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.

α-Synuclein Induces Neuroinflammation Injury through the IL6ST-AS/STAT3/HIF-1α Axis.

The aggregation of α-synuclein (α-syn) promotes neuroinflammation and neuronal apoptosis, which eventually contribute to the pathogenesis of Parkinson's disease (PD). Our microarray analysis and experimental data indicated a significant expression difference of the long noncoding RNA IL6ST-AS and its anti-sense strand, IL6ST, in α-synuclein-induced microglia, compared with unstimulated microglia. IL6ST is a key component of the IL6R/IL6ST complex in the microglial membrane, which recognizes extracellular inflammatory factors, such as IL6. Studies have shown that the binding of IL6 to the IL6R/IL6ST complex could activate the JAK2-STAT3 pathway and promote an excessive immune response in glia cells. Meanwhile, the phosphorylation and activation of STAT3 could increase the transcription of HIF1A, encoding a hypoxia-inducible factor related to cytotoxic damage. Our results indicated that the overexpression of IL6ST-AS induced by exogenous α-synuclein could inhibit the expression of IL6ST and the activation of JAK2-STAT3 pathway in HMC3 cells. In addition, a reduction in STAT3 resulted in the transcription inhibition of HIF1A and the acceleration of oxidative stress injury in SH-SY5Y cells co-cultured with α-synuclein-induced HMC3 cells. Our findings indicate that IL6ST-AS is an important factor that regulates microglia activation and neuronal necrosis in the progression of PD. In the HMC3 and SH-SY5Y cell co-culture system, the overexpression of IL6ST-AS led to microglial dysfunction and neurotoxicology through the IL6ST-AS/STAT3/HIF-1α axis. Our research revealed the relationships among α-synuclein, IL6ST, STAT3, and HIF-1α in the pathological process of PD and provided a new inflammation hypothesis for the pathogenesis of PD.

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.

Alpha-Synuclein Accumulation and Its Phosphorylation in the Enteric Nervous System of Patients Without Neurodegeneration: An Explorative Study.

Alpha-synuclein (α-Syn) is widely distributed and involved in the regulation of the nervous system. The phosphorylation of α-Syn at serine 129 (pSer129α-Syn) is known to be closely associated with α-Synucleinopathies, especially Parkinson's disease (PD). The present study aimed to explore the α-Syn accumulation and its phosphorylation in the enteric nervous system (ENS) in patients without neurodegeneration. Patients who underwent colorectal surgery for either malignant or benign tumors that were not suitable for endoscopic resection (n = 19) were recruited to obtain normal intestinal specimens, which were used to assess α-Syn immunoreactivity patterns using α-Syn and pSer129α-Syn antibodies. Furthermore, the sub-location of α-Syn in neurons was identified by α-Syn/neurofilament double staining. Semi-quantitative counting was used to evaluate the expression of α-Syn and pSer129α-Syn in the ENS. Positive staining of α-Syn was detected in all intestinal layers in patients with non-neurodegenerative diseases. There was no significant correlation between the distribution of α-Syn and age (p = 0.554) or tumor stage (p = 0.751). Positive staining for pSer129α-Syn was only observed in the submucosa and myenteric plexus layers. The accumulation of pSer129α-Syn increased with age. In addition, we found that the degenerative changes of the ENS were related to the degree of tumor malignancy (p = 0.022). The deposits of α-Syn were present in the ENS of patients with non-neurodegenerative disorders; particularly the age-dependent expression of pSer129α-Syn in the submucosa and myenteric plexus. The current findings of α-Syn immunostaining in the ENS under near non-pathological conditions weaken the basis of using α-Syn pathology as a suitable hallmark to diagnose α-Synucleinopathies including PD. However, our data provided unique perspectives to study gastrointestinal dysfunction in non-neurodegenerative disorders. These findings provide new evidence to elucidate the neuropathological characteristics and α-Syn pathology pattern of the ENS in non-neurodegenerative conditions.

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.

Curcumin-loaded PLGA-PEG nanoparticles conjugated with B6 peptide for potential use in Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder mainly characterized by ß-amyloid deposit and tau hyperphosphorylation with no curative treatments. Curcumin (Cur) has been proved to have potential use in Alzheimer's disease with its anti-amyloid, anti-inflammatory, and anti-oxidant properties, etc. However, its hydrophobicity and low bioavailability hinder its application. In this paper, we designed a novel brain-target nanoparticle, poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-PEG) conjugated with B6 peptide and was loaded with Cur (PLGA-PEG-B6/Cur) and administered it into HT22 cells and APP/PS1 Al transgenic mice. The in vitro assays including dynamic light scattering (DLS), flow cytometry (FCM), red blood cell (RBC) lysis, and thromboelastography (TEG) analysis indicated that this nanoparticle could narrow the diameter of Cur, increase its cellular uptake and possess good blood compatibility. The results from Morris water maze proved that PLGA-PEG-B6/Cur could tremendously improve the spatial learning and memory capability of APP/PS1 mice, compared with native Cur. The ex vivo assays including Bielschowsky silver staining, immunostaining, and western blotting demonstrated that PLGA-PEG-B6/Cur could reduce hippocampal ß-amyloid formation and deposit and tau hyperphosphorylation. Thus, we suggested that PLGA-PEG-B6/Cur nanoparticles would be of potential and promising use for the treatment of Alzheimer's disease.

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 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.

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.

Late-onset cystic brain necrosis after radiotherapy for nasopharyngeal carcinoma.

BACKGROUND: Cystic brain radionecrosis (CBRN) is a late-onset devastating complication after radiotherapy for head and neck neoplasms, especially for nasopharyngeal carcinoma (NPC). To our knowledge, it has scarcely been reported. METHODS: We retrospectively reviewed all available medical records of NPC patients with CBRN who were treated with surgical intervention. RESULTS: Sixteen patients were identified in this study and the mean latency of CBRN was 9.2 ± 0.9 years. The total irradiation dose of the nasopharynx ranged from 60 to 78 Gy. Cyst-like lesions were observed and there were slightly enhancements on the cyst wall in five patients on patients' brain MRI. All the included patients underwent surgical resection of the cystic necrotic lesion thought temporal approach. Specimens from surgery revealed reactive gliosis and immunopositive cytokines including TNF-α, IL-6 and HIF-2α. Only one patient experienced recurrence and received reoperation after surgery. All the other patients made a good recovery and no operation-related mortality was observed. CONCLUSIONS: CBRN is a delayed but irreversible neurological sequel in irradiated NPC patients. Post-radiotherapy follow-up is quite necessary for those with high risk of CBRN. Proper treatment is needed for early CBRN patients to suppress inflammation in the brain. Timely neurosurgery may benefit patients with late-stage CBRN by alleviating increased intracranial pressure and inflammatory responses.

Rifampicin attenuates rotenone-induced inflammation via suppressing NLRP3 inflammasome activation in microglia.

A growing body of evidence has supported that environmental factors, such as exposure to heavy metal and pesticides, play an important role in the pathogenesis of Parkinson׳s disease (PD). Rotenone, the active ingredient in various pesticides, has been identified as an inducer of PD. It has been revealed that rotenone induces activation of microglia and generation of pro-inflammatory factors in PD. Our previous studies demonstrated that rifampicin possessed neural protective effect in PD. In this study, we aimed to study the effect of rifampicin on the inflammation induced by rotenone in microglia and the underlying mechanisms. Results demonstrated that rifampicin pretreatment significantly reduced rotenone-induced cytotoxicity and gene expression of IL-1ß in BV2 microglia. Moreover, western blot analysis verified that rifampicin pretreatment suppressed NLRP3 inflammasome activation via inhibiting caspase-1 cleavage and protein expression of NLRP3. As it is indicated that reactive oxidative stress (ROS) is one of the activators for NLRP3 inflammasome, we further employed 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining and Rhodamine123 staining to detect intracellular ROS and mitochondrial membrane potential (MMP), respectively. Results confirmed that rifampicin obviously reduced intracellular ROS and reversed loss of MMP in BV2 cells treated by rotenone. Taken together, our data indicate that rifampicin pretreatment inhibits maturation of IL-1ß and neuroinflammation induced by rotenone via attenuating NLRP3 inflammasome activation. Rifampicin might emerge as a promising candidate for modulating neuroinflammation in PD.

