The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis.

Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis

Lighting-up neurons: characterization of new membrane-targeted photoswitches for geneless neural stimulation

Doctoral Program in Neuroscience Curriculum in Neuroscience and Brain Technologies XXXVI cycle Summary In the last decades, light-sensitive nanotools have emerged as a powerful approach for modulating neural circuits’ activity with exquisite precision. Among the light-sensitive approaches, membrane-targeted photoswitches hold particular promise in enabling light-induced control of neuronal activity without directly interfering with physiological mechanisms and avoiding genetic manipulation. This doctoral thesis aims to unravel the intricate dynamics driven by light in the context of four distinct newly-synthetized membrane-targeted photochromic molecules. The foundation of this research lies in the characterization of Ziapin2, a newly synthetized photoswitch that demonstrated remarkable success in inducing firing activity in response to light, both in vitro and in vivo. Ziapin2 showed a distinct affinity for lipid rafts, membrane domains characterized by high concentrations of diverse proteins, including ion channels and receptors. Building upon this knowledge, our investigation focused on the light-dependent modulation of synaptic neurotransmission in primary neurons mediated by Ziapin2, recognizing the pivotal role of lipid rafts in neurotransmitter release. This exploration involved distinguishing between excitatory and inhibitory neuronal subpopulations, considering their inherent physiological differences in protein expression and synaptic machinery. The results revealed an opposite modulation pattern in light-triggered modulation of synaptic transmission, providing insights on its potential applications in controlling neural networks. Moreover, we conducted a comprehensive characterization of 2Pyr-2Pyr, a photoswitch closely related to Ziapin2 but featuring a structural distinction that could potentially enhance its functionality. Our findings indicate that 2Pyr-2Pyr shares similar properties in modulating neuronal activity, offering the prospect of exploiting its structural difference for further engineering. Specifically, it could enable prolonged cellular membrane permanence, addressing a crucial challenge faced by Ziapin2, which is internalization through membrane recycling mechanisms over time. In addition, this thesis studies newly synthetized Push-Pull molecules, employing a distinct donor-acceptor mechanism for membrane voltage modulation, unlike Ziapin2's capacitance modulation. The characterization of these molecules not only offered insights into alternative strategies for light-induced neuronal stimulation but also enhanced our understanding of the essential patterns of membrane voltage modulation required to initiate light-dependent firing activity. Lastly, we present BV-1, a donor-acceptor photoswitch without the azobenzene core, in contrast to the previously studied photoswitches. BV-1 not only demonstrated efficient membrane voltage modulation but also exhibited the unique ability to induce light-triggered membrane poration, expanding the potential repertoire of light-sensitive tools for neuroscience research. In conclusion, this Ph.D. thesis characterizes four membrane-targeted photoswitches with a focus on their potential applications in modulating membrane voltage, neuronal activity, and cell viability, while exploring the underlying mechanisms. The findings highlight the diverse functional properties of these photoswitches and their potential applications in neuroscience. This research contributes to the growing body of knowledge in the field of optostimulation and paves the way for the development of more efficient and precise tools for neuroscience experimentation

A microfluidic-based PDAC organoid system reveals the impact of hypoxia in response to treatment

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is estimated to become the second leading cause of cancer-related deaths by 2030 with mortality rates of up to 93%. Standard of care chemotherapeutic treatment only prolongs the survival of patients for a short timeframe. Therefore, it is important to understand events driving treatment failure in PDAC as well as identify potential more effective treatment opportunities. PDAC is characterized by a high-density stroma, high interstitial pressure and very low oxygen tension. The aim of this study was to establish a PDAC platform that supported the understanding of treatment response of PDAC organoids in mono-, and co-culture with pancreatic stellate cells (PSCs) under hypoxic and normoxic conditions. Cultures were exposed to Gemcitabine in combination with molecules targeting relevant molecular programs that could explain treatment specific responses under different oxygen pressure conditions. Two groups of treatment responses were identified, showing either a better effect in monoculture or co-culture. Moreover, treatment response also differed between normoxia and hypoxia. Modulation of response to Gemcitabine was also observed in presence of a Hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) inhibitor and HIF inhibitors. Altogether this highlights the importance of adjusting experimental conditions to include relevant oxygen levels in drug response studies in PDAC

Synaptic and supra-synaptic organisation of the dopaminergic projection to the striatum

Dopamine transmission is a monoaminergic system involved in reward processing and motor control. Volume transmission is thought to be the main mechanism by which monoamines modulate effector transmission though synaptic structures are scarcely described. Here, we applied a fluorescence activated synaptosome sorting workflow to dopaminergic projections to the striatum and explored cellular and molecular features of the dopaminergic synaptome. This demonstrated that dopaminergic varicosities adhere to post-synaptic membrane baring cognate receptors. We further identified a specific bond of varicosities to glutamatergic or GABAergic synapses in structures we named dopaminergic “hub synapses”. Finally, we showed that the synaptic adhesion protein SynCAM 2 is strongly expressed at dopaminergic hub synapses. Our data strongly suggest that neuromodulation frequently operates from hub-synapses on local receptors, presumably in conjunction with extra-synaptic volume transmission. We provide a new framework for the molecular exploration of dopaminergic synapses and more generally on discrete synapse populations ex-vivo

A synaptomic analysis reveals dopamine hub synapses in the mouse striatum

International audienceDopamine transmission is involved in reward processing and motor control, and its impairment plays a central role in numerous neurological disorders. Despite its strong pathophysiological relevance, the molecular and structural organization of the dopaminergic synapse remains to be established. Here, we used targeted labelling and fluorescence activated sorting to purify striatal dopaminergic synaptosomes. We provide the proteome of dopaminergic synapses with 57 proteins specifically enriched. Beyond canonical markers of dopamine neurotransmission such as dopamine biosynthetic enzymes and cognate receptors, we validated 6 proteins not previously described as enriched. Moreover, our data reveal the adhesion of dopaminergic synapses to glutamatergic, GABAergic or cholinergic synapses in structures we named “dopamine hub synapses”. At glutamatergic synapses, pre- and postsynaptic markers are significantly increased upon association with dopamine synapses. Dopamine hub synapses may thus support local dopaminergic signalling, complementing volume transmission thought to be the major mechanism by which monoamines modulate network activity

Nanoactuator for Neuronal Optoporation

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing “on-demand” light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation

Nanoactuator for Neuronal Optoporation

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing “on-demand” light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation

Nanoactuator for Neuronal Optoporation

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing “on-demand” light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation