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Find video protocols related to scientific articles indexed in Pubmed.
Beclin 1 is required for neuron viability and regulates endosome pathways via the UVRAG-VPS34 complex.
PLoS Genet.
PUBLISHED: 10-01-2014
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Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis.
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Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits.
Neuron
PUBLISHED: 08-21-2014
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Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.
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Assessment of chloroquine treatment for modulating autophagy flux in brain of WT and HD mice.
J Huntingtons Dis
PUBLISHED: 07-27-2014
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Increasing mutant huntingtin (mHTT) clearance through the autophagy pathway may be a way to treat Huntington's disease (HD). Tools to manipulate and measure autophagy flux in brain in vivo are not well established.
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Genetic causes of Parkinson's disease and their links to autophagy regulation.
Parkinsonism Relat. Disord.
PUBLISHED: 07-12-2014
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Genetic studies over the past 15 years have revolutionized our understanding towards the etiology of Parkinson's disease (PD). These studies have discovered many disease-linked genetic loci (PARK 1 to 18), which are now being interrogated for cellular pathways contributing to PD. Various pathogenic pathways were proposed but validation of each pathway awaits rigorous experimental testing. Here we review recent progress in understanding the influence of disease risk genes on cellular functions, specifically, autophagy pathways. Autophagy is a cell self-eating, lysosomal degradation system that plays an important role in cell homeostasis and survival. Neurons are post-mitotic cells and particularly vulnerable to the impairment of autophagic degradation due to their inability to redistribute damaged proteins and organelles to daughter cells. Emerging evidence has implicated dysfunctional autophagy in a growing number of neurodegenerative diseases including PD. We will also discuss the prospect of intervening autophagy pathways as a potential strategy to treat PD.
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A Parkinson's disease gene regulatory network identifies the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity.
Hum. Mol. Genet.
PUBLISHED: 05-02-2014
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Mutations in LRRK2 are one of the primary genetic causes of Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, and familial PD mutations affect both enzymatic activities. However, the signaling mechanisms regulating LRRK2 and the pathogenic effects of familial mutations remain unknown. Identifying the signaling proteins that regulate LRRK2 function and toxicity remains a critical goal for the development of effective therapeutic strategies. In this study, we apply systems biology tools to human PD brain and blood transcriptomes to reverse-engineer a LRRK2-centered gene regulatory network. This network identifies several putative master regulators of LRRK2 function. In particular, the signaling gene RGS2, which encodes for a GTPase-activating protein (GAP), is a key regulatory hub connecting the familial PD-associated genes DJ-1 and PINK1 with LRRK2 in the network. RGS2 expression levels are reduced in the striata of LRRK2 and sporadic PD patients. We identify RGS2 as a novel interacting partner of LRRK2 in vivo. RGS2 regulates both the GTPase and kinase activities of LRRK2. We show in mammalian neurons that RGS2 regulates LRRK2 function in the control of neuronal process length. RGS2 is also protective against neuronal toxicity of the most prevalent mutation in LRRK2, G2019S. We find that RGS2 regulates LRRK2 function and neuronal toxicity through its effects on kinase activity and independently of GTPase activity, which reveals a novel mode of action for GAP proteins. This work identifies RGS2 as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients.
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Autophagy and its normal and pathogenic states in the brain.
Annu. Rev. Neurosci.
PUBLISHED: 04-21-2014
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Autophagy is a conserved catabolic process that delivers the cytosol and cytosolic constituents to the lysosome. Its fundamental role is to maintain cellular homeostasis and to protect cells from varying insults, including misfolded proteins and damaged organelles. Beyond these roles, the highly specialized cells of the brain have further adapted autophagic pathways to suit their distinct needs. In this review, we briefly summarize our current understanding of the different forms of autophagy and then offer a closer look at how these pathways impact neuronal and glial functions. The emerging evidence indicates that not only are autophagy pathways essential for neural health, but they have a direct impact on developmental and neurodegenerative processes. Taken together, as we unravel the complex roles autophagy pathways play, we will gain the necessary insight to modify these pathways to protect the human brain and treat neurodegenerative diseases.
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Enhancing depression mechanisms in midbrain dopamine neurons achieves homeostatic resilience.
Science
PUBLISHED: 04-19-2014
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Typical therapies try to reverse pathogenic mechanisms. Here, we describe treatment effects achieved by enhancing depression-causing mechanisms in ventral tegmental area (VTA) dopamine (DA) neurons. In a social defeat stress model of depression, depressed (susceptible) mice display hyperactivity of VTA DA neurons, caused by an up-regulated hyperpolarization-activated current (I(h)). Mice resilient to social defeat stress, however, exhibit stable normal firing of these neurons. Unexpectedly, resilient mice had an even larger I(h), which was observed in parallel with increased potassium (K(+)) channel currents. Experimentally further enhancing Ih or optogenetically increasing the hyperactivity of VTA DA neurons in susceptible mice completely reversed depression-related behaviors, an antidepressant effect achieved through resilience-like, projection-specific homeostatic plasticity. These results indicate a potential therapeutic path of promoting natural resilience for depression treatment.
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NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity.
Nat Commun
PUBLISHED: 03-25-2014
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The Beclin 1-Vps34 complex, the core component of the class III phosphatidylinositol-3 kinase (PI3K-III), binds Atg14L or UVRAG to control different steps of autophagy. However, the mechanism underlying the control of PI3K-III activity remains elusive. Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity. Through its microtubule interaction and trafficking (MIT) domain, NRBF2 binds Atg14L directly and enhances Atg14L-linked Vps34 kinase activity and autophagy induction. NRBF2-deficient cells exhibit enhanced vulnerability to endoplasmic reticulum (ER) stress that is reversed by re-introducing exogenous NRBF2. NRBF2-deficient mice develop focal liver necrosis and ductular reaction, accompanied by impaired Atg14L-linked Vps34 activity and autophagy, although the mice show no increased mortality. Our data reveal a key role for NRBF2 in the assembly of the specific Atg14L-Beclin 1-Vps34-Vps15 complex for autophagy induction. Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury.
