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Functional Identification of Neuroprotective Molecules

Dai, Cheng, Liang, Dong, Li, Huiwu, Sasaki, Masayuki, Dawson, Ted M., Dawson, Valina L.·2010·PLoS ONE
DOI 10.1371/journal.pone.0015008PMC PMC2991347
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Figure 1 Preconditioning induces neuroprotection and menandione kills cells via Parthanatos. (A) 15 min of OGD preconditioning induces neuroprotection 16 h and 24 h later against 90 min of lethal OGD. Primary cortical cultures were exposed to 90 min lethal OGD without preconditioning (90′ OGD) and compared to cultures exposed to 15 min of OGD and rechallenged with 90 min OGD 4 h, 6 h, 16 h and 24 h after the 15 min of OGD preconditioning. Cell viability was determined 24 h after 90 min lethal OGD by Hoescht 33342 (1 µg/ml) and prodidium iodide (7 µM) staining and unbiased computer assisted cell counting [29]. (B). Menadione induces Parthanatos (PARP-1-dependent caspase-independent) fibroblast death. Menadione (175 µM, 10 min) kills almost all of the wild type fibroblasts (WT) but only 55% or PARP-1 knockout fibroblasts (KO). Menadione induced fibroblast death cannot be blocked by the caspase inhibitor, ZVAD (100 µM) or BAF (50 µM), which can blocks staurosporine (500 nM, 24 h) induced cell death. Experiments were performed four times. Data are the mean ± SEM. *p<0.01 (Student's t-test) comparing OGD (90 min) against OGD (15 min/90 min). An average number of 500,000 cells were counted.
Figure 1 Preconditioning induces neuroprotection and menandione kills cells via Parthanatos. (A) 15 min of OGD preconditioning induces neuroprotection 16 h and 24 h later against 90 min of lethal OGD. Primary cortical cultures were exposed to 90 min lethal OGD without preconditioning (90′ OGD) and compared to cultures exposed to 15 min of OGD and rechallenged with 90 min OGD 4 h, 6 h, 16 h and 24 h after the 15 min of OGD preconditioning. Cell viability was determined 24 h after 90 min lethal OGD by Hoescht 33342 (1 µg/ml) and prodidium iodide (7 µM) staining and unbiased computer assisted cell counting [29]. (B). Menadione induces Parthanatos (PARP-1-dependent caspase-independent) fibroblast death. Menadione (175 µM, 10 min) kills almost all of the wild type fibroblasts (WT) but only 55% or PARP-1 knockout fibroblasts (KO). Menadione induced fibroblast death cannot be blocked by the caspase inhibitor, ZVAD (100 µM) or BAF (50 µM), which can blocks staurosporine (500 nM, 24 h) induced cell death. Experiments were performed four times. Data are the mean ± SEM. *p<0.01 (Student's t-test) comparing OGD (90 min) against OGD (15 min/90 min). An average number of 500,000 cells were counted.CC BY · PubMed Central
Figure 2 Functional cloning strategy. Diagram of the functional cloning strategy utilized to identify neuroprotective genes (see text for details).
Figure 2 Functional cloning strategy. Diagram of the functional cloning strategy utilized to identify neuroprotective genes (see text for details).CC BY · PubMed Central
Figure 3 Calmodulin overexpression protects against OGD and NMDA Excitotoxicity. (A and B) Representative photomicrographs of cortical cultures transfected with empty vector or calmodulin and then exposed to (A) 90 min OGD or (B) 500 µM NMDA for 5 min. Cell death was assessed 24 h later. Arrows or arrowheads indicate dead or surviving transfected neurons, respectively.
Figure 3 Calmodulin overexpression protects against OGD and NMDA Excitotoxicity. (A and B) Representative photomicrographs of cortical cultures transfected with empty vector or calmodulin and then exposed to (A) 90 min OGD or (B) 500 µM NMDA for 5 min. Cell death was assessed 24 h later. Arrows or arrowheads indicate dead or surviving transfected neurons, respectively.CC BY · PubMed Central
Figure 4 Functionally cloned genes are neuroprotective. (A and B). Primary cortical neurons were transfected with 28 of the identified genes or the empty pCI vector or the ADCGI vector. EGFP serves as the reporter for transfected cells. 48 h later, the neurons were treated with (A) OGD (90 min) or (B) NMDA (500 µM, 5 min). 24 h after treatment, neurons were stained with Hoechst 33342 and propidium iodide. Only EGFP positive cells were scored. Dead neurons were scored as those cells that were propidium iodide positive, with condensed, or fragmented nuclei. Experiments were performed at least three times. Data are the mean ± SEM. *p<0.01 (Student's t-test) comparing empty vector to each identified gene.
