From inner segment to outer segment: Palmitoylation of photoreceptor Na+, K+-ATPase and the importance of PRCD in photoreceptor outer segment morphogenesis

Photoreceptors are specialized neuroepithelial cells which are optimized for efficient capture of light and initiation of visual transduction. These cells have several compartments which are very important for proper visual function and segregation of cellular processes, including the outer segment (OS), inner segment (IS), nucleus, and synapse. The IS houses all of the cellular organelles and biosynthetic molecular machinery the cell requires and is the site of protein synthesis. The light-sensing OS is a highly modified, primary cilium, which contains many stacks of double membranous discs which house proteins required for formation and maintenance of OS structure, as well as phototransduction. These structural and phototransduction proteins are synthesized in the IS and subsequently trafficked to the OS through the narrow connecting cilium. Many of these proteins undergo post-translation lipid modifications for proper subcellular localization and association with the OS disc membranes. While the proteins involved in the phototransduction cascade are crucial for generating signals of vision, regulation of the photocurrent is needed for proper depolarization and hyperpolarization of the photoreceptor neuron, a process which is required for the cell to transduce electrical impulses to downstream neurons. Many ion channels, exchangers, and pumps, including the CNG channels and the NCKX in the OS, as well as the Na+, K+-ATPase and the HCN channel in the IS, are involved in this process of maintaining the photocurrent in both dark and light. One small OS disc-specific protein whose function has not yet been elucidated is progressive rod-cone degeneration, or PRCD. Previous studies in our lab have identified that PRCD is post-translationally lipid modified by S-palmitoylation on its sole cysteine, which is required for its stability and localization to the OS. Though PRCD has been shown to be important in OS disc morphogenesis and maintenance, its specific role in this process remains unclear. In this dissertation, I utilize a Prcd-KO animal our lab generated using CRISPR/Cas9 genome editing in order to investigate photoreceptor function, OS ultrastructure, and rhodopsin packaging into disc membranes in the absence of PRCD. Furthermore, I utilize acyl resin-assisted capture and mass spectrometry in order to identify novel proteins in the retina which undergo S-palmitoylation and validate palmitoylation profiles of several proteins using additional well-established techniques in the field. In Chapter 1 of this dissertation, I review the functional and structural needs of photoreceptor neurons, as well as the roles of both PRCD and the b2-subunit of the retinal Na+, K+-ATPase (ATP1B2) in these requirements. In Chapter 2, I characterize the functional and structural consequences of loss of PRCD in our lab-generated Prcd-KO mouse model, demonstrating through atomic force microscopy (AFM) analysis the role PRCD plays in regulation of rhodopsin incorporation into OS disc membranes. In Chapter 3, I use various techniques to reveal and validate that the ATP1B2 is palmitoylated on its N-terminal cysteine at the 10th amino acid (Cys10). Finally, in Chapter 4, I discuss the results and conclusions of my dissertation work and propose experiments to further investigate the specific role of PRCD in photoreceptors and the importance of palmitoylation of ATP1B2 in the retina. Elucidation of the specific role PRCD plays in disc morphogenesis and investigation of its interaction with rhodopsin will help to further the field and provide a deeper look into how discs are formed and maintained. Further investigation of the role palmitoylation plays in ATP1B2 function will help to establish the unique requirement of expression of ATP1B2 in the retina and identify new roles for ATP1B2 in cells where it is expressed

R17C Mutation in Photoreceptor Disc-Specific Protein, PRCD, Results in Additional Lipidation Altering Protein Stability and Subcellular Localization

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (OS) disc-specific protein essential for maintaining OS structures while contributing to rhodopsin packaging densities and distribution in disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation linked with retinitis pigmentosa (RP) in humans and dogs demonstrates the importance of palmitoylation for protein stability and trafficking to the OS. We demonstrate a mutation, in the polybasic region (PBR) of PRCD (Arg17Cys) linked with RP where an additional lipidation is observed through acyl-RAC. Immunolocalization of transiently expressed R17C in hRPE1 cells depicts similar characteristics to wild-type PRCD; however, a double mutant lacking endogenous palmitoylation at Cys2Tyr with Arg17Cys is comparable to the C2Y protein as both aggregate, mislocalized to the subcellular compartments within the cytoplasm. Subretinal injection of PRCD mutant constructs followed by electroporation in murine retina exhibit mislocalization in the inner segment. Despite being additionally lipidated and demonstrating strong membrane association, the mutation in the PBR affects protein stability and localization to the OS. Acylation within the PBR alone neither compensates for protein stability nor trafficking, revealing defects in the PBR likely lead to dysregulation of PRCD protein associated with blinding diseases

Liposomal Irinotecan Accumulates in Metastatic Lesions, Crosses the Blood-Tumor Barrier (BTB), and Prolongs Survival in an Experimental Model of Brain Metastases of Triple Negative Breast Cancer

Purpose—The blood-tumor barrier (BTB) limits irinotecan distribution in tumors of the central nervous system. However, given that the BTB has increased passive permeability we hypothesize that liposomal irinotecan would improve local exposure of irinotecan and its active metabolite SN-38 in brain metastases relative to conventional irinotecan due to enhanced-permeation and retention (EPR) effect. Methods—Female nude mice were intracardially or intracranially implanted with human brain seeking breast cancer cells (brain metastases of breast cancer model). Mice were administered vehicle, non-liposomal irinotecan (50 mg/kg), liposomal irinotecan (10 mg/kg and 50 mg/kg) intravenously starting on day 21. Drug accumulation, tumor burden, and survival were evaluated. Results—Liposomal irinotecan showed prolonged plasma drug exposure with mean residence time (MRT) of 17.7 ± 3.8 h for SN-38, whereas MRT was 3.67 ± 1.2 for non-liposomal irinotecan. Further, liposomal irinotecan accumulated in metastatic lesions and demonstrated prolonged exposure of SN-38 compared to non-liposomal irinotecan. Liposomal irinotecan achieved AUC values of 6883 ± 4149 ng-h/g for SN-38, whereas non-liposomal irinotecan showed significantly lower AUC values of 982 ± 256 ng-h/g for SN-38. Median survival for liposomal irinotecan was 50 days, increased from 37 days (