KR20170115468A - Method for diagnosis or prognosis of neurodegenerative diseases - Google Patents

Abstract

A method of diagnosing or predicting a degenerative brain disease by detecting the phosphorylation of serine 293 residue of nNOS (neuronal nitric oxide synthase), and a method of diagnosing or predicting degenerative brain disease by phosphorylating serine 293 residue of nNOS (neuronal nitric oxide synthase) Disclosed is a pharmaceutical composition for preventing or treating a degenerative brain disease comprising a CDK5 (cyclin-dependent kinase 5) inhibitor as an active ingredient.

Description

[0001] METHOD FOR DIAGNOSIS OR PROGNOSIS OF NEURODEGENERATIVE DISEASES [0002]

The present invention relates to a method for diagnosing or predicting a degenerative brain disease, and more particularly, to a method for diagnosing or predicting a degenerative brain disease through the identification of the cause of the dimerization of nNOS in a degenerative brain disease.

The prevalence of dementia, including those caused by Alzheimer's disease (AD), is increasing rapidly with prolonged life span. This is related to many socio-economic problems in developed countries. The onset of AD is due to the overproduction of amyloid beta proteins and / or tau protein, which cause senile plaques and neurofibrillary tangles, respectively. Although this molecular mechanism has been recognized as fundamental to almost all aspects of the AD invention (see non-patent reference 1), it has recently been shown that cyclin-dependent kinase 5 (CDK5), glycogen synthase 3β and mammalian target of rapamycin And various intracellular signal molecules also cause AD outbreaks (see Non-Patent Documents 2 to 4). Among these, CDK5 has been identified as a major mediator of AD (see Non-Patent Documents 3, 5 and 6).

Homozygous knockout of the CDK5 gene is perinatal lethal and these mice show defects in cortical lamination and neuronal development (see non-patent documents 7 and 8), which suggests that CDK5 Is an essential kinase for normal neuronal development during embryogenesis. CDK5 also plays an important role in neurogenesis and synaptic plasticity (see Non-Patent Documents 9 and 10). Furthermore, abnormal activation of CDK5 is associated with the onset of neuropathic diseases including AD (see non-patent document 11). For activation, CDK5 should be associated with an activating protein such as p25, a truncated form of p35 or p35. On the other hand, the CDK5 / p35 complex mediates normal physiological processes, and the association with p25 abnormally increases CDK5 activity and causes neuronal cell death and neurodegeneration (see Non-Patent Documents 12 and 13) .

NO (nitric oxide) is a gaseous signal molecule that is generated from three isoforms of NO synthase (eNOS) (endothelial NOS), nNOS (neuronal NOS) and iNOS (inducible NOS). NO obtained from such NOS enzymes plays an important role in the maintenance of vascular tone, synaptic integrity and inflammation, respectively (see Non-Patent Document 14). Among them, nNOS-derived NO has been reported to inhibit AD outbreaks (see Non-Patent Documents 16 and 17) as well as synaptic plasticity and memory formation (see Non-Patent Document 15). NOS dimerization has been established to be necessary for NOS enzymatic activity (see Non-Patent Document 14). When the NOS dimer collapses, each monomer produces a reactive oxygen species (ROS) including superoxide instead of NO (see Non-Patent Document 14). N-terminal motifs, including N-terminal leader sequences, N-terminal hooks and Zn-loops in nNOS, (See Non-Patent Document 18).

eNOS dimerization has been largely disrupted in the blood vessels of the aging mouse to provide a molecular mechanism for endothelial dysfunction in aging blood vessels (see Non-Patent Document 19), but the effect of nNOS dimerization in neurodegeneration has not been reported.

[Non-Patent Document]

- Non-Patent Document 1: Masters, C. L., Beyreuther, K., 2006. Alzheimer's centennial legacy: prospects for rational therapeutic intervention targeting the Abeta amyloid pathway. Brain 129, 2823-2839.

- Non-Patent Document 2: Amin, J., Paquet, C., Baker, A., Asuni, AA, Love, S., Holmes, C., Hugon, J., Nicoll, JA, Boche, . Effect of amyloid-beta (Abeta) immunization on hyperphosphorylated tau: a potential role for glycogen synthase kinase (GSK) -3beta. Neuropathol Appl Neurobiol 41, 445-457.

Non-Patent Document 3: Dhariwala, F.A., Rajadhyaksha, M.S., 2008. An unusual member of the Cdk family: Cdk5. Cell Mol Neurobiol 28,351-369.

Alteration of mTOR signaling occurs in the presence of mTOR signaling occurs in the absence of mTOR signaling. Early in the progression of Alzheimer disease (AD): analysis of brain from subjects with pre-clinical AD, amnestic mild cognitive impairment and late-stage AD. J Neurochem 133, 739-749.

- Non-Patent Document 5: Barnett, D. G., Bibb, J. A., 2011. The role of Cdk5 in cognition and neuropsychiatric and neurological pathology. Brain Res Bull 85, 9-13.

- Non-Patent Document 6: Crews, L., Patrick, C., Adame, A., Rockenstein, E., Masliah, E., 2011. Modulation of aberrant CDK5 signaling rescues impaired neurogenesis in models of Alzheimer's disease. Cell Death Dis 2, e120.

Non-Patent Document 7: Hirota, Y., Ohshima, T., Kaneko, N., Ikeda, M., Iwasato, T., Kulkarni, AB, Mikoshiba, K., Okano, H., Sawamoto, K., 2007. Cyclin-dependent kinase 5 is required for control of neuroblast migration in the postnatal subventricular zone. J Neurosci 27, 12829-12838.

- Non-Patent Document 8: Ohshima, T., Ward, JM, Huh, CG, Longenecker, G., Veeranna, Pant, HC, Brady, RO, Martin, LJ, Kulkarni, AB, 1996. Targeted disruption of the cyclin- dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc Natl Acad Sci U SA 93, 11173-11178.

