Vesicle Release Research Articles

Kif1a and intact microtubules maintain synaptic-vesicle populations at ribbon synapses in zebrafish hair cells.

Sensory hair cells of the inner ear utilize specialized ribbon synapses to transmit sensory stimuli to the central nervous system. This sensory transmission necessitates rapid and sustained neurotransmitter release, which relies on a large pool of synaptic vesicles at the hair-cell presynapse. Work in neurons has shown that kinesin motor proteins traffic synaptic material along microtubules to the presynapse, but how new synaptic material reaches the presynapse in hair cells is not known. We show that the kinesin motor protein Kif1a and an intact microtubule network are necessary to enrich synaptic vesicles at the presynapse in hair cells. We use genetics and pharmacology to disrupt Kif1a function and impair microtubule networks in hair cells of the zebrafish lateral-line system. We find that these manipulations decrease synaptic-vesicle populations at the presynapse in hair cells. Using electron microscopy, along with in vivo calcium imaging and electrophysiology, we show that a diminished supply of synaptic vesicles adversely affects ribbon-synapse function. Kif1a mutants exhibit dramatic reductions in spontaneous vesicle release and evoked postsynaptic calcium responses. Additionally, we find that kif1a mutants exhibit impaired rheotaxis, a behavior reliant on the ability of hair cells in the lateral line to respond to sustained flow stimuli. Overall, our results demonstrate that Kif1a-based microtubule transport is critical to enrich synaptic vesicles at the active zone in hair cells, a process that is vital for proper ribbon-synapse function.

Glutathione S-transferase omega class 1 (GSTO1)-associated large extracellular vesicles are involved in tumor-associated macrophage-mediated cisplatin resistance in bladder cancer.

Bladder cancer poses a significant challenge to chemotherapy due to its resistance to cisplatin, especially at advanced stages. Understanding the mechanisms behind cisplatin resistance is crucial for improving cancer therapy. The enzyme glutathione S-transferase omega class 1 (GSTO1) is known to be involved in cisplatin resistance in colon cancer. This study focused on its role in cisplatin resistance in bladder cancer. Our analysis of protein expression in bladder cancer cells stimulated by secretions from tumor-associated macrophages (TAMs) showed a significant increase in GSTO1. This prompted further investigation into the role of GSTO1 in bladder cancer. We found a strong correlation between GSTO1 expression and cisplatin resistance. Mechanistically, GSTO1 triggered the release of large extracellular vesicles (EVs) that promoted cisplatin efflux, thereby reducing cisplatin-DNA adduct formation and enhancing cisplatin resistance. Inhibition of EV release effectively counteracted the cisplatin resistance associated with GSTO1. In conclusion, GSTO1-mediated EV release may contribute to cisplatin resistance caused by TAMs in bladder cancer. Strategies to target GSTO1 could potentially improve the efficacy of cisplatin in treating bladder cancer.

The LRRK2-G2019S mutation attenuates repair of brain injury partially by reducing the release of osteopontin-containing monocytic exosome-like vesicles

BackgroundBrain injury has been suggested as a risk factor for neurodegenerative diseases. Accordingly, defects in the brain's intrinsic capacity to repair injury may result in the accumulation of damage and a progressive loss of brain function. The G2019S (GS) mutation in LRRK2 (leucine rich repeat kinase 2) is the most prevalent genetic alteration in Parkinson's disease (PD). Here, we sought to investigate how this LRRK2-GS mutation affects repair of the injured brain. MethodsBrain injury was induced by stereotaxic injection of ATP, a damage-associated molecular pattern (DAMP) component, into the striatum of wild-type (WT) and LRRK2-GS mice. Effects of the LRRK2-GS mutation on brain injury and the recovery from injury were examined by analyzing the molecular and cellular behavior of neurons, astrocytes, and monocytes. ResultsDamaged neurons express osteopontin (OPN), a factor associated with brain repair. Following ATP-induced damage, monocytes entered injured brains, phagocytosing damaged neurons and producing exosome-like vesicles (EVs) containing OPN through activation of the inflammasome and subsequent pyroptosis. Following EV production, neurons and astrocytes processes elongated towards injured cores. In LRRK2-GS mice, OPN expression and monocytic pyroptosis were decreased compared with that in WT mice, resulting in diminished release of OPN-containing EVs and attenuated elongation of neuron and astrocyte processes. In addition, exosomes prepared from injured LRRK2-GS brains induced neurite outgrowth less efficiently than those from injured WT brains. ConclusionsThe LRRK2-GS mutation delays repair of injured brains through reduced expression of OPN and diminished release of OPN-containing EVs from monocytes. These findings suggest that the LRRK2-GS mutation may promote the development of PD by delaying the repair of brain injury.

