Influence of spatially segregated IP3-producing pathways on spike generation and transmitter release in Purkinje cell axons.

It has been known for a long time that inositol-trisphosphate (IP3) receptors are present in the axon of certain types of mammalian neurons, but their functional role has remained unexplored. Here we show that localized photolysis of IP3 induces spatially constrained calcium rises in Purkinje cell axons. Confocal immunohistology reveals that the axon initial segment (AIS), as well as terminals onto deep cerebellar cells, express specific subtypes of Gα/q and phospholipase C (PLC) molecules, together with the upstream purinergic receptor P2Y1. By contrast, intermediate parts of the axon express another set of Gα/q and PLC molecules, indicating two spatially segregated signaling cascades linked to IP3 generation. This prompted a search for distinct actions of IP3 in different parts of Purkinje cell axons. In the AIS, we found that local applications of the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP3 photolysis led to inhibition of action potential firing. In synaptic terminals on deep cerebellar nuclei neurons, we found that photolysis of both IP3 and ATP led to GABA release. We propose that axonal IP3 receptors can inhibit action potential firing and increase neurotransmitter release, and that these effects are likely controlled by purinergic receptors. Altogether our results suggest a rich and diverse functional role of IP3 receptors in axons of mammalian neurons.

Calcium activates the light-dependent conductance in melanopsin-expressing photoreceptors of amphioxus.

Melanopsin, the photopigment of the "circadian" receptors that regulate the biological clock and the pupillary reflex in mammals, is homologous to invertebrate rhodopsins. Evidence supporting the involvement of phosphoinositides in light-signaling has been garnered, but the downstream effectors that control the light-dependent conductance remain unknown. Microvillar photoreceptors of the primitive chordate amphioxus also express melanopsin and transduce light via phospholipase-C, apparently not acting through diacylglycerol. We therefore examined the role of calcium in activating the photoconductance, using simultaneous, high time-resolution measurements of membrane current and Ca(2+) fluorescence. The light-induced calcium rise precedes the onset of the photocurrent, making it a candidate in the activation chain. Moreover, photolysis of caged Ca elicits an inward current of similar size, time course and pharmacology as the physiological photoresponse, but with a much shorter latency. Internally released calcium thus emerges as a key messenger to trigger the opening of light-dependent channels in melanopsin-expressing microvillar photoreceptors of early chordates.

Dissecting the determinants of light sensitivity in amphioxus microvillar photoreceptors: possible evolutionary implications for melanopsin signaling.

Melanopsin, a photopigment related to the rhodopsin of microvillar photoreceptors of invertebrates, evolved in vertebrates to subserve nonvisual light-sensing functions, such as the pupillary reflex and entrainment of circadian rhythms. However, vertebrate circadian receptors display no hint of a microvillar specialization and show an extremely low light sensitivity and sluggish kinetics. Recently in amphioxus, the most basal chordate, melanopsin-expressing photoreceptors were characterized; these cells share salient properties with both rhabdomeric photoreceptors of invertebrates and circadian receptors of vertebrates. We used electrophysiology to dissect the gain of the light-transduction process in amphioxus and examine key features that help outline the evolutionary transition toward a sensor optimized to report mean ambient illumination rather than mediating spatial vision. By comparing the size of current fluctuations attributable to single photon melanopsin isomerizations with the size of single-channels activated by light, we concluded that the gain of the transduction cascade is lower than in rhabdomeric receptors. In contrast, the expression level of melanopsin (gauged by measuring charge displacements during photo-induced melanopsin isomerization) is comparable with that of canonical visual receptors. A modest amplification in melanopsin-using receptors is therefore apparent in early chordates; the decrease in photopigment expression-and loss of the anatomical correlates-observed in vertebrates subsequently enabled them to attain the low photosensitivity tailored to the role of circadian receptors.

Arrestin in ciliary invertebrate photoreceptors: molecular identification and functional analysis in vivo.

