Trophoblast glycoprotein is required for efficient synaptic vesicle exocytosis from retinal rod bipolar cells

IntroductionRod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs.MethodsWe examined the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electron microscopy, electroretinogram recording, patch-clamp electrophysiology, and time-resolved membrane capacitance measurements.ResultsThe scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents.DiscussionTPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology and circuit function

Loss of the Synaptic Vesicle Protein SV2B Results in Reduced Neurotransmission and Altered Synaptic Vesicle Protein Expression in the Retina

The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Extrasynaptic NMDA receptors on rod pathway amacrine cells: molecular composition, activation, and signaling

In the rod pathway of the mammalian retina, axon terminals of glutamatergic rod bipolar cells are presynaptic to AII and A17 amacrine cells in the inner plexiform layer. Recent evidence suggests that both amacrines express NMDA receptors, raising questions concerning molecular composition, localization, activation, and function of these receptors. Using dual patch-clamp recording from synaptically connected rod bipolar and AII or A17 amacrine cells in retinal slices from female rats, we found no evidence that NMDA receptors contribute to postsynaptic currents evoked in either amacrine. Instead, NMDA receptors on both amacrine cells were activated by ambient glutamate, and blocking glutamate uptake increased their level of activation. NMDA receptor activation also increased the frequency of GABAergic postsynaptic currents in rod bipolar cells, suggesting that NMDA receptors can drive release of GABA from A17 amacrines. A striking dichotomy was revealed by pharmacological and immunolabeling experiments, which found GluN2B-containing NMDA receptors on AII amacrines and GluN2A-containing NMDA receptors on A17 amacrines. Immunolabeling also revealed a clustered organization of NMDA receptors on both amacrines and a close spatial association between GluN2B subunits and connexin 36 on AII amacrines, suggesting that NMDA receptor modulation of gap junction coupling between these cells involves the GluN2B subunit. Using multiphoton Ca2+ imaging, we verified that activation of NMDA receptors evoked an increase of intracellular Ca2+ in dendrites of both amacrines. Our results suggest that AII and A17 amacrines express clustered, extrasynaptic NMDA receptors, with different and complementary subunits that are likely to contribute differentially to signal processing and plasticity

The Impact of Steroid Activation of TRPM3 on Spontaneous Activity in the Developing Retina.

In the central nervous system, melastatin transient receptor potential (TRPM) channels function as receptors for the neurosteroid pregnenolone sulfate (PregS). The expression and function of TRPM3 has been explored in adult retina, although its role during development is unknown. We found, during the second postnatal week in mice, TRPM3 immunofluorescence labeled distinct subsets of inner retinal neurons, including a subset of retinal ganglion cells (RGCs), similar to what has been reported in the adult. Labeling for a TRPM3 promoter-driven reporter confirmed expression of the TRPM3 gene in RGCs and revealed additional expression in nearly all Müller glial cells. Using two-photon calcium imaging, we show that PregS and the synthetic TRPM3 agonist CIM0216 (CIM) induced prolonged calcium transients in RGCs, which were mostly absent in TRPM3 knock-out (KO) mice. These prolonged calcium transients were not associated with strong membrane depolarizations but induced c-Fos expression. To elucidate the impact of PregS-activation of TRPM3 on retinal circuits we took two sets of physiological measurements. First, PregS induced a robust increase in the frequency but not amplitude of spontaneous postsynaptic currents (PSCs). This increase was absent in the TRPM3 KO mice. Second, PregS induced a small increase in cell participation and duration of retinal waves, but this modulation persisted in TRPM3 KO mice, indicating PregS was acting on wave generating circuits independent of TRPM3 channels. Though baseline frequency of retinal waves was slightly reduced in the TRPM3 KO mice, other properties of waves were indistinguishable from wildtype. Together, these results indicate that the presence of neurosteroids impact spontaneous synaptic activity and retinal waves during development via both TRPM3-dependent and independent mechanisms