Galantamine suppresses α-synuclein aggregation by inducing autophagy via the activation of α7 nicotinic acetylcholine receptors.

Synucleinopathies, including Parkinson's disease and dementia with Lewy bodies, are neurodegenerative disorders characterized by the aberrant accumulation of α-synuclein (α-syn). Although no treatment is effective for synucleinopathies, the suppression of α-syn aggregation may contribute to the development of numerous novel therapeutic targets. Recent research revealed that nicotinic acetylcholine (nACh) receptor activation has neuroprotective effects and promotes the degradation of amyloid protein by activating autophagy. In an in vitro human-derived cell line model, we demonstrated that galantamine, the nAChR allosteric potentiating ligand, significantly reduced the cell number of SH-SY5Y cells with intracellular Lewy body-like aggregates by enhancing the sensitivity of α7-nAChR. In addition, galantamine promoted autophagic flux, and prevented the formation of Lewy body-resembled aggregates. In an in vivo synucleinopathy mouse model, the propagation of α-syn aggregation in the cerebral cortex was inhibited by galantamine administration for 90 days. These results suggest that α7-nAChR is expected to be a novel therapeutic target, and galantamine is a potential agent for synucleinopathies.

Ferroptosis induces nucleolar stress as revealed by live-cell imaging using thioflavin T.

Nucleolar stress induced by stressors like hypoxia, UV irradiation, and heat shock downregulates ribosomal RNA transcription, thereby impairing protein synthesis capacity and potentially contributing to cell senescence and various human diseases such as neurodegenerative disorders and cancer. Live-cell imaging of the nucleolus may be a feasible strategy for investigating nucleolar stress, but currently available nucleolar stains are limited for this application. In this study using mouse hippocampal HT22 cells, we demonstrate that thioflavin T (ThT), a benzothiazole dye that binds RNA with high affinity, is useful for nucleolar imaging in cells where RNAs predominate over protein aggregates. Nucleoli were stained with high intensity simply by adding ThT to the cell culture medium, making it suitable for use even in damaged cells. Further, ThT staining overlapped with specific nucleolar stains in both live and fixed cells, but did not overlap with markers for mitochondria, lysosomes, endoplasmic reticulum, and double-stranded DNA. Ferroptosis, an iron-dependent nonapoptotic cell death pathway characterized by lipid peroxide accumulation, reduced the number of ThT-positive puncta while endoplasmic reticulum stress did not. These findings suggest that ferroptosis is associated with oxidative damage to nucleolar RNA molecules and ensuing loss of nucleolar function.

Simple methods for measuring milk exosomes using fluorescent compound GIF-2250/2276.

Exosomes are small extracellular vesicles (EVs) found in culture supernatants, blood, and breast milk. The size of these nanocomplexes limits the methods of EV analyses. In this study, nitrobenzoxadiazole (NBD), a fluorophore, conjugated endosome-lysosome imager, GIF-2250 and its derivative, GIF-2276, were evaluated for exosome analyses. A correlation was established between GIF-2250 intensity and protein maker levels in bovine milk exosomes. We found that high-temperature sterilization milk may not contain intact exosomes. For precise analysis, we synthesized GIF-2276, which allows for the covalent attachment of NBD to the Lys residue of exosome proteins, and labeled milk exosomes were separated using a gel filtration system. GIF-2276 showed chromatographic peaks of milk exosomes containing >3 ng protein. The area (quantity) and retention time (size) of the exosome peaks were correlated to biological activity (NO synthesis suppression in RAW264.7 murine macrophages). Heat denaturation of purified milk-derived exosomes disrupted these indicators. Proteome analyses revealed GIF-2276-labeled immunomodulators, such as butyrophilin subfamily 1 member A1 and polymeric immunoglobulin receptor. The immunogenicity and quantity of these factors decreased by heat denaturation. When milk exosomes were purified from market-sourced milk we found that raw and low-temperature sterilization milk samples, contained exosomes (none in high-temperature sterilization milk). These results were also supported by transmission electron microscopy analyses. We also found that GIF-2276 could monitor exosome transportation into HEK293 cells. These results suggested that GIF-2250/2276 may be helpful to evaluate milk exosomes.

Structural features localizing the ferroptosis inhibitor GIF-2197-r to lysosomes.