Figure 4 Functionally cloned genes are neuroprotective. (A and B). Primary cortical neurons were transfected with 28 of the identified genes or the empty pCI vector or the ADCGI vector. EGFP serves as the reporter for transfected cells. 48 h later, the neurons were treated with (A) OGD (90 min) or (B) NMDA (500 µM, 5 min). 24 h after treatment, neurons were stained with Hoechst 33342 and propidium iodide. Only EGFP positive cells were scored. Dead neurons were scored as those cells that were propidium iodide positive, with condensed, or fragmented nuclei. Experiments were performed at least three times. Data are the mean ± SEM. *p<0.01 (Student's t-test) comparing empty vector to each identified gene.CC BY · PubMed Central
Figure 5 Calmodulin participates in preconditioning induced neuroprotection. Rat primary cortical neurons were treated with 15 min OGD and harvested at the indicated time points for (A) Northern blot or (B) immunoblot analysis. β-actin and β-tubulin are the loading control for Northern blot and immuonblot analysis, respectively. These experiments were conducted 3 times with similar results. (C) HEK 293 cells were transfected with calmodulin RNAi, unrelated RNAi (Lmr-1 RNAi) or empty vector. Two days later Western blot of whole-cell extracts shows reduction of calmodulin expression following calmodulin RNAi treatment but not Lmr-1 RNAi or empty vector. β–tubulin immunoreactivity was used as a loading control. (D) Primary cortical neurons were transfected for 48 h with a control vector or calmodulin RNAi along with CMV-β-Gal. Overlay of the anti-calmodulin and anti-β-Gal antibody staining. White arrows indicate transfected cells. (E and F) Primary cortical neurons were transfected with empty vector or calmodulin RNAi. EGFP serves as the reporter for transfected cells. 48 h later, the cells were preconditioned with OGD (15 min) or NMDA (50 µM, 5 min). 24 h after preconditioning, the cells were treated with OGD (90 min) or NMDA (500 µM, 5 min), respectively. 24 h after treatment, neurons were stained with Hoechst 33342 and propidium iodide. Only EGFP positive cells were scored. Dead neurons were scored as those cells that were propidium iodide positive, with condensed, or fragmented nuclei. (F) Quantification of cell viability. Experiments were performed for three times, and the data was presented as mean ± SEM. *p<0.001 (Student's t-test) when comparing RNAi to empty vector.
Figure 5 Calmodulin participates in preconditioning induced neuroprotection. Rat primary cortical neurons were treated with 15 min OGD and harvested at the indicated time points for (A) Northern blot or (B) immunoblot analysis. β-actin and β-tubulin are the loading control for Northern blot and immuonblot analysis, respectively. These experiments were conducted 3 times with similar results. (C) HEK 293 cells were transfected with calmodulin RNAi, unrelated RNAi (Lmr-1 RNAi) or empty vector. Two days later Western blot of whole-cell extracts shows reduction of calmodulin expression following calmodulin RNAi treatment but not Lmr-1 RNAi or empty vector. β–tubulin immunoreactivity was used as a loading control. (D) Primary cortical neurons were transfected for 48 h with a control vector or calmodulin RNAi along with CMV-β-Gal. Overlay of the anti-calmodulin and anti-β-Gal antibody staining. White arrows indicate transfected cells. (E and F) Primary cortical neurons were transfected with empty vector or calmodulin RNAi. EGFP serves as the reporter for transfected cells. 48 h later, the cells were preconditioned with OGD (15 min) or NMDA (50 µM, 5 min). 24 h after preconditioning, the cells were treated with OGD (90 min) or NMDA (500 µM, 5 min), respectively. 24 h after treatment, neurons were stained with Hoechst 33342 and propidium iodide. Only EGFP positive cells were scored. Dead neurons were scored as those cells that were propidium iodide positive, with condensed, or fragmented nuclei. (F) Quantification of cell viability. Experiments were performed for three times, and the data was presented as mean ± SEM. *p<0.001 (Student's t-test) when comparing RNAi to empty vector.CC BY · PubMed Central
