Targeting Tetraspanins at Cell Interfaces: Functional Modulation and Exosome-Based Drug Delivery for Precise Disease Treatment.

Tetraspanins are key players in various physiological and pathological processes, including malignancy, immune response, fertilization, and infectious disease. Affinity ligands targeting the interactions between tetraspanins and partner proteins are promising for modulating downstream signaling pathways, thus emerging as attractive candidates for interfering related biological functions. Due to the involvement in vesicle biogenesis and cargo trafficking, tetraspanins are also regarded as exosome markers, and become molecular targets for drug loading and delivery. Given the rapid development in these areas, this minireview focuses on recent advances in design and engineering of affinity binders toward tetraspanins including CD63, CD81, and CD9. Their mechanism of actions in modulating protein interactions at cell interfaces and treatment of malignant diseases are discussed. Strategies for constructing exosome-based drug delivery platforms are also reviewed, with emphasis on the important roles of tetraspanins and the affinity ligands. Finally, challenges and future development of tetraspanin-targeting therapy and exosomal drug delivery platforms are also discussed.

Association between hepatocyte TM4SF5 expression and gut microbiome dysbiosis during non-alcoholic fatty liver disease development.

Gut microbiome dysbiosis is involved in non-alcoholic fatty liver disease (NAFLD) development. Hepatic transmembrane 4 L six family member 5 (TM4SF5) overexpression promotes NAFLD. However, how gut microbiota are associated with TM4SF5-mediated NAFLD remains unexplored. We analyzed the gut microbiome using feces from hepatocyte-specific TM4SF5-overexpressing transgenic (Alb-TGTm4sf5-Flag, TG) or Tm4sf5-/- knock-out (KO) mice fed a normal chow diet (NCD), high-fat diet (HFD) for 2 weeks (HFD2W), or methionine-choline-deficient diet (MCD) for 4 weeks to investigate associations among Tm4sf5 expression, diet, and the gut microbiome. TG-NCD mice showed a higher Firmicutes-to-Bacteroidetes (F/B) ratio, with less enrichment of Akkermansia muciniphila and Lactobacillus reuteri. NASH-related microbiomes in feces were more abundant in TG-HFD2w mice than in KO-HFD2w mice. Further, TG-MCD showed a higher F/B ratio than TG-NCD or KO mice, with decreases or increases in microbiomes beneficial or detrimental to the liver, respectively. Such effects in TG-MCD animals were correlated with functional pathways producing short-chain fatty acids (SCFAs). Furthermore, potential functional pathways of the gut microbiome were metabolically parallel to NAFLD features in TG-MCD mice. These results suggest that hepatocyte Tm4sf5 supports gut microbiome dysbiosis and metabolic activity, leading to SCFA production and hepatic inflammation during NAFLD development.

Tetraspanin 5 orchestrates resilience to salt stress through the regulation of ion and reactive oxygen species homeostasis in rice.

Tetraspanins (TETs) are integral membrane proteins, characterized by four transmembrane domains and a unique signature motif in their large extracellular loop. They form dynamic supramolecular complexes called tetraspanin-enriched microdomains (TEMs), through interactions with partner proteins. In plants, TETs are involved in development, reproduction and immune responses, but their role in defining abiotic stress responses is largely underexplored. We focused on OsTET5, which is differentially expressed under various abiotic stresses and localizes to both plasma membrane and endoplasmic reticulum. Using overexpression and underexpression transgenic lines we demonstrate that OsTET5 contributes to salinity and drought stress tolerance in rice. OsTET5 can interact with itself in yeast, suggesting homomer formation. Immunoblotting of native PAGE of microsomal fraction enriched from OsTET5-Myc transgenic rice lines revealed multimeric complexes containing OsTET5, suggesting the potential formation of TEM complexes. Transcriptome analysis, coupled with quantitative PCR-based validation, of OsTET5-altered transgenic lines unveiled the differential expression patterns of several stress-responsive genes, as well as those coding for transporters under salt stress. Notably, OsTET5 plays a crucial role in maintaining the ionic equilibrium during salinity stress, particularly by preserving an elevated potassium-to-sodium (K+/Na+) ratio. OsTET5 also regulates reactive oxygen species homeostasis, primarily by modulating the gene expression and activities of antioxidant pathway enzymes and proline accumulation. Our comprehensive investigation underscores the multifaceted role of OsTET5 in rice, accentuating its significance in developmental processes and abiotic stress tolerance. These findings open new avenues for potential strategies aimed at enhancing stress resilience and making valuable contributions to global food security.

