Role of trafficking protein particle complex 2 in medaka development.

The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders.

Sarcomere level mechanics of the fast skeletal muscle of the medaka fish larva.

The medaka fish (Oryzias latipes) is a vertebrate model used in developmental biology and genetics. Here we explore its suitability as a model for investigating the molecular mechanisms of human myopathies caused by mutations in sarcomeric proteins. To this end, the relevant mechanical parameters of the intact skeletal muscle of wild-type medaka are determined using the transparent tail at larval stage 40. Tails were mounted at sarcomere length of 2.1 µm in a thermoregulated trough containing physiological solution. Tetanic contractions were elicited at physiological temperature (10°C-30°C) by electrical stimulation, and sarcomere length changes were recorded with nanometer-microsecond resolution during both isometric and isotonic contractions with a striation follower. The force output has been normalized for the actual fraction of the cross section of the tail occupied by the myofilament lattice, as established with transmission electron microscopy (TEM), and then for the actual density of myofilaments, as established with X-ray diffraction. Under these conditions, the mechanical performance of the contracting muscle of the wild-type larva can be defined at the level of the half-thick filament, where ∼300 myosin motors work in parallel as a collective motor, allowing a detailed comparison with the established performance of the skeletal muscle of different vertebrates. The results of this study point out that the medaka fish larva is a suitable model for the investigation of the genotype/phenotype correlations and therapeutic possibilities in skeletal muscle diseases caused by mutations in sarcomeric proteins.NEW & NOTEWORTHY The suitability of the medaka fish as a model for investigating the molecular mechanisms of human myopathies caused by mutations of sarcomeric proteins is tested by combining structural analysis and sarcomere-level mechanics of the skeletal muscle of the tail of medaka larva. The mechanical performance of the medaka muscle, scaled at the level of the myosin-containing thick filament, together with its reduced genome duplication makes this model unique for investigations of the genotype/phenotype correlations in human myopathies.

Deletion of IFT20 exclusively in the RPE ablates primary cilia and leads to retinal degeneration.

Vision impairment places a serious burden on the aging society, affecting the lives of millions of people. Many retinal diseases are of genetic origin, of which over 50% are due to mutations in cilia-associated genes. Most research on retinal degeneration has focused on the ciliated photoreceptor cells of the retina. However, the contribution of primary cilia in other ocular cell types has largely been ignored. The retinal pigment epithelium (RPE) is a monolayer epithelium at the back of the eye intricately associated with photoreceptors and essential for visual function. It is already known that primary cilia in the RPE are critical for its development and maturation; however, it remains unclear whether this affects RPE function and retinal tissue homeostasis. We generated a conditional knockout mouse model, in which IFT20 is exclusively deleted in the RPE, ablating primary cilia. This leads to defective RPE function, followed by photoreceptor degeneration and, ultimately, vision impairment. Transcriptomic analysis offers insights into mechanisms underlying pathogenic changes, which include transcripts related to epithelial homeostasis, the visual cycle, and phagocytosis. Due to the loss of cilia exclusively in the RPE, this mouse model enables us to tease out the functional role of RPE cilia and their contribution to retinal degeneration, providing a powerful tool for basic and translational research in syndromic and non-syndromic retinal degeneration. Non-ciliary mechanisms of IFT20 in the RPE may also contribute to pathogenesis and cannot be excluded, especially considering the increasing evidence of non-ciliary functions of ciliary proteins.

Ubiquitylation of BBSome is required for ciliary assembly and signaling.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, renal abnormalities, postaxial polydactyly, and developmental defects. Genes mutated in BBS encode for components and regulators of the BBSome, an octameric complex that controls the trafficking of cargos and receptors within the primary cilium. Although both structure and function of the BBSome have been extensively studied, the impact of ubiquitin signaling on BBSome is largely unknown. We identify the E3 ubiquitin ligase PJA2 as a novel resident of the ciliary compartment and regulator of the BBSome. Upon GPCR-cAMP stimulation, PJA2 ubiquitylates BBSome subunits. We demonstrate that ubiquitylation of BBS1 at lysine 143 increases the stability of the BBSome and promotes its binding to BBS3, an Arf-like GTPase protein controlling the targeting of the BBSome to the ciliary membrane. Downregulation of PJA2 or expression of a ubiquitylation-defective BBS1 mutant (BBS1K143R ) affects the trafficking of G-protein-coupled receptors (GPCRs) and Shh-dependent gene transcription. Expression of BBS1K143R in vivo impairs cilium formation, embryonic development, and photoreceptors' morphogenesis, thus recapitulating the BBS phenotype in the medaka fish model.

