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In recent decades, a number of novel non-visual opsin photopigments belonging to the family of G protein- coupled receptors, likely involved in a number of non-image-forming processes, have been identified and characterized in cells of the inner retina of vertebrates. It is now known that the vertebrate retina is composed of visual photoreceptor cones and rods responsible for diurnal/color and nocturnal/black and white vision, and cells like the intrinsically photosensitive retinal ganglion cells (ipRGCs) and photosensitive horizontal cells in the inner retina, both detecting blue light and expressing the photopigment melanopsin (Opn4). Remarkably, these non-visual photopigments can continue to operate even in the absence of vision under retinal degeneration. Moreover, inner retinal neurons and Müller glial cells have been shown to express other photopigments such as the photoisomerase retinal G protein-coupled receptor (RGR), encephalopsin (Opn3), and neuropsin (Opn5), all able to detect blue/violet light and implicated in chromophore recycling, retinal clock synchronization, neuron-to-glia communication, and other activities. The discovery of these new photopigments in the inner retina of vertebrates is strong evidence of novel light-regulated activities. This review focuses on the features, localization, photocascade, and putative functions of these novel non-visual opsins in an attempt to shed light on their role in the inner retina of vertebrates and in the physiology of the whole organism.

Gliomas are solid tumors of the central nervous system (CNS) that originated from different glial cells. The World Health Organization (WHO) classifies these tumors into four groups (I–IV) with increasing malignancy. Glioblastoma (GBM) is the most common and aggressive type of brain tumor classified as grade IV. GBMs are resistant to conventional therapies with poor prognosis after diagnosis even when the Stupp protocol that combines surgery and radiochemotherapy is applied. Nowadays, few novel therapeutic strategies have been used to improve GBM treatment, looking for higher efficiency and lower side effects, but with relatively modest results. The circadian timing system temporally organizes the physiology and behavior of most organisms and daily regulates several cellular processes in organs, tissues, and even in individual cells, including tumor cells. The potentiality of the function of the circadian clock on cancer cells modulation as a new target for novel treatments with a chronobiological basis offers a different challenge that needs to be considered in further detail. The present review will discuss state of the art regarding GBM biology, the role of the circadian clock in tumor progression, and new chrono-chemotherapeutic strategies applied for GBM treatment.

Natural products are an unsurpassed source of leading structures in drug discovery. The biosynthetic machinery of the producing organism offers an important source for modifying complex natural products, leading to analogs that are unattainable by chemical semisynthesis or total synthesis. In this report, through the combination of natural products chemistry and diversity-oriented synthesis, a diversity-enhanced extracts approach is proposed using chemical reactions that remodel molecular scaffolds directly on extracts of natural resources. This method was applied to subextract enriched in sesquiterpene lactones from Ambrosia tenuifolia (Fam. Asteraceae) using acid media conditions (p-toluenesulfonic acid) to change molecular skeletons. The chemically modified extract was then fractionated by a bioguided approach to obtain the pure compounds responsible for the antiglioblastoma (GBM) activity in T98G cell cultures. Indeed, with the best candidate, chronobiological experiments were performed to evaluate temporal susceptibility to the treatment on GBM cell cultures to define the best time to apply the therapy. Finally, bioinformatics tools were used to supply qualitative and quantitative information on the physicochemical properties, chemical space, and structural similarity of the compound library obtained. As a result, natural products derivatives containing new molecular skeletons were obtained, with possible applications as chemotherapeutic agents against human GBM T98G cell cultures.

The avian retina is composed of different types of photoreceptors responsible for image and non-image forming tasks: the visual photoreceptor cells (cones and rods), the melanopsin-expressing intrinsically photoresponsive retinal ganglion cells (ipRGCs) and horizontal cells. Furthermore, the non-visual opsins Opn3 (encephalopsin/panaopsin) and Opn5 (neuropsin) have been shown to be expressed in the vertebrate inner retina, responding to blue (BL) and UV light, respectively. Here we investigated the expression and localization of Opn3 and Opn5 in the developing chick retina at different embryonic days (E) as well as in primary cultures of retinal Müller glial cells (MCs). Opn3 and Opn5 mRNAs and proteins appeared as early as E10 although traces of Opn3- and Opn5-like proteins were seen earlier by E7 in the forming RGC layer and in glial cells extending throughout the developing nuclear layer. Later on, at postnatal days 1-10 (PN1-10) a significant expression of Opn3 was observed in inner retinal cells and processes in plexiform layers, together with expression of the glial markers glutamine synthetase (GS) and the glial fibrillary acidic protein (GFAP). Opn3 and Opn5 were found to be expressed in primary MC cultures prepared at E8 and kept for 2 weeks. In addition, significant effects of BL exposure on Opn3 expression and subcellular localization were observed in MCs as BL significantly increased its levels and modified its nuclear location when compared with dark controls, through a mechanism dependent on protein synthesis. More importantly, a subpopulation of MCs responded to brief BL pulses by increasing intracellular Ca levels; whereas light-responses were completely abolished with the retinal bleacher hydroxylamine pretreatment. Taken together, our findings show that these two opsins are expressed in inner retinal cells and MCs of the chicken retina at early developmental phases and remain expressed in the mature retina at PN days. In addition, the novel photic responses seen in MCs may suggest another important role for the glia in retinal physiology.

Glioblastoma multiforme is the most aggressive brain tumor, and human T98G cells constitute a useful glioblastoma multiforme model to evaluate the chemotherapeutic agents. Modern life (shiftwork, jetlag, etc.) may cause circadian disorganization promoting higher cancer risk and metabolic disorders. Although little is known about the tumor-intrinsic circadian clock function, pharmacological modulation of circadian components may offer selective anticancer strategies. REV-ERBs are heme-binding circadian clock components acting as repressors of processes involved in tumorigenesis such as metabolism, proliferation, and inflammation. A synthetic pyrrole derivative (SR9009) that acts as REV-ERBs-specific agonists exhibits potent in vivo activity on metabolism and tumor cell viability. Here, we investigated SR9009 effects on T98G cell viability, differential chemotherapy time responses, and underlying metabolic processes (reactive oxygen species [ROS] and lipid droplets [LDs]) and compared it with the proteasome inhibitor Bortezomib treatment. SR9009-treated cells exhibited significant reduction in cell viability with consequences on cell cycle progression. Dexamethasone synchronized cells displayed differential time responses to SR9009 treatment with highest responses 18 to 30 h after synchronization. SR9009 treatment decreased ROS levels while Bortezomib increased them. However, both treatments significantly increased LD levels, whereas the combined treatment showed additive or synergistic effects between both drugs. In addition, we extended these studies to HepG2 cells which also showed a significant decrease in cell viability and ROS levels and the increase in LD levels after SR9009 treatment. Our results suggest that the pharmacological modulation of the tumor-intrinsic clock by REV-ERB agonists severely affects cell metabolism and promotes cytotoxic effects on cancer cells.

To study the macroglia and microglia and the immune role in long-time light exposure in rat eyes, we performed glial cell characterization along the time-course of retinal degeneration induced by chronic exposure to low-intensity light. Animals were exposed to light for periods of 2, 4, 6, or 8 days, and the retinal glial response was evaluated by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. Retinal cells presented an increased expression of the macroglia marker GFAP, as well as increased mRNA levels of microglia markers Iba1 and CD68 after 6 days. Also, at this time-point, we found a higher number of Iba1-positive cells in the outer nuclear layer area; moreover, these cells showed the characteristic activated-microglia morphology. The expression levels of immune mediators TNF, IL-6, and chemokines CX3CR1 and CCL2 were also significantly increased after 6 days. All the events of glial activation occurred after 5–6 days of constant light exposure, when the number of photoreceptor cells has already decreased significantly. Herein, we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that occurs independently of glial-derived immune responses.

In the vertebrate retina, three types of photoreceptors—visual photoreceptor cones and rods and the intrinsically photosensitive retinal ganglion cells (ipRGCs)—converged through evolution to detect light and regulate image- and nonimage-forming activities such as photic entrainment of circadian rhythms, pupillary light reflexes, etc. ipRGCs express the nonvisual photopigment melanopsin (OPN4), encoded by two genes: the Xenopus (Opn4x) and mammalian (Opn4m) orthologs. In the chicken retina, both OPN4 proteins are found in ipRGCs, and Opn4x is also present in retinal horizontal cells (HCs), which connect with visual photoreceptors. Here we investigate the intrinsic photosensitivity and functioning of HCs from primary cultures of embryonic retinas at day 15 by using calcium fluorescent fluo4 imaging, pharmacological inhibitory treatments, and Opn4x knockdown. Results show that HCs are avian photoreceptors with a retinal-based OPN4X photopigment conferring intrinsic photosensitivity. Light responses in HCs appear to be driven through an ancient type of phototransduction cascade similar to that in rhabdomeric photoreceptors involving a G-protein q, the activation of phospholipase C, calcium mobilization, and the release of the inhibitory neurotransmitter GABA. Based on their intrinsic photosensitivity, HCs may have a key dual function in the retina of vertebrates, potentially regulating nonvisual tasks together with their sister cells, ipRGCs, and with visual photoreceptors, modulating lateral interactions and retinal processing.

Circadian rhythms orchestrate physiological processes, including immune function, across a 24‐h cycle. This study investigates the temporal distribution of immune cell populations in healthy mice entrained to a 12:12‐h light–dark cycle. Inguinal lymph node (iLN) samples were collected at zeitgeber times (ZT) 0 (lights on) and ZT12 (lights off) to assess immune cell composition. A significantly higher proportion of natural killer T (NKT) cells and neutrophils was observed at ZT0 compared to ZT12, while dendritic cells, macrophages, and natural killer (NK) cells showed no significant temporal variation. Additionally, adaptive immune cells, particularly Programmed cell Death protein 1+ (PD1+) Cluster of differentiation 4+ (CD4+) and PD1+ Cluster of differentiation 8+ (CD8+) T cells, were more abundant during the light phase. These findings suggest a diurnal pattern in immune readiness, with implications for optimizing immunotherapeutic interventions based on circadian timing. The circadian system is involved in the temporal regulation of the immune system. Our study reveals that two innate immune populations, NKT cells and neutrophils, predominate at the beginning of the day in healthy mice, highlighting how the time of day influences immune responses. These results could have implications for optimizing immunotherapeutic strategies in cancer treatment. Image adapted from Servier Medical Art (https://smart.servier.com/), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/)

The retina of vertebrates is responsible for capturing light through visual (cones and rods) and non-visual photoreceptors (intrinsically photosensitive retinal ganglion cells and horizontal cells) triggering a number of essential activities associated to image- and non-image forming functions (photic entrainment of daily rhythms, pupillary light reflexes, pineal melatonin inhibition, among others). Although the retina contains diverse types of neuronal based-photoreceptors cells, originally classified as ciliary- or rhabdomeric-like types, in recent years, it has been shown that the major glial cell type of the retina, the Müller glial cells (MC), express blue photopigments as Opn3 (encephalopsin) and Opn5 (neuropsin) and display light responses associated to intracellular Ca2 + mobilization. These findings strongly propose MC as novel retinal photodetectors (Rios et al., 2019). Herein, we further investigated the intrinsic light responses of primary cultures of MC from embryonic chicken retinas specially focused on Ca2 + mobilization by fluorescence imaging and the identity of the internal Ca2 + stores responsible for blue light responses. Results clearly demonstrated that light responses were specific to blue light of long time exposure, and that the main Ca2 + reservoir to trigger downstream responses came from intracellular stores localized in the endoplasmic reticulum These observations bring more complexity to the intrinsic photosensitivity of retinal cells, particularly with regard to the detection of light in the blue range of visible spectra, and add novel functions to glial cells cooperating with other photoreceptors to detect and integrate ambient light in the retinal circuit and participate in cell to cell communication. Summary statement: Non-neuronal cells in the vertebrate retina, Muller glial cells, express non-canonical photopigments and sense blue light causing calcium release from intracellular stores strongly suggesting a novel intrinsic photosensitivity and new regulatory events mediating light-driven processes with yet unknown physiological implications.

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca(2+) mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca(2+) levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca(2+) mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.