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells.

BACKGROUND: Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications. METHODS: In this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors. RESULTS: Fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies. CONCLUSIONS: This study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.

Tetraspanin 3 promotes NSCLC cell proliferation via regulation of ß1 integrin intracellular recycling.

BACKGROUND: The involvement of tetraspanins in cancer development has been widely implicated. In this study, the function and molecular mechanisms of tetraspanin 3 (TSPAN3) in non-small cell lung cancer (NSCLC) cells were explored. METHODS: Tissue samples from patients diagnosed with NSCLC were analyzed by immunohistochemistry, western blotting, and real-time polymerase chain reaction (PCR) to indicate the involvement of TSPAN3 in cancer progression. In the meantime, we also performed exhaustive mechanistic studies using A549 and H460 cells in vitro through a variety of methods including western blotting, real-time PCR, immunofluorescent staining, coimmunoprecipitation, cell proliferation assay, and nocodazole (NZ) washout assay. Proper statistical analysis was implemented wherever necessary in this study. RESULTS: TSPAN3 was found to be highly expressed in lung cancer cells and tissues. Moreover, high levels of TSPAN3 positively correlated with poor differentiation, lymph node involvement, advanced pathological tumor-node-metastasis stage, and poor prognosis in patients with NSCLC. TSPAN3 showed potential to promote the proliferation of NSCLC cells in vitro and in vivo. Specifically, TSPAN3 was found to interact with ß1 integrin via the LEL domain, thereby facilitating the sorting of ß1 integrin into Rab11a endosomes and promoting ß1 integrin recycling and upregulation. CONCLUSIONS: Our findings reveal TSPAN3 may represent a potentially valuable therapeutic target for NSCLC.

Possible regulation of the immune modulator tetraspanin CD81 by alpha-synuclein in melanoma.

We probed the mechanism by which the Parkinson's disease-associated protein α-synuclein (α-syn)/SNCA promotes the pathogenesis and progression of melanoma. We found that the human melanoma cell line SK-MEL-28 in which SNCA is knocked out (SNCA-KO) has low levels of tetraspanin CD81, which is a cell-surface protein that promotes invasion, migration, and immune suppression. Analyzing data from the Cancer Genome Atlas, we show that SNCA and CD81 mRNA levels are positively correlated in melanoma; melanoma survival is inversely related to the levels of SNCA and CD81; and SNCA/CD81 are inversely related to the expression of key cytokine genes (IL12A, IL12B, IFN, IFNG, PRF1 and GZMB) for immune activation and immune cell-mediated killing of melanoma cells. We propose that high levels of α-syn and CD81 in melanoma and in immune cells drive invasion and migration and in parallel cause an immunosuppressive microenvironment; these contributing factors lead to aggressive melanomas.

An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy.

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

TSPAN1 overexpression as an indicator of poor prognosis in estrogen receptor-positive breast cancer.

Background: Tetraspanin 1 (TSPAN1) is a newly discovered protein of the tetrameric protein family encoded by the TSPAN1 gene localized in the 1p34 chromosome region. TSPAN1 has been shown to be associated with various malignancies. In this study, we aimed to investigate the prognostic significance of TSPAN1 in breast cancer. Estrogen receptor-positive (ER+) breast cancer is the largest breast cancer subgroup, and ER-targeted therapies have significantly prolonged survival and improved symptoms in advanced breast cancer. TSPAN1 overexpression was found to be associated with a poor prognosis in ER+ breast cancer. Methods: We analyzed the expression of TSPAN1 in breast cancer tissues and cell lines using western blotting and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results: TSPAN1 expression was higher in breast cancer cells as compared with normal breast tissue. There was a significant association between a high TSPAN1 level and a low survival rate. Inhibition of TSPAN1 significantly reduced the proliferation and invasion of BT474 cells both in vitro and in vivo. The downregulation of TSPAN1 in breast cancer cells significantly reduced the levels of p-mitogen-activated protein kinase 1 (MEK1) (S298) and p-extracellular signal-regulating kinase (ERK) 1/2. Conclusions: TSPAN1 modulates downstream extracellular matrix (ECM) receptor signaling cascades and promotes cellular proliferation and invasion in breast cancer. TSPAN1 inhibition may be a potential new treatment strategy for breast cancer.