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Autophagy-inducing protein beclin-1 in dendritic cells regulates CD4 T cell responses and disease severity during respiratory syncytial virus infection.
J. Immunol.
PUBLISHED: 07-26-2013
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Recent work demonstrated the importance of macroautophagy in dendritic cell (DC) maturation and innate cytokine production upon viral infection through delivery of cytoplasmic viral components to intracellular TLRs. To study the functional consequences of impaired autophagosome formation during a respiratory syncytial virus (RSV) infection, mice harboring significant autophagy defects due to Beclin-1 haploinsufficiency (Beclin-1(+/-)) were used. Upon RSV infection in vivo, lungs of Beclin-1(+/-) mice showed increased Th2 cytokine production, mucus secretion, and lung infiltration of eosinophils and inflammatory DCs. Although isolated airway epithelial cells from Beclin-1(+/-) mice demonstrated little change compared with wild-type mice, Beclin-1(+/-) pulmonary and bone marrow-derived DCs showed decreased expression of MHC class II and innate cytokine production upon RSV infection. Further examination indicated that Beclin-1(+/-) DCs stimulated less IFN-? and IL-17 production by cocultured CD4(+) T cells and increased Th2 cytokine production in comparison with wild-type controls. Finally, adoptive transfer of RSV-infected Beclin-1(+/-) DCs into the airways of wild-type mice produced severe lung pathology and increased Th2 cytokine production upon subsequent RSV challenge compared with wild-type DC transfer controls. These results indicate a critical role for autophagy in DCs during pulmonary viral infection, facilitating appropriate antiviral adaptive immune responses.
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Diminished autophagy limits cardiac injury in mouse models of type 1 diabetes.
J. Biol. Chem.
PUBLISHED: 05-08-2013
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Cardiac autophagy is inhibited in type 1 diabetes. However, it remains unknown if the reduced autophagy contributes to the pathogenesis of diabetic cardiomyopathy. We addressed this question using mouse models with gain- and loss-of-autophagy. Autophagic flux was inhibited in diabetic hearts when measured at multiple time points after diabetes induction by streptozotocin as assessed by protein levels of microtubule-associated protein light chain 3 form 2 (LC3-II) or GFP-LC3 puncta in the absence and presence of the lysosome inhibitor bafilomycin A1. Autophagy in diabetic hearts was further reduced in beclin 1- or Atg16-deficient mice but was restored partially or completely by overexpression of beclin 1 to different levels. Surprisingly, diabetes-induced cardiac damage was substantially attenuated in beclin 1- and Atg16-deficient mice as shown by improved cardiac function as well as reduced levels of oxidative stress, interstitial fibrosis, and myocyte apoptosis. In contrast, diabetic cardiac damage was dose-dependently exacerbated by beclin 1 overexpression. The cardioprotective effects of autophagy deficiency were reproduced in OVE26 diabetic mice. These effects were associated with partially restored mitophagy and increased expression and mitochondrial localization of Rab9, an essential regulator of a non-canonical alternative autophagic pathway. Together, these findings demonstrate that the diminished autophagy is an adaptive response that limits cardiac dysfunction in type 1 diabetes, presumably through up-regulation of alternative autophagy and mitophagy.
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The Sac1 domain of SYNJ1 identified mutated in a family with early-onset progressive Parkinsonism with generalized seizures.
Hum. Mutat.
PUBLISHED: 04-17-2013
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This study aimed to elucidate the genetic causes underlying early-onset Parkinsonism (EOP) in a consanguineous Iranian family. To attain this, homozygosity mapping and whole-exome sequencing were performed. As a result, a homozygous mutation (c.773G>A; p.Arg258Gln) lying within the NH2 -terminal Sac1-like inositol phosphatase domain of polyphosphoinositide phosphatase synaptojanin 1 (SYNJ1), which has been implicated in the regulation of endocytic traffic at synapses, was identified as the disease-segregating mutation. This mutation impaired the phosphatase activity of SYNJ1 against its Sac1 domain substrates in vitro. We concluded that the SYNJ1 mutation identified here is responsible for the EOP phenotype seen in our patients probably due to deficiencies in its phosphatase activity and consequent impairment of its synaptic functions. Our finding not only opens new avenues of investigation in the synaptic dysfunction mechanisms associated with Parkinsonism, but also suggests phosphoinositide metabolism as a novel therapeutic target for Parkinsonism.
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Autophagy in axonal and dendritic degeneration.
Trends Neurosci.
PUBLISHED: 01-03-2013
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Degeneration of axons and dendrites is a common and early pathological feature of many neurodegenerative disorders, and is thought to be regulated by mechanisms distinct from those determining death of the cell body. The unique structures of axons and dendrites (collectively neurites) may cause them to be particularly vulnerable to the accumulation of protein aggregates and damaged organelles. Autophagy is a catabolic mechanism in which cells clear protein aggregates and damaged organelles. Basal autophagy occurs continuously as a housekeeping function, and can be acutely expanded in response to stress or injury. Emerging evidence shows that insufficient or excessive autophagy contributes to neuritic degeneration. Here, we review the recent progress that has begun to reveal the role of autophagy in neurite function and degeneration.
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HMGB1 is involved in autophagy inhibition caused by SNCA/?-synuclein overexpression: A process modulated by the natural autophagy inducer corynoxine B.
Autophagy
PUBLISHED: 01-01-2013
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SNCA/?-synuclein and its rare mutations are considered as the culprit proteins in Parkinson disease (PD). Wild-type (WT) SNCA has been shown to impair macroautophagy in mammalian cells and in transgenic mice. In this study, we monitored the dynamic changes in autophagy process and confirmed that overexpression of both WT and SNCA(A53T) inhibits autophagy in PC12 cells in a time-dependent manner. Furthermore, we showed that SNCA binds to both cytosolic and nuclear high mobility group box 1 (HMGB1), impairs the cytosolic translocation of HMGB1, blocks HMGB1-BECN1 binding, and strengthens BECN1-BCL2 binding. Deregulation of these molecular events by SNCA overexpression leads to autophagy inhibition. Overexpression of BECN1 restores autophagy and promotes the clearance of SNCA. siRNA knockdown of Hmgb1 inhibits basal autophagy and abolishes the inhibitory effect of SNCA on autophagy while overexpression of HMGB1 restores autophagy. Corynoxine B, a natural autophagy inducer, restores the deficient cytosolic translocation of HMGB1 and autophagy in cells overexpressing SNCA, which may be attributed to its ability to block SNCA-HMGB1 interaction. Based on these findings, we propose that SNCA-induced impairment of autophagy occurs, in part, through HMGB1, which may provide a potential therapeutic target for PD.
