Biology subjects

Wijnholds, J.

Publications and source records attributed to Wijnholds, J..

7 recordsLinked to original sources

bioRxiv2026

Retinal Phd2 ablation partially rescues Crb1Crb2-associated Leber congenital amaurosis and alters metabolic signaling

Leber congenital amaurosis (LCA) is a severe early-onset retinal dystrophy caused by pathogenic variants in several genes, including CRB1. CRB1-associated LCA is characterized by disrupted retinal polarity, lamination, and photoreceptor organization, with no restorative treatment. We investigated whether retinal deletion of Phd2 (Egln1), encoding prolyl hydroxylase domain protein 2 (PHD2), could provide mutation-independent neuroprotection in a severe developmental model of CRB1-associated LCA. Using conditional Crb1/Crb2-deficient mice, we additionally deleted Phd2 throughout the neural retina and assessed retinal function, structure, visually guided behavior, and glycolysis-associated gene and protein expression at one and two months. Compared with Crb1/Crb2-deficient mice, additional Phd2 deletion partially improved scotopic responses and significantly increased the optomotor spatial-frequency threshold, whereas b-wave and cone-mediated responses remained impaired. Phd2Crb1Crb2-deficient retinas displayed more organized photoreceptor rosettes containing rod- and cone-marker-positive cells, in contrast to the diffuse outer-retinal disorganization and nuclear-layer amalgamation observed in Crb1/Crb2-deficient retinas. Polarity- and synapse-associated proteins showed improved local compartmentalization within these structures. Metabolic responses were temporally selective: Glut1 and Vegfa transcript levels increased at one month, whereas GLUT1 and LDHA protein levels increased at two months; Pdk1 transcript levels decreased at 2M, whereas PDK1 protein abundance remained unchanged. Although Phd2 deletion did not correct the underlying Crumbs-complex defect or normalize retinal lamination, and elevated Vegfa coincided with persistent vascular abnormalities, these findings demonstrate that retinal Phd2 deletion partially modifies the structural and functional consequences of severe Crb1/Crb2 deficiency and supports further investigation of PHD2-associated metabolic signaling as a potential disease-modifying target for severe CRB1-associated retinal degeneration.

neuroscience↗

bioRxiv2026

USH2A High-Capacity Adenoviral Vector Restores usherin, whirlin, and ADGRV1 in Photoreceptor Cells of Retinal Organoids

Development of gene therapies for USH2A-associated diseases has been limited by the large size of the USH2A coding sequence, which exceeds the packaging capacity of adeno-associated viral vectors. Here, we evaluated high-capacity adenoviral vectors (HC-AdVs) as vehicles for a mutation-independent USH2A gene supplementation strategy in USH2A human retinal organoids. First, we generated human induced pluripotent stem cell (hiPSC) lines carrying a nonsense mutation in USH2A exon-61 (61KO). Similarly to our previously established exon-13 knockout model (13KO), 61KO hiPSC-derived retinal organoids showed reduced levels of usherin and its interacting proteins, adhesion G protein-coupled receptor V1 (ADGRV1), and whirlin, at the photoreceptor connecting cilium. Delivery of a HC-AdV5 encoding full-length USH2A under the control of the photoreceptor-specific human rhodopsin kinase (hGRK1) promoter restored usherin expression and promoted re-localization of ADGRV1 and whirlin to their physiological sites at the connecting cilium in 13KO and 61KO photoreceptors. Unexpectedly, usherin expression was also observed within mutant Muller glial cells. These findings provide proof-of-concept that HC-AdV5-mediated delivery of full-length USH2A can successfully promote the rescue of usherin and relocate its interacting partners in USH2A-defective retinal organoids, demonstrating its potential as a variant-independent therapeutic approach for inherited retinal disorders caused by USH2A mutations.

neuroscience↗

bioRxiv2026

Gene Supplementation of MYO7A or activation of Myo7b for treatment of Usher syndrome 1B

Mutations in MYO7A result in the most severe subtype of Usher syndrome, the leading genetic cause of deafblindness. The large size of MYO7A requires dual adeno-associated virus (AAV) vectors for gene transfer or alternative methods to treat retinal defects. Here, we evaluated two treatment approaches: i) Supplementation of the human MYO7A gene via dual mRNA trans-splicing AAVs, and ii) CRISPR/Cas-mediated activation of the related murine Myo7b gene. Upon MYO7A supplementation, the transgenic MYO7A transcript and protein were expressed and correctly localized in retinal pigment epithelial (RPE) and photoreceptors of mice, pigs, and human retinal organoids. In RPE-and photoreceptor-specific Myo7a knockout mice, we could restore MYO7A expression and localization of melanosomes in RPE cells to wild-type levels. Myo7b activation led to partial restoration of melanosome localization, and the localization of MYO7B protein was largely comparable to MYO7A. These findings indicate that both approaches are in principle suitable for the therapy of Usher syndrome.

