Publication Date

2012-03-09

Availability

Open access

Embargo Period

2012-03-09

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular Cell and Developmental Biology (Medicine)

Date of Defense

2012-03-01

First Committee Member

Valery I. Shestopalov

Second Committee Member

Carlos T. Moraes

Third Committee Member

John R. Bethea

Fourth Committee Member

Richard K. Lee

Abstract

Astrocytes are a critical cell type of the retina which support ganglion cell axons and maintain the blood retinal barrier. Upon ischemia reperfusion (IR) injury, astrocytes undergo rapid activation, which has been characterized by hypertrophy, proliferation and global changes in gene expression. The failure of these cells to support neurons after ischemia is thought to promote secondary injury response and retinal ganglion cell apoptosis. Therefore, promoting survival and proper function of astrocytes is critical for protecting the inner retina to ischemic injury. The activation of astrocytes and changes in gene expression in retinal IR injury has been linked to the activation of the transcription factor NF-κB. Consequently, activated astrocytes have been shown to induce the upregulation of numerous NF-κB target genes including cytokines, chemokines and notably the phagocyte NADPH oxidase (PHOX) resulting in elevated production of reactive oxygen species (ROS). The work presented in this dissertation demonstrates the regulatory mechanisms of PHOX-induced oxidative stress and NF-κB activation in astrocytes and their effects on retinal ganglion cell (RGC) loss in ischemia-reperfusion injury (IR). In chapters 2 and 3 we employed GFAP-IκBα-dn transgenic mice (TG), which results in suppression of the NF-κB canonical pathway GFAP expressing cells, primarily astrocytes, to study glial NF-κB-mediated PHOX activity and neurotoxicity. Our analysis showed that NF-κB activation in astrocytes caused increased expression of PHOX and ROS production in cells and whole retinas subjected to oxygen-glucose deprivation (OGD) and IR injury respectively. However, we found that NF-κB does not increase ROS production after injury by transcriptional upregulation of PHOX. Instead, we found constitutive down regulation of the PHOX genes p22 and p40 in TG cells, which was coincident with down regulation of PHOX proteins in vitro and in vivo. RGC loss induced by OGD in co-cultures with astroglia was blocked by inhibition of PHOX or selective blockade of NF-κB in astrocytes. Suppression of astroglial NF-κB reduced oxidative stress in ganglion layer neurons in retinal IR in vivo. These resuls demonstrate that NF-κB has a crucial role in facilitating oxidative stress through PHOX transcriptional regulation before retinal IR ensues. In chapter 4, we tested the regulatory role of toll like receptor 3 on NF-κB activation in retinal ischemia reperfusion injury. This receptor is an upstream regulator of TLR3 and is known to promote inflammation in some injury models. Using knockout animals and cells, we found that TLR3 is highly expressed in astrocytes following ischemic injury or oxygen glucose deprivation in vitro. We also found that genetic blockade of TLR3 inhibited NF-κB activation and afforded significant protection of ganglion layer neurons after retinal ischemia. Collectively, these data suggest that activation of astroglial NF-κB is detrimental by driving oxidative stress and is a crucial pathway in the pathogenesis of retinal IR injury.

Keywords

Astrocyte; oxidative stress; NADPH oxidase; NF-kappaB

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