior was considered exploratory when the mouse pointed its nose toward an object. Object preference (%) for the new object was calculated using the following formula.

[0139] [Object Preference (%) = T Novelty / (T Familiarity + T Novelty) × 100],

[0140] Here, T novelty is the time spent exploring a new object, and T familiarity is the time spent exploring a familiar object.

[0142] Manual Avoidance Test (PAT)

[0143] Hippocampus-dependent contextual fear memory involves re-exposure to an environment that received an aversive stimulus during previous training. In a hippocampus-dependent passive avoidance test, mice were placed in an isolated compartment with two rooms and received a paw strike upon entering the dark compartment, which evoked less anxiety. The following day, the mice were returned to the illuminated compartment, and their latency to enter the dark compartment was evaluated. To test this, mice were placed in the bright compartment within a chamber containing a dark compartment (Hamilton-Kinder, Poway, CA, USA). After 10 seconds of acclimatization, the bright house lights were turned on and the door leading to the dark compartment was opened. Mice preferring the dark left side generally passed through the gate quickly and entered the dark compartment; subsequently, the mice were subjected to a short paw strike (0.3 mA for 3 seconds). Each mouse underwent a test of up to 180 seconds without entering the dark compartment. After 24 hours, the mice were returned to the right compartment. After 10 seconds, the partition lights were turned on and the connecting door was opened. The time taken to re-enter the dark section was measured as a maximum of 180 seconds.

[0145] Social Interaction Test (SAT)

[0146] The device was designed to consist of a box with three rectangular illuminated chambers and an attached infrared video camera. Each chamber measured 40 cm × 60 cm × 22 cm (height), and the partition walls were made of transparent plexiglass with small square holes (5 cm wide × 3 cm high) to allow exploration of each chamber. Normal (C57BL / J) 8-week-old male mice were used as 'strangers'.

[0147] One day prior to the test, 'subject mice' were individually placed in the middle chamber and allowed to freely explore the entire apparatus for 5 minutes. Prior to the test, subject mice were placed in the middle chamber and allowed to freely explore all three chambers for 5 minutes (habituation test). For the sociability test, a strange normal male mouse ('Stranger 1') with no prior contact with the subject mice was placed in one of the side chambers. The stranger mice were enclosed in a small, round wire cage that allowed nose contact between the bars but prevented fighting. This cage was 11 cm high, 9 cm in base diameter, and had vertical bars spaced 0.5 cm apart. The subject mice were placed in the middle chamber, and Strange 1 was presented in one compartment and an empty cage in the other for 5 minutes. The time spent around each cage (Stranger 1 or empty) was measured. After the 5-minute sociability test was completed, each subject mouse was tested for 5 minutes to quantify its social preference for the new stranger (social preference test). The wire fence surrounding the familiar normal male mouse (Stranger 1) was moved to the opposite side of the room that had been empty during the sociability test. Secondly, the unfamiliar normal male mouse (Stranger 2) was placed in the other side of the same small wire fence. The subject mice were allowed to freely explore the mice from the previous sociability test (Stranger 1) and the new mice (Stranger 2). The time spent around each cage (Stranger 1 or Strange 2) was measured. Data collection and analysis were performed manually.

[0149] Golgi staining

[0150] After behavioral tests, 4–5 Plcxd3-deficient and normal mice were stained with Golgi using the FD Rapid Golgi Stain kit (FD NeuroTechnologies). The dissected brains of each mouse were cultured in solutions A and B at room temperature for 2 weeks, followed by immersion in solution C at 4°C for 24 hours. The brains were sectioned to a thickness of 150 μm using a vibratome (VT1000S; Leica). Bright-field microscopy (Axioplan 2; Zeiss) images (63x magnification) of pyramidal neurons in cortical layers V and CA1 were recorded, and the number of dendritic spines was counted blindly using ImageJ (NIH). Dendritic spines were measured on dendrites with a length greater than 20 μm.

[0152] Preparation of a brain region-specific Plcxd3 gene expression downgrade (KD) animal model

[0153] To prepare a brain region-specific animal model of Plcxd3 gene expression reduction (KD), an Adeno-Associated Virus (AAV) based on an shRNA vector labeled with a CMV promoter and eGFP was used. Normal mice were fixed to a stereotactic table, and the bregma was set as the reference point (AP 0 mm, ML 0 mm, DV 0 mm). To prepare an amygdala-specific animal model of Plcxd3 expression reduction, 0.5 μl of either the control virus (AAV-CMV-control shRNA-eGFP) or the virus for Plcxd3 expression reduction (AAV-CMV-Plcxd3 shRNA-eGFP) was injected into both amygdalae (brain 3D coordinates: AP -1.1 mm, ML - / + 3.45 mm, DV -4.5 mm from bregma) via a micropump at a flow rate of 0.25 μl / min. In addition, to prepare a hippocampal CA1-specific animal model of reduced Plcxd3 expression, 1.0 μl of either the control virus (AAV-CMV-control shRNA-eGFP) or the virus for reducing Plc-xd3 expression (AAV-CMV-Plcxd3 shRNA-eGFP) was injected into both CA1s of normal mice (brain 3D coordinates: AP -2.0 mm, ML - / + 1.5 mm, DV -1.55 mm from bregma) via a micropump at a flow rate of 0.25 μl / min. Three weeks after injection, behavioral analysis was performed to analyze the bipolar disorder-related behavioral regulatory function of Plc-xd3 in different brain regions.

