Application of PGRN in preparation of product for preventing and treating neuropsychiatric disorder related diseases caused by estrogen deficiency

Abstract

The invention discloses application of PGRN in preparation of products for preventing and treating neuropsychiatric disorder related diseases caused by estrogen deficiency. The invention provides an application of a PGRN protein or a coding gene thereof or a virus expressing the coding gene thereof in preparation of a product with any one of the following functions: 1) preventing or treating or saving neuropsychiatric disorder related diseases caused by estrogen deficiency; 2) preventing or treating perimenopausal anxiety and / or depression symptoms; and 3) relieving anxiety-like behaviors and / or depression-like behaviors caused by estrogen deficiency. The application proves that the PGRN can play a role in protecting neuron damage caused by estrogen deficiency by rescuing CTSD enzyme activity and an autophagy pathway, and prompts that related medicines and measures influencing secretion and content of the PGRN can treat perimenopausal anxiety and depressive symptoms; and a new target and a new idea are provided for clinical treatment and drug development of anxiety and depression symptoms of climacteric women.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of PGRN in the preparation of products for preventing and treating diseases related to neuropsychiatric abnormalities caused by estrogen deficiency. Background Art

[0002] Perimenopause, also known as menopause, refers to the midlife transition state in which women experience reproductive aging. When women go through perimenopause, the levels of various hormones such as estrogen in the body fluctuate, leading not only to changes in reproductive function but also to alterations in the nervous system function. A large number of clinical studies have shown that estrogen deficiency can cause neuropsychiatric symptoms such as hot flashes, sleep disorders, and anxiety and depression. Clinically, hormone replacement therapy (HRT) is often used, that is, by supplementing the body with the lacking estrogen and progesterone through drugs to treat menopausal symptoms including anxiety and depression. However, there are contraindications for estrogen supplementation, such as a history of breast cancer and coronary heart disease. In addition, there is also a "critical period" for estrogen supplementation: starting HRT treatment close to menopause can provide neuroprotective effects, but the opposite is true if treatment starts some time after menopause. Therefore, it is of great significance to find better treatment means for the anxiety and depressive symptoms caused by estrogen reduction.

[0003] Progranulin (PGRN) is a glycosylated secreted protein, also known as proepithelin (PEPI), granulin-epithelin precursor (GEP), acrogranin, or PC cell-derived growth factor (PCDGF). In the brain, PGRN protein is mostly produced by neurons and microglia.

[0004] PGRN is composed of 7.5 tandem repeat motifs. Each motif is different but consists of approximately 55 residues, including 2 (GRN G) or 4 (other granulin) double-cysteine sequences and 4 single-cysteine sequences.

[0005] PGRN protein is encoded by the GRN gene located at 17q21.31. In 2006, the GRN gene was identified as the pathogenic gene for familial frontotemporal dementia with TDP43 deposition. In addition to TDP43 deposition, the lack of PGRN also leads to pathological manifestations such as neuronal loss and lysosomal dysfunction.

[0006] The PGRN protein has two splicing modes, intracellular and extracellular, and lysosomes are the main splicing sites. After the PGRN protein is synthesized, a part directly enters the lysosome for splicing, and the other part is secreted extracellularly through vesicles. The extracellularly secreted protein part is directly spliced extracellularly, while the other part can re-enter the cell through endocytosis with the help of receptors such as sortilin. Sortilin is encoded by the SORT1 gene located at 1p13.3, belongs to the mammalian vacuolar protein sorting 10 (VPS10) family, is highly conserved among different species, and stably performs sorting and transport functions. Sortilin is mainly expressed in neurons, macrophages, hepatocytes, and white blood cells. Almost 90% of the sortilin protein is located in the late Golgi apparatus, binds to various proteins and regulates their localization, secretion, and pre-secretory degradation process in lysosomes; about 10% of sortilin is located on the cell membrane and mediates the entry of various proteins into recycling or into lysosomes for degradation as an endocytic receptor. Summary of the Invention

[0007] The object of the present invention is to provide the application of PGRN in the preparation of products for preventing and treating diseases related to neuropsychiatric abnormalities caused by estrogen deficiency.

[0008] In the first aspect, the present invention provides the application of the PGRN protein or its coding gene or a virus expressing its coding gene in the preparation of a product having any one of the following functions:

[0009] 1) Preventing or treating or rescuing diseases related to neuropsychiatric abnormalities caused by estrogen deficiency;

[0010] 2) Preventing or treating perimenopausal anxiety and / or depressive symptoms;

[0011] 3) Alleviating anxiety-like behavior and / or depressive-like behavior caused by estrogen deficiency.

[0012] The PGRN protein, the amino acid sequence of the murine PGRN protein is NP_032201.3, 09-JAN-2024; the nucleotide sequence of the murine PGRN protein-encoding gene Grn is NC_000077.7, 10-APR-2023; the amino acid sequence of the human PGRN protein is NP_002078.1, 04-JAN-2024; the nucleotide sequence of the human PGRN protein-encoding gene Grn is NC_000017.11, 07-OCT-2023.

[0013] In the second aspect, the present invention provides the application of a substance that increases the content of the PGRN protein or the expression level of the PGRN protein-encoding gene in the preparation of a product having any one of the following functions:

[0014] 1) Prevent or treat neuropsychiatric disorders caused by estrogen deficiency;

[0015] 2) prevent or treat perimenopausal anxiety and / or depression symptoms;

[0016] 3) Alleviate anxiety-like behaviors and / or depression-like behaviors caused by estrogen deficiency.

[0017] In the applications described above, the substance is an exogenous PGRN protein; in the embodiments of the present invention, the exogenous PGRN protein is a recombinant PGRN protein purchased from R&D Systems.

[0018] Alternatively, the substance is a virus that expresses the PGRN protein-encoding gene. In the embodiment of the present invention, the virus expressing the PGRN protein-encoding gene is a Grn overexpression virus (denoted as AAV-Grn), which is provided by Wuhan Shumi Brain Science and Technology Co., Ltd. The virus specifically expresses the Grn gene in neurons through the neuron-specific hSyn promoter, and the virus titer is 1.4 x 10 12 vg / mL.

[0019] In the above, the organism is a human or an animal, the animal is a mammal, and the mammal is further exemplified by a mouse.

[0020] In the above application, the neuropsychiatric disorder-related disease is manifested as at least one of the following: anxiety, depression and negative emotions.

[0021] In the above applications, the estrogen deficiency is oophorectomy or accelerated ovarian failure or mid-life menopause.

[0022] In the application described above, the promoter of the PGRN protein encoding gene in the virus expressing the PGRN protein encoding gene is a hippocampal neuron-specific promoter.

[0023] In a third aspect, the present invention provides the use of proteins that interact with PGRN protein as targets in the development or design of drugs for preventing or treating neuropsychiatric disorders related to estrogen deficiency.

[0024] In the above application, the protein that interacts with the PGRN protein is sortilin. Specifically, the amino acid sequence of the mouse Sortilin protein is AAH56343.1, 15-JUL-2006.

[0025] By searching for molecules induced by estrogen expression, the present invention discovers that the expression of the Grn gene is related to estrogen: after suckling mice consume milk containing sex hormones, the expression of the Grn gene in their hypothalamus increases significantly; on the other hand, the Grn gene is closely related to anxiety and depression: mice lacking the Grn gene exhibit anxiety-like and depression-like behaviors; supplementing the glycosylated secreted protein progranulin (PGRN) encoded by the Grn gene can rescue the anxiety-like behaviors of multiple model mice, suggesting that the PGRN protein may become an effective therapeutic target for anxiety and depressive symptoms caused by estrogen deficiency.

[0026] To explore the role of the PGRN protein in the anxiety-like and depression-like behaviors of estrogen-deficient mice, studies were conducted through experimental means such as behavioral science, molecular biology, and cell biology, and the following results were obtained: (1) There are gender differences in the expression of the Grn gene. The expression of the Grn gene in the hippocampal brain region of 1-month-old female mice representing puberty, 3-month-old female mice representing sexual maturity, and 10-month-old female mice representing middle age is negatively correlated with the anxiety-like and depression-like behaviors of the mice, while the expression of the Grn gene in the cortex of female mice and the cortex and hippocampal brain region of male mice has no obvious correlation with the anxiety-like and depression-like behaviors of the mice; (2) Estrogen affects the expression of the Grn gene: the expression of the Grn gene in the hippocampus of ovariectomized (OVX) mice increases; after treating primary cultured hippocampal neurons with estrogen, the expression of the Grn gene increases; (3) Supplementing the PGRN protein in the hippocampus or specifically overexpressing the Grn gene in hippocampal neurons can improve the negative emotions and memory disorders of multiple estrogen-deficient model mice such as OVX mice, accelerated ovarian failure (AOF) mice, and 10-month-old middle-aged female mice; (4) Reducing the expression of sortilin in hippocampal neurons can weaken the rescue effect of overexpressing the Grn gene on the anxiety-like and depression-like behaviors of OVX mice.

[0027] The experiments of the present invention prove that the expression level of the Grn gene is regulated by estrogen, and supplementing the PGRN protein in the dorsal hippocampus or overexpressing the Grn gene in hippocampal neurons has a significant rescue effect on the anxiety-like and depression-like behaviors of estrogen-deficient mice. The change in the content of PGRN caused by the fluctuation of estrogen levels is very likely to be one of the important reasons for the appearance of anxiety-like and depression-like behaviors in mice. The interaction between sortilin and PGRN in hippocampal neurons partially participates in the prevention and treatment of behavioral abnormalities in mice caused by estrogen deficiency by PGRN. The role and its molecular mechanism of PGRN in preventing and treating anxiety-like and depression-like behaviors in mice caused by estrogen deficiency are explored, suggesting that drugs and measures related to affecting the secretion and content of PGRN can treat perimenopausal anxiety and depressive symptoms, providing new targets and new ideas for the clinical treatment and drug development of anxiety and depressive symptoms in menopausal women. Description of the Drawings

[0028] Figure 1 There are gender differences in the expression of Grn gene in the cortex and hippocampus of mice of different ages; (A) RT-PCR was used to detect the expression of Grn gene in the cortex of mice of different ages and genders. (B) Expression of Grn gene in the cortex of 1-month-old male and female mice. (C) Expression of Grn gene in the cortex of 3-month-old male and female mice. (D) Expression of Grn gene in the cortex of 10-month-old male and female mice. (E) RT-PCR was used to detect the expression of Grn gene in the hippocampus of mice of different ages and genders. (F) Expression of Grn gene in the hippocampus of 1-month-old male and female mice. (G) Expression of Grn gene in the hippocampus of 3-month-old male and female mice. (H) Expression of Grn gene in the hippocampus of 10-month-old male and female mice. Samples of 1-month-old male mice, n = 3; samples of 1-month-old female mice, n = 4; samples of 3-month-old male mice, n = 8; samples of 3-month-old female mice, n = 6; samples of 10-month-old male mice, n = 18; samples of 10-month-old female mice, n = 18. *P < 0.05, **P < 0.01, ***P < 0.001; A&E, two-way ANOVA, Bonferroni post hoc test; B-D&F-H, Male vs. Female, independent samples t-test.

[0029] Figure 2 Regression analysis of Grn gene expression and behavioral results of mice of different ages; (A) Regression analysis of the expression of Grn gene in the cortex and hippocampus of 1-month-old male and female mice (B) and the number of entries into the open arms of mice in the elevated zero maze test. (C) Regression analysis of the expression of Grn gene in the cortex and hippocampus of 3-month-old male and female mice (D) and the time of entry into the open arms of mice in the elevated plus maze test. (E) Regression analysis of the expression level of Grn gene in the cortex and hippocampus of 10-month-old male and female mice (F) and the time of entry into the central area of mice in the second open field test. 1-month-old male mice, n = 9; samples of 1-month-old female mice, n = 10; samples of 3-month-old male mice, n = 8; samples of 3-month-old female mice, n = 6; 10-month-old male mice, n = 18; 10-month-old female mice, n = 18. EZM, elevated zero maze test; EPM, elevated plus maze test; OF’, second open field test. General linear regression.

[0030] Figure 3Gene expression of Grn after estrogen treatment of primary hippocampal neurons; (A) Schematic diagram of cell treatment process, estrogen treatment was given for 48 h on the seventh day after in vitro culture. (B) Gene expression of Grn after treatment with different concentrations. Control group (DMSO), n = 7; low concentration group (10 nmol / L E2), n = 8; high concentration group (100 nmol / L E2), n = 8. (C) Expression of PGRN after treatment with DMSO or 100 nmol / L E2. (D) Statistical results of PGRN protein level. *P<0.05; DMSO vs. 10 nmol / L E2 vs. 100 nmol / L E2, one-way ANOVA, Dunnett's post hoc test; DMSO vs. 100 nmol / L E2, unpaired t test.

[0031] Figure 4 Effect of estrogen receptor on Grn expression; (A) Analysis of the expression correlation between estrogen receptor gene and GRN gene in cortex and hippocampal brain regions of GTEx database using GEPIA; (B) Estrogen receptor antagonists and doses used in the experiment; (C) Gene expression of Grn in primary hippocampal neurons after treatment with different estrogen receptor antagonists. *P<0.05; one-way ANOVA, Dunnett's post hoc test.

[0032] Figure 5 Changes in Grn gene expression in cortex and hippocampus of ovariectomized mice; (A) Grn gene expression in cortex brain region of ovariectomized mice. (B) Grn gene expression in hippocampal brain region of ovariectomized mice. (C) Western Blot experimental result graph of cortex and hippocampus brain regions of OVX mice. (D-E) Statistical graphs of Western Blot results. A&B, sham operation control group (Sham), n = 8; ovariectomized group (OVX), n = 10; D&E, sham operation control group (Sham), n = 4; ovariectomized group (OVX), n = 4. *P<0.05, **P<0.01; independent samples t test.

[0033] Figure 6 Changes in Grn gene expression in cortex and hippocampus of ovariectomized mice after estrogen treatment; (A) Grn gene expression in cortex brain region of ovariectomized mice after estrogen treatment. (B) Grn gene expression in hippocampal brain region of ovariectomized mice after estrogen treatment. Ovariectomized group (OVX), n = 6; estrogen treatment group (OVX+E2), n = 10. *P<0.05; independent samples t test.

[0034] Figure 7 Experimental arrangement for PGRN protein supplementation in the hippocampus of ovariectomized mice.

[0035] Figure 8 Supplementation of hippocampal PGRN protein rescues the performance of ovariectomized mice in emotion-related behavioral tests;

[0036] (A) Total distance of movement, number of entries into the central area (B), and time (C) of mice in each group during the open field test. (D) Number of entries into the open arms and time (E) of mice in each group during the elevated zero maze test. (F) Number of entries into the open arms and time (G) of mice in each group during the elevated plus maze test. (H) Immobility time of mice in each group during the tail suspension test. (I) Immobility time of mice in each group during the forced swim test. Sham-operated group injected with saline (Sham+Saline), n = 9; ovariectomized group injected with saline (OVX+Saline), n = 7; ovariectomized group injected with progranulin protein (OVX+PGRN), n = 7. *P<0.05, **P<0.01, ***P<0.001; one-way ANOVA, Bonferroni post hoc test.

[0037] Figure 9 To detect the increase in the expression level of PGRN protein in hippocampal neurons of mice by injecting viruses.

[0038] Figure 10 Body weight and uterine organ coefficient of ovariectomized mice after injection of Grn overexpression virus into the hippocampus; (A) Experimental arrangement and timeline. (B) Changes in body weight of mice in each group. (C) Uterine atrophy and (D) uterine organ coefficient of mice in each group. Sham-operated group injected with control virus (Sham+AAV-EGFP), n = 12; ovariectomized group injected with control virus (OVX+AAV-EGFP), n = 10; ovariectomized group injected with Grn overexpression virus (OVX+AAV-Grn), n = 13. ***P<0.001; one-way ANOVA, Bonferroni post hoc test.

[0039] Figure 11 Anxiety-like behavior of ovariectomized mice after injection of Grn overexpression virus into the hippocampus; (A) Total distance of movement, number of entries into the central area (B), and time (C) of mice in each group during the open field test. (D) Trajectory map of mice in each group during the elevated zero maze test, and number of entries into the open arms (E) and time (F). (G) Trajectory map of mice in each group during the elevated plus maze test, and number of entries into the open arms (H) and time (I). Sham-operated group injected with control virus (Sham+AAV-EGFP), n = 12; ovariectomized group injected with control virus (OVX+AAV-EGFP), n = 10; ovariectomized group injected with PGRN overexpression virus (OVX+AAV-Grn), n = 13. *P<0.05; one-way ANOVA, Bonferroni post hoc test.

[0040] Figure 12 Depressive-like behaviors in ovariectomized mice after injection of Grn overexpressing virus into the hippocampus; (A) Immobility time of mice in each group during the tail suspension test. (B) Immobility time of mice in each group during the forced swimming test. Sham operation group injected with control virus (Sham+AAV-EGFP), n = 12; Ovariectomized group injected with control virus (OVX+AAV-EGFP), n = 10; Ovariectomized group injected with Grn overexpressing virus (OVX+AAV-Grn), n = 13. *P<0.05, **P<0.01, ***P<0.001; One-way ANOVA, Bonferroni post hoc test.

[0041] Figure 13 To accelerate the body weight and uterine organ coefficient of mice with accelerated ovarian failure after injection of Grn overexpressing virus into the hippocampus;

[0042] (A) Experimental arrangement and timeline. (B) Body weight changes of mice in each group. (C) Uterine atrophy of mice in each group. (D) Uterine organ coefficient of mice in each group. Control group (Sesame oil+AAV-EGFP), n = 14; Accelerated ovarian failure group injected with control virus (4-VCD+AAV-EGFP), n = 12; Accelerated ovarian failure group injected with PGRN overexpressing virus (4-VCD+AAV-Grn), n = 14. ***P<0.001; One-way ANOVA, Bonferroni post hoc test.

[0043] Figure 14 To accelerate the anxiety-like behaviors of mice with accelerated ovarian failure after injection of Grn overexpressing virus into the hippocampus; (A) Total movement distance, number of entries into the central area (B) and time (C) of mice in each group during the open field test. (D) Trajectory maps of mice in each group during the elevated zero maze test, and number of entries into the open arms (E) and time (F). (G) Trajectory maps of mice in each group during the elevated plus maze test, and number of entries into the open arms (H) and time (I). Control group (Sesame oil+AAV-EGFP), n = 14; Accelerated ovarian failure group injected with control virus (4-VCD+AAV-EGFP), n = 12; Accelerated ovarian failure group injected with Grn overexpressing virus (4-VCD+AAV-Grn), n = 14. *P<0.05, **P<0.01, ***P<0.001; One-way ANOVA, Bonferroni post hoc test.

[0044] Figure 15Accelerated depressive-like behaviors in mice with ovarian failure after injection of Grn overexpression virus into the hippocampus; (A) Immobility time of mice in each group in the tail suspension test. (B) Immobility time of mice in each group in the forced swimming test. Control group (Sesame oil+AAV-EGFP), n = 14; Accelerated ovarian failure group injected with control virus (4-VCD+

[0045] AAV-EGFP), n = 12; Accelerated ovarian failure group injected with Grn overexpression virus (4-VCD+AAV-Grn), n = 14. *P<0.05, **P<0.01, ***P<0.001; One-way ANOVA, Bonferroni post hoc test.

[0046] Figure 16 Anxiety-like behaviors in middle-aged mice after injection of Grn overexpression virus into the hippocampus; (A) Total movement distance, number of entries into the central area (B) and time (C) of mice in each group in the open field test. (D) Trajectory maps of mice in each group in the elevated zero maze test, and number of entries into the open arms (E) and time (F). (G) Trajectory maps of mice in each group in the elevated plus maze test, and number of entries into the open arms (H) and time (I). Male mice injected with control virus (Male+AAV-EGFP), n = 13; Female mice injected with control virus (Female+AAV-EGFP), n = 8; Female mice injected with Grn overexpression virus (Female+AAV-Grn), n = 10. *P<0.05, **P<0.01, ***P<0.001; One-way ANOVA, Bonferroni post hoc test.