- Non-Patent Document 9: Jessberger, S., Aigner, S., Clemenson, GD, Jr., Toni, N., Lie, DC, Karalay, O., Overall, R., Kempermann, G., Gage, FH , 2008. Cdk5 regulates the maturation of newborn granule cells in the adult hippocampus. PLoS Biol 6, e272.

- Non-patent document 10: Jessberger, S., Gage, F. H., Eisch, A. J., Lagace, D.C., 2009. Making a neuron: Cdk5 in embryonic and adult neurogenesis. Trends Neurosci 32, 575-582.

- Non-Patent Document 11: Cruz, J. C., Tsai, L. H., 2004. A Jekyll and Hyde kinase: roles for Cdk5 in brain development and disease. Curr Opin Neurobiol 14, 390-394.

Non-Patent Document 12: Lee, M.S., Kwon, Y.T., Li, M., Peng, J., Friedlander, R. M., Tsai, L. H., 2000. Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature 405, 360-364.

- Non-Patent Document 13: Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P., Tsai, L. H., 1999. Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 402, 615-622.

- Non-patent document 14: Forstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur Heart J 33, 829-837, 837a-837d.

Non-Patent Document 15: Cserep, C., Szonyi, A., Veres, JM, Nemeth, B., Szabadits, E., de Vente, J., Hajos, N., Freund, TF, Nyiri, G., 2011. Nitric oxide signaling modulates synaptic transmission during early postnatal development. Cereb Cortex 21, 2065-2074.

- Non-Patent Document 16: Harry, G. J., Sills, R., Schlosser, M.J., Maier, W.E., 2001. Neurodegeneration and glia response in rat hippocampus following nitro-L-arginine methyl ester (L-NAME). Neurotox Res 3, 307-319.

- Non-Patent Document 17: Puzzo, D., Vitolo, O., Trinchese, F., Jacob, JP, Palmeri, A., Arancio, O., 2005. Amyloid-beta peptide inhibits activation of the nitric oxide / cGMP / cAMP-responsive element-binding protein pathway during hippocampal synaptic plasticity. J Neurosci 25, 6887-6897.

Non-Patent Document 18: Panda, K., Adak, S., Aulak, KS, Santolini, J., McDonald, JF, Stuehr, DJ, 2003. Distinct influence of N-terminal elements on neuronal nitric oxide synthase structure catalysis. J Biol Chem 278, 37122-37131.

- Non-Patent Document 19: Yang, Y. M., Huang, A., Kaley, G., Sun, D., 2009. eNOS uncoupling and endothelial dysfunction in aged vessels. Am J Physiol Heart Circ Physiol 297, H1829-1836.

- Non-patent document 20: Kim, HP, Lee, JY, Jeong, JK, Bae, SW, Lee, HK, Jo, I., 1999. Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae . Biochem Biophys Res Commun 263, 257-262.

- Non-Patent Document 21: Cho, DH, Seo, J., Park, JH, Jo, C., Choi, YJ, Soh, JW, Jo, I., 2010. Cyclin-dependent kinase 5 phosphorylates endothelial nitric oxide synthase serine 116. Hypertension 55, 345-352.

- Non-Patent Document 22: Kwon, KJ, Cho, KS, Kim, JN, Kim, MK, Lee, EJ, Kim, SY, Jeon, SJ, Kim, KC, Han, JE, Kang, YS, Kim, S. , Kim, HY, Han, SH, Bahn, G., Choi, J., Shin, CY, 2013. Proteinase 3 induces oxidative stress-mediated neuronal death in rat primary cortical neurons. Neurosci Lett 548, 67-72.

- Non-Patent Document 23: Cho, WH, Park, JC, Chung, C., Jeon, WK, Han, JS, 2014. Learning strategy preference of 5XFAD transgenic mice depends on the sequence of place / maze task. Behav Brain Res 273, 116-122.

- Non-Patent Document 24: Zoubovsky, SP, Pogorelov, VM, Taniguchi, Y., Kim, SH, Yoon, P., Nwulia, E., Sawa, A., Pletnikov, MV, Kamiya, A., 2011. Working memory deficits in neuronal nitric oxide synthase knockout mice: potential impairments in prefrontal cortex mediated cognitive function. Biochem Biophys Res Commun 408, 707-712.

Non-Patent Document 25: Zhu, G., Fujii, K., Belkina, N., Liu, Y., James, M., Herrero, J., Shaw, S., 2005. Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases. J Biol Chem 280, 10743-10748.

Non-patent document 26: Crane, BR, Rosenfeld, RJ, Arvai, AS, Ghosh, DK, Ghosh, S., Tainer, JA, Stuehr, DJ, Getzoff, ED, 1999. N-terminal domain swapping and metal ion binding in nitric oxide synthase dimerization. EMBO J 18, 6271-6281.

- Non-patent document 27: Ghosh, DK, Crane, BR, Ghosh, S., Wolan, D., Gachhui, R., Crooks, C., Presta, A., Tainer, JA, Getzoff, ED, Stuehr, DJ , 1999. Inducible nitric oxide synthase: role of the N-terminal betahairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction. EMBO J 18, 6260-6270.

Non-Patent Document 28: Keynes, R. G., Garthwaite, J., 2004. Nitric oxide and its role in ischaemic brain injury. Curr Mol Med 4, 179-191.

Non-Patent Document 29: Mariotto, S., Suzuki, Y., Persichini, T., Colasanti, M., Suzuki, H., Cantoni, O., 2007. Cross-talk between NO and arachidonic acid in inflammation. Curr Med Chem 14, 1940-1944.

- Non-patent reference 30: Guglielmotto, M., Giliberto, L., Tamagno, E., Tabaton, M., 2010. Oxidative stress mediates the pathogenic effect of different Alzheimer's disease risk factors. Front Aging Neurosci 2, 3.

Non-Patent Document 31: Keller, J. N., Schmitt, F. A., Scheff, S. W., Ding, Q., Chen, Q., Butterfield, D. A., Markesbery, W. R., 2005. Evidence of increased oxidative damage in subjects with mild cognitive impairment. Neurology 64, 1152-1156.

