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Human & Experimental Toxicology Jan 2021Methylphenidate (MPH) is used as the first-line treatment for attention-deficit hyperactivity disorder. However, there are concerns that this treatment may be associated...
Methylphenidate (MPH) is used as the first-line treatment for attention-deficit hyperactivity disorder. However, there are concerns that this treatment may be associated with increased risk of retinal damage. This study was to investigate cytotoxicity of MPH on photoreceptor cells and explore its underlying mechanisms. MPH-caused cell toxicity was established in 661 W cells. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromid and lactate dehydrogenase assays. Oxidative stress was measured by the markers: glutathione (GSH) reductase, catalase, and superoxide dismutase activities as well as GSH, reactive oxygen species, and malondialdehyde levels. Gene and protein expression was detected by real-time polymerase chain reaction (PCR) and western blot, respectively. Results showed that MPH decreased 661 W cell viability, increased caspase-3/9 activities, and induced oxidative stress. Furthermore, MPH treatment increased messenger RNA (mRNA) expression of and microtubule-associated protein 1A/1B-light chain 3B (LC3B) protein expression in 661 W cells, suggesting autophagy was induced. MPH treatment also upregulated p-JAK1/p-STAT1 protein expression. These data demonstrated that MPH could increase oxidative stress in photoreceptor cells to cause cell toxicity via autophagy, providing the scientific rationale for the photoreceptor cell damage caused by the MPH administration.
Topics: Animals; Autophagy; Glutathione; Malondialdehyde; Methylphenidate; Oxidative Stress; Photoreceptor Cells; Reactive Oxygen Species
PubMed: 32748667
DOI: 10.1177/0960327120940357 -
The FEBS Journal Sep 2023The endoplasmic reticulum membrane protein complex (EMC) plays a critical role in the synthesis of multipass membrane proteins. Genetic studies indicated that mutations...
The endoplasmic reticulum membrane protein complex (EMC) plays a critical role in the synthesis of multipass membrane proteins. Genetic studies indicated that mutations in EMC1 gene were associated with retinal degeneration diseases; however, the role of EMC1 in photoreceptor has not been confirmed. Here, we show that Emc1 ablation in the photoreceptor cells of mice recapitulated the retinitis pigmentosa phenotypes, including an attenuated scotopic electroretinogram response and the progressive degeneration of rod cells and cone cells. Histopathological examination of tissues from rod-specific Emc1 knockout mice revealed mislocalized rhodopsin and irregularly arranged cone cells at the age of 2 months. Further immunoblotting analysis revealed decreased levels of membrane proteins and endoplasmic reticulum chaperones in 1-month-old rod-specific Emc1 knockout mice retinae, and this led us to speculate that the loss of membrane proteins is the main cause of the degeneration of photoreceptors. EMC1 most likely regulated the membrane protein levels at an earlier step in the biosynthetic process before the proteins translocated into the endoplasmic reticulum. The present study demonstrates the essential roles of Emc1 in photoreceptor cells, and reveals the mechanism through which EMC1 mutations are linked to retinitis pigmentosa.
Topics: Animals; Mice; Disease Models, Animal; Membrane Proteins; Mice, Knockout; Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Endoplasmic Reticulum
PubMed: 37098815
DOI: 10.1111/febs.16807 -
Pflugers Archiv : European Journal of... Sep 2021Light-evoked voltage responses of rod and cone photoreceptor cells in the vertebrate retina must be converted to a train of synaptic vesicle release events for... (Review)
Review
Light-evoked voltage responses of rod and cone photoreceptor cells in the vertebrate retina must be converted to a train of synaptic vesicle release events for transmission to downstream neurons. This review discusses the processes, proteins, and structures that shape this critical early step in vision, focusing on studies from salamander retina with comparisons to other experimental animals. Many mechanisms are conserved across species. In cones, glutamate release is confined to ribbon release sites although rods are also capable of release at non-ribbon sites. The role of non-ribbon release in rods remains unclear. Release from synaptic ribbons in rods and cones involves at least three vesicle pools: a readily releasable pool (RRP) matching the number of membrane-associated vesicles along the ribbon base, a ribbon reserve pool matching the number of additional vesicles on the ribbon, and an enormous cytoplasmic reserve. Vesicle release increases in parallel with Ca channel activity. While the opening of only a few Ca channels beneath each ribbon can trigger fusion of a single vesicle, sustained release rates in darkness are governed by the rate at which the RRP can be replenished. The number of vacant release sites, their functional status, and the rate of vesicle delivery in turn govern replenishment. Along with an overview of the mechanisms of exocytosis and endocytosis, we consider specific properties of ribbon-associated proteins and pose a number of remaining questions about this first synapse in the visual system.
