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Chaperone-mediated autophagy controls the degradation of selective cytosolic proteins and may protect neurons against degeneration. In a neuronal cell line, we found that chaperone-mediated autophagy regulated the activity of myocyte enhancer factor 2D (MEF2D), a transcription factor required for neuronal survival. MEF2D was observed to continuously shuttle to the cytoplasm, interact with the chaperone Hsc70, and undergo degradation. Inhibition of chaperone-mediated autophagy caused accumulation of inactive MEF2D in the cytoplasm. MEF2D levels were increased in the brains of α-synuclein transgenic mice and patients with Parkinson's disease. Wild-type α-synuclein and a Parkinson's disease-associated mutant disrupted the MEF2D-Hsc70 binding and led to neuronal death. Thus, chaperone-mediated autophagy modulates the neuronal survival machinery, and dysregulation of this pathway is associated with Parkinson's disease.In neurodegenerative diseases, certain populations of adult neurons are gradually lost because of toxic stress. The four myocyte enhancer factor 2 (MEF2) transcription factors, MEF2A to MEF2D, have been shown to play an important role in the survival of several types of neurons, and a genetic polymorphism of the MEF2A gene has been linked to the risk of late onset of Alzheimer's disease (1-3). In cellular models, inhibition of MEF2s contributes to neuronal death. Enhancing MEF2 activity protects neurons from death in vitro and in the substantia nigra pars compacta in a mouse model of Parkinson's disease (PD) (4). Neurotoxic insults cause MEF2 degradation in part by a caspase-dependent mechanism (5), but how MEF2 is regulated under basal conditions without overt toxicity is unknown. Autophagy refers to the degradation of intracellular components by lysosomes. Relative to macro-and microautophagy, chaperonemediated autophagy (CMA) selectively degrades cytosolic proteins (6). This process involves binding of heat shock protein Hsc70 to substrate proteins via a KFERQ-like motif and their subsequent targeting to lysosomes via the lysosomal membrane receptor Lamp2a. Dysregulation of autophagy plays a role in neurodegeneration (7-9). However, the direct mechanism by which CMA modulates neuronal survival or death is unclear.

The transcription factors in the myocyte enhancer factor 2 (MEF2) family play important roles in cell survival by regulating nuclear gene expression. Here, we report that MEF2D is present in rodent neuronal mitochondria, where it can regulate the expression of a gene encoded within mitochondrial DNA (mtDNA). Immunocytochemical, immunoelectron microscopic, and biochemical analyses of rodent neuronal cells showed that a portion of MEF2D was targeted to mitochondria via an N-terminal motif and the chaperone protein mitochondrial heat shock protein 70 (mtHsp70). MEF2D bound to a MEF2 consensus site in the region of the mtDNA that contained the gene NADH dehydrogenase 6 (ND6), which encodes an essential component of the complex I enzyme of the oxidative phosphorylation system; MEF2D binding induced ND6 transcription. Blocking MEF2D function specifically in mitochondria decreased complex I activity, increased cellular H 2 O 2 level, reduced ATP production, and sensitized neurons to stress-induced death. Toxins known to affect complex I preferentially disrupted MEF2D function in a mouse model of Parkinson disease (PD). In addition, mitochondrial MEF2D and ND6 levels were decreased in postmortem brain samples of patients with PD compared with age-matched controls. Thus, direct regulation of complex I by mitochondrial MEF2D underlies its neuroprotective effects, and dysregulation of this pathway may contribute to PD.

A eukaryotic protein is often subject to regulation by multiple modifications like phosphorylation, acetylation, ubiquitination, and sumoylation. How these modifications are coordinated in vivo is an important issue that is poorly understood but is relevant to many biological processes. We recently showed that human MEF2D (myocyte enhancer factor 2D) is sumoylated on Lys-439. Adjacent to the sumoylation motif is Ser-444, which like Lys-439 is highly conserved among MEF2 proteins from diverse species. Here we presented several lines of evidence to demonstrate that Ser-444 of MEF2D is required for sumoylation of Lys-439. Histone deacetylase 4 (HDAC4) stimulated this modification by acting through Ser-444. In addition, phosphorylation of Ser-444 by Cdk5, a cyclin-dependent kinase known to inhibit MEF2 transcriptional activity, stimulated sumoylation. Opposing the actions of HDAC4 and Cdk5, calcineurin (also known as protein phosphatase 2B) dephosphorylated Ser-444 and inhibited sumoylation of Lys-439. This phosphatase, however, exerted minimal effects on the phosphorylation catalyzed by ERK5, an extracellular signal-regulated kinase known to activate MEF2D. These results identified an essential role for Ser-444 in MEF2D sumoylation and revealed a novel mechanism by which calcineurin selectively "edits" phosphorylation at different sites, thereby reiterating that interplay between different modifications represents a general mechanism for coordinated regulation of eukaryotic protein functions in vivo.In higher eukaryotes, each cell type has a unique gene expression pattern that is ultimately determined by a specific network of transcription factors. The question how cell signaling regulates activities of transcription factors is thus of central importance to many biological processes. The MEF2 3 family of transcription factors comprises four members in mammals, MEF2A, -B, -C, and -D. They were originally identified as major transcriptional activators for muscle differentiation (1, 2). Indeed, some of them were subsequently shown to be important for cardiac myogenesis; null mutation of the murine MEF2A or MEF2C gene led to cardiac death (3-5), and a mutation on the human MEF2A gene was recently suggested to play a role in coronary artery disease (6). MEF2 proteins also have important roles in non-muscle cells by regulating growth factor response, viral gene expression, neuronal survival, T-cell apoptosis, and tumorigenesis (7-12). Consistent with this, the human MEF2D gene is rearranged in pre-B acute lymphoblastic leukemia (13,14), and large scale retrovirus-mediated insertion mutagenesis identified the mouse MEF2D gene as a potential oncogene (15, 16). Therefore, MEF2 transcription factors are key players in diverse cellular programs.At the molecular level, MEF2 is composed of a highly conserved N-terminal domain responsible for DNA recognition and a C-terminal domain with trans-acting function. Regulation of MEF2 function is complex and occurs at multiple levels, including tissue-specific expression (1, 5), altern...