piggyBac-based transgenic Helicoverpa armigera expressing the T92C allele of the tetraspanin gene HaTSPAN1 confers dominant resistance to Bacillus thuringiensis toxin Cry1Ac.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized pest control. However, the evolution of resistance by target pests poses a significant threat to the long-term success of Bt crops. Understanding the genetics and mechanisms underlying Bt resistance is crucial for developing resistance detection methods and management tactics. The T92C mutation in a tetraspanin gene (HaTSPAN1), resulting in the L31S substitution, is associated with dominant resistance to Cry1Ac in a major pest, Helicoverpa armigera. Previous studies using CRISPR/Cas9 technique have demonstrated that knockin of the HaTSPAN1 T92C mutation confers a 125-fold resistance to Cry1Ac in the susceptible SCD strain of H. armigera. In this study, we employed the piggyBac transposon system to create two transgenic H. armigera strains based on SCD: one expressing the wild-type HaTSPAN1 gene (SCD-TSPANwt) and another expressing the T92C mutant form of HaTSPAN1 (SCD-TSPANmt). The SCD-TSPANmt strain exhibited an 82-fold resistance to Cry1Ac compared to the recipient SCD strain, while the SCD-TSPANwt strain remained susceptible. The Cry1Ac resistance followed an autosomal dominant inheritance mode and was genetically linked with the transgene locus in the SCD-TSPANmt strain of H. armigera. Our results further confirm the causal association between the T92C mutation of HaTSPAN1 and dominant resistance to Cry1Ac in H. armigera. Additionally, they suggest that the piggyBac-mediated transformation system we used in H. armigera is promising for functional investigations of candidate Bt resistance genes from other lepidopteran pests.

The conformation of tetraspanins CD53 and CD81 differentially affects their nanoscale organization and interaction with their partners.

Tetraspanins, including CD53 and CD81, are four-transmembrane proteins that affect the membrane organization to regulate cellular processes including migration, proliferation, and signaling. However, it is unclear how the organizing function of tetraspanins is regulated at the molecular level. Here, we investigated whether recently proposed "open" and "closed" conformations of tetraspanins regulate the nanoscale organization of the plasma membrane of B cells. We generated conformational mutants of CD53 (F44E) and CD81 (4A, E219Q) that represent the "closed" and "open" conformation, respectively. Surface expression of these CD53 and CD81 mutants was comparable to that of WT protein. Localization of mutant tetraspanins into nanodomains was visualized by super-resolution direct stochastic optical reconstruction microscopy. Whereas the size of these nanodomains was unaffected by conformation, the clustered fraction of "closed" CD53 was higher and of "open" CD81 lower than respective WT protein. In addition, KO cells lacking CD53 showed an increased likelihood of clustering of its partner CD45. Interestingly, "closed" CD53 interacted more with CD45 than WT CD53. Absence of CD81 lowered the cluster size of its partner CD19 and "closed" CD81 interacted less with CD19 than WT CD81, but "open" CD81 did not affect CD19 interaction. However, none of the tetraspanin conformations made significant impact on the nanoscale organization of their partners CD19 or CD45. Taken together, conformational mutations of CD53 and CD81 differentially affect their nanoscale organization, but not the organization of their partner proteins. This study improves the molecular insight into cell surface nanoscale organization by tetraspanins.

Role of a novel uropod-like cell membrane protrusion in the pathogenesis of the parasite Trichomonas vaginalis.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease worldwide. As an extracellular parasite, adhesion to host cells is essential for the development of infection. During attachment, the parasite changes its tear ovoid shape to a flat ameboid form, expanding the contact surface and migrating through tissues. Here, we have identified a novel structure formed at the posterior pole of adherent parasite strains, resembling the previously described uropod, which appears to play a pivotal role as an anchor during the attachment process. Moreover, our research demonstrates that the overexpression of the tetraspanin T. vaginalis TSP5 protein (TvTSP5), which is localized on the cell surface of the parasite, notably enhances the formation of this posterior anchor structure in adherent strains. Finally, we demonstrate that parasites that overexpress TvTSP5 possess an increased ability to adhere to host cells, enhanced aggregation and reduced migration on agar plates. Overall, these findings unveil novel proteins and structures involved in the intricate mechanisms of T. vaginalis interactions with host cells.

In silico study suggests potential drugs that target CD151 to treat breast cancer and glioblastoma.

Recently tetraspanin CD151 has been identified as an important biological target involved in metastatic processes which include cell adhesion, tumor progression processes, and so forth in different types of cancers, such as breast cancer and glioblastoma. This in Silico study considered 1603 compounds from the Food and Drug Administration database, after performing an ADMET analysis; we selected 853 ligands, which were used for docking analysis. The most promising ligands were selected from docking studies, based on two criteria: (a) showed lowest affinity to the CD151 protein and (b) they interact with the QRD motif, located in the second extracellular loop. Furthermore, we investigate the stability of the protein-ligand complexes through MD simulations as well as free energy MM-PBSA calculations. From these results, loperamide and glipizide were identified as the best evaluated drugs. We suggest an in vitro analysis is needed to confirm our in silico prediction studies.

Tetraspanin proteins in membrane remodeling processes.

Membrane remodeling is a fundamental cellular process that is crucial for physiological functions such as signaling, membrane fusion and cell migration. Tetraspanins (TSPANs) are transmembrane proteins of central importance to membrane remodeling events. During these events, TSPANs are known to interact with themselves and other proteins and lipids; however, their mechanism of action in controlling membrane dynamics is not fully understood. Since these proteins span the membrane, membrane properties such as rigidity, curvature and tension can influence their behavior. In this Review, we summarize recent studies that explore the roles of TSPANs in membrane remodeling processes and highlight the unique structural features of TSPANs that mediate their interactions and localization. Further, we emphasize the influence of membrane curvature on TSPAN distribution and membrane domain formation and describe how these behaviors affect cellular functions. This Review provides a comprehensive perspective on the multifaceted function of TSPANs in membrane remodeling processes and can help readers to understand the intricate molecular mechanisms that govern cellular membrane dynamics.

Genome of tropical bed bug Cimex hemipterus (Cimicidae, Hemiptera) reveals tetraspanin expanded in bed bug ancestor.

Cimex species are ectoparasites that exclusively feed on warm-blooded animals such as birds and mammals. Three cimicid species are known to be persistent pests for humans, including the tropical bed bug Cimex hemipterus, common bed bug Cimex lectularius, and Eastern bat bug Leptocimex boueti. To date, genomic information is restricted to the common bed bug C. lectularius, which limits understanding their biology and to provide controls of bed bug infestations. Here, a chromosomal-level genome assembly of C. hemipterus (495 Mb [megabase pairs]) contained on 16 pseudochromosomes (scaffold N50 = 34 Mb), together with 9 messenger RNA and small RNA transcriptomes were obtained. In comparison between hemipteran genomes, we found that the tetraspanin superfamily was expanded in the Cimex ancestor. This study provides the first genome assembly for the tropical bed bug C. hemipterus, and offers an unprecedented opportunity to address questions relating to bed bug infestations, as well as genomic evolution to hemipterans more widely.

Genome-wide identification and phylogenetic analysis of the tetraspanin gene family in lepidopteran insects and expression profiling analysis in Helicoverpa armigera.

