Search Results (52)

The biological effects of nanoparticles are closely related to their intracellular content and location, both of which are influenced by various factors. This study investigates the effects of surface charge on the uptake, intracellular distribution, and exocytosis of CdSe/ZnS quantum dots (QDs) in Raw264.7 macrophages. Negatively charged 3-mercaptopropanoic acid functionalized QDs (QDs-MPA) show higher cellular uptake than positively charged 2-mercaptoethylamine functionalized QDs (QDs-MEA), and serum enhances the uptake of both types of QDs via protein corona-mediated receptor endocytosis. QDs-MEA primarily enter the cells through clathrin/caveolae-mediated pathways and predominantly accumulate in lysosomes, while QDs-MPA are mainly internalized through clathrin-mediated endocytosis and localize to both lysosomes and mitochondria. Exocytosis of QDs-MPA is faster and more efficient than that of QDs-MEA, though both exhibit limited excretion. In addition to endocytosis and exocytosis, cell division influences intracellular QD content over time. These results reveal the charge-dependent interactions between QDs and macrophages, providing a basis for designing biocompatible nanomaterials. Full article

Hemerocallis citrina is an herbaceous perennial plant used in Asian cuisine and Traditional Chinese Medicine. Here, we tested the therapeutic potential of extracts (HCE30%, HCE50%, and HCN) in vivo, using models of two human genetic neurodegenerative diseases—Machado–Joseph Disease/Spinocerebellar Ataxia type 3 (MJD/SCA3) and Frontotemporal Dementia with Parkinsonism associated to chromosome 17 (FTDP-17). Chronic treatment with HCE30% extract ameliorated the motor deficits typically observed in these models. Interestingly, we found that the effect on the motor phenotype of the MJD/SCA3 model was dependent on serotonergic signaling and on the action of the HLH-30/TFEB transcription factor, known to regulate the cellular response to amino acid starvation, the autophagy and mitophagy pathways, lysosome localization and biogenesis, exocytosis, and mitochondrial biogenesis. Altogether, our findings reinforce the idea that phytochemicals act through the modulation of serotonergic neurotransmission and introduce a novel layer to the HLH-30/TFEB regulatory network. Thus, it also strengthens the use of these pathways as therapeutic targets for protein-related neurodegenerative disorders and confirms the utility of medicinal plants as a source of innovation in the quest for new therapeutic agents. Full article

Mutations in MAGED2 cause transient antenatal Bartter syndrome (tBS) characterized by excessive amounts of amniotic fluid due to impaired renal salt transport via NKCC2 and NCC, high perinatal mortality, and pre-term birth. Surprisingly, renal salt handling completely normalizes after birth. Previously, we demonstrated that, under hypoxic conditions, MAGED2 depletion enhances endocytosis of GalphaS (Gαs), reducing adenylate cyclase (AC) activation and cAMP production. This impaired cAMP signaling likely contributes to the dysfunction of salt transporters NKCC2 and NCC, explaining salt wasting and the subsequent recovery with renal oxygenation after birth. In this study, we show that MAGED2 depletion significantly decreases both total cellular and plasma membrane NCC expression and activity. We further demonstrate that MAGED2 depletion disrupts NCC trafficking by reducing exocytosis, increasing endocytosis, and promoting lysosomal degradation via enhanced ubiquitination. Additionally, forskolin (FSK), which increases cAMP production by activating AC, rescues NCC expression and localization in MAGED2-depleted cells. Conversely, MAGED2 overexpression increases NCC expression and membrane localization, although this effect is diminished in Gαs-depleted cells, indicating that Gαs acts downstream of MAGED2. In summary, our findings reveal the essential role of MAGED2 in regulating NCC function and trafficking under hypoxic conditions, providing new insights into the mechanisms behind salt loss in tBS and identifying potential therapeutic targets. Full article

Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial abnormalities, cardiac defects, and gastrointestinal disorders. The Ts65Dn mouse model replicates many abnormalities of DS. We hypothesized that investigation of the cerebral cortex of fluoxetine-treated trisomic mice may provide proteomic signatures that identify therapeutic targets for DS. Subcellular fractionation of synaptosomes from cerebral cortices of age- and brain-area-matched samples from fluoxetine-treated vs. water-treated trisomic and euploid male mice were subjected to HPLC-tandem mass spectrometry. Analysis of the data revealed enrichment of trisomic risk genes that participate in regulation of synaptic vesicular traffic, pre-synaptic and post-synaptic development, and mitochondrial energy pathways during early brain development. Proteomic analysis of trisomic synaptic fractions revealed significant downregulation of proteins involved in synaptic vesicular traffic, including vesicular endocytosis (CLTA, CLTB, CLTC), synaptic assembly and maturation (EXOC1, EXOC3, EXOC8), anterograde axonal transport (EXOC1), neurotransmitter transport to PSD (SACM1L), endosomal-lysosomal acidification (ROGDI, DMXL2), and synaptic signaling (NRXN1, HIP1, ITSN1, YWHAG). Additionally, trisomic proteomes revealed upregulation of several trafficking proteins, involved in vesicular exocytosis (Rab5B), synapse elimination (UBE3A), scission of endocytosis (DBN1), transport of ER in dendritic spines (MYO5A), presynaptic activity-dependent bulk endocytosis (FMR1), and NMDA receptor activity (GRIN2A). Chronic fluoxetine treatment of Ts65Dn mice rescued synaptic vesicular abnormalities and prevented abnormal proteomic changes in adult Ts65Dn mice, pointing to therapeutic targets for potential treatment of DS. Full article

HIV-associated neurocognitive disorders (HAND) affect 15–55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1_ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND. Full article

Although it has been known for decades that lysosomes are central for degradation and recycling in the cell, their pivotal role as nutrient sensing signaling hubs has recently become of central interest. Since lysosomes are highly dynamic and in constant change regarding content and intracellular position, fusion/fission events allow communication between organelles in the cell, as well as cell-to-cell communication via exocytosis of lysosomal content and release of extracellular vesicles. Lysosomes also mediate different forms of regulated cell death by permeabilization of the lysosomal membrane and release of their content to the cytosol. In cancer cells, lysosomal biogenesis and autophagy are increased to support the increased metabolism and allow growth even under nutrient- and oxygen-poor conditions. Tumor cells also induce exocytosis of lysosomal content to the extracellular space to promote invasion and metastasis. However, due to the enhanced lysosomal function, cancer cells are often more susceptible to lysosomal membrane permeabilization, providing an alternative strategy to induce cell death. This review summarizes the current knowledge of cancer-associated alterations in lysosomal structure and function and illustrates how lysosomal exocytosis and release of extracellular vesicles affect disease progression. We focus on functional differences depending on lysosomal localization and the regulation of intracellular transport, and lastly provide insight how new therapeutic strategies can exploit the power of the lysosome and improve cancer treatment. Full article

Diabetes mellitus is associated with various complications, mainly caused by the chronic exposure of the cells to high glucose (HG) concentrations. The effects of long-term HG exposure in vitro accompanied by lipopolysaccharide (LPS) application on astrocytes are relatively unknown. We used cell medium with normal (NG, 5.5 mM) or high glucose (HG, 25 mM) for rat astrocyte cultures and measured the release of NO, IL-6, β-hexosaminidase and cell survival in response to LPS. We first demonstrated that HG long-term incubation of astrocytes increased the release of β-hexosaminidase without decreasing MTT-detected cell survival, suggesting that there is no cell membrane damage or astrocyte death but could be lysosome exocytosis. Different from what was observed for NG, all LPS concentrations tested at HG resulted in an increase in IL-6, and this was detected for both 6 h and 48 h treatments. Interestingly, β-hexosaminidase level increased after 48 h of LPS and only at HG. The NO release from astrocytes also increased with LPS application at HG but was less significant. These data endorsed the original hypothesis that long-term hyperglycemia increases proinflammatory activation of astrocytes, and β-hexosaminidase could be a specific marker of excessive activation of astrocytes associated with exocytosis. Full article