Induction of Autophagy Promotes Clearance of RHOP23H Aggregates and Protects From Retinal Degeneration.

Autophagy is a critical metabolic process that acts as a major self-digestion and recycling pathway contributing to maintain cellular homeostasis. An emerging field of research supports the therapeutic modulation of autophagy for treating human neurodegenerative disorders, in which toxic aggregates are accumulated in neurons. Our previous study identified Ezrin protein as an inhibitor of autophagy and lysosomal functions in the retina; thus, in turn, identifying it as a potential pharmacological target for increasing retinal cell clearance to treat inherited retinal dystrophies in which misfolded proteins have accumulated. This study aimed to verify the therapeutic inhibition of Ezrin to induce clearance of toxic aggregates in a mouse model for a dominant form of retinitis pigmentosa (i.e., RHOP23H/+). We found that daily inhibition of Ezrin significantly decreased the accumulation of misfolded RHOP23H aggregates. Remarkably, induction of autophagy, by a drug-mediated pulsatile inhibition of Ezrin, promoted the lysosomal clearance of disease-linked RHOP23H aggregates. This was accompanied with a reduction of endoplasmic reticulum (ER)-stress, robust decrease of photoreceptors' cell death, amelioration in both retinal morphology and function culminating in a better preservation of vision. Our study opens new perspectives for a pulsatile pharmacological induction of autophagy as a mutation-independent therapy paving the way toward a more effective therapeutic strategy to treat these devastating retinal disorders due to an accumulation of intracellular toxic aggregates.

Expression Pattern of nos1 in the Developing Nervous System of Ray-Finned Fish.

Fish have colonized nearly all aquatic niches, making them an invaluable resource to understand vertebrate adaptation and gene family evolution, including the evolution of complex neural networks and modulatory neurotransmitter pathways. Among ancient regulatory molecules, the gaseous messenger nitric oxide (NO) is involved in a wide range of biological processes. Because of its short half-life, the modulatory capability of NO is strictly related to the local activity of nitric oxide synthases (Nos), enzymes that synthesize NO from L-arginine, making the localization of Nos mRNAs a reliable indirect proxy for the location of NO action domains, targets, and effectors. Within the diversified actinopterygian nos paralogs, nos1 (alias nnos) is ubiquitously present as a single copy gene across the gnathostome lineage, making it an ideal candidate for comparative studies. To investigate variations in the NO system across ray-finned fish phylogeny, we compared nos1 expression patterns during the development of two well-established experimental teleosts (zebrafish and medaka) with an early branching holostean (spotted gar), an important evolutionary bridge between teleosts and tetrapods. Data reported here highlight both conserved expression domains and species-specific nos1 territories, confirming the ancestry of this signaling system and expanding the number of biological processes implicated in NO activities.

Autophagy in the retinal pigment epithelium: a new vision and future challenges.

The retinal pigment epithelium (RPE) is a highly specialized monolayer of polarized, pigmented epithelial cells that resides between the vessels of the choriocapillaris and the neural retina. The RPE is essential for the maintenance and survival of overlying light-sensitive photoreceptors, as it participates in the formation of the outer blood-retinal barrier, phagocytosis, degradation of photoreceptor outer segment (POS) tips, maintenance of the retinoid cycle, and protection against light and oxidative stress. Autophagy is an evolutionarily conserved 'self-eating' process, designed to maintain cellular homeostasis. The daily autophagy demands in the RPE require precise gene regulation for the digestion and recycling of intracellular and POS components in lysosomes in response to light and stress conditions. In this review, we discuss selective autophagy and focus on the recent advances in our understanding of the mechanism of cell clearance in the RPE for visual function. Understanding how this catabolic process is regulated by both transcriptional and post-transcriptional mechanisms in the RPE will promote the recognition of pathological pathways in genetic disease and shed light on potential therapeutic strategies to treat visual impairments in patients with retinal disorders associated with lysosomal dysfunction.

In Vitro and In Vivo Toxicity Evaluation of Natural Products with Potential Applications as Biopesticides.