- Non-patent document 32: Markesbery, W. R., Carney, J. M., 1999. Oxidative alterations in Alzheimer's disease. Brain Pathol 9, 133-146.

- Non-Patent Document 33: Matsumura, A., Emoto, MC, Suzuki, S., Iwahara, N., Hisahara, S., Kawamata, J., Suzuki, H., Yamauchi, A., Sato-Akaba, H , Fujii, HG, Shimohama, S., 2015. Evaluation of oxidative stress in the brain of a transgenic mouse model of Alzheimer disease by in vivo electron paramagnetic resonance imaging. Free Radic Biol Med 85, 165-173.

Non-Patent Document 34: Tamagno, E., Guglielmotto, M., Aragno, M., Borghi, R., Autelli, R., Giliberto, L., Muraca, G., Danni, O., Zhu, X. , Smith, MA, Perry, G., Jo, DG, Mattson, MP, Tabaton, M., 2008. Oxidative stress activates a positive feedback between gamma- and beta-secretase cleavages of the beta-amyloid precursor protein. J Neurochem 104, 683-695.

In order to solve the above problem, the present invention provides a method for diagnosing or predicting a degenerative brain disease by examining whether collapse of nNOS dimerization in 5x FAD (familial AD) mice causes AD onset.

Also, it is proposed to use as a pharmaceutical composition for the prevention or treatment of degenerative brain diseases by containing as an active ingredient a substance which inhibits a substance inducing collapse of nNOS dimerization which is a cause of AD.

The present invention provides a method for diagnosing or predicting a degenerative brain disease by detecting the phosphorylation of serine 293 residue of nNOS (neuronal nitric oxide synthase).

In order to solve the above-mentioned problem, the present invention provides a method for preventing or treating degenerative brain diseases comprising, as an active ingredient, a CDK5 (cyclin-dependent kinase 5) inhibitor which phosphorylates a serine 293 residue of nNOS (neuronal nitric oxide synthase) A pharmaceutical composition for therapeutic use is provided.

In the present invention, it has been found that phosphorylation of nNOS serine 293 residue inhibits nNOS dimer formation and NO production, and that the residue is actually phosphorylated by CDK5 (cyclin-dependent kinase 5). It was also confirmed that nNOS dimer and NO production were inhibited in the nNOS-S293D mutation mimicking phosphorylation. Therefore, these results suggest that phosphorylation of nNOS-Ser293 inhibits nNOS dimer formation and contributes to the development of degenerative brain diseases such as dementia. The development of a specific antibody recognizing p-nNOS-Ser293 suggests that the diagnosis or prognosis Of the total population. Furthermore, roscovitine, a CDK5 inhibitor, can be developed as a new treatment for dementia.

FIG. 1 is a graph and photographs showing experimental results on the unchanged synaptic number and synapse marker expression in the frontal cortex of a 5 × FAD mouse in the experimental example of the present invention.
FIG. 2 is a graph and a photograph showing experimental results on the collapse of nNOS dimerization and the increase of ROS production in the 5 × FAD mouse frontal cortex in the experimental example of the present invention. FIG.
FIG. 3 is a graph showing the changes in the intracellular localization of CDK5 from a diffuse cytosolic compartment to a plasma membrane in the experimental example of the present invention and the increase in the concentration of nNOS due to an increase in level of p25 protein in the frontal cortex of 5x FAD mice Graphs and photographs showing experimental results on simultaneous colocalization.
Figure 4 shows the results of experiments on nNOS-Ser ²⁹³ phosphorylation and nNOS-Ser ²⁹³ mutation degradation of nNOS in the in vitro GSP (glycine-serine-proline) motif of CDK5 in the experimental example of the present invention, Graphs and pictures.
FIG. 5 schematically illustrates the process in which phosphorylation of serine 293 residue of nNOS inhibits nNOS dimer formation and NO production in the present invention. FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Nitric oxide synthase (NOS) is an enzyme that produces NO (nitric oxide) in vivo. There are three isoforms, of which nNOS (neuronal NOS), which is mainly distributed in neurons, And it is known to play a role of fine regulation of neurotransmission in synapses. All kinds of NOS must form a dimer in order to normally produce NO. When this dimer collapses and becomes a monomer, ROS (reactive oxygen species) such as superoxide, which is very harmful to the cell instead of NO, is produced. However, to date, there has been no study on the dimerization of nNOS in degenerative brain diseases, and no amino acid residues that affect the formation of nNOS dimers have been reported. In the present invention, it has been found that phosphorylation of serine 293 residue of nNOS inhibits nNOS dimer formation and NO production, and that the residue is actually phosphorylated by CDK5 (cyclin-dependent kinase 5). It was also confirmed that nNOS dimer and NO production were inhibited in the nNOS-S293D mutation mimicking phosphorylation. Therefore, these results suggest that phosphorylation of nNOS-Ser293 inhibits nNOS dimer formation and contributes to the development of degenerative brain diseases such as dementia. The development of a specific antibody recognizing p-nNOS-Ser293 suggests that the diagnosis or prognosis Of the total population. Furthermore, roscovitine, a CDK5 inhibitor, can be developed as a new treatment for dementia.

Accordingly, the present invention provides a method of diagnosing or predicting a degenerative brain disease by detecting the phosphorylation of a serine 293 residue of nNOS (neuronal nitric oxide synthase), a method of diagnosing or predicting a degenerative brain disease, and a method of detecting neuronal nitric oxide synthase (Cyclin-dependent kinase 5) inhibiting substance which phosphorylates a residue of a protein of the present invention as an active ingredient.

Hereinafter, the present invention will be described in detail with reference to specific experimental examples.

