Parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome.

Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function. In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q10 (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells. To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ10 (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.

Hypothalamic C2-domain protein involved in MC4R trafficking and control of energy balance.

OBJECTIVE: Rates of overweight and obesity epidemic have risen significantly in the past few decades, and 34% of adults and 15-20% of children and adolescents in the United States are now obese. Melanocortin receptor 4 (MC4R), contributes to appetite control in hypothalamic neurons and is a target for future anti-obesity treatments (such as setmelanotide) or novel drug development effort. Proper MC4R trafficking regulation in hypothalamic neurons is crucial for normal neural control of homeostasis and is altered in obesity and in presence of lipids. The mechanisms underlying altered MC4R trafficking in the context of obesity is still unclear. Here, we discovered that C2CD5 expressed in the hypothalamus is involved in the regulation of MC4R endocytosis. This study unmasked a novel trafficking protein nutritionally regulated in the hypothalamus providing a novel target for MC4R dependent pathways involved in bodyweight homeostasis and Obesity. METHODS: To evaluate the expression of C2cd5, we first used in situ hybridization and RNAscope technology in combination with electronic microscopy. For in vivo, we characterized the energy balance of wild type (WT) and C2CD5 whole-body knockout (C2CD5KO) mice fed normal chow (NC) and/or western-diet (high-fat/high-sucrose/cholesterol) (WD). To this end, we performed comprehensive longitudinal assessment of bodyweight, energy balance (food intake, energy expenditure, locomotor activity using TSE metabolic cages), and glucose homeostasis. In addition, we evaluated the consequence of loss of C2CD5 on feeding behavior changes normally induced by MC4R agonist (Melanotan, MTII) injection in the paraventricular hypothalamus (PVH). For in vitro approach, we tease out the role of C2CD5 and its calcium sensing domain C2 in MC4R trafficking. We focused on endocytosis of MC4R using an antibody feeding experiment (in a neuronal cell line - Neuro2A (N2A) stably expressing HA-MC4R-GFP; against HA-tag and analyzed by flux cytometry). RESULTS: We found that 1) the expression of hypothalamic C2CD5 is decreased in diet-induced obesity models compared to controls, 2) mice lacking C2CD5 exhibit an increase in food intake compared to WT mice, 3) C2CD5 interacts with endocytosis machinery in hypothalamus, 4) loss of functional C2CD5 (lacking C2 domain) blunts MC4R endocytosis in vitro and increases MC4R at the surface that fails to respond to MC4R ligand, and, 5) C2CD5KO mice exhibit decreased acute responses to MTII injection into the PVH. CONCLUSIONS: Based on these, we conclude that hypothalamic C2CD5 is involved in MC4R endocytosis and regulate bodyweight homeostasis. These studies suggest that C2CD5 represents a new protein regulated by metabolic cues and involved in metabolic receptor endocytosis. C2CD5 represent a new target and pathway that could be targeted in Obesity.

BioID screen of Salmonella type 3 secreted effectors reveals host factors involved in vacuole positioning and stability during infection.

Many bacterial pathogens express virulence proteins that are translocated into host cells (herein referred to as effectors), where they can interact with target proteins to manipulate host cell processes. These effector-host protein interactions are often dynamic and transient in nature, making them difficult to identify using traditional interaction-based methods. Here, we performed a systematic comparison between proximity-dependent biotin labelling (BioID) and immunoprecipitation coupled with mass spectrometry to investigate a series of Salmonella type 3 secreted effectors that manipulate host intracellular trafficking (SifA, PipB2, SseF, SseG and SopD2). Using BioID, we identified 632 candidate interactions with 381 unique human proteins, collectively enriched for roles in vesicular trafficking, cytoskeleton components and transport activities. From the subset of proteins exclusively identified by BioID, we report that SifA interacts with BLOC-2, a protein complex that regulates dynein motor activity. We demonstrate that the BLOC-2 complex is necessary for SifA-mediated positioning of Salmonella-containing vacuoles, and affects stability of the vacuoles during infection. Our study provides insight into the coordinated activities of Salmonella type 3 secreted effectors and demonstrates the utility of BioID as a powerful, complementary tool to characterize effector-host protein interactions.

