-
The Journal of Biological Chemistry May 2024Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it was reported that neuronal activity affects α-syn secretion, the...
Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it was reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1 mice). Glutamate-induced α-syn and p62 secretion was suppressed by knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion was also reduced by knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.
PubMed: 38815862
DOI: 10.1016/j.jbc.2024.107419 -
BMC Cardiovascular Disorders May 2024Myocardial ischemia-reperfusion injury (I/RI) is a major cause of perioperative cardiac-related adverse events and death. Studies have shown that sevoflurane...
BACKGROUND
Myocardial ischemia-reperfusion injury (I/RI) is a major cause of perioperative cardiac-related adverse events and death. Studies have shown that sevoflurane postconditioning (SpostC), which attenuates I/R injury and exerts cardioprotective effects, regulates mitochondrial dynamic balance via HIF-1α, but the exact mechanism is unknown. This study investigates whether the PI3K/AKT pathway in SpostC regulates mitochondrial dynamic balance by mediating HIF-1α, thereby exerting myocardial protective effects.
METHODS
The H9C2 cardiomyocytes were cultured to establish the hypoxia-reoxygenation (H/R) model and randomly divided into 4 groups: Control group, H/R group, sevoflurane postconditioning (H/R + SpostC) group and PI3K/AKT blocker (H/R + SpostC + LY) group. Cell survival rate was determined by CCK-8; Apoptosis rate was determined by flow cytometry; mitochondrial membrane potential was evaluated by Mito Tracker™ Red; mRNA expression levels of AKT, HIF-1α, Opa1and Drp1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR); Western Blot assay was used to detect the protein expression levels of AKT, phosphorylated AKT (p-AKT), HIF-1α, Opa1 and Drp1.
RESULTS
Compared with the H/R group, the survival rate of cardiomyocytes in the H/R + SpostC group increased, the apoptosis rate decreased and the mitochondrial membrane potential increased. qRT-PCR showed that the mRNA expression of HIF-1α and Opa1 were higher in the H/R + SpostC group compared with the H/R group, whereas the transcription level of Drp1 was lower in the H/R + SpostC group. In the H/R + SpostC + LY group, the mRNA expression of HIF-1α was lower than the H/R + SpostC group. There was no difference in the expression of Opa1 mRNA between the H/R group and the H/R + SpostC + LY group. WB assay results showed that compared with the H/R group, the protein expression levels of HIF-1α, Opa1, P-AKT were increased and Drp1 protein expression levels were decreased in the H/R + SpostC group. HIF-1α, P-AKT protein expression levels were decreased in the H/R + SpostC + LY group compared to the H/R + SpostC group.
CONCLUSION
SpostC mediates HIF-1α-regulated mitochondrial fission and fusion-related protein expression to maintain mitochondrial dynamic balance by activating the PI3K/AKT pathway and increasing AKT phosphorylation, thereby attenuating myocardial I/R injury.
Topics: Hypoxia-Inducible Factor 1, alpha Subunit; Proto-Oncogene Proteins c-akt; Animals; Myocytes, Cardiac; Sevoflurane; Signal Transduction; Myocardial Reperfusion Injury; Mitochondrial Dynamics; Cell Line; Rats; Apoptosis; Phosphatidylinositol 3-Kinase; Mitochondria, Heart; Membrane Potential, Mitochondrial; Cell Hypoxia; Dynamins; GTP Phosphohydrolases; Phosphoinositide-3 Kinase Inhibitors; Cytoprotection; Ischemic Postconditioning; Phosphorylation
PubMed: 38811893
DOI: 10.1186/s12872-024-03868-1 -
Cell Reports May 2024Synaptic vesicle docking and priming are dynamic processes. At the molecular level, SNAREs (soluble NSF attachment protein receptors), synaptotagmins, and other factors...
Synaptic vesicle docking and priming are dynamic processes. At the molecular level, SNAREs (soluble NSF attachment protein receptors), synaptotagmins, and other factors are critical for Ca-triggered vesicle exocytosis, while disassembly factors, including NSF (N-ethylmaleimide-sensitive factor) and α-SNAP (soluble NSF attachment protein), disassemble and recycle SNAREs and antagonize fusion under some conditions. Here, we introduce a hybrid fusion assay that uses synaptic vesicles isolated from mouse brains and synthetic plasma membrane mimics. We included Munc18, Munc13, complexin, NSF, α-SNAP, and an ATP-regeneration system and maintained them continuously-as in the neuron-to investigate how these opposing processes yield fusogenic synaptic vesicles. In this setting, synaptic vesicle association is reversible, and the ATP-regeneration system produces the most synchronous Ca-triggered fusion, suggesting that disassembly factors perform quality control at the early stages of synaptic vesicle association to establish a highly fusogenic state. We uncovered a functional role for Munc13 ancillary to the MUN domain that alleviates an α-SNAP-dependent inhibition of Ca-triggered fusion.
PubMed: 38809756
DOI: 10.1016/j.celrep.2024.114026 -
Frontiers in Oncology 2024Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed by normal prostatic tissue. Therefore, molecular imaging targeting PSMA (PSMA-PET) has...
