WO2010021516A9 - Novel use of lipocalin 2 for treating brain damage - Google Patents

Abstract

The present invention relates to a composition containing a lipocalin 2 (LCN2) inhibitor for treating brain damage and a method for treating brain damage by administering the composition. When the central nervous system (CNS) is injured, astrocytes are activated and astrocytosis is induced. Astrocytosis is a main factor causing nerve cell damage and suppressing regeneration of damaged nerve cells. Since the LCN2 mainly induces astrocytosis, additional damage of nerve cells after brain damage is prevented using the LCN2 inhibitor. Also, the regeneration of damaged nerve cells can be promoted.

Description

New Uses of Lipocalin 2 to Treat Brain Injury

The present invention relates to a novel use of lipocalin 2 for the treatment of brain injury, and more particularly, a composition for treating brain injury containing a lipocalin 2 (Lipocalin 2, LCN2) inhibitor and a method for treating brain injury by administering the same. It is about.

Astrocytes are the most abundant glia cell type in the brain (Barres, BA et al. 2000, Curr Opin Neurobiol 10: 642-648 Aschner, M. 1998, Neurotoxicology 19: 269-281). Astrocytes provide neuronal metabolism and nutritional support and modulate synaptic activity. They are involved in the formation and maintenance of blood-brain barriers for neuroprotection, antioxidant defense, ion and pH homeostasis, and neuronal-glial networks. In addition to the physiological role of astrocytes in this intact central nervous system (CNS), astrocytes are key responders to damage to the CNS under various physiological conditions such as injury, ischemia, infection and neurodegeneration. (Farina, C. et al. 2007, Trends Immunol 28: 138-145). Astrocytes become reactive in response to all forms of CNS damage and undergo a process called reactive astrocytosis (Correa-Cerro, LS et al. 2007, J Neuropathol Exp Neurol 66: 169-176; Sofroniew , MV 2005, Neuroscientist 11: 400-407; Pekny, M. et al. 2005, Glia 50: 427-434). In this process, astrocytes proliferate to fill gaps, increase the amount of cytoplasm, protuberance and branching, glial fibrillary acidic protein (GFAP), nestin and nonmethine ( conventional morphological changes such as increased expression of intermediate fibers such as vimetin). Although the basic process of reactive astrocytosis is evolutionarily conserved and accompanied by all forms of nerve damage, such as neurotrauma and neurodegeneration, it is not known whether reactive astrocytosis is beneficial or detrimental. Reactive astrocytosis is associated with the protection of nerve damage and the promotion of vascular brain barrier repair. In contrast to this beneficial role, reactive astrocytosis is also associated with potentially harmful effects such as inhibiting the regeneration of axons that act as barriers and increasing the secretion of pro-inflammatory and neurotoxic mediators (Silver, J. et. al. 2004, Nat Rev Neurosci 5: 146-156). The opposite role of reactive astrocytic cells has been identified after traumatic injury of the brain (Laird, MD et al. 2008, Neurosignals 16: 154-164). Understanding the functional role of reactive astrocytosis in CNS physiology will lead to new approaches for potential therapies. To this end, it is necessary to understand the exact molecular mechanisms for reactive astrocytosis.

Recent studies suggest that inflammatory cells in the CNS become apoptized upon activation in a manner similar to activation-induced cell death (AICD) induced by lymphocyte activation (Yang, MS et al. 2002, Glia). 38: 273-280; Falsig, J. et al. 2004, J Neurochem 88: 181-193; Liu, B., et al. 2001, J Neurochem 77: 182-189; Mabuchi, T. et al. 2000, Stroke 31: 1735-1743; Suk, K. et al. 2001, Brain Res 900: 342-347; Lee, P. et al. 2001, Brain Res 892: 380-385; Suk, K. 2005, Curr Enzyme Inhibition 1: 43-50). Astrocytes as well as microglia were found to be apoptized as soon as potent inflammatory activation occurs (Ryu, JK et al. 2003, Neurobiol Dis 12: 121-132; Ferrari, D., et al. 1997, Neuropharmacology 36: 1295-). 1301; Hu, J. et al. 1996, Biochim Biophys Acta 1313: 239-245; Kingham, PJ et al. 2000, J Neurochem 74: 1452-1462; Takuma, K. et al. 2004, Prog Neurobiol 72: 111 Suk, K. et al. 2002, J Neurochem 80: 230-238; Lee, J. et al. 2001, J Biol Chem 276: 32956-32965). Apoptosis of glial cells after extensive amplification may be a means of controlling population (Jones, LL et al. 1997, J Neurocytol 26: 755-770). However, little is known about the molecular mechanisms for the autonomous apoptosis of glial cells and the termination of neuritis. Nitric oxide (N) (Lee, J. et al. 2001, J Biol Chem 276: 32956-32965), BTG1 (Lee, H. et al. 2003, J Immunol 171: 5802-5811), N-myc (Jung , DY et al. 2005, J Neurochem 94: 249-256), TLR4 (Jung, DY et al. 2005, J Immunol 174: 6467-6476), IL-4 (Yang, MS et al. 2002, Glia 38: Park, KW et al. 2005, J Neurosci Res 81: 397-402), IL-13 (Shin, WH et al. 2004, 46: 142-152), cyclooxygenase-2 (Yang, MS et al. 2006, J Immunol 177: 1323-1329) and antioxidant enzymes (Yang, MS et al. 2007, J Neurosci Res 85: 2298-2305) are known to be involved in the death of activated microglia. We have recently discovered that lipocalin 2 (lcn2) facilitates apoptosis removal of activated microglia (Lee, S. et al. 2007, J Immunol 179: 3231-3241). In addition, lcn2 is involved in mesenchymal-epithelial transition (MET) of mesenchymal cells to epithelial cells. Lcn2 is an inducer of endogenous epithelial cells (Yang, J. et al, 2002, Mol Cell 10: 1045-1056) and stimulates transformed cells to regulate invasion and metastasis (Hanai, J. et al. 2005 J Biol Chem 280: 13641-13647; Lee, HJ et al. 2006, Int J Cancer 118: 2490-249). In addition, lcn2 promotes tubulogenesis by regulating morphogenesis of epithelial cells (Lee, HJ et al. 2006, Int J Cancer 118: 2490-2497).

