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

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.

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 specific 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.

BACKGROUND: Neuroinflammatory 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. METHODS: BV2 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. RESULTS: We 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. CONCLUSIONS: Our study identifies MEF2D as a critical regulator of IL-10 gene expression that negatively controls microglia inflammation response and prevents inflammation-mediated cytotoxicity.

Myricitrin alleviates MPP⁺-induced mitochondrial dysfunction in a DJ-1-dependent manner in SN4741 cells.

Oxidative stress and mitochondrial dysfunction have been linked to Parkinson's disease. DJ-1 is a recessive familial PD gene involved in antioxidative function and mitochondrial maintenance. Myricitrin, a flavanoid isolated from the root bark of Myrica cerifera, has potent antioxidative properties. In the present study, we investigated the protective effects of myricitrin against MPP(+)-induced mitochondrial dysfunction in SN4741 cells and attempted to elucidate the mechanisms underlying this protection. The results showed that incubating SN4741 cells with myricitrin significantly reduced cell death induced by the neurotoxin MPP(+). Furthermore, myricitrin protected cells from MPP(+)-induced effects on mitochondrial morphology and function. However, these protective effects were lost under DJ-1-deficient conditions. Thus, our results suggest that myricitrin alleviates MPP(+)-induced mitochondrial dysfunction and increases cell viability via DJ-1, indicating that myricitrin is a potential beneficial agent for age-related neurodegenerative diseases, particularly Parkinson's disease.

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 Parkinson's 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.