Connexin 43 regulates astrocytic migration and proliferation in response to injury.

Marked alterations in astrocyte function are a universal response to disease or injury in the central nervous system. Affected astrocytes develop characteristic morphological changes known as "reactive astrogliosis", characterized by increased expression of the intermediate filament proteins, glial fibrillary acidic protein and vimentin. Reactive astrocytes also display alterations in other proteins including a rapid up-regulation of the gap junction protein, Connexin 43. The present study tests whether Connexin 43 is directly involved in the astrocytic response to injury. We have down regulated Connexin 43 expression using a microRNA generating plasmid and investigated the functional consequences of this treatment on the response of astrocytes in primary culture to a well-characterized scratch wound injury. The treatment resulted in more than 70% transfection efficiency and a near complete depletion of Connexin 43 in transfected cells. Compared to cells transfected with non-targeting microRNA, the cells depleted of Connexin 43 showed a slower wound closure and fewer transfected cells in the wound area. These changes were associated with decreased proliferation of the Connexin 43-depleted cells as well as shorter processes extending into the wound area suggesting a direct impairment of migration. The effects were independent of gap junction conductivity as exposure to the gap junction blocker carbenoxolone did not affect the rate of wound healing. The findings directly indicate a role for Connexin 43 in the astrocytic response to injury and suggest that modification of Connexin 43 expression might provide a therapeutic target to alter potentially deleterious astrocytic responses.

Astrocytic responses to DNA delivery using nucleofection.

Nucleofection is a powerful non-viral transfection technique that can deliver plasmid DNA with high efficiency to cells that are traditionally difficult to transfect. In this study, we demonstrate that nucleofection of astrocytes grown in primary cell culture resulted in 76 ± 9% transfected cells and low cytotoxicity. However, the nucleofected astrocytes showed a reduced re-attachment to the growth media when replated and subsequent impairment of proliferation. This led to substantially decreased cell densities during the initial 72 h following transfection. Furthermore, these cells were less efficient at producing wound closure in a scratch model of injury. Nucleofection also resulted in the generation of a small proportion of polynucleated cells. The findings demonstrate that nucleofection provides a valuable technique for delivering DNA to astrocytes in culture. However, considerable care is needed in designing and interpreting such studies because of long-lasting changes induced in key properties of these cells by the nucleofection process.