FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2130339832> ?p ?o ?g. }

• W2130339832 endingPage "6606" @default.
• W2130339832 startingPage "6594" @default.
• W2130339832 abstract "Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G1 phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca2+ and activation of calpain, a Ca2+-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca2+ and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease. Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G1 phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca2+ and activation of calpain, a Ca2+-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca2+ and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease. Increasing evidence suggests that neuronal apoptosis is involved in neurodegenerative disorders (1Stefanis L. Burke R.E. Greene L.A. Curr. Opin. Neurol. 1997; 10: 299-305Crossref PubMed Scopus (147) Google Scholar). A greater understanding of the cellular signaling pathways that regulate neuronal apoptosis may lead to novel therapeutic targets. However, the signaling pathways are not yet fully understood. Cell cycle progression is regulated through complex events controlled through the actions of cyclin-dependent kinases (CDKs) 2The abbreviations and trivial names used are: CDK, cyclin-dependent kinase; ALLN, N-acetyl-Leu-Leu-Nle-aldehyde; HD, Huntington disease; Hoechst 33342, bisbenzimide H 33342; MDL28170, Z-Val-Phe-aldehyde; MK801 maleate, (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5-10-imine maleate; MSN, medium size spiny neuron; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMDA,N-methyl-d-aspartate; 3-NP, 3-nitropropionic acid; OGB-1, Oregon Green 488 BAPTA-1, AM; PD150606, 3-(4-lodophenyl)-2-mercapto-(Z)-2-propenoic acid; Rb, retinoblastoma protein; siRNA, small interfering RNA; TBS, Tris-buffered saline; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone. and cyclins. In addition, two classes of CDK inhibitors are involved in cell cycle arrest mechanisms (2Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar). Interestingly, a growing body of work shows that cell cycle components are involved in neuronal apoptotic death. For instance, neuronal apoptosis is accompanied by changes in CDK activity and cyclin expression (3Freeman R.S. Estus S. Johnson E.M. Neuron. 1994; 12: 343-355Abstract Full Text PDF PubMed Scopus (548) Google Scholar, 4Padmanabhan J. Park D.S. Greene L.A. Shelanski M.L. J. Neurosci. 1999; 19: 8747-8756Crossref PubMed Google Scholar, 5Timsit S. Rivera S. Ouaghi P. Guischard F. Tremblay E. Ari Ben Y. Khrestchatisky M. Eur. J. Neurosci. 1999; 11: 263-278Crossref PubMed Scopus (130) Google Scholar, 6Ino H. Chiba T. J. Neurosci. 2001; 21: 6086-6094Crossref PubMed Google Scholar, 7Wen Y. Yang S. Liu R. Simpkins J.W. FEBS Lett. 2005; 579: 4591-4599Crossref PubMed Scopus (59) Google Scholar). Moreover, agents that inhibit cell cycle progression protect neuronal PC12 cells, cortical neurons, sympathetic neurons, and cerebellar granule neurons from apoptotic death (8Farinelli S.E. Greene L.A. J. Neurosci. 1996; 16: 1150-1162Crossref PubMed Google Scholar, 9Park D.S. Farinelli S.E. Greene L.A. J. Biol. Chem. 1996; 271: 8161-8169Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Similarly, overexpression of CDK inhibitors or dominant-negative CDK protect neurons from death caused by loss of trophic support (10Park D.S. Levine B. Ferrari G. Greene L.A. J. Neurosci. 1997; 17: 8975-8983Crossref PubMed Google Scholar), DNA damage (11Park D.S. Morris E.J. Padmanabhan J. Shelanski M.L. Geller H.M. Greene L.A. J. Cell Biol. 1998; 143: 457-467Crossref PubMed Scopus (243) Google Scholar), proteosomal inhibition (12Rideout H.J. Wang Q.H. Park D.S. Stefanis L. J. Neurosci. 2003; 23: 1237-1245Crossref PubMed Google Scholar), and ischemia (13Rashidian J. Iyirhiaro G. Aleyasin H. Rios M. Vincent I. Callaghan S. Bland R.J. Slack R.S. During M.J. Park D.