KR20240014449A - Use of PLCXD3 as a treatment target for memory, cognition or learning disorder - Google Patents

Abstract

The present invention relates to the use of PLCXD3 as a treatment target for memory, cognitive or learning disorders, and specifically, the present invention includes the steps of (a) measuring the expression level of PLCXD3 in a biological sample isolated from an individual; and (b) comparing the expression level of PLCXD3 in step (a) with the expression level of the normal control group, and determining that the PLCXD3 expression level is lower than the normal control group's expression level, and determining that the PLCXD3 expression level is memory, cognitive, or learning impaired. The present invention relates to a method of providing information necessary for diagnosing a disability, and also relates to an animal model with memory, cognitive or learning disabilities lacking the Plcxd3 gene (knocked out), a method for manufacturing the same, and memory using the animal model lacking the Plcxd3 gene. , relates to a screening method for a treatment for cognitive or learning disorders, and further relates to a pharmaceutical composition for the prevention or treatment of memory, cognitive or learning disorders containing PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient. In addition, the present invention confirmed a decrease in working memory and an increase in amyloid plaques and brain inflammation when PLCXD3 expression was inhibited in an animal model of Alzheimer's disease, and through this, it was identified that PLCXD3 is an important factor in the development of Alzheimer's disease. It is notable in that it has been identified that PLCXD3 expression promoter or its activator can prevent or treat Alzheimer's disease.

Description

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

The present invention relates to the use of PLCXD3 as a therapeutic target for memory, cognitive or learning disorders.

The world is rapidly entering an aging society, and as a result, humans are pursuing a high quality of life that allows them to live long and healthy lives without disease. As the elderly population increases due to the extension of lifespan due to the development of science, the incidence of degenerative brain diseases due to deterioration of brain function is rapidly increasing.

The brain has many functions, but one of the main functions is memory, cognition, and learning. Without cognitive ability, memory ability, and learning ability, it is difficult to carry out daily life and survival becomes a problem.

Meanwhile, bipolar disorder is one of the most common mental disorders whose mechanism is unknown. Bipolar disorder has a lifetime prevalence of 2-5%, and is a major mental illness that often develops in late adolescence or early adulthood and affects people throughout their lives. It is a representative mood disorder in which mood repeatedly rises and falls, and is characterized by repeated recurrence and chronicity of manic episodes, depressive episodes, and mixed episodes.

In ancestry studies of patients with bipolar disorder, it is known that the prevalence of bipolar disorder is four times higher and that of depression is twice that of the general population. In twin studies of bipolar disorder, the diagnosis was 65-100% concordant in identical twins and 10-30% concordant in fraternal twins. Considering various epidemiological studies, bipolar disorder can be considered a highly heritable disease with a heritability of 60-80%.

Many genetic studies have been conducted on bipolar disorder, but it has been reported that it may occur in various chromosomal regions, and among the various candidate genes presented, correlations were incomplete, and genes that regulate emotional states related to bipolar disorder were not identified. Despite numerous studies to confirm this, the causative genes and basic mechanisms that cause bipolar disorder have not yet been identified.

Meanwhile, Phospholipase C (PLC) reacts to external stimuli such as hormones, neurotransmitters, and nerve cell growth factors to produce the membrane phospholipid Phosphatidylinositol 4,5-bisphosphate. It is an enzyme that hydrolyzes inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Additionally, six subtypes of PLC enzymes (β, γ, δ, ε, ζ, and η) exist and they have been identified in different human tissues, including brain, heart, spleen, thymus, and hematopoietic cells. Most of the PLC subtypes (except PLC-β and PLC-γ) have a C2 domain (Core-domain) that plays an essential role in producing IP3 by interacting with the membrane-associated receptor present on the cell membrane from external stimuli. has. Additionally, all PLC subtypes show tissue-specific patterns of location of enzymes with unique functions in regulating cellular metabolism.

Recent studies have identified a class of PLC called phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC is called a PLCXD enzyme, a subgroup of the PLC family that contains only the catalytic X-domain, which has the function of producing IP3. Additionally, three different isoforms of PLCXD have been identified (PLCXD1, PLCXD2, and PLCXD3), and each enzyme is thought to have a unique function depending on its tissue distribution and cellular location, with the PLCXD enzyme responsible for the increase in inositol phosphate. It was found to function as an active phosphodiesterase.

Among these, PLCXD3 is known to exist localized in the cytoplasm and perinuclear vesicles of HeLa cells, and little research has been conducted on the specific function of PLCXD3.

Accordingly, in order to investigate the role of PLCXD3 in brain function, the present inventors created an animal model lacking the Plcxd3 gene (Knock-out, KO) using the CRISPER/Cas9 system method, and used a Plcxd3-deficient animal model and a normal animal model. Through comparative analysis, it was confirmed that deficiency of the Plcxd3 gene results in motor ability, spatial memory ability, cognitive ability, learning memory, and social imbalance.

In addition, in order to identify the role of Plc-xd3 in bipolar disorder-related behavior in each brain region in an animal model lacking the Plcxd3 gene throughout the brain, the present inventors lowered the expression of the amygdala and hippocampus-specific Plcxd3 gene (knock-down, KD) or over-expression (OE) animal models were manufactured, and as a result of analyzing each animal model, when the amygdala-specific expression of the Plcxd3 gene was decreased (KD), anxiety responses were observed compared to the normal control group. It was confirmed that this decreased, short-term spatial memory and social memory deteriorated, and fear formation did not occur normally. When amygdala-specifically overexpressing the Plcxd3 gene (OE), short-term spatial memory and social memory decreased compared to the normal control group. Deterioration was confirmed. In addition, it was confirmed that when the expression of the Plcxd3 gene was specifically lowered (KD) or overexpressed (OE) in the hippocampus CA1, hyperactivity increased compared to the normal control group, and short-term spatial memory and social memory showed no difference. did.

In addition, it was confirmed that working memory was reduced and amyloid plaques and brain inflammatory lesions were significantly increased when Plcxd3 gene was deficient in an Alzheimer's animal model (5xFAD).

Accordingly, the present inventors completed the present invention by confirming that the Plcxd3 gene can be targeted as a new target for preventing, improving, or treating memory disorders, cognitive dysfunction, brain development disorders, and degenerative brain diseases.

1. Korean Patent Publication 10-2022-0024161 2. US published patent US2020/0368239

Therefore, the object of the present invention is to measure the single expression level of PLCXD3 in a biological sample isolated from an individual; And (b) comparing the expression level of PLCXD3 in step (a) with the expression level of the normal control group, and determining that the PLCXD3 expression level is lower than that of the normal control group, determining that the PLCXD3 expression level is memory, cognitive, or learning impaired. Or, it provides a method of providing information necessary for diagnosing learning disabilities.

Additionally, the present invention provides an animal model with memory, cognitive, or learning disabilities in which the Plcxd3 gene is knocked out, and a method for producing the same.

In addition, the present invention provides a method of screening for a treatment for memory, cognitive or learning disorders using an animal model with memory, cognitive or learning disorders in which the Plcxd3 gene is knocked out.

In addition, the present invention provides a pharmaceutical composition for preventing or treating memory, cognitive or learning disorders, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving memory, cognitive or learning disorders, which contains PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

In addition, the present invention includes PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and inhibits the activity of microglia or astrocytes; and a pharmaceutical composition for preventing or treating neuroinflammation, tau-related diseases, or degenerative brain diseases, which is characterized by having amyloid plaque inhibitory activity.

Furthermore, the present invention includes PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and inhibits the activity of microglia or astrocytes; and to provide a health functional food for preventing or improving neuroinflammation, tau-related diseases, or degenerative brain diseases, which is characterized by having amyloid plaque inhibitory activity.

In order to achieve the above object, the present invention includes the steps of (a) measuring the expression level of PLCXD3 in a biological sample isolated from an individual; And (b) comparing the expression level of PLCXD3 in step (a) with the expression level of the normal control group, and determining that the PLCXD3 expression level is lower than that of the normal control group, determining that the PLCXD3 expression level is memory, cognitive, or learning impaired. Alternatively, it provides a method of providing information necessary for diagnosing a learning disability.

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.

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

In one embodiment of the present invention, the memory, cognitive or learning disorders include aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, and CNS. Hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug addiction, epilepsy, epilepsy, hippocampal sclerosis, headache, cerebral senescence, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV. , cerebrovascular disease, cranial nerve disease, cardiovascular disease, memory loss, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or memory, cognitive, or learning disability due to attention deficit.

Additionally, the present invention provides an animal model with memory, cognitive, or learning disorders in which the Plcxd3 gene is knocked out.

In one embodiment of the present invention, the lack of the Plcxd3 gene may be induced by the CRISPR/Cas9 system.

In one embodiment of the present invention, the absence of the Plcxd3 gene may be induced by targeting the TCAACGAG sequence of the Plcxd3 gene.

The present invention also provides a method for producing an animal model with memory, cognitive, or learning disorders, which includes the step of inducing knock out of the Plcxd3 gene.

In one embodiment of the present invention, the lack of the Plcxd3 gene may be induced by the CRISPR/Cas9 system by targeting the TCAACGAG sequence of the Plcxd3 gene.

