Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina.

Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1-CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1-CRB2 interactions. We observed no or a mild loss of CRB1-CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.

Rapid Variant Pathogenicity Analysis by CRISPR Activation of CRB1 Gene Expression in Patient-Derived Fibroblasts.

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of CRB1 expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the CRB1 c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected CRB1 isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.

Fine-tuning FAM161A gene augmentation therapy to restore retinal function.

For 15 years, gene therapy has been viewed as a beacon of hope for inherited retinal diseases. Many preclinical investigations have centered around vectors with maximal gene expression capabilities, yet despite efficient gene transfer, minimal physiological improvements have been observed in various ciliopathies. Retinitis pigmentosa-type 28 (RP28) is the consequence of bi-allelic null mutations in the FAM161A, an essential protein for the structure of the photoreceptor connecting cilium (CC). In its absence, cilia become disorganized, leading to outer segment collapses and vision impairment. Within the human retina, FAM161A has two isoforms: the long one with exon 4, and the short one without it. To restore CC in Fam161a-deficient mice shortly after the onset of cilium disorganization, we compared AAV vectors with varying promoter activities, doses, and human isoforms. While all vectors improved cell survival, only the combination of both isoforms using the weak FCBR1-F0.4 promoter enabled precise FAM161A expression in the CC and enhanced retinal function. Our investigation into FAM161A gene replacement for RP28 emphasizes the importance of precise therapeutic gene regulation, appropriate vector dosing, and delivery of both isoforms. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

A Precision Therapy Approach for Retinitis Pigmentosa 11 Using Splice-Switching Antisense Oligonucleotides to Restore the Open Reading Frame of PRPF31.

Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31. The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.

Investigating the Impact of Dimer Interface Mutations on Norrin's Secretion and Norrin/ß-Catenin Pathway Activation.

Purpose: This study aimed to investigate the impact of 21 NDP mutations located at the dimer interface, focusing on their potential effects on protein assembly, secretion efficiency, and activation of the Norrin/ß-catenin signaling pathway. Methods: The expression level, secretion efficiency, and protein assembly of mutations were analyzed using Western blot. The Norrin/ß-catenin signaling pathway activation ability after overexpression of mutants or supernatant incubation of mutant proteins was tested in HEK293STF cells. The mutant norrin and wild-type (WT) FZD4 were overexpressed in HeLa cells to observe their co-localization. Immunofluorescence staining was conducted in HeLa cells to analyze the subcellular localization of Norrin and the Retention Using Selective Hook (RUSH) assay was used to dynamically observe the secretion process of WT and mutant Norrin. Results: Four mutants (A63S, E66K, H68P, and L103Q) exhibited no significant differences from WT in all evaluations. The other 17 mutants presented abnormalities, including inadequate protein assembly, reduced secretion, inability to bind to FZD4 on the cell membrane, and decreased capacity to activate Norrin/ß-catenin signaling pathway. The RUSH assay revealed the delay in endoplasmic reticulum (ER) exit and impairment of Golgi transport. Conclusions: Mutations at the Norrin dimer interface may lead to abnormal protein assembly, inability to bind to FZD4, and decreased secretion, thus contributing to compromised Norrin/ß-catenin signaling. Our results shed light on the pathogenic mechanisms behind a significant proportion of NDP gene mutations in familial exudative vitreoretinopathy (FEVR) or Norrie disease.

Retinal Ciliopathies and Potential Gene Therapies: A Focus on Human iPSC-Derived Organoid Models.

The human photoreceptor function is dependent on a highly specialised cilium. Perturbation of cilial function can often lead to death of the photoreceptor and loss of vision. Retinal ciliopathies are a genetically diverse range of inherited retinal disorders affecting aspects of the photoreceptor cilium. Despite advances in the understanding of retinal ciliopathies utilising animal disease models, they can often lack the ability to accurately mimic the observed patient phenotype, possibly due to structural and functional deviations from the human retina. Human-induced pluripotent stem cells (hiPSCs) can be utilised to generate an alternative disease model, the 3D retinal organoid, which contains all major retinal cell types including photoreceptors complete with cilial structures. These retinal organoids facilitate the study of disease mechanisms and potential therapies in a human-derived system. Three-dimensional retinal organoids are still a developing technology, and despite impressive progress, several limitations remain. This review will discuss the state of hiPSC-derived retinal organoid technology for accurately modelling prominent retinal ciliopathies related to genes, including RPGR, CEP290, MYO7A, and USH2A. Additionally, we will discuss the development of novel gene therapy approaches targeting retinal ciliopathies, including the delivery of large genes and gene-editing techniques.

