Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD.

ATM-dependent phosphorylation of MEF2D promotes neuronal survival after DNA damage.

Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM-MEF2D pathway may contribute to neurodegeneration in AT.

The retroviral oncoprotein Tax targets the coiled-coil centrosomal protein TAX1BP2 to induce centrosome overduplication.

Emerging evidence suggests that supernumerary centrosomes drive genome instability and oncogenesis. Human T-cell leukaemia virus type I (HTLV-I) is etiologically associated with adult T-cell leukaemia (ATL). ATL cells are aneuploid, but the causes of aneuploidy are incompletely understood. Here, we show that centrosome amplification is frequent in HTLV-I-transformed cells and that this phenotype is caused by the viral Tax oncoprotein. We also show that the fraction of Tax protein that localizes to centrosomes interacts with TAX1BP2, a novel centrosomal protein composed almost entirely of coiled-coil domains. Overexpression of TAX1BP2 inhibited centrosome duplication, whereas depletion of TAX1BP2 by RNAi resulted in centrosome hyperamplification. Our findings suggest that the HTLV-I Tax oncoprotein targets TAX1BP2 causing genomic instability and aneuploidy.

Chronic heart failure following hemorrhagic myocardial infarction: mechanism, treatment and outlook.

Myocardial infarction (MI), the blockage of arterial blood supply of the heart, is among the most common causes of death worldwide. Even when patients receive immediate treatment by re-opening blocked arteries, they often develop chronic heart failure (CHF) in the aftermath of MI events. Yet, the factors that contribute to the development of MI-associated CHF are poorly understood. In our recent study (Nat Commun 13:6394), we link intramyocardial hemorrhage, an injury which can occur during reperfusion of areas affected by MI, to an increased risk of CHF. Mechanistically, our data suggest that an iron-induced adverse cascade of events after hemorrhagic MI drives fatty degeneration of infarcted tissue, which ultimately contributes to negative cardiac remodeling. In this Microreview, we discuss the implications of our findings regarding the molecular mechanism, more targeted treatment options as well as perspectives in the clinical care of CHF after hemorrhagic MI.

Intramyocardial hemorrhage drives fatty degeneration of infarcted myocardium.

Sudden blockage of arteries supplying the heart muscle contributes to millions of heart attacks (myocardial infarction, MI) around the world. Although re-opening these arteries (reperfusion) saves MI patients from immediate death, approximately 50% of these patients go on to develop chronic heart failure (CHF) and die within a 5-year period; however, why some patients accelerate towards CHF while others do not remains unclear. Here we show, using large animal models of reperfused MI, that intramyocardial hemorrhage - the most damaging form of reperfusion injury (evident in nearly 40% of reperfused ST-elevation MI patients) - drives delayed infarct healing and is centrally responsible for continuous fatty degeneration of the infarcted myocardium contributing to adverse remodeling of the heart. Specifically, we show that the fatty degeneration of the hemorrhagic MI zone stems from iron-induced macrophage activation, lipid peroxidation, foam cell formation, ceroid production, foam cell apoptosis and iron recycling. We also demonstrate that timely reduction of iron within the hemorrhagic MI zone reduces fatty infiltration and directs the heart towards favorable remodeling. Collectively, our findings elucidate why some, but not all, MIs are destined to CHF and help define a potential therapeutic strategy to mitigate post-MI CHF independent of MI size.

Association of the small GTPase Rheb with the NMDA receptor subunit NR3A.

The NMDAR subunit NR3A is most highly expressed during the second postnatal week, when synaptogenesis reaches peak levels. Genetic ablation or overexpression of the NR3A subunit negatively interferes with the maturation of cortical synapses and leads to changes in the shape and number of dendritic spines, the density of which is increased in NR3A knock-out mice and decreased in NR3A-overexpressing transgenic mice. Alterations in spine density have been linked to dysregulation of mTOR signaling and synaptic protein translation. Using a yeast two-hybrid system, we identified the mTOR-activating GTPase Rheb as an interacting protein of the NMDAR subunit NR3A. We confirmed the interaction in mammalian cells by expressing recombinant Rheb and NR3A and showed that Rheb and NR3A could be co-immunoprecipitated from synaptic plasma membranes from the developing rat brain. These data suggest that NR3A sequesters synaptic Rheb and might thus function as a break of the mTOR-dependent synaptic translation of protein.

NitroSynapsin therapy for a mouse MEF2C haploinsufficiency model of human autism.