In addition, the present invention includes the steps of (a) treating the animal model of the present invention with a candidate drug; and (b) screening for treatments for memory, cognitive, or learning disorders, including the step of comparing the animal model treated with the candidate drug in (a) with an untreated control group to determine whether memory, cognitive, or learning disorders are improved. Provides a way to do this.

In one embodiment of the present invention, confirmation of improvement in memory, cognitive, or learning disabilities includes a novel tank test, an alarm substance test, an open field test (OFT), Analysis may include the Y-maze, novel object recognition test (NORT), passive avoidance test (PAT), or social interaction test (SAT). there is.

In one embodiment of the present invention, in an animal model treated with the candidate drug, compared to an untreated control group, the number of dendritic spines in the cortex and hippocampus increases; Increased expression of PLC subfamilies β1 and β2; Increased production of IP3; Increased expression of learning or memory-related factors nrsn1, grun2b, and creb; Alternatively, in the case of increased expression of gng2, gpr183, and ephb2, which are top target genes of the PLC family, a step of determining the candidate drug as a treatment that can improve, prevent, or treat memory, cognitive, or learning disorders may be further included.

In one embodiment of the present invention, the memory, cognitive or learning disorders include aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, and CNS. Hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug addiction, epilepsy, epilepsy, hippocampal sclerosis, headache, cerebral senescence, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV. , cerebrovascular disease, cranial nerve disease, cardiovascular disease, memory loss, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or memory, cognitive, or learning disability due to attention deficit.

In addition, the present invention provides a pharmaceutical composition for preventing or treating memory, cognitive or learning disorders, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

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

In one embodiment of the present invention, the memory, cognitive or learning disorders include aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, bipolar disorder, head trauma, stroke, and CNS. Hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug addiction, epilepsy, epilepsy, hippocampal sclerosis, headache, cerebral senescence, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV. , cerebrovascular disease, cranial nerve disease, cardiovascular disease, memory loss, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit, or memory, cognitive, or learning disability due to attention deficit.

In addition, the present invention provides a health functional food for preventing or improving memory, cognitive or learning disorders, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

In addition, the present invention includes PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and inhibits the activity of microglia or astrocytes; and a pharmaceutical composition for preventing or treating neuroinflammation, tau-related diseases, or degenerative brain diseases, which has amyloid plaque inhibitory activity.

Furthermore, the present invention includes PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and inhibits the activity of microglia or astrocytes; and a health functional food for preventing or improving neuroinflammation, tau-related diseases, or degenerative brain diseases, which is characterized by having amyloid plaque inhibitory activity.

According to the present invention, when PLCXD3 gene is deficient, it causes imbalance in movement, space, perception, learning, memory and sociality, confirming the possibility of developing degenerative brain diseases including memory impairment, cognitive dysfunction and learning ability impairment, thereby confirming the possibility of developing Plcxd3 gene and PLCXD3 It was discovered that the protein can be used as a new target for the prevention, improvement, or treatment of these brain diseases, and therefore, the onset of brain diseases can be diagnosed by measuring the expression or activity level of PLCXD3. In addition, the Plcxd3-deficient animal model produced in the present invention can be usefully used for screening treatments for memory, cognitive, or learning disorders and related degenerative brain diseases.

Figure 1 compares zebrafish with Plcxd3 KO in an embodiment of the present invention with normal zebrafish. (A) shows the Plcxd3 gene expression pattern (top) and morphological changes 48 hours after fertilization in the zebrafish brain (top). Bottom) shows the results of analysis by in situ-hybridization, and (B) shows the normal Plcxd3 sequence in exon 2 and the mutant with + nucleotide 29 bp inserted for the production of Plcxd3 KO zebrafish. Sequences are compared, (C) is a photograph observing the phenotype of Plcxd3 KO zebrafish compared to normal zebrafish at 5 hpf (day after fertilization), and (D) and (E) are new tank tests and warnings. This shows the results of measuring the distance traveled in the material test.
Figure 2 is an analysis of the expression level of PLCXD3 in normal mouse brain regions. (A) is the result of real-time PCR analysis of the brain region-specific expression level of PLCXD3 in the brain of a 3-month-old normal mouse, (B) and ( C) shows the results of confirming the expression level of PLCXD3 protein in the brain of a 3-month-old normal mouse using immunohistochemistry, and (D) shows a photograph showing the expression level of PLCXD3 protein in the hippocampus of a 3-month-old normal mouse. will be.
Figure 3 shows that PLCXD3 is mainly expressed in excitatory and inhibitory neurons of normal mice. (A) shows the results of immunofluorescence staining using anti-PLCXD3 and anti-GluA2 antibodies in normal mice. , (B) to (G) quantitatively show the results of (A), and (H) shows the results of immunofluorescence staining using anti-PLCXD3 and anti-GAD67 antibodies in normal mice, (I) to (N) quantitatively represent the results of (H).
Figure 4 shows the process of producing Plcxd3 KO mice in one embodiment of the present invention, (A) shows the CRISPER/Cas9 system for generating Plcxd3 KO mice, and (B) shows a frame shift mutation (8 bp, Top, blue deletion) shows the target sequence of Plcxd3 for production, and confirmation of normal littermates, Plcxd3 heterozygous and KO mice through PCR, and (C) shows the mating and generation process of Plcxd3 KO mice. A schematic diagram is shown, and (D) shows the target sequence of the frame shift mutation (8 bp deletion, top, blue) of Plcxd3 and the primer sequence for detecting the Plcxd3 gene (yellow) of normal littermates and Plcxd3 KO mice, (E) is a schematic diagram of the process for confirming Plcxd3 KO mice using RT-PCR or real-time PCR, and (F) to (G) show the expression of Plcxd3 in the hippocampus of Plcxd3 KO mice and their littermates using RT- The results of analysis by PCR and real-time PCR are shown.
Figure 5 confirms the impairment of behavioral, spatial, cognitive, learning, memory and social abilities in Plcxd3 KO mice, (A) open field test, (B) Y-maze test, and (C) novel object recognition test. , (D) shows the results of the passive avoidance test, and (E) shows the results of the social relationship test.
Figure 6 confirms the decrease in the number of dendritic spines in the cortex and hippocampus in Plcxd3 KO mice. (A) shows the cortical peak slope (AO) and basal slope in the cortex and hippocampus of 3-month-old Plcxd3 KO mice and their normal littermates. The number of dendritic spines in the moving (BS) region was confirmed by Golgi staining, (B) to (C) are quantitative graphs of the results of (A), and (D) is a 7-month-old Plcxd3 KO mouse and a normal mouse. The number of dendrite spines in the cortical apex oblique (AO) and basal shift (BS) regions in the cortex and hippocampus of littermates was confirmed by Golgi staining, and (E) to (F) are quantitative graphs of the results in (D). It was angry.
Figure 7 analyzes the effect of Plcxd3 KO on the expression levels of PLC-related molecules. (A) shows the concentration of IP3 in the hippocampus of 3-month-old Plcxd3 KO mice and their normal littermates measured using ELISA. (B) is analysis of the expression levels of PLC isoforms by real-time PCR, (C) to (D) are analysis of the expression levels of nrsn1 and grun2b, factors related to learning and memory, by real-time PCR, (E ) is real-time PCR analysis of mRNA levels of PLCXD3-related upstream receptors, gng2, gpr183, and ephb2 in 7-month-old Plcxd3 KO mice and their normal littermates, and (F) is the hippocampus of 3-month-old Plcxd3 KO mice and their normal littermates. shows the results of real-time PCR analysis of the mRNA levels of creb and nrsn1 genes related to learning and memory.
Figure 8 shows changes in anxiety/hyperactivity, short-term spatial cognitive function, sociability, and fear response when amygdala-specific Plcxd3 gene expression is decreased (KD) in normal animals (normal mice). (A) is the cross maze test. , (B) shows the results of the Y-maze test, (C) the social relationship test, and (D) the fear conditioning response test.
Figure 9 shows changes in anxiety/hyperactivity, short-term spatial cognitive function, and sociability during amygdala-specific Plcxd3 gene overexpression (OE) in normal animals. (A) is a cross maze test, (B) is a Y-maze. Test (C) shows the results of the social relationship test.
Figure 10 shows changes in anxiety/hyperactivity, short-term spatial cognitive function, and sociability during decreased hippocampal CA1-specific Plcxd3 gene expression (KD) in normal animals. (A) is the cross maze test, (B) is Y. -Maze test, (C) shows the results of the social relationship test.
Figure 11 shows changes in anxiety/hyperactivity, short-term spatial cognitive function, and sociability during hippocampal CA1-specific Plcxd3 gene overexpression (OE) in normal animals. (A) is a cross maze test, (B) is a Y- Maze test, (C) shows the results of the social relationship test.
Figure 12 shows that when hippocampal CA1-specific Plcxd3 gene expression was decreased (KD) in Alzheimer's disease animals, (A) hippocampal CA1-specific Plcxd3 or control AAV virus was well infected, and (B) shows the results of the passive avoidance test. will be.
Figure 13 shows (A) PLCXD3 gene expression (B) amyloid plaque change (C) APP expression (D) BACE1 expression (E) DYRK1A expression change when hippocampal CA1-specific Plcxd3 gene expression decreases (KD) in Alzheimer's disease animals. It is shown.
Figure 14 shows changes in (AB) microglial activity (CD) and astrocyte activity when hippocampal CA1-specific Plcxd3 gene expression is decreased (KD) in Alzheimer's disease animals.

