Rifampicin Prevents SH-SY5Y Cells from Rotenone-Induced Apoptosis via the PI3K/Akt/GSK-3ß/CREB Signaling Pathway.

In addition to its original application for treating tuberculosis, rifampicin has multiple potential neuroprotective effects in chronic neurodegenerative diseases including Parkinson's disease (PD) and Alzheimer's disease. Inflammatory reactions and the PI3K/Akt pathway are strongly implicated in dopaminergic neuronal death in PD. This study aims to investigate whether rifampicin protects rotenone-lesioned SH-SY5Y cells via regulating PI3K/Akt/GSK-3ß/CREB pathway. Rotenone-treated SH-SY5Y cells were used as the cell model to investigate the neuroprotective effects of rifampicin. Cell viability and apoptosis of SH-SY5Y cells were determined by CCK-8 assay and flow cytometry, respectively. The expression of Akt, p-Akt, GSK-3ß, p-GSK-3ß, CREB and p-CREB were measured by Western blot. Our results showed that the cell viability and level of phospho-CREB significantly decreased in SH-SY5Y cells exposed to rotenone when compared to the control group. Both the cell viability and the expression of phospho-CREB in cells pretreated with rifampicin were higher than those of cells exposed to rotenone alone. Moreover, pretreatment of SH-SY5Y cells with rifampicin enhanced phosphorylation of Akt and suppressed activity of GSK-3ß. The addition of LY294002, a PI3K inhibitor, could suppress phosphorylation of Akt and CREB and activate GSK-3ß, resulting in abolishment of neuroprotective effects of rifampicin on cells exposed to rotenone. Rifampicin provides neuroprotection against dopaminergic degeneration, partially via the PI3K/Akt/GSK-3ß/CREB signaling pathway. These findings suggest that rifampicin could be an effective and promising neuroprotective candidate for treating PD.

Serum concentration of high-mobility group box 1, Toll-like receptor 4 as biomarker in epileptic patients.

Epilepsy is one of the most common neurological diseases with severe outcome. High-mobility Group Box 1/Toll-like Receptor 4 axis is proposed to participate in the epileptogenesis and correlate with seizure severity in animal models. To explore whether HMGB1 and TLR4 could serve as clinical biomarkers in epileptic patients, we recruited a total of 72 epilepsy patients and measured the serum level of HMGB1 and TLR4 by flow fluorescence technology and ELISA respectively. We found that the serum levels of HMGB1 and TLR4 were elevated in epileptic patients. The serum levels of HMGB1 and TLR4 were also significantly higher in drug-resistant group compared with drug-effective group. There was a positive linear correlation between HMGB1 and TLR4 in the study group (R2 = 0.479). The HMGB1 level was found to be related to seizures frequency (F = 6.71, P = 0.012), the duration of disease (F = 6.55, P = 0.013) and drug reactivity (F = 3.96, P = 0.024) in epileptic patients, while TLR4 level was related to seizures frequency (F = 4.68, P = 0.034) and drug reactivity (F = 3.80, P = 0.027). Our result provides experimental evidences that the expression of HMGB1 and TLR4 was correlated with clinical symptom in epileptic patients, which could be used as clinical biomarkers to monitor therapeutic effect.

Dl-3-n-butylphthalide activates Nrf2, inhibits ferritinophagy, and protects MES23.5 dopaminergic neurons from ferroptosis.