Materials and Experimental Methods

(1) Material

Antibodies against FLAG and β-actin were obtained from Sigma-Aldrich Co. (St. Louis, MO), antibodies against NeuN and PSD95 (postsynaptic density 95) were obtained from EMD Millipore (Darmstadt, Germany) And antibodies to CDK5 from Novus Biologicals (Littleton, CO), antibodies against p35 / p25 and synaptophysin from Abcam (Cambridge, MA) and BD biosciences (Franklin Lakes, NJ) 32P] -ATP from PerkinElmer Life Sciences (Boston, MA), purified recombinant CDK5 / p25 from Upstate Biotechnology Inc. (Lake Placid, NY), Protein A agarose from Thermo Scientific Inc. Collagenase type 2 from Worthington Biochemical Corporation (Freehold, NJ), MEM (Minimal Essential Medium), DPBS (Dulbecco's phosphate-buffered saline), FBS (fetal bovine serum) , Penicillin and streptomycin antibiotics, L-glutamine, trypsin-ethyle nediaminetetraacetic acid solution and a plastic instrument for cell culture were obtained from Gibco-BRL (Gaithersburg, MD) and all other materials were of pure analytical grade.

(2) site-directed mutagenesis of nNOS

The structure of rat FLAG-tagged wild-type (WT) nNOS (accession number X59949, UniProt ID: P29476) subcloned with mammalian expression vector pcDNA3 was obtained (Masatomo Watanabe, Laboratory of Molecular and Cellular Neuroscience, (Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki-city, Kagawa, Japan) (Watanabe and Itoh, 2011) and using a kit (QuikChange II Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, CA) The mutant (phosphomimetic) nNOS-S293D (serine is replaced by aspartate at serine 293) and the neutral nNOS-S293A mutant (serine is replaced by alanine) were prepared and sequenced sufficiently to obtain a successful mutation The PCR primer pairs designed to amplify each mutant were: nNOS-S293D-F, 5'-CCACCAAGAACGGCGACCCTTCCAGGTGCC-3 '; nNOS-S293D-R, 5'-GGCACCTGGAAGGGTCGCCGTTCTTGGTGG- S29 3A-F, 5'-CCACCAAGAACGGCGCCCCTTCCAGGTGCC-3 'and nNOS-S293A-R, 5'-GGCACCTGGAAGGGGCGCCGTTCTTGGTGG-3'.

(3) In silico analysis and motif scan for comparison alignment of nNOS, eNOS and iNOS.

The nNOS, eNOS and iNOS protein sequences from other species were obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein) and the accession numbers are as follows: nNOS Homo sapiens - UniProtKB / Swiss-Prot: P29475.2, nNOS Bos taurus - NCBI Reference Sequence: XP_002694631.2, nNOS Rattus norvegicus - UniProtKB / Swiss-Prot: P29476.1, nNOS Mus musculus - UniprotKB / Swiss-Prot: Q9Z0J4.1, eNOS Rattus norvegicus - UniProtKB / Swiss-Prot: Q62600.4, iNOS Rattus norvegicus - UniProtKB / Swiss-Prot: Q06518.2. For these sequences, comparative alignment analysis was performed using NPS @ (Network Protein Sequence Analysis, Combet et al., 2000). The rat nNOS sequence motifs, which seem to be the best phosphorylated by specific protein kinases, were identified using a motif scanning program (Scansite, http://scansite3.mit.edu.).

(4) Cell culture and transfection

BAEC (bovine aortic endothelial cells) was isolated by referring to a previously known method (see Non-Patent Document 20), and maintained in 5% CO ₂ and 37 ° C in air in MEM supplemented with 5% FBS. Endothelial cells were identified by a cobblestone configuration and a positive indirect immunofluorescence test of von Willebrand factor VIII during optical microscopy. In all experiments, 5 to 9 passages were used for these cells. For transfection experiments, cDNA encoding the rat WT-nNOS, nNOS-S293D or nNOS-S293A was cultured in a 60-mm culture dish using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) (confluence). Equal amounts of pcDNA3 were also infected for vector control experiments.

(5) Western blot analysis

The transfected cells were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF (10 μM β-glycerophosphate, 1 mM NaF, and 1 mM Na ₃ VO ₄ , and 1 × Protease Inhibitor Cocktail ^™ (Roche Molecular Biochemicals, Indianapolis, Ind.). Was separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under reducing conditions and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Piscataway, NJ) by electrophoresis. ), nNOS (1: 3,000 dilution), CDK5 (1: 1,000 dilution), p35 / 25 (1: 1,000 dilution), synaptophysin (1: 10,000 dilution), PSD95 1: 100,000 dilution), followed by the corresponding secondary antibody, and finally ECL (enhanced chemiluminescence) Was developed using reagents (Amersham, DE).

(6) NO emission measurement

The NO production in BAEC transfected with the nNOS structure was measured as the total nitrite concentration according to the previously known method (see non-patent reference 21). Briefly, after transduction, cells were cultured in serum-free MEM for 24 hours, and the conditioned medium was collected in a microcentrifuge tube and centrifuged. 200 μl of the supernatant sample was added to a 96-well plate, 1 μl of sulfanilamide (Sigma-Aldrich Co.) containing 50 μl of 5% phosphoric acid and 50 μl of 0.1% N- (1 (1-naphthyl) ethylenediamine) (Sigma-Aldrich Co.) was added thereto. After 10 minutes of color development at room temperature, the absorbance was measured at 520 nm using a microplate reader. Each sample was analyzed in duplicate wells. The calibration curve was plotted using a sodium nitrite solution.

(7) Measurement of nNOS dimerization using LT-PAGE (low-temperature SDS-PAGE)

LT-PAGE for detection of nNOS dimers and monomers was performed with reference to a previously known method (see Non-Patent Document 19). Briefly, cells in which BAEC was lysed with an empty vector WT-nNOS, nNOS-S293D or nNOS-S293A and then lysed with a lysis buffer, and the same amount of protein were added to a 1 * RAMEI without reducing agent such as DTT (dithiothreitol) Lt; / RTI > buffer (Laemmli buffer). SDS-PAGE was performed on the samples at 6% gel. The gel and running buffer were equilibrated prior to electrophoresis and the gel buffer temperature was maintained below 15 ° C by placing the electrophoresis buffer vessel on the ice bath. Following LT-PAGE, the gel was transferred and the blots probed according to conventional Western blot analysis. Samples treated with 100 mM DTT were used for total nNOS protein measurement.