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

Previously, there was no research on the relationship between PLCXD3 and brain diseases including memory, cognitive, or learning disorders.

While conducting research to discover new treatments for memory, cognitive or learning disorders and degenerative brain diseases, the present inventors created an animal model lacking the Plcxd3 gene, and found that the animal model lacking Plcxd3 showed hyperactivity and anxiety compared to normal animals. It was confirmed that memory disorders, cognitive disorders, or learning disorders were caused, such as enhancement, decrease in short-term/long-term memory, decrease in cognitive function, occurrence of learning disorders, and impaired social behavior.

Accordingly, the present inventors found that the Plcxd3 gene is related to brain diseases including memory, cognitive, or learning disorders, and through experiments in the examples below, the effect of PLCXD3 on brain function in zebrafish and mouse models lacking the Plcxd3 gene was confirmed. The role was identified.

According to one embodiment of the present invention, as a result of performing a novel tank test and an alarm substance test on zebrafish lacking the Plcxd3 gene and normal zebrafish, compared to normal zebrafish Zebrafish lacking Plcxd3 were found to exhibit a form of "hypermobility" in all tests, which could be considered a symptom of bipolar disorder in humans.

In another embodiment of the present invention, mice lacking the Plcxd3 gene were produced using the CRISPR/Cas9 system, and various brain function confirmation experiments were performed.

As a result, in the case of mice lacking Plcxd3, the time spent in the central area of the test space in the open field test was observed to be shorter than that of normal mice, confirming increased anxiety, and Plcxd3 expression was confirmed through the Y-maze, NORT, and PAT. Mice lacking the gene were found to have lower ability to recognize new objects and spatial recognition compared to normal mice, had lower learning ability, and were also observed to have impaired social interaction behavior.

These results imply that PLCXD3 is involved in regulating emotional behavior, spatial tasks, recognition and learning memory, and social behavior.

Additionally, in another embodiment of the present invention, the number of dendritic spines in the brain cortex and hippocampus of Plcxd3-deficient mice was analyzed, and it was found that when Plcxd3 was lacking, the number of dendritic spines was reduced. These results imply that PLCXD3 acts on dendritic spine number formation, which is involved in memory consolidation in the brain.

In addition, the present inventors analyzed whether PLCXD3 affects the expression levels of genes related to learning, cognition, and memory. As a result, in mice lacking Plcxd3, the cytoplasmic vesicle component nrsn1 and NMDA receptor subunit grun2b, which are factors related to learning and memory, were decreased. and creb mRNA levels were found to be reduced, and the expression levels of gng2, gpr183, and ephb2, which are upstream molecular targets of the PLC family, were also found to be significantly reduced. In addition, it was confirmed that when PLCXD3 expression was inhibited in Alzheimer's disease animals, working memory was decreased, amyloid plaques, and brain inflammation were increased.

This showed that PLCXD3 plays an important role in brain functions such as memory, learning, cognition, and social behavior, as well as lesions in Alzheimer's disease.

Based on the above results, the present inventors found that when the expression of PLCXD3 is reduced or suppressed in the brain, brain diseases including memory, cognitive, or learning disorders can be caused, and the expression level or activity of PLCXD3 was measured. Through this, it was found that it was possible to predict or diagnose the onset of brain diseases including memory, cognitive, or learning disorders.

Therefore, the present invention can provide an animal model with memory, cognitive or learning disorders in which the Plcxd3 gene is knocked out.

The deletion 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 TCAACGAG sequence of the Plcxd3 gene is targeted to cause deletion of the Plcxd3 gene by the CRISPR/Cas9 system.

In addition, the present invention can provide a method for producing an animal model with memory, cognitive, or learning disabilities, which includes the step of inducing knock out of the Plcxd3 gene, and the specific production method is described in the Examples below. .

In the present invention, an animal model lacking the Plcxd3 gene (knocked out) was prepared using zebrafish and mice.

In the present invention, the term “knock out” means losing the function of the target gene, and refers to removing the expression of the gene by destroying or removing the target gene using genetic engineering methods. Methods for inducing absence of a target gene may result in loss of expression and function of the target gene by deleting some base sequences of the target gene, replacing them with other base sequences, or introducing other base sequences.

When using the mouse as an animal model, the Plcxd3 gene to be deleted may be the Plcxd3 gene existing in the mouse species, and in one embodiment of the present invention, the Plcxd3 gene deficiency in mice consisting of the base sequence of SEQ ID NO. 3 is induced. did. Additionally, the amino acid sequence for the mouse PLCXD3 protein is shown in SEQ ID NO: 4.

In addition, the present invention includes the steps of (a) measuring the expression level of PLCXD3 in a biological sample isolated from an individual; and (b) comparing the expression level of PLCXD3 in step (a) with the expression level of the normal control group, and determining that the PLCXD3 expression level in the normal control group is lower than that of the normal control group, determining that the PLCXD3 expression level is memory, cognitive, or learning disorder. It can provide a method of providing information necessary for the diagnosis of learning disabilities.

The expression level of PLCXD3 may be the mRNA level of the Plcxd3 gene or the PLCXD3 protein expression level, and the PLCXD3 gene may be composed of the base sequence of SEQ ID NO: 1, and the PLCXD3 protein may be composed of the amino acid sequence of SEQ ID NO: 2.

The method of measuring the expression level of PLCXD3 is reverse transcription-polymerase chain reaction (RT-PCR), real-time polymerase chain reaction, using primers, probes, or antibodies specific for the Plcxd3 gene or PLCXD3 protein. It can be performed by a method selected from the group consisting of real time PCR, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence staining, Western blotting, and flow cytometry, but is not limited thereto. .

In addition, the present invention can provide a method of screening for a treatment for memory, cognitive, or learning disorders using the animal model prepared in the present invention, which method includes (a) memory and cognition lacking the Plcxd3 gene (knocked out); or treating a candidate drug in an animal model with learning disabilities; and (b) comparing the animal model treated with the candidate drug in (a) with an untreated control group to determine whether memory, cognitive, or learning disorders are improved.

Methods for confirming whether the memory, cognitive or learning disabilities are improved are not limited to this, and include novel tank test, alarm substance test, open field test (OFT), This can be done with the Y-maze test (Y-maze), novel object recognition test (NORT), passive avoidance test (PAT), or social interaction test (SAT).

In the above method, the criteria for judging a candidate drug as a treatment for memory, cognitive, or learning disorders are that, in the various experimental results described above, memory, cognition, or learning ability is improved or increased compared to the control group that was not treated with the candidate drug; The candidate drug can be judged as a therapeutic agent.

In addition, in animal models treated with the candidate drug, compared to the untreated control group, the number of dendritic spines in the cortex and hippocampus increased; Increased expression of PLC subfamilies β1 and β2; Increased production of IP3; Increased expression of learning or memory-related factors nrsn1, grun2b, and creb; Alternatively, if it shows increased expression of gng2, gpr183, and ephb2, which are the top target genes of the PLC family, the candidate drug can be judged as a treatment that can improve, prevent, or treat memory, cognitive, or learning disorders.

In the present invention, memory, cognitive, or learning disorders may include all brain diseases caused by deficiency of PLCXD3, but are not limited to aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, and Creutz disease. Felt-Jakob disease, depression, bipolar disorder, head trauma, stroke, CNS hypoxia, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, hypoglycemia, Wernicke-Korsakoff syndrome, drug addiction, epilepsy, epilepsy, hippocampal sclerosis, headache, brain senescence. Dementia, frontotemporal lobe degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, cranial nerve disease, cardiovascular disease, memory loss, radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, cognitive deficit or attention deficit. This may include memory, cognitive or learning difficulties.

Additionally, the brain disease may also include degenerative brain disease. Degenerative brain diseases are progressive degenerative disorders related to the central or peripheral nervous system characterized by damage to nerve structure and function, such as Alzheimer's disease and Parkinson's disease, caused by accumulation of amyloid beta, hyperphosphorylation of tau, and aggregation of α-synuclein. It is a common neurodegenerative disease that includes brain shrinkage (atrophy) and nerve depression.

Furthermore, the present invention can 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.

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 expression and activity of Plcxd3 were suppressed due to a lack of the Plcxd3 gene.

Therefore, an expression promoter that can induce the expression of the Plcxd3 gene or an activator that can promote the activity of the PLCXD3 protein prevents, improves, or treats memory, cognitive, or learning disorders, thereby improving short-term and long-term memory, cognitive ability, or learning disorders. Improvement effects can be derived.

In the present invention, "improvement of memory" and "improvement of cognitive ability" are memory loss, memory impairment, or cognitive ability that occurs when the brain atrophies and brain nerve cells are destroyed due to physical fatigue, lack of sleep, excessive alcohol consumption, dementia, etc. This refers to the effect of maintaining cognitive ability by controlling harmful substances that damage brain cells, or improving deteriorated cognitive ability by controlling neurotransmitters in the brain. The above-mentioned memory is the ability to receive necessary information, store it in the brain, and retrieve it for use when necessary, and cognitive ability refers to the ability to discern and recognize objects.