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.

Depletion of phosphatidylinositol 4-phosphate at the Golgi translocates K-Ras to mitochondria.

Ras proteins are small GTPases localized to the plasma membrane (PM), which regulate cellular proliferation, apoptosis and differentiation. After a series of post-translational modifications, H-Ras and N-Ras traffic to the PM from the Golgi via the classical exocytic pathway, but the exact mechanism of K-Ras trafficking to the PM from the ER is not fully characterized. ATP5G1 (also known as ATP5MC1) is one of the three proteins that comprise subunit c of the F0 complex of the mitochondrial ATP synthase. In this study, we show that overexpression of the mitochondrial targeting sequence of ATP5G1 perturbs glucose metabolism, inhibits oncogenic K-Ras signaling, and redistributes phosphatidylserine (PtdSer) to mitochondria and other endomembranes, resulting in K-Ras translocation to mitochondria. Also, it depletes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. Glucose supplementation restores PtdSer and K-Ras PM localization and PI4P at the Golgi. We further show that inhibition of the Golgi-localized PI4-kinases (PI4Ks) translocates K-Ras, and PtdSer to mitochondria and endomembranes, respectively. We conclude that PI4P at the Golgi regulates the PM localization of PtdSer and K-Ras.This article has an associated First Person interview with the first author of the paper.

ADAM22 and ADAM23 modulate the targeting of the Kv1 channel-associated protein LGI1 to the axon initial segment.

The distribution of the voltage-gated Kv1 K+ channels at the axon initial segment (AIS) influences neuronal intrinsic excitability. The Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) subunits are associated with cell adhesion molecules (CAMs), including Caspr2 (also known as CNTNAP2) and LGI1, which are implicated in autoimmune and genetic neurological diseases with seizures. In particular, mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). Here, by using rat hippocampal neurons in culture, we showed that LGI1 is recruited to the AIS where it colocalizes with ADAM22 and Kv1 channels. Strikingly, the missense mutations S473L and R474Q of LGI1 identified in ADLTE prevent its association with ADAM22 and enrichment at the AIS. Moreover, we observed that ADAM22 and ADAM23 modulate the trafficking of LGI1, and promote its ER export and expression at the overall neuronal cell surface. Live-cell imaging indicated that LGI1 is co-transported in axonal vesicles with ADAM22 and ADAM23. Finally, we showed that ADAM22 and ADAM23 also associate with Caspr2 and TAG-1 (also known as CNTN2) to be selectively targeted to different axonal sub-regions. Hence, the combinatorial expression of Kv1-associated CAMs may be critical to tune intrinsic excitability in physiological and epileptogenic contexts.

fam20C participates in the shell formation in the pearl oyster, Pinctada fucata.

Kinase-family with sequence similarity 20, member C (Fam20C) is a protein kinase, which can phosphorylate biomineralization related proteins in vertebrate animals. However, the function of Fam20C in invertebrate animals especially the role in biomineralization is still unknown. Herein, we cloned the cDNA of fam20C from the pearl oyster, Pinctada fucata. It is showed that the expression of fam20C in the mantle edge was much higher than other tissues. In situ hybridization showed that fam20C was expressed mostly in the outer epithelial cells of the middle fold, indicating it may play important roles in the shell formation. Besides, fam20C expression increased greatly in the D-shape stage of pearl oyster development, when the shell was first formed. During the shell repair process, the expression level of fam20C increased 1.5 times at 6 h after shell notching. Knockdown of fam20C in vivo by RNA interference resulted in abnormally stacking of calcium carbonate crystals at the edges of nacre tablets, showing direct evidence that fam20C participates in the shell formation. This study provides an insight into the role of kinase protein in the shell formation in mollusk and broaden our understanding of biomineralization mechanism.

