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.

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.

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.

Development of flavonoid probes and the binding mode of the target protein and quercetin derivatives.

This study investigated the mechanism underlying anti-cancer cell migration activity of quercetin derivatives by investigating the binding mode of the target protein. Five flavonoid probes were newly synthesized, and pull down assay using synthesized flavonoid probes indicated matrix metalloproteinase-1 (MMP-1) as the target protein of quercetin derivatives. Quercetin and 3-O-methylquercetin (3MQ) inhibited MMP-1. SPR analysis demonstrated dose dependent interaction between quercetin derivatives and recombinant MMP-1 catalytic domain. And 1H-15N heteronuclear single quantum coherence (HSQC) NMR analysis using 15N-labeled MMP-1 catalytic domain indicated that 3MQ interacted around metal ions in the MMP-1. The development of flavonoid probes can broaden the possibility to discover the new target proteins and elucidate the core mechanisms of the multi bioactivity of flavonoids.

Identification of Potential mRNA Biomarkers in Milk Small Extracellular Vesicles of Enzootic Bovine Leukosis Cattle.

Enzootic bovine leukosis (EBL) is a disease caused by bovine leukemia virus (BLV); only a small percentage of BLV-infected cattle develop EBL and present with B-cell lymphosarcoma. There is no vaccine against BLV, treatment for EBL, or method for predicting the possibility of EBL onset, thus making EBL control difficult. Herein, to explore biomarkers for EBL in milk, we examined the mRNA profiles of small extracellular vesicles (sEVs) in milk from four BLV-uninfected and four EBL cattle by microarray analysis. It was revealed that 14 mRNAs were encapsulated in significantly higher quantities, and these mRNAs were therefore selected as biomarker candidates. Primers for these mRNAs were designed, and nine primer sets were available for quantitative real-time PCR. Nine mRNAs were evaluated for their availability as biomarkers for EBL using sEVs from newly-collected milk of 7 uninfected and 10 EBL cattle. The quantities of eight mRNAs (TMEM156, SRGN, CXCL8, DEFB4A, FABP5, LAPTM5, LGALS1, and VIM) were significantly higher in milk sEVs of EBL cattle than in those of uninfected cattle. Therefore, our findings indicate that these eight mRNAs in milk sEVs can be used as potential EBL biomarkers with combination use, although single mRNA use is not enough. Consequently, cattle at risk of EBL onset can be identified by monitoring the fluctuation in quantities of these mRNAs in milk before they develop EBL.

A New Enzyme-linked Immunosorbent Assay for Serological Diagnosis of Seal Parapoxvirus Infection in Marine Mammals.

Introduction: Seal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection. Material and Methods: The gene encoding the major envelope protein of SPPV was cloned into the eukaryotic expression vector pAcGFP1-N1, which encodes the green fluorescence protein (GFP), thereby producing a fusion protein (Env-GFP). Parental and cloned vector DNA was independently transfected into cultured seal cells for the expression of GFP and Env-GFP. The wells of an ELISA plate were coated with either GFP- or Env-GFP-transfected cell lysates. The light absorbance of each serum sample was adjusted by subtracting the absorbance of GFP-coated wells from that of Env-GFP-coated wells. Sera from two spotted seals (Phoca largha), six beluga whales (Delphinapterus leucas), three Pacific white-sided dolphins (Lagenorhynchus obliquidens), and ten bottlenose dolphins (Tursiops truncatus) from an aquarium in Japan were examined using the ELISA. Results: Positive reactions were not observed, except in one preserved sample collected ten years ago from a naturally SPPV-infected spotted seal. Conclusion: The established ELISA could be useful in screening marine mammal sera for anti-SPPV antibodies.

Comparing microRNA in milk small extracellular vesicles among healthy cattle and cattle at high risk for bovine leukemia virus transmission.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by bovine leukemia virus (BLV) infection. In Japan, cattle diagnosed with EBL are not permitted for human consumption by the law, thereby causing serious economic losses to farmers. The prevalence of BLV is high in Japan (40.9% in dairy cattle and 28.7% in beef cattle, respectively), which makes it difficult to perform the test-and-slaughter of BLV-infected cattle. This necessitates preventing the spread of BLV infection in cattle by early detection, segregation, and the removal of BLV-infected cattle with high proviral load, which are considered high risk for BLV transmission. We aimed to identify cattle that were at high risk for BLV transmission by comparing microRNA (miRNA) profiles in milk small extracellular vesicles (sEV). At first, miRNA profiles in sEV were compared among 4 uninfected cattle and 4 BLV-infected cattle with high proviral load by using a microarray containing mixed probes for miRNA of cattle and humans. Significantly lower amounts of hsa-miR-557 and hsa-miR-19b-1-5p, and insignificantly but higher amounts of hsa-miR-424-5p were observed in milk sEV from BLV-infected cattle than those from uninfected cattle. Next, to evaluate the utility of the aforementioned miRNAs for the identification of cattle that were at high risk for BLV transmission, we performed quantitative real-time PCR using milk sEV newly collected from 5 uninfected cattle and 17 BLV-infected cattle with high proviral load. The cycle threshold value of hsa-miR-424-5p was significantly lower in milk sEV from BLV-infected cattle. The PCR detection was unavailable or a significant difference was not observed for hsa-miR-557 and hsa-miR-19b-1-5p, respectively. These results suggest that the amount of hsa-miR-424-5p was higher in milk sEV from BLV-infected cattle and increasing the hsa-miR-424-5p in milk sEV could be one of the characteristic trends in cattle that are high risk for BLV transmission. Moreover, assessing characteristic miRNA amounts in milk sEV, which can be recovered twice a day by milking, could be useful for the routine monitoring of cattle in dairy herds instead of blood collection.

Discovery and Structure-Based Optimization of Novel Atg4B Inhibitors for the Treatment of Castration-Resistant Prostate Cancer.

Autophagy inhibition is an attractive target for cancer therapy. In this study, we discovered inhibitors of Atg4B essential for autophagosome formation and evaluated their potential as therapeutics for prostate cancer. Seventeen compounds were identified as candidates after in silico screening and a thermal shift assay. Among them, compound 17 showed the most potent Atg4B inhibitory activity, inhibited autophagy induced by anti-castration-resistant prostate cancer (CRPC) drugs, and significantly enhanced apoptosis. Although 17 has been known as a phospholipase A2 (PLA2) inhibitor, other PLA2 inhibitors had no effect on Atg4B and autophagy. We then performed structural optimization based on molecular modeling and succeeded in developing 21f (by shortening the alkyl chain of 17), which was a potent competitive inhibitor for Atg4B (Ki = 3.1 µM) with declining PLA2 inhibitory potency. Compound 21f enhanced the anticancer activity of anti-CRPC drugs via autophagy inhibition. These findings suggest that 21f can be used as an adjuvant drug for therapy with anti-CRPC drugs.

Identification of Novel Oxindole Compounds That Suppress ER Stress-Induced Cell Death as Chemical Chaperones.

Endoplasmic reticulum (ER) stress and oxidative stress lead to protein misfolding, and the resulting accumulation of protein aggregates is often associated with the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and prion disease. Small molecules preventing these pathogenic processes may be effective interventions for such neurodegenerative disorders. In this paper, we identify several novel oxindole compounds that can prevent ER stress- and oxidative stress-induced cell death. Among them, derivatives of the lead compound GIF-0726-r in which a hydrogen atom at the oxindole ring 5 position is substituted with a methyl (GIF-0852-r), bromine (GIF-0854-r), or nitro (GIF-0856-r) group potently suppressed global ER stress. Furthermore, GIF-0854-r and -0856-r prevented protein aggregate accumulation in vitro and in cultured hippocampal HT22 neuronal cells, indicating that these two compounds function effectively as chemical chaperones. In addition, GIF-0852-r, -0854-r, and -0856-r prevented glutamate-induced oxytosis and erastin-induced ferroptosis. Collectively, these results suggest that the novel oxindole compounds GIF-0854-r and -0856-r may be useful therapeutics against protein-misfolding diseases as well as valuable research tools for studying the molecular mechanisms of ER and oxidative stress.

Novel oxindole compounds inhibit the aggregation of amyloidogenic proteins associated with neurodegenerative diseases.

Amyloidogenic proteins form aggregates in cells, thereby leading to neurodegenerative disorders, including Alzheimer's and prion's disease, amyotrophic lateral sclerosis (ALS) in humans, and degenerative myelopathy (DM) and cognitive dysfunction in dogs. Hence, many small-molecule compounds have been screened to examine their inhibitory effects on amyloidogenic protein aggregation. However, no effective drug suitable for transition to clinical use has been found. Here we examined several novel oxindole compounds (GIF compounds) for their inhibitory effects on aggregate formation of the canine mutant superoxide dismutase 1 (cSOD1 E40K), a causative mutation resulting in DM, using Thioflavin-T fluorescence. Most GIF compounds inhibited the aggregation of cSOD1 E40K. Among the compounds, GIF-0854-r and GIF-0890-r were most effective. Their inhibitory effects were also observed in cSOD1 E40K-transfected cells. Additionally, GIF-0890-r effectively inhibited the aggregate formation of human SOD1 G93A, a causative mutation of ALS. GIF-0827-r and GIF-0856-r also effectively inhibited aggregate formation of human prion protein (hPrP). Subsequently, the correlation between their inhibitory effects on cSOD1 and hPrP aggregation was shown, indicating GIF compounds inhibited the aggregate formation of multiple amyloidogenic proteins. Conclusively, the novel oxindole compounds (GIF-0827-r, GIF-0854-r, GIF-0856-r, and GIF-0890-r) are proposed as useful therapeutic candidates for amyloidogenic neurodegenerative disorders.