We previously reported that N,N-dimethylaniline derivatives are potent ferroptosis inhibitors. Among them, the novel aminoindan derivative GIF-2197-r (the racemate of GIF-2115 (R-form) and GIF-2196 (S-form)) is effective at a concentration of 0.01 µM due to its localization to lysosomes and ferrous ion coordination capacity. The current study demonstrates that the aliphatic tertiary amine moiety of GIF-2197-r is responsible for lysosomal localization. Although N,N-dimethylaniline derivatives cannot form chelate structures with Fe2+, density functional theory computation demonstrates that they can form stable monodentate complexes with a hydrated ferrous ion, likely due to the highly electron-rich nature of the (dialkylamino)phenyl ring. Furthermore, the results suggest that the aliphatic tertiary amine moiety contributes to stabilizing the complexation. These findings could prove useful for developing improved lysosomotropic ferroptosis inhibitors for neurodegenerative diseases.

Characterization of mRNA Signature in Milk Small Extracellular Vesicles from Cattle Infected with Bovine Leukemia Virus.

This study aimed to characterize the mRNA signature of milk small extracellular vesicles (sEVs) from BLV-infected cattle. A total of 23 mRNAs, which showed greater abundance in milk sEVs from BLV-infected cattle compared to those from BLV-uninfected (control) cattle, were identified through microarray analyses conducted in our previous study. To assess the significance of these differences in mRNA abundance, milk was collected from six control cattle and twenty-six cattle infected with BLV. The infected cattle were categorized into two distinct groups based on their proviral loads: a group of eight cattle with low proviral loads (LPVL), characterized by <10,000 copies per 105 white blood cells (WBC), and a group of eighteen cattle with high proviral loads (HPVL), marked by ≥10,000 copies per 105 WBC. The qPCR analysis quantified 7 out of 23 mRNAs, including BoLA, CALB1, IL33, ITGB2, MYOF, TGFBR1, and TMEM156, in the milk sEVs from control cattle, LPVL cattle, and HPVL cattle. Significantly, the average relative expression of CALB1 mRNA in milk sEVs was higher in LPVL cattle compared to HPVL cattle and control cattle (p < 0.05), while it was relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). Likewise, the average relative expression of TMEM156 mRNA in milk sEVs was significantly higher in LPVL cattle compared to HPVL cattle (p < 0.05), and relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). The results indicate distinct patterns of CALB1 and TMEM156 mRNA levels in milk sEVs, with higher levels observed in LPVL cattle and lower levels in HPVL cattle. The current study could provide essential information to comprehend the complexities during the progression of BLV infection and direct the exploration of mRNA biomarkers for monitoring the clinical stage of BLV infection.

Exploration of microRNA Biomarkers in Blood Small Extracellular Vesicles for Enzootic Bovine Leukosis.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). While most infected cattle show no clinical signs, approximately 30% of infected cattle develop persistent lymphocytosis (PL), and a small percentage may develop EBL. Currently, there is no method for predicting the possibility of EBL onset. In this study, we analyzed the microRNAs (miRNAs) encapsulated in small extracellular vesicles (sEVs) in the blood to explore the biomarkers of EBL. To identify candidate biomarkers, blood samples were collected from three BLV-uninfected and three EBL cattle. Total RNA was extracted from filtered serum and used for microarray analysis. Due to their association with cancer in human orthologs, we selected three miRNAs as candidate biomarkers, bta-miR-17-5p, bta-miR-24-3p, and bta-miR-210, which were more than twice as abundant in EBL cattle than in BLV-uninfected cattle. Quantitative real-time polymerase chain reaction (qPCR) using serum RNAs from six cattle used for the microarray analysis was carried out for the detection of the three selected miRNAs. Additionally, bta-miR-92a, whose ortholog has been associated with cancer in humans, was also examined by qPCR. bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were successfully detected, but bta-miR-210 was not. To further evaluate the utility of these three miRNAs as biomarkers, new blood samples were collected from 31 BLV-uninfected and 30 EBL cattle. The levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were significantly higher in EBL cattle than in BLV-uninfected cattle. These results suggest that increased levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a in the blood could be used as biomarkers for EBL. This study may contribute to the control of BLV infections and develop a prediction method of EBL onset.

Protein aggregation model to explain the bioactivity of condensed tannins.

