Neurogenesis promoter and use thereof

Abstract

Provided is a technology capable of promoting neurogenesis. This neurogenesis promoter contains at least one of a substance that inhibits the glutaminolysis pathway and a substance that inhibits mTOR, and is used between postnatal day 0 and corrected age of 20 months of a premature infant.

Description

Neuropoietic promoters and their uses

[0001] The present disclosure relates to a neurogenesis-promoting agent and a method for promoting neurogenesis. This application is based on Japanese Patent Application No. 2024-126455, filed on August 1, 2024, the contents of which are incorporated herein by reference.

[0002] In recent years, approximately 5% to 10% of newborns worldwide are born prematurely. It is known that premature infants are more likely to suffer from neurodevelopmental disorders than full-term infants, but the underlying pathological mechanisms and treatments have not yet been established. Generally, fundamental treatment of brain diseases requires a technology that regenerates lost nerve cells to restore brain function. One such technology, proposed for example in Patent Document 1, involves transplanting cells derived from iPS cells.

[0003] International Publication No. 2013 / 069661

[0004] The transplantation of cells generated from iPS cells raises concerns that the transplanted cells may form tumors in the brain, leaving room for improvement before they can be used clinically as regenerative medicine for brain diseases. This issue is not limited to neurodevelopmental disorders in premature infants, but is also common to other brain diseases. Therefore, through extensive research, the inventors of the present application have invented a technology that can promote neurogenesis without relying on cell transplantation.

[0005] The present invention can be realized as the following aspects.

[0006] (1) One aspect of the present invention provides a neurogenesis promoter. This neurogenesis promoter contains at least one of a substance that inhibits the glutaminolytic pathway and a substance that inhibits mTOR, and is used in premature infants from day 0 to 20 months corrected age after birth. This form of the neurogenesis promoter can promote neurogenesis.

[0007] (2) The neurogenesis promoter according to (1) above may contain a substance that inhibits mTOR. This form of the neurogenesis promoter can more effectively promote neurogenesis.

[0008] (3) In the neurogenesis promoter according to (1) or (2), the mTOR inhibitor may comprise at least one selected from the group consisting of rapamycin, everolimus, temsirolimus, eforolimus, ridaforolimus, deforolimus, zotarolimus, pimecrolimus, tacrolimus, and derivatives thereof. This form of the neurogenesis promoter can more effectively promote neurogenesis.

[0009] (4) According to another aspect of the present invention, there is provided a food or beverage containing the neurogenesis promoter according to any one of (1) to (3) above. The food or beverage of this aspect can promote neurogenesis.

[0010] (5) According to another aspect of the present invention, there is provided an infant formula for promoting neurogenesis. This infant formula is substantially free of glutamine and is administered to premature infants between day 0 and 20 months of corrected age. This form of infant formula can promote neurogenesis in infants.

[0011] (6) Another aspect of the present invention provides a method for promoting neurogenesis, comprising administering at least one of a substance that inhibits the glutaminolytic pathway and a substance that inhibits mTOR to a premature infant between day 0 and 20 months of corrected age. This method can promote neurogenesis.

[0012] The present disclosure can be realized in various forms, such as a method for producing a neurogenesis promoter, a method for producing a pharmaceutical composition, the use of a glutamine degradation pathway inhibitor in the manufacture of a therapeutic agent for premature infant brain damage or premature infant developmental disorder, the use of an mTOR inhibitor in the manufacture of a therapeutic agent for premature infant brain damage or premature infant developmental disorder, the use of a glutamine degradation pathway inhibitor in the manufacture of a neurogenesis-promoting food or beverage, the use of an mTOR inhibitor in the manufacture of a neurogenesis-promoting food or beverage, etc.

[0013] Schematic diagram of glutamine metabolism. Diagram showing an example of an experimental schema. Diagram showing the results of oxygen partial pressure levels in the V-SVZ. Diagram showing the results of principal component analysis in metabolomic analysis of the V-SVZ. Diagram showing the relative intensity of each metabolite. Diagram showing the clustering results in single-cell gene expression analysis. Violin plot showing the expression of glycolysis-related genes in RG. Diagram showing the expression of Glu and Gls. Violin plot showing the expression of Glu and Gls in RG. Dot plot showing the results of GSEA of GO terms. Dot plot showing the results of GSEA. Diagram showing the results of network analysis based on RG correlation. Diagram summarizing the results of metabolomic analysis and scRNA-seq. Representative stained image of Glu RNA in a coronal V-SVZ section. Diagram showing the dot area percentage of Glu RNA. Representative stained image of a coronal V-SVZ section from a GFAP-EGFP mouse. An illustration showing the relative expression levels of Glul in the RG. Representative staining images of coronal V-SVZ sections. ＋ pS6 ＋ Nestin ＋ An explanatory diagram showing the density of RG. An explanatory diagram showing a comparison of mTORC1 signaling activity. Representative staining images of Mash1 in coronal V-SVZ sections. Mash1 in V-SVZ. ＋ An illustration showing the number of neural progenitor cells. Representative staining images of coronal V-SVZ sections stained with Dcx. Dcx in the V-SVZ. ＋ Representative staining images of coronal sections of GCL. ＋ NewN ＋ An illustration showing the density of neurons. A representative stained image of a coronal V-SVZ section of a GFAP-EGFP mouse. An illustration showing the density of each cell type at 47.5 dpc. A representative stained image of a coronal V-SVZ section of a GFAP-CreERT2; Rosa26-tdTomato mouse. tdTomato ＋ GFAP ＋ tdTomato for NSCs ＋ Mash 1 ＋ Representative images of coronal V-SVZ sections stained with EGFP, pS6, and GFAP at P7.＋ GFAP ＋ pS6 in NSCs ＋ Representative staining images of coronal V-SVZ sections stained with EGFP, Mash1, and GFAP at P7. ＋ GFAP ＋ EGFP for NSCs ＋ Mash 1 ＋ Representative images of coronal V-SVZ sections stained with EGFP, pS6, and GFAP at P8. ＋ GFAP ＋ pS6 in NSCs ＋ Representative staining images of coronal V-SVZ sections stained with EGFP, Mash1, and GFAP at P8. ＋ GFAP ＋ EGFP for NSCs ＋ Mash 1 ＋ An explanatory diagram showing the proportion of neural progenitor cells. Representative staining images of Mash1, pS6, and Nestin in coronal V-SVZ sections at preterm P3. An explanatory diagram showing each cell in the V-SVZ at preterm P3. Representative staining images of coronal V-SVZ sections stained with EGFR, Mash1, and GFAP at preterm P29. EGFR in the V-SVZ at preterm P29. ＋ Mash 1 ＋ GFAP ＋ Schematic diagram showing the density of NSCs. Representative staining images of coronal V-SVZ sections stained with Mash1 at preterm P29. Mash1 in the V-SVZ at preterm P29. ＋ An explanatory diagram showing the number of neural progenitor cells. Representative staining images of Mash1-stained coronal V-SVZ sections at term P28. Mash1 in the V-SVZ at term P28. ＋ 1 is an explanatory diagram showing the number of neural progenitor cells, and 2 is an explanatory diagram showing a comparison between RG at full term and RG at preterm birth.

[0014] As shown in the Examples below, the present inventors have discovered that birth-induced changes in glutamine metabolism and mTOR signaling in radial glia (RG), which are neural stem cells (NSCs), are necessary for the acquisition of a quiescent state and the long-term maintenance of NSCs, and have completed the present invention based on this finding.

[0015] Resident stem cells are essential for maintaining tissue homeostasis after birth. In the adult mammalian brain, NSCs are maintained in the ventricular-subventricular zone (V-SVZ) and continue to generate new neurons that migrate to the olfactory bulb (OB). In humans, postnatal neurogenesis is thought to play a critical role in brain development and plasticity. Postnatal NSCs differentiate from embryonic NSCs (RGs). The majority of postnatal NSCs are maintained in a quiescent state, enabling their long-term maintenance. In various tissues, the stem cell niche and the metabolic profile of the stem cells themselves control whether they remain quiescent or transition to an activated and differentiated state. However, little is known about how metabolic changes associated with birth affect tissue stem cells, particularly NSCs.

[0016] FIG. 1 is a schematic diagram illustrating glutamine metabolism. In this disclosure, the term "glutaminolytic pathway" refers to the pathway in which glutamine is degraded to α-ketoglutarate (αKG) via glutamate. The series of reactions in the glutaminolytic pathway is also referred to as "glutaminolysis." After glutaminolysis, αKG can enter the tricarboxylic acid cycle. Glutamine is synthesized from glutamate by glutamate-ammonia ligase (GluI), also known as glutamine synthetase (GS). The Examples below demonstrate that birth-induced changes in glutamine metabolism, mediated by upregulation of GluI in RG, are necessary for NSCs to attain postnatal quiescence. It has also been shown that this cellular process is impaired by premature birth, resulting in depletion of the NSC pool and reduced neurogenesis in young adulthood. The present inventors have found that inhibiting at least one of the glutaminolytic pathway and its downstream mTOR signaling pathway immediately after birth in premature infants promotes the acquisition of RG quiescence immediately after birth, thereby suppressing the depletion of NSCs and thereby promoting neurogenesis.

[0017] According to one embodiment of the present disclosure, there is provided the following neurogenesis-promoting agent (a): (a) a neurogenesis-promoting agent comprising at least one of a substance that inhibits the glutaminolytic pathway and a substance that inhibits mTOR, the agent being used in premature infants from day 0 to 20 months corrected age after birth;

[0018] In the present disclosure, whether or not a substance inhibits the glutamine degradation pathway can be confirmed by measuring the glutamate concentration in the subject's body fluid using liquid chromatography. Although blood is preferably used as the body fluid, cerebrospinal fluid may also be used. Measurement can be performed, for example, by the following method. After centrifuging the collected body fluid, proteins are removed by precipitation with acid, and the supernatant is filtered. The sample is then derivatized with ortho-phthalaldehyde, separated by reverse-phase HPLC using a C18 column with ammonium acetate buffer and an organic solvent as the mobile phase, and the glutamate peak is detected and quantified using a fluorescence detector. If the glutamate concentration after administration of the substance is reduced compared to the glutamate concentration before administration, it can be determined that the substance inhibits the glutamine degradation pathway. If there is no reduction, it can be determined that the substance does not inhibit the glutamine degradation pathway. In the present disclosure, whether or not a substance inhibits mTOR can be confirmed by evaluating the expression level of phosphorylated ribosomal protein S6 (pS6) in brain tissue using immunostaining. If the expression level of pS6 after administration of a substance is reduced compared to the expression level of pS6 before administration of the substance, it can be determined that the substance inhibits mTOR, and if there is no reduction, it can be determined that the substance does not inhibit mTOR.

