KR100896237B1 - Novel use of cathepsin D for treating neurodegenerative disease - Google Patents

Abstract

The present invention relates to novel uses of cathepsin D for the treatment of neurodegenerative diseases. More specifically, the present invention is directed to a pharmaceutical composition for the prevention and treatment of degenerative neurological disorders comprising an activity inhibitor or expression inhibitor of cathepsin D and an agent for regulating the expression of cathepsin D. It relates to a method for screening a therapeutic agent.

The pharmaceutical composition of the present invention prevents the degeneration of neurodegenerative diseases caused by the neurotoxic action of microglia by inhibiting the activity of cathepsin D protein secreted from abnormally activated microglia or inhibiting the expression of cathepsin D gene. It can be cured. Cathepsin D can also be used as a target for screening therapeutic agents for neurodegenerative diseases.

Cathepsin D, microglia, degenerative neuropathy

Description

Novel use of cathepsin D for treating neurodegenerative disease

1A is the result of 2-DE analysis of lysates of LPS untreated BV-2 cells.

1B is the result of 2-DE analysis of conditioned medium (none) of LPS untreated BV-2 cells and conditioned medium (LPS) of LPS treated BV-2 cells.

Figure 2 shows the results of 1-dimensional electrophoresis and cell viability of the conditioned medium obtained from BV-2 microglia.

A: 1-D electrophoresis of BV-2 cells mixed with LPS and IFNγ in the presence of NMMA

B: Evaluation of Cell Viability by MTT Assay

The experiment was repeated three times independently and the results were expressed as mean ± standard deviation.

* Shows statistically significant difference compared to control cells not treated with LPS / IFNγ (p <0.05)

Figure 3 is a verification result of the LC-MS / MS data by Western blot analysis or enzyme activity analysis.

A: Western blot of conditioned medium of BV-2 cells activated with LPS / IFNγ

B: Western blot of conditioned medium of primary microglia culture activated with LPS / IFNγ

C: Enzyme Activity Analysis of Conditioned Medium of Activated Microglial Cells

*: Significant difference between experimental groups (p <0.05)

4A shows the number of EGFP-positive cells in co-culture of LPS and INFγ untreated BV-2 cells, LPS and IFNγ alone or mixed BV-2 microglia and B35-EGFP cells.

Figure 4b is a photograph of the co-culture of LPS untreated BV-2 cells (None) or LPS-treated BV-2 cells and B35-EGFP cells (x 200)

Figure 4c is the result of measuring the effect of LPS or INFγ on B35-EGFP cell viability by MTT analysis.

Figure 4d shows the reduction of B35-EFGP cell death following depletion of cathepsin D by neutralizing antibodies to cathepsin D in co-culture of activated BV-2 cells and B35-EFGP cells.

Figure 4e shows the reduction of B35-EFGP cell death following knockdown of cathepsin D gene expression using shRNA in BV-2 microglia in co-culture of activated BV-2 cells and B35-EFGP cells.

Figure 4f is the result confirmed by Western blot knockdown of cathepsin D gene expression by shRNs.

* Statistically significant difference (p <0.05)

The present invention relates to novel uses of cathepsin D for the treatment of neurodegenerative diseases. More specifically, the present invention is characterized by selecting a pharmaceutical composition for the prevention and treatment of degenerative neurological disorders comprising an activity inhibitor or expression inhibitor of cathepsin D and agents for controlling the activity or expression of cathepsin D. The present invention relates to a method for screening a therapeutic agent for neurological diseases.

Degenerative neuropathy is a disease that causes degenerative changes in nerve cells of the central nervous system and causes various symptoms such as impairment of higher-order causes such as impairment of motor and sensory functions, memory, learning, and computational reasoning. Representative diseases include Parkinson's disease, Alzheimer's disease and memory disorders. Degenerative neuropathy is characterized by the death of neurons by necrosis or apoptosis that progress rapidly or slowly. Therefore, understanding the mechanism of death of neurons must be made for the development of the prevention, control and treatment of central nervous system diseases.

The central nervous system consists of neurons and glial cells. Glial cells make up 90% of the brain cells and 50% of the brain. The glial cells are composed of three types of astrocytes, microglia, and oligodendroc ytes.

Among them, microglia are immune cells superimposed on the central nervous system (CNS) (Stoll and Jander, Prog Neurobiol 58: 233-247, 1999; Kim and de Vellis, J Neurosci Res 81: 302-). 313, 2005) account for 5-10% of the total brain cells. The microglia serve as the primary line of defense in the CNS. Microglia are the major intracellular source of inflammatory mediators in the CNS. Microglia are involved in neuroinflammation by producing nitric oxide (NO), reactive oxygen species (ROS), proinflammatory cytokines and prostaglandins. Activated microglia move to damaged nerve tissue regions to prey and destroy microorganisms and cell debris (Gehrmann et al., Brain Res Rev 20: 269-287, 1995). But the role of microglia as inflammatory cells is not always beneficial. Currently, unregulated microglia activation and sustained excessive neuroinflammation are thought to be the cause of various CNS pathologies, including neurodegenerative diseases (Gonzalez-Scarano and Baltuch, Annu Rev Neurosci 22: 219-240, 1999). ). That is, functionally activated microglia are known to produce and secrete inflammatory mediators resulting in neuronal cell death. Therefore, activation of microglia is associated with various types of degenerative neurological diseases. For example, it is well known that microglia play an important role in the formation of senile plague even in Alzheimer's disease (Wegiel, J. et al., Acta Neuropathol (Berl), 100 (4): 356-64, 2000). An important example is that anti-inflammatory agents, such as ibprofen, can inhibit the formation of lytic plaques and prevent the development of dementia (Weggen, S. et al., Nature , 414: 212-216, 2001; Wyss-Coray, T. and Mucke, L., Nat. Med ., 6: 973-974, 2000; Lim, GP et al., J. Neurosci 20: 5709-5714, 2000).