Ferroptosis, a newly identified iron-dependent form of cell death, has recently been implicated in the pathogenesis of Parkinson's disease (PD). Dl-3-n-butylphthalide (NBP) attenuates behavioral and cognitive deficits in animal models of PD. However, the potential of NBP to prevent dopaminergic neuron death by suppressing ferroptosis has rarely been explored. In this study, we aimed to investigate the effects of NBP on ferroptosis in erastin-induced dopaminergic neurons (MES23.5 cells) and the underlying mechanisms involved in these effects. Our results demonstrated that erastin significantly decreased viability of MES23.5 dopaminergic neurons in a dose-dependent manner, which was reversible by ferroptosis inhibitors. We further verified that NBP protected erastin-treated MES23.5 cells from death by inhibiting ferroptosis. Erastin increased the mitochondrial membrane density, caused lipid peroxidation, and decreased GPX4 expression in MES23.5 cells, which could be reversed by NBP preconditioning. NBP pretreatment suppressed erastin-induced labile iron accumulation and reactive oxygen species generation. Moreover, we demonstrated that erastin significantly reduced FTH expression, and pre-administration with NBP promoted Nrf2 translocation into the nucleus and increased the protein level of FTH. Additionally, the expression of LC3B-II in MES23.5 cells pretreated with NBP before administration of erastin was lower than that in cells treated with erastin alone. NBP reduced colocalization of FTH and autophagosomes in MES23.5 cells exposed to erastin. Finally, erastin gradually inhibited NCOA4 expression in a time-dependent manner, which was reversible by NBP pretreatment. Taken together, these results indicated that NBP suppressed ferroptosis via regulating FTH expression, which was achieved by promoting Nrf2 nuclear translocation and inhibiting NCOA4-mediated ferritinophagy. As such, NBP may be a promising therapeutic agent for the treatment of neurological diseases associated with ferroptosis.

Exploration of the α-syn/T199678/miR-519-3p/KLF9 pathway in a PD-related α-syn pathology.

BACKGROUND: Kruppel-like factor 9 (KLF9) plays a key role as an inducer of cellular oxidative stress in the modulation of cell death and in oxidant-dependent tissue injury. Our previous study indicated that lncRNA-T199678 (T199678) affected the expression of KLF9 in an α-synuclein (α-syn) induced cellular model. However, the roles of interactions among α-syn, T199678, KLF9 and related microRNAs (miRNAs) in the Parkinson's disease (PD)-related α-syn pathology are unclear and were therefore investigated in this study. METHODS: An α-syn-injected mouse model and an α-syn exposed SY-SH5Y cellular model were used in this study. We confirmed the utility of these established models with morphological and behavioral methods. We checked how expression of T199678 and KLF9 were affected by α-syn and demonstrated their interaction by fluorescence in situ hybridization (FISH) staining and western blots. We analyzed expression in ROS+ cells by immunohistochemistry. Finally, we obtained seven miRNAs through bioinformatic analysis simultaneously affected by T199678 and α-syn and verified these with RT-PCR. RESULTS: We found that expression of KLF9 was regulated by T199678, whereas expression of T199678 was not affected by KLF9 in the α-syn exposed SY-SH5Y cells. These findings suggest that KLF9 is the downstream gene regulated by T199678, whereas miR-519-3p may play a contributing role. We also confirmed that α-syn injection upregulated the expression of ROS, which could be downregulated by upregulation of T199678, indicating an anti-oxidative role of T199678 in the α-syn-related mechanisms. CONCLUSIONS: Our results indicate the existence of a potential α-syn/T199678/miR-519-3p /KLF9 pathway in PD-related α-syn pathology. This pathway might explain oxidative stress processes in α-syn-related mechanisms, which requires further verification.

Effects of miRNAs in exosomes derived from α-synuclein overexpressing SH-SY5Y cells on autophagy and inflammation of microglia.