Parallel processing of quickly and slowly mobilized reserve vesicles in hippocampal synapses.

Vesicles within presynaptic terminals are thought to be segregated into a variety of readily releasable and reserve pools. The nature of the pools and trafficking between them is not well understood, but pools that are slow to mobilize when synapses are active are often assumed to feed pools that are mobilized more quickly, in a series. However, electrophysiological studies of synaptic transmission have suggested instead a parallel organization where vesicles within slowly and quickly mobilized reserve pools would separately feed independent reluctant- and fast-releasing subdivisions of the readily releasable pool. Here, we use FM-dyes to confirm the existence of multiple reserve pools at hippocampal synapses and a parallel organization that prevents intermixing between the pools, even when stimulation is intense enough to drive exocytosis at the maximum rate. The experiments additionally demonstrate extensive heterogeneity among synapses in the relative sizes of the slowly and quickly mobilized reserve pools, which suggests equivalent heterogeneity in the numbers of reluctant and fast-releasing readily releasable vesicles that may be relevant for understanding information processing and storage.

Parallel processing of quickly and slowly mobilized reserve vesicles in hippocampal synapses

Vesicles within presynaptic terminals are thought to be segregated into a variety of readily releasable and reserve pools. The nature of the pools and trafficking between them is not well understood, but pools that are slow to mobilize when synapses are active are often assumed to feed pools that are mobilized more quickly, in a series. However, electrophysiological studies of synaptic transmission have suggested instead a parallel organization where vesicles within slowly and quickly mobilized reserve pools would separately feed independent reluctant- and fast-releasing subdivisions of the readily releasable pool. Here, we use FM-dyes to confirm the existence of multiple reserve pools at hippocampal synapses and a parallel organization that prevents intermixing between the pools, even when stimulation is intense enough to drive exocytosis at the maximum rate. The experiments additionally demonstrate extensive heterogeneity among synapses in the relative sizes of the slowly and quickly mobilized reserve pools, which suggests equivalent heterogeneity in the numbers of reluctant and fast-releasing readily releasable vesicles that may be relevant for understanding information processing and storage.

A Mutual Nexus Between Epilepsy and α-Synuclein: A Puzzle Pathway.

Alpha-synuclein (α-Syn) is a specific neuronal protein that regulates neurotransmitter release and trafficking of synaptic vesicles. Exosome-associated α-Syn which is specific to the central nervous system (CNS) is involved in the pathogenesis of epilepsy. Therefore, this review aimed to elucidate the possible link between α-Syn and epilepsy, and how it affects the pathophysiology of epilepsy. A neurodegenerative protein such as α-Syn is implicated in the pathogenesis of epilepsy. Evidence from preclinical and clinical studies revealed that upregulation of α-Syn induces progressive neuronal dysfunctions through induction of oxidative stress, neuroinflammation, and inhibition of autophagy in a vicious cycle with subsequent development of severe epilepsy. In addition, accumulation of α-Syn in epilepsy could be secondary to the different cellular alterations including oxidative stress, neuroinflammation, reduction of brain-derived neurotrophic factor (BDNF) and progranulin (PGN), and failure of the autophagy pathway. However, the mechanism of α-Syn-induced-epileptogenesis is not well elucidated. Therefore, α-Syn could be a secondary consequence of epilepsy. Preclinical and clinical studies are warranted to confirm this causal relationship.

Hippocampal excitation-inhibition balance underlies the 5-HT2C receptor in modulating depressive behaviours.