Topics: Animals; Calcium Channels; Humans; Photic Stimulation; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Synapses; Synaptic Transmission
PubMed: 33779813
DOI: 10.1007/s00424-021-02548-9 -
Nature Communications Sep 2020Short regulatory RNA molecules underpin gene expression and govern cellular state and physiology. To establish an alternative layer of control over these processes, we...
Short regulatory RNA molecules underpin gene expression and govern cellular state and physiology. To establish an alternative layer of control over these processes, we generated chimeric regulatory RNAs that interact reversibly and light-dependently with the light-oxygen-voltage photoreceptor PAL. By harnessing this interaction, the function of micro RNAs (miRs) and short hairpin (sh) RNAs in mammalian cells can be regulated in a spatiotemporally precise manner. The underlying strategy is generic and can be adapted to near-arbitrary target sequences. Owing to full genetic encodability, it establishes optoribogenetic control of cell state and physiology. The method stands to facilitate the non-invasive, reversible and spatiotemporally resolved study of regulatory RNAs and protein function in cellular and organismal environments.
Topics: Animals; Gene Expression; HEK293 Cells; Humans; MicroRNAs; Photoreceptor Cells; RNA; RNA, Small Interfering
PubMed: 32973178
DOI: 10.1038/s41467-020-18673-5 -
Biological Chemistry Apr 2020Photoreceptors are polarized neurons, with specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor... (Review)
Review
Photoreceptors are polarized neurons, with specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment (OS) where vision begins, an inner segment (IS) where protein synthesis occurs and a synaptic terminal for signal transmission to second-order neurons. The OS is a large, modified primary cilium attached to the IS by a slender connecting cilium (CC), the equivalent of the transition zone (TZ). Daily renewal of ~10% of the OS requires massive protein biosynthesis in the IS with reliable transport and targeting pathways. Transport of lipidated ('sticky') proteins depends on solubilization factors, phosphodiesterase δ (PDEδ) and uncoordinated protein-119 (UNC119), and the cargo dispensation factor (CDF), Arf-like protein 3-guanosine triphosphate (ARL3-GTP). As PDE6 and transducin still reside prominently in the OS of PDEδ and UNC119 germline knockout mice, respectively, we propose the existence of an alternate trafficking pathway, whereby lipidated proteins migrate in rhodopsin-containing vesicles of the secretory pathway.
Topics: Animals; Diffusion; Humans; Lipid Metabolism; Photoreceptor Cells; Protein Transport
PubMed: 31811799
DOI: 10.1515/hsz-2019-0375 -
The Journal of Biological Chemistry Jun 2022Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function....
Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.
Topics: 1-Acylglycerophosphocholine O-Acyltransferase; Animals; Fatty Acids; Mice; Phosphatidylcholines; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Retina; Retinal Degeneration
PubMed: 35452679
DOI: 10.1016/j.jbc.2022.101958 -
Journal of the American Association For... Jan 2023Light is an extrinsic factor that exerts widespread influence on the regulation of circadian, physiologic, hormonal, metabolic, and behavioral systems of all animals,... (Review)
Review
Light is an extrinsic factor that exerts widespread influence on the regulation of circadian, physiologic, hormonal, metabolic, and behavioral systems of all animals, including those used in research. These wide-ranging biologic effects of light are mediated by distinct photoreceptors, the melanopsin-containing intrinsically photosensitive retinal ganglion cells of the nonvisual system, which interact with the rods and cones of the conventional visual system. Here, we review the nature of light and circadian rhythms, current industry practices and standards, and our present understanding of the neurophysiology of the visual and nonvisual systems. We also consider the implications of this extrinsic factor for vivarium measurement, production, and technological application of light, and provide simple recommendations on artificial lighting for use by regulatory authorities, lighting manufacturers, designers, engineers, researchers, and research animal care staff that ensure best practices for optimizing animal health and wellbeing and, ultimately, improving scientific outcomes.