INTRODUCTION
Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed by normal prostatic tissue. Therefore, molecular imaging targeting PSMA (PSMA-PET) has gained particular interest and diffusion for PCa staging and restaging. Several factors may affect PSMA-PET results, and many tools have been proposed to improve patient selection. Furthermore, PSMA expression is not homogeneous among different tissues and within the prostate itself. The aims of this study were to evaluate immunohistochemistry (IHC) features of prostate biopsy samples and to assess their correlation with whole-mount specimens and PSMA-PET parameters.
METHODS
We included consecutive high-risk PCa patients who underwent PSMA-PET for staging proposal at our institution from January 2022 to December 2022. The PET parameters selected were SUVmax, total volume (TV), and total lesion activity (TL). Each patient underwent multiparametric MRI (mpMRI) and fusion-targeted prostate biopsy prior to surgery. IHC analyses were performed on the index lesion cores. IHC visual score (VS) (1, 2, 3) and visual pattern (VP) (membranous, cytoplasmic, and combined) and the percentage of PSMA-negative tumor areas (PSMA%neg) within biopsy cores were evaluated.
RESULTS
Forty-three patients who underwent robotic radical prostatectomy after PSMA-PET were available for analyses. Concordance between VS and VP at biopsy and final pathology showed a Cohen's kappa coefficient of 0.39 and 0.38, respectively. Patients with PSMA%neg <20% had a higher concordance in VS and VP (Cohen's kappa 0.49 and 0.4, respectively). No difference emerged in terms of median PSMA-TV ( = 0.3) and PSMA-TL ( = 0.9) according to VS at biopsy, while median SUVmax was higher in patients with VS 3 ( = 0.04). Higher SUVmax was associated with membranous and combined VP expression ( = 0.008). No difference emerged between patients with PSMA%neg <20% or PSMA%neg >20% on biopsy cores in terms of SUVmax, PSMA-TL, and PSMA-TV ( = 0.5, = 0.5, and = 0.9 respectively).
CONCLUSIONS
We found a correlation between IHC VS and VP on targeted biopsy cores and SUVmax at PSMA-PET. However, the correlation between the IHC parameters of biopsy cores and final pathology was not as high as expected. Nevertheless, the presence of PSMA%neg <20% seems to have a better concordance in terms of visual score.
PubMed: 38807770
DOI: 10.3389/fonc.2024.1324631 -
Synthetic Biology (Oxford, England) 2024Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow...
Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG-the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins-in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.
PubMed: 38807757
DOI: 10.1093/synbio/ysae007 -
BMC Ophthalmology May 2024Macular retinoschisis (MRS) and myopic macular neovascularization (mMNV) are both potentially blinding complications of high myopia. In this case report, we highlight... (Review)
Review
BACKGROUND
Macular retinoschisis (MRS) and myopic macular neovascularization (mMNV) are both potentially blinding complications of high myopia. In this case report, we highlight the progression of MRS after intravitreal anti-vascular endothelial growth factor (anti-VEGF) treatment for mMNV, as well as an extensive review of the literature on this topic.
CASE DESCRIPTION
A 49-year-old woman presented with two weeks of recent onset blurring and metamorphopsia in her right eye. She had high myopia in both eyes (right eye - 20/60 with - 16D, left eye - 20/20 with - 13D). Slit-lamp ophthalmoscopy found a normal anterior segment in both eyes. On fundus examination, features of pathological myopia with posterior staphyloma and peripapillary atrophy were observed in both eyes. An active mMNV, as well as intraretinal fluid, minimal perifoveal inner and outer MRS, and focal posterior vitreous traction along the inferotemporal retinal arcade, were detected on optical coherence tomography (OCT) of the right eye. The patient received an intravitreal injection of Aflibercept (2 mg/0.05 ml).
RESULTS
OCT scans at two- and four-month follow-up visits revealed regressed mMNV with a taut epiretinal membrane, progressive worsening of outer MRS, and the development of multiple perifoveal retinal detachment inferior to the fovea. Pars plana vitrectomy surgery was performed for the progressive MRS with good anatomical (resolved MRS) and functional outcome (maintained visual acuity at 20/60) at the last one-month post-surgery visit.
CONCLUSION
Intravitreal anti-VEGF injections for mMNV can cause vitreoretinal interface changes, exacerbating MRS and causing visual deterioration. Vitrectomy for MRS could be one of several treatment options.
Topics: Humans; Receptors, Vascular Endothelial Growth Factor; Female; Intravitreal Injections; Middle Aged; Retinoschisis; Recombinant Fusion Proteins; Myopia, Degenerative; Tomography, Optical Coherence; Visual Acuity; Angiogenesis Inhibitors; Disease Progression; Retinal Neovascularization; Fluorescein Angiography
PubMed: 38807066
DOI: 10.1186/s12886-024-03497-4 -
Light, Science & Applications May 2024Structured illumination microscopy (SIM) has emerged as a promising super-resolution fluorescence imaging technique, offering diverse configurations and computational...
