Lentiviral vector-mediated knockdown of the neuroglycan 2 proteoglycan or expression of neurotrophin-3 promotes neurite outgrowth in a cell culture model of the glial scar

Eleanor M. Donnelly, Padraig M. Strappe, Lisa M. McGinley, Nicolas N. Madigan, Elizabeth Geurts, Gemma E. Rooney, Anthony John Windebank, John Fraher, Peter Dockery, Timothy O'Brien, Siobhan S. McMahon

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Background: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). Neuroglycan 2 (NG2), one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. Methods: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury.Results and Discussion: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).

Original languageEnglish (US)
Pages (from-to)863-872
Number of pages10
JournalJournal of Gene Medicine
Volume12
Issue number11
DOIs
StatePublished - Nov 2010

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Neurotrophin 3
Neurites
Proteoglycans
Spinal Cord Injuries
Neuroglia
Astrocytes
Small Interfering RNA
Cicatrix
Chondroitin Sulfate Proteoglycans
Cell Culture Techniques
Regeneration
Growth
Nerve Tissue
Cell Line
Nerve Growth Factors
Spinal Ganglia
Coculture Techniques
Central Nervous System
Neurons
Wounds and Injuries

Keywords

  • Glial scar
  • Lentiviral vector
  • Neu7 astrocytes
  • Neurotrophin-3
  • NG2
  • Spinal cord injury

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Molecular Medicine
  • Genetics(clinical)
  • Drug Discovery

Cite this

Lentiviral vector-mediated knockdown of the neuroglycan 2 proteoglycan or expression of neurotrophin-3 promotes neurite outgrowth in a cell culture model of the glial scar. / Donnelly, Eleanor M.; Strappe, Padraig M.; McGinley, Lisa M.; Madigan, Nicolas N.; Geurts, Elizabeth; Rooney, Gemma E.; Windebank, Anthony John; Fraher, John; Dockery, Peter; O'Brien, Timothy; McMahon, Siobhan S.

In: Journal of Gene Medicine, Vol. 12, No. 11, 11.2010, p. 863-872.

Research output: Contribution to journalArticle

Donnelly, EM, Strappe, PM, McGinley, LM, Madigan, NN, Geurts, E, Rooney, GE, Windebank, AJ, Fraher, J, Dockery, P, O'Brien, T & McMahon, SS 2010, 'Lentiviral vector-mediated knockdown of the neuroglycan 2 proteoglycan or expression of neurotrophin-3 promotes neurite outgrowth in a cell culture model of the glial scar', Journal of Gene Medicine, vol. 12, no. 11, pp. 863-872. https://doi.org/10.1002/jgm.1509
Donnelly, Eleanor M. ; Strappe, Padraig M. ; McGinley, Lisa M. ; Madigan, Nicolas N. ; Geurts, Elizabeth ; Rooney, Gemma E. ; Windebank, Anthony John ; Fraher, John ; Dockery, Peter ; O'Brien, Timothy ; McMahon, Siobhan S. / Lentiviral vector-mediated knockdown of the neuroglycan 2 proteoglycan or expression of neurotrophin-3 promotes neurite outgrowth in a cell culture model of the glial scar. In: Journal of Gene Medicine. 2010 ; Vol. 12, No. 11. pp. 863-872.
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abstract = "Background: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). Neuroglycan 2 (NG2), one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. Methods: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury.Results and Discussion: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).",
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AU - Strappe, Padraig M.

AU - McGinley, Lisa M.

AU - Madigan, Nicolas N.

AU - Geurts, Elizabeth

AU - Rooney, Gemma E.

AU - Windebank, Anthony John

AU - Fraher, John

AU - Dockery, Peter

AU - O'Brien, Timothy

AU - McMahon, Siobhan S.

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N2 - Background: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). Neuroglycan 2 (NG2), one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. Methods: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury.Results and Discussion: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).

AB - Background: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). Neuroglycan 2 (NG2), one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. Methods: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury.Results and Discussion: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).

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