Bao Wang

Essential control of mitochondrial morphology and function by chaperone-mediated autophagy through degradation of PARK7

ABSTRACT As a selective degradation system, chaperone-mediated autophagy (CMA) is essential for maintaining cellular homeostasis and survival under stress conditions. Increasing evidence points to an important role for the dysfunction of CMA in the pathogenesis of Parkinson disease (PD). However, the mechanisms by which CMA regulates neuronal survival under stress and its role in neurodegenerative diseases are not fully understood. PARK7/DJ-1 is an autosomal recessive familial PD gene. PARK7 plays a critical role in antioxidative response and its dysfunction leads to mitochondrial defects. In the current study, we showed that CMA mediated the lysosome-dependent degradation of PARK7. Importantly, CMA preferentially removed the oxidatively damaged nonfunctional PARK7 protein. Furthermore, CMA protected cells from mitochondrial toxin MPP+-induced changes in mitochondrial morphology and function, and increased cell viability. These protective effects were lost under PARK7-deficiency conditions. Conversely, overexpression of PARK7 significantly attenuated the mitochondrial dysfunction and cell death exacerbated by blocking CMA under oxidative stress. Thus, our findings reveal a mechanism by which CMA protects mitochondrial function by degrading nonfunctional PARK7 and maintaining its homeostasis, and dysregulation of this pathway may contribute to the neuronal stress and death in PD pathogenesis.

Destabilization of survival factor MEF2D mRNA by neurotoxin in models of Parkinson's disease

Progressive loss of dopaminergic (DA) neurons in the substantial nigra pars compacta (SNc) is an important pathological feature in Parkinsons disease (PD). Loss of transcription factor myocyte enhancer factor 2D (MEF2D), a key neuronal survival factor, has been shown to underlie the loss of DA neurons in SNc and the pathogenic process of PD. It is known that PD‐associated neurotoxins reduce the level of MEF2D protein to trigger neuronal death. Although neurotoxins clearly destabilize MEF2D by post‐translational mechanisms, it is not known whether regulation of MEF2D mRNA contributes to neurotoxin‐induced decrease in MEF2D protein. In this work, we showed that MPP+, the toxic metabolite of MPTP, caused a significant decrease in the half‐life and total level of MEF2D mRNA in a DA neuronal cell line, SN4741 cells. Quantitative PCR analysis of the SNc DA neurons captured by immune‐laser capture microdissection showed that exposure to MPTP led to a marked reduction in the level of MEF2D mRNA in SNc DA neurons compared to controls. Down‐regulation of MEF2D mRNA alone reduced the viability of SN4741 cells and sensitized the cells to MPP+‐induced toxicity. These results suggest that destabilization and reduction in MEF2D mRNA is in part responsible for neurotoxin‐induced decrease in MEF2D protein and neuronal viability. Myocyte enhancer factor 2D (MEF2D) plays an important role in neuronal survival. How MEF2D mRNA is deregulated under toxic stress is unclear. We found that PD‐associated neurotoxins destabilize MEF2D mRNA and reduce its level in vitro and in vivo. Reduction in MEF2D mRNA is sufficient to sensitize model cells to neurotoxin‐induced toxicity, suggesting that destabilization of MEF2D mRNA is part of the mechanism by which neurotoxins trigger deregulation of neuronal survival.

Chaperone-mediated autophagy: roles in neuroprotection

Chaperone-mediated autophagy (CMA), one of the main pathways of lysosomal proteolysis, is characterized by the selective targeting and direct translocation into the lysosomal lumen of substrate proteins containing a targeting motif biochemically related to the pentapeptide KFERQ. Along with the other two lysosomal pathways, macro- and micro-autophagy, CMA is essential for maintaining cellular homeostasis and survival by selectively degrading misfolded, oxidized, or damaged cytosolic proteins. CMA plays an important role in pathologies such as cancer, kidney disorders, and neurodegenerative diseases. Neurons are post-mitotic and highly susceptible to dysfunction of cellular quality-control systems. Maintaining a balance between protein synthesis and degradation is critical for neuronal functions and homeostasis. Recent studies have revealed several new mechanisms by which CMA protects neurons through regulating factors critical for their viability and homeostasis. In the current review, we summarize recent advances in the understanding of the regulation and physiology of CMA with a specifi c focus on its possible roles in neuroprotection.