The phosphatidylinositol-3-kinase-like kinase ATM (Ataxia – telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. However, the mechanism by which DNA damage activates ATM is poorly understood. We show that Cdk5 (cyclin-dependent kinase 5), activated by DNA damage, directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM signal regulates phosphorylation and function of ATM targets p53 and H2AX. Interruption of Cdk5-ATM pathway attenuates DNA damage-induced neuronal cell cycle reentry and expression of p53 targets PUMA and Bax, protecting neurons from DNA damage-induced death. Thus, activation of Cdk5 by DNA damage serves as a critical signal to initiate ATM response and regulate ATM-dependent cellular processes.

BackgroundTraditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizaeare are prescribed together for their putative cardioprotective effects in clinical practice. However, the rationale of the combined use remains unclear. The present study was designed to investigate the cardioprotective effects of paeonol and danshensu (representative active ingredient of Cortex Moutan and Radix Salviae Milthiorrhizae, respectively) on isoproterenol-induced myocardial infarction in rats and its underlying mechanisms.MethodologyPaeonol (80 mg kg−1) and danshensu (160 mg kg−1) were administered orally to Sprague Dawley rats in individual or in combination for 21 days. At the end of this period, rats were administered isoproterenol (85 mg kg−1) subcutaneously to induce myocardial injury. After induction, rats were anaesthetized with pentobarbital sodium (35 mg kg−1) to record electrocardiogram, then sacrificed and biochemical assays of the heart tissues were performed.Principal FindingsInduction of rats with isoproterenol resulted in a marked (P<0.001) elevation in ST-segment, infarct size, level of serum marker enzymes (CK-MB, LDH, AST and ALT), cTnI, TBARS, protein expression of Bax and Caspase-3 and a significant decrease in the activities of endogenous antioxidants (SOD, CAT, GPx, GR, and GST) and protein expression of Bcl-2. Pretreatment with paeonol and danshensu combination showed a significant (P<0.001) decrease in ST-segment elevation, infarct size, cTnI, TBARS, protein expression of Bax and Caspase-3 and a significant increase in the activities of endogenous antioxidants and protein expression of Bcl-2 and Nrf2 when compared with individual treated groups.Conclusions/SignificanceThis study demonstrates the cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial infarction in rats. The mechanism might be associated with the enhancement of antioxidant defense system through activating of Nrf2 signaling and anti-apoptosis through regulating Bax, Bcl-2 and Caspase-3. It could provide experimental evidence to support the rationality of combinatorial use of traditional Chinese medicine in clinical practice.

Paeonol and danshensu is the representative active ingredient of traditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizae, respectively. Paeonol and danshensu combination (PDSS) has putative cardioprotective effects in treating ischemic heart disease (IHD). However, the evidence for the protective effect is scarce and the pharmacological mechanisms of the combination remain unclear. The present study was designed to investigate the protective effect of PDSS on isoproterenol (ISO)-induced myocardial infarction in rats and to elucidate the potential mechanism. Assays of creatine kinase-MB, cardiac troponin I and T and histopathological analysis revealed PDSS significantly prevented myocardial injury induced by ISO. The ISO-induced profound elevation of oxidative stress was also suppressed by PDSS. TUNEL and caspase-3 activity assay showed that PDSS significantly inhibited apoptosis in myocardia. In exploring the underlying mechanisms of PDSS, we found PDSS enhanced the nuclear translocation of Nrf2 in myocardial injured rats. Furthermore, PDSS increased phosphorylated PI3K and Akt, which may in turn activate antioxidative and antiapoptotic signaling events in rat. These present findings demonstrated that PDSS exerts significant cardioprotective effects against ISO-induced myocardial infarction in rats. The protective effect is, at least partly, via activation of Nrf2/HO-1 signaling and involvement of the PI3K/Akt cell survival signaling pathway.