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Short- and long-term effects of LRRK2 on axon and dendrite growth.
PLoS ONE
PUBLISHED: 01-01-2013
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Mutations in leucine-rich repeat kinase 2 (LRRK2) underlie an autosomal-dominant form of Parkinsons disease (PD) that is clinically indistinguishable from idiopathic PD. The function of LRRK2 is not well understood, but it has become widely accepted that LRRK2 levels or its kinase activity, which is increased by the most commonly observed mutation (G2019S), regulate neurite growth. However, growth has not been measured; it is not known whether mean differences in length correspond to altered rates of growth or retraction, whether axons or dendrites are impacted differentially or whether effects observed are transient or sustained. To address these questions, we compared several developmental milestones in neurons cultured from mice expressing bacterial artificial chromosome transgenes encoding mouse wildtype-LRRK2 or mutant LRRK2-G2019S, Lrrk2 knockout mice and non-transgenic mice. Over the course of three weeks of development on laminin, the data show a sustained, negative effect of LRRK2-G2019S on dendritic growth and arborization, but counter to expectation, dendrites from Lrrk2 knockout mice do not elaborate more rapidly. In contrast, young neurons cultured on a slower growth substrate, poly-L-lysine, show significantly reduced axonal and dendritic motility in Lrrk2 transgenic neurons and significantly increased motility in Lrrk2 knockout neurons with no significant changes in length. Our findings support that LRRK2 can regulate patterns of axonal and dendritic growth, but they also show that effects vary depending on growth substrate and stage of development. Such predictable changes in motility can be exploited in LRRK2 bioassays and guide exploration of LRRK2 function in vivo.
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Autophagy-mediated dendritic cell activation is essential for innate cytokine production and APC function with respiratory syncytial virus responses.
J. Immunol.
PUBLISHED: 09-12-2011
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The regulation of innate immune responses during viral infection is a crucial step to promote antiviral reactions. Recent studies have drawn attention to a strong relationship of pathogen-associated molecular pattern recognition with autophagy for activation of APC function. Our initial observations indicated that autophagosomes formed in response to respiratory syncytial virus (RSV) infection of dendritic cells (DC). To further investigate whether RSV-induced DC activation and innate cytokine production were associated with autophagy, we used several methods to block autophagosome formation. Using 3-MA, small interfering RNA inhibition of LC3, or Beclin(+/-) mouse-derived DC, studies established a relationship between RSV-induced autophagy and enhanced type I IFN, TNF, IL-6, and IL-12p40 expression. Moreover, autophagosome formation induced by starvation also promoted innate cytokine expression in DC. The induction of starvation-induced autophagy in combination with RSV infection synergistically enhanced DC cytokine expression that was blocked by an autophagy inhibitor. The latter synergistic responses were differentially altered in DC from MyD88(-/-) and TRIF(-/-) mice, supporting the concept of autophagy-mediated TLR signaling. In addition, blockade of autophagy in RSV-infected DC inhibited the maturation of DC as assessed by MHC class II and costimulatory molecule expression. Subsequently, we demonstrated that inhibition of autophagy in DC used to stimulate primary OVA-induced and secondary RSV-infected responses significantly attenuated cytokine production by CD4(+) T cells. Thus, these studies have outlined that autophagy in DC after RSV infection is a crucial mechanism for driving innate cytokine production, leading to altered acquired immune responses.
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Genetic ablation of PLA2G6 in mice leads to cerebellar atrophy characterized by Purkinje cell loss and glial cell activation.
PLoS ONE
PUBLISHED: 07-22-2011
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Infantile neuroaxonal dystrophy (INAD) is a progressive, autosomal recessive neurodegenerative disease characterized by axonal dystrophy, abnormal iron deposition and cerebellar atrophy. This disease was recently mapped to PLA2G6, which encodes group VI Ca(2+)-independent phospholipase A(2) (iPLA(2) or iPLA(2)?). Here we show that genetic ablation of PLA2G6 in mice (iPLA(2)?(-/-)) leads to the development of cerebellar atrophy by the age of 13 months. Atrophied cerebella exhibited significant loss of Purkinje cells, as well as reactive astrogliosis, the activation of microglial cells, and the pronounced up-regulation of the pro-inflammatory cytokines tumor necrosis factor-? (TNF-?) and interleukin-1? (IL-1?). Moreover, glial cell activation and the elevation in TNF-? and IL-1? expression occurred before apparent cerebellar atrophy. Our findings indicate that the absence of PLA2G6 causes neuroinflammation and Purkinje cell loss and ultimately leads to cerebellar atrophy. Our study suggests that iPLA(2)?(-/-) mice are a valuable model for cerebellar atrophy in INAD and that early anti-inflammatory therapy may help slow the progression of cerebellar atrophy in this deadly neurodegenerative disease.
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Gammaherpesvirus 68 infection of endothelial cells requires both host autophagy genes and viral oncogenes for optimal survival and persistence.
J. Virol.
PUBLISHED: 04-13-2011
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Gammaherpesvirus-associated neoplasms include tumors of lymphocytes, epithelial cells, and endothelial cells (ECs). We previously showed that, unlike most cell types, ECs survive productive gammaherpesvirus 68 (?HV68) infection and achieve anchorage-independent growth, providing a cellular reservoir for viral persistence. Here, we demonstrated autophagy in infected ECs by analysis of LC3 localization and protein modification and that infected ECs progress through the autophagosome pathway by LC3 dual fluorescence and p62 analysis. We demonstrate that pharmacologic autophagy induction results in increased survival of infected ECs and, conversely, that autophagy inhibition results in death of infected EC survivors. Furthermore, we identified two viral oncogenes, v-cyclin and v-Bcl2, that are critical to EC survival and that modify EC proliferation and survival during infection-induced autophagy. We found that these viral oncogenes can also facilitate survival of substrate detachment in the absence of viral infection. Autophagy affords cells the opportunity to recover from stressful conditions, and consistent with this, the altered phenotype of surviving infected ECs was reversible. Finally, we demonstrated that knockdown of critical autophagy genes completely abrogated EC survival. This study reveals a viral mechanism which usurps the autophagic machinery to promote viral persistence within nonadherent ECs, with the potential for recovery of infected ECs at a distant site upon disruption of virus replication.