Structural Analysis of Hen Egg Lysozyme Refolded after Denaturation at Acidic pH.

Protein structures fluctuate in solution; therefore, proteins have multiple stable structures that are slightly different from each other. In this study, we determined the crystal structure of hen egg lysozyme refolded after denaturation at acidic pH (rHEL) and found a structure different from native HEL (nHEL). The different local conformations of the peptide bond between Asp101 and Gly102 found in the crystal structure was supported by the NMR results for nHEL and rHEL. The NMR experiments also showed shifts in the heteronuclear single quantum coherence signals derived from Thr43 and Asp52. The chemical shift change of Asp52 could be explained by the crystal structure of rHEL, showing the conformational change of Tyr53, whose phenol ring directly lies on the main chain of Asp52. The catalytic activity of rHEL was similar to that of nHEL, indicating that the conformational change had little effect on activity. In contrast, conformational changes could be detected by the binding of monoclonal antibodies against HEL. Using multiple methods, we successfully detected the unusual structure of HEL, which might be another stable structure of HEL in solution.

Molecular Mechanisms of Aggregation of Canine SOD1 E40K Amyloidogenic Mutant Protein.

Canine degenerative myelopathy (DM) is a human amyotrophic lateral sclerosis (ALS)-like neurodegenerative disease. It is a unique, naturally occurring animal model of human ALS. Canine DM is associated with the aggregation of canine superoxide dismutase 1 (cSOD1), which is similar to human ALS. Almost 100% of cases in dogs are familial, and the E40K mutation in cSOD1 is a major causative mutation of DM. Therefore, it is important to understand the molecular mechanisms underlying cSOD1(E40K) aggregation. To address this, we first analyzed the structural model of wild type cSOD1. Interactions were evident between amino acid E40 and K91. Therefore, the mutation at residue E40 causes loss of the interaction and may destabilize the native structure of cSOD1. Differential scanning fluorimetry revealed that the E40K mutant was less stable than the wild type. Moreover, stability could be recovered by the E40K and K91E double mutation. Acceleration of amyloid fibril formation in vitro and aggregate formation in cells of cSOD1(E40K) was also suppressed by the introduction of this double mutation in thioflavin T fluorescence assay results and in transfectant cells, respectively. These results clearly show the importance of the interaction between amino acid residues E40 and K91 in cSOD1 for the stability of the native structure and aggregation.

Putative Internal Control Genes in Bovine Milk Small Extracellular Vesicles Suitable for Normalization in Quantitative Real Time-Polymerase Chain Reaction.

Bovine milk small extracellular vesicles (sEVs) contain many biologically important molecules, including mRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for quantifying mRNA in tissues and cells. However, the use, selection, and stability of suitable putative internal control genes in bovine milk sEVs for normalization in qRT-PCR have not yet been identified. Thus, the aim of the present study was to determine suitable putative internal control genes in milk sEVs for the normalization of qRT-PCR data. Milk sEVs were isolated from six healthy Holstein-Friesian cattle, followed by RNA extraction and cDNA synthesis. In total, 17 mRNAs were selected for investigation and quantification using qRT-PCR; they were further evaluated using geNorm, NormFinder, BestKeeper, and ∆CT algorithms to identify those that were highly stable putative internal control genes in milk sEVs. The final ranking of suitable putative internal control genes was determined using RefFinder. The mRNAs from TUB, ACTB, DGKZ, ETFDH, YWHAZ, STATH, DCAF11, and EGFLAM were detected in milk sEVs from six cattle by qRT-PCR. RefFinder demonstrated that TUB, ETFDH, and ACTB were highly stable in milk sEVs, and thus suitable for normalization of qRT-PCR data. The present study suggests that the use of these genes as putative internal control genes may further enhance the robustness of qRT-PCR in bovine milk sEVs. Since these putative internal control genes apply to healthy bovines, it would be helpful to include that the genes were stable in sEVs under "normal or healthy conditions".

Kaempferol Has Potent Protective and Antifibrillogenic Effects for α-Synuclein Neurotoxicity In Vitro.

Aggregation of α-synuclein (α-Syn) is implicated in the pathogenesis of Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Therefore, the removal of α-Syn aggregation could lead to the development of many new therapeutic agents for neurodegenerative diseases. In the present study, we succeeded in generating a new α-Syn stably expressing cell line using a piggyBac transposon system to investigate the neuroprotective effect of the flavonoid kaempferol on α-Syn toxicity. We found that kaempferol provided significant protection against α-Syn-related neurotoxicity. Furthermore, kaempferol induced autophagy through an increase in the biogenesis of lysosomes by inducing the expression of transcription factor EB and reducing the accumulation of α-Syn; thus, kaempferol prevented neuronal cell death. Moreover, kaempferol directly blocked the amyloid fibril formation of α-Syn. These results support the therapeutic potential of kaempferol in diseases such as synucleinopathies that are characterized by α-Syn aggregates.