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14080-14085Crossref PubMed Scopus (129) Google Scholar). These results suggest that cell cycle-related molecules play pivotal roles in multiple forms of neuronal cell death. 3-Nitropropionic acid (3-NP) irreversibly inhibits mitochondrial enzyme succinate dehydrogenase and interrupts electron transport, inducing impairment of energy metabolism (reviewed in Ref. 14Brouillet E. Jacquard C. Bizat N. Blum D. J. Neurochem. 2005; 95: 1521-1540Crossref PubMed Scopus (305) Google Scholar). Despite the mitochondrial inhibition broadly in brain (15Brouillet E. Guyot M.C. Mittoux V. Altairac S. Conde F. Palfi S. Hantraye P. J. Neurochem. 1998; 70: 794-805Crossref PubMed Scopus (185) Google Scholar), systemic administration of 3-NP produces selective degeneration of striatum that is reminiscent of Huntington disease (HD) (16Beal M.F. Brouillet E. Jenkins B.G. Ferrante R.J. Kowall N.W. Miller J.M. Storey E. Srivastava R. Rosen B.R. Hyman B.T. J. Neurosci. 1993; 13: 4181-4192Crossref PubMed Google Scholar, 17Wűllner U. Young A.B. Penney J.B. Beal M.F. J. Neurochem. 1994; 63: 1772-1781Crossref PubMed Scopus (124) Google Scholar, 18Brouillet E. Hantraye P. Ferrante R.J. Dolan R. Leroywillig A. Kowall N.W. Beal M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7105-7109Crossref PubMed Scopus (432) Google Scholar). In addition, post-mortem brain extracts of HD patients showed reduced activity (19Browne S.E. Bowling A.C. MacGarvey U. Baik M.J. Berger S.C. Muqit M.M. Bird E.D. Beal M.F. Ann. Neurol. 1997; 41: 646-653Crossref PubMed Scopus (747) Google Scholar, 20Gu M. Gash M.T. Mann V.M. Javoy-Agid F. Cooper J.M. Schapira A.H. Ann. Neurol. 1996; 39: 385-389Crossref PubMed Scopus (621) Google Scholar) and levels of expression (21Benchoua A. Trioulier Y. Zala D. Gaillard M.-C. Lefort N. Dufour N. Saudou F. Elalouf J.-M. Hirsch E. Hantraye P. Deglon N. Brouillet E. Mol. Biol. Cell. 2006; 17: 1652-1663Crossref PubMed Scopus (209) Google Scholar) of succinate dehydrogenase. Thus, understanding molecular machinery in 3-NP-induced selective neurodegeneration in striatum gives clues to a therapeutic strategy for HD. Although the mechanism by which 3-NP produces selective vulnerability of the striatal region is not fully understood, Galas et al. (22Galas M.C. Bizat N. Cuvelier L. Bantubungi K. Brouillet E. Schiffmann S.N. Blum D. Neurobiol. Dis. 2004; 15: 152-159Crossref PubMed Scopus (52) Google Scholar) demonstrated that calpain, a Ca2+-dependent protease, is selectively activated in striatal neurons after 3-NP administration. We previously showed that calpain activation induces cell cycle progression in glutamate-induced neurotoxicity (23Akashiba H. Matsuki N. Nishiyama N. J. Neurochem. 2006; 99: 733-744Crossref PubMed Scopus (19) Google Scholar). The first aim of the present study is to examine the involvement of aberrant cell cycle progression in 3-NP-induced neuronal cell death. Our results show that 3-NP treatment induced calpain activation and cell cycle activation in striatal but not in cortical neurons. Primarily cultured neurons derived from both striatum and cerebral cortex are known to be vulnerable to 3-NP (24Behrens M.I. Koh J. Canzoniero L.M.T. Sensi S.L. Csernansky C.A. Choi D.W. Neuroreport. 1995; 6: 545-548Crossref PubMed Scopus (104) Google Scholar). The second aim of the present study is to investigate the shared mechanisms of 3-NP neurotoxicity between cultured striatal and cortical neurons. Our results show that NMDA receptor-dependent neurotoxic mechanisms are common between neurons taken from the two regions. Thus, we here delineated both the difference and commonality in molecular machineries in 3-NP-induced death in striatal and cortical neurons. Primary Neuronal Culture—Primary neuronal cultures of rat striatum and cerebral cortex were prepared as described previously (25Okuda S. Nishiyama N. Saito H. Katsuki H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12553-12558Crossref PubMed Scopus (252) Google Scholar) with some modifications. All animal experiments conformed to the Japanese Pharmacological Society guide for the care and use of laboratory animals and the guidance of the University of Tokyo, with care to minimizing the number of animals and their suffering. In brief, whole brains were isolated from fetal rats of the Wistar strain (SLC Inc., Shizuoka, Japan) at embryonic days 17-18, and the striata and cerebral cortices were dissected out and treated with 0.25% trypsin (Difco) and 0.01% deoxyribonuclease I (Sigma) at 37 °C for 30 min. The cells were suspended in Neurobasal medium (Invitrogen) containing 10% fetal bovine serum (Biowest SAS, Nuaille, France). Then they were plated at a density of 1.0 × 105 cells/cm2 in 96-well plates (Corning), in 6-well plates (Corning), in a 35-mm dish (Corning), or on glass coverslips (Matsunami Glass Ind. Ltd., Osaka, Japan) equipped with FlexiPerm (Sartorius AG, Göttingen, Germany) precoated with 0.02% (v/v) polyethyleneimine (Sigma). Cultures were kept at 37 °C in humidified 5% CO2, 95% air. At 24 h after plating, the medium was changed to serum-free Neurobasal medium supplemented with 2% B27 (Invitrogen). Half of the culture medium was changed every 3 days. Drug Treatment—On day 8 in vitro, drugs were treated by changing half of the culture medium. 3-NP (Sigma) was dissolved in water and brought to pH 7.5 with 1 n NaOH. Caspase-3-specific inhibitor, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) (Enzyme Systems Products Inc., Livermore, CA); N-methyl-d-aspartate (NMDA) (Sigma); three calpain inhibitors (N-acetyl-Leu-Leu-Nle-CHO (ALLN) (Sigma), Z-Val-Phe-CHO (MDL28170) (Merck), and 3-(4-lodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) (Merck)); and ubiquitin-proteasome inhibitor, lactacystin (Sigma) were added to the cell cultures simultaneously as they were treated with 3-NP. NMDA receptor antagonist (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5-10-imine maleate ((+)-MK801 maleate) (TOCRIS, Park Ellisville, MO) and three pharmacological CDK inhibitors (olomoucine (Sigma), purvalanol A (Merck), and roscovitine (Merck)) were added to the cell cultures 1 h prior to the treatment of 3-NP. All drugs were treated during 3-NP exposure, with continued treatment until assay. 3-NP Injection in Vivo—Male Sprague-Dawley rats (12-16 weeks old, 300-400 g) were housed at two per cage and kept under temperature- and humidity-controlled conditions (23 ± 1 °C and 50 ± 10%, respectively). 3-NP injection was conducted by two methods. In the first group, rats were injected intraperitoneally with 10 mg/kg 3-NP (Sigma; dissolved in 0.9% (w/v) saline and pH adjusted to 7.5 with 1 n NaOH) once every 4 days for 3 weeks (26Borlongan C.V. Koutouzis T.K. Randall T.S. Freeman T.B. Cahill D.W. Sanberg P.R. Brain. Res. Bull. 1995; 36: 549-556Crossref PubMed Scopus (104) Google Scholar). In the second group, rats were injected intraperitoneally with 7.5 mg/kg 3-NP (Sigma) twice daily for 5 days (27Guyot M.-C. Hantraye P. Dolan R. Palfi S. Maziere M. Brouillet E. Neuroscience. 1997; 79: 45-56Crossref PubMed Scopus (140) Google Scholar). In both groups, control animals received 0.9% (w/v) saline. All rats survived after 3-NP injection. Assessment of Cell Viability—Neuronal cell viability was assessed with two methods. In the first method, cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in phosphate-buffered saline for 30 min at 4 °C. The cells were stained and visualized with cresyl violet (Wako Pure Chemical Industries) and also identified as neurons as judged from their morphological characteristics. The number of surviving neurons was counted and normalized by setting the number of surviving neurons in nondrug control cultures as 100%. In the second method, mitochondrial dehydrogenase activity that reduces 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to MTT formazan was used to determine