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MicroRNA-148b suppresses cell growth by targeting cholecystokinin-2 receptor in colorectal cancer.
Int. J. Cancer
PUBLISHED: 03-25-2011
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MicroRNAs (miRNAs) play an important role in the regulation of a variety of cellular processes, including cell growth, differentiation, apoptosis and carcinogenesis. The purpose of this study was to elucidate the molecular mechanisms by which miR-148b acts as a tumor suppressor in colorectal cancer. The expression of miR-148b was significantly downregulated in 96 pairs of human colorectal cancer tissues (p<0.0001) and three cell lines (p<0.01) compared with non-tumor adjacent tissues by quantitative real-time PCR. The results of in situ hybridization highlighted that miR-148b was important in the cancer transformation process. Using statistical analysis, we found that the expression level of miR-148b was associated with tumor size (p=0.033) in colorectal cancer patients. Moreover, overexpression of miR-148b in HCT-116 and HT-29 cells could inhibit cell proliferation in vitro and suppress tumorigenicity in vivo. Importantly, the result of luciferase activity assay and western blot showed that the cholecystokinin-2 receptor gene (CCK2R) was a target of miR-148b and was downregulated by miR-148b at the translational level. Then, we used siRNA, radioimmunoassay and ELISA to demonstrate that miR-148b might have an effect on cell proliferation by regulating the expression of CCK2R which functioned depending on the gastrin in colorectal cancer. Taken together, our data provides the first evidences that miR-148b acts as a tumor suppressor in colorectal cancer and should be further evaluated as a biomarker and therapeutic tool against colorectal cancer.
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Expression of leucine-rich repeat kinase 2 (LRRK2) inhibits the processing of uMtCK to induce cell death in a cell culture model system.
Biosci. Rep.
PUBLISHED: 03-05-2011
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PD (Parkinsons disease) is the most common neurodegenerative movement disorder. Mutations in LRRK2 (leucine-rich repeat kinase 2) gene are linked to the most common inherited and sporadic PD. Overexpression of LRRK2 and its mutants could induce mitochondrial-dependent neuronal apoptosis. However, the underlying mechanism remains elusive. We have identified several novel LRRK2 interacting proteins and showed that LRRK2 can interact with three components of the PTPC (permeability transition pore complex) including ANT (adenine nucleotide translocator), VDAC (voltage-dependent anion channel) and uMtCK [ubiquitous MtCK (mitochondrial creatine kinase)]. Those components have been reported to be involved in the permeability of mitochondrial membrane. We provide evidence that LRRK2 is likely to interact with uMtCK directly and expression of LRRK2 and its mutant form can suppress the processing of the immature form of uMtCK. LRRK2 expression keeps the uMtCK preprotein on the outer mitochondrial membrane instead of entering the mitochondria. In addition, the expression of both wild-type and mutant forms of LRRK2 promotes the interaction between ANT and VDAC, which plays a role in permeabilization transition pore opening. Finally, LRRK2-induced cell death can be suppressed by uMtCK. Our findings imply that LRRK2 can interact directly with uMtCK to block its entry into mitochondria and its subsequent processing, resulting in inhibition of mitochondrial energy channelling. Meanwhile, the decrease of uMtCK in mitochondria results in elevated interaction between ANT and VDAC and leads to neuronal apoptosis. Thus, our study provides the rational for clinical trials using creatine to treat PD and supports the notion of exploiting LRRK2 as a drug target for PD.
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Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice.
PLoS Genet.
PUBLISHED: 02-21-2011
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Altered protein homeostasis underlies degenerative diseases triggered by misfolded proteins, including spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by a CAG/glutamine expansion in the androgen receptor. Here we show that the unfolded protein response (UPR), an ER protein quality control pathway, is induced in skeletal muscle from SBMA patients, AR113Q knock-in male mice, and surgically denervated wild-type mice. To probe the consequence of UPR induction, we deleted CHOP (C/EBP homologous protein), a transcription factor induced following ER stress. CHOP deficiency accentuated atrophy in both AR113Q and surgically denervated muscle through activation of macroautophagy, a lysosomal protein quality control pathway. Conversely, impaired autophagy due to Beclin-1 haploinsufficiency decreased muscle wasting and extended lifespan of AR113Q males, producing a significant and unexpected amelioration of the disease phenotype. Our findings highlight critical cross-talk between the UPR and macroautophagy, and they indicate that autophagy activation accentuates aspects of the SBMA phenotype.
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Bortezomib enhances the efficacy of fulvestrant by amplifying the aggregation of the estrogen receptor, which leads to a proapoptotic unfolded protein response.
Clin. Cancer Res.
PUBLISHED: 02-03-2011
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Fulvestrant is known to promote the degradation of the estrogen receptor (ER) in the nucleus. However, fulvestrant also promotes the aggregation of the newly synthesized ER in the cytoplasm. Accumulation of protein aggregates leads to cell death but this effect is limited as a result of their elimination by the proteasome. We tested whether combining fulvestrant with the proteasome inhibitor, bortezomib, could enhance the accumulation of ER aggregates and cause apoptotic cell death.
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Genetic LRRK2 models of Parkinsons disease: Dissecting the pathogenic pathway and exploring clinical applications.
Mov. Disord.