Background: The lysosome has emerged as a promising target for overcoming chemoresistance, owing to its role in facilitating the lysosomal sequestration of drugs. The lysosomal calcium channel TRPML1 not only influences lysosomal biogenesis but also coordinates both endocytosis and exocytosis. This study explored the modulation of cisplatin sensitivity by regulating TRPML1-mediated lysosomal exocytosis and identified the metabolomic profile altered by TRPML1 inhibition. Methods: We used four types of ovarian cancer cells: two cancer cell lines (OVCAR8 and TOV21G) and two patient-derived ovarian cancer cells. Metabolomic analyses were conducted to identify altered metabolites by TRPML1 inhibition. Results: Lysosomal exocytosis in response to cisplatin was observed in resistant cancer cells, whereas the phenomenon was absent in sensitive cancer cells. Through the pharmacological intervention of TRPML1, lysosomal exocytosis was interrupted, leading to the sensitization of resistant cancer cells to cisplatin treatment. To assess the impact of lysosomal exocytosis on chemoresistance, we conducted an untargeted metabolomic analysis on cisplatin-resistant ovarian cancer cells with TRPML1 inhibition. Among the 1446 differentially identified metabolites, we focused on 84 significant metabolites. Metabolite set analysis revealed their involvement in diverse pathways. Conclusions: These findings collectively have the potential to enhance our understanding of the interplay between lysosomal exocytosis and chemoresistance, providing valuable insights for the development of innovative therapeutic strategies. Full article

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane’s raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations. Full article

Both biomedical applications and safety assessments of manufactured nanomaterials require a thorough understanding of the interaction between nanomaterials and cells, including how nanomaterials enter cells, transport within cells, and leave cells. However, compared to the extensively studied uptake and trafficking of nanoparticles (NPs) in cells, less attention has been paid to the exocytosis of NPs. Yet exocytosis is an indispensable process of regulating the content of NPs in cells, which in turn influences, even decides, the toxicity of NPs to cells. A comprehensive understanding of the mechanisms and influencing factors of the exocytosis of NPs is not only essential for the safety assessment of NPs but also helpful for guiding the design of safe and highly effective NP-based materials for various purposes. Herein, we review the current status and progress of studies on the exocytosis of NPs. Firstly, we introduce experimental procedures and considerations. Then, exocytosis mechanisms/pathways are summarized with a detailed introduction of the main pathways (lysosomal and endoplasmic reticulum/Golgi pathway) and the role of microtubules; the patterns of exocytosis kinetics are presented and discussed. Subsequently, the influencing factors (initial content and location of intracellular NPs, physiochemical properties of NPs, cell type, and extracellular conditions) are fully discussed. Although there are inconsistent results, some rules are obtained, like smaller and charged NPs are more easily excreted. Finally, the challenges and future directions in the field have been discussed. Full article

Delayed neurocognitive recovery (dNCR) is a common complication that occurs post-surgery, especially in elderly individuals. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex plays an essential role in various membrane fusion events, such as synaptic vesicle exocytosis and autophagosome–lysosome fusion. Although SNARE complex dysfunction has been observed in several neurodegenerative disorders, the causal link between SNARE-mediated membrane fusion and dNCR remains unclear. We previously demonstrated that surgical stimuli caused cognitive impairment in aged rats by inducing α-synuclein accumulation, inhibiting autophagy, and disrupting neurotransmitter release in hippocampal synaptosomes. Here, we evaluated the effects of propofol anesthesia plus surgery on learning and memory and investigated levels of SNARE proteins and chaperones in hippocampal synaptosomes. Aged rats that received propofol anesthesia and surgery exhibited learning and memory impairments in a Morris water maze test and decreased levels of synaptosome-associated protein 25, synaptobrevin/vesicle-associated membrane protein 2, and syntaxin 1. Levels of SNARE chaperones, including mammalian uncoordinated-18, complexins 1 and 2, cysteine string protein-α, and N-ethylmaleimide-sensitive factor, were all significantly decreased following anesthesia with surgical stress. However, the synaptic vesicle marker synaptophysin was unaffected. The autophagy-enhancer rapamycin attenuated structural and functional disturbances of the SNARE complex and ameliorated disrupted neurotransmitter release. Our results indicate that perturbations of SNARE proteins in hippocampal synaptosomes may underlie the occurrence of dNCR. Moreover, the protective effect of rapamycin may partially occur through recovery of SNARE structural and functional abnormalities. Our findings provide insight into the molecular mechanisms underlying dNCR. Full article