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

Solid preparations for oral administration include tablets, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose. ), gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition may be any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. It can have one dosage form.

Additionally, as described above, the pharmaceutical compositions of the present invention can be administered to treat various brain diseases, including symptoms associated with memory, cognitive, or learning disorders.

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 is determined by the type and severity of the subject, age, sex, activity of the drug, and It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects. The general dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.

The pharmaceutical composition may be administered through any general route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or rectally, depending on the purpose, but is not limited thereto. Additionally, the composition can be administered by any device capable of transporting the active agent to target cells.

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

In addition, the present invention can provide a health functional food for preventing or improving memory, cognitive or learning disorders, which contains PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient.

The health functional food of the present invention may additionally contain a foodologically acceptable carrier.

The health functional food may have a function that helps prevent, improve, or improve the disease targeted by the present invention, and the food includes the form of pills, powders, granules, needles, tablets, capsules, or liquids. , there is no particular limitation on the type of food to which the active ingredient of the present invention can be added, for example, various beverages, gum, tea, vitamin complexes, health supplements, etc.

In addition to the active ingredients of the present invention, other ingredients that do not interfere with the prevention, improvement or treatment effects can be added to the health functional food, and the types are not particularly limited. For example, like regular foods, it may contain various herbal extracts, foodologically acceptable food supplements, or natural carbohydrates as additional ingredients.

The term “health functional food” used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functional properties useful to the human body. Here, functionality means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.

There is no particular limitation on the type of health functional food of the present invention, and specific examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, There are beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and can include all health functional foods in the conventional sense.

In addition, the present invention includes PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient, and inhibits the activity of microglia or astrocytes; And a pharmaceutical composition for preventing or treating neuroinflammation, tau-related diseases, or degenerative brain diseases, characterized by having an amyloid plaque inhibitory activity, can be provided, and can be used to prevent or improve neuroinflammation, tau-related diseases, or degenerative brain diseases. Health functional foods can be provided.

According to one embodiment of the present invention, when the expression of PLCXD3 is inhibited in a mouse model inducing Alzheimer's disease, it is confirmed that the activity of microglia or astrocytes increases and amyloid plaques increase. I was able to. These results ultimately show that PLCXD3, its expression promoter, or its activator can suppress the activity of microglia or astrocytes and suppress amyloid plaques, thereby preventing neuroinflammation, tau-related diseases, or degeneration. This means that brain diseases can be prevented, improved, or treated.

Microglial cells, which play the role of macrophages in the brain, are important effector cells that regulate immune responses in the central nervous system (CNS). Their activation plays an important role in maintaining CNS homeostasis by removing foreign substances caused by drugs or toxins and secreting nerve growth factors. However, when exposed to harmful stress such as signals generated from damaged neurons, accumulation of abnormal proteins modified by external stimuli, or invasion of pathogens, the activity of microglial cells increases excessively, causing damage to nerve cells, leading to Alzheimer's disease. It can cause degenerative neurological diseases such as Parkinson's disease, multiple sclerosis, and cerebral infarction. In addition, recent research results have revealed that excessive inflammatory responses in microglia or astrocytes are the cause of degenerative brain diseases.

Astrocytes are also known to play an important role in maintaining normal brain activity. In particular, they are known to play a role in the formation of neuronal synapses, regulation of synapse number, synaptic function, and differentiation of neural stem cells into neurons. However, when these astrocytes become excessively reactive, that is, when they remain in an excessively activated state, they cause death of nerve cells and induce death of neighboring nerve cells, thereby acting as a cause of degenerative brain disease. Therefore, suppressing excessive astrocyte activation can also be a new treatment method for degenerative brain diseases.

Neuroinflammatory responses also include activation of innate immune cells (microglia), 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 thought to be a pathological marker and an important mechanism in the progression of neurodegenerative diseases. Tight regulation of microglial activity is essential to maintain brain homeostasis and prevent infectious and inflammatory diseases.

In addition, beta-amyloid (Aβ) is derived from amyloid precursor protein (APP), which is broken down by beta-secretase and gamma-secretase to produce Aβ, and is mainly found in the brains of patients with brain diseases such as Alzheimer's disease. It is the main component of plaque, and if it accumulates abnormally, it can cause various brain diseases.

Excessive accumulation of beta amyloid causes an inflammatory response in the hippocampus, cerebral cortex, and surrounding cells, which can damage nerve cells and damage the neural network that maintains normal brain function.

Additionally, tau proteins are a class of microtubule-binding proteins with a molecular weight of 50,000 to 70,000 Da and are involved in the tangle of nerve fibers. Tau protein is one of the microtubule-associated proteins present in axons of normal nerve cells. When these tau proteins accumulate excessively, microtubules collapse and the network of normal nerve cells disintegrates. It is known that the cause of tau protein accumulation is that phosphorus is excessively attached to tau protein, forming a heavy body due to phosphorylation, and the accumulation of these tau proteins and aggregation of nerve cells are known to cause various degenerative brain diseases. .

Therefore, inhibition of the activity of microglia or astrocytes; and PLCXD3, which has amyloid plaque inhibitory activity, an expression promoter thereof, or an activator thereof can be used as a substance that can prevent, improve, or treat neuroinflammation, tau-related diseases, or degenerative brain diseases.

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

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Preparation example and experiment method>

Preparation of Plcxd3 knock-out zebrafish

For the preparation of sgRNA against Plcxd3 in zebrafish, a 20bp target nucleotide sequence within exon2 was selected using the CRISPR SCAN website (http://www.crisprscan.org/). The DNA template for sgRNA was prepared by fill-in PCR and DNA purification methods according to the manufacturer's protocol. In vitro transcription of sgRNA was performed using the MaxiScript T7 kit (Ambion), and the synthesized RNA was precipitated with ammonium acetate. For the preparation of Plcxd3 mutants 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 with phenol red.

Preparation of Plcxd3 Knock-Out Mice

For the generation of Plcxd3-deficient mice, a single guide RNA (sgRNA) targeting the exon2 region was designed using the MEGAshortscript™ kit (Thermo Fisher Scientific, Waltham, MA, USA). The sequence information used to design gRNA is as follows. crRNA1: 5'-TCTGGCTGCTCCGGGCCTAC-3', crRNA2: 5'-TTCAGTGCCAAAGTCAACGA-3', and crRNA3: 5'-GGGTCTGTCGTGTTTGCCCA-3'. For microinjection of Cas9 protein and sgRNA into fertilized eggs, male C57BL6/J mice were housed individually for 1 week before mating, and female C57BL6/J mice were injected with 5 IU of pregnant mare serum gonadotropin and 5 IU of pregnant mare serum gonadotropin for superovulation. Human chorionic gonadotropin (HCG) was administered. 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 by microinjection using continuous flow injection mode. The surviving two-cell embryos were implanted into the fallopian tubes of pseudopregnant females. Founder mice (F0) were analyzed among born pups. F0 mice were crossed with C57BL6/J normal mice and F1 mice were crossed to obtain F2 homozygous mutants. To maintain a stable inbred background, female mice were backcrossed to the parental inbred lines approximately every 10 generations. The primers used for genotyping were forward: 5'-GTCCATGATTCCTTCAGCT-3' and reverse: 5'-TTTCTCTGAGTTCCACTTGCCA-3'. Deletion (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. Confirmed. Ultimately, -8 bp deletion mutant (Plcxd3 KO) mice were used in the experiment.

Immunohistochemistry (DAB staining)

The experiment was performed with approval for all animal testing methods from the Animal Care and Use Committee of the Korea Brain Research Institute (IACUC-21-00059). Normal mice aged 3 months were raised in a pathogen-free facility at 22 ± 2°C, 50 ± 5% humidity, and a 12-hour light/dark cycle, with food and water provided ad libitum. Mice were perfused and fixed with PBS and 4% paraformaldehyde. To obtain brain sections for immunohistochemical analysis, brain tissue stored in 4% paraformaldehyde for 24 hours at 4°C was immersed in 30% sucrose in PBS for 72 hours and exfoliated using a cryostat microtome. It was sectioned to a thickness of 30um using (Leica CM1850, Wetzlar, Germany). Afterwards, the slices were incubated with 30% H ₂ O ₂ in PBS and reacted with rabbit-anti-PLCXD3 antibody overnight at 4°C. Afterwards, the slices were washed with PBS buffer and biotylated for 2 hours at room temperature. Reacted with secondary antibody. After washing again with PBS buffer, ABC solution (Vector Laboratories, Burlingame, CA, USA) was added and reacted at room temperature for 1 hour. Afterwards, it was developed for 42 seconds using DAB solution (Vector Laboratories, Burlingame, CA, USA). After mounting it on a glass slide 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, USA). National Institutes of Health, Bethesda, MD, USA).

Immunofluorescence staining (IF staining)

Plcxd3-deficient mice and their normal littermates were raised in a pathogen-free facility at 22 ± 2°C, 50 ± 5% humidity, and a 12-hour light/dark cycle with food and water provided ad libitum. After behavioral testing, mice were perfused and fixed with PBS and 4% paraformaldehyde, respectively. To obtain brain sections for immunofluorescence staining, brain tissue stored in 4% paraformaldehyde for 24 hours at 4°C was immersed in PBS containing 30% sucrose for 72 hours and then transferred to a cryostat. It was sectioned to a thickness of 30 μm using a microtome (Leica CM1850, Wetzlar, Germany). Brain sections were blocked with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) at room temperature for 2 hours and then reacted with targeting antibodies overnight at 4°C. Details of the antibodies used are listed in the table below. Afterwards, the slices were washed with PBST buffer and reacted with secondary antibodies 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, MD, USA).