8 in vitro (in vitro) phosphorylation analysis

After each transfection (vector alone, WT-nNOS or nNOS-S293A), cells were lysed in lysis buffer, and the fungus was centrifuged at 12,000 * g for 10 minutes. The supernatant (400 μg) was immunoprecipitated using 4 μl of anti-FLAG antibody. As a control, 4 μl of non-immune serum (normal mouse IgG) was used. The immunoprecipitates were washed twice with lysis buffer and twice with phosphorylation assay buffer (20 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), pH 7.4, 5 mM MgCl ₂ and 1 mM DTT). Each of the immunoprecipitates (used as substrate) was resuspended in 25 μl of phosphorylation assay buffer and 2 μCi [γ-32 ^P ] -ATP and 0.2 μg of active CDK5 / p25 were added to initiate the phosphorylation assay. After incubation at 30 DEG C for 60 minutes, the reaction was terminated by adding Rammey sample buffer. The sample was boiled for 5 minutes and subjected to 10% SDS-PAGE and the dried gel was exposed to X-ray film at -80 ° C. Phosphorylation of the nNOS protein was quantitated to the corresponding band concentration measurements using an image analysis program (ImageJ; NIH, Bethesda, Md.).

(9) Animal experiments

Transgenic mice (5x FAD) were obtained from Jackson Laboratory (Bar Harbor, ME). (2 male, 6 male), 6-month-old semi-adherent 5x FAD mouse (Male 8 male, Magnetic 4 male, Male) and non-transgenic control mice (6 months old, male 9 male) were used in the experiment. Water and feed were fed unlimitedly to the experimental animals in triplicate per cage. At the beginning of the experiment, the mice were kept at Konkuk University at a temperature of 22 ± 1 ° C and a relative humidity of 50 ± 10% at 12:12 light cycle (light period 07 ~ 19). All experiments were conducted with the approval of the Konkuk University Committee (KU14163) and they complied with animal management regulations.

(10) Immunohistochemistry (IHC)

All mice were sacrificed and perfused intracardially with 10 mM cold PBS (phosphate-buffered saline) for 20 min and perfused with 4% PFA (paraformaldehyde). The mouse brain was quickly removed, immersed in a tube containing the same fixative at 4 占 폚 for 1 day, and frozen for 3 days in PBS containing 30% sucrose. The brain was then frozen in dry ice powder and stored at -80 ° C until the experiment. Coronal sections (30 μm thick) were cut through the frontal cortex using a cryostat (Leica CM1850, Wetzlar, Germany) to prepare serial sections. Well plate (Falcon, Becton Dickinson Labware SA, Le Pont-de-Claix, France) containing 1 ml of tissue stock solution (glycerol and ethylene glycol in 200 mM phosphate buffer) The sections were collected. The tissue sections of each mouse were subjected to a free-floating technique. The sections were immersed in blocking buffer (10% horse serum and 0.3% Triton X-100 in PBS) for 1 hour at room temperature. (1: 100 dilution), an anti-nNOS antibody (1: 100; Novus, Littleton, CO), anti-p35 / p25 antibody : 100 dilution). The sections were then rinsed three times for 10 minutes with wash buffer (1.5% horse serum and 0.1% Triton X-100 in PBS). Secondary antibodies conjugated with Alexa488 or Alexa594 (ThermoFisher Sci., Waltham, Mass.) Were diluted in blocking buffer and incubated at room temperature for 2 hours. After washing three times with wash buffer, brain slices were attached to a coated glass slide and mounted on a ProLong solution (ThermoFisher Sci.). The slides were observed with a confocal microscope (LSM 700 confocal microscope, Carl Zeiss, Jena, Germany) and the fluorescence intensity was measured.

(11) ROS (reactive oxygen species) level measurement

DHE (dihydroethidium, 1 mg / kg, Sigma-Aldrich Co.) intraperitoneal injection was performed according to a previously known method for measuring ROS levels in brain tissue (see non-patent reference 22). Briefly, DHE stock (1 mg / ml) was made in DMSO (dimethylsulfoxide) and DHE was added to the same volume of sterile physiological saline and injected into the lower abdomen 30 min before sacrifice. All animals were sacrificed and perfused with cold 4% PFA in PBS (pH 7.4) for 20 min. Fluorescence images were displayed on a confocal microscope (LSM 700 confocal microscope, Carl Zeiss) for brain slices.

(12) Transmission electron microscopy observation

Cerebral cortices were dissected from the 5 × FAD mouse or control and each sample was fixed with 2% glutaraldehyde-paraformaldehyde in 0.1 M PB (phosphate buffer, pH 7.4) , And washed three times in 0.1 M PB for 30 minutes. The samples were fixed after 2 hours with 1% OsO ₄ dissolved in 0.1 M PB, and the ethanol was gradually increased (50-100%) and dried and infiltrated with propylene oxide. Specimens were embedded using a kit (Poly / Bed 812, Polysciences Inc., Warrington, Pa.). After polymerization at 60 ° C for 24 hours in an embedding and electron microscope oven (TD-700, DOSAKA, Kyoto, Japan) with a pure resin, 350 nm thick sections were stained with toluidine blue for optical microscopy. For contrast staining, thin sections of 70 nm were double-stained with 7% uranyl acetate and lead citrate for 20 min. The sections were cut with a microtome (LEICA Ultracut UCT Ultra-microtome, Leica Microsystems, Austria). All thin sections were observed with a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan).

(13) Statistical analysis

All results are expressed as means ± standard deviation (SD) ( n is the number of experiments). Significant differences were identified using Student's t test for two points, and statistical significance was determined for P <0.05.

5x FAD mouse Forehead  Synaptic numbers and synapses in the cortex Marker  Unchanged expression

Recent 6-month-old 5 × FAD mice have been reported to exhibit increased accumulation of amyloid β proteins in disorders of the place / spatial training and in the frontal cortex (see non-patent reference 23) . These results indicate that the observed cognitive decline is responsible for the number of damaged synapses and / or structural integrity in the front of 5x FAD mice. Thus, in a recent study, the present inventors first examined whether the synaptic number in the frontal cortex of a 5x FAD mouse is lowered. As shown in FIG. 1A, the difference in synapse number was not significant ( P = 0.096) even though the number of synapses tended to decrease in the 5x FAD mouse. Furthermore, biochemical analysis revealed that the expression of pre- and post-synaptic marker proteins including synaptophysin and PSD95 was significantly reduced in 5 × FAD mice compared to control at 6 months of age as well as at 2 months (See FIG. 1B). These results indicate that impaired cognitive function in 6-month-old 5 × FAD mice may be due to a modification of the functional integrity of neurons prior to explicit structural changes. In FIG. 1A, the number of synapses in the frontal cortex and the control group of the 5 × FAD mouse was counted randomly at ten times at 10,000 × magnification, and the graph shows the difference in the number of synapses. In addition, densitometry was used for synaptophysin expression or PSD95 quantitation for beta -actin in Figure 1b, and the graph showed an average multiple change for the control (n = 4) Representative examples of the experiment are shown.

nNOS  Decomposition of dimerization and 5x FAD mouse Forehead  Cortical ROS  Increase production

Next, it has been attempted to confirm whether the intracellular signal pathway is related to the observed cognitive disorder (see Non-Patent Document 23). Previously, nNOS has been reported to play an important role in synaptic plasticity and memory formation as well as inhibition of the AD invention (see Non-Patent Documents 15, 16, 17 and 24). Furthermore, NOS dimerization is essential for normal enzyme function (see non-patent reference 14), eNOS dimerization in the blood vessels of older mice is disrupted and ROS production is increased, leading to vascular disease including hypertension and atherosclerosis (See Non-Patent Document 19). This is because when the NOS dimer collapses, each monomer produces ROS including superoxide instead of NO (see Non-Patent Document 14). Thus, in the present invention, we examined nNOS dimerization status in the frontal cortex of 6-month-old 5x FAD mice and clearly confirmed that nNOS dimerization was disrupted: the level of dimerization was less than 50% of that of the control group without change in total nNOS expression, The monomer was increased up to 2.7 times as compared to the control (see Fig. 2a). In contrast to 6 month old 5 × FAD mice, nNOS dimerization did not change in 2 month old 5 × FAD mice. Next, in the present invention, the level of ROS production in the 5x FAD cortex was examined because it is experimentally difficult to clearly determine the nNOS-derived NO that is not produced by other homologous proteins, particularly iNOS. As shown in FIG. 2B, ROS production was significantly higher in the frontal cortex of the 5x FAD mouse, and increased ROS was found to be significant in neurons (see arrow), indicating that the degraded nNOS plays a pathological role in 5x FAD mice In the study. In Figure 2a, total nNOS expression was measured using western blot analysis, dNTPs were used for quantification of nNOS duplexes for total nNOS, and graphs showed mean multiple changes for control (n = 4) At least four of the experiments were representative. Also, the fluorescence confocal photograph in FIG. 2B is representative of at least four experiments.

Diffused  Cytoplasmic compartment cytosolic  compartment to the plasma membrane CDK5 Intracellular  Position shifting and 5x FAD mouse Forehead  In the cortex p25  Due to the increased level of protein nNOS Simultaneous localization of colocalization )

It has been reported that amyloid beta accumulation in the frontal cortex of 6-month-old 5x FAD mice is increased (see non-patent document 23). Accumulated amyloid beta also stimulates intracellular signaling pathways and is known to be associated with p25, resulting in activation of CDK5 (see non-patent document 11). Thus, the present invention explores the role of CDK5 in reduced levels of nNOS dimers observed in the frontal cortex of 5x FAD mice. No noticeable changes in the overall CDK5 expression in the cortex of the 5x FAD mouse were found (see Figures 3a and 3b), but the IHC results suggest that the intracellular location of CDK5 diffuses from the diffuse distribution through the nucleus and the cell body, cytoplasmic membrane "(see FIG. 3A). In contrast, p25 expression was increased in the frontal cortex of the 6-month-old 5x FAD mouse by 2-fold compared to the control (see Figures 3c and 3d), indicating that CDK5 and its activation It is a role of the argument p25. Finally, in the present invention, the positions of nNOS and p25 were examined using IHC. As shown in FIG. 3E, nNOS was detected in cell bodies and cytoplasmic membranes of the frontal cortex of 5x FAD mice. Moreover, the nNOS protein was fairly co-localized with increasing levels of p25 protein in the 5x FAD cortex (see Figure 3e). These results indicate that increased p25 affects nNOS activity, including dimerization state, by constituting and activating its significant kinase, CDK5. In Figures 3b and 3d, IHC staining was representative of at least four experiments. In addition, densitometry was used to quantify the expression of CDK5 on p35 or p-actin on p35, and the graph showed a mean multiple change (n = 4) relative to the control, and the blots were representative of at least 4 experiments .

CDK5 Of in vitro ( in vitro ) GSP ( glycine - serine - proline ) In the motif nNOS - Ser ²⁹³  Phosphorylation and nNOS - Ser ²⁹³  Mutant nNOS  Dimerization collapse