[0155] Preparation of a brain region-specific Plcxd3 gene overexpression (OE) animal model

[0156] To prepare a brain region-specific animal model of Plcxd3 gene overexpression (OE), an Adeno-Associated Virus (AAV) labeled with a CMV promoter and eGFP was used. Normal mice were fixed to a stereotactic table, and the bregma was set as the reference point (AP 0 mm, ML 0 mm, DV 0 mm). To prepare an amygdala-specific animal model of Plcxd3 expression reduction, 0.5 μl of either the control virus (AAV-CMV-control-eGFP) or the Plcxd3 overexpression virus (AAV-CMV-Plc-xd3-eGFP) was injected into both amygdalae (brain 3D coordinates: AP -1.1 mm, ML - / + 3.45 mm, DV -4.5 mm from bregma) via a micropump at a flow rate of 0.25 μl / min. In addition, to prepare an animal model of hippocampal CA1-specific Plcxd3 expression reduction, 1.0 μl of either the control virus (AAV-CMV-control-eGFP) or the virus for Plcxd3 overexpression (AAV-CMV-Plc-xd3-eGFP) was injected into both CA1s of normal mice (brain 3D coordinates: AP -2.0 mm, ML - / + 1.5 mm, DV -1.55 mm from bregma) via a micropump at a flow rate of 0.25 μl / min. Three weeks after injection, behavioral analysis was performed to analyze the bipolar disorder-related behavioral regulatory function of Plc-xd3 in different brain regions.

[0158] Elevated plus maze (EPM)

[0159] Hyperactivity and abnormal anxiety responses are characteristic behaviors of bipolar disorder and can be analyzed through a cross maze test. A mouse was placed on a platform consisting of two closed arms (25x5x16 cm) positioned 50 cm above the ground and an arm (25x5x0.5 cm) open at a right angle to the other, and was allowed to move freely for 5 minutes. Behavioral performance was tracked and analyzed using video software from SMART video tracking software (SMART 3.0, Panlab, Llobregat, Spain). The time and distance spent on the closed arms and the open arms were analyzed as outcome measures.

[0161] Fear conditioning test (FCT)

[0162] FCR was performed to verify the function of amygdala-specific Plcxd3 regarding fear formation and fear memory. Experimental mice were placed in a test chamber (54 cm x 30 cm x 27 cm) and acclimatized to the surrounding environment for 3 minutes. Subsequently, an auditory stimulus of 90 dB, 2.8 kHz was administered for 30 seconds, followed by an electrical stimulus of 0.2 mA (solar electric shock) for the final 0.5 seconds. A total of 5 cycles of auditory and electrical stimulation were administered with a 1-minute interval between trials, and freezing behavior was analyzed using Packwin software (fear conditioning; analysis of fear formation was possible). After 24 hours (day 2), in order to selectively analyze only fear memories related to sound, the colors of the floor and walls of the same test chamber were changed. After the experimental mice were placed and adapted to the environment for 1 minute, sound stimuli of 90 dB, 2.8 kHz were administered 10 times for 30 seconds at intervals of 50 to 70 seconds, and freezing behavior was analyzed using Packwin software (fear retrieval). After 8 and 72 hours (days 3-4), freezing behavior was analyzed using Packwin software under the same conditions as day 2 to observe fear extinction, and thus analyzed as an outcome measure for fear memory.

[0164] Production of Alzheimer's disease mouse animal model (5x FAD mice)

[0165] F1 generation 5xFAD mice (stock number 008730, B6SJL-Tg APPSwFlLon,PSEN1*M146L*L286V6799Vas / Mmjax) were obtained from Jacson's laboratory, and transgenic male mice (5xFAD) were placed together with female C57BL / 6J mice purchased from Jacson's laboratory. 5xFAD is known to overexpress human familial Alzheimer's disease (FAD) mutations associated with human APP (695) with K670N, M671L (Swedish), I716V (Florida), and V717I (London) mutations, and human PS1 with two FAD mutations (M146L and L286V). The above transformation was regulated by the mouse Thy1 promoter to be overexpressed in the brain, and genotyping for the 5xFAD transformation was performed using the PCR method provided by the Jackson Lab's genotyping protocol.