[0019] Substances that inhibit the glutamine degradation pathway include, but are not limited to, glutaminase inhibitors, substrate analogs and intermediate analogs in the glutamine degradation pathway, and transcription inhibitors of genes encoding glutaminase (e.g., antisense nucleic acids, siRNA, ribozymes). From the viewpoint of effectively promoting neurogenesis, it is preferable to include a glutaminase inhibitor. Examples of glutaminase inhibitors include, but are not limited to, Glutaminase 1 (GLS1) inhibitors and Glutaminase 2 (GLS2) inhibitors. From the viewpoint of more effectively promoting neurogenesis, it is preferable to include a GLS1 inhibitor. Examples of GLS1 inhibitors include, but are not limited to, BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide), CB839 (Telaglenastat), Compound 968 (Glutaminase C-IN-1), etc., and from the viewpoint of more effectively promoting neurogenesis, it is preferable to include BPTES. Examples of GLS2 inhibitors include, but are not limited to, Compound 968 (Glutaminase C-IN-1), and from the viewpoint of more effectively promoting neurogenesis, it is preferable to include Compound 968 (Glutaminase C-IN-1). Substrate analogs and intermediate analogs in the glutamine degradation pathway include, but are not limited to, glutamine analogs, glutamic acid analogs, etc., and from the viewpoint of effectively promoting neurogenesis, it is preferable to include a glutamine analog. Glutamine analogs include, but are not limited to, DON (6-diazo-5-oxo-L-norleucine). Glutamic acid analogs include, but are not limited to, 4-fluoroglutamic acid.

[0020] mTOR is a serine / threonine-specific protein kinase downstream of the phosphatidylinositol 3-kinase (PI3K) / Akt (protein kinase B) pathway and has an NCBI GeneID of 2475. mTOR signaling downstream of the glutamine metabolic pathway controls NSC activation. The substance that inhibits mTOR is not particularly limited, and may be a substance that inhibits mTOR1 or a substance that inhibits mTOR2. However, from the viewpoint of effectively promoting neurogenesis, a substance that inhibits mTOR1 is preferable. Furthermore, the substance that inhibits mTOR is not particularly limited, and examples thereof include substances that inhibit mTOR activity, neutralizing antibodies against mTOR, and transcription inhibitors of the gene encoding mTOR (e.g., antisense nucleic acids, siRNA, ribozymes, etc.). Substances that inhibit the activity of mTOR include, but are not limited to, rapamycin, everolimus, temsirolimus, eforolimus, ridaforolimus, deforolimus, zotarolimus, pimecrolimus, tacrolimus, voxtalisib, omipalisib, apitolisib, gedatrisib, bistusertib, palomid 529, torin 1, torin 2, chrysophanic acid, PI-103, CC-223, INK128, AZD8055, KU 0063794, NVP-BEZ235, CZ415, torkinib (PP242), OSI-027, PF-04691502, WYE-354, GSK1059615, WYE-125132 (WYE-132), BGT226 (NVP-BGT226), PP121, WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388, and derivatives thereof.

[0021] A neurogenesis promoter (hereinafter also referred to as "neurogenesis promoter (a)") containing at least one of a substance that inhibits the glutaminolytic pathway and a substance that inhibits mTOR and is used in premature infants from day 0 to 20 months of corrected age preferably contains an mTOR inhibitor from the viewpoint of effectively promoting neurogenesis. From the viewpoints of safety and toxicity, the mTOR inhibitor is more preferably at least one selected from the group consisting of rapamycin, everolimus, temsirolimus, eforolimus, ridaforolimus, deforolimus, zotarolimus, pimecrolimus, tacrolimus, and derivatives thereof, and even more preferably at least one selected from the group consisting of rapamycin, everolimus, temsirolimus, and derivatives thereof. The neurogenesis promoter (a) may also contain both a substance that inhibits the glutaminolytic pathway and a substance that inhibits mTOR.

[0022] The neurogenesis-promoting agent of the present disclosure may be used for the treatment or prevention of at least one selected from the group consisting of premature infant brain damage and premature infant developmental disorder. The subjects to which the neurogenesis-promoting agent of the present disclosure is administered are not particularly limited, and include humans and non-human animals, but are preferably mammals, and particularly preferably humans. Non-human animals include, but are not limited to, monkeys, mice, rats, rabbits, dogs, cats, etc.

[0023] In the present disclosure, the term "preterm infant" refers to a newborn born before 36 weeks and 6 days of gestation in humans. The term "full-term infant" refers to a newborn born after 37 weeks and 0 days of gestation in humans. In the present disclosure, the term "corrected age" refers to the age in months when 40 weeks and 0 days of gestation is considered to be the corrected age 0 days in humans. Therefore, for example, "20th month corrected age" refers to the age 20 months after the expected date of birth. In the present disclosure, the term "newborn infant" refers to an infant up to 28 days after birth in humans. In the present disclosure, "treatment" refers to at least one of remission, alleviation, and delay in worsening of clinical symptoms of a disease in a subject who has developed the disease. In the present disclosure, "prevention" refers to reducing the incidence of a disease before the onset of the disease.

[0024] The amount (administration amount and intake amount) of the neurogenesis-promoting agent of the present disclosure used is not particularly limited, but is preferably 1 to 100 mg / kg body weight / day. The neurogenesis-promoting agent of the present disclosure is preferably used continuously, more preferably once every three days or more, even more preferably once every two days or more, and even more preferably every day.

[0025] As described in Sanai N, et al. Nature. 2011 Sep 28;478(7369):382-6, it has been reported that in humans, the phenomenon of neurons being generated from neural stem cells present in the subventricular zone is observed up to approximately 18 months of age, but is rarely observed thereafter. Therefore, it is speculated that neural stem cells in the subventricular zone in premature infants also have the ability to generate neurons up to approximately 18 months of corrected age. Therefore, the period during which excessive activation of neural stem cells occurs in premature infants is thought to be anywhere from day 0 to 20 months of corrected age. The neurogenesis promoter (a) is preferably used between day 0 and 18 months of corrected age in premature infants, more preferably between day 0 and 12 months of corrected age in premature infants, even more preferably between day 0 and 6 months of corrected age in premature infants, even more preferably between day 0 and 3 months of corrected age in premature infants, and even more preferably between day 0 and 1 month of corrected age in premature infants. Furthermore, the neurogenesis promoter (a) is particularly preferably used between day 0 and 7 days of postnatal life, even more preferably between day 0 and 5 days of postnatal life, and most preferably between day 0 and 3 days of postnatal life.

[0026] The neurogenesis promoting agent of the present disclosure may be contained in pharmaceuticals, quasi-drugs, foods, beverages, etc., as described below.

[0027] According to another aspect of the present disclosure, there is provided a pharmaceutical product (hereinafter also referred to as the "pharmaceutical product of the present disclosure") for treating or preventing at least one disease selected from the group consisting of premature infant brain damage and premature infant developmental disorder, comprising a neurogenesis promoter (a).

[0028] The form of the pharmaceutical of the present disclosure is not particularly limited and may be appropriately determined depending on the administration method, etc. The administration method is not particularly limited and includes, for example, oral administration, parenteral administration, etc., with oral administration being preferred. Parenteral administration is not particularly limited and includes, for example, transdermal administration, subcutaneous administration, intravenous injection, intramuscular injection, rectal administration, intraperitoneal administration, topical administration, etc. The form of the pharmaceutical of the present disclosure, in the case of oral administration, includes, for example, tablets, pills, capsules, powders, granules, liquids, syrups, etc.

[0029] The pharmaceutical product of the present disclosure may contain a pharmaceutically acceptable additive. In the present disclosure, the term "pharmaceutically acceptable additive" refers to an additive commonly used in the pharmaceutical technology field. Examples of such additives include, but are not limited to, solvents, excipients, fillers, emulsifiers, binders, disintegrants, wetting agents, suspending agents, flow regulators, lubricants, and the like. The solvent may be, for example, water or other pharmaceutically acceptable aqueous solutions, or pharmaceutically acceptable organic solvents. Examples of aqueous solutions include, but are not limited to, physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, sodium acetate buffer, citrate buffer, sodium bicarbonate buffer, and the like. Examples of adjuvants include, but are not limited to, D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.

[0030] Examples of excipients include, but are not limited to, saccharides such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides; starches such as potato starch and wheat starch; various celluloses such as crystalline cellulose; inorganic salts such as anhydrous calcium hydrogen phosphate and calcium carbonate; citric acid, tartaric acid, glycine, polyethylene glycol, kaolin, silicic acid, or combinations thereof. Examples of fillers include, but are not limited to, petrolatum, sugar, calcium phosphate, etc. Examples of emulsifiers include, but are not limited to, gum arabic, sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, propylene glycol fatty acid esters, etc. Examples of binders include, but are not limited to, crystalline cellulose, pullulan, gum arabic, sodium alginate, polyvinylpyrrolidone, macrogol, etc. Examples of disintegrants include, but are not limited to, carboxymethylcellulose, carboxymethylcellulose calcium, hydroxypropylcellulose, hydroxypropyl starch, starch, sodium alginate, etc. Examples of wetting agents include, but are not limited to, coconut oil, olive oil, sesame oil, peanut oil, soybean phospholipid, glycerin, sorbitol, etc. Examples of suspending agents include, but are not limited to, gum arabic, agar, carmellose, etc. Examples of flow additive regulators and lubricants include, but are not limited to, silicates, talc, stearates, polyethylene glycol, etc. In addition to the above, the formulation may appropriately contain solubilizers, diluents, dispersants, surfactants, soothing agents, absorption enhancers, bulking agents, moisturizers, wetting agents, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, antioxidants, fragrances, flavoring agents, sweeteners, buffers, isotonicity agents, etc. that are commonly used in pharmaceuticals.

[0031] According to another aspect of the present disclosure, a food or beverage (hereinafter also referred to as "food or beverage of the present disclosure") containing neurogenesis promoter (a) is provided. Examples of the food or beverage of the present disclosure include, but are not limited to, supplements, infant formula, nutritional supplements, nutrients, foods for specified health uses, foods with nutrient functions, and foods with functional claims. More specifically, for example, infant formula or supplements containing the neurogenesis promoter (a) are provided, which are to be consumed by premature infants between day 0 and 20 months of corrected age. The form of the food or beverage of the present disclosure is, but is not limited to, chewable tablets, powders, capsules, granules, energy drinks, tablets, candy tablets, candies, jellies, cookies, dairy drinks, soft drinks, tea drinks, and the like.