The tetraspanin gene family encodes cell-surface proteins that span the membrane 4 times and play critical roles in a wide range of biological processes across numerous organisms. Recent findings highlight the involvement of a tetraspanin of the lepidopteran pest Helicoverpa armigera in resistance to Bacillus thuringiensis Cry insecticidal proteins, which are extensively used in transgenic crops. Thus, a better understanding of lepidopteran tetraspanins is urgently needed. In the current study, genome scanning in 10 lepidopteran species identified a total of 283 sequences encoding potential tetraspanins. Based on conserved cysteine patterns in the large extracellular loop and their phylogenetic relationships, these tetraspanins were classified into 8 subfamilies (TspA to TspH). Six ancestral introns were identified within lepidopteran tetraspanin genes. Tetraspanins in TspA, TspB, TspC, and TspD subfamilies exhibit highly similar gene organization, while tetraspanins in the remaining 4 subfamilies exhibited variation in intron loss and/or gain during evolution. Analysis of chromosomal distribution revealed a lepidopteran-specific cluster of 10 to 11 tetraspanins, likely formed by tandem duplication events. Selective pressure analysis indicated negative selection across all orthologous groups, with ω values ranging between 0.004 and 0.362. However, positive selection was identified at 18 sites within TspB5, TspC5, TspE3, and TspF10. Furthermore, spatiotemporal expression analysis of H. armigera tetraspanins demonstrated variable expression levels across different developmental stages and tissues, suggesting diverse functions of tetraspanin members in this globally important insect pest. Our findings establish a solid foundation for subsequent functional investigations of tetraspanins in lepidopteran species.

Biophysical aspects of migrasome organelle formation and their diverse cellular functions.

The transient cellular organelles known as migrasomes, which form during cell migration along retraction fibers, have emerged as a crutial factor in various fundamental cellular processes and pathologies. These membrane vesicles originate from local membrane swellings, encapsulate specific cytoplasmic content, and are eventually released to the extracellular environment or taken up by recipient cells. Migrasome biogenesis entails a sequential membrane remodeling process involving a complex interplay between various molecular factors such as tetraspanin proteins, and mechanical properties like membrane tension and bending rigidity. In this review, we summarize recent studies exploring the mechanism of migrasome formation. We emphasize how physical forces, together with molecular factors, shape migrasome biogenesis, and detail the involvement of migrasomes in various cellular processes and pathologies. A comprehensive understanding of the exact mechanism underlying migrasome formation and the identification of key molecules involved hold promise for advancing their therapeutic and diagnostic applications.

Immunoassays for Extracellular Vesicle Detection via Transmembrane Proteins Using Surface Plasmon Resonance Biosensors.

Extracellular vesicles (EVs) are preeminent carriers of biomarkers and have become the subject of intense biomedical research for medical diagnostics using biosensors. To create effective EV-based immunoassays, it is imperative to develop surface chemistry approaches with optimal EV detection targeting transmembrane protein biomarkers that are not affected by cell-to-cell variability. Here, we developed a series of immunoassays for the detection of EVs derived from mouse monocyte cells using surface plasmon resonance (SPR) biosensors. We chemically immobilized antibodies onto mixed self-assembled monolayers of oligo ethylene glycol (OEG) alkanethiolates with carboxylic and hydroxylic terminal groups. The effects of antibody clonality (monoclonal vs polyclonal) and antibody surface coverage in targeting EVs via CD81 tetraspanins were investigated. We determined binding kinetic parameters, establishing trends from steric hindrance effects and epitope recognition properties of antibodies. Our results indicate that a 40% surface coverage of polyclonal antibodies covalently linked onto a mixed SAM with 10% of terminated -COOH groups yields a promising approach for EV detection with a linear range of 1.9 × 108-1.9 × 109 EVs/mL and a limit of detection of 5.9 × 106 EVs/mL. This optimal immunoassay exhibits a 1.92 nM equilibrium dissociation constant for bound EVs, suggesting a high binding affinity when CD81 is targeted. Our study provides important insights into surface chemistry development for EV detection targeted via transmembrane protein biomarkers using antibodies, which has promising applications for disease diagnostics.

CD9 Counteracts Liver Steatosis and Mediates GCGR Agonist Hepatic Effects.