[0167] Immunohistochemistry and immunofluorescence analysis

[0168] Alzheimer's disease animals were perfused and fixed using a perfusion solution (0.9% NaCl, Biosesang) and a 4% paraformaldehyde solution (Chembio). Brain tissue was sectioned using a frozen sectioner (Leica) to a thickness of 30 mm, and immunofluorescence and immunohistochemical staining were performed. For immunofluorescence staining, the tissues were washed with PBS containing 0.2% Triton X-100 (Sigma) and then blocked with 10% Normal Goat Serum Blocking Solution (Vector Laboratories) at room temperature for 2 hours. Subsequently, the primary antibody and incubation antibody were diluted with PBS containing 0.2% Triton X-100 and incubated overnight at 4°C with slow shaking. The next day, the tissues were washed with PBS containing 0.2% Triton X-100 and incubated with the secondary antibody at room temperature for 2 hours. The tissue was placed on a slide glass, DAPI-containing mounting solution (Vector Laboratories) was applied over it, covered with a cover glass, and stored at 4°C. The stained tissue was photographed using a fluorescence microscope (DMi8, Leica Microsystems).

[0170] Statistical analysis

[0171] Data analysis was performed using GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA). Unpaired two-tailed T-tests with Welch's correction were used to compare the two groups. One-way ANOVA and Tukey's test were used for multiple comparisons. A p-value of < 0.05 was considered statistically significant. Results were expressed as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

[0173] The reagents and oligonucleotide information used in the embodiments of the present invention are as shown in Table 1 below.

[0174]

[0176] <Example 1>

[0177] Expression Characteristics of PLCXD3 in Zebrafish and Behavioral Analysis Related to Bipolar Disorder

[0178] <1-1> Analysis of CNS-specific Plcxd3 expression patterns

[0179] First, the inventors analyzed the expression specificity of Plcxd3 in zebrafish. Whole-mount in situ hybridization was performed to investigate the CNS-specific expression pattern of Plcxd3 in zebrafish genes, and as a result, it was confirmed that Plcxd3 is mainly expressed in neuronal subsets of the brain and spinal cord of zebrafish, and in particular, it was confirmed that neuron-specific expression of Plcxd3 persists from 24 hpf (hours post-fertilization) to 120 hpf and into the brain of adult zebrafish (Fig. 1A).

[0181] <1-2> Analysis of Bipolar Disorder-Related Behaviors in Plcxd3 KO Zebrafish

[0182] In addition, to confirm the physiological role of PLCXD3, the inventors produced PLCXD3-deficient (KO) zebrafish using the CRISPR / Cas9 system, selected PLCXD3 KO mutants, and finally established an allele with +29 bp inserted (Fig. 1B). In this experiment, the +29 bp inserted mutant (PLcxd3 KO zebrafish) was used. Furthermore, as a result of morphological comparative analysis between the mutant and normal, as shown in Fig. 1C, it was confirmed that the PLCXD3 KO zebrafish showed no morphological defects compared to normal zebrafish embryos up to 5 dpf (days after fertilization).

[0184] Furthermore, to determine whether Plcxd3 KO zebrafish exhibit behavioral patterns seen in bipolar disorder, such as 'hyperactivity,' new tank tests and warning substance tests were performed.

[0185] As a result, it was confirmed that Plcxd3 KO adult zebrafish exhibited a 'hyperkinetic' pattern in all tests compared to normal zebrafish (Figs. 1D and 1E).

[0186] Through these results, the inventors were able to see that Plcxd3 KO zebrafish exhibit behaviors similar to human patients with bipolar disorder, and that PLCXD3 is associated with bipolar disorder.

[0188] <Example 2>

[0189] Analysis of PLCXD3 expression patterns in the hippocampus of normal mouse brains

[0190] The expression pattern of Plcxd3 in mouse brains was analyzed. To this end, real-time PCR was performed using primers specific to the Plcxd3 gene of normal mice.

[0192] As a result, it was confirmed that the mRNA levels of Plcxd3 were highly expressed in the cerebellum (CB), hypothalamus, and hippocampal CA1 and CA3 of normal mice (Fig. 2A). In addition, through immunohistochemistry, we were able to identify PLCXD3 protein highly expressed in the CB, hypothalamus, and hippocampal CA1 and CA3 regions of normal mouse brains (Figs. 2B, 2C). In particular, it was confirmed that it was mainly expressed in the pyramidal and molecular layers of CA1 and the pyramidal layer of CA3 (Figs. 2C, 2D).

[0194] <Example 3>

[0195] Analysis of PLCXD3 Expression Patterns by Neuron Type in Normal Mice

[0196] To determine which cell types specifically express PLCXD3, double fluorescent staining was performed on normal mice using anti-PLCXD3 and anti-GluA2 antibodies (excitatory neuron markers) or anti-PLCXD3 and anti-GAD67 antibodies (inhibitory neuron markers).

[0198] As a result, Figures 3A and 3H show the proportion of co-localized cells stained with PLCXD3 antibodies and excitatory or inhibitory neuronal markers, and PLCXD3-expressing cells were found to be co-localized with GluA1-positive excitatory neurons in CA1 (44.35%), DG (38%), and CA3 (35%) (Figures 3B–3G). Additionally, PLCXD3-expressing cells were found to be co-localized with GAD67-positive neurons in CA1 (77.55%), DG (92.34%), and CA3 (87.83%) of the hippocampus (Figures 3I–3N).

[0199] These results showed that PLCXD3 is expressed in both excitatory and inhibitory neurons, and in particular, that PLCXD3 is predominantly expressed in inhibitory neurons in the hippocampus of normal mouse brains.