Reverse transcription PCR

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 using QIAzol Lysis Reagent (Qiagen, Cat No. 79306), and 30-cycle RT-PCR was performed with Prime Taq Premix (GeNetBio). The sequences of the primers used are shown in the table below. The amplicon was separated by electrophoresis on a 2% agarose gel supplemented with EcoDye (1:5000, Biofact, Daejeon, Korea), and gel imaged using the software Fusion Capt Advance (Vilber Lourmat, Eberhardzell, Germany). was analyzed.

Real time PCR

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

IP ₃ Assay

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, homogenized in PBS buffer, and left on ice for 1 hour. Then, the supernatant was collected by centrifugation at 10,000 rpm at 4°C for 15 minutes. The concentration of total protein was measured using the BCA protein assay, and the expression level of IP3 was measured using an ELISA kit (Cat no. CSB-E13410m, CUSABIO, Huston, TX, USA).

New tank testing

Zebrafish are known to live at the bottom of a fish tank and display anxious behavior when exposed to an unfamiliar environment. Therefore, to investigate behavioral differences between Plcxd3-deficient mutants and normals, each zebrafish was placed in a new tank (24 cm × 15 cm × 15 cm), the water level of the tank was filled to 12 cm, and the temperature was maintained at 28°C. Zebrafish behavior was recorded for 30 minutes using a video camera (Sony, HDRCX190, Japan). Image tracking of video recordings was performed using EthoVision XT 7.0 motion tracking software (Noldus).

Alarm substance test

It is known that when a zebrafish is harmed by a predator, warning substances are released from its skin to signal danger to other fish. Zebrafish exhibit behaviors such as panic and freezing when exposed to warning substances. Therefore, we performed an experiment to confirm whether these behavioral changes occur in mutants lacking Plcxd3. The warning substance was prepared from zebrafish skin extract. The tank was prepared under the same conditions as the one used in the new tank test described previously (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 min using a video camera (Sony, HDRCX190, Japan), and image tracking was performed using EthoVision XT 7.0 movement tracking software (Noldus) by dividing the height of the tank into thirds.

Open field test (OFT)

The open field consisted of a brightly illuminated enclosure (40 × 40 cm) with a white plastic floor and transparent Plexiglas walls (Kinder Scientific, Poway, CA, USA). On the day of testing, mice were allowed to explore the enclosure for 5 min, and 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 exploration time in the center or periphery of the enclosure were analyzed as outcome measures. After evaluating the activity in the open field, the equipment was washed with 70% ethanol to remove residual odor.

Y-maze test

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

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

New Object Recognition Test (NORT)

To confirm the effect of Plcxd3 on long-term memory and recognition memory, a NOR test was performed. The apparatus for the NOR test used an open box with a side length of 40 cm and a height of 25 cm. The NOR test consisted of two stages: training and testing. Training and testing phases were performed 24 hours apart. In the training phase, two identical objects were placed in a box and the mouse was allowed to explore the box for 5 minutes. In the test phase, mice were returned to a box containing one familiar object and one novel object for 5 minutes. During the test 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 odor. Exploration times were calculated manually by reviewing video recordings of each test. Behavior was considered exploratory when the mouse pointed its nose toward an object. The object preference (%) for the new object was calculated using the following formula.

[Object preference (%) = T novelty/(T familiarity + T novelty) × 100],

Here, T novelty is the exploration time for a new object, and T familiarity is the exploration time for a familiar object.

Passive Avoidance Test (PAT)

Hippocampus-dependent contextual fear memory involves re-exposure to an environment that received aversive stimulation during previous training. In the hippocampus-dependent passive avoidance test, mice were placed in a separate compartment with two rooms and given a foot shock when entering the less anxiety-inducing dark compartment. The next day, mice were returned to the illuminated compartment and latency to enter the dark compartment was assessed. To test this, mice were placed in a bright compartment in 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 that prefer the dark left side generally passed through the gate quickly and entered the dark compartment. Afterwards, the mouse received a brief foot shock (0.3 mA for 3 seconds). Each mouse was tested for up to 180 seconds without entering the dark compartment. After 24 hours, the mouse was placed back into the right compartment. 10 seconds later, the partition light turned on and the connecting door opened. The time to re-enter the dark compartment was measured at a maximum of 180 seconds.

Social Interaction Test (SAT)

The device consisted of a box with three rectangular illuminated chambers and an attached infrared video camera. The size of each room was 40 cm × 60 cm × 22 cm (height), the partition wall was made of transparent plexiglass, and a small square hole (5 cm wide × 3 cm high) was created to allow exploration of each room. A normal (C57BL/J) 8-week-old male mouse was used as a ‘stranger’.

One day before testing, 'subject mice' were individually placed in the middle chamber and allowed to freely explore the entire apparatus for 5 minutes. Before the test, the subject mouse was placed in the middle chamber and allowed to freely explore all three chambers for 5 minutes (habituation test). In the sociability test, an unfamiliar normal male mouse (‘stranger 1’) with no prior contact with the subject mouse was placed in one of the side chambers. Stranger mice were housed in small, round wire cages that allowed nose contact between the bars but prevented fighting. The cage was 11 cm high, had a base diameter of 9 cm, and had vertical bars spaced 0.5 cm apart. The subject mouse was placed in the middle room, and unfamiliar object 1 was presented in one compartment and an empty cage in the other compartment for 5 minutes. The time spent around each cage (stranger 1 or empty) was measured. At the end of the 5-minute sociability test, each subject mouse was tested for 5 minutes to quantify social preference for a new, unfamiliar object (social preference test). The barbed wire surrounding a familiar normal male mouse (Stranger 1) was moved to the opposite side of the room that was empty during the sociability test. Second, an unfamiliar normal male mouse (Stranger 2) was placed in a room on the other side of the same small wire cage. Subject mice were allowed to freely explore the mouse (stranger object 1) and the new mouse (stranger object 2) from the previous sociability test. The time spent around each cage (Stranger 1 or Stranger 2) was measured. Data collection and analysis were performed manually.

Golgi staining

After behavioral testing, 4-5 Plcxd3 null mice and normal mice were Golgi stained using the FD Rapid Golgi Stain kit (FD NeuroTechnologies). The dissected brain from each mouse was cultured in solutions A and B for 2 weeks at room temperature and then immersed in solution C for 24 hours at 4°C. The brain was sectioned at a thickness of 150 μm using a vibratome (VT1000S; Leica). Bright-field microscopy (Axioplan 2; Zeiss) images (63× magnification) of pyramidal neurons in cortical layers V and CA1 were recorded, and dendritic spine numbers were counted in a blinded manner using ImageJ (NIH). Dendrite spine visibility was measured in dendrites with dendrite lengths longer than 20 μm.

Preparation of a brain region-specific low Plcxd3 gene expression (KD) animal model

To prepare an animal model for brain-specific Plcxd3 gene knockdown (KD), an Adeno-Associated Virus (AAV) based on shRNA vector labeled with CMV promoter and eGFP was used. A normal mouse was fixed on a stereotaxic operating table, bregma was set as a reference point (AP 0 mm, ML 0 mm, DV 0 mm), and both amygdala (brain) were used to prepare an amygdala-specific Plcxd3 expression animal model. 3D coordinates: AP -1.1 mm, ML -/+ 3.45 mm, DV -4.5 mm from bregma) with control virus (AAV-CMV-control shRNA-eGFP) or virus for reducing Plcxd3 expression (AAV-CMV-Plcxd3 shRNA- eGFP) was injected at a rate of 0.5 μl through a micropump at a flow rate of 0.25 μl/min. In addition, to prepare an animal model with hippocampal CA1-specific Plcxd3 expression reduction, a control virus (AAV) was administered to both CA1 (brain 3D coordinates: AP -2.0 mm, ML -/+ 1.5 mm, DV -1.55 mm from bregma) of normal mice. -CMV-control shRNA-eGFP) or Plc-xd3 expression reduction virus (AAV-CMV-Plcxd3 shRNA-eGFP) was injected at a rate of 1.0 μl through a micropump at a flow rate of 0.25 μl/min. Behavioral analysis was conducted 3 weeks after injection to analyze the bipolar disorder-related behavioral regulation function of Plc-xd3 in each brain region.

Preparation of brain region-specific-Plcxd3 gene overexpression (OE) animal model

To prepare a brain region-specific Plcxd3 gene overexpression (OE) animal model, Adeno-Associated Virus (AAV) labeled with the CMV promoter and eGFP was used. A normal mouse was fixed on a stereotaxic operating table, bregma was set as a reference point (AP 0 mm, ML 0 mm, DV 0 mm), and both amygdala (brain) were used to prepare an amygdala-specific Plcxd3 expression animal model. 3D coordinates: AP -1.1 mm, ML -/+ 3.45 mm, DV -4.5 mm from bregma) with control virus (AAV-CMV-control-eGFP) or virus for Plcxd3 overexpression (AAV-CMV-Plc-xd3-eGFP). ) was injected at a volume of 0.5 μl at a flow rate of 0.25 μl/min through a micropump. In addition, to prepare an animal model with hippocampal CA1-specific Plcxd3 expression reduction, a control virus (AAV- CMV-control-eGFP) or Plcxd3 overexpression virus (AAV-CMV-Plc-xd3-eGFP) was injected at a rate of 1.0 μl through a micropump at a flow rate of 0.25 μl/min. Behavioral analysis was conducted 3 weeks after injection to analyze the bipolar disorder-related behavioral regulation function of Plc-xd3 in each brain region.