Glucagon receptor (GCGR) agonism offers potentially greater effects on the mitigation of hepatic steatosis. However, its underlying mechanism is not fully understood. Here, it screened tetraspanin CD9 might medicate hepatic effects of GCGR agonist. CD9 is decreased in the fatty livers of patients and upregulated upon GCGR activation. Deficiency of CD9 in the liver exacerbated diet-induced hepatic steatosis via complement factor D (CFD) regulated fatty acid metabolism. Specifically, CD9 modulated hepatic fatty acid synthesis and oxidation genes through regulating CFD expression via the ubiquitination-proteasomal degradation of FLI1. In addition, CD9 influenced body weight by modulating lipogenesis and thermogenesis of adipose tissue through CFD. Moreover, CD9 reinforcement in the liver alleviated hepatic steatosis, and blockage of CD9 abolished the remission of hepatic steatosis induced by cotadutide treatment. Thus, CD9 medicates the hepatic beneficial effects of GCGR signaling, and may server as a promising therapeutic target for hepatic steatosis.

Elucidating hippocampal proteome dynamics in moderate hepatic encephalopathy rats: insights from high-resolution mass spectrometry.

Hepatic encephalopathy (HE) is a debilitating neurological disorder associated with liver failure and characterized by impaired brain function. Decade-long studies have led to significant advances in our understanding of HE; however, effective therapeutic management of HE is lacking, and HE continues to be a significant cause of morbidity and mortality in patients, underscoring the need for continued research into its pathophysiology and treatment. Accordingly, the present study provides a comprehensive overview aimed at elucidating the molecular underpinnings of HE and identifying potential therapeutic targets. A moderate-grade HE model was induced in rats using thioacetamide, which simulates the liver damage observed in patients, and its impact on cognitive function, neuronal arborization, and cellular morphology was also evaluated. We employed label-free LC-MS/MS proteomics to quantitatively profile hippocampal proteins to explore the molecular mechanism of HE pathogenesis; 2175 proteins were identified, 47 of which exhibited significant alterations in moderate-grade HE. The expression of several significantly upregulated proteins, such as FAK1, CD9 and Tspan2, was further validated at the transcript and protein levels, confirming the mass spectrometry results. These proteins have not been previously reported in HE. Utilizing Metascape, a tool for gene annotation and analysis, we further studied the biological pathways integral to brain function, including gliogenesis, the role of erythrocytes in maintaining blood-brain barrier integrity, the modulation of chemical synaptic transmission, astrocyte differentiation, the regulation of organ growth, the response to cAMP, myelination, and synaptic function, which were disrupted during HE. The STRING database further elucidated the protein‒protein interaction patterns among the differentially expressed proteins. This study provides novel insights into the molecular mechanisms driving HE and paves the way for identifying novel therapeutic targets for improved disease management.

Appearance of small extracellular vesicles in the mouse pregnant serum and the localization in placentas.

Exosomes or small extracellular vesicles (sEVs) are present in the blood of pregnant mice and considered to be involved in pregnancy physiology. Although sEVs in pregnant periods are proposed to be derived from placentas, sEVs-producing cells are not well known in mouse placentas. We studied the dynamics and localization of sEVs in pregnant serum and placentas, and examined gestational variation of microRNA (miRNA). Serums and placentas were collected from non-pregnant (NP) and pregnant mice throughout the entire gestational day (Gd). EVs were purified from serums and total RNA was isolated from EVs. Nanoparticle-tracking assay (NTA) revealed that the rates of sEVs in EVs are 53% at NP, and increased to 80.1% at Gd 14.5 and 97.5% at Gd 18.5. Western blotting on EVs showed positive reactivity to the tetraspanin markers and clarified that the results using anti-CD63 antibody were most consistent with the sEVs appearance detected by NTA. Serum EVs also showed a positive reaction to the syncytiotrophoblast marker, syncytin-1. Immunohistostaining using anti-CD63 antibody showed positive reactions in mouse placentas at the syncytiotrophoblasts and endothelial cells of the fetal capillaries. Quantitative PCR revealed that significantly higher amounts of miRNAs were included in the sEVs of Gd 18.5. Our results suggested that sEVs are produced in the mouse placenta and transferred to maternal or fetal bloodstreams. sEVs are expected to have a miRNA-mediated physiological effect and become useful biomarkers reflecting the pregnancy status.