PUBLISHED: 02-02-2011
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Dominantly inherited mutations in leucine-rich repeat kinase 2 are the most common cause of familial Parkinsons disease. Understanding leucine-rich repeat kinase 2 biology and pathophysiology is central to the elucidation of Parkinsons disease etiology and development of disease intervention. Recently, a number of genetic mouse models of leucine-rich repeat kinase 2 have been reported utilizing different genetic approaches. Some similarities in Parkinsons disease-related pathology emerge in these genetic models despite lack of substantial neuropathology and clinical syndromes of Parkinsons disease. The systematic characterization of these models has begun to shed light on leucine-rich repeat kinase 2 biology and pathophysiology and is expected to offer the identification and validation of drug targets. In this review, we summarize the progress of genetic leucine-rich repeat kinase 2 mouse models and discuss their utility in understanding much needed knowledge regarding early-stage (presymptomatic) disease progression, identifying drug targets, and exploring the potential to aid compound screening focused on inhibitors of kinase activity of leucine-rich repeat kinase 2.
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Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinsons disease.
PLoS ONE
PUBLISHED: 01-20-2011
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Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinsons disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown.
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A role for autophagic protein beclin 1 early in lymphocyte development.
J. Immunol.
PUBLISHED: 01-14-2011
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Autophagy is a highly regulated and evolutionarily conserved process of cellular self-digestion. Recent evidence suggests that this process plays an important role in regulating T cell homeostasis. In this study, we used Rag1(-/-) (recombination activating gene 1(-/-)) blastocyst complementation and in vitro embryonic stem cell differentiation to address the role of Beclin 1, one of the key autophagic proteins, in lymphocyte development. Beclin 1-deficient Rag1(-/-) chimeras displayed a dramatic reduction in thymic cellularity compared with control mice. Using embryonic stem cell differentiation in vitro, we found that the inability to maintain normal thymic cellularity is likely caused by impaired maintenance of thymocyte progenitors. Interestingly, despite drastically reduced thymocyte numbers, the peripheral T cell compartment of Beclin 1-deficient Rag1(-/-) chimeras is largely normal. Peripheral T cells displayed normal in vitro proliferation despite significantly reduced numbers of autophagosomes. In addition, these chimeras had greatly reduced numbers of early B cells in the bone marrow compared with controls. However, the peripheral B cell compartment was not dramatically impacted by Beclin 1 deficiency. Collectively, our results suggest that Beclin 1 is required for maintenance of undifferentiated/early lymphocyte progenitor populations. In contrast, Beclin 1 is largely dispensable for the initial generation and function of the peripheral T and B cell compartments. This indicates that normal lymphocyte development involves Beclin 1-dependent, early-stage and distinct, Beclin 1-independent, late-stage processes.
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The class IA phosphatidylinositol 3-kinase p110-beta subunit is a positive regulator of autophagy.
J. Cell Biol.
PUBLISHED: 11-08-2010
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Autophagy is an evolutionarily conserved cell renewal process that depends on phosphatidylinositol 3-phosphate (PtdIns(3)P). In metazoans, autophagy is inhibited by PtdIns(3,4,5)P(3), the product of class IA PI3Ks, which mediates the activation of the Akt-TOR kinase cascade. However, the precise function of class IA PI3Ks in autophagy remains undetermined. Class IA PI3Ks are heterodimeric proteins consisting of an 85-kD regulatory subunit and a 110-kD catalytic subunit. Here we show that the class IA p110-? catalytic subunit is a positive regulator of autophagy. Genetic deletion of p110-? results in impaired autophagy in mouse embryonic fibroblasts, liver, and heart. p110-? does not promote autophagy by affecting the Akt-TOR pathway. Rather, it associates with the autophagy-promoting Vps34-Vps15-Beclin 1-Atg14L complex and facilitates the generation of cellular PtdIns(3)P. Our results unveil a previously unknown function for p110-? as a positive regulator of autophagy in multicellular organisms.
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From a global view to focused examination: understanding cellular function of lipid kinase VPS34-Beclin 1 complex in autophagy.
J Mol Cell Biol
PUBLISHED: 09-15-2010
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Phosphoinositide 3 kinase Class III (PIK3C3) or VPS34-Beclin 1 complex plays a key role in the autophagy-lysosome pathway. Previous identification of numerous binding partners for VPS34-Beclin 1 suggested a complex scheme of the autophagy control mechanism. Recent large-scale screening of autophagy network and signaling pathways in mammalian cells not only confirms the previous binding partners, but also reveals additional interactors and intricate connections of VPS34-Beclin 1 complex to other functional groups of autophagy, yielding a wealth of information that will direct future detailed study of the central control mechanism of autophagy mediated by VPS34-Beclin 1 and other regulators.
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Association of heparanase gene (HPSE-1) single nucleotide polymorphisms with gastric cancer.
J Surg Oncol
PUBLISHED: 06-26-2010
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Heparanase activity plays a decisive role in biological processes associated with remodeling of the extracellular matrix (e.g., cancer metastasis, angiogenesis, and inflammation). Heparanase gene overexpression has been associated with advanced stage and poor survival in several cancers. We investigated the potential association between single nucleotide polymorphisms (SNPs) of the HPSE-1 gene, tumor susceptibility, clinicopathological parameters, and survival with gastric cancer among the Han population in northern China.
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Aberrant expression of miR-203 and its clinical significance in gastric and colorectal cancers.
J. Gastrointest. Surg.
PUBLISHED: 05-24-2010
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MicroRNAs (miRNAs) are small, non-coding RNAs of endogenous origin, they have been increasingly shown to have aberrant expression in many tumor types. miR-203 has not been comprehensively investigated in gastric and colorectal cancers.
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Autophagy in lurcher mice: indicted but yet to be acquitted for the death of Purkinje cells.
Autophagy
PUBLISHED: 05-16-2010
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A recent study published in the Journal of Neuroscience by Nishiyama et al., has revisited an autophagy-neurodegeneration model of lurcher (Lc) mice and promoted further discussion regarding the "autophagic cell death" hypothesis. While the study confirmed the previous report by Yue et al., that GluRD2Lc induces autophagy both in vitro and in vivo, it also suggests that GluRD2 (Lc)-mediated autophagy and cell death occur via pathways outside the nPIST-Beclin 1 pathway. For example, the study makes an interesting observation that GluRD2 (Lc)-induced degeneration is associated with energy crisis and an aberrant AMPK activity. The result provides insight into the downstream events induced by GluRD2 (Lc); however, it is not surprising considering that constitutive ion influx caused by the Lc mutation is expected to cause activation of multiple cellular pathways or responses. In conclusion, the authors state that "constitutive ion flux causes cell death with, but not by, autophagy." The conclusion appears consistent with the primary function of autophagy, from an evolutionary point of view, as a survival mechanism.