molecular biology↗

bioRxiv2026

Loss of ABCA4 from photoreceptor discs triggers changes in glial cell homeostasis

Loss-of-function mutations in the ABCA4 gene cause Stargardt disease (STGD1), the most common inherited macular dystrophy leading to progressive central vision loss. Here, we generated hiPSC-derived retinal organoids harboring a premature stop codon in exon-24 of ABCA4 to evaluate the impact of this mutation on mRNA and protein levels in a human model. Immunofluorescence analysis revealed the absence of ABCA4 protein in the mutant photoreceptor outer segment discs, while single-cell RNA sequencing detected no major transcriptional alterations in rods and cones. Unexpectedly, differential gene expression and pathway enrichment analyses of Muller glial cells (MGCs) and astrocytes highlighted disruption of neuronal development, microenvironment of glial cells, intercellular communication, and programmed cell death pathways. These findings suggest that ABCA4 might play a role in maintaining the retinal microenvironment homeostasis, and that the early transcriptomic response of MGCs and astrocytes preceding photoreceptor degeneration could contribute to Stargardt disease development. Significance StatementHuman induced pluripotent stem cell (hiPSC)-derived retinal organoids provide a powerful platform to investigate inherited retinal diseases. In this study, we generated ABCA4-mutant hiPSC lines and differentiated them into retinal organoids to model Stargardt disease. Despite complete loss of ABCA4 protein from the photoreceptor outer segment discs, rods and cones exhibited minimal transcriptional alterations. In contrast, the ABCA4 variant triggered changes in the glial cell homeostasis, suggesting that Muller glial cells and astrocytes might exhibit an early response to photoreceptor dysfunction in the absence of ABCA4. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/718110v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1d8dbf6org.highwire.dtl.DTLVardef@51239corg.highwire.dtl.DTLVardef@f8fbdborg.highwire.dtl.DTLVardef@5f0b44_HPS_FORMAT_FIGEXP M_FIG Human induced pluripotent stem cells (hiPSCs) were engineered to generate ABCA4-mutant cell lines, later differentiated into retinal organoids as a model of Stargardt disease. The organoids showed loss of ABCA4 from the outer segment discs of rod and cone photoreceptors, while the mutant Muller glial cells and astrocytes exhibited transcriptional changes in pathways involved in neuronal development, microenvironment, and programmed cell death. C_FIG

neuroscience↗

bioRxiv2023

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

Large genes including several CRISPR-Cas modules, such as gene activators (CRISPRa), require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, intravitreal supplementation of ABCA4 via REVeRT improves retinal degeneration and function in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

bioengineering↗

bioRxiv2019

CRB2 controls primary ciliogenesis in association with PAR6α, HOOK2 and RAB8A

Ciliogenesis is an essential process in animal development and physiology and when affected, the origin of severe pathologies called ciliopathies. CRB2 has been recently implicated when mutated in several pathologies such as retina degeneration and a ciliopathy-like syndrome. Here, we have investigated the role of CRB2 in primary ciliogenesis and showed that CRB2 depletion blocks the formation of the ciliary vesicle after activation of the mother centriole, indicating a defect in either transport or anchoring of the ciliary vesicle. CRB2 is accumulated in intracellular vesicles in the vicinity of the centrosome and we showed here that CRB2 forms a complex made of HOOK2 and PAR6, two proteins important for primary ciliogenesis or centrosome integrity. In addition, CRB2 interacts with RAB8A which over-expression rescued CRB2 knockdown. All together, these findings indicate that CRB2 regulates a membrane transport step essential for the formation of the ciliary vesicle.

cell biology↗

bioRxiv2019

Crumbs 2 mediates ventricular layer remodelling to form the adult spinal cord central canal

In the spinal cord, the adult central canal forms through a poorly-understood process termed dorsal collapse that involves attrition and remodelling of the pseudostratified dorsal ventricular layer. Here we show, in mouse, that dorsal ventricular layer cells adjacent to midline Nestin(+) radial glia downregulate the apical polarity proteins Crumbs2 (CRB2) and aPKC and delaminate in a step-wise manner; concomitantly, Nestin(+) radial glial end-feet ratchet down, to repeat this process. Nestin(+) radial glia secrete a factor that promotes cell delamination. This activity is mimicked by a secreted variant of CRB2 (CRB2S), which is specifically expressed by dorsal midline Nestin(+) radial glia. In cultured cells, CRB2S associated with apical membranes and decreased cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice further confirmed an essential role for CRB2 in dorsal collapse. We propose a model in which CRB2S promotes the progressive attrition of the ventricular layer without loss of overall integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.

developmental biology↗