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Functional identification of neuroprotective molecules.

Dai C, Liang D, Li H, Sasaki M, Dawson TM, Dawson VL - PLoS ONE (2010)

Bottom Line: The central nervous system has the capacity to activate profound neuroprotection following sub-lethal stress in a process termed preconditioning.These results reveal that the brain possesses a wide and diverse repertoire of neuroprotective genes.Further characterization of these and other protective signals could provide new treatment opportunities for neurological injury from ischemia or neurodegenerative disease.

View Article: PubMed Central - PubMed

Affiliation: Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The central nervous system has the capacity to activate profound neuroprotection following sub-lethal stress in a process termed preconditioning. To gain insight into this potent survival response we developed a functional cloning strategy that identified 31 putative neuroprotective genes of which 28 were confirmed to provide protection against oxygen-glucose deprivation (OGD) or excitotoxic exposure to N-methyl-D-aspartate (NMDA) in primary rat cortical neurons. These results reveal that the brain possesses a wide and diverse repertoire of neuroprotective genes. Further characterization of these and other protective signals could provide new treatment opportunities for neurological injury from ischemia or neurodegenerative disease.

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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.
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pone-0015008-g005: 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.

Mentions: We next evaluated whether the neuroprotective genes identified could be involved in the phenomena of preconditioning. Calmodulin, a gene of known function, and NPG2, a gene of unknown function, were selected for further characterization. In primary cortical neurons exposed to the preconditioning stimulus of 15 min OGD constitutively expressed calmodulin is upregulated at both the mRNA and protein levels (Fig. 5A, B). Because overexpression of calmodulin is neuroprotective against OGD and NMDA, toxicity experiments were conducted to determine if the up-regulation of calmodulin participates in the preconditioning induced neuroprotection. RNAi to calmodulin was designed to knock down the expression of calmodulin. Calmodulin RNAi was introduced into HEK 293 cells and primary cortical neurons via lipofectamine transfection. Knock down of calmodulin in HEK 293 cells was confirmed by immunoblot analysis (Fig. 5C) Transfection with empty vector or an un-related RNAi (Lmr-1 RNAi) had no effect on calmodulin expression showing the specificity of the RNAi to calmodulin (Fig. 5C). Efficacy of knockdown of calmodulin via calmodulin RNAi in cortical neurons was assessed via immunocytochemistry by co-transfecting calmodulin RNAi with β-galactosidase (β-Gal) (Fig. 5D). In cells expressing β-Gal, calmodulin expression is substantially reduced (Fig. 5D). To analyze the effects of calmodulin knock down on the protective effects of preconditioning, primary cortical neurons were co-transfected with calmodulin RNAi and β-galactosidase and compared to empty vector controls prior to the preconditioning exposure. 48 h later, the cells were preconditioned with OGD (15 min) or NMDA (50 µM, 5 min) and 24 hr later the cultures were exposed to a lethal treatment of OGD (90 min) or NMDA (500 µM, 5 min), respectively. 24 h later, the cells were fixed and stained with X-Gal and surviving neurons were scored as those cells that displayed normal morphology. Knocking down calmodulin expression by RNAi treatment during the preconditioning phase is sufficient to block the survival of cortical neurons triggered by OGD or NMDA preconditioning (Fig. 5E, F). These data indicate that calmodulin is a potential important component in the survival response activated by preconditioning.


Functional identification of neuroprotective molecules.

Dai C, Liang D, Li H, Sasaki M, Dawson TM, Dawson VL - PLoS ONE (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991347&req=5

pone-0015008-g005: 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.
Mentions: We next evaluated whether the neuroprotective genes identified could be involved in the phenomena of preconditioning. Calmodulin, a gene of known function, and NPG2, a gene of unknown function, were selected for further characterization. In primary cortical neurons exposed to the preconditioning stimulus of 15 min OGD constitutively expressed calmodulin is upregulated at both the mRNA and protein levels (Fig. 5A, B). Because overexpression of calmodulin is neuroprotective against OGD and NMDA, toxicity experiments were conducted to determine if the up-regulation of calmodulin participates in the preconditioning induced neuroprotection. RNAi to calmodulin was designed to knock down the expression of calmodulin. Calmodulin RNAi was introduced into HEK 293 cells and primary cortical neurons via lipofectamine transfection. Knock down of calmodulin in HEK 293 cells was confirmed by immunoblot analysis (Fig. 5C) Transfection with empty vector or an un-related RNAi (Lmr-1 RNAi) had no effect on calmodulin expression showing the specificity of the RNAi to calmodulin (Fig. 5C). Efficacy of knockdown of calmodulin via calmodulin RNAi in cortical neurons was assessed via immunocytochemistry by co-transfecting calmodulin RNAi with β-galactosidase (β-Gal) (Fig. 5D). In cells expressing β-Gal, calmodulin expression is substantially reduced (Fig. 5D). To analyze the effects of calmodulin knock down on the protective effects of preconditioning, primary cortical neurons were co-transfected with calmodulin RNAi and β-galactosidase and compared to empty vector controls prior to the preconditioning exposure. 48 h later, the cells were preconditioned with OGD (15 min) or NMDA (50 µM, 5 min) and 24 hr later the cultures were exposed to a lethal treatment of OGD (90 min) or NMDA (500 µM, 5 min), respectively. 24 h later, the cells were fixed and stained with X-Gal and surviving neurons were scored as those cells that displayed normal morphology. Knocking down calmodulin expression by RNAi treatment during the preconditioning phase is sufficient to block the survival of cortical neurons triggered by OGD or NMDA preconditioning (Fig. 5E, F). These data indicate that calmodulin is a potential important component in the survival response activated by preconditioning.

Bottom Line: The central nervous system has the capacity to activate profound neuroprotection following sub-lethal stress in a process termed preconditioning.These results reveal that the brain possesses a wide and diverse repertoire of neuroprotective genes.Further characterization of these and other protective signals could provide new treatment opportunities for neurological injury from ischemia or neurodegenerative disease.

View Article: PubMed Central - PubMed

Affiliation: Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The central nervous system has the capacity to activate profound neuroprotection following sub-lethal stress in a process termed preconditioning. To gain insight into this potent survival response we developed a functional cloning strategy that identified 31 putative neuroprotective genes of which 28 were confirmed to provide protection against oxygen-glucose deprivation (OGD) or excitotoxic exposure to N-methyl-D-aspartate (NMDA) in primary rat cortical neurons. These results reveal that the brain possesses a wide and diverse repertoire of neuroprotective genes. Further characterization of these and other protective signals could provide new treatment opportunities for neurological injury from ischemia or neurodegenerative disease.

Show MeSH
Related in: MedlinePlus