Since chronic neuritis affects neurodegenerative diseases, much attention has been paid to microglia which lead to neuroinflammatory responses. To better work the role of microglia in the pathophysiology of neurodegenerative diseases, a systemic biological approach has recently been used (Suk, Expert Opin. Ther. Patents 2006, 16, 237-247). Transcriptomic or proteomic analysis of microglia produced a large amount of information that was previously unavailable. On the other hand, single step analysis of the entire transcript or protein has its own limitations. For example, low abundance transcripts or proteins are masked by large amounts of transcripts or proteins that are not detected. In addition, the complexity of the transcript or proteome is another reason for the limitation. More recently, subcellular or targeted proteomics (subproteomics) have emerged as an alternative (Wu et al., Am J Physiol Lung Cell Mol Physiol 2005, 288, L585-595; Dreger, Mass Spectrom). Rev 2003, 22, 27-56; Jung et al., Electrophoresis 2000, 21,3369-3377). Secrotom analysis (so-called secretomixes) is one example. Since microglia and neurons interact with each other through the secretion protein, analysis of microglia secrotome can provide insight into how microglia affect neuronal function (Suk, Expert Opin). . Ther. Patents 2006, 16, 237-247). Nevertheless, no secretory analysis of microglia has ever been performed.

Secretory proteins play a direct role in controlling and regulating many physiological and disease processes (Grimmond et al., Genome Res 2003, 13, 1350-1359; Greenbaum et al., Genome Res 2001, 11, 1463-). 1468). Secretory proteins can regulate cell growth, division, differentiation and apoptosis as intracellular signals in multicells. Secretory proteins can also regulate key processes in the pathogenesis of cancer, immune disease, cardiovascular disease and degenerative neuropathy. Since secreted proteome (secreto) is the most likely biomarker for the diagnosis and prognosis of diseases, the recent research on the cytochrome has attracted much attention. Although computer analysis of the genome has been used for indirect identification of the secrotom based on amino acid sequence information (Grimmond et al., Genome Res 2003, 13, 1350-1359), proteomic approaches have provided a wide range of cell types and tissues. To directly test secreted proteins (Thouvenot et al., Proteomics 2006, 6, 5941-5952; Khwaja et al., Oncogene 2006, 25, 7650-7661; Pellitteri-Hahn et al., J Proteome Res 2006, 5, 2861-2864; Zvonic et al., Mol Cell Proteomics 2007, 6, 18-28; Dupont et al., Proteomics 2005, 5, 585-596; Dupont et al., Proteomics 2004, 4, 1761-1778; Lafon-Cazal et al., J Biol Chem 2003, 278, 24438-24448; Delcourt et al., Mol Cell Proteomics 2005, 4, 1085-1094) .

The present inventors have analyzed the protein secreted from microglia to determine the neurotoxic mechanism of microglia, and as a result, various experiments have confirmed that cathepsin D is secreted from microglia to mediate neurotoxicity. It was.

It is therefore an object of the present invention to provide novel uses of cathepsin D for the treatment of neurodegenerative diseases.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for the prevention and treatment of degenerative neurological diseases, including the inhibitor or expression inhibitor of cathepsin D.

The present invention also provides a method for screening a therapeutic agent for neurodegenerative diseases, characterized by selecting an agent that modulates the expression of cathepsin D.

Hereinafter, the present invention will be described in detail.

The present invention is based on the finding that cathepsin D is secreted from activated microglia to mediate neurotoxicity of microglia.

Proteases of the cathepsin family are lysosomal hydrolases that degrade peptides and proteins in lysosomes at acidic pH. Cathepsin is divided into three subgroups depending on their active site amino acids; That is, cysteine cathepsin (B, C, H, F, K, L, O, S, V and W), aspartic acid cathepsin (D and E) or serine cathepsin (G). Cathepsin D as an aspartic acid protease has two aspartic acid residues within the deep gaps of the active site. The main function of cathepsin D is intracellular catabolism in the lysosome compartment, but not only the processing of hormones, antigens and neuropeptides, but also programmed cell death (Liaudet-Coopman, Cancer Lett 2006, 237, 167-179; Fusek et. al., Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2005, 149, 43-50), also involved in CNS disorders such as Alzheimer's disease (AD) and epilepsy (Dreyer et al., Eur J Biochem 1994, 224, 265-). 271; Hetman et al., Exp Neurol 1995, 136,53-63). In addition, research has shown that cathepsin D and its pro-enzyme have ligand-like functions under pathological conditions. Catalytically inactive cathepsin D precursors have been shown to secrete and promote proliferation of epithelial breast cancer cells (Laurent-Matha et al., J Cell Biol 2005, 168, 489-499). In addition, the action of secreted cathepsin D in other types of cancer progression and metastasis has been reported (Sebzda et al., J Exp Ther Oncol 2005, 5, 145-150; Merseburger et al., Eur Urol 2005). , 48, 519-526; Losch et al., Gynecol Oncol 2004, 92, 545-552). The literature reports that abnormal secretion of cathepsin D is due to defective acidification of intracellular organs (Kokkonen et al., J Biol Chem 2004, 279, 39982-39988). It can be assumed that cell surface receptors, which have not yet been identified, mediate the extracellular action of cathepsin D.

Microglial-regulated inflammatory responses have both neuroprotective and neurotoxic effects in the central nervous system. On the other hand, excessive and chronic activity of microglia can be shifted towards neurotoxic effects. In this respect, proteins secreted from activated microglia are expected to play an important role in neurotoxic effects, and these proteins have the potential to be applied to the diagnosis and treatment of inflammatory mediated neurodegenerative diseases.

Thus, in one embodiment of the present invention, in order to characterize the secreted protein (secrotom) of the activated microglia, conditioned media obtained from BV-2 mouse microglia or primary microglia culture Analysis by liquid chromatography combined with two-dimensional gel electrophoresis or tandem mass spectrometry (see Example 1).