[0032] Another aspect of the present disclosure provides an infant formula for promoting neurogenesis (hereinafter also referred to as "the infant formula of the present disclosure"). This infant formula is substantially free of glutamine and is to be consumed by premature infants between day 0 and 20 months of corrected age. In the present disclosure, "substantially free" means that glutamine may be present to an extent that is unavoidable during the manufacturing process. The concentration of glutamine in the formula, after dilution or other appropriate adjustment, is preferably 1 μg / mL or less, and more preferably 100 ng / mL or less. The form of the infant formula of the present disclosure is not particularly limited, and may be a powdered milk formula or a liquid milk formula. The infant formula of the present disclosure may be taken orally, via nasal feeding, tube feeding, or the like. Because the infant formula of the present disclosure is substantially free of glutamine, a substrate for the glutamine degradation pathway, it promotes the acquisition of RG quiescence immediately after birth, suppressing the depletion of NSCs, and thereby promoting neurogenesis.

[0033] According to another aspect of the present disclosure, there is provided a method for promoting neurogenesis, comprising administering at least one of a substance that inhibits the glutaminolytic pathway and a substance that inhibits mTOR to a premature infant between day 0 and 20 months of corrected age. The substance used in this step corresponds to the substance described above in the section on neurogenesis-promoting agent (a).

[0034] According to another aspect of the present disclosure, a method for promoting neurogenesis is provided. The method includes a step of raising premature infants in an environment with an oxygen concentration of 30% to 50% from day 0 after birth until 20 months of corrected age. This step may be achieved by various forms, such as an incubator control system or an incubator control method. In the examples described below, it has been shown that full-term birth induces a transition from anaerobic to aerobic metabolism in RG and upregulation of GluI expression. It has also been reported that glutaminolysis is activated under hypoxic conditions (CT Taylor & CC Scholz, Nat Rev Nephrol 18, 573-587 (2022) and L. Xiang et al., Cell Death Dis 10, 40 (2019)). Therefore, raising premature infants in an environment with an increased oxygen concentration from day 0 after birth until 20 months of corrected age can reduce glutaminolysis and, consequently, αKG. As a result, the enhancement of mTOR signaling can be suppressed, preventing neural stem cells from attaining quiescence, and as a result, the depletion of neural stem cells can be suppressed, thereby suppressing the decrease in neurogenesis in young adults.

[0035] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0036] 1. Materials and Methods (1) Samples: Wild-type (WT) Institute for Cancer Research (ICR) mice and C57BL6 / J mice were purchased from Japan SLC. GFAP-EGFP mice were obtained from MMRRC (Mutant Mouse Regional Resource Center, Stock No. 000315-MU) and maintained on an ICR background. GFAP-CreERT2 mice expressing tamoxifen-dependent Cre recombinase under the human gfap gene promoter were obtained from MMRRC (Stock No. 016992-MU) and maintained on an ICR background. R26-tdTomato mice (The Jackson Laboratory, Stock No. 7914) on a C57BL6 / J background were provided by Dr. Masahiro Yamaguchi (Kochi Medical University). Healthy male and female mice were used in all animal experiments. WT mice on an ICR background were used unless otherwise noted. Mice were housed in cages lined with chip bedding in a specific pathogen-free facility under a controlled environment (23±1°C, 12-hour light / dark cycle switched at 8:00 AM) with free access to water and food (MF, Oriental Yeast Co., Ltd.). Timed mating was performed by placing one male mouse and two female mice in a cage from the evening until the following morning. The morning a vaginal plug was observed was designated as embryonic day (E) 0.5, or 0.5 dpc. In the following description, "dpc" refers to days post-fertilization (days post-coitus).

[0037] (2) Induction of Preterm Birth. Figure 2 shows an example of the experimental scheme. Preterm birth was induced in mice by mifepristone treatment. Mifepristone administration was performed using a modified version of a previously published method (DJ Dudley et al., Biol Reprod 55, 992-995 (1996). and T. Toda et al., Dev Cell 27, 32-46 (2013)). 150 μg of mifepristone (Sigma-Aldrich) or phosphate-buffered saline (PBS) as a control was administered subcutaneously at embryonic day 17.5 (E17.5). Mice were born at 18.5 dpc or 19.5 dpc, resulting in preterm or full-term births, respectively. FIG. 2 shows that before birth (P0), mice exist in an intrauterine environment, and after birth (P0), they exist in an extrauterine environment.

[0038] (3) Injection of EdU and BrdU To label proliferating cells, mice were intraperitoneally injected twice with EdU (5-ethynyl-2'-deoxyuridine, 50 mg / kg, dissolved in PBS (Abcam)) and BrdU (bromodeoxyuridine, 50 mg / kg, dissolved in PBS (Sigma-Aldrich)) at 2-hour intervals.

[0039] (3) Tamoxifen Treatment: Tamoxifen (Sigma-Aldrich) was dissolved in a solvent (5 mg / ml tamoxifen, 90% sesame oil, 6% ethanol, 4% dimethyl sulfoxide (DMSO, Sigma-Aldrich)). Tamoxifen (50 mg / kg) was administered intraperitoneally to GFAP-CreERT2;R26-tdTomato mice at 47.5 days after conception.

[0040] (4) In vivo pO in the V-SVZ ２ Measurement of oxygen partial pressure (pO) in mice on embryonic day 18.5 (E18.5) ２To measure pO in pregnant mice, pregnant mice were anesthetized with a mixed anesthetic (0.75 mg / kg medetomidine, 4 mg / kg midazolam, and 5 mg / kg butorphanol). After laparotomy, the uterus was incised to expose the fetal head with the placenta still attached. pO in full-term day 2 (P2) and preterm day 3 (P3) mice was measured. ２ For the measurements, pups were also anesthetized with the same anesthetic mixture. After removing the skull, a carbon fiber electrode needle (IPC-020, tip diameter 300 μm, manufactured by Intermedical) was manually inserted into the V-SVZ. pO ２ was measured using IMP-211 (Intermedical). After the experiment, the brain was fixed and the insertion path of the electrode needle was examined. Only animals in which the tip of the electrode needle reached the V-SVZ were used for analysis.

[0041] (5) Probe electrospray ionization / tandem mass spectrometry (PESI / MS / MS) and data analysis. An LCMS-8040 triple quadrupole tandem mass spectrometer (Shimadzu Corporation) equipped with an ESI light source was used. A probe needle (tip diameter 700 nm, Shimadzu Corporation) was used, and a new needle was replaced for each sample analysis. V-SVZ tissue was dissected as previously reported (Z. Mirzadeh et al., J Vis Exp 10.3791 / 1938, (2010)). Bilateral V-SVZ tissues excised from mice were placed on a sample plate (Shimadzu Corporation) and stored at -80°C until analysis. To increase ionization efficiency, 15 μL of 50% ethanol solution was added to the sample. Triplicate analyses were performed for each sample. Following a previous study (K. Zaitsu et al., Anal Chem 92, 8514-8522 (2020)), 65 metabolites primarily related to central energy metabolism, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, and the β-oxidation pathway, were selected. Peak area integration for each metabolite was performed using the built-in LabSolutions software (version 5.86, Shimadzu Corporation). High-throughput direct metabolomics was performed using an analytical platform called PiTMaP. Metabolites with a variable importance in projection (VIP) value above a set threshold (1.0) were selected as statistical families according to a previous study (K. Zaitsu et al., Anal Chem 92, 8514-8522 (2020)). The p-values ​​obtained by Welch's t-test were adjusted for the false discovery rate (FDR) using the procedure proposed by Benjamini and Hochberg, and the adjusted p-values ​​were described as q-values.

[0042] (6) Single-Cell Isolation. V-SVZ tissues from C57BL6 / J mice were dissected for single-cell gene expression analysis (scRNA-seq). Bilateral V-SVZ tissues from four littermates were pooled to form one sample. Two independent samples were generated from different litters at each time point as biological replicates (embryonic day E18.5, E18.5-1, E18.5-2; full-term P2, P2-1, P2-2; preterm P0, P0-1, P0-2; preterm P3, P3-1, P3-2). Samples were generated over the following three days: 1), 2), and 3), each generated on the same day. 1) embryonic day E18.5-1, term day P2-1, and preterm day P0-1; 2) embryonic day E18.5-2, term day P2-2, and preterm day P0-2; and 3) preterm day P3-1 and preterm day P3-2. Dissected V-SVZ tissues were digested with papain-EBSS (LK003150, Worthington) for 20 min at 37°C. After an initial 10 min of incubation in papain-EBSS solution, the tissues were mechanically dissociated with a P1000 pipette for 20 s, followed by a P200 pipette for 20 s. Cells were filtered through 70 μm and 40 μm cell strainers (Corning Life Sciences, Corning) to remove cell clumps, then centrifuged at 400 g for 5 min at room temperature and resuspended in DNase solution containing ovomucoid inhibitor according to the manufacturer's instructions (Worthington). The cells were again centrifuged at 100 g for 6 minutes at room temperature and then resuspended at a density of 1,000 cells / μL in RNase-free PBS (Invitrogen) containing 0.04% ultrapure bovine serum albumin (Invitrogen) and 0.1 U / mL recombinant RNase inhibitor (Invitrogen). Cell viability was measured using trypan blue (Thermo Fisher Scientific) and maintained above 90%.

[0043] (7) Single-Cell Library Preparation. A suspension of 10,000 single cells was loaded onto a Chromium Next GEM Chip G (PN-2000177, 10x Genomics). cDNA synthesis and library construction were performed according to the manufacturer's Chromium Single Cell 3'v3.1 protocol (PN-1000128, 10x Genomics). cDNA synthesis and library construction were performed over three days, with each of the following runs performed on the same chip on the same day: 1) embryonic day E18.5-1, full-term day P2-1, and preterm day P0-1; 2) embryonic day E18.5-2, full-term day P2-2, and preterm day P0-2; and 3) preterm day P3-1 and preterm day P3-2. The libraries were sequenced using an Illumina sequencing platform (NovaSeq 6000, Illumina) to generate 150-bp paired-end reads.

[0044] (8) Analysis of scRNA-seq Data. Primary data analysis was performed using cellranger v5.0.0 (10x Genomics) to generate fastq files (cellranger mkfastq) and a count matrix (cellranger count). The mouse reference transcriptome used in cellranger count was downloaded from 10x Genomics (https: / / cf.10xgenomics.com / supp / cell-exp / refdata-gex-mm10-2020-A.tar.gz). Secondary analysis was performed on the filtered feature barcode matrix.

[0045] (9) Sample Integration and Clustering. Each dataset was normalized (min.cells.per.gene = 3) using Pagoda2 (N. Barkas et al., pagoda2: Single Cell Analysis and Differential Expression. R package (2021); https: / / cran.r-project.org / web / packages / pagoda2 / pagoda2.pdf). Sample integration was then performed using Conos (N. Barkas et al., Nat Methods 16, 695-698 (2019)). To ensure sample alignment, the alignment.strength parameter was set to 0.3 when integrating all four sample time points together. UMAP embeddings were estimated for each sample set, and clustering was performed using the leiden.community method. To increase the clustering resolution, the resolution parameter was set to 4 or 5, and the min.group.size was set to 15. The final clusters were generated using a combination of manual selection and the findSubcommunities function in Conos to further split the clusters.

[0046] (10) Compositional Data Analysis. To perform compositional data analysis, we used the Cacoa function estimateCellLoadings (V. Petukhov et al., bioRxiv 10.1101 / 2022.03.15.484475, 2022.2003.2015.484475 [Preprint] (2022)). First, an isometric log-ratio (ILR) transformation was applied to the cell type fractions, followed by canonical discriminant analysis (CDA) to calculate weighted contrasts between cell types. To assess the robustness and significance of the separation coefficients, we performed a random subsampling of 1,000 cells and analyzed 1,000 randomly selected cells from each group.

[0047] (11) Gene Expression Plot A seurat (Y. Hao et al., Cell 184, 3573-3587 e3529 (2021)) object was created from the normalized and filtered count matrix, and a gene expression plot was created using the VlnPlot function.

[0048] (12) Gene Expression Analysis. Cacoa (V. Petukhov et al., bioRxiv 10.1101 / 2022.03.15.484475, 2022.2003.2015.484475 [Preprint] (2022).) (https: / / github.com / kharchenkolab / cacoa) was used for differentially expressed gene (DEG) analysis. The Cacoa function estimatePerCellTypeDE was applied with default parameters to estimate differentially expressed genes between two different conditions for each cell type. This function is based on the DESeq2 package and collapses gene expression per sample and cell type into a pseudo-bulk gene expression level. DESeq2 estimates differentially expressed genes using the Wald test.

[0049] (13) Gene Set Enrichment Analysis. GSEA (Gene Set Enrichment Analysis) was performed using the Cacoa function estimateOntology with default settings. This function is based on the clusterProfiler package. Dot plots of specific GO terms were generated using the dotplot function in the enrichplot package (G. Yu. enrichplot: Visualization of Functional Enrichment Result. R package version 1.18.3 (2022); https: / / yulab-smu.top / biomedical-knowledge-mining-book / ). Only GO terms belonging to the category "biological process" were included. The words "cell," "regulation," and "process" were excluded from the collapsed GO terms.

[0050] (14) Analysis of Active and Quiescent Signatures. To perform GSEA of the active and quiescent signatures of RG, we first defined a unique gene set using the active and quiescent marker genes from Cheung and Rando (T.H. Cheung, T.A. Rando, Nat Rev Mol Cell Biol 14, 329-340 (2013)). The list of differentially expressed genes in RG, sorted by log2 fold change, was used as input for the GSEA function in the clusterProfiler package (G. Yu et al., OMICS 16, 284-287 (2012)). Dot plots were generated using the dotplot function in the clusterProfiler package.

[0051] (15) Correlation-Based Network Analysis. To avoid overcomplicating single-cell transcriptome data, 2,237 metabolome-related genes were selected using Gene ID and the UniProt website before correlation-based network analysis. Based on the clustering results from UMAP, transcriptome data for each cell cluster was extracted using R software (ver. 4.2.1) (transcriptome data for 21 cell clusters). The transcriptome data for each cell cluster was then filtered using metabolome-related genes. These cells were then assigned to each scRNA-seq experiment. Because the transcriptome data for each cell cluster contained a large number of cells, the transcriptome data for each cell cluster in each scRNA-seq experiment was divided into three cohorts. The average value was considered to represent the gene expression level for each cohort. Three average gene expression datasets were obtained for each scRNA-seq experiment (n = 2), resulting in six transcriptome datasets for each cell cluster (i.e., pseudo n = 6 for E18.5, term P2, preterm P0, and preterm P3, respectively). After excluding unexpressed genes from the transcriptome data for each cell cluster, the metabolomic data (n = 6 for E18.5, term P2, preterm P0, and preterm P3) were combined with the transcriptome datasets for each cell cluster (i.e., pseudo n = 6 for E18.5, term P2, preterm P0, and preterm P3) using R software. Following previous studies (Y. Hibino et al., Toxicol Appl Pharmacol 10.1016 / j.taap.2022.116316, 116316 (2022); K. Zaitsu et al., ACS Omega 7, 23717-23726 (2022); K. Zaitsu et al., Life Sci 207, 550-561 (2018)), correlation-based network analysis was applied to the integrated data of each cell cluster. Correlations were considered significant at R > 0.75, and the size of each node (circle) was considered to reflect the betweenness centrality value.In this analysis, we sought to determine the correlation between the amount of metabolites in the V-SVZ and the expression levels of metabolic genes in each cell type. This analysis was performed using the igraph package in R software.

[0052] (16) RNAscope. For RNAscope, brains (three animals per group) were cryosectioned at 10 μm thickness using a Cryostar NX70 (Epredia) and thaw-mounted onto glass slides. RNAscope in situ hybridization, optimized for fresh-frozen samples, was performed as previously described (B. Jablonska et al., Nat Commun 13, 4771 (2022)). Specifically, the RNAscope multiplex fluorescent reagent kit V2 (323100) from Advanced Cell Diagnostics was used according to the manufacturer's instructions. To detect glu l mRNA, RNAscope Probe-Mm-Glul (426231-C2, Advanced Cell Diagnostics) was used. Chromogenic detection was performed using a horseradish peroxidase (HPR) construct and fluorescent Opal 520 reagent (1:1,000, FP1487001KT, Perkin Elmer). Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). Images were acquired using an LSM700 confocal laser scanning microscope (Carl Zeiss) equipped with a 40x objective, scanning at 1 μm intervals. Analysis was performed using ImageJ version 1.54i software (National Institutes of Health). The V-SVZ was traced based on DAPI staining, and the total area of ​​the V-SVZ was measured. RNAscope signals in the V-SVZ were identified, and the area of ​​each RNAscope signal was calculated. The ratio of the total area of ​​the V-SVZ RNAscope signals to the area of ​​the V-SVZ was also calculated.

[0053] (17) Immunohistochemistry: Immunohistochemistry was performed as previously described (H. Jinnou et al., Cell Stem Cell 22, 128-137 e129 (2018)). Brains were perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) and immersed in the same perfusion solution overnight at 4°C. 60 μm-thick coronal sections were prepared using a vibratome (VT1200S, Leica). Sections were incubated with primary antibodies in blocking solution (10% normal donkey serum (Millipore) and 0.5% Triton X-100 in PBS) for 40 minutes at 4°C overnight, followed by incubation with Alexa Fluor-conjugated secondary antibodies (1:500, Invitrogen) for 2 hours at room temperature. The anti-nestin antibody used was AffiniPure donkey anti-chicken IgY secondary antibody (Jackson ImmunoResearch Laboratory Inc.). Signal amplification was performed using a biotinylated secondary antibody (Jackson ImmunoResearch Laboratory Inc.) and a Vectastain Elite ABC kit (Vector Laboratories). Signals were visualized using the TSA Fluorescence System (PerkinElmer). For BrdU staining, H ２ O ２Before processing, sections were treated with 1 M HCl for 30 min at 37°C. The following primary antibodies were used: rabbit anti-glutamine synthetase (Glu) (1:500, Abcam), chicken anti-nestin (1:1,000, Aves Labs), rabbit anti-Mash1 (1:1,000, Abcam), mouse anti-Mash1 (1:000, Santa Cruz Biotechnology), rabbit anti-phospho-S6 ribosomal protein (pS6) (1:500, Cell Signaling Technology), and rabbit anti-phospho-p70 S6 kinase (Thr421) (1:500, Thermo Fisher Scientific). Antibodies used were: guinea pig anti-Dcx (1:500, Millipore), mouse anti-NeuN (1:100, Millipore), rat anti-BrdU (1:100, Abcam), sheep anti-BrdU (1:1,000, Fitzgerald), rat anti-GFP (1:500, Nacalai Tesque), rabbit anti-EGFR (1005) (1:100, Santa Cruz Biotechnology), rabbit anti-DsRed (1:1,200, Takara Bio), and chicken anti-glial ciliary acidic protein (GFAP) (1:1,000, Abcam). For EdU staining, the medium was 150 mM NaCl, 50 mM Tris buffer (pH 7.5), 2 mM CuSO. ４ (II) The sections were incubated with 2 μM Alexa Fluor azide (Life Technologies) and 10 mM sodium ascorbate in distilled water for 2 hours at room temperature and then washed with PBS. Hoechst 33342 (1:3,000, Thermo Fisher Scientific) was used for nuclear staining.

[0054] (18) Confocal Imaging and Quantification. Images were acquired using an LSM700 confocal laser scanning microscope (Carl Zeiss) or an FV3000 confocal microscope (Olympus) with 20x and 40x objectives. The scanning intervals were 0.9 μm (Figures 27, 28(B), and 30), 0.96 μm (Figures 28(C) and 29), 1 μm (Figures 16-20, 21, 22, and 31-46), and 2 μm (Figures 23-26 and 28(A)). For quantitative analysis of V-SVZ cells at 47.5, 48.5, and 52.5 dpc, sections covering the coronal plane of the V-SVZ (+1.2 to +0.1 mm relative to bregma) were examined. For quantitative analysis at 18.5 dpc, 21.5 dpc, 26.5 dpc, and 34.5 dpc, areas corresponding to those observed at 47.5 dpc were examined.

[0055] To quantify the cell density of immunoreactive RG, all immunoreactive cells in the lateral wall of the lateral ventricle were counted in two consecutive 60 μm-thick coronal sections in Figures 19, 20, 40(A), and 40(B). The cell density in the V-SVZ was calculated by dividing the cell density by the V-SVZ area measured using Zen software (Carl Zeiss). In Figure 22 (21.5, 26.5, and 34.5 dpc) and Figure 40(C), Mash1 was used. ＋ For the analysis of neural progenitor cells, all immunopositive cells in two consecutive 60 μm-thick coronal sections were counted for every six sections, and the sum was multiplied by 6 to estimate the total number of cells per hemisphere. ＋ For the analysis of neural progenitor cells, all immunopositive cells in three consecutive sections were counted for every six 60 μm-thick coronal sections, and the sum was multiplied by six to estimate the total number per hemisphere. ＋ For neuroblast analysis, the number of immunopositive cells in one 2-μm-thick z-plane was counted and multiplied by 10 to estimate the total number of cells per section (mounted sections: 20 μm thick). Furthermore, the total number of cells per hemisphere was estimated by multiplying the sum of the cell counts in three 60-μm-thick coronal sections by six. BrdU in the GCL of the OB shown in Figure 26. ＋ NewN＋ For neuronal density analysis, immunoreactive cells were counted in five consecutive 60-μm coronal sections (+3.5 mm to +5.3 mm relative to bregma) and divided by the volume of the GCL analyzed using Zen software (Carl Zeiss). For NSC density quantification in Figure 28(A), immunoreactive cells were counted in three consecutive 60-μm coronal sections and divided by the V-SVZ area to calculate cell density. For activated NSC density quantification in Figure 28(B) and Figure 42, immunoreactive cells were counted in nine consecutive 60-μm coronal sections (every two sections). Cell density was calculated by dividing by the V-SVZ area. For tdTomato in Figure 28(C), immunoreactive cells were counted in nine consecutive 60-μm coronal sections. ＋ Mash 1 ＋ Neural progenitor cells and tdTomato ＋ GFAP ＋ For the analysis of the ratio to NSCs, all immunopositive cells were counted in three consecutive 60-μm coronal sections per six sections, and the ratio was calculated. For the analysis of Glul-KD and Glul-OE in the RG in Figures 32, 34, 36, and 38, all immunopositive cells were counted in two consecutive 60-μm coronal sections per six sections. The fluorescence intensity of Glul in the RG was analyzed using GFAP-EGFP mice (Figures 16 and 17). The cell outline of the RG was traced based on EGFP staining, and the Glul-KD and Glul-OE in the RG were analyzed using ZEN software (Carl Zeiss). ＋ The fluorescence intensity of the signal was quantified. 20 cells were randomly selected for analysis from each individual. A total of 60 cells were analyzed at each time point. Relative intensity was calculated by setting the Glu intensity of RG at E18.5 as 1 (Figure 17).

[0056] (19) Classification of V-SVZ Cells. At 18.5, 21.5, and 26.5 dpc, cells with apical contacts to the lateral ventricle and nestin expression on long basal fibers were classified as RG (Figures 18-20, 39, 40(A), and 40(B)). In GFAP-EGFP mice, EGFP cells with apical contacts to the lateral ventricle and nestin expression on long basal fibers were classified as RG. ＋At 18.5 and 21.5 dpc, cells were classified as RG (Fig. 16, Fig. 17). In GFAP-EGFP mice, EGFP cells with apical contacts in the lateral ventricle were identified at 47.5 dpc. ＋ The cells were classified as NSCs (Fig. 27, Fig. 28(A)). ＋ EGFR ＋ Mash 1 ＋ The cells were classified as active neurogenic NSCs (Fig. 27, Fig. 28(B)). In R26-tdTomato mice, tdTomato cells had apical contacts with the lateral ventricles and expressed GFAP in their long basal processes. ＋ The cells were classified as NSCs and ＋ Mash 1 ＋ The cells were classified as neural progenitor cells (Fig. 28(C), Fig. 29). In the Glul-KD and Glul-OE experiments, EGFP cells with apical contacts in the lateral ventricle and GFAP expression in their long basal processes were identified. ＋ The cells were classified as NSCs (Figures 31 to 38). ＋ Mash 1 ＋ The cells were classified as neural progenitor cells (Figures 33, 34, 37, 38). In ICR mice, EGFR cells have apical contacts in the lateral ventricle and express GFAP on their long basal processes. ＋ Mash 1 ＋ The cells were classified as activated neurogenic NSCs (FIGS. 41 and 42).

[0057] (20) Viral Vectors and Plasmids. pCMV6-Glul was purchased from OriGene Technologies, Inc. (MR205788). The CSII lentiviral expression vector was provided by Dr. Hiroyuki Miyoshi (RIKEN Tsukuba BioResource Center). To construct pENTR-D-TOPO-Glul, Glul cDNA was amplified from pCMV6-Glul by PCR and inserted into the pENTR-D-TOPO vector by In-Fusion cloning (Takara Bio). The knockdown (KD) vector was constructed as previously described (M. Sawada et al., EMBO J 37, e97404 (2018)). The target sequence of the mouse glu gene was inserted into a modified Block-iT Pol II miR RNAi entry vector containing EmGFP (Invitrogen). A lacZ target sequence was used as a control. The following CSII lentiviral expression vectors were constructed using the Gateway System (Invitrogen): CSII-EF-EmGFP-miR-lacZ, CSII-EF-EmGFP-miR-glul, and pCSII-EF-Glul-IRES2-Venus. These viral vectors and packaging vectors (pCAG-HIVgp and pCMV-VSV-G-RSV-Rev) were cotransfected into HEK293T cells to produce lentiviral particles. The culture supernatants were then concentrated by centrifugation at 8,000 rpm at 4°C for 16 hours in an MX-307 refrigerated microcentrifuge (Tomy).

[0058] (21) Cell Culture, In Vitro Viral Infection, and Measurement of Intracellular Concentrations of Glutamate and Glutamate. V-SVZ tissue from ICR P0 mice was detached in L-15 medium (Invitrogen) and dissociated with trypsin-EDTA (Invitrogen). Cells were washed with L-15 medium (Invitrogen) containing 40 μg / ml DNase I (Roche Diagnostics), plated in 24-well plates, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin B (Thermo Fisher Scientific), and 2 mM GlutaMAX (Thermo Fisher Scientific). The medium also contained human epidermal growth factor (EGF, 20 ng / ml; AF-100-15, Peprotech) and human fibroblast growth factor (FGF)-basic (20 ng / ml; 100-18B, Peprotech). Ten microliters of lentivirus suspension was added per well. The plates were incubated at 37°C, 5% CO. ２ The cells were maintained in a humidified incubator. Two days after virus infection, the medium was replaced. Four days later, the glutamine and glutamic acid contents in the cell lysates were assessed by luminescence detection using the Glutamine / Glutamate-Glo Assay (Promega) according to the manufacturer's protocol.

[0059] (22) Immunocytochemistry. V-SVZ cells cultured on coverslips were rinsed with PBS (pH 7.4) and fixed with 4% PFA in 0.1 M PBS for 30 minutes at room temperature. The cells were then incubated in blocking solution (10% normal donkey serum and 0.5% Triton X-100 in PBS) for 30 minutes, incubated with primary antibodies overnight at 4°C, and then incubated with Alexa Fluor-conjugated secondary antibodies (1:1,000, Invitrogen) for 2 hours at room temperature. The following primary antibodies were used: chicken anti-nestin (1:2,000, Aves Labs), guinea pig anti-Dcx (1:800, Millipore), and rabbit anti-Sox2 (1:1,000, Millipore). Hoechst 33342 (1:3,000, Thermo Fisher Scientific) was used for nuclear staining. The labeled cells were observed using an LSM700 confocal laser scanning microscope (Carl Zeiss) with 20x and 40x objective lenses.

[0060] (23) In Vivo Viral Infection. For Glu-KD experiments, pregnant mice at E16.5 were anesthetized with a mixed anesthetic (0.75 mg / kg medetomidine, 4 mg / kg midazolam, and 5 mg / kg butorphanol). After laparotomy, the uterus of the pregnant animals was exposed, and 2 μL of lentivirus suspension was manually injected into the lateral ventricle of the right fetal hemisphere. The surgical incision was closed with sutures. For Glu-OE experiments, preterm mice at P0 were anesthetized with hypothermia (5 min) and secured to the platform of a stereotaxic injection apparatus (David Kopf Instruments) with skull clamps. Two microliters of lentivirus suspension was injected into the lateral ventricles of both cerebral hemispheres (1.5 mm anterior and 0.8 mm lateral to lambda, 2.5 mm deep).

[0061] (24) Rapamycin Treatment: Rapamycin (Tokyo Chemical Industry Co., Ltd.) was dissolved in 1.25% DMSO (Sigma-Aldrich), 15% PEG-400 (Sigma-Aldrich), and 5% Tween-20 (Sigma-Aldrich) to prepare a stock solution, which was then dissolved in PBS for injection. Rapamycin was intraperitoneally injected at 0.25 mg / kg into preterm mice at P0 (18.5 dpc) and P2 (20.5 dpc). Rapamycin was also intraperitoneally injected at 0.25 mg / kg into term mice at P0 (19.5 dpc) and P2 (21.5 dpc).

[0062] (25) Experimental Design and Statistical Analysis: Statistical analysis was performed using EZR (Y. Kanda, Bone Marrow Transplant 48, 452-458 (2013)) as previously reported (K. Fujikake et al., J Neurosci 38, 4598-4609 (2018)). Experiments were not randomized. Sample size was not predetermined but was selected based on previous studies (H. Jinnou et al., Cell Stem Cell 22, 128-137 e129 (2018) and M. Matsumoto et al., J Neurosci 39, 9967-9988 (2019)). All numerical data were bilateral and presented as mean ± SEM (standard error of the mean). Normality of the data was analyzed using the Kolmogorov-Smirnov test. For normally distributed data, homogeneity of variance was examined using the F test, and comparisons of means between two independent groups were performed using the unpaired t-test or Welch's t-test. Comparisons of means between three independent groups were performed using one-way analysis of variance followed by the Tukey-Kramer test. For data that were not normally distributed, comparisons of medians between groups were performed using the Mann-Whitney U test or the Kruskal-Wallis test followed by the Steel-Dwass test. *p<0.05, **p<0.01, and ***p<0.001 are indicated in the figures.

[0063] 2. Experimental content and results <Confirmation of oxygen partial pressure level in V-SVZ> The oxygen partial pressure (pO ２ ) levels were measured. To evaluate the effects of birth and its timing, full-term mice and mifepristone-induced preterm mice were used. Full-term and preterm mice were compared at the same day per cycle (dpc). The number and weight of offspring produced by preterm mice were similar to those of full-term mice.

