In primary open-angle glaucoma, the major site of damage is within the lamina cribrosa (LC) region of the optic nerve head (ONH). The LC consists of a connective tissue framework through which millions of unmyelinated retinal ganglion cell axons exit the eye and form the optic nerve. Non-neural cells within the LC are closely associated with individual axons and are know to synthesize ECM components. Neurotrophins (NT) include NGF, BDNF, NT-3, and NT-4 and are known to signal via high affinity Trk receptors. While originally thought to only regulate neuronal survival, non-neuronal cells have recently been shown to express NT and signal via Trk receptors. This raises the possibility that non-neuronal cells within the human LC may synthesize and secrete NT locally to control the microenvironment and/or maintain the viability of RGC. Our long-term research objective is to increase our knowledge of the physiological roles of NT within the normal and glaucomatous human LC. The hypothesis of this proposal is that NT expression and paracrine signaling within the human LC is decreased in glaucoma and under experimental glaucomatous conditions. The following specific aims will be used to test the hypothesis: (1) isolate and characterize non-neural cells from normal and glaucomatous human LC tissues, (2) compare and contrast NT and Trk receptor mRNA and protein expression in cells isolated from the LC of normal donors, (3) demonstrate that ONH astrocytes and LC cells secrete and respond to NT, (4) compare and contrast NT and Trk receptor mRNA and protein expression and NT secretion in cells from glaucomatous donors, (5) compare and contrast functional responses (e.g. cell proliferation and ECM modulator expression) to exogenous NT in cells from normal and glaucomatous donors, and (6) using ischemic and hypoxic conditions, compare and contrast NT and Trk receptor expression, NT secretion, and functional responses in ONH cells from normal and glaucomatous donors. To achieve the specific aims we will use RT-PCR to determine gene expression, Real-time Quantitative RT-PCR to determine mRNA levels, cell proliferation assays, Western-blotting and immunohistochemistry to demonstrate protein expression, ELISA immunoassay to determine NT, MMP and TIMP secretion and phosphorylation assays to demonstrate Trk activation. This study is significant because it will increase our knowledge of the role of NT in the human LC and may lead to alternative treatment modalities in glaucoma.