Transcription factor myocyte enhancer factor 2D regulates interleukin-10 production in microglia to protect neuronal cells from inflammation-induced death

BackgroundNeuroinflammatory responses have been recognized as an important aspect in the pathogenesis of Parkinson’s disease (PD). Transcriptional regulation plays a critical role in the process of inflammation. Transcription factor myocyte enhancer factor 2D (MEF2D) is identified as a central factor in transmission of extracellular signals and activation of the genetic programs in response to a wide range of stimuli in several cell types, including neurons. But its presence and function in microglia have not been reported. We therefore investigated the effect of MEF2D in activated microglia on the progress of neuroinflammation and the survival of neurons.MethodsBV2 cells and primary cultured glial cells were stimulated with lipopolysaccharide (LPS). Samples from cells were examined for MEF2D expression, interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α) by immunoblotting, quantitative real-time PCR (qPCR) or enzyme-linked immunosorbent assay (ELISA). The activity of MEF2D was examined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Recombinant lentivirus expressing shRNA specific to MEF2D was used to silence MEF2D expression in BV2 cells. The role of IL-10 transcriptionally induced by MEF2D on neuronal survival was assessed by anti-IL-10 neutralizing antibody. The survival of neurons was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Male C57bl/6 mice were used to establish an acute PD model. Brain sections and cell slides were tested by immunofluorescence.ResultsWe demonstrated that MEF2D was present in microglia. Activation of microglia was associated with an increase in MEF2D level and activity in response to different stimuli in vivo and in vitro. MEF2D bound to a MEF2 consensus site in the promoter region of IL-10 gene and stimulated IL-10 transcription. Silencing MEF2D decreased the level of IL-10, increased the TNF-α mRNA, and promoted inflammation-induced cytotoxicity, consistent with the result of inhibiting IL-10 activity with an anti-IL-10 neutralizing antibody.ConclusionsOur study identifies MEF2D as a critical regulator of IL-10 gene expression that negatively controls microglia inflammation response and prevents inflammation-mediated cytotoxicity.

Salidroside Protects Against 6-Hydroxydopamine-Induced Cytotoxicity by Attenuating ER Stress

Parkinson’s disease (PD) is a neurodegenerative disease characterized by a persistent decline of dopaminergic (DA) neurons in the substantia nigra pars compacta. Despite its frequency, effective therapeutic strategies that halt the neurodegenerative processes are lacking, reinforcing the need to better understand the molecular drivers of this disease. Importantly, increasing evidence suggests that the endoplasmic reticulum (ER) stress-induced unfolded protein response is likely involved in DA neuronal death. Salidroside, a major compound isolated from Rhodiola roseaL., possesses potent anti-oxidative stress properties and protects against DA neuronal death. However, the underlying mechanisms are not well understood. In the present study, we demonstrate that salidroside prevents 6-hydroxydopamine (6-OHDA)-induced cytotoxicity by attenuating ER stress. Furthermore, treatment of a DA neuronal cell line (SN4741) and primary cortical neurons with salidroside significantly reduced neurotoxin-induced increases in cytoplasmic reactive oxygen species and calcium, both of which cause ER stress, and cleaved caspase-12, which is responsible for ER stress-induced cell death. Together, these results suggest that salidroside protects SN4741 cells and primary cortical neurons from 6-OHDA-induced neurotoxicity by attenuating ER stress. This provides a rationale for the investigation of salidroside as a potential therapeutic agent in animal models of PD.

The Ras/Raf/Erk Pathway Mediates the Subarachnoid Hemorrhage-Induced Apoptosis of Hippocampal Neurons Through Phosphorylation of p53

Apoptosis plays a crucial role in the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH). However, the exact molecular mechanisms underlying neuronal apoptosis in EBI after SAH have not been fully elucidated. The present study showed that EBI induced significantly neuronal apoptosis activation of Ras/Raf/Erk signals in hippocampus after SAH. Intracisternal administration of PD98059, an inhibitor of Erk1/2, decreased the hippocampal neuronal apoptosis and alleviated the cognitive deficits induced by SAH. Interestingly, an increase in phosphorylation of p53 was paralleled with p-Erk, and PD98059 also blocked the level of p-p53. In primary cultures, oxyhemoglobin (OxyHb) treatment significantly increased p-Erk, p-p53, and apoptosis, which was used to mimic the pathological injury of SAH. Both p53 small interfering RNA (siRNA) and PD98059 reduced the OxyHb-induced apoptosis. Moreover, PD98059 significantly decreased the levels of p-Erk and p-p53; however, p53 siRNA had little effect on the level of p-Erk. Taken together, our study implicates that the Ras/Raf/Erk signals contribute to neuronal death through the phosphorylation of p53 in hippocampus after SAH and also suggests Erk/p53 as a potential target for clinical drug treatment of SAH.

Iron accumulation and microglia activation contribute to substantia nigra hyperechogenicity in the 6-OHDA-induced rat model of Parkinson's disease

INTRODUCTION This study aims to explain the mechanisms for the formation of sonographic features of Parkinsons disease (PD) using a 6-hydroxydopamine (6-OHDA) rat model of PD. The iron chelator deferiprone (DFP) was used in the PD model rat to examine the relationship between iron and the echo signal. METHODS Rat models were created using stereotactic injections of 6-OHDA. DFP was administered intragastrically. Transcranial sonography (TCS) was performed to observe the substantia nigra (SN) echo signal of the brain. Immunofluorescence and iron staining were performed to observe the histological characteristics of the hyperechogenic area. The imaging findings were compared with the histopathological findings. RESULTS The PD model rat presented a large area of hyperechogenicity in the SN. Ferric ion accumulation and microglia proliferation occurred in the hyperechogenic area. DFP inhibited dopaminergic (DA) neuron necrosis, ferric ion accumulation and microglia proliferation and reduced the hyperechogenic area of the SN. CONCLUSIONS Both iron aggregation and gliosis contribute to the formation of substantia nigra hyperechogenicity (SNH) in PD. DFP exhibits a neuroprotective effect by inhibiting SNH. Iron deposit and the SNH are correlated with DA neuron necrosis.