Figure 6 NPG2 participates in preconditioning induced neuroprotection. Primary cortical neurons were treated with 15 min OGD and harvested at the indicated time for (A) Northern blot analysis. Lower panel shows loading of RNA. These experiments were conducted three times with similar results. (B) HEK 293 cells were transfected with myc-tagged NPG2 and non-silencing control siRNA (nsRNA) or four different siRNA to NPG2 (siRNA1-4). Western blots for anti-myc show siRNA 2-4 effectively knock-down NPG2 expression. β–tubulin immunoreactivity was used as a loading control. (C) QT-PCR of Rat-2 cells transfected for 48 h with a control vector or siRNA to NPG2 shows that siRNA2 effectively knocks down endogenous NPG2 transcript. (D and E) Primary cortical neurons were transfected with an empty vector or NPG2 siRNA along with EGFP. 48 h later, the cells were preconditioned with OGD (15 min) or NMDA (50 µM, 5 min). 24 h after preconditioning, the cells were treated with OGD (90 min) or NMDA (500 µM, 5 min), respectively. 24 h later, the cells were stained with Hoescht 33342 and propidium iodide. Only EGFP positive neurons were scored. (D) Viable neurons were scored as those cells that displayed normal morphology. Dead neurons were scored as those cells that were propidium iodide positive, with condensed, or fragmented nuclei. (E) Quantification of cell viability. Experiments were performed three times, and the data was presented as mean ± SEM. *p<0.001 (Student's t test) when comparing siRNA to empty vector.
Figure 6 NPG2 participates in preconditioning induced neuroprotection. Primary cortical neurons were treated with 15 min OGD and harvested at the indicated time for (A) Northern blot analysis. Lower panel shows loading of RNA. These experiments were conducted three times with similar results. (B) HEK 293 cells were transfected with myc-tagged NPG2 and non-silencing control siRNA (nsRNA) or four different siRNA to NPG2 (siRNA1-4). Western blots for anti-myc show siRNA 2-4 effectively knock-down NPG2 expression. β–tubulin immunoreactivity was used as a loading control. (C) QT-PCR of Rat-2 cells transfected for 48 h with a control vector or siRNA to NPG2 shows that siRNA2 effectively knocks down endogenous NPG2 transcript. (D and E) Primary cortical neurons were transfected with an empty vector or NPG2 siRNA along with EGFP. 48 h later, the cells were preconditioned with OGD (15 min) or NMDA (50 µM, 5 min). 24 h after preconditioning, the cells were treated with OGD (90 min) or NMDA (500 µM, 5 min), respectively. 24 h later, the cells were stained with Hoescht 33342 and propidium iodide. Only EGFP positive neurons were scored. (D) Viable neurons were scored as those cells that displayed normal morphology. Dead neurons were scored as those cells that were propidium iodide positive, with condensed, or fragmented nuclei. (E) Quantification of cell viability. Experiments were performed three times, and the data was presented as mean ± SEM. *p<0.001 (Student's t test) when comparing siRNA to empty vector.CC BY · PubMed Central

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Citations

Cites

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    David, K. K. (2009) · Frontiers in Bioscience · doi:10.2741/3297 · Cited by 450 · cited 1×

    “In the nervous system NMDA excitotoxicity is mediated largely by parthanatos, poly (ADP-ribose) polymerase 1 (PARP-1) dependent cell death [2,3].”
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Metadata

Authors
Dai, C., Liang, D., Li, H., Sasaki, M., Dawson, T. M., & Dawson, V. L.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2010-11-24
Volume
5
Issue
11
Pages
e15008
ISSN
1932-6203
DOI
10.1371/journal.pone.0015008
PMC
PMC2991347
Cited by
29
References
1
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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