piRNAs in the human retina and retinal pigment epithelium reveal a potential role in intracellular trafficking and oxidative stress.

Long considered active only in the germline, the PIWI/piRNA pathway is now known to play a significant role in somatic cells, especially neurons. In this study, piRNAs were profiled in the human retina and retinal pigment epithelium (RPE). Furthermore, RNA immunoprecipitation with HIWI2 (PIWIL4) in ARPE19 cells yielded 261 piRNAs, and the expression of selective piRNAs in donor eyes was assessed by qRT-PCR. Intriguingly, computational analysis revealed complete and partial seed sequence similarity between piR-hsa-26131 and the sensory organ specific miR-183/96/182 cluster. Furthermore, the expression of retina-enriched piR-hsa-26131 was positively correlated with miR-182 in HIWI2-silenced Y79 cells. In addition, the lnc-ZNF169 sequence matched with two miRNAs of the let-7 family, and piRNAs, piR-hsa-11361 and piR-hsa-11360, which could modulate the regulatory network of retinal differentiation. Interestingly, we annotated four enriched motifs among the piRNAs and found that the piRNAs containing CACAATG and CTCATCAKYG motifs were snoRNA-derived piRNAs, which are significantly associated with developmental functions. However, piRNAs consisting of ACCACTANACCAC and AKCACGYTCSC motifs were mainly tRNA-derived fragments linked to stress response and sensory perception. Additionally, co-expression network analysis revealed cell cycle control, intracellular transport and stress response as the important biological functions regulated by piRNAs in the retina. Moreover, loss of piRNAs in HIWI2 knockdown ARPE19 confirmed altered expression of targets implicated in intracellular transport, circadian clock, and retinal degeneration. Moreover, piRNAs were dysregulated under oxidative stress conditions, indicating their potential role in retinal pathology. Therefore, we postulate that piRNAs, miRNAs, and lncRNAs might have a functional interplay during retinal development and functions to regulate retinal homeostasis.

Protein Drug Delivery Using a Novel Maxillofacial Technique Targeting the Visual Pathway in the Brain, the Optic Nerve, and the Retina.

Protein drugs are used for treating many diseases of the eye and the brain. The formidable blood neural barriers prevent the delivery of these drugs into the eye and the brain. Hence, there is a need for a protein drug delivery system to deliver large proteins across blood-neural barriers. Low half-life, poor penetration of epithelial barriers, low stability, and immunogenicity limit the use of non-invasive systemic routes for delivering proteins. In this pre-clinical study, the efficacy of a new maxillofacial route for administering protein drugs using a novel drug delivery system is compared with systemic administration through intra-peritoneal injection and ocular administration through topical eye drops and subconjunctival and intravitreal injections. Bevacizumab and retinoschisin proteins were administered using the maxillofacial technique along with systemic and ocular routes in wild-type male C57BL/6J mice. Liquid chromatography with tandem mass spectrometry and western blot was used to detect bevacizumab in tissue samples. Furthermore, immunohistochemistry was performed to detect the presence and localization of bevacizumab and retinoschisin in the retina and brain. The maxillofacial route of delivery could target the brain including regions involved in the visual pathway and optic nerve. The maxillofacial technique and intravitreal injection were effective in delivering the drugs into the retina. A new concept based on the glymphatic pathway, cerebrospinal fluid drug distribution, and the crossover of ipsilateral optic nerve fibers at optic chiasma is proposed to explain the presence of the drug in contralateral eye following maxillofacial administration and intravitreal injection.

Retinoschisis and Norrie disease: a missing link.

OBJECTIVE: Retinoschisis and Norrie disease are X-linked recessive retinal disorders caused by mutations in RS1 and NDP genes respectively. Both are likely to be monogenic and no locus heterogeneity has been reported. However, there are reports showing overlapping features of Norrie disease and retinoschisis in a NDP knock-out mouse model and also the involvement of both the genes in retinoschisis patients. Yet, the exact molecular relationships between the two disorders have still not been understood. The study investigated the association between retinoschisin (RS1) and norrin (NDP) using in vitro and in silico approaches. Specific protein-protein interaction between RS1 and NDP was analyzed in human retina by co-immunoprecipitation assay and MALDI-TOF mass spectrometry. STRING database was used to explore the functional relationship. RESULT: Co-immunoprecipitation demonstrated lack of a direct interaction between RS1 and NDP and was further substantiated by mass spectrometry. However, STRING revealed a potential indirect functional association between the two proteins. Progressively, our analyses indicate that FZD4 protein interactome via PLIN2 as well as the MAP kinase signaling pathway to be a likely link bridging the functional relationship between retinoschisis and Norrie disease.

SARS-CoV-2: The Road Less Traveled-From the Respiratory Mucosa to the Brain.

Neurological manifestations have been reported in COVID-19; however, the route used by SARS-CoV-2 to enter the brain is still under debate. Recent studies have focused on the olfactory route. SARS-CoV-2 viral proteins were also detected in the glossopharyngeal and vagal nerves originating from the lower brainstem and in isolated cells of the brainstem. Our proof of concept in vivo real-time imaging study of mice using an indocyanine green dye indicated that the neurovascular component of the connective tissue of the respiratory mucosa can also provide an alternate route to the brain.

Understanding variable disease severity in X-linked retinoschisis: Does RS1 secretory mechanism determine disease severity?

X-linked retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in RS1 gene leading to splitting of retinal layers (schisis) which impairs visual signal processing. Retinoschisin (RS1) is an adhesive protein which is secreted predominantly by the photoreceptors and bipolar cells as a double-octameric complex. In general, XLRS patients show wide clinical heterogeneity, presenting practical challenges in disease management. Though researchers have attempted various approaches to offer an explanation for clinical heterogeneity, the molecular basis has not been understood yet. Therefore, this study aims at establishing a link between the phenotype and genotype based on the molecular mechanism exerted by the mutations. Twenty seven XLRS patients were enrolled, of which seven harboured novel mutations. The mutant constructs were genetically engineered and their secretion profiles were studied by in vitro cell culture experiments. Based on the secretory profile, the patients were categorized as either secreted or non-secreted group. Various clinical parameters such as visual acuity, location of schisis, foveal thickness and ERG parameters were compared between the two groups and control. Although the two groups showed severe disease phenotype in comparison with control, there was no significant difference between the two XLRS groups. However, the secreted group exhibited relatively severe disease indications. On the other hand molecular analysis suggests that most of the RS1 mutations result in intracellular retention of retinoschisin. Hence, clinical parameters of patients with non-secreted profile were analyzed which in turn revealed wide variability even within the group. Altogether, our results indicate that disease severity is not merely dependent on secretory profile of the mutations. Thus, we hypothesize that intricate molecular detail such as the precise localization of mutant protein in the cell as well as its ability to assemble into a functionally active oligomer might largely influence disease severity among XLRS patients.

Clinical and genetic analysis of Indian patients with NDP-related retinopathies.

PURPOSE: NDP-related retinopathies are a group of X-linked disorders characterized by degenerative and proliferative changes of the neuroretina, occasionally accompanied with varying degrees of mental retardation and sensorineural hearing loss. NDP is the predominant gene associated with NDP-related retinopathies. The purpose of this study was to report the clinical and genetic findings in three unrelated patients diagnosed with NDP-related retinopathies. METHODS: The patients underwent complete ophthalmic examination followed by genetic analyses. NDP gene was screened by direct sequencing approach. Targeted resequencing of several other ocular genes was carried out in patient samples that either indicated NDP gene deletion or tested negative for NDP mutation. Gene quantitation analysis was performed using real-time PCR. RESULTS: The whole NDP gene was deleted in patient I, while a missense NDP mutation, c.205T>C, was identified in patient II, and both had classical Norrie disease ocular phenotype (with no other systemic defects). Patient III who was diagnosed with familial exudative vitreoretinopathy did not show any mutation in the known candidate genes as well as in other ocular genes tested. CONCLUSIONS: The patient with whole NDP gene deletion did not exhibit any apparent extraocular defects (like mental retardation or sensorineural hearing loss) during his first decade of life, and this is considered to be a notable finding. Our study also provides evidence emphasizing the need for genetic testing which could eliminate ambiguities in clinical diagnosis and detect carrier status, thereby aiding the patient and family members during genetic counseling.

PIWI-like protein, HIWI2 is aberrantly expressed in retinoblastoma cells and affects cell-cycle potentially through OTX2.

Retinoblastoma (RB), a childhood cancer, is caused by biallelic mutation of the RB1 gene, but its development is not clearly understood. Furthermore, the presence of a cancer stem cell subpopulation in RB might impact its treatment. PIWI protein, known for its role in stem cell self-renewal, is aberrantly expressed in cancers. We examined the role of the PIWI-like protein HIWI2 in RB and its effect on the stem cell markers in cells of the RB line, Y79. The expression of HIWI2 is significantly increased in Y79 compared with its level in HeLa and ARPE19 cells. The stem cell markers Oct-3/4, Nanog and Sox-2 were not altered upon HIWI2 knockdown in Y79 cells. Interestingly, OTX2 was significantly downregulated in the absence of HIWI2. Otx2 transcripts also decreased in HIWI2-silenced Y79 and ARPE19 cells. Moreover, silencing HIWI2 in Y79 accumulated the cells at G2-M phase and reduced the levels of proliferating cell nuclear antigen (PCNA) and the tumor suppressor, p16. Our results demonstrate that HIWI2 is aberrantly expressed in Y79 cells and silencing of HIWI2 downregulates OTX2, suggesting that HIWI2 might play a role in the progression of RB.

Genetic studies in a patient with X-linked retinoschisis coexisting with developmental delay and sensorineural hearing loss.