The implication of 5-hydroxytryptamine 2C receptor (5-HT2CR) in depression is a topic of debate, and the underlying mechanisms remain largely unclear. We now elucidate hippocampal excitation-inhibition (E/I) balance underlies the regulatory effects of 5-HT2CR in depression. Molecular biological analyses showed that chronic mild stress (CMS) reduced the expression of 5-HT2CR in hippocampus. We revealed that inhibition of 5-HT2CR induced depressive-like behaviors, reduced GABA release and shifted the E/I balance towards excitation in CA3 pyramidal neurons by using behavioral analyses, microdialysis coupled with mass spectrum, and electrophysiological recording. Moreover, 5-HT2CR modulated neuronal nitric oxide synthase (nNOS)-carboxy-terminal PDZ ligand of nNOS (CAPON) interaction through influencing intracellular Ca2+ release, as determined by fiber photometry and coimmunoprecipitation. Notably, disruption of nNOS-CAPON by specific small molecule compound ZLc-002 or AAV-CMV-CAPON-125C-GFP, abolished 5-HT2CR inhibition-induced depressive-like behaviors, as well as the impairment in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly-mediated GABA vesicle release and a consequent E/I imbalance. Importantly, optogenetic inhibition of CA3 GABAergic neurons prevented the effects of AAV-CMV-CAPON-125C-GFP on depressive behaviors in the presence of 5-HT2CR antagonist. Conclusively, our findings disclose the regulatory role of 5-HT2CR in depressive-like behaviors and highlight the hippocampal nNOS-CAPON coupling-triggered E/I imbalance as a pivotal cellular event underpinning the behavioral consequences of 5-HT2CR inhibition.

Metabolic regulation of single synaptic vesicle exo- and endocytosis in hippocampal synapses

Glucose has long been considered a primary energy source for synaptic function. However, it remains unclear to what extent alternative fuels, such as lactate/pyruvate, contribute to powering synaptic transmission. By detecting individual release events in hippocampal synapses, we find that mitochondrial ATP production regulates basal vesicle release probability and release location within the active zone (AZ), evoked by single action potentials. Mitochondrial inhibition shifts vesicle release closer to the AZ center and alters the efficiency of vesicle retrieval by increasing the occurrence of ultrafast endocytosis. Furthermore, we uncover that terminals can use oxidative fuels to maintain the vesicle cycle during trains of activity. Mitochondria are sparsely distributed along hippocampal axons, and we find that terminals containing mitochondria display enhanced vesicle release and reuptake during high-frequency trains. Our findings suggest that mitochondria not only regulate several fundamental features of synaptic transmission but may also contribute to modulation of short-term synaptic plasticity.

Skeletal Muscle Extracellular Vesicles Enhance Mitochondrial Movement and have a Dose-dependent Effect on Increasing Mitochondrial Respiration

