A Novel and Efficient Gene Transfer Strategy Reduces Glial Reactivity and Improves Neuronal Survival and Axonal Growth In Vitro

PLoS ONE, 2009 · DOI: 10.1371/journal.pone.0006227 · Published: July 14, 2009

Simple Explanation

The study addresses the challenge of axonal regeneration in the central nervous system, which is often hindered by the formation of glial scars. These scars are largely composed of reactive astrocytes that overproduce GFAP and vimentin. The study aimed to develop a gene therapy strategy using lentiviral vectors to reduce astrocyte reactivity by silencing GFAP and vimentin expression. Lentiviral vectors (Lv-shGFAP and Lv-shVIM) were designed to deliver RNA interference (RNAi) to silence GFAP and vimentin in cultured astrocytes. This approach aimed to reduce astrogliosis, improve neuronal survival, and enhance axonal growth in an in vitro model. The study also used a scratch assay to mimic injury and assess the impact of gene silencing on glial scar formation. The study found that lentiviral-mediated knockdown of GFAP and vimentin in astrocytes reduced glial reactivity, decreased glial scarring, and improved neuronal survival and neurite outgrowth in vitro. These findings suggest that targeting GFAP and vimentin could be a promising therapeutic approach for promoting axonal regeneration after CNS injuries.

Study Duration

Not specified

Participants

Primary cultures of murine astrocytes, embryonic day-14 wild-type neocortical neurons, HEK 293T cells

Evidence Level

In vitro study

Key Findings

1
Lv-shGFAP and Lv-shVIM vectors effectively silenced GFAP and vimentin expression in cultured astrocytes.
2
Knockdown of GFAP and vimentin reduced astrocytic reactivity and glial scarring in a scratch wound assay.
3
GFAP and vimentin silencing increased neuronal survival and neurite outgrowth in a heterotopic coculture model.

Research Summary

This study demonstrates that lentiviral-mediated RNAi can efficiently silence GFAP and vimentin expression in cultured astrocytes. The use of Lv-shGFAP and Lv-shVIM vectors resulted in a significant decrease in protein levels, reducing astrocytic reactivity and glial scarring in vitro. In a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. This indicates a potential for enhancing neuronal plasticity through targeted gene silencing in astrocytes. The findings suggest that manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions. GFAP is identified as a key target for modulating reactive gliosis and monitoring neuron/glia interactions.

Practical Implications

Therapeutic Potential

Targeting GFAP and vimentin in reactive astrocytes may offer a novel approach to promote axonal regeneration after CNS injuries.

Gene Therapy Strategy

Lentiviral-mediated RNAi can be used to modulate the intrinsic behavior of reactive astrocytes and prevent glial scar formation.

Clinical Applications

The strategy is appropriate for application in animal models of CNS injury and could be of value for adjunct therapy in several CNS pathological conditions.

Study Limitations

1
The study was conducted in vitro, and the results may not directly translate to in vivo conditions.
2
The long-term effects of GFAP and vimentin silencing were not fully explored.
3
The study did not address the potential off-target effects of RNAi.

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