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The Beclin 1-VPS34 complex--at the crossroads of autophagy and beyond.
Trends Cell Biol.
PUBLISHED: 03-05-2010
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An increasing body of research on autophagy provides overwhelming evidence for its connection to diverse biological functions and human diseases. Beclin 1, the first mammalian autophagy protein to be described, appears to act as a nexus point between autophagy, endosomal, and perhaps also cell death pathways. Beclin 1 performs these roles as part of a core complex that contains vacuolar sorting protein 34 (VPS34), a class III phosphatidylinositol-3 kinase. The precise mechanism of Beclin 1-mediated regulation of these cellular functions is unclear, but substantial progress has recently been made in identifying new players and their functions in Beclin 1-VSP34 complexes. Here we review emerging studies that are beginning to unveil the physiological functions of Beclin 1-VPS34 in the central control of autophagic activity and other trafficking events through the formation of distinct Beclin 1-VPS34 protein complexes.
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Development of a mechanism-based high-throughput screen assay for leucine-rich repeat kinase 2--discovery of LRRK2 inhibitors.
Anal. Biochem.
PUBLISHED: 02-22-2010
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LRRK2 is a large and complex protein that possesses kinase and GTPase activities and has emerged as the most relevant player in PD pathogenesis possibly through a toxic gain-of-function mechanism. Kinase activity is a critical component of LRRK2 function and represents a viable target for drug discovery. We now report the development of a mechanism-based TR-FRET assay for the LRRK2 kinase activity using full-length LRRK2. In this assay, PLK-peptide was chosen as the phosphoryl acceptor. A combination of steady-state kinetic studies and computer simulations was used to calculate the initial concentrations of ATP and PLK-peptide to generate a steady-state situation that favors the identification of ATP noncompetitive inhibitors. The assay was also run in the absence of GTP. Under these conditions, the assay was sensitive to inhibitors that directly interact with the kinase domain and those that modulate the kinase activity by directly interacting with other domains including the GTPase domain. The assay was optimized and used to robustly evaluate our compound library in a 384-well format. An inhibitor identified through the screen was further characterized as a noncompetitive inhibitor with both ATP and PLK-peptide and showed similar inhibition against LRRK2 WT and the mutant G2019S.
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Kinetic mechanistic studies of wild-type leucine-rich repeat kinase 2: characterization of the kinase and GTPase activities.
Biochemistry
PUBLISHED: 02-12-2010
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Recent studies have identified mutations in the leucine-rich repeat kinase2 gene (LRRK2) in the most common familial forms and some sporadic forms of Parkinsons disease (PD). LRRK2 is a large and complex protein that possesses kinase and GTPase activities. Some LRRK2 mutants enhance kinase activity and possibly contribute to PD through a toxic gain-of-function mechanism. Given the role of LRRK2 in the pathogenesis of PD, understanding the kinetic mechanism of its two enzymatic properties is critical for the discovery of inhibitors of LRRK2 kinase that would be therapeutically useful in treating PD. In this report, by using LRRK2 protein purified from murine brain, first we characterize kinetic mechanisms for the LRRK2-catalyzed phosphorylation of two peptide substrates: PLK-derived peptide (PLK-peptide) and LRRKtide. We found that LRRK2 follows a rapid equilibrium random mechanism for the phosphorylation of PLK-peptide with either ATP or PLK-peptide being the first substrate binding to the enzyme, as evidenced by initial velocity and inhibition mechanism studies with nucleotide analogues AMP and AMP-PNP, product ADP, and an analogue of the peptide substrate. The binding of the first substrate has no effect on the binding affinity of the second substrate. Identical mechanistic conclusions were drawn when LRRKtide was the phosphoryl acceptor. Next, we characterize the GTPase activity of LRRK2 with a k(cat) of 0.2 +/- 0.02 s(-1) and a K(m) of 210 +/- 29 microM. A SKIE of 0.97 +/- 0.04 was measured on k(cat) for the GTPase activity of LRRK2 in a D(2)O molar fraction of 0.86 and suggested that the product dissociation step is rate-limiting, of the steps governed by k(cat) in the LRRK2-catalyzed GTP hydrolysis. Surprisingly, binding of GTP, GDP, or GMP has no effect on kinase activity, although GMP and GDP inhibit the GTPase activity. Finally, we have identified compound LDN-73794 through screen of LRRK2 kinase inhibitors. Our study revealed that this compound is a competitive inhibitor of the binding of ATP and inhibits the kinase activity without affecting the GTPase activity.
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Altered expression of MiR-148a and MiR-152 in gastrointestinal cancers and its clinical significance.
J. Gastrointest. Surg.
PUBLISHED: 02-05-2010
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MicroRNAs are endogenous small noncoding RNAs that aberrantly expressed in various carcinomas. MiR-148a and miR-152, which have the same "seed region", have not been comprehensively investigated in gastrointestinal cancers.
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Enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial Parkinsons disease mutation G2019S.
J. Neurosci.
PUBLISHED: 02-05-2010
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PARK8/LRRK2 (leucine-rich repeat kinase 2) was recently identified as a causative gene for autosomal dominant Parkinsons disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events.
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Cell "self-eating" (autophagy) mechanism in Alzheimers disease.
Mt. Sinai J. Med.