[0201] <Example 4>

[0202] Preparation of Plcxd3-deficient (KO) mice and confirmation of Plcxd3 deficiency

[0203] To investigate the physiological function of PLCXD3 in the brain, PLCXD3-deficient (KO) mice were created using the CRISPER / Cas9 system, and various insertion or deletion mutations were identified in the created mice. Mutations identified included a mutant with a +1 bp insertion, a mutant with a -8 bp deletion, and a mutant with two -10 bp deletions. In this experiment, the mutant with an -8 bp deletion (Plcxd3 KO) was used (Fig. 4B). The -8 bp deletion was predicted to be the cause of a frameshift mutation resulting in the premature cleavage of the PLCXD3 protein.

[0204] Then, Plcxd3+ / + homozygotes of normal mice and Plcxd3- / - homozygotes of Plcxd3 KO mice (F1) were crossed to obtain Plcxd3+ / - heterozygous mice (F2), and the heterozygotes of F2 mice were crossed and bred to obtain F3 mice, which are Plcxd3+ / + homozygotes, Plcxd3+ / - heterozygotes, and Plcxd3- / - homozygous mice according to Mendelian genetics (Fig. 4C). In this experiment, Plcxd3- / - mice of F3, which are the offspring of Plcxd3+ / - heterozygotes, were used as Plcxd3-deficient mice.

[0205] In addition, to determine whether the Plcxd3 gene was deleted in the brains of Plcxd3 KO mice, genotyping analysis was performed using Plcxd3 gene-specific primers, and as a result, it was confirmed that the Plcxd3 gene was not detected in the hippocampus of Plcxd3 KO mice (Figs. 4D–4F). Real-time PCR results also showed that the Plcxd3 mRNA level in the hippocampus was significantly reduced in Plcxd3 KO mice (Plcxd3- / -) compared to normal F3 mice (Plcxd3+ / +), and it was confirmed that the gene was deficient to the extent that it was almost not expressed (Fig. 4F).

[0207] <Example 5>

[0208] Confirmation of impairment in learning / memory, cognitive function, and social behavior due to Plcxd3 deficiency

[0209] <5-1> Open Field Test

[0210] The inventors conducted experiments to determine whether PLCXD3 is associated with learning, memory, cognitive function, and social behavior. To this end, animal behavior experiments were performed using 3-month or 7-month-old Plcxd3 KO mice and their littermates.

[0212] As a result, compared to normal mice with no change in total travel distance, 3-month-old Plcxd3 KO mice showed a significant decrease in the central zone search time (Fig. 5A), and similarly, 7-month-old Plcxd3 KO mice also showed a tendency for the central zone search time to decrease, but showed no significant change in total travel distance (Fig. 5A).

[0213] Through these results, the inventors were able to determine that Plcxd3 KO mice feel more anxiety compared to normal individuals, which means that the Plcxd3 gene plays a role in regulating anxiety-related behaviors.

[0215] <5-2> Y-Maze, NORT, and PAT Test

[0216] Next, to investigate the effects of PLCXD3 on spatial, recognition, and learning memory, Y-maze, NORT (Novel Object Recognition Test), and PAT (Passive Avoidance Test) were performed, respectively.

[0218] As a result, the degree of spontaneous change was found to be significantly reduced in 3-month and 7-month-old Plcxd3 KO mice compared to normal mice of the same litter (F3) (Fig. 5B). In addition, affinity for new objects was found to be significantly suppressed in 7-month-old Plcxd3 KO mice (Fig. 5C). In PAT analysis, the waiting time in the right cue induction room was found to be significantly reduced in 3-month and 7-month-old Plcxd3 KO mice (Fig. 5D). These results indicate that PLCXD3 plays an important role in maintaining homeostasis of spatial, cognitive, and learning memory.

[0220] <5-3> Social Behavior Test

[0221] Experiments were conducted to determine whether PLCXD3 is involved in social behavior. Social interaction tests were analyzed using 3-month and 7-month-old Plcxd3 KO mice and their littermates (F3).

[0223] As a result, sociability and social preference were found to be significantly reduced in 3-month and 7-month Plcxd3 KO mice compared to normal mice, which means that social interaction behaviors were impaired in Plcxd3 KO mice (Fig. 5E). Through these results, the inventors were able to determine that PLCXD3 is involved in emotional behavior, spatial work, perception, learning memory, and the regulation of social behavior.

[0225] <Example 6>

[0226] Confirmation of reduction in cortical and hippocampal dendritic spines due to Plcxd3 deficiency

[0227] To determine whether PLCXD3 affects the formation of dendritic spines associated with memory integration in the brain, Golgi staining was performed. Golgi staining was performed using 3- or 7-month-old Plcxd3 KO mice and their littermates (F3).

[0229] As a result, it was confirmed that dendritic spines were significantly reduced in the cortical apex slope (AO) and basal shift (BS) regions of 3-month-old Plcxd3 KO mice (Figs. 6A, 6B). However, dendritic spines in the hippocampus were significantly reduced only in the cortical apex slope (AO) region of the hippocampus in 3-month-old PLCxd3 KO mice, and showed no significant reduction in the basal shift (BS) region (Figs. 6A, 6C). Additionally, the number of dendritic spines was found to be significantly reduced in the cortical apex slope (AO) and basal shift (BS) regions of the cortex and hippocampus in 7-month-old Plcxd3 KO mice (Figs. 6D, 6E).

