Excess phosphoserine-129 α-synuclein induces synaptic vesicle trafficking and declustering defects at a vertebrate synapse.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein aberrantly accumulates throughout neurons, including at synapses. During neuronal activity, α-synuclein is reversibly phosphorylated at serine 129 (pS129). While pS129 comprises ∼4% of total α-synuclein under physiological conditions, it dramatically increases in PD and DLB brains. The impacts of excess pS129 on synaptic function are currently unknown. We show here that compared with wild-type (WT) α-synuclein, pS129 exhibits increased binding and oligomerization on synaptic membranes and enhanced vesicle "microclustering" in vitro. Moreover, when acutely injected into lamprey reticulospinal axons, excess pS129 α-synuclein robustly localized to synapses and disrupted SV trafficking in an activity-dependent manner, as assessed by ultrastructural analysis. Specifically, pS129 caused a declustering and dispersion of SVs away from the synaptic vicinity, leading to a significant loss of total synaptic membrane. Live imaging further revealed altered SV cycling, as well as microclusters of recently endocytosed SVs moving away from synapses. Thus, excess pS129 caused an activity-dependent inhibition of SV trafficking via altered vesicle clustering/reclustering. This work suggests that accumulation of pS129 at synapses in diseases like PD and DLB could have profound effects on SV dynamics.

Differential Gene and Protein Expression of Conjunctival Bleb Hyperfibrosis in Early Failure of Glaucoma Surgery.

The early failure of glaucoma surgery is mainly caused by over-fibrosis at the subconjunctival space, causing obliteration of the filtration bleb. Because fibrosis has a suspected basis of genetic predisposition, we have undertaken a prospective study to identify upregulated profibrotic genes in a population of glaucoma patients with signs of conjunctival fibrosis and early postoperative surgical failure. Clinical data of re-operated fibrosis patients, hyperfibrosis patients who re-operated more than once in a short time, and control patients with no fibrosis were recorded and analyzed at each follow-up visit. Conjunctival-Tenon surgical specimens were obtained intraoperatively to evaluate the local expression of a panel of genes potentially associated with fibrosis. In order to correlate gene expression signatures with protein levels, we quantified secreted proteins in primary cultures of fibroblasts from patients. Expression of VEGFA, CXCL8, MYC, and CDKN1A was induced in the conjunctiva of hyperfibrosis patients. VEGFA and IL8 protein levels were also increased in fibroblast supernatants. We propose that an increase in these proteins could be useful in detecting conjunctival fibrosis in glaucoma patients undergoing filtering surgery. Molecular markers could be crucial for early detection of patients at high risk of failure of filtration surgery, leading to more optimal and personalized treatments.

Blue prescription: A pilot study of health benefits for oncological patients of a short program of activities involving the sea.

Performing outdoor activities in blue spaces can help improve human health and mental well-being by reducing stress and promoting social relationships. The number of people surviving cancer has increased globally to experience this disease as a life-changing and chronic condition with physical and psychosocial symptoms that have negative impacts on their quality of life. While there has been a growth of programs in green spaces to meet the needs of cancer patients, such as follow-up post-treatment care, support groups and physical activity programs, very few studies have examined the effects of activities involving the sea for the health and well-being of oncology patients. This is the first study to evaluate whether different outdoor activities in blue spaces can benefit oncological patients' physical and mental health using smartwatches, sphygmomanometers and Profile of Mood States (POMS) questionnaires. We assessed changes in blood pressure, heart rate, sleep quality and mental health of 16 patients after twelve sessions of three different activities (walking, beach and snorkelling) and four sessions of a control activity. While no significant differences between activities were observed in terms of the data gathered by the smartwatches, a gradient of positive results for human mental health was observed towards exposure to a blue space, assessed through POMS questionnaires. Results show that exposure to blue spaces contributes to tension and anger reduction and improves the vigour mood state of oncology patients. No significant increases in patients' heart rate were recorded after the beach and snorkelling activities, with results similar to the control activity, suggesting that the contribution may be to participants' relaxation.

