Eye on a Dish Models to Evaluate Splicing Modulation

Inherited retinal dystrophies, such as Leber congenital amaurosis, Stargardt disease, and retinitis pigmentosa, are characterized by photoreceptor dysfunction and death and currently have few treatment options. Recent technological advances in induced pluripotent stem cell (iPSC) technology and differentiation methods mean that human photoreceptors can now be studied in vitro. For example, retinal organoids provide a platform to study the development of the human retina and mechanisms of diseases in the dish, as well as being a potential source for cell transplantation. Here, we describe differentiation protocols for 3D cultures that produce retinal organoids containing photoreceptors with rudimentary outer segments. These protocols can be used as a model to understand retinal disease mechanisms and test potential therapies, including antisense oligonucleotides (AONs) to alter gene expression or RNA processing. This "retina in a dish" model is well suited for use with AONs, as the organoids recapitulate patient mutations in the correct genomic and cellular context, to test potential efficacy and examine off-target effects on the translational path to the clinic

Inhibition of VCP preserves retinal structure and function in autosomal dominant retinal degeneration

Due to continuously high production rates of rhodopsin (RHO) and high metabolic activity, photoreceptor neurons are especially vulnerable to defects in proteostasis. A proline to histidine substitution at position 23 (P23H) leads to production of structurally misfolded RHO, causing the most common form of autosomal dominant Retinitis Pigmentosa (adRP) in North America. The AAA-ATPase valosin-containing protein (VCP) extracts misfolded proteins from the ER membrane for cytosolic degradation. Here, we provide the first evidence that inhibition of VCP activity rescues degenerating P23H rod cells and improves their functional properties in P23H transgenic rat and P23H knock-in mouse retinae, both in vitro and in vivo. This improvement correlates with the restoration of the physiological RHO localization to rod outer segments (OS) and properly-assembled OS disks. As a single intravitreal injection suffices to deliver a long-lasting benefit in vivo, we suggest VCP inhibition as a potential therapeutic strategy for adRP patients carrying mutations in the RHO gene

Modeling and Rescue of RP2 Retinitis Pigmentosa Using iPSC-Derived Retinal Organoids

RP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2-associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene-edited isogenic RP2 knockout (RP2 KO) induced pluripotent stem cells (iPSCs) and RP2 patient-derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient-derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. Adeno-associated virus-mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell deat

The modulation of tau proteostasis in tauopathies by NUB1/NUB1L interaction with the autophagy-lysosomal pathway

NEDD8 ultimate buster 1 (NUB1) and its longer isoform NUB1L are ubiquitin-like (UBL)/ ubiquitin-associated (UBA) proteins that target the downregulation of ubiquitin-like modifiers and aggregation-prone proteins leading to neurodegeneration (synphilin-1, huntingtin and tau). Previous data revealed that the NUB1-mediated reduction in tau phosphorylation and aggregation was enhanced upon proteasome inhibition, suggesting a switch in NUB1 function from targeted proteasomal degradation to a role in autophagy. The aim of this study was to test this hypothesis and understand the molecular mechanisms involved. NUB1 expression and localization was localized in the hippocampus of transgenic mice models of tauopathy. In wild type animals, NUB1 localized to the nuclei of neurons but also co-localized with tau along neuronal axons. In pathological models at later stages, NUB1 localized in cytosolic inclusions positive for pathological forms of tau and components of the autophagy-lysosomal machinery (p62 and LAMP1). From 12 months, NUB1 expression decreased in animal models with a severe pathology. To investigate the role of NUB1 in vitro, a GFP-tau inducible neuroblastoma cell line was generated and treated with proteasome inhibitor to induce the formation of aggresomes. NUB1 significantly decreased the ratio of phosphorylated tau to tau, particularly following proteasomal inhibition. Moreover, analysis of phosphorylated tau fractionating to the detergent insoluble fraction revealed that NUB1 targets aggregation-prone phosphorylated tau. Interestingly, upon proteasome inhibition, NUB1/NUB1L increased the levels of the autophagosome marker LC3BII and increased the size and numbers of LC3B puncta. Moreover, NUB1 increased the recruitment of LAMP1 positive vesicles around the aggresomes and interacted with p62 in a manner dependent on proteasome inhibition. Autophagy flux assays revealed that NUB1/NUB1L blocked the autophagy-lysosomal pathway downstream of autophagosome formation. Assays conducted with NUB1L lacking the UBA or UBL domain revealed that while both these domains were required for the block in autophagy, the UBA domain was primarily involved in this function. Accordingly, the increase in the level of autophagosomes and interaction with p62 upon proteasome inhibition was compromised by deletion of the UBA domain

A Proximity Complementation Assay to Identify Small Molecules That Enhance the Traffic of ABCA4 Misfolding Variants

ABCA4-related retinopathy is the most common inherited Mendelian eye disorder worldwide, caused by biallelic variants in the ATP-binding cassette transporter ABCA4. To date, over 2200 ABCA4 variants have been identified, including missense, nonsense, indels, splice site and deep intronic defects. Notably, more than 60% are missense variants that can lead to protein misfolding, mistrafficking and degradation. Currently no approved therapies target ABCA4. In this study, we demonstrate that ABCA4 misfolding variants are temperature-sensitive and reduced temperature growth (30 °C) improves their traffic to the plasma membrane, suggesting the folding of these variants could be rescuable. Consequently, an in vitro platform was developed for the rapid and robust detection of ABCA4 traffic to the plasma membrane in transiently transfected cells. The system was used to assess selected candidate small molecules that were reported to improve the folding or traffic of other ABC transporters. Two candidates, 4-PBA and AICAR, were identified and validated for their ability to enhance both wild-type ABCA4 and variant trafficking to the cell surface in cell culture. We envision that this platform could serve as a primary screen for more sophisticated in vitro testing, enabling the discovery of breakthrough agents to rescue ABCA4 protein defects and mitigate ABCA4-related retinopathy.Medicine, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofReviewedFacultyResearche