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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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Related in: MedlinePlus

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.
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pone-0015008-g006: 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.

Mentions: Following exposure to 15 min OGD, NPG2 mRNA expression is induced in primary cortical cultures (Fig. 6A). Four siRNAs to NPG2 were tested for their efficacy in knocking down overexpressed NPG2 epitope tagged with myc at the N–terminus. HEK 293 cell cultures were transfected with the myc-tagged NPG2 in the presences or absence of each of the NPG2 siRNAs. Three out of four NPG2 siRNAs blocked myc-tagged NPG2 expression (Fig. 6B). To test the efficacy of the NPG2 siRNAs to knock down endogenous NPG2, Rat-2 cell cultures were transfected with each of the siRNAs to NPG2 and knock down was assessed by quantitative RT-PCR analysis. Of the four siRNAs, siRNA2 knocked down endogenous NPG2 (Fig. 6C). NPG2 siRNA2 was used to knock down the induction of NPG2 in primary neurons in response to the preconditioning stimulus of 15 min OGD or 50 µM NMDA for 5 min. NPG2 siRNA2 and pCI-EGFP were co-transfected at ratio of 1∶4 for pCI-EGFP and the NPG2 siRNA2, respectively. Scrambled siRNA was used as a control. One day after the preconditioning event the cultures were exposed to lethal OGD or NMDA and 24 hr later, the neurons were stained with Hoechst 33342 and propidium iodide to assess cell viability. Since only 1–2% of the cells in primary cortical cultures are transfected, only EGFP positive cells were assessed for cell viability. Dead neurons were scored as those cells that displayed propidium iodide positive, condensed or fragmented nuclei (Fig. 6D). NPG2 siRNA 2 is sufficient to prevent the neuronal survival induced by OGD and NMDA preconditioning, but scrambled siRNA had no effect (Fig. 6E). These results taken together indicate that NPG2 is a neuroprotective gene that can participate in preconditioning.


Functional identification of neuroprotective molecules.

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991347&req=5

pone-0015008-g006: 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.
Mentions: Following exposure to 15 min OGD, NPG2 mRNA expression is induced in primary cortical cultures (Fig. 6A). Four siRNAs to NPG2 were tested for their efficacy in knocking down overexpressed NPG2 epitope tagged with myc at the N–terminus. HEK 293 cell cultures were transfected with the myc-tagged NPG2 in the presences or absence of each of the NPG2 siRNAs. Three out of four NPG2 siRNAs blocked myc-tagged NPG2 expression (Fig. 6B). To test the efficacy of the NPG2 siRNAs to knock down endogenous NPG2, Rat-2 cell cultures were transfected with each of the siRNAs to NPG2 and knock down was assessed by quantitative RT-PCR analysis. Of the four siRNAs, siRNA2 knocked down endogenous NPG2 (Fig. 6C). NPG2 siRNA2 was used to knock down the induction of NPG2 in primary neurons in response to the preconditioning stimulus of 15 min OGD or 50 µM NMDA for 5 min. NPG2 siRNA2 and pCI-EGFP were co-transfected at ratio of 1∶4 for pCI-EGFP and the NPG2 siRNA2, respectively. Scrambled siRNA was used as a control. One day after the preconditioning event the cultures were exposed to lethal OGD or NMDA and 24 hr later, the neurons were stained with Hoechst 33342 and propidium iodide to assess cell viability. Since only 1–2% of the cells in primary cortical cultures are transfected, only EGFP positive cells were assessed for cell viability. Dead neurons were scored as those cells that displayed propidium iodide positive, condensed or fragmented nuclei (Fig. 6D). NPG2 siRNA 2 is sufficient to prevent the neuronal survival induced by OGD and NMDA preconditioning, but scrambled siRNA had no effect (Fig. 6E). These results taken together indicate that NPG2 is a neuroprotective gene that can participate in 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