[0047] Figure 17 Depressive-like behaviors in middle-aged mice after injection of Grn overexpression virus into the hippocampus; (A) Immobility time of mice in each group in the tail suspension test. (B) Immobility time of mice in each group in the forced swimming test. Black represents male mice injected with control virus (Male+AAV-EGFP), n = 13; Female mice injected with control virus (Female+

[0048] AAV-EGFP), n = 8; Female mice injected with Grn overexpression virus (Female+AAV-Grn), n = 10. *P<0.05, **P<0.01, ***P<0.001; One-way ANOVA, Bonferroni post hoc test.

[0049] Figure 18Gene expression of Grn and related receptors in the hippocampus of mice with different estrogen deficiency models; (A) Gene expression of Grn, Psap gene (B), Sort1 gene (C), Lrp1 gene (D) and M6pr gene (E) in the hippocampal region of mice with different estrogen deficiency models. *P<0.05, **P<0.01, ***P<0.001; one-way ANOVA, Bonferroni post hoc test.

[0050] Figure 19 Anxiety-like behaviors of ovariectomized mice after injection of Grn overexpression virus and Sort1 interfering virus; (A) Total movement distance, number of entries into the central area (B) and time (C) of mice in each group during the open field test. (D) Trajectory maps of mice in each group during the elevated zero maze test and elevated plus maze test. (E) Number of entries into the open arms and time (F) of mice in each group during the elevated zero maze test. (G) Number of entries into the open arms and time (H) of mice in each group during the elevated plus maze test. Black represents the sham operation group (Sham+AAV-mCherry), n = 10; ovariectomized group injected with control virus (OVX+AAV-mCherry), n = 10; ovariectomized group injected with Grn overexpression virus (OVX+AAV-Grn+

[0051] AAV-mCherry), n = 12; ovariectomized group injected with Grn overexpression virus and Sort1 interfering expression virus simultaneously (OVX+AAV-Grn+AAV-sh-Sort1), n = 11. *P<0.05, **P<0.01, ***P<0.001; one-way ANOVA, Bonferroni post hoc test.

[0052] Figure 20 Depression-like behaviors of ovariectomized mice after injection of Grn overexpression virus and Sort1 interfering virus; (A) Immobility time of mice in each group during the tail suspension test. (B) Immobility time of mice in each group during the forced swimming test. Black represents the sham operation group (Sham+AAV-mCherry), n = 10; ovariectomized group injected with control virus (OVX+

[0053] AAV-mCherry), n = 10; ovariectomized group injected with Grn overexpression virus (OVX+AAV-Grn+

[0054] AAV-mCherry), n = 12; ovariectomized group injected with Grn overexpression virus and Sort1 interfering expression virus simultaneously (OVX+AAV-Grn+AAV-sh-Sort1), n = 11. *P<0.05, **P<0.01; one-way ANOVA, Bonferroni post hoc test.

[0055] Figure 21 It is the map of the AAV-Grn viral vector PT-3344.

[0056] Figure 22 They are the vector maps of AAV-Grn viral vector PT-5809 before inserting the target fragment (A) and after inserting the target fragment (B). Specific implementation manners

[0057] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0058] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0059] In the following examples, for the PGRN protein, the amino acid sequence of the murine PGRN protein is NP_032201.3, 09-JAN-2024; the nucleotide sequence of the murine PGRN protein-encoding gene Grn is NC_000077.7, 10-APR-2023; the amino acid sequence of the human PGRN protein is NP_002078.1, 04-JAN-2024; the nucleotide sequence of the human PGRN protein-encoding gene GRN is NC_000017.11, 07-OCT-2023.

[0060] The amino acid sequence of the murine Sortilin protein is AAH56343.1, 15-JUL-2006; the nucleotide sequence of the murine Sortilin protein-encoding gene Sort1 is NC_000069, 10-APR-2023.

[0061] The primer sequences used in the following examples are shown in Table 1.

[0062] Table 1 is the primer sequences

[0063]

[0064]

[0065] In the above table, the sequences from top to bottom are Sequence 1 - Sequence 14.

[0066] In the following examples, the animal behavior tests are all recorded by an infrared camera (supplemented with infrared light for illumination), and analyzed using SMART software (version 2.5.21, Panlab, Spain) unless otherwise specified. The mice to be tested are placed in the test room 1 h before each test to adapt to the environment. During the test, the room is kept quiet, and all the equipment used is thoroughly cleaned and dried with 75% ethanol.

[0067] 1) Open field (OF) experiment

[0068] The open field test box is made of plexiglass with dimensions of 100 cm × 100 cm × 50 cm and is equally divided into 4 small grids. The central area is 1 / 4 of the area of each grid. Each test mouse is placed in the center of each grid of the open field and allowed to explore freely for 30 minutes. Analyze and count the total movement distance of the mouse within 30 minutes, as well as the number of times and the staying time the mouse enters the central area in the first 10 minutes.

[0069] 2) Elevated zero maze (EZM) experiment and elevated plus maze (EPM) experiment

[0070] The elevated zero maze consists of a circular ring. There are 20-cm-high walls on both sides of the two 1 / 4 circular closed arms, and the device is 60 cm above the ground.

[0071] The elevated plus maze is a cross-shaped device. Each arm is 30 cm long, and there are 20-cm-high walls on both sides of the closed arms. The device is 60 cm above the ground.

[0072] During the experiment, the mouse is placed facing the open arm and allowed to explore freely for 10 minutes. Analyze the movement trajectory of the mouse, and analyze and count the number of times the mouse enters the open arm and the staying time in the open arm.

[0073] 3) Tail suspension test (TST): Fix the mouse's tail (about 2 cm from the tip of the tail) with medical tape to maintain the hanging state, and the mouse's head is about 15 cm from the tabletop. The experiment lasts for 5 minutes, and analyze and count the immobility time of the mouse.

[0074] 4) Forced swimming test (FST): The forced swimming device is a cylindrical transparent plastic water container filled with about 20 cm deep water at about 22 °C. Place the mouse in the water, and the experiment lasts for 6 minutes and 30 seconds. Analyze and count the immobility time of the mouse in the last 5 minutes.

[0075] 5) Novel location recognition (NLR) experiment

[0076] The novel object recognition experiment uses the above-mentioned open field test box and is carried out on the day after the open field experiment.

[0077] In stage 1, the test mouse is allowed to adapt to the open field environment for 10 minutes and then put back into the original cage.

[0078] In Phase 2, two identical objects are placed at any two adjacent corners of the open field as the familiar positions. Subsequently, the mouse to be tested is placed in, and it freely explores for 10 minutes;

[0079] In Phase 3, after a 10-minute interval, the open field and the objects are thoroughly cleaned with 75% ethanol and dried. Meanwhile, the video of Phase 2 is analyzed using SMART software;

[0080] In Phase 4, the object that the mouse explored less is moved to a new position (novel), and the other object remains in its original position. The object at the new position and the familiar object are placed diagonally. The mouse to be tested is placed in and freely explores for 10 minutes.

[0081] For the video of Phase 4, analyze and count the sniffing time of the mouse to be tested for the familiar object and the object at the new position. Calculate the recognition index according to the following formula:

[0082]

[0083] 6) Novel object recognition (NOR) experiment

[0084] The requirements for the novel object recognition experiment are the same as those for the novel position recognition experiment. The experiment is carried out on the second day after the novel position recognition experiment.

[0085] In Phase 1, the objects used in the novel position recognition experiment are placed at the diagonals of the open field as the familiar objects. Subsequently, the mouse to be tested is placed in, and it freely explores for 10 minutes;

[0086] In Phase 2, after a 10-minute interval, the open field and the objects are thoroughly cleaned with 75% ethanol and dried. Meanwhile, the video of Phase 1 is analyzed using SMART software;

[0087] In Phase 3, the object that the mouse explored less is replaced with a novel object, and the mouse to be tested is placed in and freely explores for 10 minutes.

[0088] For the video of Phase 3, analyze and count the sniffing time of the mouse to be tested for the familiar object and the novel object. Calculate the recognition index according to the following formula:

[0089]

[0090] The mice in the following examples are SPF-grade healthy male and female C57BL / 6 mice (all female mice are not distinguished by estrous cycle). They are purchased from the Experimental Animal Science Department of Peking University Health Science Center and are housed in groups of 4 - 6 in a clean-class IVC independent ventilation system under a 12 / 12 h cycle of day-night alternating light environment, with free access to water and food. All animal experiments comply with the regulations of the Animal Ethics Review Committee of Peking University Health Science Center.

[0091] Example 1: There is a correlation between the expression levels of the Grn gene in the cerebral cortex and hippocampus and the behavior of mice

[0092] To explore whether there is an association between the expression level of the Grn gene in the brain and the behavior of mice, behavioral studies and Grn expression level detection were first performed on the mice

[0093] 1. There are gender differences in the expression of the Grn gene in the cortex and hippocampus of mice

[0094] Analogous to human age, 1-month-old mice were selected to represent human adolescence, 3-month-old mice to represent human adulthood, and 10-month-old mice to represent the middle-aged period of human females experiencing menopause

[0095] The expression of the Grn gene in the cerebral cortex and hippocampus of mice of different ages was detected by RT-PCR as follows

[0096] Total RNA of the hippocampus or cortex tissues of mice of different months of age was extracted according to the Tissue / Cell RNA Rapid Extraction Kit (purchased from Beijing Aidlab Biotechnologies Co., Ltd., product number RN28) as follows

[0097] For the hippocampus or cortex tissues, after adding 300 μL of lysis buffer, the tissues were thoroughly lysed by shaking

[0098] The remaining steps were performed according to the instructions of the Tissue / Cell RNA Rapid Extraction Kit (purchased from Beijing Aidlab Biotechnologies Co., Ltd., product number RN28) to obtain the total RNA solution

[0099] cDNA was obtained by reverse transcription as a template, and then real-time fluorescence quantitative PCR was performed using the primers of Mouse Grn (Table 1). Rpl13a was used as an internal reference (the primers were Mouse Rpl13a, Table 1), and the expression level of the target gene was calculated by the 2-ΔΔCT method; taking the mean value of the expression level of the target gene in the control group as 1, the expression levels of the other groups were standardized to obtain the expression level of the Grn gene. The samples were subjected to 3 repeated experiments, and only the results with stability were included in the statistical analysis

[0100] The results are as Figure 1 shown, as Figure 1 A and Figure 1 E show that with the change of age, the expression of the Grn gene in the cortex and hippocampus of male mice and in the hippocampus of female mice all showed a trend of first increasing and then decreasing; in the cortex of female mice, the expression of the Grn gene was the highest during puberty and maintained a relatively low level after sexual maturity. The expression of the Grn gene in the same brain regions of male and female mice of the same age was analyzed( Figure 1In (B-D, 1F-H), gender differences in Grn gene expression were found in the cortex and hippocampus of 3-month-old mice, as well as in the hippocampus of 10-month-old mice.