PUBLISHED: 01-27-2010
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The autophagy pathway is the major degradation pathway of the cell for long-lived proteins and organelles. Dysfunction of autophagy has been linked to several neurodegenerative disorders that are associated with an accumulation of misfolded protein aggregates. Alzheimers disease, the most common neurodegenerative disorder, is characterized by 2 aggregate forms, tau tangles and amyloid-beta plaques. Autophagy has been linked to Alzheimers disease pathogenesis through its merger with the endosomal-lysosomal system, which has been shown to play a role in the formation of the latter amyloid-beta plaques. However, the precise role of autophagy in Alzheimers disease pathogenesis is still under contention. One hypothesis is that aberrant autophagy induction results in an accumulation of autophagic vacuoles containing amyloid-beta and the components necessary for its generation, whereas other evidence points to impaired autophagic clearance or even an overall reduction in autophagic activity playing a role in Alzheimers disease pathogenesis. In this review, we discuss the current evidence linking autophagy to Alzheimers disease as well as the uncertainty over the exact role and level of autophagic regulation in the pathogenic mechanism of Alzheimers disease.
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LRRK2 in Parkinsons disease: in vivo models and approaches for understanding pathogenic roles.
FEBS J.
PUBLISHED: 10-05-2009
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The recent discovery of the genetic causes for Parkinsons disease (PD) is fruitful; however, the continuing revelation of PD-related genes is rapidly outpacing the functional characterization of the gene products. Although the discovery of multiple PD-related genes places PD as one of the most complex multigenetic diseases of the brain, it will undoubtedly facilitate the unfolding of a central pathogenic pathway and an understanding of the etiology of PD. Recent findings of pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2) (PARK8) that are linked to the most common familial forms and some sporadic forms of PD provide a unique opportunity to gain insight into the pathogenesis of PD. Despite rapid growth in biochemical, structural and in vitro cell culture studies of LRRK2, the in vivo characterizations of LRRK2 function generally fall short and are largely limited to invertebrates. The investigation of LRRK2 or homologs of LRRK2 in nonmammalian models provides important clues with respect to the cellular functions of LRRK2, but an elucidation of the physiology and pathophysiology of LRRK2 relevant to PD would still depend on mammalian models established by multiple genetic approaches, followed by rigorous examination of the models for pathological process. This minireview summarizes previous studies of genes for ROCO and LRRK2 homologs in slime mold, nematode worms and fruit flies. It also discusses the results obtained from available mouse models of LRRK2 that begin to provide information for understanding LRRK2-mediated pathogenesis in PD.
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Pagets disease is associated with eleven cancerous regions: a case report and therapeutic strategy.
J. Huazhong Univ. Sci. Technol. Med. Sci.
PUBLISHED: 06-19-2009
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Pagets disease of the breast is an uncommon disorder that accounts for 1% to 3% of all mammary tumors. The incidence of underlying carcinoma associated with Pagets disease has been reported in 82% to 100% of cases. The finding of underlying carcinoma reaches almost 100% when a palpable lump is also present. In this rare case, we described a patient presenting with Pagets disease but no palpable lump. However, we found 11 independent regions which were all invasive ductal carcinoma after the operation. Considering this patient, we should pay more attention to a multifocal and multicentric breast carcinoma associated with Pagets disease. Furthermore, we believe the mammography examination and a modified radical mastectomy are the most appropriate treatments for this population in clinical practice.
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Using genetic mouse models to study the biology and pathology of autophagy in the central nervous system.
Meth. Enzymol.
PUBLISHED: 02-17-2009
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Autophagy is a cellular self-eating process that plays an important role in neuroprotection as well as neuronal injury and death. The detailed pathway of autophagy in these two opposing functions remains to be elucidated. Neurons are highly specialized, postmitotic cells that are typically composed of a soma (cell body), a dendritic tree, and an axon. Here, we describe methods for studying autophagy in the central nervous system (CNS). The first involves the use of recently developed transgenic mice expressing the fluorescent autophagosome marker, GFP-LC3. Although CNS neurons show little evidence for the presence of GFP-LC3-containing puncta under normal conditions, under pathological conditions such neurons exhibit many GFP-LC3 puncta. The onset and density of GFP-LC3 puncta have been found to vary significantly in the subcompartments of the affected neurons. These studies suggest that autophagy is distinctly regulated in CNS neurons and that neuronal autophagy can be highly compartmentalized. While transgenic mice expressing GFP-LC3 are a valuable tool for assessing autophagic activity in the CNS, caution needs to be taken when interpreting results solely based on the presence of GFP-LC3 puncta. Therefore, traditional ultrastructural analysis using electron microscopy remains an important tool for studying autophagosomes in vivo. Additional reporters of autophagy are constantly being sought. For example, recently a selective substrate of autophagy p62/SQSTM1 has been shown to be specifically regulated by autophagic activity. Therefore, p62/SQSTM1 protein levels can be used as an additional reporter for autophagic activity.
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Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex.
Nat. Cell Biol.
PUBLISHED: 02-03-2009
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Beclin 1, a mammalian autophagy protein that has been implicated in development, tumour suppression, neurodegeneration and cell death, exists in a complex with Vps34, the class III phosphatidylinositol-3-kinase (PI(3)K) that mediates multiple vesicle-trafficking processes including endocytosis and autophagy. However, the precise role of the Beclin 1-Vps34 complex in autophagy regulation remains to be elucidated. Combining mouse genetics and biochemistry, we have identified a large in vivo Beclin 1 complex containing the known proteins Vps34, p150/Vps15 and UVRAG, as well as two newly identified proteins, Atg14L (yeast Atg14-like) and Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein). Characterization of the new proteins revealed that Atg14L enhances Vps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces Vps34 activity and downregulates autophagy. We show that Beclin 1 and Atg14L synergistically promote the formation of double-membraned organelles that are associated with Atg5 and Atg12, whereas forced expression of Rubicon results in aberrant late endosomal/lysosomal structures and impaired autophagosome maturation. We hypothesize that by forming distinct protein complexes, Beclin 1 and its binding proteins orchestrate the precise function of the class III PI(3)K in regulating autophagy at multiple steps.
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The cellular pathways of neuronal autophagy and their implication in neurodegenerative diseases.