Lcn2 is a member of the lipocalin family and binds to or carries lipids and other hydrophobic molecules (Flower, DR et al. 2000, Biochim Biophys Acta 1482: 9-24; Kjeldsen, L. et al. 2000, Biochim Biophys Acta) 1482: 272-283. Lcn2 also contains 24p3 (Flower, DR, et al. 1991. Biochem Biophys Res Commun 180: 69-74), SIP24 (24 kDa superinducible protein) (Hamilton, RT et al. 1985, J Cell Physiol 123: 201-208 ) And NGAL (neutrophil gelatinase-associated lipocalin, a human homologue of lcn2) (Borregaard, N. et al. 2006, Biometals 19: 211-215 ; Kjeldsen, L. et al. 1993, J Biol Chem 268: 10425-10432 Is known as Lcn2 has a variety of features. In vitro studies have shown that lcn2 is important in both apoptosis and survival of cells (Nelson, AM et al. 2008, J Clin Invest 118: 1468-1478; Yousefi, S. et al. 2002, Cell Death Differ 9: 595-597; Devireddy, LR et al. 2005, Cell 123: 1293-1305; Devireddy, LR et al. 2001, Science 293: 829-834; Tong, Z. et al. 2003, Biochem J 372: 203-210 Tong, Z. et al. 2005, Biochem J 391: 441-448). Plays a central role in inducing cell differentiation in the kidney during embryogenesis (Yang, J. et al. 2002, Mol Cell 10: 1045-1056) and protects the kidney from ischemic damage (Mishra, J. et. al. 2004, J Am Soc Nephrol 15: 3073-3082; Mori, K. et al. 2005, J Clin Invest 115: 610-621). lcn2 has been suggested as an indicator of active kidney injury (Mori, K. et al. 2007, Kidney Int 71: 967-970). In various forms of gastrointestinal damage, lcn2 facilitates cell regeneration by promoting cell migration (Playford, RJ et al. 2006, Gastroenterology 131: 809-817). However, in vivo studies in mice lacking lcn2 refute the role of lcn2 in protecting kidneys (Berger, T. et al. 2006, Proc Natl Acad Sci USA 103: 1834-1839). In addition, mice lacking lcn2 did not have iron sequestration, which increased the susceptibility to bacterial infection. This suggests an important role of lcn2 in protection against viral infections (Flo, TH et al. 2004, Nature 432: 917-921). Previous studies have shown that lcn2 prevents degradation of NACP (Yan, L. et al. 2001, J Biol Chem 276: 37258-37265), and acute phase protein (Liu, Q. et al. 1995, J Biol Chem 270: 22565 -22570) and has been suggested to act as adipokine associated with insulin resistance (Yan, QW et al. 2007, Diabetes 56: 2533-2540; Zhang, J. et al. 2008, Mol Endocrino l 22: 1416-1426). Recently two cellular receptors for lcn2 have been identified. Megalin, a member of the low density lipoprotein receptor family, has been shown to bind to human lcn2 and mediate its influx into cells (Hvidberg, V. et al. 2005, FEBS Lett 579: 773-777). Another cell surface receptor for mouse lcn2, which has been shown to selectively mediate apoptosis (Devireddy, LR et al. 2001, Science 293: 829-834). Despite receptor identification, the exact role of lcn2 in cell survival and death has not been identified.

The present inventors have confirmed that LCN2 is proactively involved in astrocytosis, which inhibits the regeneration of neurons, and thus, by inhibiting LCN2, it is possible to promote the regeneration of neurons after brain injury and to complete the present invention. Reached.

In one aspect, the present invention provides a composition for treating brain injury containing an LCN2 inhibitor.

In another aspect, the present invention relates to a method of treating brain injury by administering a composition for treating brain injury containing an LCN2 inhibitor.

Astrocytes, one of the brain's most abundant glial cells, provide neuronal metabolic and nutritional support and modulate synaptic activity. In response to brain damage, astrocytes amplify, increase the expression of intermediate filament proteins, and become hypertrophic. This process is called (reactive) astrocytosis.

Lipocalin 2 (LCN2) is a member of the lipocalin family that binds to small hydrophobic molecules and is involved in various physiological processes and can be highly induced in inflammatory and neoplastic diseases. According to the present invention LCN2 is considered to be an autocrine mediator of reactive astrocytosis. This conclusion is based on the results confirming that LCN2 is involved in the regulation of apoptosis, morphology and migration of astrocytes as follows.

LCN2 increases the apoptosis sensitivity of astrocytes to cytotoxic substances. That is, it accelerates the cell death of astrocytes by cytotoxic substances. LCN2 accelerates astrocytic cell death induced by NO, as well as necrosis cell death induced by H ₂ O ₂ or paraquat.

On the other hand, the influx of iron into the cell reduces the amount of expression of Bim protein, a proapoptosis protein. Conversely, iron depletion in cells leads to up-regulation of Bim. Examples of the present invention confirmed that the sensitization of astrocytic cells induced by lcn2 to apoptosis was counteracted by addition of a siderophore-iron complex (ie, by the supply of iron). This means that the sensitization of astrocytes to apoptosis induced by lcn2 is associated with the Bim protein involved in iron transport and metabolism.

In addition, the expression of lcn2 is strongly induced by lipopolysaccharide (LPS) and TNF-α and weakly by a mixture of serum withdrawal, PMA, INF-γ and gangliosides. This means that the expression and secretion of lcn2 in astrocytes increases under inflammatory conditions in the CNS.

In addition, exposure of LCN2 to astrocytes increases the number of cell processes of astrocytes. As such, the morphological changes of astrocytes induced by LCN2 are dose and time dependent. Changes in the cell processes induced by LCN2 are made by upregulation of GFAP (glial fibrillary acidic protein) expression, which is also dependent on the dose and time of LCN2.

On the other hand, it was confirmed through the embodiments of the present invention that Forskolin and dbcGMP, which induce morphological changes of astrocytes similar to those induced by LCN2, also increase the sensitivity to apoptosis. This means that morphological changes of astrocytes and susceptibility to cell death are closely related to each other.

In addition, NO amplifies the expression of GFAP in astrocytes (Brahmachari, S. et al. 2006. I J Neurosci 26: 4930-4939). Since the present invention confirmed that LCN2 can also induce the expression of GFAP, it was investigated whether NO is involved in LCN2 action in astrocytes. LCN2 induces an increase in NO production in astrocytes, similar to that of LPS. Expression of GFAP induced by LCN2 by the NOS inhibitor NMMA is blocked, and the sensitivity to cell death induced by LCN2 is attenuated. This means that NO plays an important role in cell death, GFAP expression and morphological changes of astrocytes mediated by LCN2.

On the other hand, the Rho subfamily of small G proteins is involved in the regulation of astrocyte morphology (Boran, MS et al. 2007. J Neurochem 102: 216-230; Chen, CJ et al. 2006. stellation. Eur J Neurosci 23: 1977-1987; Suidan, HS et al. 1997. Glia 21: 244-252; John, GR et al. 2004. J Neurosci 24: 2837-2845; Ramakers, GJ et al. 1998. Exp Cell Res 245: 252- 262; Holtje, M. et al. 2005. J Neurochem 95: 1237-1248; Hall, A. 2005. Biochem Soc Trans 33: 891-895) Rho protein's relevance in morphological changes of astrocytes induced by LCN2 Was investigated. LCN2 induces activation of Rho, while ROCK inhibitors block morphological changes induced by LCN2 and inhibit the expression of GFAP. This means that the Rho / ROCK pathway is involved in morphological regulation of astrocytes.

In addition, the effect of LCN2 on astrocyte migration was investigated because cell migration is a phenotype of cells that is closely related to cell morphology. In vitro wound treatment assays and Boyden chamber assays confirmed that LCN2 increased stellate cell mobility.

As a result, it can be seen that LCN2, a secretory protein of astrocytes, performs two functions that determine the pathway for the shape and function of activated astrocytes (FIG. 36).

In conclusion, LCN2 is a protein that leads to the process of astrocytes in astrocytes, so it can be controlled by controlling this protein. Astrocytes are a major cause of inhibition of regeneration of CNS neurons. Therefore, it is possible to suppress astrocytosis by inhibiting the expression of the lcn2 gene or inhibiting the activity of the LCN2 protein, and promote regeneration of CNS neurons as astrocytosis is suppressed.

If neural cell regeneration is required, ie brain injury treatment is required for all types of brain injury, it is not particularly limited, but degenerative nerves, including, for example, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease and multiple sclerosis Diseases, stroke, trauma, brain infections, Creutzfeldt-Jakob disease, brain inflammatory diseases, and the like.

Accordingly, the present invention provides a composition for treating brain injury containing an inhibitor of LCN2. Herein, the inhibitor of LCN2 may be an activity inhibitor of LCN2 or an expression inhibitor of LCN2.

In the present invention, the activity inhibitor of LCN2 may be, for example, but not limited to, peptides, polypeptides, proteins, peptide replicas, compounds, and biologics. Preferably it may be an anti-LCN2 antibody capable of neutralizing the activity of LCN2.

In the present invention, the anti-LCN2 antibody may be a polyclonal antibody or a monoclonal antibody. Antibodies of the invention can be prepared by conventional methods well known in the art of immunology using LCN2 protein as an antigen.

Polyclonal antibodies can be used in one of ordinary skill in the art from several warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats. That is, the antigen is immunized to the animal via intraperitoneal, intramuscular, intraocular or subcutaneous injection. Immunity to the antigen can be increased using an adjuvant, for example Freund's complete adjuvant or incomplete adjuvant. Following booster immunization, a small sample of serum is collected and tested for reactivity to the desired antigen. Once the titer of the animal reaches a steady state in terms of its reactivity to the antigen, large amounts of polyclonal immune serum can be obtained by bleeding weekly or by bleeding the animal.

Monoclonal antibodies can also be generated using known techniques (Kennettm McKearn and Bechtol (eds.), Monoclonal Antibodies, Hybridomas; A New Dimension in Biological Analyses , Plenum Press, 1980). Monoclonal antibodies immunize animals with LCN2 protein as an immunogen, fusion of splenocytes of the immunized animal with myeloma cells to produce hybridomas, select hybridomas that selectively recognize LCN2 protein, and select high It can be prepared by culturing the bridoma and separating the antibody from the culture medium of the hybridoma. In addition, the monoclonal antibody of the present invention can be prepared by injecting the above-mentioned hybridomas producing an anti-LCN2 antibody selectively recognizing LCN2 protein into an animal and separating it from the ascites of the recovered animal after a certain period of time after the injection. Can be.

In the present invention, "inhibition of gene expression" includes inhibition of gene transcription and translation into protein. In addition, not only the gene expression is completely stopped, but also the expression is reduced.

Antisense molecules are most commonly used as a method of inhibiting gene expression. Antisense molecules inhibit the expression of target genes by inhibiting transcriptional initiation by triple chain formation, transcriptional inhibition by hybridization at sites where local open loop structure is formed by RNA polymerase, and in RNA where synthesis is in progress. Inhibition of transcription by hybrid formation, inhibition of splicing by hybridization at the junction of introns and exons, inhibition of splicing by hybridization at the site of splicosomal formation, transition from nucleus to cytoplasm by hybridization with mRNA Inhibition of translation initiation by hybridization at the site of translation initiation factor binding. They inhibit the expression of target genes by inhibiting transcription, splicing or translation processes.

The antisense molecule used in the present invention may inhibit the expression of the target gene by any of the above actions. Representative antisense molecules include triplets, ribozymes, RNAi, or antisense nucleic acids. The triple agent allows the initiation of transcription to be suppressed by winding around double helix DNA to form a three-stranded helix (Maher et al., Antisense Res. And Dev ., 1 (3): 227, 1991; Helene, C., Anticancer Drug Design , 6 (6): 569, 1991).

Ribozymes are RNA enzymes that possess the ability to specifically cleave single-stranded RNA. Ribozymes inhibit protein expression of target genes by recognizing and site-specific cleavage of specific nucleotide sequences in target RNA molecules (Cech, J. Amer. Med. Assn ., 260: 3030, 1998; Sarver et al., Science 247: 1222-1225, 1990).

RNAi (RNA interference) is a method of inhibiting gene expression at the transcriptional level or at the post-transcriptional level by using RNA of hairpin type small molecule that acts in sequence (Mette et al., EMBO J. , 19: 5194-). 5201, 2000). The small molecule RNA used in the RNAi method is a double-stranded RNA molecule having homology with the target gene.

As a method of preparing the RNA molecule in the above, known chemical synthesis methods and enzymatic methods can be used. For example, the chemical synthesis of RNA molecules can use the methods described in the literature (Verma and Eckstein, Annu. Rev. Biochem. 67, 99-134, 1999), and the enzymatic synthesis of RNA molecules is T7, T3. And phage RNA polymerases such as SP6 RNA polymerase are disclosed in the literature (Milligan and Uhlenbeck, Methods Enzymol . 180: 51-62, 1989).

Antisense nucleic acids refer to DNA or RNA molecules that are at least partially complementary to a target mRNA molecule (Weintraub, Scientific American , 262: 40, 1990). In cells, antisense nucleic acids hybridize with their corresponding mRNAs to form double-stranded molecules that inhibit protein translation by inhibiting mRNA translation of target genes (Marcus-Sakura, Anal. Biochem ., 172: 289, 1988). The antisense nucleic acid may be prepared by any suitable method known in the art, preferably in the form of oligonucleotides. The antisense oligonucleotides may be used in chemical synthesis, for example, such as phosphoramidite chemistry, which is sulfided with tetraethylthiuram disulfide in acetonitrile as described in Tetrahedron Lett ., 1991, 32, 30005-30008. It can manufacture very easily.

In the present invention, the LCN2 inhibitor may be administered by any route as long as the LCN2 inhibitor can be directed to the lesion, i. Accordingly, the compositions of the present invention may be used in various forms including topical (including buccal, sublingual, skin and intraocular), parenteral (including subcutaneous, intradermal, intravascular and intraarticular) or transdermal administration. Routes may also be administered, preferably parenterally, most preferably directly at the site where the beta amyloid has been deposited.

In one embodiment, the LCN2 inhibitor can be administered to a subject by suspending in a suitable diluent, which diluent is used for the purpose of protecting and maintaining the cells and facilitating use when infused into the desired brain tissue. The diluent may include physiological saline, PBS, HBSS buffer solution, plasma, cerebrospinal fluid or blood components.

In another embodiment, the LCN2 inhibitor may be used in admixture with a pharmaceutically acceptable carrier according to conventional methods. Examples of suitable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. If necessary, the composition may further include a filler, an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

Compositions of the invention can be formulated using methods well known in the art to provide rapid or delayed release of the active ingredient after administration to a subject. The formulations may be in the form of tablets, powders, pills, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like. The formulation will be convenient in a single dosage form.

The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease, and an effective dose level may include the severity of the disease; The age, body weight, health and sex of the patient; Sensitivity to the drug of the patient; Time of administration, route of administration, and rate of excretion; Duration of treatment; It may be determined according to factors including drugs used in combination or coincidental with the composition of the present invention and other factors known in the medical field. Preferably, the pharmaceutical composition of the present invention may vary the effective amount depending on the extent of the disease, preferably 1 to 10,000 g / weight kg / day, more preferably 10 to 1000 mg / kg / day The effective amount may be repeated several times a day.

Because LCN2 inhibitors can inhibit astrocytosis, which inhibits the regeneration of neurons, they can promote neuronal regeneration.

As such, LCN2 inhibitors can be used to treat a variety of diseases that require neuronal regeneration, ie, any disease associated with brain damage.

In particular, LCN2 is involved in the upper stages in the process of astrocytosis, so LCN2 inhibitors can effectively inhibit astrocytosis and promote neuronal regeneration.

Figure 1 shows the results of analysis of protein expression and changes in lcn2 mRNA in C6 cells transfected with sense or antisense lcn2 cDNA (S3: sense lcn2 transfectants; AS7: antisense lcn2 transfectants).

Figure 2 shows the change in cell viability by SNP, H ₂ O ₂ and paraquat in C6 cells transfected with sense or antisense lcn2 cDNA (S3: sense lcn2 transfectant; AS7: antisense lcn2 transfectant).