Secretomes include proteins such as actin and lactate dehydrogenase and are classified as "cytoplasmic" in organs without showing a leader sequence or transmembrane domain. Previously, secretos were considered to be a subset of proteins secreted through the classical secretion pathway. At present, in a broader sense, secretos include proteins secreted through a variety of mechanisms, such as classical secretion, nonclassical secretion pathways and secretion through exosomes. Exosomes are secretory membrane vesicles first mentioned in specialized antigen presenting cells such as B cells and dendritic cells. Recently, microglia have been shown to provide a home for exosomes (Potolicchio, J Immunol 2005, 175, 2237-2243). LC-MS / MS analysis of microglia exosomes revealed that cytoskeleton, cytoskeleton related proteins, chaperones and several other cytoplasmic proteins make up the exosomes. Thus, proteins of cytoplasmic origin can be found in microglia secretos. In addition, intracellular proteins may be derived from necrotic cells because treatment of BV-2 cells with LPS / IFNγ for 8 hours or more under serum-free conditions leads to a significant decrease in cell viability (FIG. 2B).

On the other hand, relatively long periods of stimulation are required for optimal accumulation of secreted proteins in conditioned medium. Therefore, microscopic analysis of microglia was performed at three different time points (4, 12 and 18 hours) (Table 1). The longer the microglia stimulated, the higher the number of intracellular proteins detected. Significant increases in cytosolic protein levels in conditioned media after long periods of culture have been previously reported (Zwickl, Electrophoresis 2005, 26, 2779-2785). Removal of serum albumin by washing is inversely proportional to cell viability, because strict washing can damage cells prior to the secrotom assay. For optimal recovery of secreted proteins, culture and wash conditions for minimal cell lysis were established (Pellitteri-Hahn, J Proteome Res 2006, 5, 2861-2864). At least 12 hours of stimulation and rigorous washing have been shown to be optimal for secrotom analysis of LPS / IFNγ-activated BV-2 microglia cultures.

BV-2 cells were treated or untreated with LPS / IFNγ under serum-free conditions and incubated for 4-18 hours before conditioned medium of the culture was recovered for LC-MS / MS analysis. Mixture of LPS and IFNγ was used to maximize activation of microglia. Since LPS / IFNγ treatment is known to induce activation and apoptosis of microglia through the production of nitric oxide (NO), the NO synthase (NOS) inhibitor NMMA is combined with LPS / IFNγ to minimize microglia death. It was added together in the culture. NMMA can rule out the possibility of NO effects by blocking inducible NOS (iNOS) -derived NO production in activated microglia.

In another embodiment of the present invention, several proteins that are upregulated in activated microglia based on the results obtained by LC-MS / MS were verified by Western blot analysis or enzyme activity analysis using specific antibodies ( See Example 2).

As a result, the levels of cathepsin D precursor and haptoglobin precursors were found to increase in the conditioned medium of LPS / IFNγ stimulated BV-2 microglia or primary microglia culture compared to LPS / IFNγ unstimulated cells (FIG. A and B).

In addition, an increase in aminopeptidase B activity was observed in conditioned medium and primary microglia culture of BV-2 cells stimulated with LPS / IFNγ (FIG. 3C).

Heptoglobin is an acute phase protein. It exhibits extensive anti-inflammatory activity and acts as bacteriostatic and antioxidant by binding to free hemoglobin (Lim, Redox Rep 2001, 6, 219-227). Free hemoglobin released from red blood cells induces iron precipitation, which can increase oxidation and inflammation. Heptoglobin hemoglobin complexes are removed by the reticuloendothelial system, thereby inhibiting the oxidative and proinflammatory effects of free hemoglobin. Various immunomodulatory effects are attributes of heptoglobin. Heptoglobin is considered as a regulator of Th1 / Th2 balance in human and rat immune systems (Guetta et al., Atherosclerosis 2006; Arredouani et al., Redox Rep 2001, 6, 369-371). Specific phenotypes of heptoglobin are associated with a variety of common diseases (eg, cardiovascular diseases, autoimmune diseases and malignancies) (Wassell, Clin Lab 2000, 46, 547-552). Heptoglobin is produced primarily by hepatocytes, but recent studies have shown that granulocytes also produce the protein (Theilgaard-Monch et al., Blood 2006, 108, 353-361). Currently, the present invention shows that the protein is produced by activated microglia. Heptoglobin produced by activated microglia is believed to play an important role in the regulation of neuroinflammatory. Recently, an increase in heptoglobin protein levels has been detected in CSF of AD patients (Zhang et al., J Alzheimers Dis 2005, 7, 125-133; discussion 173-180). Since the chronic activity of microglia is associated with the disease process of AD and other types of neurodegenerative diseases, these experimental results support that activated microglia may be a source of upregulated heptoglobin in patients. .

Aminopeptidase is an exopeptidase that promotes hydrolysis of amino acid residues from the N-terminus of a peptide or protein. Changes in the function and regulation of aminopeptidase are associated with cancer and cardiovascular disease. Aminopeptidase B (EC 3.4.11.6) that specifically cleaves N-terminal Arg or Lys residues from peptides among twelve or more members of the family is involved in inflammatory processes and tumor progression (Diaz-Perales et al, J Biol Chem 2005, 280, 14310-14317; Fukasawa et al., Biochemistry 2006, 45, 11425-11431). As shown in the enzyme assay, the activity of aminopeptidase B was increased in the conditioned medium of LPS / IFNγ stimulated microglia. These results indicate that the secretion of aminopeptidase B is increased after inflammatory activity of microglia and plays an important role in microglia-regulated neuronal inflammation.

Furthermore, in another embodiment of the present invention, the present inventors have determined whether cathepsin D, one of the relatively increased proteins in the conditioned medium of activated microglial cells, mediates the neurotoxicity of microglial cells. Confirmation through the culture experiment (see Example 3). As a result, it was confirmed that neurotoxicity of activated glioblastoma neuroblastoma was reduced due to depletion of cathepsin D and neutralization of cathepsin D gene expression in microglia using neutralizing antibodies (D, E and FIG. 4). F).