[0101] The above results suggest that the Grn gene plays an important role in the gender differences of mouse behavior.

[0102] 2. There is a correlation between Grn gene expression and mouse behavior

[0103] Although there are gender differences in both the mouse behavior results and Grn gene expression, the relationship between Grn gene expression and mouse behavior performance is not clear. To further explore the connection between the two, regression analysis was performed on the mouse behavior test results (elevated zero maze (EZM) experiment, elevated plus maze (EPM) experiment, and stage 1 of the novel location recognition behavior test) of different age mice and the Grn gene expression levels in the mouse cortex and hippocampus.

[0104] Representative results are as Figure 2 shown, where OF’ represents the analysis result of the open field test conducted in stage 1 of the novel location recognition behavior test. It can be seen that for mice of the same age, although the specific behavioral paradigms and indicators showing significant correlations are different, female mice all showed the phenomenon that the higher the Grn gene expression level in the hippocampus, the lower the anxiety level of the mice; compared with the cortex, the Grn gene expression level in the hippocampal brain region is more correlated with the behavioral performance of female mice.

[0105] The above results suggest that the Grn gene in the hippocampal brain region is very likely to be involved in regulating the behavioral performance related to the emotions of female mice.

[0106] II. Regulatory effect of estrogen on Grn gene expression

[0107] It has been found in a study that after giving neonatal rats milk containing sex hormones, the granulin content in their hypothalamus increases. To explore whether PGRN is involved in the rescue effect of estrogen replacement therapy on the behavior of ovariectomized mice, whether estrogen affects Grn gene expression and its mechanism were explored. Through in vivo animal experiments and in vitro cell experiments, the Grn gene expression levels under different estrogen stimulations were detected, and the results showed that estrogen promoted Grn gene expression.

[0108] 1. Estrogen treatment of primary hippocampal neurons induces Grn gene expression

[0109] To explore whether estrogen affects Grn gene expression, estrogen stimulation was first given to primary cultured hippocampal neurons, and the changes in Grn gene expression levels were detected by RT-PCR.

[0110] Hippocampal neurons were isolated from ICR pregnant mice (E16.5 days) purchased from the Experimental Animal Science Department of Peking University Health Science Center and cultured in complete neuronal medium (Neurobasal phenol red-free medium produced by Gibco, catalog number 12348017 + B-27 produced by Gibco, catalog number A3582801 + GlutaMAX produced by Gibco, catalog number 35050061, 100:2:1) containing cytosine arabinoside (final concentration 10 μmol / L). On the seventh day of primary hippocampal neuron culture, the pre-prepared estrogen solution working solution (1:1000, denoted as 10 nmol / L E2 or 100 nmol / L E2 in the figure) or an equal volume of DMSO (denoted as DMSO in the figure) was added to the medium, and markings were made. The medium was gently shaken to disperse the drug evenly, and then the culture dish was returned to the incubator. After 48 h, the culture dish was taken out of the incubator, the medium was aspirated and discarded, and RNA extraction (Tissue / Cell RNA Rapid Extraction Kit) or protein extraction was performed according to experimental requirements.

[0111] The above estrogen solution working solution was prepared as follows:

[0112] β-Estradiol was purchased from Sigma-Aldrich, catalog number E8875;

[0113] Dimethyl sulfoxide (DMSO) was purchased from Yeasen Biotech Co., Ltd., catalog number 60313ES60;

[0114] 2.7238 mg of β-estradiol was dissolved in DMSO and made up to 10 mL to prepare a stock solution of 1000 μmol / L. The stock solution was serially diluted to obtain working solutions with concentrations of 100 μmol / L and 10 μmol / L, and stored in the dark.

[0115] The experimental procedure was as Figure 3 shown in A. Primary hippocampal neurons were cultured from the hippocampi of E16.5 fetal mice. The enzymes and medium used in this experiment were both phenol red-free.

[0116] The RNA of cells after estrogen stimulation was extracted according to the previous method to detect the expression level of the Grn gene.

[0117] The RT-PCR results ( Figure 3 B) showed that after estrogen treatment, the level of Grn mRNA in primary hippocampal neurons increased significantly;

[0118] The proteins of cells after estrogen stimulation were extracted according to the previous method and subjected to Western Blot detection. The antibodies are shown in Tables 2 and 3 below:

[0119] Table 2 shows antibody information

[0120]

[0121] Table 3 shows antibody information

[0122]

[0123] Western Blot results ( Figure 3 C, D) showed that after estrogen treatment, the PGRN protein level also increased significantly.

[0124] These results suggest that estrogen can induce the expression of PGRN protein by the Grn gene.

[0125] 2. Estrogen induces Grn gene expression through ERα and GPER receptors, but is not dependent on the ERβ receptor

[0126] Estrogen exerts its effects through its receptors. Currently, the estrogen receptors that have been studied more are ERα (gene: ESR1), ERβ (gene: ESR2), and GPER (gene: GPER1). To explore whether different estrogen receptors have different effects on Grn gene expression, the relevant data in the GTEx database (https: / / www.gtexportal.org / home / index.html) of healthy human tissue samples were preliminarily analyzed using the GEPIA website, and it was found that in human samples, the expression of GRN gene in the cortex and hippocampus was correlated with the expression of all three estrogen receptors ( Figure 4 A).

[0127] On the seventh day of culturing the primary hippocampal neurons of mice obtained in 1 above, the pre-prepared antagonist working solution (1:1000) or an equal volume of DMSO was added to the culture medium, and marks were made. The culture medium was gently shaken to disperse the drug evenly, and then the culture dish was placed back into the incubator. After 1 h, a 100 μmol / L estrogen solution working solution (E2, 1:1000) (the estrogen receptor antagonists and doses used in the experiment are as Figure 4 shown in B) was added to the culture medium. The culture medium was gently shaken to disperse the drug evenly, and then the culture dish was placed back into the incubator. After 48 h, the culture medium was aspirated and discarded, and the samples were retained.

[0128] The above estrogen receptor antagonists and agonists are shown in Table 4 below:

[0129] Table 4 shows estrogen receptor antagonist and agonist information

[0130]

[0131] The above drugs were dissolved in DMSO and fixed to volume to prepare a 1000 μmol / L stock solution. The stock solution was serially diluted to obtain working solutions with concentrations of 100 μmol / L and 10 μmol / L, and stored in the dark.

[0132] RNA was extracted from the sample cells, and the expression of Grn gene was detected by RT-PCR according to the previous method.

[0133] The results are as follows Figure 4 As shown in Figure C, compared with the control (denoted as E2 + DMSO), administration of an ERα antagonist (denoted as E2 + MPP) and a GPER antagonist (denoted as E2 + G-15) attenuated the estrogen-stimulating effect on Grn gene expression, whereas an ERβ antagonist (denoted as E2 + PHTPP) had no effect on the effect of estrogen on Grn gene expression. This is consistent with the results from the GTEx database for the hippocampus, which showed a relatively weak correlation between ESR2 and GRN expression (R = 0.2).

[0134] These results suggest that estrogen induces Grn gene expression through ERα and GPER receptors, but is independent of ERβ receptors. The specific regulatory mechanism remains to be explored.

[0135] 3. Compensatory increase in Grn gene expression in the brain of ovariectomized mice

[0136] Construction of ovariectomy (OVX) mouse model:

[0137] Nine-week-old female C57BL / 6 mice were used for model establishment.

[0138] (1) 8-week-old female C57BL / 6 mice were purchased and placed in a clean-grade IVC independent ventilation system for 1 week of adaptive breeding;

[0139] (2) Anesthetics, sutures, and surgical instruments should be prepared one day before surgery, and all instruments should be sterilized using high pressure to reduce the risk of infection;

[0140] (3) Anesthesia was performed by intraperitoneal injection of 0.5% sodium pentobarbital injection;

[0141] (4) The mouse was fixed in a supine position, and the abdominal hair of the mouse was carefully trimmed with curved-tip surgical scissors and wiped with alcohol cotton for disinfection;

[0142] (5) Use surgical scissors to make a 1-1.5 cm opening in the middle of the abdomen, 3-4 cm from the vaginal opening. After opening the abdominal cavity, white adipose tissue can be seen under the stereoscope. The ovaries can be seen by pushing aside the adipose tissue.

[0143] (6) Ligation of fat and fallopian tubes, followed by separation and removal of the ovaries, and return of the uterine horns, followed by suturing;

[0144] (7) Immediately after the operation, penicillin was intramuscularly injected into the mice. The mice were placed on a heating pad until they woke up, and then put back into their original cages to obtain ovariectomized mice.

[0145] Sham operation group (sham group): The surgical procedure for the mice was the same as that for the above-mentioned ovariectomized mice, but the fallopian tubes were not ligated and the ovaries were not removed.

[0146] The relationship between estrogen and the expression of Grn gene in ovariectomized mice under in vivo conditions was explored as follows:

[0147] RNA was extracted from the cerebral cortex and hippocampal brain regions of mice before ovariectomy (represented by the sham operation group Sham) and ovariectomized mice (OVX), and RT-PCR was performed to detect the Grn gene.

[0148] The RT-PCR results showed that there was no significant change in the mRNA content of the Grn gene in the cerebral cortex of mice before and after ovariectomy ( Figure 5 A), but the mRNA content of the Grn gene in the hippocampal brain region of ovariectomized mice was higher than that of the sham operation group ( Figure 5 B).

[0149] Proteins were extracted from the cerebral cortex and hippocampal brain regions of mice before ovariectomy (represented by the sham operation group Sham) and ovariectomized mice (OVX), and Western blot was used to detect the expression of PGRN protein.

[0150] The results showed that as Figure 5 C-E, the content of PGRN protein in the cerebral cortex ( Figure 5 D) and hippocampus ( Figure 5 E) of ovariectomized mice was higher than that of the sham operation group.

