Biology subjects

Guarascio, R.

Publications and source records attributed to Guarascio, R..

5 recordsLinked to original sources

bioRxiv2026

Pharmacological rescue of cilia trafficking defects in IFT140 retinal organoid and RPE models of retinal dystrophy

Pathogenic variants in IFT140 are associated with a spectrum of syndromic and non-syndromic ciliopathies, with retinal degeneration as a common feature. Despite advances in understanding IFT140 function across various tissues, human retina-specific models are lacking. Here, we show that knock-in mice homozygous for the IFT140 patient variant c.932A>G (p.Y311C) did not develop retinal degeneration, while mice with the homozygous variant c.1451C>T (p.T484M), associated with non-syndromic retinal dystrophy, were embryonic lethal. Therefore, to understand the effect of these variants on retinal homeostasis, we generated novel human in vitro models of IFT140-associated retinal dystrophy, including CRISPR/Cas9 IFT140 knock-out (IFT140KO) induced pluripotent stem cells (iPSC) and patient-derived iPSC retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-ROs). IFT140KO iPSC-RPE cells display stubby cilia compared to isogenic controls, while IFT140T484M/T484Mpatient-derived iPSC-RPE cells exhibit slightly shorter cilia and cilia tip protein accumulation. Both IFT140KO and IFT140T484M/T484M iPSC-ROs show accumulation of cilia proteins at the connecting cilium and outer segment of photoreceptors, and mislocalization of rhodopsin to the inner segments and outer nuclear layer. Pharmacological screening of compounds previously reported to improve cilia structure identified the flavonoid eupatilin as the most effective molecule. Treatment with eupatilin improved cilium length and IFT traffic in iPSC-RPE, and IFT traffic and rhodopsin localization in iPSC-ROs. These findings emphasize the importance of human stem cell derived models to investigate tissue specific disease mechanisms and highlight the therapeutic potential of eupatilin to ameliorate cilia defects in retinal tissue.

neuroscience↗

bioRxiv2025

Preventing light-induced toxicity in a new mouse model of rhodopsin sector retinitis pigmentosa

Retinitis Pigmentosa (RP) is an inherited retinal dystrophy characterized by the progressive loss of rod photoreceptors. Sector RP is a form of RP, where degeneration originates in the inferior retina, mainly influenced by light exposure. Over 200 RHO variants are pathogenic and associated with autosomal dominant RP. RHOM39Ris one of the most common RHO variants linked to sector RP in the UK. A knock-in (KI) mouse model expressing RhoM39R was generated and characterized to investigate the mechanisms of degeneration associated with this variant and explore novel therapeutic strategies for rhodopsin sector RP. Under ambient light, RhoM39R/+ KI mice exhibited impaired retinal function by ERG, with some defects in OS ultrastructure, but retained normal outer nuclear layer (ONL) thickness. Repeated exposure to bright light led to photoreceptor loss. In contrast, RhoM39R/M39R KI mice in ambient light displayed severe retinal dysfunction, ONL thinning, and grossly abnormal OS ultra structure. In homozygous mice, a single bright light exposure significantly reduced ONL thickness within 48 h. The rescue of these models was achieved through reduced light exposure and pharmacological intervention. Rearing in dim red light (red cage condition) restored ERG responses in RhoM39R/+ KI mice and improved ONL thickness in RhoM39R/M39R KI mice. Transcriptomic analysis in RhoM39R/M39R KI mice revealed upregulation of Sphingosine 1-P Receptor (S1PR) transcripts. Treatment with the S1PR agonist Fingolimod (FTY720) before bright light exposure significantly reduced degeneration, demonstrating a protective effect in both heterozygous and homozygous models and suggesting potential a therapeutic approach for sector RP patients.

neuroscience↗

bioRxiv2024

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 2,200 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{degrees}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.

molecular biology↗

bioRxiv2021

The role of Musashi-1 in CEP290 c.2991+1655A>G cryptic exon splicing in Leber Congenital Amaurosis

Human photoreceptors maximise alternative exon splicing to generate a unique set of gene isoforms. Conversely, the inclusion of a cryptic exon caused by the c.2991+1655A>G deep intronic change in CEP290 occurs in the human retina leading to Leber Congenital Amaurosis (LCA10). The RNA-binding protein Musashi-1 (MSI1) is a key component of alternative splicing in the developing mouse retina. Here we investigated the role of MSI1 in human photoreceptor-specific splicing and its potential role in CEP290 aberrant splicing disease. Alternative splicing was studied using human induced pluripotent stem cell derived 3D retinal organoid and RPE RNA-seq datasets and several photoreceptor gene isoforms were identified. Their temporal expression was resolved in control 3D retinal organoids in comparison to development and differentiation markers. Morpholino knockdown of MSI1 in control retinal organoids reduced the expression of several photoreceptor differentiation markers and the inclusion of photoreceptor-specific exons. Nonetheless, MSI1 knockdown in homozygous CEP290 c.2991+1655A>G LCA10 retinal organoids did not affect the inclusion of the LCA10-associated cryptic exon. These results show that while MSI1 is important for photoreceptor alternative splicing and homeostasis, it is not a major driver of the recognition of the CEP290 cryptic splice site and the manifestation of LCA10. HIGHLIGHTS{blacksquare} The human retina expresses a unique set of gene isoforms {blacksquare}Musashi-1 regulates alternative splicing in 3D human retinal organoids {blacksquare}Musashi-1 knockdown in 3D retinal organoids affects gene splicing and homeostasis in photoreceptors {blacksquare}Musashi-1 may regulate alternative splicing of cryptic exons in retina but not in LCA10

cell biology↗

bioRxiv2020

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.

neuroscience↗