It is known that N-terminal sequences including N-terminal hooks and Zn-loops play an important role in nNOS dimerization and normal NO production (see Non-Patent Document 18) The present invention suggests the role of CDK5 in the collapse of nNOS dimers in the frontal cortex of 5x FAD mice. Moreover, CDK5 is categorized as one of the CMGC kinase superfamily, wherein CMGC is a member of the CDK family, including CDK, mitogen-activated protein kinases, glycogen synthase kinases and CDK-like kinases CDK-like kinase) (see Non-Patent Document 25). CMGC kinase, including CDK5, recognizes and phosphorylates S / T in the XS / TP sequence, where X represents any amino acid (see Non-Patent Documents 21 and 25). Based on this fact, the present invention sought to find an XS / TP sequence at the N-terminus of nNOS. For comparative alignment, similarity and heterology in rat nNOS, eNOS and iNOS protein sequences were analyzed using NPS @ (Network Protein Sequence Analysis, Combet et al., 2000). As shown in Fig. 4A, a new GSP motif was found in Ser293 located upstream of the N-terminal hook of rat nNOS that is not present in eNOS and iNOS. Moreover, GSP motifs in nNOS are well conserved across all species (see FIG. 4A), which implies a role in nNOS dimerization. Next, in the present invention, another in silico analysis was performed using a motif scan (http://scansite3.mit.edu) to predict whether CDK5 could phosphorylate nNOS-Ser ²⁹³ in the GSP motif. As shown in Figure 4b, the motif scan predicted that CDK5 could phosphorylate nNOS-Ser ²⁹³ at the highest probability of the four motif sequences located at the N-terminus. Based on in silico analysis predictions, the present invention experimentally tested this possibility. For the experiments, nNOS-S293D (phosphomimetic form) and nNOS-S293A (neutral form) were made as two mutants for rat nNOS-Ser ²⁹³ . As shown in Figure 4c, the in vitro phosphorylation assay clearly showed that CDK5 / p25 definitely phosphorylates nNOS-Ser ²⁹³ . The in silico assay predicted at least 9 motif sequences for the CDK5 substrate in the entire nNOS sequence, but the CDK5 / p25-mediated phosphorylation for the nNOS-S293A mutation was reduced to less than 50% compared to WT-nNOS (see Figure 4c) , Indicating that nNOS-Ser ²⁹³ in the GSP motif is at least one of the major substrate residues for CDK5 in vitro. Next, in the present invention, it was examined whether rat nNOS-Ser ²⁹³ plays a significant role in the regulation of nNOS dimerization. To illustrate this, the mutant constructs were infected with BAEC with the cellular machinery required for eNOS regulation and LT-PAGE was performed to assess nNOS dimerization. As shown in FIG. 4d, the nNOS dimerization of nNOS-S293D or nNOS-S293A was reduced by 50% compared to the WT-nNOS control, while the monomers of these mutants were increased by 3-fold compared to the WT-nNOS control. Moreover, NO production of nNOS-S293D or nNOS-S293A was reduced by 50% compared to the WT-nNOS control, similar to the collapse of nNOS dimerization (see FIG. 4e). These results indicate that any modification, including phosphorylation, in Ser293 of nNOS disrupts nNOS dimer formation and consequently reduces NO production. The expression of FLAG-tagged nNOS transduced in Figure 4c was determined using western blot analysis and densitometry was used to quantify the optical density of radioactive ³² P contained in each nNOS to total nNOS, (N = 4), and autoradiograms and blots were representative of at least four experiments. In addition, the expression of FLAG-tagged nNOS transduced in Figure 4d was measured using Western blot analysis and nNOS duplexes for all nNOS or densitometry for monomer quantification were used and the graph shows the mean multiple change over WT (N = 4), and the blots were representative of at least 4 experiments. In addition, the expression of FLAG-tagged nNOS transduced in Figure 4e was measured using Western blot analysis, and the graph shows the NO accumulation to WT by mean multiple (n = 4), and the blot was tested at least 4 times Representative examples are shown.

As a clear evidence, nNOS-derived NO contributes to the precise regulation of synaptic plasticity (see non-patent references 15 and 24) and reduces the incidence of AD (see non-patent document 17). The formation of NOS homodimers is necessary to maintain physiological NO levels because the collapsed NOS monomers generate superoxides that are very harmful ROS instead of NO (see Non-Patent Document 14). For example, eNOS dimerization has been reported to cause endothelial dysfunction and hypertension in the blood vessels of old mice (see Non-Patent Document 19). However, up to now there has been no report on the effects of nNOS dimerization and the onset of neurodegenerative diseases such as AD. In this respect, the most important finding in the present invention is that nNOS dimerization is reduced without changing the overall expression in the cortex of 6-month-old 5x FAD mice (see Fig. 2a). Furthermore, the expression of synaptic numbers and synaptic marker proteins, which demonstrate no clear structure and no pathological damage in the mouse, was unchanged (see Figures 1a and 1b. According to previous studies, 6 month old 5x FAD mice showed cued and place / spatial water maze tasks, whereas 5 × FAD mice prefer to be given a signal in the symbol experiment, the expression of the signaled cognition (See Non-Patent Document 23). Thus, the present invention suggests that a weak disorder of cognitive function in AD is caused by a disruption of nNOS dimerization prior to clear pathological signs and signs of dementia .

Excessive production of amyloid beta protein in neuronal cells contributes to the onset of AD (see Non-Patent Document 1). When the amyloid-beta protein increases, the protein binds to a receptor located in the cell membrane and mediates cytotoxic cascade, resulting in the death of neuronal cells (see Non-Patent Document 1). Of the many signal transducers, CDK5 is central to AD development and induces a variety of neurotoxic effects through the activation of abnormal CDK5 by p25 produced by the division of the normal binding partner (p35) See Patent Documents 3, 5, 6, 12 and 13). Consistent with the role of this molecular mechanism in the pathogenesis of AD, it has been reported that the accumulation of amyloid beta protein is increased in the frontal cortex of 5x FAD mice (see non-patent reference 23) (See Figures 3c and 3d). &Lt; / RTI &gt; Furthermore, in the present invention, it was confirmed that the increased p25 protein in the 5x FAD mouse cortex is highly localized with the plasma membrane and cytoplasmic nNOS (see Fig. 3e), and the intracellular localization of CDK5 also shifts from the diffusion cytosolic expression to the plasma membrane (See Fig. 3A). The increase in p25 is well known to constitute CDK5 as a specific intracellular compartment and abnormal activation thereof, resulting in various cytotoxic effects (see Non-Patent Documents 3, 5, 6, 12 and 13). Thus, the present invention sees that increased levels of p25 in the frontal cortex of 5x FAD mice also constitute CDK5 and subsequently phosphorylation of nNOS, which in turn may affect nNOS dimerization. In the present invention, nNOS dimerization was disrupted in 5x FAD mice (see Fig. 2a), and in the silico analysis and in vitro phosphorylation assay, GSP motifs including nNOS-Ser ²⁹³ were shown to be actually phosphorylated by active CDK5 4c). Also, modification of nNOS-Ser ²⁹³ , including substitution mimicking phosphorylation of serine residues, inhibited nNOS dimer formation (see FIG. 4d). Thus, these results strongly support the possibility that the increased p25 protein constitutes and binds CDK5 and that the abnormally activated CDK5 phosphorylates nNOS-Ser ²⁹³ , resulting in the collapse of the nNOS dimer.