ADF10 shapes the overall organization of apical actin filaments by promoting their turnover and ordering in pollen tubes.

Here, we show that Arabidopsis ADF10 plays an important role in shaping the overall organization of apical actin filaments by promoting their turnover and ordering. ADF10 severs and depolymerizes actin filaments in vitro and is distributed throughout the entire pollen tube. In adf10 mutants, severing and monomer dissociation events for apical actin filaments are reduced, and the apical actin structure extends further toward the tube base than in wild-type tubes. In particular, the percentage of apical actin filaments that form large angles to the tube growth axis is much higher in adf10 pollen tubes, and the actin filaments are more randomly distributed, implying that ADF10 promotes their ordering. Consistent with the role of apical actin filaments in physically restricting the movement of vesicles, the region in which apical vesicles accumulate is enlarged at the tip of adf10 pollen tubes. Both tipward and backward movements of small vesicles are altered within the growth domain of adf10 pollen tubes. Thus, our study suggests that ADF10 shapes the organization of apical actin filaments to regulate vesicle trafficking and pollen tube growth.

Reactive oxygen species trigger Parkin/PINK1 pathway-dependent mitophagy by inducing mitochondrial recruitment of Parkin.

Defective mitophagy linked to dysfunction in the proteins Parkin and PTEN-induced putative kinase 1 (PINK1) is implicated in the pathogenesis of Parkinson's disease. Although the mechanism by which Parkin mediates mitophagy in a PINK1-dependent manner is becoming clearer, the triggers for this mitophagy pathway remain elusive. Reactive oxygen species (ROS) have been suggested as such triggers, but this proposal remains controversial because ROS scavengers fail to retard mitophagy. Here we demonstrate that the role of ROS in mitophagy has been underappreciated as a result of the inefficiency of ROS scavengers to control ROS bursts after high-dose treatment with carbonyl cyanide m-chlorophenylhydrazone. Supporting this, combinatorial treatment with N-acetyl-l-cysteine and catalase substantially inhibited the ROS upsurge and PINK1-dependent Parkin translocation to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone treatment. In addition to the chemical mitophagy inducer, overexpression of voltage-dependent anion channel 1 (VDAC1) induced Parkin translocation to mitochondria, presumably by stimulating ROS generation. Similarly, combined N-acetyl-l-cysteine and catalase treatment also suppressed VDAC1-induced redistribution of Parkin. Alongside these observations, we also found that the elevated protein level of PINK1 was not necessary for Parkin translocation to mitochondria. Thus, our data suggest that ROS may act as a trigger for the induction of Parkin/PINK1-dependent mitophagy. In addition, our study casts doubt on the importance of protein quantity of PINK1 in the recruitment of Parkin to mitochondria.

Neuroplastin Isoform Np55 Is Expressed in the Stereocilia of Outer Hair Cells and Required for Normal Outer Hair Cell Function.

UNLABELLED: Neuroplastin (Nptn) is a member of the Ig superfamily and is expressed in two isoforms, Np55 and Np65. Np65 regulates synaptic transmission but the function of Np55 is unknown. In an N-ethyl-N-nitrosaurea mutagenesis screen, we have now generated a mouse line with an Nptn mutation that causes deafness. We show that Np55 is expressed in stereocilia of outer hair cells (OHCs) but not inner hair cells and affects interactions of stereocilia with the tectorial membrane. In vivo vibrometry demonstrates that cochlear amplification is absent in Nptn mutant mice, which is consistent with the failure of OHC stereocilia to maintain stable interactions with the tectorial membrane. Hair bundles show morphological defects as the mutant mice age and while mechanotransduction currents can be evoked in early postnatal hair cells, cochlea microphonics recordings indicate that mechanontransduction is affected as the mutant mice age. We thus conclude that differential splicing leads to functional diversification of Nptn, where Np55 is essential for OHC function, while Np65 is implicated in the regulation of synaptic function. SIGNIFICANCE STATEMENT: Amplification of input sound signals, which is needed for the auditory sense organ to detect sounds over a wide intensity range, depends on mechanical coupling of outer hair cells to the tectorial membrane. The current study shows that neuroplastin, a member of the Ig superfamily, which has previously been linked to the regulation of synaptic plasticity, is critical to maintain a stable mechanical link of outer hair cells with the tectorial membrane. In vivo recordings demonstrate that neuroplastin is essential for sound amplification and that mutation in neuroplastin leads to auditory impairment in mice.