Transcription factor MEF2C regulates multiple genes linked to autism spectrum disorder (ASD), and human MEF2C haploinsufficiency results in ASD, intellectual disability, and epilepsy. However, molecular mechanisms underlying MEF2C haploinsufficiency syndrome remain poorly understood. Here we report that Mef2c +/-(Mef2c-het) mice exhibit behavioral deficits resembling those of human patients. Gene expression analyses on brains from these mice show changes in genes associated with neurogenesis, synapse formation, and neuronal cell death. Accordingly, Mef2c-het mice exhibit decreased neurogenesis, enhanced neuronal apoptosis, and an increased ratio of excitatory to inhibitory (E/I) neurotransmission. Importantly, neurobehavioral deficits, E/I imbalance, and histological damage are all ameliorated by treatment with NitroSynapsin, a new dual-action compound related to the FDA-approved drug memantine, representing an uncompetitive/fast off-rate antagonist of NMDA-type glutamate receptors. These results suggest that MEF2C haploinsufficiency leads to abnormal brain development, E/I imbalance, and neurobehavioral dysfunction, which may be mitigated by pharmacological intervention.

Mitochondrial targeting of growth suppressor protein DLC2 through the START domain.

Deleted in liver cancer 2 (DLC2) is a candidate tumor suppressor frequently found to be deleted in hepatocellular carcinoma. In this study, we determined the subcellular localization of DLC2. Co-localization and biochemical fractionation studies revealed that DLC2 localized to mitochondria. In addition, the DLC2-containing cytoplasmic speckles were in proximity to lipid droplets. A DLC2 mutant containing the steroidogenic acute regulatory protein-related lipid transfer (START) domain only showed a localization pattern identical to that of DLC2. Taken together, we have provided the first evidence for mitochondrial localization of DLC2 through the START domain. These findings might have implications in liver physiology and carcinogenesis.

Transcriptional profiling of MEF2-regulated genes in human neural progenitor cells derived from embryonic stem cells.

[Briefly describe the contents of the Data in Brief article. Tell the reader the repository and reference number for the data in the abstract to.] The myocyte enhancer factor 2 (MEF2) family of transcription factors is highly expressed in the brain, and constitutes a key determinant of neuronal survival, differentiation, and synaptic plasticity. However, genome-wide transcriptional profiling of MEF2-regulated genes has not yet been fully elucidated, particularly at the neural stem cell stage. Here we report the results of microarray analysis comparing mRNAs isolated from human neural progenitor/stem cells (hNPCs) derived from embryonic stem cells expressing a control vector versus progenitors expressing a constitutively-active form of MEF2 (MEF2CA), which increases MEF2 activity. Microarray experiments were performed using the Illumina Human HT-12 V4.0 expression beadchip (GEO#: GSE57184). By comparing vector-control cells to MEF2CA cells, microarray analysis identified 1880 unique genes that were differentially expressed. Among these genes, 1121 genes were upregulated and 759 genes were down-regulated. Our results provide a valuable resource for identifying transcriptional targets of MEF2 in hNPCs.

Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain.

NR3A is a developmentally regulated N-methyl-D-aspartate receptor (NMDAR) subunit that was previously known as NMDAR-L or chi-1. Unlike other NMDAR subunits, NR3A inhibits the NMDAR-associated ion channel in a novel manner, and a role in synaptogenesis has been suggested for this subunit. Here, we report a comprehensive study to delineate the temporal and anatomic expression of NR3A protein in the mammalian brain by using a monoclonal anti-NR3A antibody. NR3A protein was found to peak at postnatal day (P) 8, and to decrease gradually from P12 to adulthood in the rat central nervous system. Moreover, NR3A protein was heavily expressed in all areas of the isocortex, portions of the amygdaloid nuclei, and selective cell layers and nuclei of the hippocampus, thalamus, hypothalamus, brainstem, and spinal cord. NR3A protein was also expressed in the cerebellar cortex, whereas only weak signal was detected in the previous in situ studies by using riboprobes. At an ultrastructural level, NR3A was associated specifically with asymmetrical synapses and localized to postsynaptic membranes. This information will facilitate future research on NMDARs by providing clues to possible inclusion of the NR3A subunit in NMDARs in many brain regions.

S-nitrosylation-mediated redox transcriptional switch modulates neurogenesis and neuronal cell death.

Redox-mediated posttranslational modifications represent a molecular switch that controls major mechanisms of cell function. Nitric oxide (NO) can mediate redox reactions via S-nitrosylation, representing transfer of an NO group to a critical protein thiol. NO is known to modulate neurogenesis and neuronal survival in various brain regions in disparate neurodegenerative conditions. However, a unifying molecular mechanism linking these phenomena remains unknown. Here, we report that S-nitrosylation of myocyte enhancer factor 2 (MEF2) transcription factors acts as a redox switch to inhibit both neurogenesis and neuronal survival. Structure-based analysis reveals that MEF2 dimerization creates a pocket, facilitating S-nitrosylation at an evolutionally conserved cysteine residue in the DNA binding domain. S-Nitrosylation disrupts MEF2-DNA binding and transcriptional activity, leading to impaired neurogenesis and survival in vitro and in vivo. Our data define a molecular switch whereby redox-mediated posttranslational modification controls both neurogenesis and neurodegeneration via a single transcriptional signaling cascade.