Thus, aspartic acid endoprotease cathepsin D was shown to mediate microglia cytotoxicity based on the following results among the many proteins identified in the secrotom of activated microglia: 1) Western blot analysis of conditioned medium Expression of cathepsin D was significantly increased in non-stimulated microglia in lipopolysaccharide (LPS) / interferon-γ-stimulated microglia cells (see Example <2-1>); 2) neurotoxicity of microglia was reduced in co-culture of BV-2 microglia and B35 neuroblastoma by depletion of cathepsin D using neutralizing antibodies (see Example <3-2>); 3) Knockdown of cathepsin D expression in microglia using shRNA eliminates neurotoxicity in co-culture of BV-2 microglia and B35 neuroblastoma (see Example <3-3>).

In conclusion, cathepsin D plays a pivotal role in microglia neurotoxicity, which can be a biomarker or pharmaceutical target for the diagnosis and treatment of degenerative neuropathy and secondary neurotoxic inflammation associated with excessive microglia activation.

Accordingly, the present invention provides a composition for preventing and treating neurodegenerative diseases, including an inhibitor of cathepsin D activity or expression inhibitor.

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

In the present invention, the anti-cathepsin D 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 cathepsin D 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 with 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 cathepsin D protein as an immunogen, fusion of splenocytes of the immunized animal with myeloma cells to produce hybridomas, and selection of hybridomas that selectively recognize cathepsin D protein. It can be produced by culturing the selected hybridoma and separating the antibody from the hybridoma culture solution. In addition, the monoclonal antibody of the present invention is injected from the above-mentioned hybridomas producing an anti-cathepsin D antibody selectively recognizing cathepsin D protein, and from a plurality of animals recovered after a certain period of time after injection. It can manufacture by separating.

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, nuclei to cytoplasm by hybridization with mRNA Inhibition of translation, inhibition of translation initiation by hybridization at the translational initiator binding site, and the like. 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 users 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 acid refers to a DNA or RNA molecule that is at least partially complementary to a target mRNA molecule (Weintraub, Scientific American , 262: 40, 1990). In cells, antisense nucleic acids hybridize with corresponding mRNAs to form double-stranded molecules that inhibit mRNA translation of the target gene, thereby inhibiting protein expression (Marcus-Sakura, Anal. Biochem ., 172: 289, 1988). The antisense nucleic acid may be particularly useful as an inhibitor for inhibiting the expression of cathepsin D of the present invention. The antisense nucleic acid may be prepared by any suitable method known in the art, preferably in the form of oligonucleotides. The antisense oligonucleotides are 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.

Preferably, RNAi can inhibit the expression of the cathepsin D gene. Methods for inducing RNAi in mammals include siRNA transformation and shRNA vector transformation. In one embodiment of the present invention, the cathepsin D gene was knocked out by preparing a shRNA (short hairpin RNA) vector specific to the cathepsin D gene and transforming it into microglia.

Preferred shRNAs that can be used in the present invention are single strands that include approximately 10-30 base pairs in length and may optionally have one or more free or looped ends. For example, the shRNA of the present invention may have a base corresponding to 660-678 nucleotides of cathepsin D cDNA.

Meanwhile, an RNA pol III promoter such as an H1 promoter or a U6 promoter is mainly used as a shRNA vector for transforming an shRNA vector, and at this time, the shRNA is designed to be converted into siRNA in a cell. These include shRNA vectors based on plasmids and PCR expression cassettes, and shRNA vectors based on viruses.

In one embodiment of the present invention, shRNAs were prepared by cloning the shRNA corresponding to nucleotides 660-678 of cathepsin D cDNA, 9 base loops, and reverse repeat regions into siExpress ^TM vectors (Mirus; Madison, WI).

On the other hand, the pharmaceutical composition according to the present invention can be formulated in a suitable form with a pharmaceutically acceptable carrier. 'Pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not cause an allergic or similar reaction, such as gastrointestinal disorders, dizziness or the like, when administered to a human. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, and parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. And the like may further comprise stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical compositions of the present invention may be prepared in various parenteral or oral administration forms according to known methods. As representative of formulations for parenteral administration, isotonic aqueous solutions or suspensions are preferred for injectable formulations. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, oral dosage forms include, but are not limited to, powders, granules, tablets, pills and capsules.

Pharmaceutical compositions formulated in such a manner can be administered in a effective amount via several routes, including oral, transdermal, subcutaneous, intravenous or intramuscular.

As used herein, the term 'effective amount' refers to an amount of a compound or extract that exhibits a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected depending on the route of administration, subject to administration, age, gender weight, individual difference and disease state. Preferably, the pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the degree of disease, preferably 1 to 10,000 g / weight kg / day, more preferably 10 to 1000 mg / weight kg The effective dose of / day may be repeated several times a day.

The pharmaceutical composition of the present invention prepared as described above can be effectively used for the prevention and treatment of neurodegenerative diseases. In the present invention, "neurodegenerative disease" includes all diseases caused by a disorder of neurotransmission as neurons in a part of the brain gradually degenerate. Examples of degenerative neurological diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, and multiple sclerosis.

Furthermore, the present invention provides a method for screening a therapeutic agent for neurodegenerative diseases.

More specifically, the method for screening a therapeutic agent for neurodegenerative diseases of the present invention comprises the steps of: (a) contacting a test substance with activated microglia; And

(b) measuring the expression level of cathepsin D from the microglia of step (a); And

(c) checking whether the test substance of step (a) promotes or inhibits expression of cathepsin D.

In addition, the screening method of the neurodegenerative agent for neurodegenerative disease of the present invention comprises the steps of: (a) co-culturing a test substance on activated microglia and neuroblastoma;

(b) measuring the survival rate of the neuroblastoma of step (a);

(c) checking whether the test substance of step (a) reduces or increases the survival rate of neuroblastoma.