Arabidopsis COG Complex Subunits COG3 and COG8 Modulate Golgi Morphology, Vesicle Trafficking Homeostasis and Are Essential for Pollen Tube Growth.

Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, γ-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth.

Gypenoside XVII Enhances Lysosome Biogenesis and Autophagy Flux and Accelerates Autophagic Clearance of Amyloid-ß through TFEB Activation.

A strategy for activating transcription factor EB (TFEB) to restore autophagy flux may provide neuroprotection against Alzheimer's disease. Our previous study reported that gypenoside XVII (GP-17), which is a major saponin abundant in ginseng and Panax notoginseng, ameliorated amyloid-ß (Aß)25-35-induced apoptosis in PC12 cells by regulating autophagy. In the present study, we aimed to determine whether GP-17 has neuroprotective effects on PC12 cells expressing the Swedish mutant of APP695 (APP695swe) and APP/PS1 mice. We also investigated the underlying mechanism. We found that GP-17 could significantly increase Atg5 expression and the conversion of LC3-I to LC3-II in APP695 cells, which was associated with a reduction in p62 expression. GP-17 also elevated the number of LC3 puncta in APP695 cells transduced with pCMV-GFP-LC3. GP-17 promoted the autophagy-based elimination of AßPP, Aß40, and Aß42 in APP695swe cells and prevented the formation of Aß plaques in the hippocampus and cortex of APP/PS1 mice. Furthermore, spatial learning and memory deficits were cured. Atg5 knockdown could abrogate the GP-17-mediated removal of AßPP, Aß40, and Aß42 in APP695swe cells. GP-17 upregulated LAMP-1, increased LysoTracker staining, and augmented LAMP-1/LC3-II co-localization. GP-17 could release TFEB from TFEB/14-3-3 complexes, which led to TFEB nuclear translocation and the induction of autophagy and lysosome biogenesis and resulted in the amelioration of autophagy flux. The knockdown of TFEB could abolish these effects of GP-17. In summary, these results demonstrated that GP-17 conferred protective effects to the cellular and rodent models of Alzheimer's disease by activating TFEB.

Yin Yang-1 increases apoptosis through Bax activation in pancreatic cancer cells.

The transcriptional regulator Yin Yang-1 (YY1) is a tumor suppressor known to be overexpressed in pancreatic cancer. We found that overexpression of YY1 promoted apoptosis and increased the expression and mitochondrial localization of the pro-apoptotic Bax protein in pancreatic cancer cell lines. Luciferase reporter, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays revealed binding of YY1 to the BAX promoter. Moreover, YY1 promoted pancreatic cancer cell apoptosis through Bax transcriptional activation and subsequent translocation of Bax to the mitochondrial membrane, leading to cytochrome c release, and caspase activation.YY1 and BAX are co-expressed in pancreatic cancer tissues and higher BAX expression predicts better outcomes for patients. The ability of YY1 to promote apoptosis in pancreatic cancer cells suggests it may represent a valuable diagnostic and therapeutic target.

Oxytocin Regulates Stress-Induced Crf Gene Transcription through CREB-Regulated Transcription Coactivator 3.

