Yuanyuan Chen

Current treatments targeting individual protein quality control pathways have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common druggable target capable of global proteostasis modulation. Building upon our prior research establishing nuclear speckles as pivotal membrane-less organelles for transcriptional control of proteostasis, we aim to alleviate proteinopathies through nuclear speckle rehabilitation. We identify pyrvinium pamoate as a nuclear speckle rehabilitator that enhances protein quality control gene expression and suppresses YAP1 transcriptional activity via decreasing the surface/interfacial tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, nanomolar pyrvinium pamoate protects against retinal degeneration and tauopathy mainly by promoting autophagy and ubiquitin-proteasome activity in a SON-dependent manner without causing stress. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity are observed in human tauopathies. Our study provides proof-of-principle of targeting nuclear speckles to ameliorate proteinopathies.

Vision decline in the elderly, often due to retinal aging, predisposes individuals to pathologies like age-related macular degeneration. Currently, there are few effective oral treatments for this condition. Our study introduces an oral agent, 8-aminoguanine (8-AG), which targets age-related retinal degeneration using an aged Fischer 344 rat model. When administered in drinking water at a low dose for 8 weeks starting at 22 months of age, 8-AG significantly preserves retinal structure and function, as evidenced by increased retinal thickness, enhanced photoreceptor integrity, and improved electroretinogram responses. 8-AG reduces apoptosis, oxidative damage, and microglial/macrophage activation in aging retinae. 8-AG also mitigates retinal inflammation at transcriptional and cytokine levels. Extending treatment to 17 weeks further amplifies these protective effects. Given its efficacy in various disease models, 8-AG shows great promise as an anti-aging compound with the potential to mitigate common hallmarks of aging.

The retinal explant culture system is a valuable tool for studying the pharmacological, toxicological, and developmental aspects of the retina. It is also used for translational studies such as gene therapy. While no photoreceptor-like cell lines are available for in vitro studies of photoreceptor cell biology, the retinal explant culture maintains the laminated retinal structure ex vivo for as long as a month. Human and nonhuman primate (NHP) postmortem retinal explants cut into small pieces offer the possibility of testing multiple conditions for safety and adeno-associated viral (AAV) vector optimization. In addition, the cone-enriched foveal area can be studied using the retinal explants. Here, we present a detailed working protocol for retinal explant isolation and culture from mouse, human, and NHP for testing drug efficacy and AAV transduction. Future applications of this protocol include combining live imaging and multiwell retinal explant culture for high-throughput drug screening systems in rodent and human retinal explants to identify new drugs against retinal degeneration.

Visual decline in the elderly is often attributed to retinal aging, which predisposes the tissue to pathologies such as age-related macular degeneration. Currently, effective oral pharmacological interventions for retinal degeneration are limited. We present a novel oral intervention, 8-aminoguanine (8-AG), targeting age-related retinal degeneration, utilizing the aged Fischer 344 rat model. A low-dose 8-AG regimen (5 mg/kg body weight) via drinking water, beginning at 22 months for 8 weeks, demonstrated significant retinal preservation. This was evidenced by increased retinal thickness, improved photoreceptor integrity, and enhanced electroretinogram responses. 8-AG effectively reduced apoptosis, oxidative damage, and microglial/macrophage activation associated with aging retinae. Age-induced alterations in the retinal purine metabolome, characterized by elevated levels of inosine, hypoxanthine, and xanthine, were partially mitigated by 8-AG. Transcriptomics highlighted 8-AG's anti-inflammatory effects on innate and adaptive immune responses. Extended treatment to 17 weeks further amplified the retinal protective effects. Moreover, 8-AG showed temporary protective effects in the Rho^P23H/+ mouse model of retinitis pigmentosa, reducing active microglia/macrophages. Our study positions 8-AG as a promising oral agent against retinal aging. Coupled with previous findings in diverse disease models, 8-AG emerges as a promising anti-aging compound with the capability to reverse common aging hallmarks.

Current treatments targeting individual protein quality control pathways have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common druggable target capable of global proteostasis modulation. Building upon our prior research establishing nuclear speckles as pivotal membrane-less organelles for transcriptional control of proteostasis, we aim to alleviate proteinopathies through nuclear speckle rehabilitation. We identified pyrvinium pamoate as a nuclear speckle rehabilitator that enhances protein quality control gene expression and suppresses YAP1 transcriptional activity via decreasing the surface/interfacial tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, nanomolar pyrvinium pamoate protected against retinal degeneration and tauopathy mainly by promoting autophagy and ubiquitin-proteasome activity in a SON-dependent manner without causing stress. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity were observed in human tauopathies. Our study provides proof-of-principle of targeting nuclear speckles to ameliorate proteinopathies.

Rhodopsin -associated ( RHO -associated) retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified nonretinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules toward the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Furthermore, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in Rho ^P23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL), indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the Rho ^P23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in Rho ^P23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in Rho ^P23H/+ mice. Collectively, we provide proof of principle that nonretinoid chaperones are promising drug candidates in treating RHO -associated RP.

Neural Regen Res, Official publication of Publishing House of Neural Regeneration Research China Association of Rehabilitation Medicine,China

Rhodopsin mutation and misfolding is a common cause of autosomal dominant retinitis pigmentosa (RP). Using a luciferase reporter assay, we undertook a small‐molecule high‐throughput screening (HTS) of 68, 979 compounds and identified nine compounds that selectively reduced the misfolded P23H rhodopsin without an effect on the wild type (WT) rhodopsin protein. Further, we found five of these compounds, including methotrexate (MTX), promoted P23H rhodopsin degradation that also cleared out other misfolded rhodopsin mutant proteins. We showed MTX increased P23H rhodopsin degradation via the lysosomal but not the proteasomal pathway. Importantly, one intravitreal injection (IVI) of 25 pmol MTX increased electroretinogram (ERG) response and rhodopsin level in the retinae of Rho ^P23H/+ knock‐in mice at 1 month of age. Additionally, four weekly IVIs increased the photoreceptor cell number in the retinae of Rho ^P23H/+ mice compared to vehicle control. Our study indicates a therapeutic potential of repurposing MTX for the treatment of rhodopsin‐associated RP.