[0064] Figure 3 is an explanatory diagram showing the results of oxygen partial pressure levels in V-SVZ. In Figure 3, the vertical axis represents pO ２ The pO (mmHg) of the V-SVZ in full-term mice on postnatal day 2 (P2) is shown. ２ The levels were significantly higher in the V-SVZ at postnatal day 3 (P3) in preterm mice compared to embryonic day 18.5 (E18.5), which is before full-term birth. ２ Levels were unchanged compared to postnatal day 2 (P2) in full-term mice, leading us to hypothesize that parturition causes changes in the metabolic profile of the V-SVZ.

[0065] To verify the above hypothesis, metabolomic analysis was performed on the entire V-SVZ in the lateral wall of the lateral ventricle using PESI / MS / MS. The V-SVZ was excised and analyzed at each time point.

[0066] Figure 4 shows the results of principal component analysis of the V-SVZ metabolome analysis. As shown in Figure 4, the metabolic profile in the V-SVZ of E18.5 mice was clearly different from that of full-term P2 mice. Furthermore, preterm mice exhibited a distinct metabolic profile on both the day of birth (P0) and postnatal day 3 (P3). Metabolomic analysis revealed that the levels of metabolites involved in glycolysis, such as fructose 1,6-bisphosphate and glyceraldehyde 3-phosphate, and many amino acids, such as glutamine and glutamic acid, were significantly lower in the V-SVZ of full-term P2 mice compared with the V-SVZ of E18.5 mice (not shown).

[0067] Figure 5 shows the relative intensities of each metabolite. Figure 5 shows the relative intensities of glutamine, glutamate, α-ketoglutarate (αKG), and lactate at each time point. The V-SVZ of full-term mice (P2) had significantly lower levels of glutamine, glutamate, αKG, and lactate than the V-SVZ of E18.5 mice. These results suggest that during full-term birth, a decrease in amino acids and a metabolic shift to aerobic conditions occur in the V-SVZ, resulting in decreased glutaminolysis. Furthermore, the levels of amino acids, including glutamine, at 18.5 dpc were significantly lower in the V-SVZ of preterm mice (P0) than in the V-SVZ of E18.5 mice. This suggests that parturition itself induces a decrease in amino acids in the V-SVZ. When comparing term and preterm births, the levels of fructose 1,6-bisphosphate and glyceraldehyde 3-phosphate at 21.5 dpc were significantly higher in the V-SVZ of preterm P0 mice than in the V-SVZ of term P2 mice (not shown). Furthermore, the levels of glutamate, αKG, and lactate at 21.5 dpc were significantly higher in the V-SVZ of preterm P0 mice than in the V-SVZ of term P2 mice. These results suggest that birth-related changes in pO ２ Despite elevated glutamate levels, preterm births were associated with persistent anaerobic conditions in the V-SVZ. Furthermore, it was also suggested that glutamine-to-glutamate conversion was enhanced in the V-SVZ during preterm births. This suggests that preterm births result in an incomplete reduction in glutaminolysis in the V-SVZ. Furthermore, we also analyzed mice administered mifepristone at 18.5 days after birth (full-term mice administered mifepristone to their mothers) born at 19.5 days after birth. We found that mifepristone, a labor-inducing agent, did not significantly affect the metabolomic profile of the V-SVZ (not shown).

[0068] Single-cell gene expression analysis: To characterize birth-related changes in V-SVZ cells, we performed scRNA-seq across the entire V-SVZ, the lateral wall of the lateral ventricle, in E18.5 mice, full-term mice at P2, preterm mice at P0, and preterm mice at P3. A total of 50,389 cells were analyzed: 10,331 cells in E18.5, 14,826 cells in full-term mice at P2, 14,026 cells in preterm mice at P0, and 11,206 cells in preterm mice at P3. Sequenced V-SVZ cells were clustered and assigned to cell types based on the expression of known marker genes.

[0069] Figure 6 shows the clustering results of single-cell gene expression analysis. Radial glia (RG), a neural stem cell in the fetal stage, were identified as a unique cluster. To estimate the metabolic state of RG, we analyzed the expression of genes related to glycolysis and glutamine metabolism.

[0070] Figure 7 is a violin plot showing the expression of glycolysis-related genes in the RG. The expression of glycolysis-related genes, such as Gapdh, Ldha, and Eno1, was found to decrease at term. In general, the expression of these genes increases in cells under anaerobic conditions. Therefore, a metabolic shift to aerobic conditions occurs in the RG itself, and the pO ２ At 21.5 days after birth, the expression of Gapdh, Ldha, and Eno1 was elevated in preterm mice, suggesting that the RG in preterm mice is under persistent anaerobic conditions. To estimate the level of glutamine metabolism in the RG, we analyzed the expression of GluI and Gls.

[0071] Figure 8 is an explanatory diagram showing the expression of Glul and Gls visualized by UMAP. Figure 9 is a violin plot showing the expression of Glul and Gls in RG. Glul is a gene encoding an enzyme that converts glutamate to glutamine. Gls is a gene encoding an enzyme that converts glutamine to glutamate. In RG, the expression level of Gls was negligible, but Glul was specifically expressed. Furthermore, Glul expression increased at full term, suggesting that glutamine synthesis from glutamate occurs cell-autonomously in full-term RG. When compared at 21.5 dpc, Glul expression was reduced in preterm mice, suggesting that changes in glutamine metabolism after birth are incomplete in RG.

[0072] <GSEA in RG> To investigate how preterm birth affects the biological properties of RG, GSEA (Gene Set Enrichment Analysis) was performed.

[0073] Figure 10 shows dot plots of GSEA results for GO terms related to ribosome and ribonucleoprotein synthesis. In Figure 10 and Figure 11 (discussed later), gene ratios indicate the proportion of significant genes associated with a given GO (Gene Ontology) term, dot size indicates the number of genes, and color indicates the adjusted p-value (Benjamini-Hochberg adjustment). GO terms related to ribosome and ribonucleoprotein synthesis were enriched in E18.5 RG compared with full-term P2 RG. These GO terms are characteristic of activated NSCs. The decrease in expression of genes involved in ribosome and ribonucleoprotein synthesis at term suggests a transition from an activated to a quiescent state during term birth. These activated NSC characteristics were also enriched in preterm P3 RG compared with the levels in full-term P2 RG.

[0074] Figure 11 shows dot plots of GSEA results for active and quiescent stem cell signatures in RG. Figure 11(A) shows GSEA results showing GO terms related to mitochondrial respiratory chain complexes. Figure 11(B) shows GSEA results using custom-defined gene sets for stem cell activation and quiescence signatures, comparing E18.5RG and preterm P2RG. Figure 11(C) shows GSEA results using custom-defined gene sets for stem cell activation and quiescence signatures, comparing E18.5RG and preterm P0RG. Figure 11(D) shows GSEA results using custom-defined gene sets for active and quiescent stem cell signatures. As shown in Figure 11(A), GO terms related to the mitochondrial respiratory chain were also enriched in preterm P3 RGs compared with full-term P2 RGs, suggesting that preterm birth causes changes in mitochondrial function in RGs. Furthermore, to comprehensively evaluate RG activity and quiescence, we performed GSEA using stem cell activity and quiescence marker genes. As shown in Figure 11(B), the quiescent stem cell signature was enriched in full-term P2 RGs compared with E18.5 RGs. As shown in Figure 11(C), the quiescent stem cell signature was also enriched in preterm P0 RGs compared with E18.5 RGs. However, as shown in Figure 11(D), it was suppressed at 21.5 dpc, and the active stem cell signature was enriched in preterm P3 RGs compared with full-term P2 RGs. These results suggest that birth induces a transition from an active to a quiescent state in RG, while preterm birth impairs this transition.

[0075] <RG Correlation Network Analysis> To investigate the correlation between changes in metabolite levels in the V-SVZ and gene expression in each cell type, a correlation network analysis was performed integrating metabolomics data and scRNA-seq data.

[0076] Figure 12 is an explanatory diagram showing the results of network analysis based on RG correlation. In Figure 12, correlations are considered significant when R > 0.75, and the size of each node is considered to reflect the value of betweenness centrality. αKG and Glu are tightly bound via several RG molecules. Therefore, it was found that there is a strong correlation between changes in Glu expression in RG and changes in the amount of αKG metabolites.

[0077] Figure 13 is an explanatory diagram summarizing the results of metabolomic analysis and scRNA-seq in the V-SVZ. The above results indicate that parturition induces a transition from anaerobic to aerobic metabolism in the RG and upregulation of GluI expression, suggesting that changes in glutamine metabolism are involved. Full-term birth increases GluI expression in the RG, resulting in decreased glutaminolysis and a decrease in αKG. As a result, neural stem cells attain quiescence. In contrast, preterm birth results in a poor increase in GluI expression in the RG, resulting in an incomplete decrease in glutaminolysis in the RG and an increase in αKG. As a result, neural stem cells are unable to attain quiescence. This results in an activated state compared to full-term birth.

[0078] <Confirmation of Glul Expression in RG> To confirm the in vivo expression of Glul in RG, RNA scope and immunohistochemical staining were performed.

[0079] Figure 14 shows a representative staining image of Glul RNA in a coronal V-SVZ section. A 20 μm scale bar is also shown in Figure 14. Figure 15 is an explanatory diagram showing the percentage of dot area of ​​Glul RNA in the V-SVZ region. Figure 15 shows the results of a one-way analysis of variance followed by a Tukey-Kramer test (n=3). Figure 16 shows a representative staining image of a coronal V-SVZ section from a GFAP-EGFP mouse. Figure 16 shows the staining results for EGFP, Glul, and Nestin, as well as merged images of these results. The trace outline of the RG based on EGFP staining is indicated by a dashed line, and a 20 μm scale bar is also shown. Figure 17 is an explanatory diagram showing the relative expression level of Glul in the RG. Figure 17 shows the results of a Kruskal-Wallis test followed by a Steel-Dwass test for n = 60 cells from three mice. These results indicate that Glu expression in the RG increases during the course of full-term birth, but decreases at 21.5 dpc in preterm mice.