The major regulator of the neuroendocrine stress response in the brain is corticotropin releasing factor (CRF), whose transcription is controlled by CREB and its cofactors CRTC2/3 (TORC2/3). Phosphorylated CRTCs are sequestered in the cytoplasm, but rapidly dephosphorylated and translocated into the nucleus following a stressful stimulus. As the stress response is attenuated by oxytocin (OT), we tested whether OT interferes with CRTC translocation and, thereby, Crf expression. OT (1 nmol, i.c.v.) delayed the stress-induced increase of nuclear CRTC3 and Crf hnRNA levels in the paraventricular nucleus of male rats and mice, but did not affect either parameter in the absence of the stressor. The increase in Crf hnRNA levels at later time points was parallel to elevated nuclear CRTC2/3 levels. A direct effect of Thr(4) Gly(7)-OT (TGOT) on CRTC3 translocation and Crf expression was found in rat primary hypothalamic neurons, amygdaloid (Ar-5), hypothalamic (H32), and human neuroblastoma (Be(2)M17) cell lines. CRTC3, but not CRCT2, knockdown using siRNA in Be(2)M17 cells prevented the effect of TGOT on Crf hnRNA levels. Chromatin-immunoprecipitation demonstrated that TGOT reduced CRTC3, but not CRTC2, binding to the Crf promoter after 10 min of forskolin stimulation. Together, the results indicate that OT modulates CRTC3 translocation, the binding of CRTC3 to the Crf promoter and, ultimately, transcription of the Crf gene. SIGNIFICANCE STATEMENT: The neuropeptide oxytocin has been proposed to reduce hypothalamic-pituitary-adrenal (HPA) axis activation during stress. The underlying mechanisms are, however, elusive. In this study we show that activation of the oxytocin receptor in the paraventricular nucleus delays transcription of the gene encoding corticotropin releasing factor (Crf), the main regulator of the stress response. It does so by sequestering the coactivator of the transcription factor CREB, CRTC3, in the cytosol, resulting in reduced binding of CRTC3 to the Crf gene promoter and subsequent Crf gene expression. This novel oxytocin receptor-mediated intracellular mechanism might provide a basis for the treatment of exaggerated stress responses in the future.

Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation.

Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic L-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions.

Pharmacologic rescue of an enzyme-trafficking defect in primary hyperoxaluria 1.

Primary hyperoxaluria 1 (PH1; Online Mendelian Inheritance in Man no. 259900), a typically lethal biochemical disorder, may be caused by the AGT(P11LG170R) allele in which the alanine:glyoxylate aminotransferase (AGT) enzyme is mistargeted from peroxisomes to mitochondria. AGT contains a C-terminal peroxisomal targeting sequence, but mutations generate an N-terminal mitochondrial targeting sequence that directs AGT from peroxisomes to mitochondria. Although AGT(P11LG170R) is functional, the enzyme must be in the peroxisome to detoxify glyoxylate by conversion to alanine; in disease, amassed glyoxylate in the peroxisome is transported to the cytosol and converted to oxalate by lactate dehydrogenase, leading to kidney failure. From a chemical genetic screen, we have identified small molecules that inhibit mitochondrial protein import. We tested whether one promising candidate, Food and Drug Administration (FDA)-approved dequalinium chloride (DECA), could restore proper peroxisomal trafficking of AGT(P11LG170R). Indeed, treatment with DECA inhibited AGT(P11LG170R) translocation into mitochondria and subsequently restored trafficking to peroxisomes. Previous studies have suggested that a mitochondrial uncoupler might work in a similar manner. Although the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited AGT(P11LG170R) import into mitochondria, AGT(P11LG170R) aggregated in the cytosol, and cells subsequently died. In a cellular model system that recapitulated oxalate accumulation, exposure to DECA reduced oxalate accumulation, similar to pyridoxine treatment that works in a small subset of PH1 patients. Moreover, treatment with both DECA and pyridoxine was additive in reducing oxalate levels. Thus, repurposing the FDA-approved DECA may be a pharmacologic strategy to treat PH1 patients with mutations in AGT because an additional 75 missense mutations in AGT may also result in mistrafficking.

Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency.

Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5' sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.

Wogonoside induces cell cycle arrest and differentiation by affecting expression and subcellular localization of PLSCR1 in AML cells.