cell survival in a quantitative colorimetric assay, a biochemical index for cellular viability (28Abe K. Matsuki N. Neurosci. Res. 2000; 38: 325-329Crossref PubMed Scopus (175) Google Scholar). The cells were incubated with 0.25 mg/ml MTT (Sigma) for 35 min at 37 °C in humidified 5% CO2, 95% air. The reaction was stopped by a solution (pH 4.7) containing 50% dimethylformamide (Wako Pure Chemical Industries) and 20% SDS (Wako Pure Chemical Industries). The amount of intracellular MTT formazan product was quantified spectrophotometrically using a microplate reader (model 550; Bio-Rad) at an excitation wavelength of 570 nm and an emission wavelength of 655 nm. Immunocytochemistry—After fixation as described above, cells were washed and permeabilized with 0.1% Triton X-100 (Wako Pure Chemical Industries) in Tris-buffered saline (TBS) (25 mm Tris (pH 8.0), 125 mm NaCl) for 15 min. The cultures were incubated with TBS containing 5% goat serum (Vector Laboratories Inc., Burlingame, CA) for 1 h at room temperature and then with a primary antibody overnight at 4 °C. The primary antibodies used were anti-p27 (mouse, 1:200 dilution; BD Transduction Laboratories), anti-MAP2 (microtubule-associated protein-2) (rabbit, 1:1000; Chemicon International, Inc., Temecula, CA), and anti-DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After washing, the cells were incubated with a fluorochrome-conjugated secondary antibody (Alexa 488-conjugated anti-mouse or anti-goat IgG, 1:1000 dilution; Alexa 568-conjugated anti-rabbit IgG, 1:1000 dilution; Molecular Probes Inc., Eugene, OR) for 1 h at room temperature. For the last 5 min, cells were incubated with the bisbenzimide, Hoechst 33342 (Sigma), at 5 μg/ml and a fluorescent probe was intercalated into nuclear DNA. After washing, images were acquired with a cooled CCD camera (Hamamatsu ORCA II; Hamamatsu Photonics KK, Hamamatsu, Japan) and a ×40 objective lens equipped with an inverted microscope (Eclipse TE300; Nikon Corp., Tokyo, Japan). Those images were analyzed using an Aqua-Cosmos system (Hamamatsu Photonics). Immunoblotting—In in vitro analysis, cells were washed with cold TBS and lysed for 30 min on ice in radioimmunoprecipitation buffer (10 mm Na2HPO4, 300 mm NaCl, 0.1% (w/v) SDS, 1% Nonidet P-40, 0.1% (w/v) sodium deoxycholate, 2 mm EDTA disodium salt dihydrate, pH 7.0) containing 0.5 μg/ml leupeptin (Peptide Institute Inc., Osaka, Japan), 1 μg/ml pepstatin A (Peptide Institute), 1 mm NaF (Wako Pure Chemical Industries), and 1 mm Na3VO4 (Wako Pure Chemical Industries). In in vivo analysis, brains were removed and sectioned at 400-μm thickness, and striatum and cerebral cortex were dissected under a microscope, homogenized, and lysed for 1 h on ice in radioimmunoprecipitation buffer, adding 10 mm aprotinin (Roche Applied Science) and 0.01% deoxyribonuclease I (Sigma). Proteins were denatured by heating at 65 °C in sample buffer (10 mm Tris-HCl (pH 6.8), 10 mm dithiothreitol, 2% (w/v) SDS, and 0.01% w/v bromphenol blue) for 10 min. Protein concentration was measured using a bicinchoninic acid protein assay kit according to the manufacturer's instructions (Pierce). Samples (20-50 μg) were separated electrophoretically and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 4% nonfat milk in TBS with 0.1% Tween 20 for 2 h and then incubated overnight with primary antibodies at 4 °C on a rotary platform with gentle agitation. The primary antibodies used were anti-p27 (mouse, 1:1000 dilution; BD Transduction Laboratories), anti-β-actin (mouse, 1:2000 dilution; Sigma), and anti-phosphoretinoblastoma protein (Rb) (Ser795) antibody (rabbit, 1:2000 dilution; New England Biolabs, Beverly, MA). They were subsequently probed with secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (diluted 1:1000-1:2000; Sigma). After washing, detection was performed using the enhanced chemiluminescence assay (Amersham Biosciences). Equal protein loading was confirmed using Coomassie