Figure 3 shows the results of the analysis of lcn2 expression in C6 cells transfected with adenovirus vectors containing lcn2 DNA fused with GFP.

Figure 4 shows the change in cell viability by NO donors SNP, H ₂ O ₂ and paraquat in C6 cells transfected with adenovirus vectors comprising lcn2 cDNA fused with GFP.

5 shows recombinant mouse LCN2 protein.

FIG. 6 shows changes in cell viability when stellate cells were treated with LCN2 alone and when treated with SNP, H ₂ O ₂ or paraquat.

Figure 7 shows the changes in necrosis and apoptosis cell death rate of astrocytes when LCN2 was treated with astrocytes alone and when treated with SNP, H ₂ O ₂ or paraquat.

8 shows the change in astrocyte death rate according to the dose of LCN2 protein.

Figure 9 shows the results confirming whether the LCN2 protein affects the cell cycle distribution of C6 cells and astrocytes.

FIG. 10 shows changes in cell viability when astrocytes and C6 cells were treated with iron chelator (DFO) alone and when treated with NO donor SNAP.

11 shows changes in cell viability when treated with iron donors (FC) alone and when treated with SNAP in astrocytes and C6 cells.

FIG. 12 shows the change in cell viability when C6 cells were treated with LCN2 and SNAP and co-treated with the cyder pores and iron complexes.

FIG. 13 shows changes in Bim RNA and protein amounts in culture of C6 cells treated with LCN2.

Figure 14 shows the changes in the amount of Bim RNA and protein in the culture of astrocytes treated with LCN2.

Figure 15 shows the results of analyzing the change in the amount of LCN2 expression by the mixture of SNP, LPS, serum clearance (SW), PMA, IFN-γ, TNF-α and gangliosides.

Figure 16 shows the results of analyzing the change in the secretion amount of LCN2 by LPS.

Figure 17 shows the results of analyzing the change in the expression amount of LCN2 receptor lcn2R / 24p3R in C6 cells and astrocytes.

Figure 18 shows the results of analyzing the changes in morphological changes, protuberance length and cell viability when astrocytes were exposed to LCN2, Forskolin or dbcGMP.

Figure 19 shows the morphological changes of astrocytes with the dose of LCN2 and the time of exposure to LCN2.

FIG. 20 shows the results of analyzing changes in GFAP mRNA and protein expression levels of astrocytes with the dose of LCN2 and time exposed to LCN2.

Figure 21 shows the change in cell viability when co-treatment with SNP and LCN2, Forskolin or dbcGMP in astrocytes.

Figure 22 shows the results of analyzing the changes in the LCN2 protein or GFAP expression when treated with Forskolin or dbcGMP in astrocytes.

Figure 23 shows the results of analyzing the changes in GFAP mRNA and protein levels by SNP.

Figure 24 shows the effect of LCN2 on the NO production of astrocytes.

Figure 25 shows the effect of polymyxin B (PB) on the NO production of astrocytes induced by LCN2.

Figure 26 shows the effect of the NOS inhibitor NMMA on GFAP expression in astrocytes induced by LCN2.

27 shows the effect of NMMA on cell death of astrocytic cells induced by LCN2.

28 shows the effect of ROCK inhibitor Y27632 on the morphological changes of astrocytes induced by LCN2.

29 shows the effect of Y27632 on the expression of GFAP induced by LCN2.

30 shows the effect of LCN2 on the activation of Rho.

Figure 31 shows the results confirmed the effect of LCN2, Forskolin or dbcGMP on the migration of astrocytes using an in vitro wound treatment assay.

32 is a result confirming the effect of LCN2, Forskolin or dbcGMP on the migration of astrocytes using the Boyden chamber assay.

33 shows the results of LCN2 expression in the brain and spine of zebrafish embryos at 3 days after fertilization.

Fig. 34 shows the result of confirming the effect on the generation of radial glial cells by injecting LCN2 mRNA into the zebrafish embryo.

Figure 35 shows the change in the projection thickness, length and number of radial glial cells by injecting the mRNA of LCN2 into the embryo of zebrafish.

36 is a schematic showing that LCN2 is involved at higher stages in sensitization and morphological changes of astrocytes to cell death.

37 shows that lipocalin 2 antibody inhibits astrocytosis of astrocytes.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art to which the present invention pertains. Will be self-evident.

Example 1: Obtaining Reagents and Cells

Lipopolysaccharide (LPS) obtained from E. coli 0111: B4 was prepared by phenol extraction and gel filtration chromatography. Sodium nitroprusside dihydrate (SNP), S-nitroso- N -acetylpenicillamine (SNAP), phorbol 12-myristate 13-acetate (PMA), paraquat dichloride, ganglioside mixture, H ₂ O ₂ , NMMA (N ^G -Methyl- L -arginine), forskolin, dibutyryl cyclic GMP (dbcGMP), deferoxamine mesylate, ferric citrate, and polymyxin B are all Sigma Chemical Co. (St Louis, MO). Rho Kinase (ROCK) inhibitor Y27632 was purchased from Calbiochem (La Jolla, Calif.). Recombinant human TNF-a and mouse IFN-g proteins were purchased from R & D Systems (Minneapolis, MN). Iron-saturated enterochelin (0.7 kDa) was purchased from EMC Microcollections GmbH (Tuebingen, Germany). All other compounds were purchased from Sigma Chemical Co. unless otherwise noted.

C6 rat glioma cells were maintained in DMEM (Dulbecco's modified Eagle medium) supplemented with 5% heat inactivated fetal bovine serum (FBS) (GibcoBRL Gaithersburg, MD), gentamicin (50 mg / ml). Astrocyte cultures were prepared from the brains of three-day-old ICR mice (Samtako Co .; Osan, Korea) by the method of McCarthy and de Vellis. Whole brains were disassociated in DMEM supplemented with 10% FBS, 100 U / ml penicillin and 100 mg / ml streptomycin (Gibco-BRL). Cells were seeded in 75 cm ² tissue culture flasks coated with poly D-lysine (Falcon, Becton Dickinson and Company Franklin Lakes, NJ). Cells were incubated at 37 ° C. in 5% CO ₂ humid air. The culture medium was changed after every 3 days thereafter. Secondary pure cultures of astrocytes were obtained by shaking the culture of mixed glial cells at 250 rpm overnight and the culture medium was removed. Astrocytes were harvested using trypsin-EDTA and then centrifuged at 1,000 rpm for 10 minutes. Cells were resuspended in DMEM supplemented with 10% FBS, 100 U / ml penicillin and 100 mg / ml streptomycin, inoculated at 1 x 10 ⁵ cells / ml in 6-spheres coated with poly D-lysine, and then 4 Incubated for days. Purity of astrocytic cultures was greater than 95% as determined by GFAP immunocytochemistry (data not shown). Animals used in this example were obtained and managed according to the guidelines published in the NIH Guide for the Management and Use of Laboratory Animals.

Example 2: Cytotoxicity Assessment by MTT Assay

Cells were inoculated with 5 × 10 ⁴ cells at 200 ml / sphere in 96-spheres and treated with various stimulants for a designated time interval. After treatment, the medium was removed, 3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT; 0.5 mg / ml) was added, followed by CO ₂ incubator at 37 ° C. for 2 hours. Let it stand. Insoluble crystals were completely dissolved in DMSO and then absorbed at 570 nm using a microplate reader (Anthos Labtec Instruments GmbH Salzburg, Austria).