Tannins are involved in the taste of foods and multi bioactivity of traditional herbal medicines. The characteristics of tannins are believed to derive from their connectivity with proteins. However, the mode of interaction between proteins and tannins is not yet understood because of the complexity of the tannin structure. Then this study aimed to elucidate the detail binding mode of tannin and protein by the 1H-15N HSQC NMR method using the 15N-labeled MMP-1that have not been used so far. The HSQC results suggested cross-link sites between MMP-1s, which cause protein aggregation and inhibit MMP-1 activity. This study presents the first 3D protein aggregation model of condensed tannins, which is important for understanding the bioactivity of polyphenols. Furthermore, it can broaden the understanding of the range of interactions between other proteins and polyphenols.

Identification of Suitable Internal Control miRNAs in Bovine Milk Small Extracellular Vesicles for Normalization in Quantitative Real-Time Polymerase Chain Reaction.

This study aimed to identify a suitable RNA extraction kit and stable internal control microRNA (miRNA) in bovine milk small extracellular vesicles (sEVs) for a quantitative polymerase chain reaction (qPCR) analysis. Two RNA extraction kits, miRNeasy Micro Kit, and Maxwell RSC miRNA Tissue Kit, were compared and evaluated using bovine milk sEVs via qPCR analysis. Five miRNAs, bta-miR-29a, bta-miR-200a, bta-miR-26b, hsa-miR-27b-3p, and hsa-miR-30b-5p, were selected by microarray analyses, and their cycle threshold (Ct) values were further evaluated mathematically using geNorm, NormFinder, BestKeeper, and ∆Ct algorithms. The results revealed that both the miRNeasy Micro Kit and Maxwell RSC miRNA Tissue Kit are useful for the efficient recovery of RNA from bovine milk sEVs. According to the final stability ranking analyzed by RefFinder, hsa-miR-27b-3p and bta-miR-29a can be used as suitable internal control miRNAs in bovine milk sEVs. The study also indicated that using a suitable internal control miRNA may improve the reliability and accuracy of the qPCR analysis for normalization in bovine milk sEVs. To the best of our knowledge, this is the first study to uncover the suitable internal control miRNAs in bovine milk sEVs.

Characterization of miRNAs in Milk Small Extracellular Vesicles from Enzootic Bovine Leukosis Cattle.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). Most BLV-infected cattle show no clinical signs and only some develop EBL. The pathogenesis of EBL remains unclear and there are no methods for predicting EBL before its onset. Previously, it was reported that miRNA profiles in milk small extracellular vesicles (sEVs) were affected in cattle in the late stage of BLV infection. It raised a possibility that miRNA profile in milk sEVs from EBL cattle could be also affected. To characterize the difference in milk of EBL cattle and healthy cattle, we examined the miRNA profiles in milk sEVs from four EBL and BLV-uninfected cattle each using microarray analysis. Among the detected miRNAs, three miRNAs-bta-miR-1246, hsa-miR-1290, and hsa-miR-424-5p-which were detectable using quantitative real-time PCR (qPCR) and are associated with cancers in humans-were selected as biomarker candidates for EBL. To evaluate the utility of these miRNAs as biomarkers for EBL, their levels were measured using milk that was freshly collected from 13 EBL and seven BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p, but not hsa-miR-1290, were detected using qPCR and their levels in milk sEVs from EBL cattle were significantly higher than those in BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p in sEVs may promote metastasis by targeting tumor suppressor genes, resulting in increased amounts in milk sEVs in EBL cattle. These results suggest that bta-miR-1246 and hsa-miR-424-5p levels in milk sEVs could serve as biomarkers for EBL.

Evaluation of multi-specificity of antibody G2 using its single-chain Fv and its covalently linked antigen peptides.

The antibody G2 specifically binds to four peptides with different amino acid sequences: Pep18mer, Pep8, Pep395, and PepH4P6. To elucidate the multi-specificity of G2, we generated a G2 single-chain Fv (scFv) antibody and analyzed its binding thermodynamics and kinetics to antigen peptides. Our results clearly showed that the recognition of PepH4P6 was similar to that of Pep18mer, to which G2 could obtain binding ability through the deletion of Pro95 at light chain on the affinity maturation process. The covalent linking of peptides could increase the thermal stability of G2 scFv due to intramolecular antigen binding. In the effects of respective peptides, the increased thermal stability of G2 scFv linked to Pep8 was significant, possibly due to the rapid dissociation. Binding experiments of G2 scFv linked to peptides to other peptides showed decreased association rates relative to those of antigen-free G2 scFv while the dissociation rates were almost unchanged.