Our previous study has revealed that GFP-α-synuclein overexpressing SH-SY5Y cells-derived exosomes (GFP-SNCA Exo) decrease autophagy in microglia via their load of miRNAs. However, it is unclear whether GFP-SNCA Exo can affect microglial inflammation via modulation of autophagy. In order to investigate the effects of miRNAs carried by GFP-SNCA Exo on autophagy and inflammation of microglia. SH-SY5Y cells were transfected with lentivirus expressing α-synuclein and then their exosomes were collected. Western blot and laser confocal images showed that α-synuclein transferred between SH-SY5Y cells and microglia through exosomes. Differentially expressed miRNAs between GFP-SNCA Exo and the vector exosomes were detected by microarray analysis. After bioinformatics analysis of the differentially expressed miRNAs, we found that their target genes were enriched in the MAPK and autophagy-associated signaling pathway. The expression of P62, p-JNK/JNK, and p-ERK/ERK and the release of IL-6 significantly increased whereas LC3 II/I decreased in microglia exposed to GFP-SNCA Exo for 48 h when compared to the control group. But rapamycin could reverse the increasing expression of p-JNK/JNK, p-ERK/ERK and the release of IL-6 induced by GFP-SNCA Exo. Dual immunofluorescence staining for LC3B and LAMP1 showed that the fluorescence density of LC3B decreased and the fluorescence of LC3B and LAMP1 were not co-located in microglia after 48 h co-culture with GFP-SNCA Exo compared with the control group, which indicated that these exosomes decreased autophagy and impaired the autophagy flux in recipient microglia. Taken together, our results indicate that GFP-SNCA Exo activate the MAPK signaling pathway and inflammation by decreasing autophagy in microglia.

LncRNA-T199678 Mitigates α-Synuclein-Induced Dopaminergic Neuron Injury via miR-101-3p.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by dopaminergic neuron death and the abnormal accumulation and aggregation of α-synuclein (α-Syn) in the substantia nigra (SN). Although the abnormal accumulation of α-Syn can solely promote and accelerate the progress of PD, the underlying molecular mechanisms remain unknown. Mounting evidence confirms that the abnormal expression of long non-coding RNA (lncRNA) plays an important role in PD. Our previous study found that exogenous α-Syn induced the downregulation of lncRNA-T199678 in SH-SY5Y cells via a gene microarray analysis. This finding suggested that lncRNA-T199678 might have a potential pathological role in the pathogenesis of PD. This study aimed to explore the influence of lncRNA-T199678 on α-Syn-induced dopaminergic neuron injury. Overexpression of lncRNA-T199678 ameliorated the neuron injury induced by α-Syn via regulating oxidative stress, cell cycle, and apoptosis. Studies indicate lncRNAs could regulate posttranscriptional gene expression via regulating the downstream microRNA (miRNA). To discover the downstream molecular target of lncRNA-T199678, the following experiment found out that miR-101-3p was a potential target for lncRNA-T199678. Further study showed that the upregulation of lncRNA-T199678 reduced α-Syn-induced neuronal damage through miR-101-3p in SH-SY5Y cells and lncRNA-T199678 was responsible for the α-Syn-induced intracellular oxidative stress, dysfunction of the cell cycle, and apoptosis. All in all, lncRNA-T199678 mitigated the α-Syn-induced dopaminergic neuron injury via targeting miR-101-3p, which contributed to promote PD. Our results highlighted the role of lncRNA-T199678 in mitigating dopaminergic neuron injury in PD and revealed a new molecular target for PD.

Rifampicin attenuates rotenone-treated microglia inflammation via improving lysosomal function.

Mounting evidence suggests that lysosome dysfunction promotes the progression of several neurodegenerative diseases via hampering autophagy flux. While regulation of autophagy in microglia may affect chronic inflammation involved in Parkinson's disease (PD). Our previous studies have reported rifampicin inhibits rotenone-induced microglia inflammation by enhancing autophagy, however the precise mechanism remains unclear. Human microglia (HM) cells were pretreated with 100 µM rifampicin for 2 h followed by exposure to 0.1 µM rotenone. We found that rifampicin pretreatment suppressed the gene expression of IL-1ß and IL-6 via inhibiting activation of JNK after rotenone induction, but the anti-inflammatory effect of rifampicin was reversed by chloroquine. Moreover, rifampicin pretreatment not only improved the ratio of LC3-II/LC3-I in rotenone-treated cells, but also increased autolysosomes and decreased autophagosomes in RFP-GFP-LC3B transfected HM cells exposed to rotenone, thus indicating rifampicin improves autophagy flux in rotenone-treated HM cells. Finally, we verified rifampicin pretreatment enhanced ATP6V0A1 expression when compared to that exposed to rotenone alone. ATP6V0A1 knockdown inhibited the effect of rifampicin on maintaining lysosome acidification and autophagosome-lysosome fusion in rotenone-treated microglia. Taken together, our results indicated that rifampicin attenuates rotenone-induced microglia inflammation partially via elevating ATP6V0A1. Modulation of lysosomal function by rifampicin may be a novel therapeutic strategy for PD.