Synuclein Regulates Synaptic Vesicle Clustering and Docking at a Vertebrate Synapse.

Neurotransmission relies critically on the exocytotic release of neurotransmitters from small synaptic vesicles (SVs) at the active zone. Therefore, it is essential for neurons to maintain an adequate pool of SVs clustered at synapses in order to sustain efficient neurotransmission. It is well established that the phosphoprotein synapsin 1 regulates SV clustering at synapses. Here, we demonstrate that synuclein, another SV-associated protein and synapsin binding partner, also modulates SV clustering at a vertebrate synapse. When acutely introduced to unstimulated lamprey reticulospinal synapses, a pan-synuclein antibody raised against the N-terminal domain of α-synuclein induced a significant loss of SVs at the synapse. Both docked SVs and the distal reserve pool of SVs were depleted, resulting in a loss of total membrane at synapses. In contrast, antibodies against two other abundant SV-associated proteins, synaptic vesicle glycoprotein 2 (SV2) and vesicle-associated membrane protein (VAMP/synaptobrevin), had no effect on the size or distribution of SV clusters. Synuclein perturbation caused a dose-dependent reduction in the number of SVs at synapses. Interestingly, the large SV clusters appeared to disperse into smaller SV clusters, as well as individual SVs. Thus, synuclein regulates clustering of SVs at resting synapses, as well as docking of SVs at the active zone. These findings reveal new roles for synuclein at the synapse and provide critical insights into diseases associated with α-synuclein dysfunction, such as Parkinson's disease.

Effects of Excess Brain-Derived Human α-Synuclein on Synaptic Vesicle Trafficking.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle trafficking under physiological conditions. However, in several neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, α-synuclein accumulates throughout the neuron, including at synapses, leading to altered synaptic function, neurotoxicity, and motor, cognitive, and autonomic dysfunction. Neurons typically contain both monomeric and multimeric forms of α-synuclein, and it is generally accepted that disrupting the balance between them promotes aggregation and neurotoxicity. However, it remains unclear how distinct molecular species of α-synuclein affect synapses where α-synuclein is normally expressed. Using the lamprey reticulospinal synapse model, we previously showed that acute introduction of excess recombinant monomeric or dimeric α-synuclein impaired distinct stages of clathrin-mediated synaptic vesicle endocytosis, leading to a loss of synaptic vesicles. Here, we expand this knowledge by investigating the effects of native, physiological α-synuclein isolated from the brain of a neuropathologically normal human subject, which comprised predominantly helically folded multimeric α-synuclein with a minor component of monomeric α-synuclein. After acute introduction of excess brain-derived human α-synuclein, there was a moderate reduction in the synaptic vesicle cluster and an increase in the number of large, atypical vesicles called "cisternae." In addition, brain-derived α-synuclein increased synaptic vesicle and cisternae sizes and induced atypical fusion/fission events at the active zone. In contrast to monomeric or dimeric α-synuclein, the brain-derived multimeric α-synuclein did not appear to alter clathrin-mediated synaptic vesicle endocytosis. Taken together, these data suggest that excess brain-derived human α-synuclein impairs intracellular vesicle trafficking and further corroborate the idea that different molecular species of α-synuclein produce distinct trafficking defects at synapses. These findings provide insights into the mechanisms by which excess α-synuclein contributes to synaptic deficits and disease phenotypes.

The Roses Ocean and Human Health Chair: A New Way to Engage the Public in Oceans and Human Health Challenges.