[0230] Through these results, the inventors were able to determine that PLCXD3 plays a significant role in the formation of dendritic spines associated with memory integration in the brain, and that a deficiency in the expression of the PLCXD3 gene or protein can reduce the formation of dendritic spines in the brain, thereby causing brain dysfunction; conversely, they were able to determine that increasing the expression and activity of the PLCXD3 gene or protein can improve, prevent, or treat brain dysfunction.

[0232] <Example 7>

[0233] Analysis of changes in expression of molecules related to memory, learning, or cognitive function due to Plcxd3 deficiency

[0234] PLC is a type of membrane-bound enzyme that hydrolyzes PIP2 into IP3 and DAG in the brain. Accordingly, the inventors performed an IP3 analysis on hippocampal tissue of Plcxd3 KO mice to determine whether PLC activity is regulated by the deficiency of the Plcxd3 gene.

[0236] As a result, the concentration of IP3 was found to be significantly reduced in the hippocampus of Plcxd3 KO mice compared to normal littermates, which confirmed that PLCXD3 activity is significantly lower in Plcxd3 KO mice (Fig. 7A).

[0238] In addition, the inventors performed real-time PCR to determine whether Plcxd3 KO mice affect the expression of isoforms of the PLC family.

[0239] As a result, it was found that the expression of β1 and β2, subfamilies of PLC, was significantly reduced in 3-month-old Plcxd3 KO mice (Fig. 7B), indicating that the lack of Plcxd3 affects the expression of PLC subfamilies.

[0241] In addition, to determine whether PLCXD3 also affects the expression levels of learning and memory-related genes, real-time PCR was performed, and the results showed that the mRNA levels of the cytoplasmic vesicle component nrsn1 (neurensin 1) and the NMDA receptor subunit glun2b (NMDA receptor 2B), which are learning and memory-related factors, were significantly reduced in 3-month-old Plcxd3 KO mice (Figs. 7C, 7D).

[0242] In addition, in 7-month-old Plcxd3 KO mice, the expression levels of upstream molecular target factors of the PLC family, gng2 (G protein subunit gamma 2), gpr183 (Orphan G-protein coupled receptor 183), and ephb2 (Eph receptor B2), were all found to be significantly reduced (Fig. 7E), and the expression levels of memory-related genes creb and nrsn1 were also found to be significantly reduced (Fig. 7F).

[0244] Through these results, the inventors were able to determine that PLCXD3 plays an important role in brain function by regulating the expression of molecules related to memory, learning, cognition, and social behavior, and ultimately, that increasing the expression or activity of PLCXD3 can effectively improve, prevent, or treat disorders of memory, learning ability, and cognitive function.

[0246] <Example 8>

[0247] Confirmation of hyperactive / anxious behaviors, short-term spatial cognitive ability, sociality, and fear conditioning responses according to the expression levels of amygdala-specific Plcxd3

[0249] <8-1> Confirmation of response to decreased expression of amygdala-specific Plcxd3

[0250] ① Cross Maze Test (EPM)

[0251] The inventors conducted experiments to determine whether amygdala-specific Plcxd3 is associated with hyperactive / anxious behavior. For this purpose, experimental mice were used in which the amygdala was injected with a control virus or a virus designed to reduce Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP).

[0252] As a result, experimental mice with reduced expression of amygdala-specific plcxd3 showed a significantly increased time spent in the open arm and a significantly decreased time spent in the closed arm compared to the normal control group, confirming that the anxiety response was lower than normal levels (Fig. 8A). Since there was no difference between the two groups in the distance traveled between the two arms, it could be inferred that amygdala-specific Plcxd3 is involved in the anxiety response but not in hyperactivity.

[0254] ② Y-Maze Test (Y maze)

[0255] The inventors conducted experiments to determine whether amygdala-specific Plcxd3 is associated with short-term spatial cognitive behavior. For this purpose, experimental mice were used in which a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) was injected into the amygdala.

[0256] As a result, it was confirmed that cognitive deficits occurred because spontaneous alteration was significantly reduced in experimental mice with reduced amygdala-specific plcxd3 expression compared to normal control groups (Fig. 8B).

[0258] ③ Social Behavior Test (SAT)

[0259] Experiments were conducted to determine whether amygdala-specific Plcxd3 is involved in social behavior. For this purpose, experimental mice were used in which the amygdala was injected with either a control virus or a virus designed to reduce Plcxd3 expression (AAV-CMV-Plcxd3 shRNA-eGFP).

[0260] As a result, it was confirmed that in experimental mice with reduced amygdala-specific plcxd3 expression, there was no difference in sociability compared to normal control groups, and social preference / social interaction ability was reduced (Fig. 8C).