Arrestin was identified in ciliary photoreceptors of Pecten irradians, and its role in terminating the light response was established electrophysiologically. Downstream effectors in these unusual visual cells diverge from both microvillar photoreceptors and rods and cones; the finding that key regulatory mechanisms of the early steps of visual excitation are conserved across such distant lineages of photoreceptors underscores that a common blueprint for phototransduction exists across metazoa. Arrestin was detected by Western blot analysis of retinal lysates, and localized in ciliary photoreceptors by immunostaining of whole-eye cryosections and dissociated cells. Two arrestin isoforms were molecularly identified by PCR; these present the canonical N- and C-arrestin domains, and are identical at the nucleotide level over much of their sequence. A high degree of homology to various ß-arrestins (up to 70% amino acid identity) was found. In situ hybridization localized the two transcripts within the retina, but failed to reveal finer spatial segregation, possibly because of insufficient differences between the riboprobes. Intracellular dialysis of anti arrestin antibodies into voltage-clamped ciliary photoreceptors produced a gradual slow-down of the photocurrent falling phase, leaving a tail that decayed over many seconds after light termination. The antibodies also caused spectrally neutral flashes to elicit prolonged aftercurrents in the absence of large metarhodopsin accumulation; such aftercurrents could be quenched by chromatic illumination that photoconverts metarhodopsin back to rhodopsin. These observations indicate that the antibodies depleted functionally available arrestin, and implicate this molecule in the deactivation of the photoresponse at the rhodopsin level.

Light-transduction in melanopsin-expressing photoreceptors of Amphioxus.

Spatial vision in different organisms is mediated by 2 classes of photoreceptors: microvillar and ciliary. Recently, additional photosensitive cells implicated in nonvisual light-dependent functions have been identified in the mammalian retina. A previously undescribed photopigment, melanopsin, underlies these photoresponses, and it has been proposed that its transduction mechanisms may be akin to the lipid-signaling scheme of invertebrate microvillar receptors, rather than the cyclic-nucleotide cascade of vertebrates. Melanopsin has an ancient origin in deuterostomia, and expresses in 2 morphologically distinct classes of cells in the neural tube of Amphioxus, the most basal extant chordate: pigmented ocelli, and Joseph cells. However, to our knowledge, their physiology and alleged photosensitivity had never been investigated. We dissociated both types of cells, and conclusively demonstrated by patch-electrode recoding that they are primary photoreceptors; their receptor potential is depolarizing, accompanied by an increase in membrane conductance. The action spectrum peaks in the blue region, approximately 470 nm, similar to the absorption of melanopsin in vitro. The light-dependent conductance rectifies inwardly; Na and Ca are differentially implicated in the 2 cell types. Fluorescence Ca imaging reveals that photostimulation rapidly mobilizes calcium from internal stores. Intracellular 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate severely impairs the photoresponse, indicating that light-evoked Ca elevation is an important event in photoexcitation. These observations support the notion that the lineage of microvillar photoreceptors and its associated light-signaling pathway also evolved in the chordates. Thus, Joseph cells and pigmented ocelli of the Amphioxus may represent a link between ancestral rhabdomeric-like light sensors present in prebilaterians and the circadian photoreceptors of higher vertebrates.

Modulation of Gq/PLC-Mediated Signaling by Acute Lithium Exposure.