Example 3: Nitric Acid Quantification

Cells were treated with a stimulus on 96 platelets and then NO in supernatant of the culture.₂                 ^-                  NO production was evaluated by measuring. 50 μl of the sample drop in 96 spheres was mixed with Greases reagent (1% sulfanilamide / 0.1% naphthylethylene diamine dihydrochloride / 2% phosphoric acid) and left at 25 ° C. for 10 minutes. Absorbance at 540 nm was measured with a microplate reader (Anthos Labtec Instruments GmbH). NaNO₂NO₂                 ^-                  It was used as a reference for calculating the concentration.

Example 4 Morphological Analysis of Astrocytes by Immunofluorescence

Immunofluorescence assays were performed as known (Seo, MC et al. 2008. J Neurosci Res 86: 1087-1095.).

That is, astrocytes were inoculated at 1 x 10 ⁵ cells per sphere on sterile cover slips on a 24 sphere plate, then fixed for 4 minutes with 4% formaldehyde and washed twice with PBS. Samples were blocked with 1% BSA dissolved in PBS-Tween 20 and placed in PBS containing 3% BSA and mouse anti-GFAP antibody (1:30 dilution) (Biogenex; San Ramon, CA). The spheres were washed twice with PBS-Tween 20 followed by 2.5 mg / ml of Hoechst 33342 fluorochrome (Molecular Probes Eugene, OR) and anti-mouse IgG-fluorescein isothiocyanate (FITC) -conjugated secondary antibody (BD Biosciences; San Jose , CA). Samples were observed using a fluorescence microscope (Olympus BX50 Tokyo, Japan). Microscopic images were processed with MetaMorph Imaging System (Molecular Devices; Sunnyvale, Calif.). Treatment of astrocytes was performed with a slight modification of known methods (Wilhelmsson, U. et al. 2004. J Neurosci 24: 5016-5021; Boran, MS et al. 2007. J Neurochem 102: 216-230). The average projection length is based on the longest projection for each cell from five microscopic regions containing at least 100 cells optionally selected.

Example 5: Flow cytometry of apoptosis and cell cycle

Astrocytes were detached using trypsin-EDTA and washed twice with cold PBS. The cells were then resuspended in 250 ml of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl ₂ pH 7.4) and then placed in 3 μl of FITC-conjugated annexin V (Molecular Probes) according to the manufacturer's instructions. It was. The cells were then vortexed lightly and left under dark conditions for 15 minutes at room temperature. After adding Propidium iodide (20 mg / ml), flow cytometry was performed for 1 hour with FACSAria (BD Biosciences).

Cells were suspended by PBS-5 mM EDTA and analyzed by adding 100% ethanol drops for analysis of cell cycle distribution. RNase A (40 mg / ml) was added to the resuspended cells and then left at room temperature for 30 minutes. Propidium iodide (100 mg / ml) was added and left to stand for 30 minutes. The percentage of cells at each stage of the cell cycle was determined by flow cytometry using FACSCalibur (BD Biosciences).

Example 6: Reverse Transcription Polymerase Reaction (RT-PCR)

Total RNA was extracted according to the manufacturer's protocol by TRIzol reagent (Invitrogen Carlsbad, Calif.) From C6 cells or primary astrocytic cells on the sixth plaque. Reverse transcription was performed using Superscript (Gibco-BRL) and oligo (dT) primers. PCR amplification using specific primer sets was performed 25 times at annealing temperatures of 60 ° C. The nucleotide sequence of the primers was constructed with reference to known cDNA sequences (Table 1). PCR reactions were performed using a DNA Engine Tetrad Peltier Thermal Cycler (MJ Research Waltham, Mass.). For the analysis of PCR products 10 μl of PCR reactions were electrophoresed on 1% agarose gel and detected under UV light. β-actin was used as internal control.

Example 7: Western Blot Essay

Cells in 6-spheres were lysed in three lysis solutions (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride). Protein concentration in cell lysate was determined using Bio-Rad Protein Assay Kit (Bio-Rad; Hercules, CA). For each sample, the same amount of protein was separated by 12% SDS-PAGE and transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences Piscataway, NJ). Membrane blocked with 5% scheme milk followed by primary polyclonal anti-LCN2 antibody (R & D systems) rabbit polyclonal anti-LCN2 / NGAL antibody (Santa Cruz Biotech) rat monoclonal anti-BIM antibody (Calbiochem) mouse monoclonal anti-GFAP antibody (Biogenex) monoclonal anti-a-tubulin clone B-5-1-2 mouse ascites fluid (Sigma)] and HRP-conjugated secondary antibody (anti-goat, anti-rabbit, anti-rat, and anti-mouse) ECL detection (Amersham Biosciences) was performed after standing with IgG; Amersham Biosciences).

Western blot analysis was performed on the culture medium to detect secreted LCN2 protein. That is, astrocyte cultures cultured in a 100 mm culture dish were first washed five times with PBS. Cells were then covered with minimal amount of culture medium and treated with stimulant at 37 ° C. The conditioned medium was recovered and centrifuged at 2,000 x g (5 minutes) and 15,600 x g (10 minutes) to remove non-adherent cells and cell debris. Samples were precipitated with a mixture of trichloroacetic acid (TCA) and acetone (10% TCA and 10 mM DTT in acetone) overnight at -20 ° C. Precipitated protein was subjected to SDS-PAGE and ECL detection.

Example 8: Rho GTPase Activity Assay

Cells were treated with stimulants in a 100 mm petri dish and washed with cold PBS, followed by lysis buffer [20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl ₂ , 1% Nonidet P-40, 10% glycerol , and 1 x protease inhibitor mixture (Roche Molecular Biochemicals Mannheim, Germany). After lysing the lysates and normalizing the protein concentration, Rho bound to GTP in the lysates was measured by effector pull-down assay with EZ-Detect ^™ Rho activation kit (Pierce Rockford, IL). That is, the cell lysate containing a similar amount of Rho was left standing with GST-Rhotekin immobilized on agarose, and the coprecipitate was subjected to anti-Rho Western blot assay to evaluate the amount of Rho protein bound to GTP. It is known that the anti-Rho antibodies used herein can recognize RhoA, RhoB and RhoC.

Example 9 Gateway Cloning and Stable Transfection of lcn2 cDNA

RT-PCR was performed using total RNA isolated from C6 rat glioma cells. Lcn2 cDNA sequences of rats with sense or antisense orientation were PCR amplified from pooled cDNA using target sequence specific primers by Gateway cloning (Invitrogen). The sequence of the PCR primer for gateway cloning is as follows: sense lcn2 forward, 5'-GGGG ACA AGT TTG TAC AAA AAA GCA GGCT CCA CC ATG GGC CTG GGT GTC CTG TGT -3 '(SEQ ID NO: 1) sense lcn2 reverse , 5'-GGGG ACC ACT TTG TAC AAG AAA GCT GGG TTG TT GTC AAT GCA TTG GTC GGT -3 '(SEQ ID NO: 2) antisense lcn2 forward, 5'-GGGG ACA AGT TTG TAC AAA AAA GCA GGCT CCA CC ATG TCA GTT GTC AAT GCA TTG GTC -3 '(SEQ ID NO: 3) Antisense lcn2 Reverse, 5'-GGGG ACC ACT TTG TAC AAG AAA GCT GGG TTG TT ATG GGC CTG GGT GTC CTG TG T-3' (SEQ ID NO: 4) . The forward primer was used by introducing the attB1 sequence (underlined) followed by the Kozak sequence (CCACC) and the gene specific sequence (bold). Similarly, reverse primers were used by introducing an attB2 sequence (underlined) followed by a gene specific sequence (bold). PCR products were cloned into the pDONR207 dornor vector (Invitrogen), and the sequence was reconfirmed with (Macrogen Inc. Seoul, Korea) and then converted to the pDS-GFP-XB destination vector (Invitrogen). In 6-plates, C6 cells were transformed with lipofectAMINE reagent (Invitrogen) with sense or antisense rat lcn2 cDNA with 4 μg of GFP (Green fluorescent protein) tag. An empty pEGFP vector was used as a control for stable expression of lcn2. Stable transformants were selected in the presence of G418 (800 μg / ml) 2 days after transformation. Up or down regulation of lcn2 mRNA or protein in stable transformants was confirmed by RT-PCR or Western blot.