Chronic exercise leads to systemic health benefits across tissues, including an increase in mitochondrial function in skeletal muscle. One potential mechanism is via the release of extracellular vesicles (EVs), which can transfer functional cargo to recipient cells. Previously, we have shown that using an in vitro model of exercise, chronic contractile activity (CCA)-derived EVs (CCA-EV) increased mitochondrial biogenesis in healthy myoblasts. Here, we hypothesize that CCA-EVs will have a concentration-dependent effect on mitochondrial respiration, and also regulate mitochondrial dynamics in myoblasts. C2C12 myoblasts were differentiated into myotubes, and electrically paced (3hrs/day, 4 days, 14V, C-PACE EM, IonOptix). EVs from control and CCA-stimulated myotubes were isolated from conditioned media using differential ultracentrifugation and biophysically characterized by size and concentration using TRPS (Izon). C2C12 myoblasts were treated daily for 4 days using three different concentrations: 1.0E4; 2.0E4 and 4.0E4 EVs/cell (N=4-5). Basal and maximal oxygen consumption rates (OCR) were measured using the Seahorse XFe24 (Agilent). We used live-cell fluorescence microscopy and Mitometer software to access mitochondrial movement: area/volume, displacement, distance, length, perimeter, speed and velocity. Average size was unchanged between control-EVs (123±10.8 nm) and CCA-EVs (123±11.9 nm) (p=0.9137, N=3), and 2.3-fold more CCA-EVs were released vs. control-EVs (p=0.2072, N=3). Lowest EV concentration (1.0E+4 EVs/cell, N=5) had no effect on basal (p=0.4226) and maximal (p=0.1835) OCR. Intermediate dosage of CCA-EVs (2.0E+4 EVs/cell, N=5) increased basal OCR by 20% (p=0.0037) and maximal OCR by 18% (p=0.0095), while the highest concentration (4.0E+4 EVs/cell, N=4) decreased basal OCR by 9% (p=0.0001) and maximal OCR by 29% (p=0.0009), compared to control-EVs. Mitochondrial motility (N=484-976) in myoblasts treated daily after each day of CCA showed enhanced mitochondrial dynamics with CCA-EVs: increased area/volume (p<0.0001), displacement (p<0.0001), distance (p<0.0001), mean intensity (p<0.0001), perimeter/surface area (p=0.0137) and speed (p<0.0001), compared to control-EVs. In summary, CCA-EVs induced concentration-dependent improvements in mitochondrial respiration in healthy myoblasts, concomitant with enhanced mitochondrial movement. These results open exciting avenues for the therapeutic potential of CCA-EVs in rescuing mitochondrial dysfunction. TFGS is funded by a Research Manitoba Postdoctoral Fellowship. Grants from CHRIM, CHF, DREAM and NSERC to AS funded the research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

K+ Channel Activation Inhibits Allergen-Stimulated ATP Release by Human Bronchial Epithelial Cells

Allergens derived from the fungus Alternaria alternata stimulate the release of multiple alarmins including ATP, IL-33 and DNA fragments from human bronchial epithelial cells (hBECs). Oxidative stress induced by Alternaria extract activates ATP release by opening voltage-dependent anion channels (VDAC-1) in the apical membrane and by stimulating exocytosis of ATP containing membrane vesicles. The present study shows that pretreatment of hBECs with K2P channel inhibitors prior to Alternaria exposure significantly increases the initial rate of ATP release and inhibition of ATP loading into membrane vesicles with bafilomycin A1 blocks the response. Furthermore, pretreatment with the K+ channel openers, EBIO (KCa3.1 channels), NS11021 (KCa1.1 channels) and pinacidil (KATP channels) each inhibited the bafilomycin A1-dependent component of ATP release, reducing the initial rate of ATP secretion by ~50%. K2P channel blockers caused depolarization whereas K+ channel openers produced hyperpolarization of the cell membrane. Additionally, K2P channel inhibitors also increased Alternaria-stimulated Ca2+ uptake into hBECs while K+ channel openers reduced the Alternaria response by ~50%. Imaging experiments involving visualization of fluorescently labeled MARCKS proteins in the apical membrane showed that treatment with Alternaria caused a significant decrease in fluorescence intensity, however pretreatment with K+ channel openers, but not K2P channel blockers, inhibited the effect. These results are consistent with recent findings showing that MARCKS proteins bind to the plasma membrane through an electrostatic association with PIP2 and that phosphorylation by PKC or membrane hyperpolarization following K+ channel activation disrupts this interaction resulting in MARCKS detachment from the membrane. MARCKS detachment uncouples the membrane from the actin cytoskeleton, facilitating vesicle docking, fusion and release of ATP. This study was supported by NIH grants (AI128729 and AI169530) to HK and SMO and by USDA-AES 0016134 to SMO. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19

BackgroundCOVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. ObjectivesFrom the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. MethodsWe utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. ResultsCompared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. ConclusionWe conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.