Brilliant Blue staining of the gels or probing with anti-β-actin. To provide semiquantitative analysis, band densitometry analysis of the membrane was performed using scanned images of nonsaturated immunoblot films, using software (Scion Image, version Beta 4.0.2; Scion Corp., Frederick, MD). Pixel intensities of the bands obtained in each experiment were normalized using β-actin signals and then calculated as a percentage of control bands in the same membrane. Assessment of Calpain Activity—Assessment of calpain activity was performed by detecting the calpain-mediated breakdown products of α-spectrin in immunoblotting. Cell lysates were prepared as described above; samples were processed for gel electrophoresis and blotting. The primary antibody used was anti-α-spectrin (mouse, 1:1000 dilution; Chemicon). (Pixel intensity × area of 145-kDa α-spectrin breakdown product)/(pixel intensity × area of (145-, 150-, and 280-kDa α-spectrin)) was measured densitometrically using software (Scion Image, version Beta 4.0.2; Scion Corp.). The individual band density was calculated as a percentage of control signals. In Vitro Degradation of p27 with Purified Calpain—On days 7-8 in vitro, striatal cells were washed with cold TBS and then lysed for 30 min on ice in radioimmunoprecipitation buffer containing 1 μg/ml pepstatin A (Peptide Institute), 1 mm NaF (Wako Pure Chemical Industries), and 1 mm Na3VO4 (Wako Pure Chemical Industries). Calpain inhibitors (ALLN, MDL28170, and PD150606) or ubiquitin-proteasome inhibitor (lactacystin) were included in the radioimmunoprecipitation buffer. The cell lysates were incubated with 3.8 units/ml of calpain 1 (Merck) at 25 °C for 10 min. Calcium chloride dihydrate (10 μm) was added to the reaction mixture to increase the free Ca2+ concentration. After the reaction, sample buffer was added. The samples were processed for gel electrophoresis and immunoblotting. Control experiments verified that the addition of CaCl2 to the radioimmunoprecipitation buffer over the range used in this study did not measurably alter the buffer pH. Small Interfering RNA (siRNA) Preparation and Transfection—siRNAs were prepared and transfected as previously described (29Akashiba H. Matsuki N. Nishiyama N. Cell. Mol. Life. Sci. 2006; 63: 2397-2404Crossref PubMed Scopus (20) Google Scholar). siRNAs were synthesized by Qiagen (Hilden, Germany). The target sequences were as follows. Control siRNA was 5′-AATTCTCCGAACGTGTCACGT-3′. The 3′-sense strand was labeled with Alexa Fluor-488. Scrambled p27 siRNA was 5′-AAGACCGAGCCATTGAGGTAA-3′. p27 siRNA was 5′-AAGCACUGCCGAGAUAUGGAA-3′. Transfection was performed with Lipofectamine 2000 (Invitrogen). Briefly, Lipofectamine 2000 (5 μl/well) and siRNA (5 μl/well) were incubated at 25 °C for 5 min, and both were mixed at 25 °C for 20 min. The siRNA-lipid mixture was added to the cultures. The final amount of the siRNAs was 100 pmol/well. siRNA was removed by changing the medium at 4 h after the transfection. Ca2+ Imaging—On day 8 in vitro, cells in a 35-mm dish were filled with 2 ml of dye solution and incubated for 1 h in a humidified incubator at 37 °C in 5% CO2, 95% air as previously described (30Sasaki T. Matsuki N. Ikegaya Y. J. Neurosci. 2007; 27: 517-528Crossref PubMed Scopus (141) Google Scholar). The dye solution was Neurobasal medium (Invitrogen) containing 10 μl of 0.1% Oregon Green 488 BAPTA-1, AM (OGB-1; Invitrogen)/Me2SO. Images (16-bit intensity) were captured with a Nipkow-disk confocal unit (CSU10; Yokogawa Electric, Tokyo, Japan), a cooled CCD camera (Cascade 512B/F, Roper Scientific, Tucson, AZ), an upright microscope (AxioSkop2; Zeiss, Oberkochen, Germany), water immersion objective (×40, Achroplan; Zeiss), and MetaMorph software (Molecular Devices, Union City, CA). Fluorophores were excited at 488 nm with an argon-krypton laser (15 milliwatts, 641-YB-A01; Melles Griot, Carlsbad, CA) and visualized with a 507-nm long pass emission filter. For quantitative