Topics: Animals; Lighting; Retinal Ganglion Cells; Photoreceptor Cells; Circadian Rhythm
PubMed: 36755210
DOI: 10.30802/AALAS-JAALAS-23-000003 -
The Journal of Thoracic and... Dec 2020
Topics: Ebstein Anomaly; Humans; Retinal Cone Photoreceptor Cells
PubMed: 32682585
DOI: 10.1016/j.jtcvs.2020.06.049 -
International Journal of Molecular... Nov 2022Retinal organoids (ROs) are three-dimensional retinal tissues, which are differentiated in vitro from induced pluripotent stem cells (iPSC), ultimately forming all main...
Retinal organoids (ROs) are three-dimensional retinal tissues, which are differentiated in vitro from induced pluripotent stem cells (iPSC), ultimately forming all main retinal cell types under defined culture conditions. ROs show several highly specialized retinal features, including the outgrowth of photoreceptor outer segments (OSs). In vivo, the photoreceptor OSs are enveloped and maintained by protrusions of retinal pigment epithelium (RPE) cells, the so-called apical microvilli, while ROs fail to recapitulate this critical interaction in culture development. Here, we define specific co-culture conditions aiming to compensate for the missing physical proximity of RPE and OSs in RO development. Accordingly, functional RPE cells and ROs were differentiated simultaneously from the same iPSC clone, the former resulting in byproduct RPE or bRPE cells. While some co-culture approaches indicated a temporary functional interaction between bRPE and RO photoreceptors, they did not improve the photoreceptor histoarchitecture. In contrast, embedding ROs in a basement membrane extract without bRPE cells showed a robust improvement in the rate of photoreceptor OS retention. RO embedding is a quick and easy method that greatly enhances the preservation of photoreceptor OSs, an important structure for modelling retinal diseases with the involvement of photoreceptors.
Topics: Retinal Pigment Epithelium; Organoids; Retina; Induced Pluripotent Stem Cells; Cell Differentiation; Photoreceptor Cells
PubMed: 36499228
DOI: 10.3390/ijms232314893 -
Advanced Biology May 2021Optogenetics harnesses natural photoreceptors to non-invasively control selected processes in cells with previously unmet spatiotemporal precision. Linking the activity... (Review)
Review
Optogenetics harnesses natural photoreceptors to non-invasively control selected processes in cells with previously unmet spatiotemporal precision. Linking the activity of a protein of choice to the conformational state of a photosensor domain through allosteric coupling represents a powerful method for engineering light-responsive proteins. It enables the design of compact and highly potent single-component optogenetic systems with fast on- and off-switching kinetics. However, designing protein-photoreceptor chimeras, in which structural changes of the photoreceptor are effectively propagated to the fused effector protein, is a challenging engineering problem and often relies on trial and error. Here, recent advances in the design and application of optogenetic allosteric switches are reviewed. First, an overview of existing optogenetic tools based on inducible allostery is provided and their utility for cell biology applications is highlighted. Focusing on light-oxygen-voltage domains, a widely applied class of small blue light sensors, the available strategies for engineering light-dependent allostery are presented and their individual advantages and limitations are highlighted. Finally, high-throughput screening technologies based on comprehensive insertion libraries, which could accelerate the creation of stimulus-responsive receptor-protein chimeras for use in optogenetics and beyond, are discussed.
Topics: Light; Optogenetics; Photoreceptor Cells; Protein Engineering; Proteins
PubMed: 33107225
DOI: 10.1002/adbi.202000181