As used herein, the term "test agent" includes any substance, molecule, element, compound, entity, or combination thereof. For example, but not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, synthetic compound or chemical compound or a combination of two or more substances. Unless otherwise specified, agents, materials, and compounds may be used interchangeably.

Test substances that can be screened or identified by the methods of the invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines (benzodiazepines), oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines or derivatives thereof, structural analogs or combinations thereof. The test substance can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Preferably, the test substance may be a peptide, such as a peptide having about 5-30, preferably about 5-20, more preferably about 7-15 amino acids. The peptide may be a cleavage of a naturally occurring protein, random peptide or “biased” random peptide.

The test substance may also be "nucleic acid." Nucleic acid test agents can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids.

In addition, the test substance may be a small molecule (eg, a molecule having a molecular weight of about 1,000 or less). The method for screening small molecule modulating agents may preferably be subjected to a high throughput assay.

The microglial cells may be derived from a mammal. Preferably it may be derived from a mouse. Activation of microglia can be maximized by mixing LPS and IFNγ. On the other hand, since LPS / IFNγ treatment is known to induce activation and apoptosis of microglial cells through the production of nitric oxide (NO), the NO synthase (NOS) inhibitor NMMA is used to minimize microglial cell death. It can be added in the culture with IFNγ.

Neuroblastoma in the above may be derived from a mammal. Preferably it may be derived from a mouse. Survival of neuroblastomas can be measured by MTT assays known in the art or by transforming the neuroblastomas with pEGFPN1 plasmids to count the number of EGFP positive cells under fluorescence microscopy.

The expression level of cathepsin D may be measured by immunoassay, hybridization and amplification at the protein and / or mRNA level. For example, Western blot analysis, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunosorbent assay (ELISA), immunoblotting, farr assay, immunity Sedimentation, latex aggregation, erythrocyte aggregation, turbidity, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion, immunochromatography, protein chips and immunofluorescence can be used.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

<Example 1>

Identification of Proteins Secreted from Activated Microglial Cells

2-DE (two-dimensional gel electrophoresis) followed by matrix-assisted laser desorption and ionization-elapsed time (MALDI-TOF) analysis in conditioned medium of BV-2 mouse microglia culture Protein secreted from was identified.

<1-1> Preparation of Sample

BV-2 mouse microglial cell line (Blasi et al., J Neuroimmunol 1990, 27, 229-237) contains DMEM containing 5% fetal bovine serum (FBS), 2 mM glutamine and penicillin-streptomycin (Gibco, Gaithersburg, MD) Incubated at.

Primary cultures of mouse microglia were prepared by mild trypsinization with a slight modification of previously known methods (Saura, Glia 2003, 44, 183-189). That is, the whole brain of a newborn ICR rat was chopped, and then chopped and disassembled by mechanical rupture using a nylon mesh. The cells were seeded in poly-L-lysine coated flasks. After incubation for 10-14 days in vitro , microglia were isolated from glial mixed culture by mild trypsin treatment. The glial mixed cultures were incubated for 30-60 minutes in trypsin solution (0.25% trypsin dissolved in HBSS, 1 mM EDTA) diluted 1: 4 with PBS containing 1 mM CaCl ₂ . By the culture, the astrocytes are separated into the upper layer, and the microglia are attached to the bottom of the culture flask. Therefore, the astrocyte layer was aspirated and removed, and the remaining microglia were used for the experiment. Purity of microglia cultures was greater than 95% as determined by isolectin B4 staining.

Microglia-conditioned medium or cell extracts were prepared by slightly modifying previously known methods (Lafon-Cazal et al., J Biol Chem 2003, 278, 24438-24448). That is, microglia cultured in a 100-mm culture dish were washed five times with serum-free culture medium to remove all serum proteins. Cells were then filled with minimal volume of serum free medium and LPS (100 ng / ml) alone or LPS and IFNγ (recombinant mouse interferon-γ, R & D Systems, Minneapolis) in the presence of N-monomethyl L-arginine (NMMA; 0.5 mM). , MN) (50 units / ml) at 37 ° C. for 4-8 hours. The conditioned medium was recovered and subsequently centrifuged at 2,000 x g (5 minutes) and 15,600 x g (10 minutes) to remove unattached cells and debris. The sample was then precipitated overnight at -20 ° C with a trichloroacetic acid (TCA) and acetone mixture (10% TCA and 10 mM DTT dissolved in acetone). After removal of the conditioned medium, residual cells were scraped in PBS to 2,000 × g Centrifuge at (10 min). The cells were then cultured in cold lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40) containing a protease inhibitor mixture (Amersham Biosciences, Piscataway, NJ). , 0.5% sodium deoxycholate). After cell lysis, the samples were centrifuged at 13,400 x g (10 minutes) and the supernatant was used for analysis. Protein concentrations in conditioned medium and cell extracts were measured using the Bio-Rad Protein Assay Kit.

<1-2> Two-Dimensional Gel Electrophoresis (2-DE) and Protein Identification

Conditioned medium of cell culture under BV-2 cell lysate or serum-free conditions prepared in Example <1-1> was analyzed with 2-DE and MALDI-TOF to compare the proteome of cell lysate and culture medium.