analysis, OGB-1 fluorescence intensity in the soma area of each neuron (data in nonneuronal cells were excluded by phase-contrast image) were measured using software (Image J, version 1.371.20; Microsoft Java, Redmond, WA) and then calculated as a percentage of control in nontreated cultures. Statistical Evaluation—Data are shown as mean ± S.E. To avoid possible variation of the cell cultures, data were pooled from at least two independent experiments. For statistical analyses, one-way analysis of variance was followed by Tukey's test. 3-NP Induces Cell Death both in Cultured Striatal and Cortical Neurons—3-NP treatment of cultured striatal cells resulted in concentration- and time-dependent reduction in the number of surviving neurons, as observed in cresyl violet (Nissl) staining (Fig. 1A). 3-NP did not affect neuronal viability at 0.1 and 0.3 mm, but marked cell death was apparent in cultures treated with 3-NP at concentrations as low as 1 mm (Fig. 1A). In the presence of 1 and 3 mm 3-NP, about 60 and 70% of total neurons degenerated at 48 h, respectively. Similarly, exposure of cortical neurons to 3-NP resulted in concentration- and time-dependent reduction in the number of surviving neurons (Fig. 1B). 3-NP did not show toxic effect at 0.1 and 0.3 mm, but marked cell death was apparent in cultures treated with 3-NP at more than 1 mm (Fig. 1B). In the presence of 1 and 3 mm 3-NP, about 30 and 40% of total cortical neurons degenerated at 48 h, respectively. In both striatal and cortical cultures, the surviving neurons, as judged from the cresyl violet-positive staining, displayed deformed morphology (Fig. 1, C-F). We also evaluated cell survival by MTT assay. The results obtained with this method showed concentration- and time-dependent toxicity of 3-NP both in cultured striatal and cortical neurons, which were almost identical with the results of the cresyl violet staining method (Fig. 1, G and H). Because the two methods gave similar results, the following experiments to assess neuronal viability were performed with MTT assay. Next we examined whether striatal neurons sensitive to 3-NP were medium size spiny neurons (MSNs). We performed immunocytochemical analysis using labeling for DARPP-32, a molecule enriched in MSNs (31Bogush A. Pedrini S. Pelta-Heller J. Chan T. Yang Q. Mao Z. Sluzas E. Gieringer T. Ehrlich M.E. J. Biol. Chem. 2007; 282: 7352-7359Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Under control conditions, about 90% of striatal neurons were DARPP-32-positive in our experiment (data not shown). In cultures exposed to 3-NP (1 mm) for 48 h, the number of DARPP-32-positive cells decreased by about 30% compared with nontreated controls (Fig. 1, I-K). 3-NP Decreases p27 Expression in Cultured Striatal but Not in Cortical Neurons—Our previous work suggests that among CDK inhibitors involved in cell cycle arrest, p27 plays a critical role in regulation of cell cycle progression and neuronal survival (29Akashiba H. Matsuki N. Nishiyama N. Cell. Mol. Life. Sci. 2006; 63: 2397-2404Crossref PubMed Scopus (20) Google Scholar). Immunocytochemical analysis using triple labeling for a neuronal marker MAP2, p27, and a nuclear marker Hoechst dye revealed that p27 is localized in the nucleus of striatal neurons under control conditions (Fig. 2, A-D). This pattern of p27 expression in cultured striatal neurons is similar to that in cultured cortical neurons, as shown in our previous research (23Akashiba H. Matsuki N. Nishiyama N. J. Neurochem. 2006; 99: 733-744Crossref PubMed Scopus (19) Google Scholar). Next, we analyzed the p27 expression after 3-NP treatment in both cultures with an immunoblotting method. 3-NP treatment decreased the expression level of p27 concentration- and time-dependently in striatal neurons (Fig. 2, E and F). However, p27 expression in cortical neurons was unchanged by 3-NP treatment at all concentrations and times examined (Fig. 2, G and H). Transfection of p27 siRNA Induces Cell Death in Cultured Striatal Neurons—We previously showed that transfection of p27 siRNA induced cell death through elevating cell cycle activity in cultured cortical neurons, suggesting that reduction of endogenous p27 is sufficient for cell death in cortical neurons (29Akashiba H. Matsuki N. Nishiyama N. Cell. Mol. Life. Sci. 2006; 63: 2397-2404Crossref PubMed Scopus (20) Google Scholar). The same p27 siRNA was transfected to cultured striatal neurons to examine its effect on cell cycle progression and cell viability. The expression level of p27 was decreased at 48 h after the transfection of p27 siRNA at 100 pmol/well, whereas control siRNA or scrambled p27 siRNA had no effect on p27 expression (Fig. 3A). When the cell cycle transits from G1 to S phase, tumor suppressor Rb is phosphorylated by CDKs (32Liu D.X. Greene L.A. Cell Tissue Res. 2001; 305: 217-228Crossref PubMed Scopus (213) Google Scholar). To examine whether reduction of p27 is critical for cell cycle progression, we analyzed the Rb phosphorylation status on a CDK consensus site, Ser-795, by p27 knockdown. Transfection of p27 siRNA increased the expression level of phosphorylated Rb at 48 h after transfection, whereas control siRNA or scrambled p27 siRNA had no effect on Rb phosphorylation (Fig. 3A). p27 siRNA reduced striatal neuronal viability at about 55% at 48 h after the transfection, whereas control siRNA or scrambled p27 siRNA did not affect the viability of striatal neurons (Fig. 3B). 3-NP Induces Rb Phosphorylation in Cultured Striatal but Not in Cortical Neurons—Next, we analyzed the Rb phosphorylation status after 3-NP treatment using the phosphoepitope antibody in cultured striatal and cortical neurons. Only a trace level of phosphorylated Rb immunoreactivity was detected in nontreated cultures from both regions (Fig. 4, A-D). The expression level of phosphorylated Rb was up-regulated concentration- and time-dependently after 3-NP treatment in striatal neurons (Fig. 4, A and B). On the other hand, expression level of phosphorylated Rb did not increase after 3-NP treatment in cortical neurons at all concentrations and times examined (Fig. 4, C and D). CDK and NMDA Receptor Activities Are Cooperatively Involved in 3-NP-induced Cell Death in Cultured Striatal Neurons, whereas Only NMDA Receptor Activity Is Involved in 3-NP-induced Cell Death in Cultured Cortical Neurons—We next examined whether CDK activity is involved in the 3-NP-induced cell death in striatal and cortical cultures with pharmacological experiments. Three pharmacological CDK inhibitors, olomoucine (0.1-1 μm), purvalanol A (1-3 μm), and roscovitine (0.3-1 μm), partially but significantly protected striatal neurons from 3-NP-induced cell death (Fig. 5A). These pharmacological CDK inhibitors, however, did not exert a protective effect against 3-NP toxicity in cortical neurons (Fig. 5B). 3-NP-induced blockade of ATP production may hamper sodium-potassium-ATPase pumps and lead to reduced plasma membrane potentials (33Beal M.F. Ann. Neurol. 1992; 31: 119-130Crossref PubMed Scopus (899) Google Scholar). This would produce relief of voltage-dependent Mg2+ blockade of NMDA receptors" @default.
• W2130339832 created "2016-06-24" @default.
• W2130339832 creator A5010795802 @default.
• W2130339832 creator A5017351608 @default.
• W2130339832 creator A5022467762 @default.
• W2130339832 creator A5088683710 @default.
• W2130339832 date "2008-03-01" @default.
• W2130339832 modified "2023-10-03" @default.
• W2130339832 title "Differential Involvement of Cell Cycle Reactivation between Striatal and Cortical Neurons in Cell Death Induced by 3-Nitropropionic Acid" @default.
• W2130339832 cites W1509584111 @default.
• W2130339832 cites W1563847798 @default.
• W2130339832 cites W1626297236 @default.
• W2130339832 cites W1646788158 @default.
• W2130339832 cites W1733854047 @default.
• W2130339832 cites W1755608157 @default.
• W2130339832 cites W1825468095 @default.