We previously demonstrated that mice with targeted deletion of the leucine repeat rich kinase 1 (Lrrk1) gene were osteopetrotic due to the failure of osteoclasts to resorb bone. To determine how LRRK1 regulates osteoclast activity, we examined the intracellular and extracellular acidification with an acidotropic probe, acridine orange, in live osteoclasts on bone slices. We examined lysosome distribution in osteoclasts by localization of LAMP-2, cathepsin K, and v-ATPase by immunofluorescent staining with specific antibodies. We found that both vertical and horizontal cross-sectional images of the wild-type (WT) osteoclasts showed orange-staining of the intracellular acidic vacuoles/lysosomes dispersed to the ruffled border. By contrast, the LRRK1 deficient osteoclasts exhibited fluorescent orange staining in the cytoplasm away from the extracellular lacunae because of an altered distribution of the acidic vacuoles/lysosomes. In addition, WT osteoclasts displayed a peripheral distribution of LAMP-2 positive lysosomes with a typical actin ring. The clustered F-actin constitutes a peripheral sealing zone and a ruffled border which was stretched out into a resorption pit. The LAMP-2 positive lysosomes were also distributed to the sealing zone, and the cell was associated with a resorption pit. By contrast, LRRK1-deficient osteoclasts showed diffused F-actin throughout the cytoplasm. The sealing zone was weak and not associated with a resorption pit. LAMP-2 positive lysosomes were also diffuse in the cytoplasm and were not distributed to the ruffled border. Although the LRRK1-deficient osteoclast expressed normal levels of cathepsin K and v-ATPase, the lysosomal-associated cathepsin K and v-ATPase were not accumulated at the ruffled border in Lrrk1 KO osteoclasts. Our data indicate that LRRK1 controls osteoclast activity by regulating lysosomal distribution, acid secretion, and protease exocytosis. Full article

The COVID-19 (Coronavirus Disease 2019), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), severely affects mainly individuals with pre-existing comorbidities. Here our aim was to correlate the mTOR (mammalian/mechanistic Target of Rapamycin) and autophagy pathways with the disease severity. Through western blotting and RNA analysis, we found increased mTOR signaling and suppression of genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2 as well as in transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients. Immunofluorescence co-localization assays also indicated that SARS-CoV-2 colocalizes within autophagosomes but not with a lysosomal marker. Our findings indicate that SARS-CoV-2 can benefit from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling. Full article

Nanoplastics, one component of plastic pollution, can enter human bodies via inhalation and thus threaten human health. However, the knowledge about the uptake and exocytosis of nanoplastics in cells of human lung organs is still very limited. Herein, we investigated the endocytosis, distribution, and exocytosis of polystyrene nanoparticles (PS NPs) of 50 nm (G50PS) and 100 nm (R100PS) in A549 cells and BEAS-2B cells. We found that both the cellular uptake of PS NPs increased positively with exposure time and dose, and A549 cells ingested more PS NPs than BEAS-2B cells did. In addition, the intracellular content of G50PS was higher than that of R100PS except at a higher dose and longer time. The ingested PS NPs were distributed mainly in lysosomes, while many G50PS appeared around the cell membrane, and R100PS also accumulated in mitochondria in BEAS-2B cells. As for the exocytosis, R100PS was more difficult to excrete than G50PS. Lysosomes in A549 cells and actin and microtubule in BEAS-2B cells were involved in the exocytosis of the PS NPs. These findings provide detailed information about the translocation of nanoplastics in lung cells, which is valuable for the safety assessment of nanoplastics in the environment. Full article

Significant advances have been made in the morphological observations of the dorsal gland (DG), an oval organ/tissue which lies on both sides of the dorsal midline of the crocodilian. In the current study, RNA sequencing (RNA-seq) was used to identify the changing patterns of Alligator sinesis DGs at different timepoints from the 31st embryonic day (E31) to the newly hatched 1st day (NH1). A comprehensive transcriptional changes of differentially expression gene (DEGs) involved in the melanogenesis, cholesterol metabolism, and cell apoptosis pathways suggested that the DG might serves as a functional secretory gland in formation, transport and deposition of pigment, and lipids secretion via lysosomal exocytosis. Furthermore, the remarkable immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and B-cell lymphoma 2 (Bcl-2)-positive signals in the basilar cells, in parallel with the immuno-reactive TdT-mediated dUTP nick-End labeling(TUNEL) within suprabasal cells, provided direct molecular evidence supporting for the speculation that DG serves as a holocrine secretion mode. Finally, subsequent phylogenetic and immunohistochemical analysis for the PITX2, the identified DEGs in the RNA-seq, was helpful to further elucidate the transcriptional regulatory mechanism of candidate genes. In conclusion, the current results are of considerable importance in enriching our understanding of the intrinsic relationship between the skin derivatives and lifestyles of newborn Alligator sinesis. Full article