Isoelectirc focusing (IEF) was performed using the IPGphor / IsoDalt system (Amersham Biosciences) at 20 ° C. with slight modifications to previously known methods (West, K. A et al., Exp Eye Res 2001, 73, 479-). 491). IPG gel strips (Amersham Biosciences) were recovered at 20 ° C. for 12 h in swelling solution (7M urea, 2M thiourea, 2% CHAPS, 100 mM DTT, 0.5% IPG buffer and bromophenol blue) containing 100-500 μg protein. Mad. IEF was carried out for 1 hour at 200V, 1 hour at 500V, 1 hour at 1000V, 1 hour at 1000V, 30 minutes at 8000V, and 45000Vhr. The IPG strip was then 15 min in 50 mM Tris-HCl (pH 8.8), 6M urea, 30% glycerol, 2% SDS, 10 mg / ml DTT and bromophenol blue, followed by 50 mM Tris-HCl (pH 8.8, Equilibrate for 15 minutes in 6M urea, 30% glycerol, 2% SDS, 2% iodineacetamide and bromophenol blue For the two-dimensional analysis, 1.5 mm of vertical gel (12% SDS PAGE) was prepared and equilibrated IPG gel The strip was placed on top of the gel filled with 0.5% agarose solution Gel electrophoresis was performed for 1 hour at 16 ° C., 5 mA / cm and at 10 mA / cm until bromophenol blue reached the bottom of the gel. The gel was fixed with 50% methanol and 12% acetic acid for 1.5 hours The protein was detected by silver staining according to a previously known method Scanned gel images were obtained from PDQuest software (Bio-Rad, Hercules, CA). Analysis using standard protocols for protein identification Voyager-DE Using STR bio spectrometer Mitt Lee workstation (Applied Biosystems, Foster City, CA) was performed by MALDI-TOF (Matrix Assisted Laser Desorption / Ionization- Time of).

As a result of 2-DE analysis of BV-2 cell lysate, many spots were identified by MALDI-TOF spectrometer (FIG. 1A). From this, it could be confirmed that the sample prepared in Example <1-2> was complete. On the other hand, there was no similar spot pattern between cell lysate and conditioned medium (FIGS. 1A and 1B). This indicates that the conditioned medium is not significantly contaminated by proteins isolated from necrotic cells.

Since the spot pattern between the cell lysate and the conditioned medium is not similar, the spot pattern of the conditioned media obtained from LPS treated BV-2 cells and LPS treated cells was compared. LPS is well known as a substance that induces inflammatory activation of microglia. However, the spot pattern or intensity between the two samples did not show a significant difference and only a slight difference (FIG. 1B). In addition, because of the small amount of protein analyzed, it was not possible to identify several spots with minor differences. Due to this limitation, we performed LC-MS / MS analysis as a more sensitive method.

<1-3> 1-D electrophoresis (1-DE) and Coomassie staining

Proteins were isolated by first performing 1-DE for LC-MS / MS analysis. The conditioned medium of Example <1-2> was subjected to reduced SDS sample buffer (63 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue and 1:20 (v / v) β-mer Captoethanol) for 5 minutes and then separated by electrophoresis on a 10% polyacrylamide gel. After electrophoresis, the proteins were visualized by Coomassie Brilliant Blue G-250 staining solution. The entire gel lane was then cut into 1-15 pieces of equal size.

Analysis of the protein obtained from the conditioned medium by 1-DE revealed that bovine serum albumin (BSA), a major serum protein, was not detected (FIG. 2A). The cultured cells were confirmed to be sufficiently washed to remove all the fetal bovine serum (FBS) components in the preparation of the conditioned medium.

<1-4> Cell Viability Assay

Of Example <1-1> The survival rate over time of LPS / IFNγ-treated microglia and untreated cells was measured by MTT assay. MTT analysis was performed in the following manner. Cells (5 × 10 ⁴ cells in 200 μl / well) were seeded in 96-well plates and treated with LPS and / or IFNγ. After treatment, the medium was removed and 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT; 0.5 mg / ml) was added followed by 37 ° C. in a CO ₂ incubator. Incubated for 2 hours. Insoluble crystals were completely dissolved in DMSO and then absorbance was measured at 570 nm using a microplate reader (Antos Labtec Instruments GmbH, Salzburg, Austria). The experiment was repeated three times independently and the experimental results were expressed as mean ± standard deviation. Statistical comparisons between different groups were performed with the Student T test or the one-way ANOVA test along with the Duncan multiple comparison test using the GraphPad Prism program (GraphPad Software, Inc.). If the p value was less than 0.05, it was regarded as having a statistically significant difference.

As a result, 12 hours of LPS / IFNγ showed a significant decrease in cell viability (FIG. 2B).

<1-5> In-gel trypsin digestion

The gel slices cut in the above Example <1-3> were obtained using residual 50% (v / v) acetonitrile dissolved in 0.1M ammonium bicarbonate and 0.1M ammonium bicarbonate, and remaining SDS and Coomassie blue. Wash until the dye is completely removed. Desalted gel pieces were then dehydrated in acetonitrile and shrunk and dried in a vacuum centrifuge. Proteins in the shrunken gel pieces were digested with trypsin overnight at a substrate to enzyme ratio of 10: 1 (wt / wt) in 25 mM ammonium bicarbonate, pH 8.0. The enzymatic reaction was terminated by addition of 0.1% formic acid dissolved in water. Gel fragment derived peptides were sonicated for 10 minutes to extract and the supernatant containing peptides was transferred to a new tube.

<1-6> Liquid Chromatography and Tandem Mass Spectrometry (μLC-ESI-MS / MS) Analysis

Proteins in conditioned medium were analyzed by LC-MS / MS after 1-DE separation. A 2D-LC-MS / MS system equipped with reverse phase liquid chromatography (RP-LC) was used for proteome analysis. The system consists of a Pinigan LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose) with a Surveyor MS pump (Thermo Electron, San Jose, CA), a spark automatic sampler (EMMEN, The Netherlands), and a nano spray ionization (NSI) source. , CA). Trypsin digested peptide dissolved in 12 μl of 0.1% formic acid was injected for LC-MS / MS analysis. Digested samples were first injected into peptide CapTrap cartridges for peptide concentration and desalting and, depending on their hydrophobicity, were applied to reversed-phase (RP) columns filled with 5 μm, 300 mm pore size C18 silica gel for peptide separation. The RP column was installed directly in front of the ion transfer tube for electrospray. The solutions used as mobile phases were A (H ₂ O) and B (acetonitrile, ACN), respectively, which all contained 0.1% (v / v) formic acid. The flow rate was maintained at 200 nl / min. The solvent gradient started at 5% B and reached a linear gradient up to 43 minutes 65% B and then raised to 80% B in 7 minutes and then to 100% A in 10 minutes. The mass spectrometer follows each full MS scan followed by five MS / MS scans, with the five most peptide molecular ions from previous scans for collision-induced decomposition (CID) using a standardized collision energy of 35%. It was operated in a dynamically selected data-dependent mode (m / z 300-1800). The temperature and electrospray voltage of the heated capillary were 195 ° C. and 2.0 kV, respectively. Relative abundance was based on the number of peptides matched in LPS / IFNγ untreated cells and treated cells.

