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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.

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.

Functional cloning strategy.Diagram of the functional cloning strategy utilized to identify neuroprotective genes (see text for details).
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pone-0015008-g002: Functional cloning strategy.Diagram of the functional cloning strategy utilized to identify neuroprotective genes (see text for details).

Mentions: To identify endogeneous brain protective genes, we generated a retroviral cDNA expression library from primary cortical neurons preconditioned with OGD and conducted a functional cloning screen for protective molecules (Fig. 2). The mRNA for the library was obtained from cortical neurons 16 h after a preconditioning stimulus of 15 min of OGD, which leads to sustained and profound protection against a subsequent lethal 90 min OGD treatment (see Fig. 1A). The cDNA library from OGD preconditioned cortical cultures with an average insert size of 2.5 Kb was cloned into the pFB retrovirus vector and virus was generated. Primary mouse fibroblasts were infected with the retrovirus and 48 h after infection the fibroblasts were challenged with menadione (175 µM, 10 min) to induce parthanatos. Cells that survived were grown another 9–13 days, and then re-challenged with menadione to confirm cytoprotection and eliminate false positives. 113 cell clones survived the initial menadione treatment. Of these, 95 survived the menadione re-challenge. The protective genes were recovered from the surviving cell clones by PCR with oligonucleotide primers that recognize regions flanking the multiple cloning site of the pFB vector (Fig. 2). A total of 31 independent genes were recovered and sequenced from the 95 cell clones (Table 1) Thirteen of the genes were designated neuroprotective genes (NGP1 to NPG13) and the sequences were deposited in the Genbank database. Accession numbers are indicated in Table 1.

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Functional Identification of Neuroprotective Molecules

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

Functional cloning strategy.Diagram of the functional cloning strategy utilized to identify neuroprotective genes (see text for details).
© Copyright Policy
pone-0015008-g002: Functional cloning strategy.Diagram of the functional cloning strategy utilized to identify neuroprotective genes (see text for details).
Mentions: To identify endogeneous brain protective genes, we generated a retroviral cDNA expression library from primary cortical neurons preconditioned with OGD and conducted a functional cloning screen for protective molecules (Fig. 2). The mRNA for the library was obtained from cortical neurons 16 h after a preconditioning stimulus of 15 min of OGD, which leads to sustained and profound protection against a subsequent lethal 90 min OGD treatment (see Fig. 1A). The cDNA library from OGD preconditioned cortical cultures with an average insert size of 2.5 Kb was cloned into the pFB retrovirus vector and virus was generated. Primary mouse fibroblasts were infected with the retrovirus and 48 h after infection the fibroblasts were challenged with menadione (175 µM, 10 min) to induce parthanatos. Cells that survived were grown another 9–13 days, and then re-challenged with menadione to confirm cytoprotection and eliminate false positives. 113 cell clones survived the initial menadione treatment. Of these, 95 survived the menadione re-challenge. The protective genes were recovered from the surviving cell clones by PCR with oligonucleotide primers that recognize regions flanking the multiple cloning site of the pFB vector (Fig. 2). A total of 31 independent genes were recovered and sequenced from the 95 cell clones (Table 1) Thirteen of the genes were designated neuroprotective genes (NGP1 to NPG13) and the sequences were deposited in the Genbank database. Accession numbers are indicated in Table 1.

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.

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

ABSTRACT

Background: 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.

View Similar Images In: Results  - Collection
View Article: Pubmed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2991347&rFormat=json&query=null&req=5