Elevated plus maze (EPM)

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

Fear conditioning test (FCT)

FCR was performed to confirm the function of amygdala-specific Plcxd3 on fear formation and fear memory. Place the experimental mouse in the test chamber (54cm were imposed together. A total of 5 sound and electrical stimulations were applied with an interval of 1 minute, and freezing behavior was analyzed using packwin software (fear conditioning; fear formation analysis possible). 24 hours later (day 2), in order to selectively analyze fear memories of sound, the colors of the floor and walls of the same test chamber were changed, and the experimental mouse was placed in the environment for 1 minute to adapt to the environment, and then exposed to the environment for 50 to 70 seconds. At intervals, a sound stimulus of 90 dB, 2.8 kHz was applied 10 times for 30 seconds, and freezing behavior was analyzed using packwin software (fear retrieval). After 8 and 72 hours (day 3-4), freezing behavior was analyzed using packwin software under the same conditions as day 2 to observe fear extinction, and was analyzed as an outcome measure for fear memory.

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

5xFAD mice (stock no. 008730, B6SJL-Tg APPSwFlLon,PSEN1*M146L*L286V6799Vas/Mmjax) of the F1 generation were obtained from the Jacson laboratory, and transgenic male mice (5xFAD) were placed together with female C57BL/6J purchased from the Jacson laboratory. . 5xFAD is a human familial Alzheimer's disease (695) with mutant human APP (695) with K670N, M671L (Swedish), I716V (Florida), and V717I (London) and human PS1 with two FAD mutations (M146L and L286V). It is known to overexpress the Familial Alzheimer's Disease (FAD) mutation. The transformation was controlled by the mouse Thy1 promoter to cause overexpression in the brain, and genotyping for the 5xFAD transformation was performed using the PCR method provided by Jackson Lab's genotyping protocol.

Immunohistochemistry and immunofluorescence analysis

Alzheimer's animals were perfused and fixed using perfusion solution (0.9% NaCl, Biosesang) and 4% paraformaldehyde solution (Chembio), and brain tissue was cut with a frozen tissue slicer (Leica) (30 mm thick) and immunohistochemistry. Fluorescence and immunohistochemical staining were performed. For immunofluorescence staining, the cells were washed with PBS containing 0.2% Triton The incubated antibodies 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 secondary antibodies for 2 hours at room temperature. The tissue was placed on a glass slide, DAPI-containing mounting solution (Vector Laboratories) was added thereto, covered with a cover glass, and refrigerated at 4 degrees. The stained tissue was photographed using a fluorescence microscope (DMi8, Leica Microsystems).

Statistical analysis

Data analysis was performed with GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA). For comparison of the two groups, an unpaired two-tailed T-test with Welch's correction was used. For multiple comparisons, one-way ANOVA and Tukey's test were used. And if p < 0.05, it was considered significant. Results are expressed as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

Information on reagents and oligonucleotides used in the examples of the present invention is shown in Table 1 below.

<Example 1>

Expression characteristics of PLCXD3 in zebrafish and analysis of bipolar disorder-related behavior

<1-1> CNS-specific Plcxd3 expression pattern analysis

First, the present inventors analyzed the expression specificity of Plcxd3 in zebrafish. To investigate the CNS-specific expression pattern of Plcxd3 in the zebrafish gene, whole-mount in situ hybridization was performed, and the results confirmed that Plcxd3 is mainly expressed in a subset of neurons in the zebrafish brain and spinal cord. In particular, it was confirmed that neuron-specific expression of Plcxd3 persisted from 24 hpf (hours post fertilization) to 120 hpf and in the adult zebrafish brain (Figure 1A).

<1-2> Analysis of bipolar disorder-related behavior in Plcxd3 KO zebrafish

In addition, in order to confirm the physiological role of PLCXD3, the present inventors used the CRISPR/Cas9 system to produce Plcxd3-deficient (KO) zebrafish, selected PLCXD3 KO mutants, and finally added +29 bp inserted. The allele was established (Figure 1B), and the +29 bp insertion mutant (Plcxd3 KO zebrafish) was used in this experiment. In addition, as a result of morphological comparative analysis between the mutant and normal, as shown in Figure 1C, it was confirmed that compared to normal zebrafish embryos, Plcxd3 KO zebrafish had no morphological defects up to 5 dpf (days after fertilization).

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

As a result, it was confirmed that Plcxd3 KO adult zebrafish showed a form of 'hyperactivity' in all tests compared to normal zebrafish (Figures 1D and 1E).

Through these results, the present inventors were able to find that Plcxd3 KO zebrafish showed similar behavior to human bipolar disorder patients and that PLCXD3 was related to bipolar disorder.

<Example 2>

Analysis of the expression pattern of PLCXD3 in the hippocampus of normal mouse brain

The expression pattern of Plcxd3 was analyzed in mouse brain. For this purpose, real-time PCR was performed using specific primers for the Plcxd3 gene of normal mice.

As a result, it was confirmed that the mRNA level of Plcxd3 was highly expressed in the cerebellum (CB), hypothalamus, hippocampus CA1, and CA3 of normal mice (Figure 2A). Additionally, we were able to identify PLCXD3 protein, which was highly expressed in CB, hypothalamus, hippocampus CA1, and CA3 regions in normal mouse brain through immunohistochemistry (Figures 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 (Figures 2C, 2D).

<Example 3>

Analysis of the expression pattern of PLCXD3 by nerve cell type in normal mice

To determine in which cell types PLCXD3 is specifically expressed, anti-PLCXD3 and anti-GluA2 (excitatory neuron markers) or anti-PLCXD3 and anti-GAD67 antibodies (inhibitory neuron markers) were applied to normal mice. Double fluorescence staining was performed using

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

These results showed that PLCXD3 was expressed in both excitatory and inhibitory neurons, and in particular, PLCXD3 was found to be predominantly expressed in inhibitory neurons in the hippocampus of normal mouse brain.

<Example 4>

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

To characterize the physiological function of PLCXD3 in the brain, Plcxd3-deficient (KO) mice were generated using the CRISPER/Cas9 system, and several insertion or deletion mutations were identified in the generated mice. As mutations, a mutant with a +1 bp insertion, a mutant with a -8 bp deletion, and two mutants with a -10 bp deletion were identified. In this experiment, a mutant with -8 bp deletion (Plcxd3 KO) was used (Figure 4B). The -8 bp deletion was predicted to cause a frameshift mutation resulting in premature truncation of the PLCXD3 protein.

Then, Plcxd3+/+ homozygotes from normal mice were crossed with Plcxd3-/- homozygotes from Plcxd3 KO mice (F1) to obtain Plcxd3+/- heterozygous mice (F2), and the heterozygotes from F2 mice were crossed with each other and bred. According to Mendelian genetics, F3 mice, Plcxd3+/+ homozygous, Plcxd3+/- heterozygous, and Plcxd3-/- homozygous mice were obtained (Figure 4C). In this experiment, F3 Plcxd3-/- mice, which are littermates of Plcxd3+/- heterozygotes, were used as Plcxd3-deficient mice.

Additionally, to confirm whether the Plcxd3 gene was deleted in the Plcxd3 KO mouse brain, genotyping 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 mouse. (Figures 4D-4F). Real-time PCR results also showed that the level of Plcxd3 mRNA in the hippocampus was significantly reduced in Plcxd3-/- KO mice (Plcxd3-/-) compared to F3 normal mice (Plcxd3+/+), and was almost completely absent. It was confirmed that there was (Figure 4F).

<Example 5>

Confirmation of impairment in learning/memory, cognitive function, and social behavior due to lack of Plcxd3

<5-1> Open field test

The present inventors conducted an experiment to determine whether PLCXD3 is related to learning, memory, cognitive function, and social behavior. For this purpose, animal behavioral experiments were performed using 3- or 7-month-old Plcxd3 KO mice and their littermates.

As a result, compared to normal mice with no change in total distance traveled, 3-month-old Plcxd3 KO mice showed a significant decrease in exploration time in the central zone (Figure 5A), and similar to these results, 7-month-old Plcxd3 KO mice Although the search time in the central area of the province tended to decrease, there was no significant change in the total travel distance (Figure 5A).

Through these results, the present inventors were able to see that Plcxd3 KO mice felt more anxious than normal individuals, which means that the Plcxd3 gene plays a role in regulating anxiety-related behaviors.

<5-2> Y-maze, NORT and PAT tests

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.