Neuroprotective effects of nitidine in Parkinson's disease models through inhibiting microglia activation: role of the Jak2–Stat3 pathway

Parkinsons disease (PD) is the second most common neurodegenerative disorder; currently, no effective therapy is available to halt the progression of this disease. Neuroinflammation significantly contributes to the pathogenesis of PD. Recent studies have demonstrated that nitidine possesses anti-inflammatory activity. However, the role of nitidine in neuroinflammation of PD and the mechanism of its action, if any, are still unknown. In the present study, our results showed that nitidine significantly suppressed neurotoxin induced microglial activation in vitro. Further, we demonstrated that the inhibitory effect of nitidine on microglial activation is mediated by the Jak2–Stat3 pathway and that nitidine can suppress the nuclear translocation of p-Stat3 via enhancing the binding activity of αB-crystalline (CRYAB). Importantly, nitidine could inhibit reactive microgliosis and protect dopaminergic neurons in two Parkinsons disease animal models. Finally, our data showed that nitidine significantly improved neurobehavioral activity in PD animal models. Our results indicated that nitidine could significantly suppress microglial activation via the Jak2–Stat3 pathway and obviously improve behavioral function in PD animal models, which sheds some light on a promising therapeutic strategy for PD.

Efficacy and Safety of Continuous Micro-Pump Infusion of 3% Hypertonic Saline combined with Furosemide to Control Elevated Intracranial Pressure.

Background Elevated intracranial pressure is one of the most common problems in patients with diverse intracranial disorders, leading to increased morbidity and mortality. Effective management for increased intracranial pressure is based mainly on surgical and medical techniques with hyperosmolar therapy as one of the core medical treatments. The study aimed to explore the effects of continuous micro-pump infusions of 3% hypertonic saline combined with furosemide on intracranial pressure control. Material/Methods We analyzed data on 56 eligible participants with intracranial pressure >20 mmHg from March 2013 to July 2014. The target was to increase and maintain plasma sodium to a level between 145 and 155 mmol/L and osmolarity to a level of 310 to 320 mOsmol/kg. Results Plasma sodium levels significantly increased from 138±5 mmol/L at admission to 151±3 mmol/L at 24 h (P<0.01). Osmolarity increased from 282±11 mOsmol/kg at baseline to 311±8 mOsmol/kg at 24 h (P<0.01). Intracranial pressure significantly decreased from 32±7 mmHg to 15±6 mmHg at 24 h (P<0.01). There was a significant improvement in CPP (P<0.01). Moreover, central venous pressure, mean arterial pressure, and Glasgow Coma Scale slightly increased. However, these changes were not statistically significant. Conclusions Continuous infusion of 3% hypertonic saline + furosemide is effective and safe for intracranial pressure control.

Neuroprotection effect of Y-27632 against H2O2-induced cell apoptosis of primary cultured cortical neurons

Oxidative stress-mediated neuron damage is believed to contribute greatly to the pathogenesis and outcome of ischemia. Y-27632, a Rho-associated kinase (ROCK) inhibitor, has been reported to protect various cells from oxidative injury. However, whether a Rho-kinase inhibitor can directly protect neurons against oxidative damage and the molecular mechanisms underlying it is poorly understood. In the present study, we investigated the potential protective effect of Y-27632 against H2O2-induced apoptosis in cultured rat cortical neurons and potential mechanisms underlying it. Both the cell viability tests and cell apoptosis assays showed that Y-27632 effectively protected the cultured rat cortical neurons from H2O2-induced injury. Furthermore, the mechanisms underlying the protective effect were determined next. Our results revealed that Y-27632 pretreatment regulated the apoptosis-related proteins by inhibiting the H2O2-induced decrease in anti-apoptotic protein Bcl-2 and an increase in the level of pro-apoptotic protein Bax. Meanwhile, Y-27632 also significantly reduced oxidative stress and the activation of JNK1/2/3 and p38 MAPKs induced by H2O2, without affecting the phosphorylation of ERK1/2. Moreover, inhibiting the JNK and p38 pathways by SP600125 and SB203580 respectively alleviated the cell viability loss induced by H2O2 and markedly disputed the neuroprotective effect of Y-27632 against H2O2-induced apoptosis. Taken together, these results demonstrate that Y-27632 can directly protect cultured cortical neurons from H2O2-induced apoptosis by inhibiting oxidative stress and the activation of JNK and p38 MAPKs pathways. Our study provides useful insights into the therapeutic mechanisms of Y-27632 and further supports that ROCK is a promising drug target for neurological diseases.