Structured illumination microscopy (SIM) has emerged as a promising super-resolution fluorescence imaging technique, offering diverse configurations and computational strategies to mitigate phototoxicity during real-time imaging of biological specimens. Traditional efforts to enhance system frame rates have concentrated on processing algorithms, like rolling reconstruction or reduced frame reconstruction, or on investments in costly sCMOS cameras with accelerated row readout rates. In this article, we introduce an approach to elevate SIM frame rates and region of interest (ROI) coverage at the hardware level, without necessitating an upsurge in camera expenses or intricate algorithms. Here, parallel acquisition-readout SIM (PAR-SIM) achieves the highest imaging speed for fluorescence imaging at currently available detector sensitivity. By using the full frame-width of the detector through synchronizing the pattern generation and image exposure-readout process, we have achieved a fundamentally stupendous information spatial-temporal flux of 132.9 MPixels · s, 9.6-fold that of the latest techniques, with the lowest SNR of -2.11 dB and 100 nm resolution. PAR-SIM demonstrates its proficiency in successfully reconstructing diverse cellular organelles in dual excitations, even under conditions of low signal due to ultra-short exposure times. Notably, mitochondrial dynamic tubulation and ongoing membrane fusion processes have been captured in live COS-7 cell, recorded with PAR-SIM at an impressive 408 Hz. We posit that this novel parallel exposure-readout mode not only augments SIM pattern modulation for superior frame rates but also holds the potential to benefit other complex imaging systems with a strategic controlling approach.
PubMed: 38806501
DOI: 10.1038/s41377-024-01464-8 -
Nature Communications May 2024Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable...
Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable membrane fusion in-vivo through specific molecules such as SNAREs, controlling the fusion in-vitro while fabricating artificial membrane structures in physiological ionic solutions without fusion proteins has been a challenge, becoming a significant obstacle to practical applications. We present an approach consisting of an electric field and a few kPa hydraulic pressure as an additional variable to physically control the fusion, enabling tuning of the shape and size of the 3D freestanding lipid bilayers in physiological ionic solutions. Mechanical model analysis reveals that pressure-induced parallel/normal tensions enhance fusion among membranes in the microwell. In-vitro peptide-membrane assay, mimicking vesicular transport via pressure-assisted fusion, and stability of 38 days with in-chip pressure control via pore size-regulated hydrogel highlight the potential for diverse biological applications.
Topics: Lipid Bilayers; Membrane Fusion; Ions; Membranes, Artificial; Hydrogels; Pressure; Peptides
PubMed: 38806492
DOI: 10.1038/s41467-024-48875-0 -
Frontiers in Cellular Neuroscience 2024The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) superfamily plays a pivotal role in cellular trafficking by facilitating... (Review)
Review
The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) superfamily plays a pivotal role in cellular trafficking by facilitating membrane fusion events. These SNARE proteins, including syntaxins, assemble into complexes that actively facilitate specific membrane fusion events. Syntaxins, as integral components of the SNARE complex, play a crucial role in initiating and regulating these fusion activities. While specific syntaxins have been extensively studied in various cellular processes, including neurotransmitter release, autophagy and endoplasmic reticulum (ER)-to-Golgi protein transport, their roles in the retina remain less explored. This review aims to enhance our understanding of syntaxins' functions in the retina by shedding light on how syntaxins mediate membrane fusion events unique to the retina. Additionally, we seek to establish a connection between syntaxin mutations and retinal diseases. By exploring the intricate interplay of syntaxins in retinal function and health, we aim to contribute to the broader comprehension of cellular trafficking in the context of retinal physiology and pathology.
PubMed: 38799985
DOI: 10.3389/fncel.2024.1380064 -
ACS Omega May 2024Cells inhabit a mechanical microenvironment that they continuously sense and adapt to. The plasma membrane (PM), serving as the boundary of the cell, plays a pivotal... (Review)
Review
Cells inhabit a mechanical microenvironment that they continuously sense and adapt to. The plasma membrane (PM), serving as the boundary of the cell, plays a pivotal role in this process of adaptation. In this Review, we begin by examining well-studied processes where mechanoregulation proves significant. Specifically, we highlight examples from the immune system and stem cells, besides discussing processes involving fibroblasts and other cell types. Subsequently, we discuss the common molecular players that facilitate the sensing of the mechanical signal and transform it into a chemical response covering integrins YAP/TAZ and Piezo. We then review how this understanding of molecular elements is leveraged in drug discovery and tissue engineering alongside a discussion of the methodologies used to measure mechanical properties. Focusing on the processes of endocytosis, we discuss how cells may respond to altered membrane mechanics using endo- and exocytosis. Through the process of depleting/adding the membrane area, these could also impact membrane mechanics. We compare pathways from studies illustrating the involvement of endocytosis in mechanoregulation, including clathrin-mediated endocytosis (CME) and the CLIC/GEEC (CG) pathway as central examples. Lastly, we review studies on cell-cell fusion during myogenesis, the mechanical integrity of muscle fibers, and the reported and anticipated roles of various molecular players and processes like endocytosis, thereby emphasizing the significance of mechanoregulation at the PM.
PubMed: 38799362
DOI: 10.1021/acsomega.4c01962