Involving and engaging stakeholders is crucial for studying and managing the complex interactions between marine ecosystems and human health and wellbeing. The Oceans and Human Health Chair was founded in the town of Roses (Catalonia, Spain, NW Mediterranean) in 2018, the fruit of a regional partnership between various stakeholders, and for the purpose of leading the way to better health and wellbeing through ocean research and conservation. The Chair is located in an area of the Mediterranean with a notable fishing, tourist, and seafaring tradition and is close to a marine reserve, providing the opportunity to observe diverse environmental conditions and coastal and maritime activities. The Chair is a case study demonstrating that local, collaborative, transdisciplinary, trans-sector, and bottom-up approaches offer tremendous opportunities for engaging coastal communities to help support long-lasting solutions that benefit everyone, and especially those living by the sea or making their living from the goods and services provided by the sea. Furthermore, the Chair has successfully integrated most of its experts in oceans and human health from the most prestigious institutions in Catalonia. The Chair focuses on three main topics identified by local stakeholders: Fish and Health; Leisure, Health, and Wellbeing; and Medicines from the Sea. Led by stakeholder engagement, the Chair can serve as a novel approach within the oceans and human health field of study to tackle a variety of environmental and public health challenges related to both communicable and non-communicable diseases, within the context of sociocultural issues. Drawing on the example provided by the Chair, four principles are established to encourage improved participatory processes in the oceans and human health field: bottom-up, "think local", transdisciplinary and trans-sectorial, and "balance the many voices".

Outcomes of Trabeculectomy With and Without Mitomycin C in Pseudoexfoliative Glaucoma Compared With Mitomycin C in Primary Open Angle Glaucoma.

The aim of this study was to evaluate the outcomes of trabeculectomy with mitomycin C (MMC) in patients with Pseudoexfoliative Glaucoma (PXG) and compare the results with the outcomes of trabeculectomy without MMC in PXG and with MMC in Primary Open Angle Glaucoma (POAG). Ninety eyes (76 patients) submitted to trabeculectomy were included in a one-year retrospective study. Fifty-eight eyes with PXG were divided into group 1 (28 eyes) and group 2 (30 eyes), with and without MMC application respectively. Then, the group 1 results were compared with 32 eyes with POAG that performed trabeculectomy with MMC (group 3). Main outcome measures were intraocular pressure (IOP), number of IOP lowering medications, rate of bleb failure (encapsulation, flattening and/or vascularization) and the number of eyes submitted to surgical procedures after trabeculectomy (needling, 5-fluorouracil (5FU) or 2nd trabeculectomy). Results revealed that compared to trabeculectomy with MMC in POAG and trabeculectomy with MMC in PXG, trabeculectomy without MMC in PXG leads to higher IOP (preoperative mean ± standard deviation [SD] was 28.6 ± 5.4 mmHg in group 1, 32.2 ± 8.2 mmHg in group 2 and 26.1 ± 6.5 mmHg in group 3; and after one year was 13.9 ± 3.9 mmHg in group 1, 16.1 ± 5.9 mmHg in group 2 and 12.5 ± 4.0 mmHg in group 3); higher number of IOP lowering medications (preoperative mean ± SD was 3.1 ± 0.60 in group 1, 2.8 ± 0.81 in group 2 and 3.4 ± 0.76 in group 3; and after one year was 1.1 ± 1.1 in group 1, 1.1 ± 1.0 in group 2 and 0.33 ± 0.89 in group 3); higher prevalence of bleb failure (47% in group 1, 53% in group 2, and 18% in group 3); and increased participation in surgical procedures following trabeculectomy (47% in group 1, 57% in group 2, and 6% in group 3). We concluded that trabeculectomy without MMC in PXG had the worst surgical outcome. Thus, PXG appears to be a potential risk factor for filtration bleb failure. Therefore, it could be considered in surgical protocols of MMC application.