Astragaloside IV inhibits colorectal cancer metastasis by reducing extracellular vesicles release and suppressing M2-type TAMs activation

Ethnopharmacological relevanceTumour-derived extracellular vesicles (TEVs) have been confirmed to facilitate colorectal cancer (CRC) metastasis by remodelling the tumour microenvironment (TME). Drugs targeted TEVs is considered as a promising therapeutic strategy for cancer treatment. Traditional Chinese medicine (TCM) plays a vital role in improving the prognosis of CRC patients and eventually CRC patients with distant metastasis. Although the anti-tumour effects of active compounds from TCM prescriptions are observed widely, the molecular mechanisms remain unknown. Aim of the studyThis study aims to investigate the effects of active compounds in our library of TCM on preventing CRC metastasis, and also explore the potential mechanisms from the perspective of TEVs. Materials and methods: The effects of active compounds on the proliferation of CRC cells were determined by CCK-8 assay. TEVs were extracted from MC38 cells by ultracentrifugation and characterized by electron microscopy, Nanosight NS300 and western blotting. The TEV particles were quantified by Nanosight NS300. The potential mechanism by which astragaloside IV (ASIV) reduced TEV secretion was determined by western blotting. RAW264.7 cells were cocultured with the conditioned medium (CM) of MC38 cells treated with or without ASIV, and the activation of tumour-associated macrophages (TAMs) was assessed by immunofluorescence and quantitative polymerase chain reaction (qPCR). The migration of CRC cells was measured by wound healing and Transwell assay. A spleen-to-liver metastasis model of colorectal cancer was used to confirm the efficiency of ASIV in vivo. Liver metastatic tumours of the mice were used for liver weight measures and H&E staining. Immunofluorescence was applied to observe the infiltration of TAMs, the expression of neutral sphingomyelinase 2 (nSMase2) and Rab27a. ResultsBy screening our TCM monomer library, we found that ASIV, which is mainly extracted from Radix Astragali, reduced the release of TEVs from CRC cells in a time- and concentration-dependent manner. Mechanistically, ASIV inhibited the production and secretion of TEVs by downregulating nSMase2 and Rab27a expression in CRC cells. CM from ASIV-treated CRC cells reshaped the polarization of TAMs by decreasing M2-type polarization, increasing M1-type polarization. Consequently, the repolarization of M2-type to M1-type macrophages led to reduced invasion and migration of CRC cells. Moreover, we confirmed that ASIV inhibited the liver metastasis of CRC, reduced M2-type macrophage infiltration and decreased the expression of nSMase2 and Rab27a in liver metastases. ConclusionsASIV inhibited CRC metastasis by reducing EVs release and suppressing M2-type TAMs activation. All these findings reveal a new insight into the mechanisms of ASIV in preventing CRC progression and provide a promising approach for anti-tumour therapy.

Measuring Presynaptic Calcium Influx at the Drosophila Larval Neuromuscular Junction.

Synaptic transmission plays a critical role in information processing and storage within the nervous system. The triggering of action potentials activates voltage-gated calcium channels at presynaptic active zones, facilitating the calcium-dependent release of synaptic vesicles. Homeostatic mechanisms are crucial in stabilizing synaptic function. At the Drosophila neuromuscular junction, a compensatory increase in presynaptic neurotransmitter release occurs when postsynaptic glutamate receptor function is pharmacologically or genetically impaired, thereby stabilizing synaptic output. This adaptation is known as presynaptic homeostatic potentiation (PHP). Recent advancements, including confocal and super-resolution imaging techniques, have demonstrated an increase in presynaptic calcium influx during both the rapid induction and long-term maintenance of PHP. These observations indicate that the abundance and structural organization of presynaptic calcium channels, along with various active zone components, undergo modifications following the suppression of postsynaptic glutamate receptors. Such findings underscore the critical roles of trafficking and stabilization of presynaptic calcium channels and active zone proteins in homeostatic plasticity. This protocol describes using calcium indicators and confocal imaging methods to measure single-action potential-evoked presynaptic calcium influx during PHP.

Measuring the Readily Releasable Synaptic Vesicle Pool at the Drosophila Larval Neuromuscular Junction.