Although lithium has long been one of the most widely used pharmacological agents in psychiatry, its mechanisms of action at the cellular and molecular levels remain poorly understood. One of the targets of Li+ is the phosphoinositide pathway, but whereas the impact of Li+ on inositol lipid metabolism is well documented, information on physiological effects at the cellular level is lacking. We examined in two mammalian cell lines the effect of acute Li+ exposure on the mobilization of internal Ca2+ and phospholipase C (PLC)-dependent membrane conductances. We first corroborated by Western blots and immunofluorescence in HEK293 cells the presence of key signaling elements of a muscarinic PLC pathway (M1AchR, Gq, PLC-ß1, and IP3Rs). Stimulation with carbachol evoked a dose-dependent mobilization of Ca, as determined with fluorescent indicators. This was due to release from internal stores and proved susceptible to the PLC antagonist U73122. Li+ exposure reproducibly potentiated the Ca response in a concentration-dependent manner extending to the low millimolar range. To broaden those observations to a neuronal context and probe potential Li modulation of electrical signaling, we next examined the cell line SHsy5y. We replicated the potentiating effects of Li on the mobilization of internal Ca, and, after characterizing the basic properties of the electrical response to cholinergic stimulation, we also demonstrated an equally robust upregulation of muscarinic membrane currents. Finally, by directly stimulating the signaling pathway at different links downstream of the receptor, the site of action of the observed Li effects could be narrowed down to the G protein and its interaction with PLC-ß. These observations document a modulation of Gq/PLC/IP3-mediated signaling by acute exposure to lithium, reflected in distinct physiological changes in cellular responses.

New STAG3 gene variant as a cause of premature ovarian insufficiency / Nueva variante del gen STAG3 causante de insuficiencia ovárica prematura

Objectives: To describe a case of ovarian failure secondary to a homozygous pathogenic variant in the STAG3 gene not previously reported. Materials and Methods: A 16-year-old patient with primary amenorrhea and absence of secondary sexual characteristics, with documented autoimmune hypothyroidism, poor genital and gonadal streak development which prompted the performance of clinical exome sequencing. A homozygous pathogenic variant not previously reported in the STAG3 gene, which has been associated with premature ovarian insufficiency (POI), was identified. Conclusions: In this case, clinical exome sequencing was key for identifying a STAG gene abnormality, probably associated with POI and long term prognosis for the patient. A new pathogenic variant c.2773delT; p.Ser925Profs*6 of the STAG3 gene associated with POI was established.

Objetivos: describir un caso de falla ovárica secundaria a una variante patogénica homocigota en el gen STAG3 no reportada previamente. Materiales y métodos: paciente de 16 años con amenorrea primaria y ausencia de características sexuales secundarias, en quien se documentó hipotiroidismo autoinmune, pobre desarrollo genital y cintilla gonadal, por lo cual se realizó secuenciación de exoma clínico. Se identificó una variante homocigota patogénica previamente no reportada en el gen STAG3, el cual ha sido relacionado con insuficiencia ovárica prematura (IOP). Conclusiones: en este caso, la realización de exoma clínico fue determinante para identificar una alteración del gen STAG, probablemente asociada a la IOP y el pronóstico a largo plazo de la paciente. Se establece una nueva variante patogénica c.2773delT; p.Ser925Profs*6 del gen STAG3 asociada a la IOP.

Juvenile polyautoimmunity in a rheumatology setting.

Overt polyautoimmunity (PolyA) corresponds to the presence of more than one well-defined autoimmune disease (AD) manifested clinically in a single patient. The current study aimed to describe the main characteristics of juvenile PolyA in a pediatric rheumatology setting and analyze the chronological aspects, index cases, familial autoimmunity, and clustering pattern. This was a cross-sectional and multicenter study in which 313 children with overt PolyA were included. Patients were systematically interviewed and their medical records reviewed using a questionnaire that sought information about demographic, clinical, immunological, and familial characteristics. A hierarchical cluster analysis was done to determine similarities between autoimmune diseases based on PolyA. PolyA occurred simultaneously in 138 (44%) patients. Multiple autoimmune syndrome was observed in 62 (19.8%) patients. There were 25 index diseases of which, systemic lupus erythematosus (SLE, n = 134, 42.8%), juvenile idiopathic arthritis (JIA, n = 40, 12.7%), Hashimoto's thyroiditis (HT, n = 24, 7.66%), immune thrombocytopenic purpura (ITP n = 20, 6.39%), antiphospholipid syndrome (APS, n = 15, 4.79%), and vitiligo (VIT, n = 15, 4.79%) were the most frequent and represented 79.23% of the total number of patients. Familial autoimmunity influenced PolyA. A high aggregation of autoimmunity was observed (λr = 3.5). Three main clusters were identified, of which SLE and APS were the most similar pair of diseases (based on the Jaccard index) followed by HT and JIA, which were related to ITP and Sjögren's syndrome. The third cluster was composed of localized scleroderma and VIT. Our findings may assist physicians to make an early diagnosis of this frequent condition. Pediatric patients with ADs should be systematically assessed for PolyA.