[0080] <Histological confirmation of changes in RG neurogenic activity> Figure 18 shows representative staining images of coronal V-SVZ sections. In Figure 18, staining results for pS6, Mash1, and Nestin are shown. ＋ pS6 － Nestin ＋ RG is indicated by a white arrow, and Mash1 － pS6 ＋ Nestin ＋ RG is indicated by a gray arrow, and Mash1 ＋ pS6 ＋ Nestin ＋ The RG is indicated by an arrowhead, and a 50 μm scale bar is also shown. In Figure 18, the area indicated by the dashed line in the image on the left side of the page is shown enlarged on the right side of the page. Figure 19 shows the Mash1 of the V-SVZ. ＋ pS6 ＋ Nestin ＋Figure 19 shows the density of RGs. Figure 19 shows the results of an unpaired t-test using n = 4 mice at 18.5 dpc, 21.5 dpc, and 26.5 dpc. All RGs expressed Nestin in their long fibers, regardless of neurogenic activity. The density of Mash1+Nestin+ neurogenic RGs in the lateral wall decreased after full-term birth, but transiently increased in preterm mice at 21.5 dpc compared with full-term mice. In the dorsal and medial walls, preterm birth significantly increased Mash1+Nestin+. ＋ Nestin ＋ Since preterm birth did not induce a transient increase in neurogenic RGs (not shown), suggesting that preterm birth specifically impairs the acquisition of quiescence in lateral RGs, we examined the activity of mTORC1 signaling in RGs by immunostaining for phosphorylated ribosomal protein S6 (pS6).

[0081] Figure 20 is an explanatory diagram showing a comparison of mTORC1 signaling activity between full-term and preterm births. ＋ Nestin ＋ The density of RG is shown, and the results of an unpaired t-test using n = 3 mice at 18.5 dpc, 21.5 dpc, and 26.5 dpc are shown. Figure 20(B) shows the density of RG at 21.5 dpc. ＋ Nestin ＋ RG and pS6 － Nestin ＋ Mash1 in RG ＋ The percentage of cells is shown, and the results of a Mann-Whitney U test using n = 4 mice for each group are shown. ＋ Mash 1 ＋ Nestin ＋ The density of RG is shown, and the results of an unpaired t-test using n = 4 mice are shown. As shown in Figure 18 and Figure 20(A), pS6 ＋ Nestin ＋ The density of RG decreased after full-term birth. At 18.5 days after birth, pS6 ＋ Nestin ＋The density of RG was significantly lower in preterm P0 mice than in embryonic E18.5 mice, suggesting that birth itself induces downregulation of mTORC1 signaling in RG. ＋ Nestin ＋ The density of RG transiently increased in preterm mice at 21.5 days of age compared to full-term mice. As shown in Figure 20(B), pS6 － RG hardly expressed Mash1, but pS6 ＋ RGs expressed Mash1 more frequently, suggesting that mTORC1 signaling in RGs is involved in differentiation into neural progenitor cells. ＋ Mash1 in RG ＋ The proportion of pS6+Mash1+Nestin+RG cells did not differ between full-term and preterm mice at 21.5 dpc. As shown in Figure 20(C), the density of pS6+Mash1+Nestin+RG cells was also increased in preterm mice at 21.5 dpc. These results suggest that in preterm mice, RG cells transiently enter a neurogenic state via mTORC1 signaling, concomitant with insufficient upregulation of Glul.

[0082] <Confirmation of the effect of preterm birth on postnatal neurogenesis> Figure 21 shows representative images of Mash1 staining of coronal V-SVZ sections at 21.5, 26.5, 34.5, and 47.5 dpc. In Figure 21, LV stands for lateral ventricle, and Str stands for striatum. A scale bar of 50 μm is also shown in Figure 21. Figure 22 shows Mash1 staining of V-SVZ sections at 21.5, 26.5, 34.5, and 47.5 dpc. ＋ This is an explanatory diagram showing the number of neural progenitor cells. Figure 22 shows the results of an unpaired t-test using mice born at 21.5 dpc (n = 6), 26.5 dpc (n = 8), 34.5 dpc (n = 6), 47.5 dpc (n = 8) full-term, and 47.5 dpc (n = 6) preterm. In preterm mice, Mash1 in the lateral V-SVZ was detected at 26.5 dpc. ＋The number of neural progenitor cells was increased compared to full-term mice. This result was consistent with the results in Figure 19, which showed that preterm RG transiently enters a neurogenic state. However, in preterm mice, Mash1 ＋ The number of neural progenitor cells was reduced at 34.5 and 47.5 dpc.

[0083] Figure 23 shows representative staining images of coronal V-SVZ sections stained with Dcx at 47.5 dpc. In Figure 23, LV is the lateral ventricle and Str is the striatum. A 50 μm scale bar is also shown in Figure 23. Figure 24 shows the staining images of Dcx in the V-SVZ at 47.5 dpc. ＋ Figure 23 shows the number of newborn neurons in the lateral V-SVZ at 47.5 days after birth. Figure 24 shows the results of an unpaired t-test using n = 5 mice. As shown in Figures 23 and 24, the number of newborn neurons in the lateral V-SVZ was reduced at 47.5 days after birth in preterm mice. This result indicates that neurogenesis in young adulthood is reduced in preterm mice.

[0084] To assess the number of newly generated neurons reaching the olfactory bulb (OB), full-term and preterm mice were intraperitoneally injected with BrdU (bromodeoxyuridine) at 47.5 days after birth (dpc) and fixed at 75.5 days after birth (dpc).

[0085] Figure 25 shows representative staining images of coronal sections of the olfactory bulb granule cell layer (GCL) at 75.5 dpc. In Figure 25, staining for NeuN and BrdU was performed. ＋ NewN ＋ Neurons are indicated by arrowheads, and a 50 μm scale bar is also shown. In Figure 25, the area indicated by the dashed line in the image on the left side of the page is shown enlarged on the right side of the page. Figure 26 shows the BrdU signaling in the GCL at 75.5 dpc. ＋ NewN ＋ Fig. 26 shows the density of neurons. In preterm mice, BrdU in the GCL of OB was significantly increased. ＋NewN ＋ Therefore, preterm birth may reduce neurogenesis in the V-SVZ, leading to a decrease in BrdU expression in the olfactory bulb. ＋ NewN ＋ The number of neurons, namely mature neurons, was shown to decrease. Next, the number of NSCs was evaluated using GFAP-EGFP mice.

[0086] Figure 27 shows representative staining images of coronal V-SVZ sections of GFAP-EGFP mice stained for EGFP, Mash1, and EGFR at 47.5 dpc. ＋ EGFR － Mash 1 － NSCs are indicated by white arrowheads, and EGFP, which are NSCs in the activated and neural development stages, ＋ EGFR ＋ Mash 1 ＋ NSCs are indicated by gray arrowheads. In Figure 27, the area indicated by the dashed line in the image on the left side of the page is shown enlarged on the right side of the page. Figure 28 is an explanatory diagram showing the density of each cell type at 47.5 dpc. Figure 28(A) shows the density of EGFP cells. ＋ The density of NSCs is shown in Fig. 28(B). ＋ EGFR ＋ Mash 1 ＋ The density of NSCs is shown in Fig. 28(C). ＋ EGFR in NSCs ＋ Mash 1 ＋ Fig. 28(A) shows the results of an unpaired t-test using n = 3 mice, and Fig. 28(B) and (C) show the results of an unpaired t-test using n = 5 mice. As shown in Fig. 27 and Fig. 28(A), EGFP, which is a neural stem cell, ＋ The density of NSCs was not significantly different between full-term and preterm mice at 47.5 days after birth. However, as shown in Figures 27 and 28(B), the density of EGFP-positive NSCs, which are activated neural stem cells that produce neurons, was significantly higher than that of the control mice. ＋ EGFR ＋ Mash 1 ＋The density of NSCs was decreased in preterm mice. ＋ EGFP in NSCs ＋ EGFR ＋ Mash 1 ＋ The proportion of NSCs was also reduced in preterm mice, suggesting a depletion of activated neural stem cells that generate neurons.

[0087] To examine the ability of NSCs to generate neural progenitor cells, we used the GFAP-CreERT2;R26-tdTomato double transgenic mouse strain. In this strain, tamoxifen administration induces the production of GFAP ＋ tdTomato expression is induced in NSCs and their progeny. Term and preterm mice were administered tamoxifen at 47.5 dpc. At 48.5 dpc (1 day after tamoxifen administration), tdTomato expression in the V-SVZ was significantly increased. ＋ Approximately half of the cells are GFAP ＋ None of the NSCs expressed Mash1, indicating specific Cre-mediated recombination in NSCs.

[0088] Figure 29 shows representative staining images of coronal V-SVZ sections of GFAP-CreERT2;Rosa26-tdTomato mice stained with Mash1, tdTomato, and GFAP at 52.5 dpc. ＋ GFAP ＋ NSCs are indicated by white arrowheads, and tdTomato ＋ Mash 1 ＋ Neural progenitor cells are indicated by gray arrowheads and a 20 μm scale bar is also shown. ＋ GFAP ＋ tdTomato for NSCs ＋ Mash 1 ＋ 29 and 30, in prematurely born mice at 52.5 dpc (5 days after tamoxifen administration), tdTomato was expressed in the 29th and 30th mice, respectively. ＋ GFAP＋ tdTomato for NSCs ＋ Mash 1 ＋ The proportion of neural progenitor cells was decreased, suggesting a reduced capacity of NSCs to generate neural progenitor cells. These results suggest that preterm birth transiently activates RG early after birth, resulting in neural stem cell depletion and reduced postnatal neurogenesis in young adults.

[0089] To inhibit the decline in glutaminolysis in RG associated with term birth, lentivirus expressing a GluI knockdown (KD) vector was injected into the lateral ventricle of embryonic day E16.5 (GluI-KD). These mice were born at term, and the fate of RG was analyzed on postnatal day 7 (P7).

[0090] Figure 31 shows representative staining images of coronal V-SVZ sections stained with EGFP, pS6, and GFAP at P7. ＋ pS6 － GFAP ＋ NSCs are indicated by white arrowheads, and EGFP ＋ pS6 ＋ GFAP ＋ NSCs are indicated by gray arrowheads, and a 20 μm scale bar is also shown. In Figure 31, the area indicated by the dashed line in the image on the left side of the page is shown enlarged on the right side of the page. Figure 32 shows the EGFP expression at P7. ＋ GFAP ＋ pS6 in NSCs ＋ Figure 32 shows the results of an unpaired t-test using n=5 mice. Figure 33 shows representative staining images of coronal V-SVZ sections stained with EGFP, Mash1, and GFAP at P7. In Figure 33, EGFP ＋ GFAP ＋ NSCs are indicated by white arrowheads, and EGFP ＋ Mash 1 ＋Neural progenitor cells are indicated by gray arrowheads, and a 20 μm scale bar is also shown. In Figure 33, the area indicated by the dashed line in the image on the left side of the page is shown enlarged on the right side of the page. Figure 34 shows EGFP at P7. ＋ GFAP ＋ EGFP for NSCs ＋ Mash 1 ＋ Fig. 34 shows the proportion of neural progenitor cells. Figure 34 shows the results of an unpaired t-test using n=5 mice. Glul-KD increased pS6 ＋ GFAP ＋ NSCs, i.e., neural stem cells positive for pS6, an mTOR signaling marker, are increased. Glul-KD also increases Mash1 expression in the RG. ＋ These results suggest that the decline in glutaminolysis in the RG at term leads to the acquisition of neural stem cell quiescence via the suppression of mTOR signaling.