BACKGROUND: In this study, we present a juvenile retinoschisis patient with developmental delay, sensorineural hearing loss, and reduced axial tone. X-linked juvenile retinoschisis (XLRS) is a retinal dystrophy, most often not associated with systemic anomalies and also not showing any locus heterogeneity. Therefore it was of interest to understand the genetic basis of the condition in this patient. MATERIALS AND METHODS: RS1 gene screening for XLRS was performed by Sanger sequencing. Whole genome SNP 6.0 array analysis was carried out to investigate gross chromosomal aberrations that could result in systemic phenotype. In addition, targeted next generation sequencing (NGS) was employed to determine any possible involvement of X-linked syndromic and non-syndromic mental retardation genes. This NGS panel consisted of 550 genes implicated in several other rare inherited diseases. RESULTS: RS1 gene screening revealed a pathogenic hemizygous splice site mutation (c.78+1G>T), inherited from the mother. SNP 6.0 array analysis did not indicate any significant chromosomal aberrations that could be disease-associated. Targeted resequencing did not identify any mutations in the X-linked mental retardation genes. However, variations in three other genes (NSD1, LARGE, and POLG) were detected, which were all inherited from the patient's unaffected father. CONCLUSIONS: Taken together, RS1 mutation was found to segregate with retinoschisis phenotype while none of the other identified variations were co-segregating with the systemic defects. Hereby, we infer that the multisystemic defects harbored by the patient are a rare coexistence of XLRS, developmental delay, sensorineural hearing loss, and reduced axial tone reported for the first time in the literature.

Proteomic profiling of human intraschisis cavity fluid.

BACKGROUND: X-linked retinoschisis (XLRS) is a vitreoretinal degenerative disorder causing vision deterioration, due to structural defects in retina. The hallmark of this disease includes radial streaks arising from the fovea and splitting of inner retinal layers (schisis). Although these retinal changes are attributed to mutations in the retinoschisin gene, schisis is also observed in patients who do not carry mutations. In addition, the origin of intraschisis fluid, the triggering point of schisis formation and its progression are largely unknown still. So far, there is no report on the complete proteomic analysis of this fluid. Schisis fluid proteome could reflect biochemical changes in the disease condition, helping in better understanding and management of retinoschisis. Therefore it was of interest to investigate the intraschisis fluid proteome using high-resolution mass spectrometry. METHODS: Two male XLRS patients (aged 4 and 40 years) underwent clinical and genetic evaluation followed by surgical extraction of intraschisis fluids. The two fluid samples were resolved on a SDS-PAGE and the processed peptides were analyzed by Q-Exactive plus hybrid quadrupole-Orbitrap mass spectrometry. Functional annotation of the identified proteins was performed using Ingenuity pathway analysis software. RESULTS: Mass spectrometry analysis detected 770 nonredundant proteins in the intraschisis fluid. Retinol dehydrogenase 14 was found to be abundant in the schisis fluid. Gene ontology based analysis indicated that 19% of the intraschisis fluid proteins were localized to the extracellular matrix and 15% of the proteins were involved in signal transduction. Functional annotation identified three primary canonical pathways to be associated with the schisis fluid proteome viz., LXR/RXR activation, complement system and acute phase response signalling, which are involved in immune and inflammatory responses. Collectively, our results show that intraschisis fluid comprises specific inflammatory proteins which highly reflect the disease environment. CONCLUSION: Based on our study, it is suggested that inflammation might play a key role in the pathogenesis of XLRS. To our knowledge, this is the first report describing the complete proteome of intraschisis fluid, which could serve as a template for future research and facilitate the development of therapeutic modalities for XLRS.

Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway.

PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3ß in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/ß in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/ß signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.

Phenotypic characterization of X-linked retinoschisis: Clinical, electroretinography, and optical coherence tomography variables.

AIMS: To study the phenotypic characteristics of X-linked retinoschisis (XLRS) and report the clinical, electroretinogram (ERG), and optical coherence tomography (OCT) variables in Indian eyes. DESIGN: A retrospective study. MATERIALS AND METHODS: Medical records of 21 patients with retinoschisis who were genetically confirmed to have RS1 mutation were reviewed. The phenotype characterization included the age of onset, best-corrected visual acuity, refractive error, fundus findings, OCT, and ERG. STATISTICAL ANALYSIS USED: Data from both the eyes were used for analysis. A P < 0.05 was set as statistical significance. Data were not normally distributed (P < 0.05, Shapiro wilk); hence, nonparametric tests were used for statistical analysis. RESULTS: All were males whose mean age of presentation was 9 years. Visual acuity was moderately impaired (median 0.6 logMAR, interquartile range: 0.47, 1) in these eyes with a hyperopic refractive error of median +1.75 Ds (interquartile range: +0.50 Ds, +4.25 Ds). About 54.7% of the eyes had both foveal and peripheral schisis, isolated foveal schisis was seen in 28.5% of the eyes, and schisis with retinal detachment was seen in 16.6% of the eyes. The inner nuclear layer was found to be commonly involved in the schisis, followed by outer nuclear and plexiform layers as evident on OCT. On ERG, a- and b-wave amplitudes were significantly reduced in eyes with foveal and peripheral schisis when compared to the eyes with only foveal schisis (P < 0.05). CONCLUSIONS: XLRS has phenotypic heterogeneity as evident on OCT, ERG, and clinical findings.