[0262] ④ Fear Conditioning Test (CFT)

[0263] Experiments were conducted to analyze the effects of amygdala-specific Plcxd3 on vacuole formation / clearance. For this purpose, experimental mice were used in which the amygdala was injected with either a control virus or a virus designed to reduce Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP).

[0264] As a result, experimental mice with reduced amygdala-specific plcxd3 expression showed lower fear responses compared to normal controls, and fear memory and extinction also showed different patterns from normal mice due to abnormal fear formation (Fig. 8D).

[0266] <8-2> Confirmation of Response to Amygdala-Specific Plcxd3 Overexpression

[0267] ① Cross Maze Test (EPM)

[0268] Having confirmed a low-anxiety response upon reduction of amygdala-specific Plcxd3, the inventors conducted an experiment to determine whether there were changes in hyperactive / anxious behavior upon overexpression of amygdala-specific Plcxd3. For this purpose, experimental mice were used in which a control virus or a virus for Plcxd3 overexpression (AAV-CMV-Plcxd3-eGFP) was injected into the amygdala.

[0269] As a result, in experimental mice overexpressing the amygdala-specific plcxd3 gene, there was no difference in the time spent in the open and closed arms and the distance traveled compared to the normal control group (Fig. 9A).

[0271] ② Y-Maze Test (Y maze)

[0272] The inventors conducted experiments to determine whether amygdala-specific Plcxd3 overexpression is associated with short-term spatial cognitive behavior. For this purpose, experimental mice were used in which a control virus or a virus for Plcxd3 overexpression (AAV-CMV-Plc-xd3-eGFP) was injected into the amygdala.

[0273] As a result, cognitive deficits were confirmed as spontaneous alteration was significantly reduced in experimental mice with overexpression of amygdala-specific plcxd3 compared to normal control groups (Fig. 9B).

[0275] ③ Social Behavior Test (SAT)

[0276] Experiments were conducted to determine whether amygdala-specific Plcxd3 overexpression is involved in social behavior. For this purpose, experimental mice injected with a control virus or a virus for Plcxd3 overexpression (AAV-CMV-Plcxd3-eGFP) into the amygdala were used.

[0277] As a result, it was confirmed that in experimental mice with overexpression of amygdala-specific plcxd3, there was no difference in sociability compared to normal control groups, while social preference / social interaction ability was reduced (Fig. 9C).

[0279] Through the above results, the inventors found that amygdala-specific Plcxd3 regulates anxiety responses, short-term spatial memory, social memory, and fear formation processes, and that if the expression homeostasis of Plcxd3 is not maintained, that is, if expression is reduced or overexpressed, these functional deficiencies are caused.

[0281] <Example 9>

[0282] Analysis of hyperactive / anxious behavior, short-term spatial cognitive ability, and social interaction according to hippocampal CA1-specific Plcxd3 expression levels

[0284] <9-1> Confirmation of behavioral changes following decreased expression of hippocampal CA1-specific Plcxd3

[0285] ① Cross Maze Test (EPM)

[0286] The inventors conducted experiments to determine how hippocampal CA1-specific Plcxd3 regulates hyperactive / anxious behaviors. For this purpose, experimental mice were used in which a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) was injected into hippocampal CA1.

[0287] As a result, experimental mice with reduced hippocampus-specific plcxd3 expression showed significantly increased distance traveled in both the open arm and the full arm compared to normal control groups, confirming hyperactivity (Fig. 10A). Since there was no difference between the two groups in the time spent in both arms, it was found that hippocampus CA1-specific Plcxd3 is involved in hyperactivity but not in anxiety responses.

[0289] ② Y-Maze Test (Y maze)

[0290] The inventors conducted experiments to determine whether hippocampal CA1-specific Plcxd3 is associated with short-term cognitive function. For this purpose, experimental mice were used in which hippocampal CA1 was injected with a control virus or a virus designed to reduce Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP).

[0291] As a result, it was confirmed that there was no change in spontaneous alteration in experimental mice with reduced hippocampal CA1-specific plcxd3 expression compared to normal controls (Fig. 10B).

[0293] ③ Social Behavior Test (SAT)

[0294] To conduct experiments to determine whether hippocampal CA1-specific Plcxd3 is involved in social behavior, experimental mice injected with a control virus or a virus to depressant Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) into hippocampal CA1 were used.

[0295] As a result, experimental mice with reduced hippocampal CA1-specific plcxd3 expression showed no deficiency in sociability and social preference compared to normal controls (Fig. 10C).

[0297] <9-2> Confirmation of behavioral changes following overexpression of hippocampal CA1-specific Plcxd3

[0298] ① Cross Maze Test (EPM)

[0299] Experiments were conducted to determine whether there were changes in hyperactive / anxious behaviors upon hippocampal CA1-specific Plcxd3 overexpression. For this purpose, experimental mice injected with a control virus or a virus for Plcxd3 overexpression (AAV-CMV-Plcxd3-eGFP) into hippocampal CA1 were used.

[0300] As a result, experimental mice overexpressing the hippocampal CA1-specific plcxd3 gene showed a significantly increased distance traveled on the open arm compared to the normal control group, while there was no difference in the time spent on both arms (Fig. 11A).