Rifampicin improves neuronal apoptosis in LPS-stimulated co­cultured BV2 cells through inhibition of the TLR-4 pathway.

Agents inhibiting microglial activation are attracting attention as candidate drugs for neuroprotection in neurodegenerative diseases. Recently, researchers have focused on the immunosuppression induced by rifampicin. Our previous study showed that rifampicin inhibits the production of lipopolysaccharide (LPS)-induced pro-inflammatory mediators and improves neuron survival in inflammation; however, the mechanism through which rifampicin inhibits microglial inflammation and its neuroprotective effects are not completely understood. In this study, we examined the effects of rifampicin on morphological changes induced by LPS in murine microglial BV2 cells. Then we investigated, in BV2 microglia, the effects of rifampicin on two signaling pathway componentss stimulated by LPS, the Toll­like receptor-4 (TLR-4) and the nuclear factor-κB (NF-κB). In addition, we co-cultured BV2 microglia and neurons to observe the indirect neuroprotective effects of rifampicin. Rifampicin inhibited LPS-stimulated expression of the TLR-4 gene. When neurons were co-cultured with LPS-stimulated BV2 microglia, pre-treatment with rifampicin increased neuronal viability and reduced the number of apoptotic cells. Taken together, these findings suggest that rifampicin, with its anti-inflammatory properties, may be a promising agent for the treatment of neurodegenerative diseases.

Investigations into the role of 26S proteasome non-ATPase regulatory subunit 13 in neuroinflammation.

OBJECTIVE: To investigate 26S proteasome non-ATPase regulatory subunit 13 (PSMD13) gene silencing as a potential treatment for neuroinflammatory disorders via regulation of microglial activation and production of inflammatory mediators. METHODS: RNA interference was used to knockdown PSMD13 gene expression, followed by inhibitors of κB (IκBα) protein degradation and nuclear factor κB (NF-κB) activity measurement in lipopolysaccharide (LPS)-stimulated BV2 microglia. Nitrite (Griess) assay, reporter gene assay, enzyme-linked immunosorbent assay and Western blot were used to investigate the role of PSMD13 in microglial activation and inflammation. RESULTS: PSMD13 gene knockdown significantly reduced IκBα degradation and NF-κB activation in LPS-stimulated murine BV2 microglia. It also decreased the production of LPS-induced proinflammatory mediators, such as inducible nitric oxide synthase, nitric oxide, cyclooxygenase-2 and prostaglandin E2. CONCLUSIONS: PSMD13 gene silencing suppressed the production of proinflammatory mediators by modulating ubiquitin-proteasome system-mediated neuroinflammation via the downregulation of IκBα degradation and NF-κB activation in LPS-stimulated BV2 microglia. PSMD13 gene knockdown may have therapeutic implications for the treatment of neuroinflammatory disorders.

Rifampicin protects PC12 cells from rotenone-induced cytotoxicity by activating GRP78 via PERK-eIF2α-ATF4 pathway.

Rifampicin has been proposed as a therapeutic candidate for Parkinson's disease (PD). We previously showed that rifampicin was neuroprotective in PD models in vivo and in vitro. However, the molecular mechanisms underlying are not fully elucidated. In this study, using the comprehensive proteomic analysis, we identified that the 78 kDa glucose-regulated protein (GRP78), a hallmark of the unfolded protein response (UPR), was upregulated in rifampicin-treated PC12 cells. Western blot analysis confirmed GRP78 activation. GRP78 functions cytoprotectively in stressed cells, therefore, we hypothesized that GRP78 mediated rifampicin-induced neuroprotection. Using RNA interference, we found that GRP78 gene knockdown significantly attenuated the neuroprotective effects of rifampicin. Next, we examined three UPR transducers, namely, protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol requiring kinase α (IREα) and activating transcription factor 6 (ATF 6), and how they regulated rifampicin-stimulated GRP78 expression. Our results showed that PERK, eukaryotic initiation factor 2α (eIF2α), and activating transcription factor 4 (ATF4) were activated in rifampicin-treated PC12 cells. Silencing the ATF4 gene using RNAi inhibited GRP78 stimulation. Interestingly, we did not detect significant IREα activation, X-box binding protein 1 mRNA splicing, or ATF6 cleavage up to 24 h after rifampicin treatment. Taken together, our data suggested that rifampicin induced GRP78 via the PERK-eIF2α-ATF4 pathway to protect neurons against rotenone-induced cell damage. Targeting molecules in this pathway could be a novel therapeutic approach for PD treatment.

Rifampicin and Parkinson's disease.

Rifampicin is a macrocyclic antibiotic used extensively for the treatment of Mycobacterium tuberculosis and other mycobacterial infections. Recently, it was discovered that rifampicin exhibits neuroprotective effects. It has been shown to protect PC12 cells against MPP(+)-induced apoptosis and inhibit the expression of α-synuclein multimers. In in vitro studies, rifampicin pretreatment protects PC12 cells against rotenone-induced cell death. Qualitative and quantitative analyses uncover that rifampicin significantly suppresses rotenone-induced apoptosis by ameliorating mitochondrial oxidative stress. It reduces microglial inflammation and improves neuron survival. Our results indicate that rifampicin is cytoprotective under a variety of experimental conditions, and suggest that it may be useful in PD therapeutics. It is the aim of this paper to review the experimental neuroprotection data reported using rifampicin with a focus on the molecular and cellular mechanisms of cytoprotective effect in in vitro models of PD.

Inhibition of 26S protease regulatory subunit 7 (MSS1) suppresses neuroinflammation.

Recently, researchers have focused on immunosuppression induced by rifampicin. Our previous investigation found that rifampicin was neuroprotective by inhibiting the production of pro-inflammatory mediators, thereby suppressing microglial activation. In this study, using 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we discovered that 26S protease regulatory subunit 7 (MSS1) was decreased in rifampicin-treated microglia. Western blot analysis verified the downregulation of MSS1 expression by rifampicin. As it is indicated that the modulation of the ubiquitin-26S proteasome system (UPS) with proteasome inhibitors is efficacious for the treatment of neuro-inflammatory disorders, we next hypothesized that silencing MSS1 gene expression might inhibit microglial inflammation. Using RNA interference (RNAi), we showed significant reduction of IkBα degradation and NF-kB activation. The production of lipopolysaccharides-induced pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide, cyclooxygenase-2, and prostaglandin E(2) were also reduced by MSS1 gene knockdown. Taken together, our findings suggested that rifampicin inhibited microglial inflammation by suppressing MSS1 protein production. Silencing MSS1 gene expression decreased neuroinflammation. We concluded that MSS1 inhibition, in addition to anti-inflammatory rifampicin, might represent a novel mechanism for the treatment of neuroinflammatory disorders.

Delivery of cationic polymer-siRNA nanoparticles for gene therapies in neural regeneration.

The therapeutic applications of neural stem cells (NSCs) have potential to promote recovery in many obstinate diseases in central nervous system. Regulation of certain gene expressions using siRNA may have significant influence on the fate of NSC. To achieve the optimum gene silencing effect of siRNA, non-viral vector polyethylene glycol-polyethyleneimine (PEG-PEI) was investigated in the delivery of siRNA to NSCs. The characteristics of PEG-PEI/siRNA polyplexes were detected by scanning electron microscopy (SEM). The effects of nanoparticles on cell viability were measured via CCK-8 assay. In addition, the transfection efficiency was evaluated by fluorescence microscope and flow cytometry, and real-time PCR and Western Blot were employed to detect the gene inhibition effect of siRNA delivered by PEG-PEI. The SEM micrographs showed that PEG-PEI could condense siRNA to form diffuse and spherical nanoparticles. The cytotoxicity of PEG-PEI/siRNA nanocomplexes (N/P=15) was significantly lower when compared with that of Lipofectamine 2000/siRNA (P<0.05). Moreover, the highest transfection efficiency of PEG-PEI/siRNA nanoparticles was obtained at an N/P ratio of 15, which was better than that achieved in the transfection using Lipofectamine 2000 (P<0.05). Finally, the gene knockdown effect of PEG-PEI/siRNA nanoparticles was verified at the levels of mRNA and protein. These results suggest that PEG-PEI may potentially be used as a siRNA delivery vector for neural regeneration therapy.