The use of natural products in agriculture as pesticides has been strongly advocated. However, it is necessary to assess their toxicity to ensure their safe use. In the present study, mammalian cell lines and fish models of the zebrafish (Danio rerio) and medaka (Oryzias latipes) have been used to investigate the toxic effects of ten natural products which have potential applications as biopesticides. The fungal metabolites cavoxin, epi-epoformin, papyracillic acid, seiridin and sphaeropsidone, together with the plant compounds inuloxins A and C and ungeremine, showed no toxic effects in mammalian cells and zebrafish embryos. Conversely, cyclopaldic and α-costic acids, produced by Seiridium cupressi and Dittrichia viscosa, respectively, caused significant mortality in zebrafish and medaka embryos as a result of yolk coagulation. However, both compounds showed little effect in zebrafish or mammalian cell lines in culture, thus highlighting the importance of the fish embryotoxicity test in the assessment of environmental impact. Given the embryotoxicity of α-costic acid and cyclopaldic acid, their use as biopesticides is not recommended. Further ecotoxicological studies are needed to evaluate the potential applications of the other compounds.

Pomegranate Peel Extract as an Inhibitor of SARS-CoV-2 Spike Binding to Human ACE2 Receptor (in vitro): A Promising Source of Novel Antiviral Drugs.

Plant extracts are rich in bioactive compounds, such as polyphenols, sesquiterpenes, and triterpenes, which potentially have antiviral activities. As a consequence of the coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, thousands of scientists have been working tirelessly trying to understand the biology of this new virus and the disease pathophysiology, with the main goal of discovering effective preventive treatments and therapeutic agents. Plant-derived secondary metabolites may play key roles in preventing and counteracting the rapid spread of SARS-CoV-2 infections by inhibiting the activity of several viral proteins, in particular those involved in the virus entry into the host cells and its replication. Using in vitro approaches, we investigated the role of a pomegranate peel extract (PPE) in attenuating the interaction between the SARS-CoV-2 Spike glycoprotein and the human angiotensin-converting enzyme 2 receptor, and on the activity of the virus 3CL protease. Although further studies will be determinant to assess the efficacy of this extract in vivo, our results opened new promising opportunities to employ natural extracts for the development of effective and innovative therapies in the fight against SARS-CoV-2.

The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation.

The primary cilium is a microtubule-based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X-linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin-proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G-protein-coupled receptor (GPCR)-cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2-UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non-phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders.

Loss of Ciliary Gene Bbs8 Results in Physiological Defects in the Retinal Pigment Epithelium.

Primary cilia are sensory organelles vital for developmental and physiological processes. Their dysfunction causes a range of phenotypes including retinopathies. Although primary cilia have been described in the retinal pigment epithelium (RPE), little is known about their contribution to biological processes within this tissue. Ciliary proteins are increasingly being identified in non-ciliary locations and might carry out additional functions, disruption of which possibly contributes to pathology. The RPE is essential for maintaining photoreceptor cells and visual function. We demonstrate that upon loss of Bbs8, predominantly thought to be a ciliary gene, the RPE shows changes in gene and protein expression initially involved in signaling pathways and developmental processes, and at a later time point RPE homeostasis and function. Differentially regulated molecules affecting the cytoskeleton and cellular adhesion, led to defective cellular polarization and morphology associated with a possible epithelial-to-mesenchymal transition (EMT)-like phenotype. Our data highlights the benefit of combinatorial "omics" approaches with in vivo data for investigating the function of ciliopathy proteins. It also emphasizes the importance of ciliary proteins in the RPE and their contribution to visual disorders, which must be considered when designing treatment strategies for retinal degeneration.

Emerging Lysosomal Functions for Photoreceptor Cell Homeostasis and Survival.

Lysosomes are membrane-bound cell organelles that respond to nutrient changes and are implicated in cell homeostasis and clearance mechanisms, allowing effective adaptation to specific cellular needs. The relevance of the lysosome has been elucidated in a number of different contexts. Of these, the retina represents an interesting scenario to appreciate the various functions of this organelle in both physiological and pathological conditions. Growing evidence suggests a role for lysosome-related mechanisms in retinal degeneration. Abnormal lysosomal activation or inhibition has dramatic consequences on photoreceptor cell homeostasis and impacts extensive cellular function, which in turn affects vision. Based on these findings, a series of therapeutic methods targeting lysosomal processes could offer treatment for blindness conditions. Here, we review the recent findings on membrane trafficking, subcellular organization, mechanisms by which lysosome/autophagy pathway impairment affects photoreceptor cell homeostasis and the recent advances on developing efficient lysosomal-based therapies for retinal disorders.