[0302] ② Y-Maze Test (Y maze)

[0303] The inventors conducted experiments to determine whether hippocampal CA1-specific Plcxd3 overexpression is associated with short-term spatial cognitive behavior. For this purpose, experimental mice were used in which a control virus or a virus for Plcxd3 overexpression (AAV-CMV-Plc-xd3-eGFP) was injected into hippocampal CA1.

[0304] As a result, there was no significant change in spontaneous alteration in experimental mice overexpressing hippocampal CA1-specific plcxd3 compared to normal controls (Fig. 11B).

[0306] ③ Social Behavior Test (SAT)

[0307] Experiments were conducted to determine whether hippocampal CA1-specific-Plcxd3 overexpression is involved in social behavior. For this purpose, experimental mice injected with a control virus or a virus for Plcxd3 overexpression (AAV-CMV-Plcxd3-eGFP) into hippocampal CA1 were used.

[0308] As a result, it was confirmed that there was no change in sociability and social preference in experimental mice overexpressing hippocampal CA1-specific plcxd3 compared to normal controls (Fig. 11C).

[0310] <Example 10>

[0311] Analysis of working memory following inhibition of hippocampal CA1-specific Plcxd3 expression in animals with Alzheimer's disease

[0312] After injecting a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) into the hippocampal CA1 of 3-month-old and 6-month-old Alzheimer's disease animals, a passive avoidance experiment was performed.

[0313] As a result, it was confirmed that the control virus or the virus for reducing Plcxd3 expression successfully infected the mice, and working memory was significantly reduced in experimental mice with reduced hippocampal CA1-specific plcxd3 expression compared to normal controls (Figs. 12A, 12B).

[0315] <Example 11>

[0316] Analysis of changes in amyloid plaques following inhibition of hippocampal CA1-specific Plcxd3 expression in animals with Alzheimer's disease

[0317] After injecting a control virus or a virus for reducing PLCXD3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) into the hippocampal CA1 of 6-month-old Alzheimer's disease animals, respectively, changes in PLCXD3 expression were analyzed by real-time PCR, and it was confirmed that PLCXD3 expression was significantly reduced in Alzheimer's disease animals administered with the virus for reducing PLCXD3 expression (Fig. 13A).

[0318] In addition, to confirm changes in amyloid lesions due to the inhibition of PLCXD3 expression, a control virus or a virus for inhibiting PLCXD3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) was injected into the hippocampal CA1 of 6-month-old Alzheimer's disease animals, and immunofluorescence staining was performed using antibody-6E10. As a result, it was confirmed that amyloid plaques were significantly increased in Alzheimer's disease animals administered with the virus for inhibiting PLCXD3 expression (Fig. 13B).

[0319] Furthermore, the molecular mechanisms related to amyloid lesions in animals with Alzheimer's disease following the inhibition of PLCXD3 expression were confirmed using real-time PCR, and it was found that APP, BACE1, and DYRK1A expression were significantly increased in animals with Alzheimer's disease administered a virus to reduce PLCXD3 expression (Fig. 13C).

[0321] <Example 12>

[0322] Analysis of changes in brain inflammation following inhibition of hippocampal CA1-specific Plcxd3 expression in animals with Alzheimer's disease

[0323] To confirm changes in amyloid lesions due to inhibition of PLCXD3 expression, 6-month-old Alzheimer's disease animals were injected with a control virus or a virus for inhibiting PLCXD3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) into the hippocampal CA1, and then immunofluorescence staining was performed using antibody-Iba-1 and antibody-GFAP.

[0324] As a result, it was confirmed that brain inflammation was significantly increased in experimental mice with reduced hippocampal CA1-specific plcxd3 expression compared to normal controls (Figs. 14A, 14B).

[0326] Through these results, the inventors were able to determine that hippocampal CA1 and amygdala (BLA)-specific Plcxd3 regulates hyperactivity, and that if homeostasis is not maintained in the expression of Plcxd3—that is, if expression is reduced or overexpressed—the aforementioned functional deficiency is caused. Furthermore, through these results, the inventors were able to determine that PLCXD3 not only affects memory, learning, cognition, and social behavior but is also associated with Alzheimer's disease. In particular, they were able to determine that if plcxd3 expression is reduced compared to the normal group, it can cause brain diseases, such as leading to impairments in memory, learning, cognition, and social behavior, and exacerbating the symptoms of Alzheimer's disease.

[0328] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 A method for providing information necessary for the diagnosis of memory, cognitive, or learning disabilities, comprising: (a) a step of measuring the expression level of PLCXD3 in a biological sample isolated from an individual; and (b) a step of comparing the PLCXD3 expression level of step (a) with the expression level of a normal control group. Claim 2 A method for providing information necessary for the diagnosis of memory, cognitive, or learning disabilities, characterized in that, in claim 1, the expression level is the mRNA level of the Plcxd3 gene or the expression level of the PLCXD3 protein. Claim 3 A method for providing information necessary for the diagnosis of memory, cognition, or learning disabilities, characterized in that, in paragraph 2, the Plcxd3 gene is composed of the nucleotide sequence of SEQ ID NO. 1 and the PLCXD3 protein is composed of the amino acid sequence of SEQ ID NO.