N-terminal motifs including N-terminal lead sequences, N-terminal hooks and Zn-loop sequences in nNOS have been reported to be very important for duplexing (see non-patent document 18). There are homologous homologous proteins in comparable sequence, but nNOS has a unique additional N-terminal sequence that is different from other homologous proteins. Unlike eNOS and iNOS, deletion and / or point mutations in these sequences are well known to affect nNOS dimerization (see non-patent references 18, 26 and 27). In this regard, the present invention uses silicic acid analysis to confirm interesting results. In the present invention, a new GSP motif that was well conserved across all species was identified in nNOS, which was completely deleted in eNOS and iNOS (see FIG. 4A). Interestingly, the GSP motif is located very early in the N-terminal hook sequence (see FIG. 4A). Based on these facts, nNOS-Ser ²⁹³ is expected to contribute to nNOS dimer formation in GSP motifs. It is further supported by this possibility that the modification of the residue in the present invention disrupts the nNOS dimerization.

Activation of nNOS in experiments using slice cultures produces low levels of NO, which has a large neuroprotective effect on ischemic brains (see non-patent reference 28). On the other hand, physiological NO production from nNOS in neurons and astrocytes has anti-inflammatory activity by inhibiting the activity of astrocytes and microglia (see Non-Patent Document 29), and chronic nNOS inhibition is inhibited in vivo (see non-patent reference 16) in vivo in inflammatory neurodegeneration. On the other hand, neuronal oxidative stress induced by ROS certainly contributes to the onset of AD and is known as an early event in the AD invention (see Non-Patent Documents 30 to 33). Furthermore, it has been reported that increased ROS production activates amyloid precursor proteins that process enzymes such as [beta] -secretase, which trigger an extreme cycle of A [beta] production, to worsen A [beta] production 34). The collapsed NOS monomer is well known to produce superoxide instead of NO (see Non-Patent Document 14). Consistent with this, it has been shown clearly in the present invention that nNOS monomer and ROS production levels are increased in 5x FAD mice (see Figure 2) and nNOS dimerization and NO production of nNOS-S293D or nNOS-S293A is reduced 4c and 4e). These results suggest that neurodegenerative diseases such as AD progression, CDK5-induced phosphorylation of nNOS-Ser ²⁹³ lead to an increase in nNOS monomers and promote the production of ROS, including superoxide, instead of NO. As a result, increased ROS can damage many cellular constituents and cause damage to synaptic integrity, eventually leading to the initiation of a severe cycle leading to neurodegeneration. This hypothesis can explain whether weak cognitive impairment and memory impairment occurs before apparent dementia.

In conclusion, the present invention is the first to disclose the reduction of nNOS dimerization levels in the 5x FAD mouse cortex. Further, the present invention identified novel regulatory motif sequences in nNOS-Ser ²⁹³ , which were well conserved across all species, phosphorylated by CDK5, and involved in nNOS dimerization and NO production. These results can contribute to prediction of the diagnosis or prognosis of neurodegenerative diseases associated with nNOS such as AD (see FIG. 5).

Hereinafter, a CDK5 (cyclin-dependent kinase 5) inhibitor which phosphorylates a serine 293 residue of nNOS (neuronal nitric oxide synthase) according to the present invention is used as a preventive or therapeutic agent for degenerative brain diseases containing roscovitine as an active ingredient The application form of the pharmaceutical composition will be described, but is not limited thereto.

Formulation Example 1: Powder preparation

Table 1 below shows an acid composition containing roscovitine as an active ingredient. Powders can be prepared by mixing the following ingredients and filling the airtight container.

Formulation Example 2: Preparation of tablets

Table 2 below shows a tablet composition containing roscovitine as an active ingredient. The tablet may be prepared by mixing the following ingredients and then tableting according to a conventional tablet preparation method.

Formulation Example 3: Capsule preparation

Table 3 below illustrates the composition of capsules containing roscovitine as an active ingredient. The capsules may be prepared by mixing the following ingredients according to the conventional method for preparing a capsule, and filling the capsules with gelatin.

Formulation Example 4: Injection preparation

Table 4 below shows the composition of injections containing roscovitine as an active ingredient. The injectable preparation can be prepared in the following ingredient contents per ampoule (2 ml) according to the usual injection preparation method.

Formulation Example 5: Liquid preparation

Table 5 below shows the composition of the liquid containing roscovitine as an active ingredient. The liquid preparation is prepared by adding and dissolving each of the following ingredients in purified water according to a conventional liquid preparation method, mixing the following components after adding a proper amount of lemon flavor, adding purified water to adjust the total volume to 100 ml, filling and sterilizing in a brown bottle .

The preferred embodiments of the present invention have been described in detail with reference to the drawings. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing detailed description, and all changes or modifications derived from the meaning, range, and equivalence of the claims are included in the scope of the present invention Should be interpreted.

Claims (2)

A method for detecting the collapse of dimerization of neuronal nitric oxide synthase (nNOS) in a brain tissue of a patient to provide information necessary for diagnosis or prognosis prediction of degenerative brain disease. The method according to claim 1, wherein the degradation of the dimerization of the nNOS is detected using LT-PAGE (low-temperature SDS-PAGE).

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