<1-7> Bioinformatics

All MS / MS data were searched against the IPI mouse protein database using the SEQUEST algorithm (Thermo Electron, San Jose, CA), which is incorporated in BioWorks software (version 3.2). This database search allows for various modifications of carboxyamidomethylation on cysteine residues (57 Da) and oxidation of methionine residues (16 Da), peptide mass tolerance for 2 Da, and fragment mass tolerance for 1 Da. The database search is limited to peptides produced by trypsin cleavage. SEQUEST results were filtered by Xcorr for charge state. Xcorr values were used as 1.9 for single charge ions and 3.75 for 2.2 triple charge ions. Delta Cn ≧ 0.1, Rsp ≦ 4 and probability score ≧ 1.0E-3. Protein identification was performed based on peptide identification. Proteins with two or more peptides were considered positive. After protein identification, a subtracted proteome analysis was performed for each data set using ProtAn, an in-house analytic program. The intracellular location of the protein was based on ExPASy proteomics server (http://www.expasy.org) or bioinformation harvester (http://harvester.embl.de). The identification results of the proteins are shown in Table 1.

a: SWISS-PROT, TrEMBL or IPI approval number

b: BV-2 cells treated with LPS (100 ng / ml) and IFNγ (50 uints / ml) for 4, 12 or 18 hours in the presence of NMMA (0.5 mM) under serum free conditions

c: ratio of matched peptide number (T) in LPS / IFNγ treated cells to matched peptide number (U) in LPS / IFNγ untreated cells; T / U

d: intracellular location based on web sites at www.expasy.org and http://harvester.embl.de

e: not detected

f: not applicable

<Example 2>

Validation of LC-MS / MS Results

High-efficiency cytochrome analysis of microglia has identified a number of proteins involved in neuroinflammatory and other cellular responses. On the other hand, these proteins require validation before further studies are conducted with potential biomarkers or drug targets. Based on the results obtained by LC-MS / MS, several proteins upregulated in activated microglial cells were verified by Western blot analysis or enzymatic activity analysis with specific antibodies. Since it is not possible to verify all of the proteins shown in Table 1, one protein subset was chosen for analysis depending on neurological inflammation and potential associations.

<2-1> Western blot analysis

Protein concentration in microglia conditioned medium was determined using the Bio-Rad Protein Assay Kit. The same amount of protein was separated by 12% SDS-PAGE for each sample and transferred to a high bond ECL nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% scheme milk and primary antibodies (goat anti-cathepsin D polyclonal antibody (Santa Cruz Biotech; Santa Cruz, CA), both anti-haptoglobin polyclonal antibodies (Abcam; Cambridge, UK )] And HRP-conjugated secondary antibody (anti-goat, or -sheep IgG; Amersham Biosciences) followed by sequential incubation followed by ECL detection (Amersham Biosciences) using LAS. Monoclonal antibodies against α-tubulin (clone B-5-1-2; Sigma) were used as controls for cathepsin D neutralization.

Experimental results showed that the levels of cathepsin D precursor and haptoglobin precursors were increased in the conditioned medium of LPS / IFNγ stimulated BV-2 microglia or primary microglia culture compared to LPS / IFNγ unstimulated cells (FIG. 3). A and B).

<2-2> Aminopeptidase B Activity Assay

The aminopeptidase B enzyme activity of the conditioned medium was analyzed using AMC-linked arginine according to previously known methods (Diaz-Perales J Biol Chem 2005, 280, 14310-14317). The analysis was performed at 37 ° C. with a substrate concentration of 5 μM in a buffer containing 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The mixture was incubated for 30 minutes and the fluorescence in the reaction was measured at 460 nm. The excitation wavelength was 360 nm. The fluorescence signal was calculated using a known AMC concentration. The experiment was repeated three times independently and the experimental results were expressed as mean ± standard deviation. Statistical comparisons between different groups were performed with the Student T test or the one-way ANOVA test along with the Duncan multiple comparison test using the GraphPad Prism program (GraphPad Software, Inc.). If the p value was less than 0.05, it was regarded as having a statistically significant difference.

As a result, it was observed that aminopeptidase B activity was increased in conditioned medium and primary microglia culture of BV-2 cells stimulated with LPS / IFNγ (FIG. 3C). These results indicate that the secretion of aminopeptidase B can be increased after inflammatory activity of microglia and play an important role in microglia-regulated neuronal inflammation.

<Example 3>

To investigate the role of cathepsin D as a mediator of microglia neurotoxicity

Cathepsin D is one of the relatively increased proteins in the conditioned medium of activated microglia. Since cathepsin D is known to be secreted to regulate cell growth and survival / apoptosis, coculture of microglia and neurons was used to investigate whether cathepsin D mediates neurotoxicity of microglia. A coculture system of BV-2 microglia and B35 neuroblastoma was used as a model of microglia / neuron coculture. Subclones of B35 cells stably expressing EGFP (B35-EGFP) were used for simple differentiation of microglia derived neuroblastoma in coculture. We hypothesized that cathepsin D secreted from activated microglia exerts cytotoxicity on neurons. This hypothesis confirmed the knockdown of cathepsin D gene expression in microglial cells prior to either co-culture or depletion of cathepsin D using neutralizing antibodies in the co-culture system.