Detección y medición de la enfermedad funcional y orgánica en población general: desarrollo y validación de la escala clínica TOPYPS / Detecting and measuring functional and organic disease in general population: Development and validation of TOPYPS clinical scale

OBJETIVO: Desarrollar y validar la escala Trastorno Psiquiátrico y Patología Somática (TOPYPS), un instrumento diseñado para: a) detectar, con un alto grado de sospecha, las patologías funcionales más frecuentes según los criterios diagnósticos estandarizados, y b) evaluar de forma rápida, global y fiable el estado de salud física en población general. DISEÑO: Validación de una escala. Emplazamiento: Centro de atención primaria, Barcelona. PARTICIPANTES: La escala se administró a 67 adultos seleccionados al azar. Mediciones: La escala TOPYPS cuenta con 6 secciones en base a los sistemas corporales, y cada una de ellas se puntúa en función del grado de interferencia en la actividad diaria, el tipo de tratamiento recibido y el pronóstico de las enfermedades orgánicas y/o funcionales recogidas en cada sección. La fiabilidad test-retest se realizó en 2 ocasiones, con una semana de diferencia. La validez se comprobó comparando los resultados de la escala con el examen clínico realizado por 2 especialistas diferentes en medicina general (gold standard). RESULTADOS: La repetibilidad (test-retest) y el acuerdo entre los evaluadores fueron satisfactorios para cada una de las 6 secciones, para la puntuación total y para la valoración por sospecha diagnóstica de enfermedades funcionales. La validez en general fue aceptable tanto para contenido como para constructo, de acuerdo a la correlación con el gold standard. CONCLUSIONES: La escala TOPYPS muestra buenas propiedades psicométricas. Es una herramienta adecuada para detectar y medir las enfermedades funcionales y orgánicas en población general

OBJECTIVE: To develop and validate the TOPYPS scale, an instrument designed to: (I) detect with a high degree of suspicion the most frequent functional pathologies according to standard diagnostic criteria, and (II) to assess the physical health in the general population quickly, comprehensive and reliable. DESIGN: Validation of a scale. LOCATION: Primary Care Centre, Barcelona. PARTICIPANTS: The scale was administered to 67 randomly selected adults. Measurements: TOPYPS scale was administered to 67 adults randomly selected from a primary care setting in Barcelona, Spain. TOPYPS has six sections based on body systems, each one scored according to the degree of interference in daily activity, type of treatment received, and prognostic of the reported illnesses in each section. Test-retest reliability completions were on two separate occasions one week apart. Validity was then tested by comparing the results with the clinical examination conducted by two different specialists in general practice (gold standard). RESULTS: Repeatability (test-retest) and inter-rater agreement for each of the six sections and for the total score were satisfactory. Validity was acceptable both for content and construct, according to their correlation with the gold standard. CONCLUSIONS: TOPYPS displayed good psychometrical properties. It is a suitable tool to detect and measure functional and organic diseases in general population

Retromer stops beta-arrestin 1-mediated signaling from internalized cannabinoid 2 receptors.

G protein-coupled receptors mediate their complex functions through activation of signaling cascades from receptors localized at the cell surface and endosomal compartments. These signaling pathways are modulated by heterotrimeric G proteins and the scaffold proteins beta-arrestin 1 and 2. However, in contrast to the events occurring at the cell surface, our knowledge of the mechanisms controlling signaling from receptors localized at intracellular compartments is still very limited. Here we sought to investigate the intracellular signaling from cannabinoid 2 receptor (CB2R). First, we show that receptor internalization is required for agonist-induced phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Then we demonstrate that ERK1/2 activation is mediated by beta-arrestin 1 from receptors localized exclusively at Rab4/5 compartments. Finally, we identify the retromer complex as a gatekeeper, terminating beta-arrestin 1-mediated ERK phosphorylation. These findings extend our understanding of the events controlling signaling from endocytosed receptors and identify the retromer as a modulator of beta-arrestin-mediated signaling from CB2R.

[Detecting and measuring functional and organic disease in general population: Development and validation of TOPYPS clinical scale]. / Detección y medición de la enfermedad funcional y orgánica en población general: desarrollo y validación de la escala clínica TOPYPS.