Presynaptic homeostatic potentiation (PHP) is a type of homeostatic regulation that stabilizes synaptic output under conditions where postsynaptic receptor function is impaired. PHP manifests as a significant increase in presynaptic neurotransmitter release, compensating for decreased postsynaptic receptor activity and thus maintaining stable excitation levels in postsynaptic cells. Presynaptic neurotransmitter release is calcium-dependent, initiated by calcium influx through voltage-gated calcium channels localized at the presynaptic active zones. This calcium influx triggers the fusion of vesicles from the readily releasable vesicle pool (RRP) that are ready for immediate release. Two key presynaptic cellular mechanisms are essential for PHP's induction and maintenance. First, a compensatory rise in the abundance of presynaptic calcium channels (and consequently, an increase in calcium influx) occurs when postsynaptic glutamate receptors are suppressed. Second, the RRP size enlarges during PHP. PHP is disrupted if either of these processes is impaired. This protocol outlines the use of the two-electrode voltage-clamp technique for assessing the RRP during PHP, induced either pharmacologically or genetically, at the Drosophila neuromuscular junction (NMJ). Electrophysiological recordings typically take place at the NMJ of muscle 6 in abdominal segments A2 and A3.

Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Exploring the effects of the acaricide cyflumetofen on the vital organs of the honey bee Apis mellifera (Hymenoptera: Apidae) workers

Bees are important for maintaining ecosystems, pollinating crops and producing marketable products. In recent years, a decline in bee populations has been reported, with multifactorial causes, including the intensification of pesticide use in agriculture. Among pesticides, cyflumetofen is an insecticide and acaricide used in apple, coffee and citrus crops, whose main pollinator is the honey bee Apis mellifera. Therefore, this bee is a potential target of cyflumetofen during foraging. This study evaluated the histopathological and cytological damage in the midgut, hypopharyngeal glands and fat body of A. mellifera workers exposed to LC50 of cyflumetofen. The midgut epithelium of exposed bees presented cytoplasmic vacuolization, release of vesicles and cell fragments, which indicate autophagy, increased production of digestive enzymes and cell death, respectively. The cytological analysis of the midgut revealed the dilation of the basal labyrinth and the presence of spherocrystals in the digestive cells. The hypopharyngeal glands produced greater amounts of secretion in treated bees, whereas no changes were observed in the fat body. The results indicate that acute exposure to cyflumetofen negatively affect A. mellifera, causing damage to the midgut and changes in the hypopharyngeal glands, which may compromise the survival and foraging of this pollinator.

Autophagy Deregulation in HIV-1-Infected Cells Increases Extracellular Vesicle Release and Contributes to TLR3 Activation.

Human immunodeficiency virus type 1 (HIV-1) infection can result in HIV-associated neurocognitive disorder (HAND), a spectrum of disorders characterized by neurological impairment and chronic inflammation. Combined antiretroviral therapy (cART) has elicited a marked reduction in the number of individuals diagnosed with HAND. However, there is continual, low-level viral transcription due to the lack of a transcription inhibitor in cART regimens, which results in the accumulation of viral products within infected cells. To alleviate stress, infected cells can release accumulated products, such as TAR RNA, in extracellular vesicles (EVs), which can contribute to pathogenesis in neighboring cells. Here, we demonstrate that cART can contribute to autophagy deregulation in infected cells and increased EV release. The impact of EVs released from HIV-1 infected myeloid cells was found to contribute to CNS pathogenesis, potentially through EV-mediated TLR3 (Toll-like receptor 3) activation, suggesting the need for therapeutics to target this mechanism. Three HIV-1 TAR-binding compounds, 103FA, 111FA, and Ral HCl, were identified that recognize TAR RNA and reduce TLR activation. These data indicate that packaging of viral products into EVs, potentially exacerbated by antiretroviral therapeutics, may induce chronic inflammation of the CNS observed in cART-treated patients, and novel therapeutic strategies may be exploited to mitigate morbidity.

Podocyte-specific silencing of acid sphingomyelinase gene to abrogate hyperhomocysteinemia-induced NLRP3 inflammasome activation and glomerular inflammation.

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.

Role of the dynamin-related protein 2 family and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana.

Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.

Extracellular Vesicles from Scedosporium apiospermum Mycelial Cells: Implication for Fungal-Host Interplays.

The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs' protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.