Example 10 Viruses and Infections

Recombinant adenovirus (Ad-lcn2-GFP) expressing rat lcn2-GFP was developed by Newgex Inc. Produced by (Seoul, Korea). That is, cDNA encoding rat lcn2-GFP was inserted into pShuttle-cytomegalovirus vector (Clontech Palo Alto, Calif.). The pShuttle-cytomegalovirus vector containing rat lcn2-GFP was linearized with Pme I and cotransformed with BJ5183 E. coli with a vector of pAdEasy-1 (Clontech) adenovirus. Transformed cells were placed on agarose plates containing kanamycin and each colony was checked for the presence of a suitable transformant. After sequence identification, stocks of recombinant adenovirus were expanded by infection and extraction with HEK-293A cells. Adenovirus was semi-purified from high titer supernatants of infected HEK-293A cells. Supernatants were clarified by centrifugation to remove cell debris and stored at -80 ° C. C6 cells were infected with adenovirus expressing GFP or rat lcn2-GFP for 2 days and observed by fluorescence microscopy. More than 100 cells were identified from several randomly selected regions. Adenoviruses expressing GFP were used as controls.

Example 11: Purification of Recombinant Lipocalin 2 Protein

Recombinant mouse LCN2 protein was prepared as known (Yang, J. et al. 2002,Mol cell10: 1045-1056). In other words, the recombinant LCN2 protein does not synthesize siderophores.E. coliFrom the BL21 strain It was expressed as glutathione S-transferase (GST) fusion protein. Proteins were purified by using glutathione-Sepharose 4B beads (Amersham Biosciences) and then eluting with thrombin or glutathione. For loading of iron and enterochelin, enterokeline (EMC Microcollections GmbH) saturated with 5-fold molar excess of ions was mixed with recombinant LCN2 protein.

Example 12 Phagocytosis Essay

Primary astrocytes were seeded at 6 x platelets at a concentration of 1 x 10 ⁶ cells per sphere. After removing the medium, 20 mg / ml of fluorescent zymosan particles [zymosan A (S. cerevisiae) BioParticles, Alexa Fluor 594 conjugate (Molecular Probes)] were added to each sphere, allowed to settle by gravity and left for 1-3 hours. Unbound particles were washed three times with cold PBS to remove the whole and the medium was replaced. The samples were observed by fluorescence microscopy (Olympus BX50) or analyzed by flow cytometry using FACSAria (BD Biosciences).

Example 13: Cell Migration Assay

Cell migration was measured using a 48-ball Boyden chamber (Neuro Probe, Inc. Gaithersburg, MD). Various concentrations of LCN2 protein or compound in DMEM were placed on a lower wall separated from the upper wall by a polycarbonate filter (8-mm pore size, 25 x 80 mm Neuro Probe, Inc.) without polyvinylpyrrolidone. Located. Cells were recovered by treatment with trypsin, resuspended in DMEM and added to the upper chamber at 1 × 10 ⁴ cells / sphere. Cells were left at 37 ° C. under 5% CO ₂ . Cells were then fixed with methanol for 10 minutes and stained with modified Giemsa stain (Sigma) for 1 hour. Cells on top of the membrane were removed using cotton wool. The migrated cells were counted under a light microscope (Olympus CK2; Tokyo, Japan) (magnifications, x100). All migrated cells were counted and the results were expressed as the mean ± SD of 3 replicates (total number of cells moved).

For in vitro wound healing assays, scratch wounds were made using a 10 μl pipette tip in a confluent cell monolayer on a 24-neck culture plate and 10% FBS containing 100 U / ml penicillin and 100 μg / ml streptomycin. Refilled with DMEM including. Cells were left at 37 ° C. under 5% CO ₂ while the monolayer moved to the wound area. The wound area was observed under a microscope (Olympus CK2) (magnification, x 100). Relative cell migration distance was determined by measuring the width of the wound and subtracting it from the initial value (Bassi, R. et al. 2008, J Neurooncol 87: 23-33). A total of three regions were randomly selected and calculated for each sphere. T results are expressed as multiples of the increase in travel distance.

Example 14 Zebrafish Analysis

14-1: Zebrafish Breeding and Management

Embryos were recovered from crosses and raised in egg water at 28.5 ° C. and developed according to days after fertilization (Park, HC et al. J Neurosci 25: 6836-6844; Kucenas, S. et al. 2008. Nat Neurosci 11: 143 -151). In this example, AB and Tg (GFAP: egfp) fish (Bernardos, RL et al. 2006, Gene Expr Patterns 6: 1007-1013) expressing EGFP under the control of the GFAP promoter were used.

14-2: RNA injection

Rat lcn2 cDNA, including full-length open reading frame, was subcloned into the pCSDest vector for mRNA injection (Villefranc, JA et al. 2007, Dev Dyn 236: 3077-3087.) mRNA was expressed in the Message Machine Kit (Ambion Austin, TX). Produced as. The production of lcn2 mRNA was confirmed by gel electrophoresis of transcriptional reactants (data not shown). 100 pg of lcn2 mRNA was injected into egg yolk at one to two cell stages. Expression of LCN2 protein in embryos injected with mRNA was confirmed by immunocytochemistry (data not shown).

14-3: Immunocytochemistry

The embryos were fixed at 4 ° C. overnight with AB Fix (4% paraformaldehyde, 8% sucrose, 1 × PBS), embedded in 1.5% agarose / 30% sucrose and immersed in liquid nitrogen and frozen in frozen 2-methyl butane. A 10 μm transreverse section was recovered using a cryostat microtome. The following primary antibodies were used for immunocytochemistry: mouse antibody against Zrf-1 (1: 400 dilution, University of Oregon Monoclonal Antibody Facility), rabbit polyclonal anti-LCN2 / NGAL antibody (1: 100 dilution, Santa Cruz Biotech). The length, thickness and number of projections of the zebrafish radial glial cells were quantified as follows: The projections of the five longest and thickest cells for each section were measured. The total number of protrusions was counted for each section. Seven zebrafish sections were quantified for each treatment. Results were normalized to the diameter of the spine [(longest vertical diameter + longest horizontal diameter) / 2) and expressed as mean ± SEM (n = 7).

The following examples are the results analyzed by at least one of the experimental methods described in Examples 1-14 described above.

Example 15 LCN2 Increases Sensitivity of Astrocytes to Cytotoxic Stimulators

Immune activation of cultured microglia and astrocytes is known to induce their apoptosis (Suk, K. et al. 2002. J Neurochem 80: 230-238; Lee, J. et al. 2001. J Biol Chem 276: 32956-32965. Since lcn2 is involved in the survival and apoptosis of various types of cells, the following three methods were used to investigate how lcn2 is involved in the apoptosis of activated astrocytes: 1) Transfection of C6 glial cells with either sense or antisense lcn2 cDNA Stable overexpression or knockdown of lcn2 by; 2) transient expression mediated by adenovirus of lcn2 in C6 cells; And 3) treatment of primary astrocytic cultures or C6 with recombinant LCN2 protein.