Molecular and functional identification of a novel photopigment in Pecten ciliary photoreceptors.

The two basic animal photoreceptor types, ciliary and microvillar, use different light-transduction schemes: their photopigments couple to Gt versus Gq proteins, respectively, to either mobilize cyclic nucleotides or trigger a lipid signaling cascade. A third class of photoreceptors has been described in the dual retina of some marine invertebrates; these present a ciliary morphology but operate via radically divergent mechanisms, prompting the suggestion that they comprise a novel lineage of light sensors. In one of these organisms, an uncommon putative opsin was uncovered that was proposed to signal through Go Orthologues subsequently emerged in diverse phyla, including mollusks, echinoderms, and chordates, but the cells in which they express have not been identified, and no studies corroborated their function as visual pigments or their suggested signaling mode. Conversely, in only one invertebrate species, Pecten irradians, have the ciliary photoreceptors been physiologically characterized, but their photopigment has not been identified molecularly. We used the transcriptome of Pecten retina to guide the cloning by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) extensions of a new member of this group of putative opsins. In situ hybridization shows selective transcription in the distal retina, and specific antibodies identify a single band of the expected molecular mass in Western blots and distinctly label ciliary photoreceptors in retina sections. RNA interference knockdown resulted in a reduction in the early receptor current-the first manifestation of light transduction-and prevented the prolonged aftercurrent, which requires a large buildup of activated rhodopsin. We also obtained a full-length clone of the α-subunit of a Go from Pecten retina complementary DNA and localized it by in situ hybridization to the distal photoreceptors. Small interfering RNA targeting this Go caused a specific depression of the photocurrent. These results establish this novel putative opsin as a bona fide visual pigment that couples to Go to convey the light signal.

Light control of G protein signaling pathways by a novel photopigment.

Channelopsins and photo-regulated ion channels make it possible to use light to control electrical activity of cells. This powerful approach has lead to a veritable explosion of applications, though it is limited to changing membrane voltage of the target cells. An enormous potential could be tapped if similar opto-genetic techniques could be extended to the control of chemical signaling pathways. Photopigments from invertebrate photoreceptors are an obvious choice-as they do not bleach upon illumination -however, their functional expression has been problematic. We exploited an unusual opsin, pScop2, recently identified in ciliary photoreceptors of scallop. Phylogenetically, it is closer to vertebrate opsins, and offers the advantage of being a bi-stable photopigment. We inserted its coding sequence and a fluorescent protein reporter into plasmid vectors and demonstrated heterologous expression in various mammalian cell lines. HEK 293 cells were selected as a heterologous system for functional analysis, because wild type cells displayed the largest currents in response to the G-protein activator, GTP-γ-S. A line of HEK cells stably transfected with pScop2 was generated; after reconstitution of the photopigment with retinal, light responses were obtained in some cells, albeit of modest amplitude. In native photoreceptors pScop2 couples to Go; HEK cells express poorly this G-protein, but have a prominent Gq/PLC pathway linked to internal Ca mobilization. To enhance pScop2 competence to tap into this pathway, we swapped its third intracellular loop-important to confer specificity of interaction between 7TMDRs and G-proteins-with that of a Gq-linked opsin which we cloned from microvillar photoreceptors present in the same retina. The chimeric construct was evaluated by a Ca fluorescence assay, and was shown to mediate a robust mobilization of internal calcium in response to illumination. The results project pScop2 as a potentially powerful optogenetic tool to control signaling pathways.