• W2130339832 cites W1893523030 @default.
• W2130339832 cites W1924264746 @default.
• W2130339832 cites W1951782199 @default.
• W2130339832 cites W1966611928 @default.
• W2130339832 cites W1970458627 @default.
• W2130339832 cites W1972228250 @default.
• W2130339832 cites W1977032113 @default.
• W2130339832 cites W1978190113 @default.
• W2130339832 cites W1981680830 @default.
• W2130339832 cites W1983729920 @default.
• W2130339832 cites W1992517022 @default.
• W2130339832 cites W1998470223 @default.
• W2130339832 cites W2006916686 @default.
• W2130339832 cites W2007439129 @default.
• W2130339832 cites W2007706798 @default.
• W2130339832 cites W2015066175 @default.
• W2130339832 cites W2022212670 @default.
• W2130339832 cites W2024861994 @default.
• W2130339832 cites W2030047809 @default.
• W2130339832 cites W2030258195 @default.
• W2130339832 cites W2030968933 @default.
• W2130339832 cites W2031825480 @default.
• W2130339832 cites W2034103190 @default.
• W2130339832 cites W2036627529 @default.
• W2130339832 cites W2048646985 @default.
• W2130339832 cites W2049387807 @default.
• W2130339832 cites W2050122964 @default.
• W2130339832 cites W2052722987 @default.
• W2130339832 cites W205780862 @default.
• W2130339832 cites W2061316875 @default.
• W2130339832 cites W2061462069 @default.
• W2130339832 cites W2063351395 @default.
• W2130339832 cites W2065691470 @default.
• W2130339832 cites W2069491929 @default.
• W2130339832 cites W2073947266 @default.
• W2130339832 cites W2074410961 @default.
• W2130339832 cites W2077298522 @default.
• W2130339832 cites W2077445707 @default.
• W2130339832 cites W2078747214 @default.
• W2130339832 cites W2080973723 @default.
• W2130339832 cites W2091403769 @default.
• W2130339832 cites W2095025678 @default.
• W2130339832 cites W2102430316 @default.
• W2130339832 cites W2111853402 @default.
• W2130339832 cites W2113149589 @default.
• W2130339832 cites W2116508440 @default.
• W2130339832 cites W2120739159 @default.
• W2130339832 cites W2122956388 @default.
• W2130339832 cites W2139454515 @default.
• W2130339832 cites W2139826556 @default.
• W2130339832 cites W2145213346 @default.
• W2130339832 cites W2145413598 @default.
• W2130339832 cites W2149313534 @default.
• W2130339832 cites W2154439306 @default.
• W2130339832 cites W2169436026 @default.
• W2130339832 cites W4232493852 @default.
• W2130339832 doi "https://doi.org/10.1074/jbc.m707730200" @default.
• W2130339832 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18182390" @default.
• W2130339832 hasPublicationYear "2008" @default.
• W2130339832 type Work @default.
• W2130339832 sameAs 2130339832 @default.
• W2130339832 citedByCount "23" @default.
• W2130339832 countsByYear W21303398322016 @default.
• W2130339832 countsByYear W21303398322018 @default.
• W2130339832 countsByYear W21303398322020 @default.
• W2130339832 countsByYear W21303398322021 @default.
• W2130339832 countsByYear W21303398322022 @default.
• W2130339832 countsByYear W21303398322023 @default.
• W2130339832 crossrefType "journal-article" @default.
• W2130339832 hasAuthorship W2130339832A5010795802 @default.
• W2130339832 hasAuthorship W2130339832A5017351608 @default.
• W2130339832 hasAuthorship W2130339832A5022467762 @default.
• W2130339832 hasAuthorship W2130339832A5088683710 @default.
• W2130339832 hasBestOaLocation W21303398321 @default.
• W2130339832 hasConcept C1491633281 @default.
• W2130339832 hasConcept C169760540 @default.
• W2130339832 hasConcept C185592680 @default.
• W2130339832 hasConcept C190283241 @default.
• W2130339832 hasConcept C29537977 @default.
• W2130339832 hasConcept C2983650178 @default.
• W2130339832 hasConcept C31573885 @default.
• W2130339832 hasConcept C55493867 @default.

• next