By stable transfection of the sense or antisense lcn2 cDNA, C6 cells with increased or decreased expression of lcn2 were obtained. Protein expression and changes in lcn2 mRNA in stable transfectants (lcn2 sense transfectants, S3; lcn2 antisense transfectants, AS7) were confirmed by Western blot analysis and RT-PCR, respectively (FIG. 1). Stable overexpression of lcn2 increased the sensitivity of C6 glial cells to NO donors SNP, H ₂ O ₂ and paraquat (FIG. 2), which are known to induce apoptosis of astrocytes (Suk, K. et. 2001. Brain Res 900: 342-347; Son, E. et al. 2005. Biochem Pharmacol 70: 590-597; Kim, S. et al. 2008. J Neurosci Res 86: 2062-2070). In contrast, stable knockdown of lcn2 by transfection of antisense lcn2 constructs reduced the sensitivity of glial cells to cytotoxic substances (FIG. 2).

Transient overexpression of lcn2 was achieved by using an adenovirus vector containing lcn2 cDNA fused with GFP (FIG. 3). Expression of lcn2 increased the sensitivity of C6 glial cells to NO donors SNP, H ₂ O ₂ and paraquat (FIG. 4).

Recombinant mouse LCN2 protein was prepared and tested for potential cytotoxic effects (FIG. 5). LCN2 protein sensitized primary astrocytic cultures to cell death, whereas LCN2 protein alone did not affect astrocytic survival (FIG. 6). Similar results were obtained from C6 glial cells (data not shown). In order to confirm the properties of cell death, the effect of increasing the cell death of LCN2 protein was evaluated by staining with propidium iodide (PI) and annexin V and flow cytometry (FIG. 7). Treatment of LCN2 protein increased the sensitivity of apoptosis (PI ⁻ / annexin V ⁺ or PI ⁺ / annexin V ⁺ ) as well as necrosis (PI ⁺ / annexin V ⁻ ) of astrocytes. LCN2 sensitized astrocytes with necrosis cell death induced by H ₂ O ₂ or paraquat as well as apoptosis cell death induced by NO. The effect of increasing cell death of LCN2 protein was observed from a dose dependent and statistically significant effect of 0.1 ng / ml of LCN2 (FIG. 8). However, there was no significant effect on cell cycle distribution of C6 glial or primary astrocytic cells (FIG. 9).

Example 16: Characterization of Iron and Bim in Apoptosis Sensitization of LCN2

It is known that the effect of preapoptosis of lcn2 is associated with iron metabolism (Devireddy, LR et al. 2005. Cell 123: 1293-1305), the iron chelator deferoxamine (DFO) or iron donor FC on survival of astrocytes. The effect of ferric citrate was investigated. It is known that lcn2, including the iron complex of bacterial cyderpore, supplies iron to cells through the lcn2 receptor (lcn2R / 24p3R) (Devireddy, LR et al. 2005. Cell 123: 1293-1305). Internalization of lcn2 and its receptors leads to the influx of iron from the siderophore-iron complex. The iron supply to the cells reduces the amount of transferrin receptor (TfR1) expression and increases the amount of ferritin. In addition, the influx of iron into the cell reduces the amount of expression of Bim protein, a proapoptosis protein. Conversely, when lcn2 binds to lcn2R / 24p3R without iron complex and internalizes intracellularly, intracellular mammalian ciderpore iron complex binds to lcn2 and is released out of the cell by exocytosis. Depletion of iron in cells leads to upregulation of Bim, a proapoptotic molecule.

The role of iron and BIM proteins in the apoptosis sensitization effect of lcn2 was investigated in astrocytes. Iron deferoxamine (DFO) alone is weakly toxic to astrocytes and C6 glial cells, but as a further effect increased the killing of astrocytes and C6 glial cells induced by NO donors (SNAP) (FIG. 10). . In contrast, ferric citrate (FC) partially inhibited cell death induced by NO donors (FIG. 11). In addition, cell death sensitization induced by lcn2 was canceled by the simultaneous addition of the cider pore-iron complex (FIG. 12). The amount of Bim mRNA and protein in primary astrocytic cultures as well as C6 glial cells was significantly increased by LCN2 protein treatment (FIGS. 13 and 14).

Example 17: Expression and Control of lcn2 and lcn2 Receptors (lcn2R / 24p3R) in Astrocytes

lcn2 has been proposed as an acute phase protein (Liu, Q. et al. 1995. J Biol Chem 270: 22565-22570) and expression of lcn2 was regulated by immune stimulators in macrophages (Liu, Q. et al. 1995 J Biol Chem 270: 22565-22570; Meheus, LA et al. 1993. J Immunol 151: 1535-1547; Cowland, JB et al. 2003. J Immunol 171: 6630-6639. Therefore, it was confirmed whether the expression of lcn2 is regulated by immunity or other stimulator in the phase.

Expression of lcn2 was strongly increased by lipopolysaccharide (LPS) and TNF-α (FIG. 15). A mixture of serum clearance (SW), PMA, IFN-γ and gangliosides induced a slight increase in lcn2 expression (FIG. 15). Secretion of lcn2 was increased by LPS as determined by Western blot analysis of astrocytic cultures (FIG. 16). These results indicate that the expression and secretion of lcn2 in astrocytes increases under inflammatory conditions in the CNS. Expression of lcn2 receptor (lcn2R / 24p3R), which appears to mediate lcn2 induced cell death (Devireddy, LR et al. 2005, Cell 123: 1293-1305), was detected in primary astrocytes and C6 glial cells (FIG. 17).

Example 18: Identification of Morphological Changes of Astrocytes Induced by LCN2

LCN2 induced morphological changes in astrocytes in addition to apoptosis sensitization effects. When astrocytes were exposed to LCN2 protein, the number of cell processes increased without affecting cell viability (FIG. 18). Because cyclic AMP (cAMP) and cyclic GMP (cGMP) are known to induce similar morphological changes (Boran, MS et al. 2007. J Neurochem 102: 216-230; Hu, W. et al. 2008. Forskolin and dibutyryl cyclic GMP (dbcGMP) were used for comparison ( Cell Mol Neurobiol 28: 519-528) (FIG. 18).

Morphological changes in astrocytes induced by LCN2 were dose and time dependent (FIG. 19). Significant changes occurred at 12 ng / ml of LCN2 and 12 hours after exposure thereto. They have been shown to increase the expression of lcn2 in astrocytes (FIGS. 15 and 16) and to induce activation of glial cells (Jou, I. et al. 2006. Am J Pathol 168: 1619-1630; Yoon, HJ et al. 2008. Mol Cells 25: 99-104).

Changes in the cell processes induced by LCN2 are made by upregulation of the expression of GFAP mRNA and protein, which is dependent on the dose and time of LCN2 (FIG. 20). Morphological changes in this type of astrocytes are similar to those occurring in in vivo reactive astrocytes (Sofroniew, MV 2005. Neuroscientist 11: 400-407).

Hypertrophy and increased GFAP expression are two features of reactive astrocytic cells that appear after all forms of neuronal damage in vivo (Wilhelmsson, U. et al. 2004. J Neurosci 24: 5016-5021). Changes in astrocyte morphology induced by LCN2 were confirmed to be associated with phenotypes sensitive to cell death. Forskolin and dbcGMP, which induce changes in astrocytic morphology similar to those induced by LCN2, also led to phenotypes sensitive to cell death (FIG. 21). dbcGMP increased LCN2 expression while forskolin did not (FIG. 22). However, both forskolin and dbcGMP increased the expression of GFAP to some extent (FIG. 22). Although the effect of dbcGMP on GFAP expression was weak, it was reproducibly observed in repeated experiments (data not shown). dbcGMP and forskolin mean that morphological changes can be induced in LCN2 dependent and independent manner. These results also indicate that morphological changes in astrocytes and phenotypes sensitive to cell death are closely related to each other, and that LCN2 plays a dual role in the regulation of apoptosis and morphology of astrocytes.

Example 19: Role of NO in the Effects of LCN2

Recently, NO has been found to amplify the expression of GFAP in astrocytes (Brahmachari, S. et al. 2006. I J Neurosci 26: 4930-4939). Since it was previously confirmed that LCN2 can also induce the expression of GFAP (FIG. 20), it was confirmed whether NO is involved in LCN2 action in astrocytes. GFAP expression induced by NO was reconfirmed at the mRNA and protein levels using NO donor SNP (FIG. 23). The expression of nonmethine used for comparison was not affected. The effect of LCN2 on NO production of astrocytes was confirmed. LCN2 induced a significant increase in NO production in astrocytes (FIG. 24). The amount of NO produced induced by LCN2 was similar to that of LPS and was not offset by polymyxin B treatment to rule out the possibility of LPS contamination in recombinant LCN2 preparations (FIG. 25). Recombinant GST protein prepared in the same manner as LCN2 was also used as a control to exclude the possibility of contamination of LPS (FIG. 25). NO production induced by LPS / IFN-γ was completely offset by polymyxin B treatment. Expression of GFAP induced by LCN2 was blocked by the NOS inhibitor NMMA (FIG. 26). The increase in cell death sensitivity induced by LCN2 was attenuated by NMMA (FIG. 27). The above results indicate that NO plays an important role in astrocytic cell death, GFAP expression and morphological changes mediated by LCN2.

Example 20: Characterization of Rho / ROCK Pathway in Changes in Astrocytes Induced by LCN2

Since the Rho subfamily of small G proteins is known to be involved in the regulation of astrocyte morphology (Boran, MS et al. 2007. J Neurochem 102: 216-230; Chen, CJ et al. 2006. stellation. Eur J Neurosci 23 : 1977-1987; Suidan, HS et al. 1997. Glia 21: 244-252; John, GR et al. 2004. J Neurosci 24: 2837-2845; Ramakers, GJ et al. 1998. Exp Cell Res 245: 252 Hol262, M. et al. 2005. J Neurochem 95: 1237-1248; Hall, A. 2005. Biochem Soc Trans 33: 891-895). The association of Rho protein in the morphological changes of astrocytes induced by LCN2 was investigated. The Rho / ROCK pathway appears to play an important role in the action of LCN2 on astrocyte morphology according to the following results: 1) ROCK inhibitor Y27632 partially blocked morphological changes induced by LCN2 (FIG. 28); 2) Y27632 also inhibited the expression of GFAP induced by LCN2 (FIG. 29) and 3) LCN2 induced the activation of Rho (FIG. 30). Y27632 had no effect on cell viability at the concentrations used in this example (data not shown). Activation of Rho induced by LCN2 was initiated 1 hour after LCN2 stimulation and continued up to 24 hours (FIG. 30).

Because cell migration is a phenotype of cells that is closely related to cell morphology, the effects of LCN2 on astrocytic migration were investigated. As expected, the stellate cell mobility was increased in the in vitro wound treatment assay and in the Boyden chamber assay (FIG. 31) (FIG. 32). LCN2 accelerated wound closure (FIG. 31) and migration through the membrane (FIG. 32).

Example 21 Expression and Functional Analysis of lcn2 in Zebrafish

A zebrafish model was used to confirm the expression of lcn2 and its functional role in in vivo reactive astrocytes. First, the expression of lcn2 in zebrafish CNS was examined. Labeling of zebrafish embryos with anti-LCN2 polyclonal antibodies showed no signal on day 2 after fertilization (data not shown), but LCN2 ⁺ cells were identified in the whole brain and spine on day 3 (A, B in FIG. 33). ). LCN2 antibodies in whole brain sections labeled microglia-like cells in the form of microglia (arrows in A of FIG. 33). This is consistent with previous reports that on day 3 all macrophages in the zebrafish embryo's brain undergo transformation with specific phenotypes to early microglia (Herbomel, P. et al. 2001. Dev Biol 238: 274-). 288). Interestingly, LCN2 antibodies in the spinal column of zebrafish embryos on day 3 after fertilization labeled cells with features of radial glial cells, ie long radial projections from the central duct of the spine to the surface of the smoke. In order to analyze cell types labeled by LCN2 antibodies, markers of LCN2 antibodies and radial glial cells (Inagaki, M., Y. Nakamura, M. Takeda, T. Nishimura, and N. Inagaki. 1994. Glial fibrillary acidic protein Brain Pathol 4: 239-243) Spinal sections were labeled with anti-GFAP antibodies. All LCN2 ⁺ processes expressed GFAP, meaning that all LCN2 ⁺ cells were radial glial cells in the spine of the zebrafish embryo (FIG. 33C, D).

To investigate the role of lcn2 in the generation of radial glial cells, synthetic mNRAs encoding rat LCN2 proteins were injected at one cell level of Tg (gfap-egfp) embryos, which were anti-Zrf-1 markers of radial glial protuberances. It was labeled with an antibody. Tg (gfap-egfp) embryos express EGFP in radial glial cells under the control of the gfap promoter (Bernardos, RL et al. 2006. Gene Expr Patterns 6: 1007-1013). By labeling the processes (Trevarrow, B. et al. 1990. Neuron 4: 669-679), the Zrf-1 ⁺ processes of EGFP ⁺ cell bodies and radial glial cells could be observed simultaneously (A and C in FIG. 34). The number and thickness of the protrusions of the spine increased in most of the embryos injected with lcn2 mRNA, consistent with in vitro experiments, while the length of the protrusions was unaffected (FIGS. 34B and 35, 35). Since most glial processes extend from the central tube of the spine to the surface of the smoke, the length of the processes cannot be a suitable means for measuring the change in the phenotype of radial glial cells.

Example 22 Inhibition of Astrocytes by LCN 2 Antibodies

In vitro astrocytes were treated with LCN 2 alone, while astrocytes were treated with LCN 2 and antibodies against them (Ab), and then the number and shape of cell processes of astrocytes were observed.

As a result, when the astrocytes were exposed to the LCN2 protein, the number of cell protrusions was increased, and when the LCN2 antibody was treated, the number of cell protrusions was confirmed to decrease. In other words, it was found that astrocytosis is suppressed by the LCN2 antibody (FIG. 37).

The compositions of the present invention can be used to treat brain damage.

Claims (7)

A composition for treating brain damage containing an inhibitor of lipocalin 2. The method of claim 1, Inhibitor of lipocalin 2 is a composition for treating brain damage, characterized in that it inhibits astrocytosis of astrocytes. The method according to claim 1 or 2, Inhibitor of lipocalin 2 is a composition for treating brain damage, characterized in that the antibody against lipocalin 2. The method according to claim 1 or 2, Inhibitor of lipocalin 2 is a composition for treating brain damage, characterized in that the antisense molecule for the gene of lipocalin 2. The method according to claim 1 or 2, Brain damage is a composition for treating brain damage, characterized in that selected from the group consisting of neurodegenerative diseases, stroke, trauma, brain infection diseases, Creutzfeldt Jacob disease, brain inflammatory diseases. The method of claim 5, The neurodegenerative disease is a composition for treating brain damage, characterized in that selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease and multiple sclerosis. A method of treating brain injury by administering a composition for treating brain damage according to claim 1.

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