Wogonoside is the main flavonoid component derived from the root of Scutellaria baicalensis Georgi. It is a popular Chinese herbal medicine with the potential to treat hematologic malignancies. In this study, we investigated the anticancer effects of wogonoside in acute myeloid leukemia (AML) cell lines and primary patient-derived AML cells. Wogonoside exerted antiproliferative properties both in vitro and in vivo. Furthermore, it efficiently inhibited the proliferation of U937 and HL-60 cells through the induction of G1 phase arrest and the promotion of differentiation. We also demonstrated that wogonoside significantly increased the transcription of phospholipid scramblase 1 (PLSCR1) due to its influence on the expression of cell cycle- and differentiation-related genes, including the upregulation of p21waf1/cip1 and downregulation of the oncogenic protein c-Myc. Wogonoside also promoted PLSCR1 trafficking into the nucleus and facilitated its binding to the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) promoter, thus increasing the expression of IP3R1. Finally, inhibition of PLSCR1 expression with small interfering RNA partially blocked wogonoside-induced cell cycle arrest and differentiation and disturbed the wogonoside-associated molecular events. The results of this study therefore suggest that wogonoside may represent a therapeutic candidate for the treatment of AML.

Adiponectin inhibits KISS1 gene transcription through AMPK and specificity protein-1 in the hypothalamic GT1-7 neurons.

Adiponectin secreted from adipose tissues plays a role in the regulation of energy homeostasis, food intake, and reproduction in the hypothalamus. We have previously demonstrated that adiponectin significantly inhibited GNRH secretion from GT1-7 hypothalamic GNRH neuron cells. In this study, we further investigated the effect of adiponectin on hypothalamic KISS1 gene transcription, which is the upstream signal of GNRH. We found that globular adiponectin (gAd) or AICAR, an artificial AMPK activator, decreased KISS1 mRNA transcription and promoter activity. Conversely, inhibition of AMPK by Compound C or AMPKα1-SiRNA augmented KISS1 mRNA transcription and promoter activity. Additionally, gAd and AICAR decreased the translocation of specificity protein-1 (SP1) from cytoplasm to nucleus; however, Compound C and AMPKα1-siRNA played an inverse role. Our experiments in vivo demonstrated that the expression of Kiss1 mRNA was stimulated twofold in the Compound C-treated rats and decreased about 60-70% in gAd- or AICAR-treated rats compared with control group. The numbers of kisspeptin immunopositive neurons in the arcuate nucleus region of Sprague Dawley rats mimicked the same trend seen in Kiss1 mRNA levels in animal groups with different treatments. In conclusion, our results provide the first evidence that adiponectin reduces Kiss1 gene transcription in GT1-7 cells through activation of AMPK and subsequently decreased translocation of SP1.

Dynein and dynactin components modulate neurodegeneration induced by excitotoxicity.

Glutamate excitotoxicity causes neuronal dysfunction and degeneration. It is implicated in chronic disorders, including Alzheimer's disease, and in acute CNS insults such as ischemia. These disorders share prominent morphological features, including axon degeneration and cell body death. However, the molecular mechanism underlying excitotoxicity-induced neurodegeneration remains poorly understood. A key molecular feature of neurodegeneration is deficits in microtubule-based cargo transport that plays a pivotal role in maintaining the balance of survival and stress signaling in the axon. We developed an excitotoxicity-induced neurodegeneration system in primary neuronal cultures. We find that excitotoxicity generates a C-terminal truncated form of p150Glued, a major component of the dynactin complex, which exacerbates axon degeneration. This p150Glued truncated form was identified in brain tissues of patients with Alzheimer's disease. Overexpression of wild-type (WT) dynein intermediate chain (DIC), a dynein component that interacts with p150Glued and links dynein and dynactin complexes, DIC (S84D) mutant, and WT p150Glued suppressed axon degeneration. These modulating effects of p150Glued and DIC on excitotoxicity-induced axon degeneration are also observed in apoptosis and cell body death. Thus, our findings identify retrograde transport proteins, p150Glued and DIC, as novel modulators of neurodegeneration induced by glutamate excitotoxicity.