[0151] The above results suggest that the expression of Grn gene in the cerebral cortex and hippocampal brain regions of ovariectomized mice increases compensatorily.

[0152] 4. Estrogen replacement therapy promotes the expression of Grn gene in the brain of ovariectomized mice

[0153] To further explore whether estrogen affects the expression of Grn gene, the expression of Grn gene in the cerebral cortex and hippocampal brain regions of ovariectomized mice after estrogen replacement therapy was detected as follows:

[0154] Estrogen replacement therapy for mice: The Co60 irradiated mouse breeding feed and the estrogen-added customized feed were provided by Spf (Beijing) Biotechnology Co., Ltd.

[0155] β-Estradiol was purchased from Sigma-Aldrich, catalog number E8875; it was incorporated into the feed at a concentration of 0.15 mg / kg and then pressed into shape to obtain estrogen feed.

[0156] The ovariectomized mice obtained above were fed estrogen feed since the ovariectomy surgery, allowed to eat and drink freely until sampling, and were denoted as the estrogen supplementation group (denoted as OVX+E2 in the figure).

[0157] Feeding normal feed served as the non-supplemented group (denoted as OVX in the figure).

[0158] RNA was extracted from the cortex and hippocampus of ovariectomized mice in the estrogen supplementation group and the non-supplemented group respectively, and RT-PCR was used to detect the expression level of the Grn gene after estrogen supplementation.

[0159] The results were as Figure 6 shown. The RT-PCR results showed that the content of Grn mRNA in the cortex brain region ( Figure 6 A) of ovariectomized mice in the estrogen supplementation group was higher than that in the non-supplemented group; although the content of Grn mRNA in the hippocampus brain region ( Figure 6 B) had an increasing trend, it was not significant (P = 0.107).

[0160] The above results indicate that estrogen promotes the expression of the Grn gene.

[0161] Example 2. Application of PGRN in rescuing the anxious-like and depressive-like behaviors of ovariectomized mice, accelerating the ovarian failure model mice and perimenopausal model mice

[0162] (I) Application of PGRN in rescuing the anxious-like and depressive-like behaviors of ovariectomized mice

[0163] I. Application of PGRN in rescuing the anxious-like and depressive-like behaviors of ovariectomized mice

[0164] 1. Method for locally administering PGRN to the hippocampus to rescue the behavioral changes of ovariectomized mice

[0165] The previous results showed that the expression levels of the Grn gene in the cortex and hippocampus of female mice were correlated with the anxiety and depression levels of the mice, and the changes in estrogen in the mice were accompanied by the changes in the expression of the Grn gene in the cortex and hippocampus, suggesting that the Grn gene may be involved in affecting the behavioral performance of ovariectomized mice. Since the expression level of the Grn gene in the hippocampus of female mice is correlated with the anxiety level and depression level of the mice, the hippocampus brain region was selected for the rescue experiment in the subsequent experiments.

[0166] PGRN is a secreted protein. Therefore, the cannula administration method was used to explore whether direct supplementation of PGRN protein could rescue the abnormal behavioral manifestations in estrogen-deficient mice represented by ovariectomized mice.

[0167] The above PGRN administration method is as follows:

[0168] 1) Cannula implantation and administration in the hippocampal brain region

[0169] Cannula size: The injection cannula was customized and provided by Changsha Maiyue Biotechnology Co., Ltd. The cannula size is as follows:

[0170] P 3.5mm; L1 1.8mm; L3 0.5mm; L4 0mm.

[0171] 2) Cannula implantation

[0172] Use 0.5% sodium pentobarbital injection to deeply anesthetize the mice by intraperitoneal injection. After shaving the mice with a curved-tip surgical scissors, fix the mice on a stereotaxic apparatus. Apply an appropriate amount of erythromycin ointment to the eyes of the mice. Wipe and disinfect the head skin with 75% alcohol, and cut open the scalp along the midline to expose the skull. Wipe off the tissue on the surface of the skull with a clean cotton swab, clearly expose the anterior and posterior fontanelles and adjust them to the same horizontal plane (the deviation of the M / L axis is less than 0.02mm). Taking the anterior fontanelle coordinates as the zero point, select the injection coordinates (A / P - 2.20mm, M / L ± 1.75mm, D / V - 1.80mm), and gently drill through the skull with a cranial drill. Use a bone drill (Φ = 0.8mm) to drill two holes symmetrically around the implantation position and implant the cranial nails (pre-soaked in 75% alcohol in advance). Use a gripper to hold the cannula base to ensure that the two injection inner tubes are at the same height and the same front-back position. Place the cannula according to the target coordinates (A / P - 2.20mm, M / L ± 1.75mm, D / V - 1.80mm) using the stereotaxic apparatus, and then fix the cannula with dental cement. After the first layer of dental cement solidifies, perform a second layer of dental cement sealing and reinforcement. After the fixation is completed, remove the mice from the stereotaxic apparatus, insert the catheter, and tighten the catheter cap. Immediately inject penicillin intramuscularly after the operation, place the mice on a heating pad until they wake up, and then put them back into the original cage.

[0173] 3) Brain administration

[0174] Recombinant PGRN protein solution: The recombinant PGRN protein was purchased from R&D Systems, catalog number 2557-PG-050; it was prepared into a stock solution with a final concentration of 500 μg / mL with 1×PBS, filtered and sterilized, and stored at -20°C after aliquoting.

[0175] The administration process is as follows:

[0176] (1) Wrap the mouse with a clean and odorless soft towel in advance and grasp it multiple times to make it adapt.

[0177] (2) Connect the injection inner tube, PE tube, and microsyringe in sequence. Tighten the nut sleeve on the PE tube, hold the microsyringe on the microinjection pump, and fill the system with mineral oil.

[0178] (3) After checking the airtightness of the system, aspirate 400 nL of the recombinant PGRN protein solution into each injection tube, and adjust the position for standby.

[0179] (4) Wrap the mouse with a soft towel. When it stops moving, gently unscrew the catheter cap, pull out the catheter, then insert the injection inner tube and tighten the locking nut. Place the mouse in a new clean cage to allow it to move freely.

[0180] (5) Push the drug at a speed of 80 nL / min. After the injection, leave the injection inner tube in place for 5 minutes. Then wrap and fix the mouse with a soft towel again, slowly pull out the injection inner tube and reinsert the catheter, and tighten the catheter cap. Then place the mouse back into the original cage.

[0181] The experimental arrangement is as Figure 7 . Six-week-old female mice were raised for one week for adaptation, and then underwent bilateral hippocampal cannula implantation surgery. After the mice recovered for one week, 400 nL of the recombinant PGRN protein solution (denoted as PGRN in the figure; 500 μg / mL) was injected unilaterally into the hippocampus for the first time, and the control mice were injected with an equal volume of normal saline (denoted as saline in the figure). Then, the PGRN protein was supplemented every two weeks. When the mice were 9 weeks old, ovariectomy was performed according to the method of the ovariectomy (OVX) mouse model in Example 1. After one month of recovery, the behavioral experiments were started. There were 3 groups: sham + saline (ovariectomy sham operation group injected with normal saline), OVX + saline (ovariectomy group injected with normal saline), OVX + PGRN (ovariectomy group injected with PGRN).

[0182] 2. Supplementing PGRN protein in the hippocampus rescues the anxiety-like and depression-like behaviors of ovariectomized mice

[0183] To investigate whether directly supplementing PGRN protein has a rescue effect on the anxiety-like and depression-like behaviors of ovariectomized mice, the above-mentioned mice in each group were subjected to relevant behavioral tests reflecting the emotions of the mice, including open field, elevated zero maze, elevated plus maze, tail suspension, and forced swimming.

[0184] The results are as Figure 8As shown, in the open field test, the locomotor distance of ovariectomized mice decreased, and the number of times and the time spent entering the central area were significantly reduced, indicating that ovariectomized mice exhibited obvious anxiety-like behaviors. After supplementing PGRN protein in the hippocampal brain region, the number of times and the time spent by the mice entering the central area increased significantly ( Figure 8 A-C).

[0185] In the elevated zero maze experiment ( Figure 8 D,E), consistent with the previous results, compared with sham-operated mice, the anxiety level of ovariectomized mice was significantly increased, manifested as a significant decrease in the number of times and the time spent entering the open arms. After injecting PGRN protein, the number of times and the time spent by ovariectomized mice entering the open arms increased significantly, indicating that the anxiety-like behaviors of the mice were rescued.

[0186] In the elevated plus maze experiment ( Figure 8 F,G), consistent with the previous results, compared with sham-operated mice, the time spent by ovariectomized mice entering the open arms was significantly reduced; however, supplementing PGRN protein did not affect the behavioral performance of ovariectomized mice in the elevated plus maze experiment.

[0187] In the tail suspension test ( Figure 8 H) reflecting the depressive level of mice, there was no significant difference in the immobile time among the groups of mice. The reason might be that the part of the mouse with a head catheter affected the movement of the mouse. But in the forced swimming test ( Figure 8 I), consistent with the previous results, the immobile time of ovariectomized mice increased significantly, indicating that the depressive level of ovariectomized mice increased; after injecting PGRN protein, the immobile time of the mice decreased significantly, indicating that the depressive-like behaviors of the mice were alleviated.

[0188] The above results indicate that supplementing PGRN protein in the hippocampus can alleviate the anxiety-like and depressive-like behaviors of ovariectomized mice.

[0189] II. Specific overexpression of the Grn gene in hippocampal neurons alleviates anxiety-like and depressive-like behaviors in estrogen-deficient model mice

[0190] 1. Specific overexpression of the Grn gene in hippocampal neurons alleviates anxiety-like and depressive-like behaviors in estrogen-deficient model mice

[0191] Since the content of PGRN in neurons mainly affects the overall content of PGRN in the brain tissue, combined with the change in the content of PGRN in the brains of ovariectomized mice, neurons were selected for further study.

[0192] 1) Increasing the expression level of the Grn gene in mouse hippocampal neurons by injecting viruses

[0193] (1). Construction and synthesis of adeno-associated virus (AAV) vectors

[0194] The AAV virus serotype was AAV 2 / 9, and plasmid construction, synthesis, and virus packaging services were provided by Wuhan Shumi Brain Science Technology Co., Ltd. (Table 5). The company synthesized the Grn overexpression virus (denoted as AAV-Grn) and the control virus (denoted as AAV-EGFP).