[0091] <Experiment on the Decrease of Glutaminolysis After Preterm Birth> Based on the above results, the inventors hypothesized that the decrease in glutaminolysis in RG in preterm newborns is insufficient, resulting in increased activity, and tested this hypothesis. To sufficiently decrease glutaminolysis in RG after preterm birth, a lentivirus expressing a GluI overexpression vector was injected intracerebroventricularly on postnatal day 0 (P0) (GluI-OE). The fate of RG was analyzed on postnatal day 8 (P8).

[0092] Figure 35 shows representative staining images of coronal V-SVZ sections stained with EGFP, pS6, and GFAP at P8. ＋ pS6 － GFAP ＋ NSCs are indicated by white arrowheads, and EGFP ＋ pS6 ＋ GFAP ＋ NSCs are indicated by gray arrowheads, and a 20 μm scale bar is also shown. In Figure 35, the area indicated by the dashed line in the image on the left side of the page is shown enlarged on the right side of the page. Figure 36 shows the EGFP expression at P8. ＋GFAP ＋ pS6 in NSCs ＋ Figure 36 shows the results of an unpaired t-test using n=5 mice. Figure 37 shows representative staining images of coronal V-SVZ sections stained with EGFP, Mash1, and GFAP at P8. In Figure 37, EGFP ＋ GFAP ＋ NSCs are indicated by white arrowheads, and EGFP ＋ Mash 1 ＋ Neural progenitor cells are indicated by gray arrowheads, and a 20 μm scale bar is also shown. In Figure 37, the area indicated by the dashed line in the image on the left side of the page is shown enlarged on the right side of the page. Figure 38 shows EGFP at P8. ＋ GFAP ＋ EGFP for NSCs ＋ Mash 1 ＋ Fig. 38 shows the proportion of neural progenitor cells. In Fig. 38, the results of an unpaired t-test using n = 4 mice are shown. Glul-OE in the preterm RG is expressed in pS6 ＋ GFAP ＋ Glu-OE in preterm RG decreased NSCs. ＋ These results suggest that the reduction of glutaminolysis in RGs at the appropriate time of birth is important for the acquisition of neural stem cell quiescence.

[0093] <Administration experiment of mTORC1 signal inhibitor> We investigated a clinically applicable therapeutic strategy targeting excessive mTORC1 signaling in the RG of preterm newborns. Rapamycin, an mTORC signal inhibitor, was intraperitoneally injected at 0.25 mg / kg at P0 and P2 in preterm mice. A vehicle without rapamycin was also administered as a control.

[0094] Figure 39 shows representative staining images of Mash1, pS6, and Nestin in coronal V-SVZ sections at preterm P3. ＋ pS6 ＋ Nestin ＋The V-SVZ is indicated by a gray arrowhead, and a 50 μm scale bar is also shown. Figure 40 is an explanatory diagram showing each cell in the V-SVZ of preterm P3. Figure 40 (A) shows the Mash1 ＋ Nestin ＋ The density of the V-SVZ is shown in Fig. 40(B). ＋ pS6 ＋ Nestin ＋ The density of the V-SVZ is shown in Fig. 40(C), and the Mash1 of the V-SVZ is shown in Fig. 40(D). ＋ The numbers of neural progenitor cells are shown. Figures 40(A) and (B) show the results of an unpaired t-test using n = 5 mice, and Figure 40(C) shows the results of an unpaired t-test using n = 4 mice. As shown in Figures 39, 40(A) and (B), at preterm birth P3, Mash1 ＋ Nestin ＋ RG and Mash1 ＋ pS6 ＋ Nestin ＋ Both Mash1 and RG were reduced by rapamycin treatment. ＋ The number of neural progenitor cells was also reduced. These results suggest that the increased expression of RG in preterm mice was suppressed by rapamycin treatment.

[0095] Figure 41 shows representative staining images of coronal V-SVZ sections stained for EGFR, Mash1, and GFAP at preterm birth P29. ＋ Mash 1 ＋ GFAP ＋ NSCs are indicated by gray arrowheads, and a 50 μm scale bar is also shown. In Figure 41, the area indicated by the dashed line in the image on the left side of the page is shown enlarged on the right side of the page. Figure 42 shows EGFR in the V-SVZ at preterm birth P29. ＋ Mash 1 ＋ GFAP ＋Figure 43 shows representative staining images of coronal V-SVZ sections stained with Mash1 at preterm P29. The 50 μm scale bar is also shown in Figure 43. Figure 44 shows the density of NSCs in the V-SVZ at preterm P29. ＋ 42 and 44 show the results of an unpaired t-test using n=5 mice. As shown in Fig. 41 and Fig. 42, administration of rapamycin to prematurely born mice increased the number of activated neural stem cells (GFAP) that produce neurons. ＋ EGFR ＋ Mash 1 ＋ Furthermore, as shown in Figures 43 and 44, administration of rapamycin to preterm mice increased neural progenitor cells (Mash1-positive cells) in the V-SVZ. These results suggest that administration of an mTOR inhibitor can prevent the depletion of neural stem cells caused by preterm birth and other conditions.

[0096] For comparison, a similar experiment was performed on full-term mice. 0.25 mg / kg of rapamycin was intraperitoneally injected into full-term mice at P0 and P2, and the mice were fixed at P28 (47.5 days after birth). A vehicle containing no rapamycin was also administered as a control.

[0097] Figure 45 shows representative staining images of Mash1-stained coronal V-SVZ sections at term P28. The 50 μm scale bar is also shown in Figure 45. Figure 46 shows Mash1 staining of the V-SVZ at term P28. ＋ Fig. 46 shows the number of neural progenitor cells. Fig. 46 shows the results of an unpaired t-test using n=5 mice. As shown in Fig. 45 and Fig. 46, administration of rapamycin to full-term mice did not significantly increase the number of Mash1 cells in the young adult stage. ＋ There was no effect on neural progenitor cell numbers.

[0098] 3. Summary Figure 47 is an explanatory diagram comparing RG in full-term birth and RG in preterm birth. In mammals, the fetus in the uterus is physiologically in a hypoxic state, and birth is accompanied by an escape from this state. In the above example, the pO２ It has been shown that the level of RG increases in normal full-term birth, and RG shifts to an aerobic state. Furthermore, metabolomics has revealed that birth itself causes a decrease in amino acids in the V-SVZ. This is thought to be due to the cessation of amino acid supply via the placenta. Even in preterm birth, the pO ２ Although levels increased to those observed in full-term births, scRNA-seq results indicated that RG remained anaerobic, and GSEA results suggested that preterm birth caused mitochondrial dysfunction in RG, leading to reduced oxygen availability.

[0099] The above examples demonstrate that birth triggers RG quiescence, resulting in increased Glu expression and altered glutamine metabolism. Furthermore, we also found that the expression of genes associated with stem cell activation and quiescence, downstream of mTORC1 signaling, dramatically changes in RG after birth. The birth-induced upregulation of Glu in RG is thought to be necessary for postnatal NSCs to acquire quiescence through the suppression of mTORC1 signaling. This may enable long-term maintenance of NSCs in the V-SVZ and appropriate postnatal neurogenesis. As shown in the above examples, preterm birth transiently upregulated mTORC1 signaling in RG, increasing the production of neural progenitor cells. Consequently, preterm birth depleted the NSC pool and reduced neurogenesis in young adulthood.

[0100] In humans, RGs persist until late gestation and, similar to mice, transform into postnatal NSCs after birth (AM Coletti et al., Development 145, dev170100 (2018) and S. Malik et al., J Neurosci 33, 411-423 (2013)). Postnatally generated neurons are thought to migrate to the prefrontal cortex and contribute to higher brain functions in humans (MF Paredes et al., Science 354, aaf7073 (2016) and N. Sanai et al., Nature 478, 382-386 (2011)). Given that mouse young adulthood corresponds to human infancy and childhood, reduced postnatal neurogenesis may contribute to the poor neurodevelopmental outcomes of preterm infants. The results of the above examples suggest that the postnatal decline in neurogenesis can be ameliorated by suppressing mTOR signaling in RG, and thus by suppressing the glutaminolytic pathway.

[0101] According to the present disclosure, neurogenesis can be promoted, and it is expected that the differentiation, maturation, and migration of neurons to the cerebral cortex, etc., can improve or restore brain function. For example, it may be possible to improve the neurodevelopmental prognosis of premature infants. Furthermore, because neurogenesis can be promoted without cell transplantation, it is minimally invasive and reduces the risk of safety problems. Furthermore, for example, neurogenesis promoters can be administered to experimental animals, such as brain disease model animals, for use in research.

[0102] The present invention is not limited to the above-described embodiments and can be realized in various configurations without departing from the spirit of the present invention. For example, the technical features in the embodiments and examples corresponding to the technical features in each aspect described in the Summary of the Invention section can be appropriately replaced or combined to solve some or all of the above-described problems or achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

Claims

1. A neurogenesis promoter comprising at least one of a substance that inhibits the glutamine degradation pathway and a substance that inhibits mTOR, and which is used in premature infants from day 0 to 20 months of corrected age.

2. The neurogenesis promoter according to claim 1, comprising a substance that inhibits mTOR.

3. The neurogenesis promoter according to claim 2, wherein the substance that inhibits mTOR comprises at least one selected from the group consisting of rapamycin, everolimus, temsirolimus, eforolimus, ridaforolimus, deforolimus, zotarolimus, pimecrolimus, tacrolimus, and derivatives thereof.

4. A food or beverage comprising the neurogenesis promoting agent according to any one of claims 1 to 3.

5. An infant formula for promoting neurogenesis, which is substantially free of glutamine and is ingested by premature infants between day 0 and 20 months of corrected age.

6. A method for promoting neurogenesis, comprising the step of administering at least one of a substance that inhibits the glutamine degradation pathway and a substance that inhibits mTOR to a premature infant between day 0 and 20 months of corrected age.

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