α-synuclein accumulation in SH-SY5Y cell impairs autophagy in microglia by exosomes overloading miR-19a-3p.

Aims: To reveal whether miRNAs in exosomes from α-synuclein transgenic SH-SY5Y cells are able to regulate autophagy in recipient microglia. Materials & methods: Microarray analysis and experimental verification were adopted to assess the significance of autophagy-associated miRNAs in exosomes from neuronal model of α-synucleinopathies. Results: We found that miR-19a-3p increased remarkably in the exosomes from α-synuclein gene transgenic SH-SY5Y cells. Further study inferred that α-synuclein gene transgenic SH-SY5Y cell-derived exosomes and miR-19a-3p mimic consistently inhibited the expression of phosphatase and tensin homolog and increased the phosphorylation of AKT and mTOR, both of which ultimately lead to the dysfunction of autophagy in recipient microglia. Conclusion: The data suggested that enhanced expression of miR-19a-3p in exosomes suppress autophagy in recipient microglia by targeting the phosphatase and tensin homolog/AKT/mTOR signaling pathway.

Chronic back pain cured by low-dose levodopa: is it a variant of restless legs syndrome?

Chronic back pain is one of the most common reasons for missed work and visits to the doctor. This report presents 2 interesting cases of chronic back pain that were effectively relieved by low-dose levodopa. These 2 patients showed no sign of anatomical problem of the spine or relative structures, but the discomforts on the back manifested some characteristics resembling those in restless legs syndrome (RLS), and one of them actually developed RLS after many years of back problem. We believe that this type of chronic back pain might be a variant of RLS, which we would like to call "restless back", and it can be effectively treated by dopaminergic drugs.

Rifampicin inhibits rotenone-induced microglial inflammation via enhancement of autophagy.

Mitochondrial and autophagic dysfunction, as well as neuroinflammation, are associated with the pathophysiology of Parkinson's disease (PD). Rotenone, an inhibitor of mitochondrial complex I, has been associated as an environmental neurotoxin related to PD. Our previous studies reported that rifampicin inhibited microglia activation and production of proinflammatory mediators induced by rotenone, but the precise mechanism has not been completely elucidated. BV2 cells were pretreated for 2h with rifampicin followed by 0.1µM rotenone, alone or in combination with chloroquine. Here, we demonstrate that rifampicin pretreatment alleviated rotenone induced release of IL-1ß and IL-6, and its effects were suppressed when autophagy was inhibited by chloroquine. Moreover, preconditioning with 50µM rifampicin significantly increased viability of SH-SY5Y cells cocultured with rotenone-treated BV2 cells in the transwell coculture system. Chloroquine partially abolished the neuroprotective effects of rifampicin pretreatment. Rifampicin pretreatment significantly reversed rotenone-induced mitochondrial membrane potential reduction and reactive oxygen species accumulation. We suggest that the mechanism for rifampicin-mediated anti-inflammatory and antioxidant effects is the enhancement of autophagy. Indeed, the ratio of LC3-II/LC3-I in rifampicin-pretreated BV2 cells was significantly higher than that in cells without pretreatment. Fluorescence and electron microscopy analyses indicate an increase of lysosomes colocalized with mitochondria in cells pretreated with rifampicin, which confirms that the damaged mitochondria were cleared through autophagy (mitophagy). Taken together, the data provide further evidence that rifampicin exerts neuroprotection against rotenone-induced microglia inflammation, partially through the autophagy pathway. Modulation of autophagy by rifampicin is a novel therapeutic strategy for PD.