Pathogenic variants in IMPG1 cause autosomal dominant and autosomal recessive retinitis pigmentosa.

BACKGROUND: Inherited retinal disorders are a clinically and genetically heterogeneous group of conditions and a major cause of visual impairment. Common disease subtypes include vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). Despite the identification of over 90 genes associated with RP, conventional genetic testing fails to detect a molecular diagnosis in about one third of patients with RP. METHODS: Exome sequencing was carried out for identifying the disease-causing gene in a family with autosomal dominant RP. Gene panel testing and exome sequencing were performed in 596 RP and VMD families to identified additional IMPG1 variants. In vivo analysis in the medaka fish system by knockdown assays was performed to screen IMPG1 possible pathogenic role. RESULTS: Exome sequencing of a family with RP revealed a splice variant in IMPG1. Subsequently, the same variant was identified in individuals from two families with either RP or VMD. A retrospective study of patients with RP or VMD revealed eight additional families with different missense or nonsense variants in IMPG1. In addition, the clinical diagnosis of the IMPG1 retinopathy-associated variant, originally described as benign concentric annular macular dystrophy, was also revised to RP with early macular involvement. Using morpholino-mediated ablation of Impg1 and its paralog Impg2 in medaka fish, we confirmed a phenotype consistent with that observed in the families, including a decreased length of rod and cone photoreceptor outer segments. CONCLUSION: This study discusses a previously unreported association between monoallelic or biallelic IMPG1 variants and RP. Notably, similar observations have been reported for IMPG2.

MLL4-associated condensates counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki syndrome.

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.

MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B.

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.

Retinal Degeneration in MPS-IIIA Mouse Model.

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo A) is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development.

Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance.

Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are controlled by light is largely unknown. Here, we show that the light-responsive miR-211 controls lysosomal biogenesis at the beginning of light-dark transitions in the RPE by targeting Ezrin, a cytoskeleton-associated protein essential for the regulation of calcium homeostasis. miR-211-mediated down-regulation of Ezrin leads to Ca2+ influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light-mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR-211-/- mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR-211-/- phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.

AAV-miR-204 Protects from Retinal Degeneration by Attenuation of Microglia Activation and Photoreceptor Cell Death.

Inherited retinal diseases (IRDs) represent a frequent cause of genetic blindness. Their high genetic heterogeneity hinders the application of gene-specific therapies to the vast majority of patients. We recently demonstrated that the microRNA miR-204 is essential for retinal function, although the underlying molecular mechanisms remain poorly understood. Here, we investigated the therapeutic potential of miR-204 in IRDs. We subretinally delivered an adeno-associated viral (AAV) vector carrying the miR-204 precursor to two genetically different IRD mouse models. The administration of AAV-miR-204 preserved retinal function in a mouse model for a dominant form of retinitis pigmentosa (RHO-P347S). This was associated with a reduction of apoptotic photoreceptor cells and with a better preservation of photoreceptor marker expression. Transcriptome analysis showed that miR-204 shifts expression profiles of transgenic retinas toward those of healthy retinas by the downregulation of microglia activation and photoreceptor cell death. Delivery of miR-204 exerted neuroprotective effects also in a mouse model of Leber congenital amaurosis, due to mutations of the Aipl1 gene. Our study highlights the mutation-independent therapeutic potential of AAV-miR204 in slowing down retinal degeneration in IRDs and unveils the previously unreported role of this miRNA in attenuating microglia activation and photoreceptor cell death.

The Impact of miRNAs in Health and Disease of Retinal Pigment Epithelium.

MicroRNAs (miRNAs), a class of non-coding RNAs, are essential key players in the control of biological processes in both physiological and pathological conditions. miRNAs play important roles in fine tuning the expression of many genes, which often have roles in common molecular networks. miRNA dysregulation thus renders cells vulnerable to aberrant fluctuations in genes, resulting in degenerative diseases. The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells that resides between the light-sensitive photoreceptors (PR) and the choriocapillaris. The demanding physiological functions of RPE cells require precise gene regulation for the maintenance of retinal homeostasis under stress conditions and the preservation of vision. Thus far, our understanding of how miRNAs function in the homeostasis and maintenance of the RPE has been poorly addressed, and advancing our knowledge is central to harnessing their potential as therapeutic agents to counteract visual impairment. This review focuses on the emerging roles of miRNAs in the function and health of the RPE and on the future exploration of miRNA-based therapeutic approaches to counteract blinding diseases.