Primary Ciliary Dyskinesia and Retinitis Pigmentosa: Novel RPGR Variant and Possible Modifier Gene.

We report a novel RPGR missense variant co-segregated with a familial X-linked retinitis pigmentosa (XLRP) case. The brothers were hemizygous for this variant, but only the proband presented with primary ciliary dyskinesia (PCD). Thus, we aimed to elucidate the role of the RPGR variant and other modifier genes in the phenotypic variability observed in the family and its impact on motile cilia. The pathogenicity of the variant on the RPGR protein was evaluated by in vitro studies transiently transfecting the mutated RPGR gene, and immunofluorescence analysis on nasal brushing samples. Whole-exome sequencing was conducted to identify potential modifier variants. In vitro studies showed that the mutated RPGR protein could not localise to the cilium and impaired cilium formation. Accordingly, RPGR was abnormally distributed in the siblings' nasal brushing samples. In addition, a missense variant in CEP290 was identified. The concurrent RPGR variant influenced ciliary mislocalisation of the protein. We provide a comprehensive characterisation of motile cilia in this XLRP family, with only the proband presenting PCD symptoms. The variant's pathogenicity was confirmed, although it alone does not explain the respiratory symptoms. Finally, the CEP290 gene may be a potential modifier for respiratory symptoms in patients with RPGR mutations.

Protective roles of peroxiporins AQP0 and AQP11 in human astrocyte and neuronal cell lines in response to oxidative and inflammatory stressors.

In addition to aquaporin (AQP) classes AQP1, AQP4 and AQP9 known to be expressed in mammalian brain, our recent transcriptomic analyses identified AQP0 and AQP11 in human cortex and hippocampus at levels correlated with age and Alzheimer's disease (AD) status; however, protein localization remained unknown. Roles of AQP0 and AQP11 in transporting hydrogen peroxide (H2O2) in lens and kidney prompted our hypothesis that up-regulation in brain might similarly be protective. Established cell lines for astroglia (1321N1) and neurons (SHSY5Y, differentiated with retinoic acid) were used to monitor changes in transcript levels for human AQPs (AQP0 to AQP12) in response to inflammation (simulated with 10-100 ng/ml lipopolysaccharide [LPS], 24 h), and hypoxia (5 min N2, followed by 0 to 24 h normoxia). AQP transcripts up-regulated in both 1321N1 and SHSY5Y included AQP0, AQP1 and AQP11. Immunocytochemistry in 1321N1 cells confirmed protein expression for AQP0 and AQP11 in plasma membrane and endoplasmic reticulum; AQP11 increased 10-fold after LPS and AQP0 increased 0.3-fold. In SHSY5Y cells, AQP0 expression increased 0.2-fold after 24 h LPS; AQP11 showed no appreciable change. Proposed peroxiporin roles were tested using melondialdehyde (MDA) assays to quantify lipid peroxidation levels after brief H2O2. Boosting peroxiporin expression by LPS pretreatment lowered subsequent H2O2-induced MDA responses (∼50%) compared with controls; conversely small interfering RNA knockdown of AQP0 in 1321N1 increased lipid peroxidation (∼17%) after H2O2, with a similar trend for AQP11 siRNA. Interventions that increase native brain peroxiporin activity are promising as new approaches to mitigate damage caused by aging and neurodegeneration.

Intraocular pressure across the lifespan of Tg-MYOCY437H mice.

Transgenic C57BL/6 mice expressing human myocilinY437 (Tg-MYOCY437H) are a well-established model for primary open-angle glaucoma (POAG). While the reduced trabecular meshwork (TM) cellularity due to severe endoplasmic reticulum (ER) stress has been characterized as the etiology of this model, there is a limited understanding of how glaucomatous phenotypes evolve over the lifespan of Tg-MyocY437H mice. In this study, we compiled the model's intraocular pressure (IOP) data recorded in our laboratory from 2017 to 2023 and selected representative eyes to measure the outflow facility (Cr), a critical parameter indicating the condition of the conventional TM pathway. We found that Tg-MYOCY437H mice aged 4-12 months exhibited significantly higher IOPs than age-matched C57BL/6 mice. Notably, a decline in IOP was observed in Tg-MYOCY437H mice at 17-24 months of age, a phenomenon not attributable to the gene dosage of mutant myocilin. Measurements of the Cr of Tg-MYOCY437H mice indicated that the age-related IOP reduction was not a result of ongoing TM damage. Instead, Hematoxylin and Eosin staining, immunohistochemistry analysis, and transmission electron microscopic examination revealed that this reduction might be induced by degenerations of the non-pigmented epithelium in the ciliary body of aged Tg-MYOCY437H mice. Overall, our findings provide a comprehensive profile of mutant myocilin-induced ocular changes over the Tg-MYOCY437H mouse lifespan and suggest a specific temporal window of elevated IOP that may be ideal for experimental purposes.