[0195] Grn overexpression virus (denoted as AAV-Grn) specifically expresses the Grn gene in neurons through the neuron-specific hSyn promoter, with a viral titer of 1.4 x 10 12 vg / mL.

[0196] The specific virus construction is as follows:

[0197] AAV-Grn viral vector and AAV-sh-Sort1 viral vector were both constructed and provided by Wuhan Shumi Brain Science Technology Co., Ltd.

[0198] The above-mentioned AAV-Grn viral vector PT-3344 contains the Grn gene (NM_008175.5), and its vector is shown in FIG. Figure 21 As shown, this plasmid expresses the Grn gene.

[0199] The above-mentioned AAV-sh-Sort1 viral vector PT-5809 (such as Figure 22 B) is to insert shRNA (Sort1) into PT-4614 (as shown in FIG Figure 22 The vector obtained by adding BbsI (shown in A).

[0200] The target gene-specific sequence was selected using the Sort1 gene (NM_001271599.1) as a template, restriction sites were added at both ends, and forward and reverse mixed annealing was performed using the following 5809-F and 5809-R primers to obtain shRNA (Sort1);

[0201] 5809-F:

[0202] gcgAGGACATGGTCTTCATGCATGTTAGTGAAGCCACAGATGTAACATGCATGAAGACCATGTCC

[0203] 5809-R:

[0204] gcaCGGACATGGTCTTCATGCATGTTACATCTGTGGCTTCACTAACATGCATGAAGACCATGTCC

[0205] Transfect pHelper (carrying adenovirus-derived genes) (AF369965, 18-DEC-2018), pAAV-RC (carrying AAV replication and capsid genes) plasmid (AF369963, 17-DEC-2018) and each of the above viral vectors into AAV-293 cells at a mass ratio of 1:1:1, collect the supernatant, and purify to obtain Grn overexpression virus (denoted as AAV-Grn) and mouse Sort1 gene interference expression virus (denoted as AAV-sh-Sort1).

[0206] Table 5 shows the virus information

[0207]

[0208] (2) Stereotaxic injection into the brain

[0209] Use 0.5% sodium pentobarbital injection to deeply anesthetize mice by intraperitoneal injection. After shaving the mice with a curved-tip surgical scissors, fix the mice on a stereotaxic apparatus. Apply an appropriate amount of erythromycin ointment to the eyes of the mice. Wipe and disinfect the head skin with 75% alcohol, and cut open the scalp along the midline to expose the skull. Wipe off the tissue on the surface of the skull with a clean cotton swab, clearly expose the anterior and posterior fontanelles and adjust them to the same horizontal plane (the deviation of the M / L axis is less than 0.02 mm). Taking the anterior fontanelle coordinates as the zero point, select the injection coordinates (A / P - 2.20 mm, M / L ± 1.75 mm, D / V - 1.80 mm), and gently drill through the skull with a cranial drill. Use a glass electrode filled with mineral oil (Sigma-Aldrich) to aspirate 0.3 μL of the virus, vertically insert the glass electrode according to the injection coordinates, and use a Nanoliter2000 injection pump to inject the virus (0.06 μL / min). After the injection, leave it for 5 minutes, and then slowly pull out the glass electrode. Ensure that the glass electrode is unobstructed before and after injection. After suturing, remove the mice from the stereotaxic apparatus, immediately inject penicillin intramuscularly, place the mice on a heating pad until they wake up, and then put them back into the original cage.

[0210] The experimental arrangement is as Figure 10 shown in A. Purchase 6-week-old female mice, adaptively raise them in a new environment for one week, and then perform stereotaxic injection into the mouse brain, injecting the Grn overexpression virus AAV-Grn and the control virus AAV-EGFP into the bilateral hippocampi of the mice respectively.

[0211] Extract the proteins in the hippocampi of the mice 5 weeks after injecting the virus, and perform Western blot to detect the expression of PGRN protein.

[0212] The results are as Figure 9 shown. It can be seen that compared with the mice injected with the control virus AAV-EGFP, the hippocampi of the mice injected with the Grn overexpression virus AAV-Grn express PGRN protein.

[0213] Two weeks after the mice recovered (the day of virus injection was recorded as the first day of recovery), ovariectomy was performed for modeling, and behavioral tests were conducted one month after ovariectomy.

[0214] There were 3 groups as follows: sham+AAV-EGFP, OVX+AAV-EGFP, OVX+AAV-Grn.

[0215] 2. Detection of the alleviation of behavioral disorders in ovariectomized mice by specific overexpression of Grn in hippocampal neurons

[0216] Comparing the behavioral performance of mice after administration of PGRN protein, it was suggested that different PGRN supplementation methods would also affect its rescue effect on the behavior of ovariectomized mice; however, supplementing PGRN in different ways could effectively rescue the anxious-like behavior and depressive-like behavior of ovariectomized mice.

[0217] 1) Specific overexpression of Grn in hippocampal neurons does not affect uterine atrophy in ovariectomized mice

[0218] The body weights and uteri of the 3 groups of mice sham+AAV-EGFP, OVX+AAV-EGFP, and OVX+AAV-Grn obtained in 1 above were detected.

[0219] The results were as follows: Compared with sham+AAV-EGFP, after ovariectomy for modeling, the body weight of OVX+AAV-EGFP mice showed an increasing trend (P = 0.107), while after injection of the Grn overexpression virus into the hippocampus, the increased body weight of OVX+AAV-Grn ovariectomized mice was alleviated (P = 0.186)( Figure 10 B). Compared with the sham operation group (sham+AAV-EGFP), uterine atrophy occurred in OVX+AAV-EGFP ovariectomized mice, and injection of the Grn overexpression virus into the hippocampus had no obvious effect on uterine atrophy in OVX+AAV-Grn ovariectomized mice( Figure 10 C and 10D).

[0220] The above results suggested that specific overexpression of Grn in hippocampal neurons could partially restore the increased body weight of ovariectomized mice, but had no obvious effect on uterine atrophy in mice; at the same time, it indicated that the ovariectomy modeling was successful in the ovariectomy group injected with the control virus (OVX+AAV-EGFP) and the ovariectomy group injected with the Grn overexpression virus (OVX+AAV-Grn).

[0221] 2) Specific overexpression of Grn in hippocampal neurons alleviates the anxious-like behavior of ovariectomized mice

[0222] To investigate the effect of increasing the expression level of Grn gene in mouse hippocampal neurons on the behavior of ovariectomized mice, the three groups of mice obtained above 1 were first subjected to open field, elevated zero maze, and elevated plus maze behavioral tests to reflect the anxiety-like level of the mice.

[0223] The results are as Figure 11 shown in A - C. In the open field test, the total movement distance of ovariectomized mice injected with the overexpression virus (OVX+AAV-Grn) was higher than that of ovariectomized mice injected with the control virus (OVX+AAV-EGFP), suggesting an increase in the activity of mice after injection of the overexpression virus. The number of times ovariectomized mice entered the central area was significantly reduced, and the number of times ovariectomized mice entered the central area showed an increasing trend after injection of the overexpression virus. There was no significant change in the time of each group of mice entering the central area.

[0224] In the elevated zero maze test ( Figure 11 D - F), there was no significant difference in the number of times ovariectomized mice (OVX+AAV-EGFP) and sham-operated mice (Sham+AAV-EGFP) entered the open arms, but the time of ovariectomized mice entering the open arms showed a decreasing trend. The number of times and the time of ovariectomized mice injected with the overexpression virus (OVX+AAV-Grn) entering the open arms increased significantly, indicating that the anxiety level of ovariectomized mice was alleviated.

[0225] In the elevated plus maze test with a higher stress level ( Figure 11 G - I), ovariectomized mice (OVX+AAV-EGFP) showed anxiety-like behaviors, manifested as a significant decrease in the number of times and the time of entering the open arms; while the anxiety level of ovariectomized mice injected with the Grn overexpression virus (OVX+AAV-Grn) was significantly alleviated, manifested as an increase in the time and the number of times of entering the open arms.

[0226] The above results indicate that the anxiety level of ovariectomized mice shows an increasing trend, and specific overexpression of Grn in hippocampal neurons can alleviate the anxiety-like behaviors of ovariectomized mice.

[0227] 3) Specific overexpression of Grn in hippocampal neurons alleviates the depressive-like phenotype of ovariectomized mice

[0228] To investigate the effect of increasing the expression level of Grn gene in mouse hippocampal neurons on the depressive-like behaviors of ovariectomized mice, the three groups of mice injected with different viruses obtained above 1 were subjected to tail suspension and forced swimming behavioral tests.

[0229] The results are as Figure 12 shown. Compared with sham-operated mice (Sham+AAV-EGFP), ovariectomized mice (OVX+AAV-EGFP) in the tail suspension ( Figure 12 A) and forced swimming ( Figure 12B) During the tests, the immobility time significantly increased in all cases, indicating an elevated level of depressive-like behavior in the ovariectomized mice. In the ovariectomized mice injected with Grn overexpressing virus into the hippocampus (OVX+AAV-Grn), the immobility time significantly decreased, indicating a remission of the depressive state in the mice.

[0230] The above results indicate that the depressive level of ovariectomized mice is significantly elevated, and specific overexpression of Grn in hippocampal neurons can alleviate the depressive-like behavior of ovariectomized mice.

[0231] (II) Application of PGRN in alleviating the anxious-like and depressive-like behaviors of mice with accelerated ovarian failure

[0232] Since the ovariectomized mice are surgically modeled, the estrogen level in the body drops rapidly after the operation, which is different from the slow change process of estrogen in human females during perimenopause. Therefore, a 15-day injection of 4-VCD was used to establish a model of accelerated ovarian failure, and the expression level of Grn gene in hippocampal neurons of mice was increased by injecting virus.