<3-1> Cell Viability Assessment

Treated BV-2 microglia with LPS (100 ng / ml) and IFNγ (50 units / ml) alone or in combination and B35- co-cultured by reduction of EGFP-positive cell count 48 hours after co-culture with B35-EGFP cells It was confirmed that it induced a significant decrease in EGFP cell viability. B35 rat neuroblastoma (provided by Professor Park, Yong-yong, School of Medicine, Gyeongsang National University) was cultured in DMEM containing 10% FBS, 2 mM glutamine and penicillin-streptomycin. B35 neuroblastoma was stably transformed with pEGFPN1 plasmid (Clontech, Mountain View, Calif.). Stable transformants were selected and maintained in the presence of G418. Expression of EGFP in stable transformants (B35-EGFP) was confirmed by examining the cells under fluorescence microscopy. More than 99% of the cells were EGFP positive.

After co-culturing B35-EGFP cells and BV-2 microglia at a ratio of 2.5: 1, the viability of B35-EGFP cells was evaluated by counting EFGP positive cells under fluorescence microscopy. Five microscopic fields were randomly selected for the evaluation of cell viability and the percentage of cell numbers calculated.

In addition, B35-EGFP cells were treated with LPS and IFNγ alone or together and the effects on the cell death were examined by MTT analysis in the same manner as in Example <1-4>.

Statistical comparisons between different groups were performed with the Student T test or the one-way ANOVA test along with the Duncan multiple comparison test using the GraphPad Prism program (GraphPad Software, Inc.). If the p value was less than 0.05, it was regarded as having a statistically significant difference.

As a result, activation of BV-2 microglia by treatment with LPS and IFNγ alone or together induced death of co-cultured B35-EGFP cells (FIGS. 4A and B). On the other hand, MTT assay, LPS or IFNγ itself did not affect B35-EGFP cell death (Fig. 4c). This suggests that the decrease in B35-EFGP cell viability is due to LPS or IFNγ-activated microglia and not to LPS or IFNγ itself. In addition, it was confirmed by counting B35-EFGP cells that LPS / IFNγ is not toxic to pure B35-EFGP cells (results not shown).

<3-2> Depletion of Cathepsin D Using Neutralizing Antibodies

Goat anti-cathepsin D polyclonal antibody, which is stimulated by treating LPS (100 ng / ml) and IFNγ (50 units / ml) alone or in combination with BV-2 microglia and cathepsin D when co-cultured with B35-EGFP cells ((Santa Cruz Biotech; Santa Cruz, CA) (20 μg / ml) was treated and cultured for 48 hours, and the number of B35-EFGP cells was counted in the same manner as in Example <3-1>. Statistical comparisons were performed with the Student T test or the one-way ANOVA test with Duncan's multiple comparison test using the GraphPad Prism program (GraphPad Software, Inc.) A statistically significant difference was observed when the p-value was less than 0.05. It was.

As a result, antibody mediated neutralization of cathepsin D reduced the toxic effect of activated microglia on co-cultured B35-EGFP cells (FIG. 4D).

<3-3> Knockout of Cathepsin D Gene Expression Using shRNA

BV-2 cells in 6-well plates were transformed with 2 μg of mouse cathepsin D specific shRNA constructs using lipofectamine reagent (Invitrogen). ShRNA corresponding to nucleotides 660-678 of cathepsin D cDNA, 9 base loop and reverse repeat site were cloned into siExpress ^™ vector (Mirus; Madison, WI). The sequence of shRNA is as shown in SEQ ID NO: 1. Vectors encoding scrambled RNA were used as controls for stable expression of cathepsin D specific shRNA. After 2 days of transformation stable transformants were selected in the presence of G418 (400 μg / ml). Downregulation of cathepsin D protein in stable transformants was confirmed by Western blot analysis in the same manner as in Example <2-1>. Statistical comparisons between different groups were performed with the Student T test or the one-way ANOVA test along with the Duncan multiple comparison test using the GraphPad Prism program (GraphPad Software, Inc.). If the p value was less than 0.05, it was regarded as having a statistically significant difference.

As a result, knockout of cathepsin D expression in BV-2 cells using shRNA significantly attenuated the neurotoxicity of microglia (FIG. 4E). Knockout of cathepsin D expression by shRNA was confirmed by Western blot analysis of conditioned media of LPS / IFNγ untreated or treated cells (FIG. 4F). Compared to scrambled shRNA transformed cells, cathepsin D-specific shRNA transformants showed significantly lower levels of cathepsin D precursors in conditioned media. These results show that cathepsin D plays a pivotal role in the neurotoxic effects of activated microglia. These results also suggest that cathepsin D precursors are secreted from activated microglia under inflammatory conditions in the CNS in vivo, which kill or damage neurons or nearby glial cells, leading to degenerative neuropathy.

As described above, the pharmaceutical composition of the present invention is due to the neurotoxic action of microglia by inhibiting the activity of cathepsin D protein secreted from abnormally activated microglia or inhibiting the expression of cathepsin D gene. It can prevent and treat the degenerative neurological diseases caused. Cathepsin D can also be used as a target for screening therapeutic agents for neurodegenerative diseases.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Novel use of cathepsin D for treating neurodegenerative disease <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> shRNA <400> 1 gctggtggac aagaacatc 19  

Claims (7)

Composition for the prevention and treatment of degenerative neurological diseases comprising shRNA for cathepsin D represented by SEQ ID NO: 1 as an active ingredient. delete delete delete The composition of claim 1, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, and multiple sclerosis. (a) contacting the test substance with activated microglia; And (b) measuring the expression level of cathepsin D from the microglia of step (a); And (c) a method for screening a therapeutic agent for neurodegenerative diseases comprising the step of confirming whether the test substance of step (a) promotes or inhibits the expression of cathepsin D. delete

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