The results showed that the degree of spontaneous alterations was significantly reduced in 3- and 7-month-old Plcxd3 KO mice compared to normal mice from their littermates (F3) (Figure 5B). Additionally, liking for novel objects was found to be significantly suppressed in 7-month-old Plcxd3 KO mice (Figure 5C). PAT analysis showed that latency to the right cue induction chamber was significantly reduced in 3- and 7-month-old Plcxd3 KO mice ( Fig. 5D ). These results imply that PLCXD3 plays an important role in maintaining homeostasis of spatial, recognition, and learning memory.

<5-3> Social behavior test

An experiment was conducted to determine whether PLCXD3 is also involved in social behavior. Social interaction tests were analyzed using 3- and 7-month-old Plcxd3 KO mice and their littermates (F3).

As a result, sociability and social preference were found to be significantly reduced in 3- and 7-month-old Plcxd3 KO mice compared to normal mice, indicating that social interaction behavior was impaired in Plcxd3 KO mice (Figure 5E) . Through these results, the present inventors found that PLCXD3 is involved in regulating emotional behavior, spatial tasks, recognition and learning memory, and social behavior.

<Example 6>

Identification of dendritic spines in cortex and hippocampus due to lack of Plcxd3

To determine whether PLCXD3 affects dendritic spine formation related to memory consolidation in the brain, Golgi staining was performed. Golgi staining was performed using 3- or 7-month-old Plcxd3 KO mice and their littermates (F3).

As a result, it was confirmed that dendritic spines were significantly reduced in the cortical apex oblique (AO) and basal shift (BS) regions in 3-month-old Plcxd3 KO mice (Figures 6A, 6B). However, dendritic spine spines in the hippocampus were significantly reduced only in the hippocampal cortex apical oblique (AO) region and not in the basal shift (BS) region in 3-month-old PLCXD3 KO mice (Figures 6A, 6C). Additionally, the number of dendritic spines was significantly reduced in the cortical apical oblique (AO) and basal shift (BS) regions of the cortex and hippocampus of 7-month-old Plcxd3 KO mice (Figure 6D, 6E).

Through these results, the present inventors were able to see that PLCXD3 plays an important role in the formation of dendritic spines related to memory consolidation in the brain, and the lack of expression of the Plcxd3 gene or protein reduces the formation of dendritic spines in the brain. It was found that it could cause functional disorders, and on the other hand, it was found that increasing the expression and activity of the Plcxd3 gene or protein could improve, prevent, or treat brain dysfunction.

<Example 7>

Analysis of expression changes of molecules related to memory, learning, or cognitive functions due to lack of Plcxd3

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

As a result, the concentration of IP3 was found to be significantly reduced in the hippocampus of Plcxd3 KO mice compared to normal littermates, and through this, it was confirmed that PLCXD3 activity was significantly low in Plcxd3 KO mice (Figure 7A).

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

As a result, the expression of β1 and β2, subfamilies of PLC, was found to be significantly reduced in 3-month-old Plcxd3 KO mice (Figure 7B), indicating that the lack of Plcxd3 affects the expression of subfamilies of PLC. there was.

In addition, to determine whether PLCXD3 affects the expression levels of genes related to learning and memory, real-time PCR was performed and the results showed that cytoplasmic vesicle components nrsn1 (neurensin 1) and NMDA, factors related to learning and memory, were expressed in 3-month-old Plcxd3 KO mice. The mRNA level of the receptor subunit grun2b (NMDA receptor 2B) was found to be significantly reduced (Figures 7C, 7D).

In addition, in 7-month-old Plcxd3 KO mice, the expression levels of gng2 (G protein subunit gamma 2), gpr183 (Orphan G-protein coupled receptor 183), and ephb2 (Eph receptor B2), which are upstream molecular target markers of the PLC family, were all significant. It was found to be significantly decreased (Figure 7E), and the expression levels of memory-related genes creb and nrsn1 were also found to be significantly decreased (Figure 7F).

Through these results, the present inventors found that PLCXD3 plays an important role in brain function by regulating the expression of molecules related to memory, learning, cognition, and social behavior. Ultimately, increasing the expression or activity of PLCXD3 improves memory, It was found that learning ability and cognitive dysfunction diseases can be effectively improved, prevented, or treated.

<Example 8>

Confirmation of hyper/anxious behavior, short-term spatial cognitive ability, sociability, and fear conditioning response according to the expression level of amygdala-specific Plcxd3.

<8-1> Confirmation of response due to decreased expression of amygdala-specific Plcxd3

① Cross maze test (EPM)

We conducted experiments to determine whether amygdala-specific Plcxd3 is associated with hyperactive/anxious behavior. For this purpose, experimental mice injected into the amygdala with a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) were used.

As a result, in experimental mice with reduced amygdala-specific plcxd3 expression, the time spent in the open arm was significantly increased compared to the normal control group, and the time spent in the closed arm was significantly decreased, indicating that the anxiety response was lower than the normal value. Confirmed (Figure 8A). There was no difference in the distance traveled in both arms between the two groups, making it possible to infer that amygdala-specific Plcxd3 is involved in anxiety responses but not hyperactivity.

② Y-maze test (Y maze)

The present inventors conducted an experiment to determine whether amygdala-specific Plcxd3 is related to short-term spatial cognitive behavior. For this purpose, experimental mice injected into the amygdala with a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) were used.

As a result, it was confirmed that cognitive deficits occurred in experimental mice with decreased amygdala-specific plcxd3 expression, as spontaneous alterations were observed to be significantly reduced compared to the normal control group (Figure 8B).

③ Social Behavior Test (SAT)

An experiment was performed to determine whether amygdala-specific Plcxd3 is involved in social behavior. For this purpose, experimental mice injected into the amygdala with a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plcxd3 shRNA-eGFP) were used.

As a result, it was confirmed that in experimental mice with reduced amygdala-specific plcxd3 expression, there was no difference in sociability and social interaction ability/social memory (social preference) was decreased compared to the normal control group (Figure 8C).

④ Fear conditioning test (CFT)

An experiment was performed to analyze the effect of amygdala-specific Plcxd3 on fear formation/extinction. For this purpose, experimental mice injected into the amygdala with a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) were used.

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

<8-2> Confirmation of response to amygdala-specific overexpression of Plcxd3

① Cross maze test (EPM)

The present inventors, who confirmed a low-anxiety response when amygdala-specific Plcxd3 was lowered, conducted an experiment to determine whether there was a change in hyper/anxious behavior when amygdala-specific Plcxd3 was overexpressed. For this purpose, experimental mice injected with a control virus or Plcxd3 overexpression virus (AAV-CMV-Plcxd3-eGFP) into the amygdala were used.

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

② Y-maze test (Y maze)

We conducted an experiment to determine whether amygdala-specific Plcxd3 overexpression is related to short-term spatial cognitive behavior. For this purpose, experimental mice injected into the amygdala with a control virus or a virus for overexpressing Plcxd3 (AAV-CMV-Plc-xd3-eGFP) were used.

As a result, in experimental mice overexpressing amygdala-specific plcxd3, a significant decrease in spontaneous alteration was observed compared to the normal control group, confirming cognitive deficit (Figure 9B).

③ Social Behavior Test (SAT)

An experiment was performed to determine whether amygdala-specific Plcxd3 overexpression is involved in social behavior. For this purpose, experimental mice injected with a control virus or Plcxd3 overexpression virus (AAV-CMV-Plcxd3-eGFP) into the amygdala were used.

As a result, it was confirmed that in experimental mice overexpressing amygdala-specific plcxd3, there was no difference in sociability compared to the normal control group, and social interaction ability/social memory (social preference) was decreased (Figure 9C).

Through the above results, the present inventors have shown that amygdala-specific Plcxd3 regulates the anxiety response, short-term spatial memory, social memory, and fear formation process, and that when the expression homeostasis of Plcxd3 is not maintained, that is, when the expression is decreased or overexpressed, these functions It was found that a deficiency was occurring.

<Example 9>

Analysis of hyper/anxious behavior, short-term spatial cognitive ability, and social interaction according to the expression level of hippocampal CA1-specific Plcxd3

<9-1> Confirmation of behavioral changes due to decreased expression of hippocampal CA1-specific Plcxd3

① Cross maze test (EPM)

The present inventors performed an experiment to determine how hippocampal CA1-specific Plcxd3 regulates hyperactive/anxious behavior. For this purpose, experimental mice injected with a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) into the hippocampal CA1 were used.

As a result, experimental mice with decreased hippocampus-specific plcxd3 expression were confirmed to have hyperactivity as the distance moved in the open arm and the entire arm was significantly increased compared to the normal control group (Figure 10A). There was no difference between the two groups in the time spent in both arms, indicating that hippocampal CA1-specific Plcxd3 is involved in hyperactivity but not anxiety response.

② Y-maze test (Y maze)

The present inventors performed an experiment to determine whether hippocampal CA1-specific Plcxd3 is related to short-term cognitive function. For this purpose, experimental mice injected with a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) into the hippocampal CA1 were used.

As a result, it was confirmed that there was no change in spontaneous alteration in experimental mice with decreased hippocampal CA1-specific plcxd3 expression compared to the normal control group (FIG. 10B).

③ Social Behavior Test (SAT)

To perform experiments to determine whether hippocampal CA1-specific Plcxd3 is involved in social behavior, we used experimental mice injected with control virus or Plcxd3 down-expression virus (AAV-CMV-Plc-xd3 shRNA-eGFP) into hippocampal CA1. did.