2. Claim 4 A method for providing information necessary for the diagnosis of memory, cognitive, or learning disorders according to claim 1, wherein the memory, cognitive, or learning disorder is a memory, cognitive, or learning disorder caused by aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, CNS hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug intoxication, epilepsy, seizures, hippocampal sclerosis, headache, cerebral senility, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, neurological disease, cardiovascular disease, amnesia, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or attention deficit. Claim 5 Animal models, excluding humans, with memory, cognitive, or learning disabilities in which the Plcxd3 gene is knocked out. Claim 6 An animal model, excluding humans, having memory, cognitive, or learning disabilities, characterized in that, in claim 5, the deficiency of the Plcxd3 gene is induced by the CRISPR / Cas9 system. Claim 7 An animal model, excluding humans, having memory, cognitive, or learning disabilities, characterized in that, in claim 6, the TCAACGAG sequence of the Plcxd3 gene is targeted to induce a deficiency of the Plcxd3 gene. Claim 8 A method for producing an animal model having memory, cognitive, or learning disabilities, comprising a step of inducing a knockout of the Plcxd3 gene in animals other than humans. Claim 9 A method for producing an animal model having memory, cognitive, or learning disabilities, wherein, in claim 8, the deficiency of the Plcxd3 gene is induced by a CRISPR / Cas9 system targeting the TCAACGAG sequence of the Plcxd3 gene. Claim 10 A method for screening a therapeutic agent for memory, cognitive, or learning disabilities, comprising: (a) a step of treating an animal model of claim 5 with a candidate drug; and (b) a step of comparing the animal model treated with the candidate drug in (a) with an untreated control group to determine whether there is an improvement in memory, cognitive, or learning disabilities. Claim 11 A method for screening a treatment for memory, cognitive, or learning disabilities according to claim 10, characterized in that determining whether there is improvement in memory, cognitive, or learning disabilities is performed by analyzing the Novel Tank Test, Alarm Substance Test, Open Field Test (OFT), Y-Maze Test, Novel Object Recognition Test (NORT), Passive Avoidance Test (PAT), or Social Interaction Test (SAT). Claim 12 A method for screening a therapeutic agent for memory, cognitive, or learning disorders according to claim 10, further comprising the step of determining that the candidate drug is a therapeutic agent capable of improving, preventing, or treating memory, cognitive, or learning disorders if, in an animal model treated with the candidate drug, compared to an untreated control group, the candidate drug shows an increase in the number of dendritic spines in the cortex and hippocampus; an increase in the expression of β1 and β2, which are PLC subfamily members; an increase in the production of IP3; an increase in the expression of nrsn1, glun2b, and creb, which are learning or memory-related factors; or an increase in the expression of gng2, gpr183, and ephb2, which are upstream target factors of the PLC family. Claim 13 A method for screening a treatment for memory, cognitive, or learning disorders according to claim 10, wherein the memory, cognitive, or learning disorder is a memory, cognitive, or learning disorder caused by aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, CNS hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug intoxication, epilepsy, seizures, hippocampal sclerosis, headache, cerebral senility, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, neurological disease, cardiovascular disease, amnesia, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or attention deficit. Claim 14 A pharmaceutical composition for the prevention or treatment of memory, cognitive, or learning disorders, comprising as an active ingredient a viral vector loaded with PLCXD3 protein or PLCXD3 consisting of the nucleotide sequence of SEQ ID NO.

1. Claim 15 A pharmaceutical composition for the prevention or treatment of memory, cognitive, or learning disorders, characterized in that, in claim 14, the PLCXD3 protein comprises the amino acid sequence of SEQ ID NO.

2. Claim 16 A pharmaceutical composition for the prevention or treatment of memory, cognitive, or learning disorders according to claim 14, wherein the memory, cognitive, or learning disorder is a memory, cognitive, or learning disorder caused by aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, CNS hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug intoxication, epilepsy, seizures, hippocampal sclerosis, headache, cerebral senility, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, neurological disease, cardiovascular disease, amnesia, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or attention deficit. Claim 17 A health functional food for the prevention or improvement of memory, cognitive, or learning disabilities, comprising as an active ingredient a viral vector loaded with PLCXD3 protein or PLCXD3 consisting of the nucleotide sequence of SEQ ID NO.

1. Claim 18 A pharmaceutical composition for the prevention or treatment of Alzheimer's disease, characterized by comprising, as an active ingredient, a viral vector loaded with PLCXD3 protein or PLCXD3 consisting of the nucleotide sequence of SEQ ID NO. 1, and having inhibition of the activity of microglia or astrocytes; and inhibition of amyloid plaques. Claim 19 A health functional food for the prevention or improvement of Alzheimer's disease, characterized by containing as an active ingredient a viral vector loaded with PLCXD3 protein or PLCXD3 consisting of the nucleotide sequence of SEQ ID NO. 1, inhibiting the activity of microglia or astrocytes; and having amyloid plaque inhibitory activity.

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