The Role of RAL-Interacting Protein of 76 kDa (RLIP76) in Development of Ocular Tissues

Mukesh Kumar Sahu. THE ROLE OF RAL-INTERACTING PROTEIN OF 76 kDa (RLIP76) IN THE DEVELOPMENT OF OCULAR TISSUES. August 2013. Total number of pages – 114; 17 illustrations; 4 tables; 137 references. Abstract: RalBP1/RLIP76 is a ubiquitously expressed protein, involved in the promotion and regulation of functions initiated by Ral and R-Ras small GTPases. Presence of multiple domains in its structure enables RLIP76 to be involved in a number of physiological processes such as endocytosis, exocytosis, mitochondrial fission, actin cytoskeleton remodeling, and transport of exogenous and endogenous toxicants. Previous studies in Dr. Awasthi’s laboratory have established that RLIP76 provides protection to ocular tissues against oxidative stress by transporting the glutathione-conjugates (GSH-conjugates) of the toxic, electrophilic products of lipid peroxidation (e.g. 4-Hydroxynonenal) generated during oxidative stress. In particular it was demonstrated that by transporting the GSH-conjugates of 4-Hydroxynonenal (GS-HNE), RLIP76 protects ocular tissues against oxidative stress. Rationale: It was reasoned that lens specific RLIP76 Tg mice due to their enhanced capability to detoxify 4-HNE would be more resistant to HNE-induced cataract formation. We engineered lens specific RLIP76 transgenic mice (RLIP76 Tg) to delineate the role of RLIP76 as a protective mechanism in the lens. Results: Surprisingly, lens specific RLIP76 Tg mice showed impairment in the development of lens and eye, and a phenotype with small eyes similar to that observed in microphthalmia. I report here for the first time a novel mouse model of human genetic disorder microphthalmia. In this dissertation, I report engineering of lens specific RLIP76 Tg mice, characterization of the associated phenotype, and the possible molecular mechanisms that lead to the impaired 2 development of lens and eye in these mice. Briefly, the lens specific RLIP76 Tg mice show remarkably small eyes with impaired lens development, disrupted cytoskeleton organization and aberrant fiber cells differentiation. The results of microarray analysis indicated that genes involved in pathways for G-Protein signaling, actin reorganization, endocytosis, and apoptosis are affected in these transgenic mice. The expression of transcription factors, Pax6, Hsf1, and Hsf4b known to be involved in lens development was down regulated in the lens of these Tg mice. The expression of heat shock proteins (HSPs), the downstream targets of Hsfs was differentially affected in the lens showing down regulated expression of Hsp27 and Hsp40, up-regulated expression of Hsp60, and no effect on the expression of Hsp70, and Hsp90. Our results show that the disruption in the organization of actin cytoskeleton of these Tg mice was associated with the inhibition of the activation of Cdc42 that is known to regulate the organization of actin cytoskeleton. Fiber cells differentiation is required for development, growth, and transparency of the lens. Our data suggest disrupted cytoskeleton organization in Tg mice is caused by aberrant differentiation of lens epithelial cells into lens fiber cells. Conclusions and significance: Present studies show that lens specific RLIP76 Tg mice show phenotype similar to microphthalmia and these mice may provide useful animal model for elucidating the mechanisms of lens development, and etiology of microphthalmia.