AAV-RPGR Gene Therapy Rescues Opsin Mislocalisation in a Human Retinal Organoid Model of RPGR-Associated X-Linked Retinitis Pigmentosa.

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial.

Deciphering a crucial dimeric interface governing Norrin dimerization and the pathogenesis of familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disease that could cause blindness. It has been established that Norrin forms dimers to activate ß-catenin signaling, yet the core interface for Norrin dimerization and the precise mechanism by which Norrin dimerization contributes to the pathogenesis of FEVR remain elusive. Here, we report an NDP variant, c.265T>C (p.Phe89Leu), that interrupted ß-catenin signaling by disrupting Norrin dimerization. Structural and functional analysis revealed that the Phe-89 of one Norrin monomer interacts with Pro-98, Ser-101, Arg-121, and Ile-123 of another, forming two core symmetrical dimerization interfaces that are pivotal for the formation of a "hand-by-arm" dimer. Intriguingly, we proved that one of the two core symmetrical interfaces is sufficient for dimerization and activation of ß-catenin signaling, with a substantial contribution from the Phe-89/Pro-98 interaction. Further functional analysis revealed that the disruption of both dimeric interfaces eliminates potential binding sites for LRP5, which could be partially restored by over-expression of TSPAN12. In conclusion, our findings unveil a core dimerization interface that regulates Norrin/LRP5 interaction, highlighting the essential role of Norrin dimerization on ß-catenin signaling and providing potential therapeutic avenues for the treatment of FEVR.

Sources of Variance in Human Tear Proteomic Samples: Statistical Evaluation, Quality Control, Normalization, and Biological Insight.

Human tear fluid contains numerous compounds, which are present in highly variable amounts owing to the dynamic and multipurpose functions of tears. A better understanding of the level and sources of variance is essential for determining the functions of the different tear components and the limitations of tear samples as a potential biomarker source. In this study, a quantitative proteomic method was used to analyze variations in the tear protein profiles of healthy volunteers. High day-to-day and inter-eye personal variances were observed in the tear volumes, protein content, and composition of the tear samples. Several normalization and outlier exclusion approaches were evaluated to decrease variances. Despite the intrapersonal variances, statistically significant differences and cluster analysis revealed that proteome profile and immunoglobulin composition of tear fluid present personal characteristics. Using correlation analysis, we could identify several correlating protein clusters, mainly related to the source of the proteins. Our study is the first attempt to achieve more insight into the biochemical background of human tears by statistical evaluation of the experimentally observed dynamic behavior of the tear proteome. As a pilot study for determination of personal protein profiles of the tear fluids of individual patients, it contributes to the application of this noninvasively collectible body fluid in personal medicine.

Enhancement of Haloperidol-Induced Catalepsy by GPR143, an L-Dopa Receptor, in Striatal Cholinergic Interneurons.

Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene-deficient (Gpr143-/y ) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143-/y mice. The phenotypic defect in Gpr143-/y mice was mimicked in cholinergic interneuron-specific Gpr143-/y (Chat-cre;Gpr143flox/y ) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre;Gpr143flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs.

LncRNA SOX9-AS1 triggers a transcriptional program involved in lipid metabolic reprogramming, cell migration and invasion in triple-negative breast cancer.

At the molecular level, triple-negative breast cancer (TNBC) is frequently categorized as PAM50 basal-like subtype, but despite the advances in molecular analyses, the clinical outcome for these subtypes is uncertain. Long non-coding RNAs (lncRNAs) are master regulators of genes involved in hallmarks of cancer, which makes them suitable biomarkers for breast cancer (BRCA) diagnosis and prognosis. Here, we evaluated the regulatory role of lncRNA SOX9-AS1 in these subtypes. Using the BRCA-TCGA cohort, we observed that SOX9-AS1 was significantly overexpressed in basal-like and TNBC in comparison with other BRCA subtypes. Survival analyzes showed that SOX9-AS1 overexpression was associated with a favorable prognosis in TNBC and basal-like patients. To study the functions of SOX9-AS1, we determined the expression levels in a panel of nine BRCA cell lines finding increased levels in MDA-MB-468 and HCC1187 TNBC. Using subcellular fractionation in these cell lines, we ascertained that SOX9-AS1 was located in the cytoplasmic compartment. In addition, we performed SOX9-AS1 gene silencing using two short-harping constructs, which were transfected in both cell models and performed a genome-wide RNA-seq analysis. Data showed that 351 lncRNAs and 740 mRNAs were differentially expressed in MDA-MB-468 while 56 lncRNAs and 100 mRNAs were modulated in HCC1187 cells (Log2FC < - 1.5 and > 1.5, p.adj value < 0.05). Pathway analysis revealed that the protein-encoding genes potentially regulate lipid metabolic reprogramming, and epithelial-mesenchymal transition (EMT). Expression of lipid metabolic-related genes LIPE, REEP6, GABRE, FBP1, SCD1, UGT2B11, APOC1 was confirmed by RT-qPCR. Functional analysis demonstrated that the knockdown of SOX9-AS1 increases the triglyceride synthesis, cell migration and invasion in both two TNBC cell lines. In conclusion, high SOX9-AS1 expression predicts an improved clinical course in patients, while the loss of SOX9-AS1 expression enhances the aggressiveness of TNBC cells.