[0233] 1. Construction of a mouse model of accelerated ovarian failure (AOF)

[0234] Five-week-old female C57BL / 6 mice were used for modeling, and the specific modeling method is as follows:

[0235] 1) Preparation of main reagents and solutions

[0236] (1) Reagents used:

[0237] Sesame oil, purchased from SantaCruz Company, product number sc-215848;

[0238] 4-Vinylcyclohexene diepoxide (4-VCD) (CAS#106-87-6), purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., product number BD100534;

[0239] (2) Preparation of drugs:

[0240] In a fume hood, 587 μL of 4-VCD (ρ = 1.09 g / mL) was mixed with sesame oil and made up to 10 mL. Gently invert up and down to mix evenly, seal with a sealing film, and store at 4°C for a maximum of 7 days.

[0241] 2). Modeling

[0242] The mice were weighed daily and intraperitoneally injected with 4-VCD solution (160 mg / kg) balanced to room temperature or an equal volume of sesame oil (the injection volume was converted to 2.5 mL / kg). The injection for modeling was carried out at the same time every day for 15 consecutive days. Note that the injection site was changed daily to avoid injury, and mice with accelerated ovarian failure were obtained.

[0243] 2. Specific overexpression of Grn in hippocampal neurons alleviates the behavioral disorders in mice with accelerated ovarian failure

[0244] 1) Specific overexpression of Grn in hippocampal neurons has no obvious alleviating effect on uterine atrophy in mice with accelerated ovarian failure

[0245] The experimental arrangement was as Figure 13 shown in A. Female mice at 5 weeks of age were purchased. While being adaptively raised, corresponding volumes of the solvent sesame oil (recorded as sesame oil) or the 4-VCD drug were injected intraperitoneally every day for 15 days of modeling. When the mice were 9 weeks old, stereotaxic injection into the mouse brain was performed, and the Grn overexpression virus AAV-Grn and the control virus AAV-EGFP were respectively injected into the bilateral hippocampi of the mice according to the method in (2) of (1) above. After the mice recovered for one month, behavioral tests were started. There were 3 groups in total: sesame oil + AAV-EGFP, 4-VCD + AAV-EGFP, 4-VCD + AAV-Grn.

[0246] When the behavioral tests were started, the mice were in the peri-ovarian failure (peri-AOF) stage similar to the perimenopausal period in humans, and the estrogen levels in their bodies fluctuated.

[0247] Different from the ovariectomized mice, after the establishment of the accelerated ovarian failure model, the body weight of the mice did not change significantly ( Figure 13 B). However, similar to the ovariectomized mice, compared with the control group (sesame oil + AAV-EGFP), the accelerated ovarian failure mice (4-VCD + AAV-EGFP) also showed uterine atrophy; the overexpression of Grn virus injection into the hippocampus of the accelerated ovarian failure mice (4-VCD + AAV-Grn) had no obvious effect on uterine atrophy ( Figure 13 C and 13D).

[0248] The above results suggest that specific overexpression of Grn in hippocampal neurons neither affects the body weight of mice with accelerated ovarian failure nor affects the uterine atrophy of mice; at the same time, it indicates that the accelerated ovarian failure model group mice injected with the control virus (4-VCD + AAV-EGFP) and the accelerated ovarian failure model group mice injected with the Grn overexpression virus (4-VCD + AAV-Grn) were successfully modeled.

[0249] 2) Specific overexpression of Grn in hippocampal neurons alleviates anxiety-like behavior in mice with accelerated ovarian failure

[0250] To investigate the effect of increasing the expression level of Grn gene in mouse hippocampal neurons on the behavior of mice with accelerated ovarian failure, anxiety-like behavior tests were first performed on the three groups of mice sesame oil+AAV-EGFP, 4-VCD+AAV-EGFP, and 4-VCD+AAV-Grn obtained in 1) above.

[0251] The results are as Figure 14 shown in A-C. In the open field test, there was no significant difference in the total movement distance of mice in each group, indicating that there was no significant change in the activity of mice in each group; there were no significant differences in the number of times and time of mice entering the central area in each group.

[0252] In the elevated zero maze test ( Figure 14 D-F), the number of times and time of accelerated ovarian failure mice (4-VCD+AAV-EGFP) entering the open arms decreased significantly, and the number of times and time of accelerated ovarian failure mice (4-VCD+AAV-Grn) entering the open arms increased significantly after hippocampal injection of Grn overexpressing virus.

[0253] In the elevated plus maze test ( Figure 14 G-I), accelerated ovarian failure mice (4-VCD+AAV-EGFP) showed obvious anxiety-like behavior, manifested as a significant decrease in the number of times and time of entering the open arms; the number of times and time of accelerated ovarian failure mice (4-VCD+AAV-Grn) entering the open arms increased significantly after hippocampal injection of Grn overexpressing virus.

[0254] The above results indicate that mice in the ovarian failure model show obvious anxiety-like behavior, and specific overexpression of Grn in hippocampal neurons can rescue anxiety-like behavior in mice with accelerated ovarian failure.

[0255] 3) Specific overexpression of Grn in hippocampal neurons alleviates depressive-like behavior in mice with accelerated ovarian failure

[0256] To investigate the effect of increasing the expression level of Grn gene in mouse hippocampal neurons on the depressive level of mice with accelerated ovarian failure, tail suspension and forced swimming behavior tests reflecting the depressive level were performed on the three groups of mice sesame oil+AAV-EGFP, 4-VCD+AAV-EGFP, and 4-VCD+AAV-Grn obtained by injecting different viruses in 1) above.

[0257] The results are as Figure 15 shown. Different from ovariectomized mice, accelerated ovarian failure mice (4-VCD+AAV-EGFP) in the tail suspension ( Figure 15A) There was no increase in the immobility time during the experiment. Consistent with ovariectomized mice, compared with the control group (sesame oil + AAV-EGFP), the mice with accelerated ovarian failure had no increase in immobility time during the forced swimming Figure 15 B) There was a significant increase in the immobility time during the test, indicating an elevated level of depressive-like behavior in mice after the establishment of the accelerated ovarian failure model. The immobility time of the mice with accelerated ovarian failure (4-VCD + AAV-Grn) after hippocampal injection of the Grn overexpression virus was significantly reduced in the forced swimming test, indicating the alleviation of depressive-like behavior in the mice.

[0258] The above results indicate that the mice in the ovarian failure model showed obvious depressive-like behavior in the forced swimming test, and hippocampal neuron-specific overexpression of Grn could alleviate the depressive-like behavior in mice with accelerated ovarian failure.

[0259] (III) Hippocampal neuron-specific overexpression of Grn alleviates behavioral disorders in middle-aged female mice

[0260] Although the accelerated ovarian failure model is closer to the natural perimenopausal hormone changes compared with the ovariectomy model, it is not clear whether 4-VCD has other effects. To further clarify the effect of hippocampal neuron-specific overexpression of Grn on the behavioral improvement of estrogen-deficient mice, middle-aged mice (10 months old) of the same age analogous to the middle-aged period when humans are about to experience perimenopause were selected for behavioral tests to explore the effect of hippocampal neuron-specific overexpression of Grn on the behavior of mice.

[0261] 1. Hippocampal neuron-specific overexpression of Grn alleviates the anxiety-like behavior of middle-aged female mice

[0262] Nine-month-old retired breeder female mice that had experienced normal reproduction and male mice of the same age were purchased. After confirming that the basic conditions of the mice were good and the female mice were not pregnant, stereotaxic injection into the mouse brain was performed, and the Grn overexpression virus AAV-Grn and the control virus AAV-EGFP were injected into the bilateral hippocampi of the mice respectively.

[0263] After a one-month recovery period, the mice started the behavioral tests. There were 3 groups in total: Male + AAV-EGFP, Female + AAV-EGFP, Female + AAV-Grn.

[0264] The above 3 groups of mice were subjected to tests related to anxiety-like behavior:

[0265] In the open field test ( Figure 16 A-C)), there were no gender differences in the behavioral performance of middle-aged mice injected with the control virus; while hippocampal injection of the Grn overexpression virus did not affect the total movement distance, the number of times and time of entering the central area of middle-aged female mice; indicating that there were no significant differences in the anxiety levels shown by each group of mice in the open field test.

[0266] In the elevated zero maze test ( Figure 16 D - F) and the elevated plus maze test ( Figure 16 G - I), consistent with the behavioral results of 3 - month - old mice, there were gender differences in the anxiety levels of middle - aged mice. Female mice (Female+AAV - EGFP) spent less time entering the open arms in both the elevated zero maze test and the elevated plus maze test compared to male mice (Male+AAV - EGFP). Female mice (Female+AAV - Grn) injected with Grn - overexpressing virus into the hippocampus showed a significant increase in the number and time of entering the open arms in the elevated zero maze test and the time of entering the open arms in the elevated plus maze test, indicating that the anxiety level of female mice was alleviated.

[0267] The above results indicate that there are gender differences in the anxiety levels of middle - aged mice, and specific overexpression of Grn in hippocampal neurons can alleviate anxiety - like behaviors in middle - aged female mice.

[0268] 2. Specific overexpression of Grn in hippocampal neurons alleviates depressive - like manifestations in middle - aged female mice

[0269] To explore the effect of increasing the expression level of the Grn gene in mouse hippocampal neurons on the depressive - like level of middle - aged female mice, the above three groups of mice were subjected to tail suspension and forced swimming behavioral tests.

[0270] The results are as Figure 17 shown. Consistent with the behavioral results of 3 - month - old mice, there were gender differences in the depressive levels of middle - aged mice. Female mice (Female+AAV - EGFP) had longer immobility times compared to male mice (Male+AAV - EGFP) in the tail suspension ( Figure 17 A) and forced swimming ( Figure 17 B) tests. Middle - aged female mice (Female+AAV - Grn) injected with Grn - overexpressing virus into the hippocampus showed significantly reduced immobility times in both the tail suspension ( Figure 17 A) and forced swimming ( Figure 17 B) tests, indicating that the depressive condition of the mice was alleviated.

[0271] The above results indicate that there are gender differences in the depressive levels of middle - aged mice, and specific overexpression of Grn in hippocampal neurons can alleviate depressive - like behaviors in middle - aged female mice.

[0272] Example 3. Effect of changes in sortilin content in hippocampal neurons on the rescue effect of overexpressing Grn on abnormal behaviors in estrogen - deficient mice

[0273] PGRN is a secreted protein. After the synthesis of the precursor protein, there are two cleavage modes, intracellular and extracellular, and lysosomes are the main cleavage sites. When the PGRN precursor protein is synthesized, a part of it is secreted extracellularly through the Golgi apparatus, and a part directly enters the lysosome for cleavage. The PGRN precursor protein secreted extracellularly is partially cleaved extracellularly, while another part re-enters the cell through endocytosis with the help of receptors such as sortilin and is cleaved in the lysosome. Current studies have found that the molecules affecting the uptake of PGRN into neurons include: prosaposin, sortilin, mannose 6-phosphate receptor (M6PR), and low-density lipoprotein receptor-related protein 1 (LRP1).

[0274] Functionally, the PGRN precursor protein and granulin monomer protein may play different or even opposite roles. For example, in terms of inflammation, PGRN as a whole tends to inhibit inflammation, but the GRNB monomer can stimulate the production of the pro-inflammatory chemokine interleukin-8 (CXCL8). Thus, in the effects on the behavior of estrogen-deficient mice, the PGRN precursor protein and monomer protein may play different roles. According to the RT-PCR results, the expression of neuronal sortilin was interfered by injecting virus to explore whether the process of sortilin-mediated PGRN entry into the cell and into the lysosome affects the rescue effect of PGRN on the behavior of estrogen-deficient mice.

[0275] The experimental results showed that in ovariectomized mice and middle-aged female mice, after simultaneously injecting the Sort1 interfering expression virus to reduce the process of sortilin-mediated PGRN entry into the cell and into the lysosome, the rescue effect of PGRN on the behavior of mice was partially weakened; while in accelerated ovarian failure mice, reducing sortilin-mediated PGRN entry into the cell enhanced the rescue effect of PGRN on the behavior of mice.

[0276] 1. Expression of PGRN and related receptor genes in different estrogen-deficient model mice

[0277] To explore whether the process of PGRN entry into the cell and into the lysosome is affected in different estrogen-deficient model mice, first, RT-PCR was used to explore the expression of PGRN and related receptor genes in different estrogen-deficient model mice.

[0278] RNA was extracted from the hippocampi of mice in the three models of Example 2: sham+AAV-EGFP, OVX+AAV-EGFP, OVX+AAV-Grn, sesame oil+AAV-EGFP, 4-VCD+AAV-EGFP, 4-VCD+AAV-Grn, Male+AAV-EGFP, Female+AAV-EGFP, Female+AAV-Grn. cDNA was obtained by reverse transcription and used as a template for RT-PCR to detect the expression of each gene (the primer sequences are shown in Table 1).

[0279] The results are as Figure 18 shown. It can be seen that in different estrogen-deficient mouse models, due to different hormone change patterns, the content of hippocampal Grn mRNA shows different changes due to possible feedback regulation. Specifically, in the ovariectomy model and the middle-aged female mouse model, the content of Grn mRNA shows a decreasing trend; in the accelerated ovarian failure mice, the content of Grn mRNA shows an increasing trend ( Figure 18 A). When comparing the expression of different molecules in the same model, the change trend of Grn expression level is consistent with the change trend of the content of molecules affecting its entry into cells, suggesting that the process of PGRN entry and cleavage is also affected in different estrogen-deficient mouse models. It is very likely that PGRN precursor and granulins monomers play different roles in regulating mouse behavior. Among the three different estrogen-deficient mouse models, the molecules with significant differences are sortilin and prosaposin.

[0280] Previous studies have shown that the processes of PGRN entering lysosomes mediated by prosaposin and sortilin are independent of each other. After prosaposin binds to PGRN, it enters the cell through LRP1 and M6PR. In both Sort1 knockout mice and Psap knockout mice, the ratio of granulin monomers to PGRN precursor protein in the brain decreases. In microglia, knocking down Sort1 does not affect the content of PGRN, while knocking down Psap increases the content of PGRN. Since the function of PGRN in neurons is mainly concerned, sortilin, which has no obvious effect on PGRN in microglia, was selected as the object for further study.

[0281] 2. Effect of knocking down neuronal sortilin on the rescue effect of behavior by specifically overexpressing Grn in the hippocampus of ovariectomized mice

[0282] 1) Knocking down sortilin in hippocampal neurons weakens the rescue effect of overexpressing Grn on the anxiety-like behavior of OVX mice

[0283] The various combinations of the two viruses AAV-sh-Sort1, AAV-mCherry, and AAV-Grn in (1) of Example 2 were respectively injected into the bilateral hippocampi of mice; two weeks after the mice recovered, ovariectomy was performed for modeling, and behavioral tests were conducted one month after ovariectomy.

[0284] There were 4 groups in total: sham+AAV-mCherry (the sham operation group was injected with AAV-mCherry), OVX+AAV-mCherry (the ovariectomy group was injected with AAV-mCherry), OVX+AAV-Grn+AAV-mCherry (the ovariectomy group was injected with AAV-mCherry and AAV-Grn), OVX+AAV-Grn+AAV-sh-Sort1 (the ovariectomy group was injected with AAV-sh-Sort1 and AAV-Grn; the sortilin gene was knocked out)

[0285] In order to explore the effect of the process of PGRN endocytosis and lysosomal entry mediated by sortilin in hippocampal neurons on the rescue of anxiety-like behavior in OVX mice by overexpressing Grn in hippocampal neurons, anxiety-like behavior tests were conducted on mice in each group.

[0286] In the open field test ( Figure 19 A-C), there were no significant differences in the total movement distance of mice in each group; compared with the sham operation group (sham+AAV-mCherry), the time and frequency of the ovariectomized mice injected with the control virus (OVX+AAV-mCherry) entering the central area were significantly reduced, indicating successful modeling. There were no obvious changes in the time and frequency of the ovariectomized mice in each group entering the central area in the open field test, indicating that PGRN and sortilin do not significantly affect anxiety-like behavior under low stress levels.

[0287] In the elevated zero maze test ( Figure 19 D, E, F), the number of times and time of the OVX mice injected with the Grn overexpression virus (OVX+AAV-Grn+AAV-mCherry) entering the open arms increased significantly, indicating the rescue effect of overexpressing Grn in neurons on the anxiety-like behavior of OVX mice; at the same time, the number of times and time of the OVX mice injected with the Sort1 interfering expression virus (OVX+AAV-Grn+AAV-sh-Sort1) entering the open arms were not significantly different from those of the OVX mice injected with the control virus (OVX+AAV-mCherry), and the time of entering the open arms was significantly lower than that of the mice injected with the AAV-Grn virus, indicating that the rescue effect of neuron-specific overexpression of Grn was interfered.

[0288] In the elevated plus maze experiment ( Figure 19In (D, G, H), the number of entries into the open arms and the time spent in the open arms of OVX mice injected with the control virus (OVX+AAV-mCherry) were reduced compared to those of the control group mice (sham+AAV-mCherry), indicating successful modeling. The number of entries into the open arms of OVX mice injected with the Grn overexpression virus (OVX+AAV-Grn+AAV-mCherry) increased significantly, and the time spent in the open arms showed an increasing trend; in OVX mice co-injected with the Sort1 interfering expression virus (OVX+AAV-Grn+AAV-sh-Sort1), the rescue effect of neuron-specific overexpression of Grn was interfered with.

[0289] The above results indicate that specific knockdown of sortilin in hippocampal neurons can weaken the rescue effect of neuron overexpression of Grn on the anxiety-like behavior of OVX mice.

[0290] 2) Knockdown of sortilin in hippocampal neurons does not affect the rescue effect of neuron-specific overexpression of Grn on the depressive-like behavior of OVX mice

[0291] To investigate whether the process of PGRN entry into cells and into lysosomes mediated by sortilin in hippocampal neurons affects the rescue effect of overexpression of Grn on the depressive-like behavior of OVX mice, the above 4 groups of mice obtained in 1) injected with different viruses were subjected to behavioral tests related to the depressive level of tail suspension and forced swimming.

[0292] In the tail suspension experiment ( Figure 20 A) and the forced swimming experiment ( Figure 20 B), the immobile time of OVX mice injected with the control virus (OVX+AAV-mCherry) increased significantly, indicating successful modeling. In OVX mice injected with the Grn overexpression virus into the hippocampus (OVX+AAV-Grn+AAV-mCherry), the immobile time in both tests decreased significantly, indicating that the depressive condition of the mice was alleviated; in OVX mice co-injected with the Grn overexpression virus and the Sort1 interfering expression virus (OVX+AAV-Grn+AAV-sh-Sort1), there was no significant difference in the immobile time compared to OVX mice injected only with the Grn overexpression virus (OVX+AAV-Grn+AAV-mCherry) in the tail suspension experiment and the forced swimming experiment.

[0293] The above results indicate that specific knockdown of sortilin in hippocampal neurons does not affect the rescue effect of neuron-specific overexpression of Grn on the depressive-like behavior of OVX mice.

Claims

1. Use of PGRN protein, or its encoding gene, or a virus expressing its encoding gene in the preparation of a product having any of the following functions: 1) Preventing, treating, or rescuing diseases related to neuropsychiatric abnormalities caused by estrogen deficiency; 2) Preventing or treating perimenopausal anxiety and / or depressive symptoms; 3) Alleviating anxiety-like behavior and / or depressive-like behavior caused by estrogen deficiency.

2. Use of a substance that increases the content of PGRN protein or the expression level of the PGRN protein-encoding gene in the preparation of a product having any of the following functions: 1) Preventing or treating diseases related to neuropsychiatric abnormalities caused by estrogen deficiency; 2) Preventing or treating perimenopausal anxiety and / or depressive symptoms; 3) Alleviating anxiety-like behavior and / or depressive-like behavior caused by estrogen deficiency.

3. The application according to claim 2, wherein: The substance is exogenous PGRN protein or a virus expressing the PGRN protein-encoding gene.

4. The application according to any one of claims 1-3, characterized in that: The diseases related to neuropsychiatric abnormalities are manifested as at least one of the following: anxiety, depression, and negative emotions.

5. The application according to any one of claims 1-4, characterized in that: The estrogen deficiency is caused by ovariectomy, accelerated ovarian failure, or midlife menopause.

6. Use of a protein that interacts with PGRN protein as a target in the development or design of a drug for preventing or treating diseases related to neuropsychiatric abnormalities caused by estrogen deficiency.

7. The application according to claim 6, characterized in that: The protein that interacts with PGRN protein is sortilin.

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