Biology subjects

Deng, W.-T.

Publications and source records attributed to Deng, W.-T..

5 recordsLinked to original sources

bioRxiv2025

Super-resolution microscopy reveals a Rab6a-dependent trafficking hub for rhodopsin at the mammalian rod photoreceptor Golgi

Rod photoreceptor stability is critical for retinal health and lifelong vision. The proper intracellular trafficking of the photopigment receptor rhodopsin (Rho) is essential for normal rod homeostasis, as Rho mislocalization precedes rod cell death in inherited retinal disorders such as retinitis pigmentosa. Despite its importance, the molecular mechanisms of Rho trafficking in mammalian rods remain largely undefined. In this study, we combined multiple Rho-labeling strategies with super-resolution microscopy to investigate the subcellular organization of Rho in the Golgi complex of mammalian rods. Using stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM) super-resolution imaging modalities, we mapped the localization of Golgi proteins with Rho in mouse and macaque rods and found that Rho specifically colocalizes with Rab6a in the trans-Golgi. To test the functional significance of this interaction, we utilized a dominant-negative Rab6a mutant in both HEK293T cells and mouse rods. We demonstrated that the dominant negative Rab6a significantly inhibits Rho secretion in cell culture, causing intracellular retention. In mouse rods, we found that this mutant similarly causes significant Rho retention in the trans-Golgi. However, surprisingly, a majority of Rho protein still escaped the Golgi and reached the outer segment. Together, these findings uncover critical new subcellular details about Rho organization at the Golgi and establish a role for Rab6a as a regulator of Rho protein release from the trans-Golgi in mammalian rods. Our results provide critical insight into the protein trafficking mechanisms that must be sustained and regulated in mammalian rods for long-term retinal health.

cell biology↗

bioRxiv2025

Gene Therapy Rescues Cone Function in an All-cone Retina Mouse Model for Blue Cone Monochromacy with the Most Common C203R Missense Mutation

Blue cone monochromacy (BCM) is an X-linked cone dystrophy characterized by loss of long- (L) and medium-wavelength (M) cone function. A common cause is the C203R missense mutation, which occurs in both OPN1LW and OPN1MW, or in hybrid OPN1LW/OPN1MW opsin genes. Because BCM primarily affects foveal cones, we generated Opn1mwC198R/Opn1sw-/-/Nrl-/- (C198RAC) mice carrying the murine equivalent of the human C203R mutation on an all-cone retinal background. C198RAC mice exhibited absent photopic ERG responses and significantly shortened cone outer segments, recapitulating foveal cone deficits in BCM. Metabolomic profiling further revealed altered retinal metabolism, including reduced cGMP and elevated oxidative stress-related metabolites. To evaluate therapy, we delivered AAV8-Y733F expressing human L-opsin (OPN1LW) cDNA under the cone-specific PR2.1 promoter at 1 and 5 months of age. Treatment restored cone function, regenerated outer segment structures, and provided rescue for at least 5 months post-injection in both early- and late-treatment groups. These results demonstrate that densely packed cones expressing only the C198R mutant opsin remain viable targets for gene therapy. Together, this study establishes the C198RAC mouse as a cone-rich model of BCM and provides compelling preclinical evidence that AAV-mediated gene augmentation can restore cone structure and function, supporting the feasibility of gene therapy for BCM.

molecular biology↗

bioRxiv2025

HSP90α is specifically required for rod photoreceptor function and cannot be replaced by HSP90β.

Heat Shock Protein 90 (HSP90) is a critical molecular chaperone that exists as two cytosolic paralogs, HSP90 and HSP90{beta}, which share high sequence identity but may perform non-redundant functions in vivo. Loss of HSP90 in mice results in progressive rod photoreceptor degeneration despite normal retinal development and expression of HSP90{beta}. To investigate whether HSP90{beta} can substitute for HSP90 in photoreceptors, we generated adeno-associated virus (AAV) vectors expressing HA-tagged HSP90 or HSP90{beta} under the control of a short rhodopsin promoter. In Hsp90 -/- mice, subretinal delivery of AAV-Hsp90aa1 (HSP90) restored rod function and prevented photoreceptor degeneration, as measured by electroretinography (ERG). In contrast, AAV-mediated expression of HSP90{beta} failed to rescue rod function despite comparable expression levels. Overexpression of either paralog in wild-type mice had no adverse effects on retinal function. These findings reveal a paralog-specific and intrinsic requirement for HSP90 in rod photoreceptors, demonstrating that HSP90{beta} cannot compensate for its loss despite structural similarity.

biochemistry↗

bioRxiv2025

Molecular Mechanisms Limiting the Therapeutic Window of AAV Gene Therapy in Mouse Models of Blue Cone Monochromacy

Blue cone monochromacy (BCM) is an X-linked retinal disorder caused by mutations in the OPN1LW/OPN1MW gene locus, resulting in impaired cone function and structural degeneration. We conducted a comparative analysis of AAV-mediated gene therapy in Opn1lw/Opn1mw double knockout (DKO) and Opn1mwC198R/Opn1sw-/- (C198R) BCM mouse models and evaluated the therapeutic window, efficacy, and longevity. Our results demonstrate that the AAV8-Y733F capsid achieved superior cone rescue compared to AAV5. While both DKO and C198R models showed similar therapeutic windows and rescue longevity, treatment efficacy decreased markedly in older mutant mice. Structural analysis revealed that aged cones in both models displayed degenerative changes, including mislocalized mitochondria and compromised connecting cilia. At the molecular level, we observed reduced AAV-mediated transgene expression in DKO and C198R older cones, which may result from decreased transduction efficiency, decreased circular episome stability, genome-wide transcription/translation downregulation, targeted mRNA/protein degradation, or overall cone degeneration. Notably, the cone-specific promoters for Pde6c and Cngb3 maintained robust activity in degenerating cones. These findings suggest that combining an efficient AAV serotype with an optimized cone promoter could be a viable approach to extend the therapeutic window and enhance treatment longevity for BCM patients.

molecular biology↗

bioRxiv2024

ER Aggregation Causes Synaptic Protein Imbalance in Retinitis Pigmentosa Mutant Photoreceptor Neurons

Rod photoreceptor neurons in the retina detect scotopic light through the visual pigment rhodopsin (Rho) in their outer segments (OS). Efficient Rho trafficking to the OS through the inner rod compartments is critical for long-term rod health. Given the importance of protein trafficking to the OS, less is known about the trafficking of rod synaptic proteins. Furthermore, the subcellular impact of Rho mislocalization on rod synapses (i.e., "spherules") has not been investigated. In this study we used super-resolution and electron microscopies, along with proteomics, to perform a subcellular analysis of Rho synaptic mislocalization in P23H-Rho-RFP mutant mice. We discovered that mutant P23H-Rho-RFP protein mislocalized in distinct ER aggregations within the spherule cytoplasm, which we confirmed with AAV overexpression. Additionally, we found synaptic protein abundance differences in P23H-Rho-RFP mice. By comparison, Rho mislocalized along the spherule plasma membrane in WT and rd10 mutant rods, in which there was no synaptic protein disruption. Throughout the study, we also identified a network of ER membranes within WT rod presynaptic spherules. Together, our findings indicate that photoreceptor synaptic proteins are sensitive to ER dysregulation. Summary StatementThis study examines the impact of rhodopsin mislocalization on rod photoreceptor synaptic structures and synaptic protein levels using P23H rhodopsin and other retinitis pigmentosa mouse models.

neuroscience↗