Short peptides derived from pigment epithelium-derived factor attenuate retinal ischemia reperfusion injury through inhibition of apoptosis and inflammatory response in rats.

Pigment epithelium-derived factor (PEDF) is widely recognized as a neuroprotective factor expressed in the retina and has shown therapeutic potential in several retinal diseases. Our study aimed to identify the neuroprotective fragment in PEDF and investigate its protective activity in retinas under ischemia-reperfusion (IR) condition. We synthesized a series of shorter synthetic peptides, 6-mer (Ser93-Gln98) and its d-form variant (6 dS) derived from the 44-mer (Val78-Thr121; a PEDF neurotrophic fragment), to determine their cytoprotective activity in IR injury, which was induced in rat retinas by injection of saline into the anterior chamber to increase the intraocular pressure (IOP) followed by reperfusion. We found the cytoprotective effect of 6-mer on glutamate-treated Neuro-2a cells and tert-butyl hydroperoxide (tBHP)-treated 661W cells were 2.6-fold and 1.5-fold higher than the 44-mer, respectively. The cytoprotective effect was blocked by a chemical inhibitor atglistatin and blocking antibody targeting PEDF receptor (PEDF-R). IR induced several impairments in retina, including cell apoptosis, activation of microglia/macroglia, degeneration of retinal capillaries, reduction in electroretinography (ERG) amplitudes, and retinal atrophy. Such IR injuries were ameliorated by treatment with 6-mer and 6 dS eye drops. Also, the neuroprotective activity of 6-mer and 6 dS in ischemic retinas were dramatically reversed by atglistatin preconditioning. Taken together, our data demonstrate smallest neuroprotective fragment of PEDF has potential to treat retinal degeneration-related diseases.

The role of phosphorylation in calmodulin-mediated gating of human AQP0.

Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis.

Pigment epithelium-derived factor (PEDF) represses the glucose transporter 1 (GLUT1) mRNA expression and may be a potential therapeutic agent in psoriasis: a case-control and experimental study.

We investigated the whole blood GLUT1 mRNA expression and serum pigment epithelium-derived factor (PEDF), interleukin-6 (IL-6), fetuin-A, and pentraxin-3 (PTX3) levels in psoriatic patients and tested their correlations with the severity of psoriasis using the psoriasis area and severity index (PASI) score. Also, we tested the GLUT1 mRNA expression after an in vitro treatment of human skin fibroblast (HSF) cell lines with PEDF. The case-control part of the study recruited 74 participants (44 psoriatic patients and 30 healthy volunteers). Whole blood GLUT1 mRNA fold changes were estimated by RT-PCR, and serum PEDF, IL-6, fetuin-A, and PTX3 levels were measured by ELISA kits. In the experimental part, the HSF cell lines were treated with different concentrations of PEDF for different times to test its effect on the GLUT1 mRNA expression. The whole blood GLUT 1 expression significantly increased in psoriatic patients and correlated positively with serum IL-6, fetuin-A, PTX3 levels and with the severity of psoriasis while negatively with serum PEDF levels. The PEDF-treated HSF cell lines showed a time- and dose-dependent decline in the GLUT 1 mRNA expression. The whole blood GLUT 1 mRNA is a non-invasive biomarker that is associated with the severity of psoriasis. PEDF represses GLUT 1 expression and may be a potential therapeutic agent in psoriasis.Trial registration: ClinicalTrials.gov Identifier: NCT04242082.

Pathogenic Variants in USH1G/SANS Alter Protein Interaction with Pre-RNA Processing Factors PRPF6 and PRPF31 of the Spliceosome.

Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf-blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology.

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina.

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.