OBJECTIVE: To develop and validate the TOPYPS scale, an instrument designed to: (i)detect with a high degree of suspicion the most frequent functional pathologies according to standard diagnostic criteria, and (ii)to assess the physical health in the general population quickly, comprehensive and reliable. DESIGN: Validation of a scale. LOCATION: Primary Care Centre, Barcelona. PARTICIPANTS: The scale was administered to 67 randomly selected adults. MEASUREMENTS: TOPYPS scale was administered to 67 adults randomly selected from a primary care setting in Barcelona, Spain. TOPYPS has six sections based on body systems, each one scored according to the degree of interference in daily activity, type of treatment received, and prognostic of the reported illnesses in each section. Test-retest reliability completions were on two separate occasions one week apart. Validity was then tested by comparing the results with the clinical examination conducted by two different specialists in general practice (gold standard). RESULTS: Repeatability (test-retest) and inter-rater agreement for each of the six sections and for the total score were satisfactory. Validity was acceptable both for content and construct, according to their correlation with the gold standard. CONCLUSIONS: TOPYPS displayed good psychometrical properties. It is a suitable tool to detect and measure functional and organic diseases in general population.

Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons.

Total internal reflection fluorescence (TIRF) microscopy allows probing the cellular events occurring close and at the plasma membrane. Over the last decade, we have seen a significant increase in the number of publications applying TIRF microscopy to unravel some of the fundamental biological questions regarding G protein-coupled receptors (GPCRs) function such as the mechanisms controlling receptor trafficking, quaternary structure, and signaling among others. Most of the published work has been performed in heterologous systems such as HEK293 and CHO cells, where the imaging surface available is higher and smoother when compared with the narrow processes or the smaller cell bodies of neurons. However, some publications have expanded our understanding of these events to primary cell cultures, mostly rat hippocampal and striatal neuronal cultures. Results from these cells provide a bona fide model of the complex events controlling GPCR function in living cells. We believe more work needs to be performed in primary cultures and eventually in intact tissue to complement the knowledge obtained from heterologous cell models. Here, we described a step-by-step protocol to investigate the surface trafficking and signaling from GPCRs in rat hippocampal and striatal primary cultures.

Real-time imaging of mu opioid receptors by total internal reflection fluorescence microscopy.

Receptor trafficking and signaling are intimately linked, especially in the Mu opioid receptor (MOR) where ligand-dependent endocytosis and recycling have been associated with opioid tolerance and dependence. Ligands of MOR can induce receptor endocytosis and recycling within minutes of exposure in heterologous systems and cultured neurons. Endocytosis removes desensitized receptors after their activation from the plasma membrane, while recycling promotes resensitization by delivering functional receptors to the cell surface. These rapid mechanisms can escape traditional analytical methods where only snapshots are obtained from highly dynamic events.Total internal reflection fluorescence (TIRF) microscopy is a powerful tool that can be used to investigate, in real time, surface trafficking events at the single molecule level. The restricted excitation of fluorophores located at or near the plasma membrane in combination with high sensitivity quantitative cameras makes it possible to record and analyze individual endocytic and recycling event in real time. In this chapter, we describe a TIRF microscopy protocol to investigate in real time, the ligand-dependent MOR trafficking in Human Embryonic Kidney 293 cells and dissociated striatal neuronal cultures. This approach can provide unique spatio-temporal resolution to understand the fundamental events controlling MOR trafficking at the plasma membrane.

Imaging of kiss-and-run exocytosis of surface receptors in neuronal cultures.

Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis.

Ocular Toxicity Secondary to Asclepias physocarpa: The Balloon Plant.

We report a case of a 65-year-old woman with symptoms of blurred vision and ocular irritation a few hours after accidental contact of the right eye with Asclepias physocarpa milky latex. Observation showed a diffuse conjunctival hyperemia and stromal corneal edema with Descemet's membrane folds. Recovery was fast and apparently complete in less than one month. However, specular microscopy at 6-months follow-up showed an abnormal endothelial morphology as sequelae, suggesting this condition is not as innocuous as it has been suggested.

Fast modulation of µ-opioid receptor (MOR) recycling is mediated by receptor agonists.

The µ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.