S-Nitrosylation of parkin as a novel regulator of p53-mediated neuronal cell death in sporadic Parkinson's disease.

BACKGROUND: Mutations in the gene encoding parkin, a neuroprotective protein with dual functions as an E3 ubiquitin ligase and transcriptional repressor of p53, are linked to familial forms of Parkinson's disease (PD). We hypothesized that oxidative posttranslational modification of parkin by environmental toxins may contribute to sporadic PD. RESULTS: We first demonstrated that S-nitrosylation of parkin decreased its activity as a repressor of p53 gene expression, leading to upregulation of p53. Chromatin immunoprecipitation as well as gel-shift assays showed that parkin bound to the p53 promoter, and this binding was inhibited by S-nitrosylation of parkin. Additionally, nitrosative stress induced apoptosis in cells expressing parkin, and this death was, at least in part, dependent upon p53. In primary mesencephalic cultures, pesticide-induced apoptosis was prevented by inhibition of nitric oxide synthase (NOS). In a mouse model of pesticide-induced PD, both S-nitrosylated (SNO-)parkin and p53 protein levels were increased, while administration of a NOS inhibitor mitigated neuronal death in these mice. Moreover, the levels of SNO-parkin and p53 were simultaneously elevated in postmortem human PD brain compared to controls. CONCLUSIONS: Taken together, our data indicate that S-nitrosylation of parkin, leading to p53-mediated neuronal cell death, contributes to the pathophysiology of sporadic PD.

MEF2C enhances dopaminergic neuron differentiation of human embryonic stem cells in a parkinsonian rat model.

Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to treat Parkinson's disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in vitro. Conversely, RNAi knockdown of MEF2C in NPCs decreases neuronal differentiation and dendritic length. When we inject MEF2CA-programmed NPCs into 6-hydroxydopamine-lesioned parkinsonian rats in vivo, the transplanted cells survive well, differentiate into tyrosine hydroxylase-positive neurons, and improve behavioral deficits to a significantly greater degree than non-programmed cells. The enriched generation of dopaminergic neuronal lineages from hESCs by forced expression of MEF2CA in the proper context may prove valuable in cell-based therapy for CNS disorders such as PD.

Deleted in liver cancer (DLC) 2 encodes a RhoGAP protein with growth suppressor function and is underexpressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major malignancy in many parts of the world, especially in Asia and Africa. Loss of heterozygosity (LOH) on the long arm of chromosome 13 has been reported in HCC. In search of tumor suppressor genes in this region, here we have identified DLC2 (for deleted in liver cancer 2) at 13q12.3 encoding a novel Rho family GTPase-activating protein (GAP). DLC2 mRNA is ubiquitously expressed in normal tissues but was significantly underexpressed in 18% (8/45) of human HCCs. DLC2 is homologous to DLC1, a previously identified tumor suppressor gene at 8p22-p21.3 frequently deleted in HCC. DLC2 encodes a novel protein with a RhoGAP domain, a SAM (sterile alpha motif) domain related to p73/p63, and a lipid-binding StAR-related lipid transfer (START) domain. Biochemical analysis indicates that DLC2 protein has GAP activity specific for small GTPases RhoA and Cdc42. Expression of the GAP domain of DLC2 sufficiently inhibits the Rho-mediated formation of actin stress fibers. Introduction of human DLC2 into mouse fibroblasts suppresses Ras signaling and Ras-induced cellular transformation in a GAP-dependent manner. Taken together, our findings suggest a role for DLC2 in growth suppression and hepatocarcinogenesis.

Characterization and comparison of the NR3A subunit of the NMDA receptor in recombinant systems and primary cortical neurons.

Recently, we cloned and began to characterize a new N-methyl-D-aspartate receptor (NMDAR) subunit, NR3A. Here we extend our earlier findings by showing that recombinantly expressed NR3A in COS cells is biochemically associated with both NR1 and NR2 subunits. In the oocyte or HEK 293 cell expression systems, co-injection of NR3A with NR1/NR2 subunits acts in a dominant-interfering manner, resulting in a decrease in NMDAR unitary conductance, decrease in Ca(2+) permeability, decrease in Mg(2+) sensitivity, and slight increase in mean open time compared with NR1/NR2 channels. The smaller unitary conductance channel has also been observed in primary cortical neurons cultured from wild-type rodent on postnatal day 8 (P8) and similarly found to be relatively insensitive to Mg(2+) block. Consistent with these findings, whole cell NMDA-evoked currents are larger in NR3A-deficient mice compared with wild-type mice, and this effect follows a developmental pattern similar to that of NR3A protein expression on Western blots, with peak expression at P8. Finally, a new longer splice variant of NR3A has been cloned and found to be expressed in rodent cortical neurons by single-cell RT-PCR and in situ hybridization.