As a result, it was confirmed that experimental mice with reduced hippocampal CA1-specific plcxd3 expression did not have deficiencies in sociability and social interaction ability/social memory (social preference) compared to normal controls (FIG. 10C).

<9-2> Confirmation of behavioral changes due to overexpression of hippocampal CA1-specific Plcxd3

① Cross maze test (EPM)

An experiment was performed to determine whether there were hyper/anxious behavioral changes 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.

As a result, the distance moved in the open arm of the experimental mouse overexpressing the hippocampal CA1-specific plcxd3 gene was significantly increased compared to the normal control group, and there was no difference in the time spent in both arms (Figure 11A).

② Y-maze test (Y maze)

We conducted an experiment to determine whether hippocampal CA1-specific Plcxd3 overexpression is related to short-term spatial cognitive behavior. For this purpose, experimental mice injected with a control virus or a virus for Plcxd3 overexpression (AAV-CMV-Plc-xd3-eGFP) into the hippocampus CA1 were used.

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

③ Social Behavior Test (SAT)

An experiment was performed 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.

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).

<Example 10>

Working memory analysis following hippocampal CA1-specific inhibition of Plcxd3 expression in Alzheimer's disease animals

A control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) was injected into the hippocampus CA1 of 3-month-old and 6-month-old Alzheimer's disease animals, and then a passive avoidance experiment was performed.

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

<Example 11>

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

After injecting control virus or Plcxd3 expression reduction virus (AAV-CMV-Plc-xd3 shRNA-eGFP) into the hippocampus CA1 of 6-month-old Alzheimer's disease animals, Plcxd3 expression change was analyzed by real-time PCR, and Plcxd3 expression was reduced. It was confirmed that PLCXD3 expression was significantly decreased in Alzheimer's disease animals administered the dragon virus (Figure 13A).

In addition, to confirm changes in amyloid lesions due to inhibition of PLCXD3 expression, a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) was injected into the hippocampus CA1 of 6-month-old Alzheimer's disease animals. As a result of immunofluorescence staining using antibody-6E10, it was confirmed that amyloid plaques were significantly increased in Alzheimer's disease animals administered a virus for reducing Plcxd3 expression (Figure 13B).

Furthermore, as a result of confirming the molecular mechanism related to amyloid lesions in Alzheimer's disease animals due to inhibition of PLCXD3 expression using real-time PCR, the expression of APP, BACE1, and DYRK1A was significantly increased in Alzheimer's disease animals administered a virus for reducing Plcxd3 expression. This could be confirmed (Figure 13C).

<Example 12>

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

To confirm changes in amyloid lesions due to inhibition of PLCXD3 expression, a control virus or a virus for reducing Plcxd3 expression (AAV-CMV-Plc-xd3 shRNA-eGFP) was injected into the hippocampal CA1 of 6-month-old Alzheimer's disease animals, and then antibody Immunofluorescence staining was performed using -Iba-1 and antibody-GFAP.

As a result, it was confirmed that brain inflammation significantly increased in experimental mice with decreased hippocampal CA1-specific plcxd3 expression compared to the normal control group (Figures 14A and 14B).

Through these results, the present inventors found that hippocampus CA1 and amygdala (BLA)-specific Plcxd3 regulates hyperactivity, and that if homeostasis is not maintained in the expression of Plcxd3, that is, when expression is decreased or overexpressed, the above functional deficiency occurs. It was found that it was possible. In addition, through the above results, the present inventors were able to see that PLCXD3 not only affects memory, learning, cognition, and social behavior, but is also related to Alzheimer's disease. In particular, when plcxd3 expression is lowered compared to the normal group, memory , it was found that it can cause learning, cognitive and social behavior disorders and can worsen the symptoms of Alzheimer's disease, causing brain diseases.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (19)

(a) measuring the expression level of PLCXD3 in a biological sample isolated from an individual; and
(b) comparing the PLCXD3 expression level in step (a) with the expression level of the normal control group and determining that the PLCXD3 expression level is lower than the normal control group's expression level, determining that the PLCXD3 expression level is memory, cognitive, or learning disorder;
How to provide information needed to diagnose a memory, cognitive, or learning disability. According to paragraph 1,
A method of providing information necessary for diagnosis of memory, cognitive or learning disorders, wherein the expression level is the mRNA level of the Plcxd3 gene or the PLCXD3 protein expression level. According to paragraph 2,
A method of providing information necessary for the diagnosis of memory, cognitive, or learning disorders, wherein the Plcxd3 gene consists of the base sequence of SEQ ID NO: 1, and the PLCXD3 protein consists of the amino acid sequence of SEQ ID NO: 2. According to paragraph 1,
The memory, cognitive or learning disorders include 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 addiction, epilepsy, epilepsy, hippocampal sclerosis, headache, cerebral senescence, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, cranial nerve disease, cardiovascular disease Information necessary for the diagnosis of memory, cognitive or learning disorders characterized by memory, cognitive or learning disorders due to related diseases, memory loss, radiation exposure, metabolic diseases, hypothyroidism, mild cognitive impairment, cognitive deficits, or attention deficits. How to provide. An animal model with memory, cognitive, or learning disorders in which the Plcxd3 gene is knocked out. According to clause 5,
An animal model with memory, cognitive or learning disorders, wherein the lack of the Plcxd3 gene is induced by the CRISPR/Cas9 system. According to clause 6,
An animal model with memory, cognitive or learning disorders, characterized in that it induces absence of the Plcxd3 gene by targeting the TCAACGAG sequence of the Plcxd3 gene. A method for producing an animal model with memory, cognitive or learning disorders, comprising the step of inducing knock out of the Plcxd3 gene. According to clause 8,
A method for producing an animal model with memory, cognitive or learning disorders, characterized in that the lack of the Plcxd3 gene is induced by the CRISPR/Cas9 system by targeting the TCAACGAG sequence of the Plcxd3 gene. (a) treating the candidate drug in the animal model of paragraph 5; and
(b) comparing the animal model treated with the candidate drug in (a) with the untreated control group to determine whether memory, cognitive, or learning disorders are improved,
Methods for screening treatments for memory, cognitive, or learning disorders. According to clause 10,
Tests for improvement in memory, cognitive, or learning difficulties include the novel tank test, alarm substance test, open field test (OFT), and Y-maze test. ), a method for screening treatments for memory, cognitive or learning disorders, characterized by analysis with the Novel Object Recognition Test (NORT), Passive Avoidance Test (PAT) or Social Interaction Test (SAT). According to clause 10,
Compared to the untreated control group in the animal model treated with the candidate drug,
Increased number of dendritic spines in cortex and hippocampus;
Increased expression of PLC subfamilies β1 and β2;
Increased production of IP3;
Increased expression of learning or memory-related factors nrsn1, grun2b, and creb; or
In the case of increased expression of gng2, gpr183, and ephb2, which are the top target genes of the PLC family, determining the candidate drug as a treatment that can improve, prevent, or treat memory, cognitive, or learning disorders. Characterized by further comprising the step of determining , a method for screening treatments for memory, cognitive, or learning disorders. According to clause 10,
The memory, cognitive or learning disorders include 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 addiction, epilepsy, epilepsy, hippocampal sclerosis, headache, cerebral senescence, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, cranial nerve disease, cardiovascular disease Method for screening for a treatment for memory, cognitive or learning disorders, characterized by memory, cognitive or learning disorders due to related diseases, memory loss, radiation exposure, metabolic diseases, hypothyroidism, mild cognitive impairment, cognitive deficits, or attention deficits. . A pharmaceutical composition for preventing or treating memory, cognitive or learning disorders, comprising PLCXD3, an expression promoter thereof, or an activator thereof as an active ingredient. According to clause 14,
The Plcxd3 gene is composed of the base sequence of SEQ ID NO: 1, and the PLCXD3 protein is composed of the amino acid sequence of SEQ ID NO: 2. A pharmaceutical composition for preventing or treating memory, cognitive, or learning disorders. According to clause 14,
The memory, cognitive or learning disorders include 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 addiction, epilepsy, epilepsy, hippocampal sclerosis, headache, cerebral senescence, dementia, frontotemporal degeneration, tumor, normal pressure hydrocephalus, HIV, cerebrovascular disease, cranial nerve disease, cardiovascular disease Pharmaceuticals for the prevention or treatment of memory, cognitive or learning disorders, characterized by memory, cognitive or learning disorders due to related diseases, memory loss, radiation exposure, metabolic diseases, hypothyroidism, mild cognitive impairment, cognitive deficits or attention deficits. enemy composition. A health functional food for preventing or improving memory, cognitive or learning disorders, containing PLCXD3, its expression promoter or its activator as an active ingredient. Contains PLCXD3, its expression promoter or its activator as an active ingredient, and inhibits the activity of microglia or astrocytes; And characterized by having amyloid plaque inhibitory activity,
Pharmaceutical composition for preventing or treating neuroinflammation, tau-related diseases, or degenerative brain diseases. Contains PLCXD3, its expression promoter or its activator as an active ingredient, and inhibits the activity of microglia or astrocytes; And characterized by having amyloid plaque inhibitory activity,
Health functional food for preventing or improving neuroinflammation, tau-related diseases, or degenerative brain diseases.