Biochim. Biophys. Acta
PUBLISHED: 01-24-2009
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Autophagy is a tightly regulated cell self-eating process. It has been shown to be associated with various neuropathological conditions and therefore, traditionally known as a stress-induced process. Recent studies, however, reveal that autophagy is constitutively active in healthy neurons. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree, and an axon. Despite the vast growth of our current knowledge of autophagy, the detailed process in such a highly differentiated cell type remains elusive. Current evidence strongly suggests that autophagy is uniquely regulated in neurons and is also highly adapted to local physiology in the axons. In addition, the molecular mechanism for basal autophagy in neurons may be significantly divergent from "classical" induced autophagy. A considerable number of studies have increasingly shown an important role for autophagy in neurodegenerative diseases and have explored autophagy as a potential drug target. Thus, understanding the neuronal autophagy process will ultimately aid in drug target identification and rational design of drug screening to combat neurodegenerative diseases.
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Recent patents and patent applications relating to mTOR pathway.
Recent Pat DNA Gene Seq
PUBLISHED: 01-20-2009
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The mammalian target of rapamycin (mTOR) plays an important role in cell growth. Dysfunction of mTOR has been linked to many human diseases, such as cancers, obesity, diabetes, cardiovascular diseases and neurological disorders. Currently, the mTOR inhibitor rapamycin and its analogs have been vigorously evaluated and developed as anticancer drugs. Here, we will review the most recent patents and patent applications relating to mTOR pathway. Moreover, we will discuss the patents and patent applications on the treatment of mTOR-associated metabolic diseases and cancers.
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Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations.
Sci Transl Med
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Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinsons disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser(1292) occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser(1292) autophosphorylation. Mutation of Ser(1292) to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser(1292) as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser(1292) autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser(1292) autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.
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Impaired autophagy in neurons after disinhibition of mammalian target of rapamycin and its contribution to epileptogenesis.
J. Neurosci.
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Certain mutations within the mammalian target of rapamycin (mTOR) pathway, most notably those affecting the tuberous sclerosis complex (TSC), lead to aberrant activation of mTOR and result in a high incidence of epilepsy in humans and animal models. Although hyperactivation of mTOR has been strongly linked to the development of epilepsy and, conversely, inhibition of mTOR by rapamycin treatment is protective against seizures in several models, the downstream epileptic mechanisms have remained elusive. Autophagy, a catabolic process that plays a vital role in cellular homeostasis by mediating the turnover of cytoplasmic constituents, is negatively regulated by mTOR. Here we demonstrate that autophagy is suppressed in brain tissues of forebrain-specific conditional TSC1 and phosphatase and tensin homlog knock-out mice, both of which display aberrant mTOR activation and seizures. In addition, we also discovered that autophagy is suppressed in the brains of human TSC patients. Moreover, conditional deletion of Atg7, an essential regulator of autophagy, in mouse forebrain neurons is sufficient to promote development of spontaneous seizures. Thus, our study suggests that impaired autophagy contributes to epileptogenesis, which may be of interest as a potential therapeutic target for epilepsy treatment and/or prevention.
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Guidelines for the use and interpretation of assays for monitoring autophagy.
Daniel J Klionsky, Fábio C Abdalla, Hagai Abeliovich, Robert T Abraham, Abraham Acevedo-Arozena, Khosrow Adeli, Lotta Agholme, Maria Agnello, Patrizia Agostinis, Julio A Aguirre-Ghiso, Hyung Jun Ahn, Ouardia Ait-Mohamed, Slimane Ait-Si-Ali, Takahiko Akematsu, Shizuo Akira, Hesham M Al-Younes, Munir A Al-Zeer, Matthew L Albert, Roger L Albin, Javier Alegre-Abarrategui, Maria Francesca Aleo, Mehrdad Alirezaei, Alexandru Almasan, Maylin Almonte-Becerril, Atsuo Amano, Ravi Amaravadi, Shoba Amarnath, Amal O Amer, Nathalie Andrieu-Abadie, Vellareddy Anantharam, David K Ann, Shailendra Anoopkumar-Dukie, Hiroshi Aoki, Nadezda Apostolova, Giuseppe Arancia, John P Aris, Katsuhiko Asanuma, Nana Y O Asare, Hisashi Ashida, Valerie Askanas, David S Askew, Patrick Auberger, Misuzu Baba, Steven K Backues, Eric H Baehrecke, Ben A Bahr, Xue-Yuan Bai, Yannick Bailly, Robert Baiocchi, Giulia Baldini, Walter Balduini, Andrea Ballabio, Bruce A Bamber, Edward T W Bampton, Gábor Bánhegyi, Clinton R Bartholomew, Diane C Bassham, Robert C Bast, Henri Batoko, Boon-Huat Bay, Isabelle Beau, Daniel M Béchet, Thomas J Begley, Christian Behl, Christian Behrends, Soumeya Bekri, Bryan Bellaire, Linda J Bendall, Luca Benetti, Laura Berliocchi, Henri Bernardi, Francesca Bernassola, Sébastien Besteiro, Ingrid Bhatia-Kiššová, Xiaoning Bi, Martine Biard-Piechaczyk, Janice S Blum, Lawrence H Boise, Paolo Bonaldo, David L Boone, Beat C Bornhauser, Karina R Bortoluci, Ioannis Bossis, Fréderic Bost, Jean-Pierre Bourquin, Patricia Boya, Michaël Boyer-Guittaut, Peter V Bozhkov, Nathan R Brady, Claudio Brancolini, Andreas Brech, Jay E Brenman, Ana Brennand, Emery H Bresnick, Patrick Brest, Dave Bridges, Molly L Bristol, Paul S Brookes, Eric J Brown, John H Brumell, Nicola Brunetti-Pierri, Ulf T Brunk, Dennis E Bulman, Scott J Bultman, Geert Bultynck, Lena F Burbulla, Wilfried Bursch, Jonathan P Butchar, Wanda Buzgariu, Sérgio P Bydlowski, Ken Cadwell, Monika Cahova, Dongsheng Cai, Jiyang Cai, Qian Cai, Bruno Calabretta, Javier Calvo-Garrido, Nadine Camougrand, Michelangelo Campanella, Jenny Campos-Salinas, Eleonora Candi, Lizhi Cao, Allan B Caplan, Simon R Carding, Sandra M Cardoso, Jennifer S Carew, Cathleen R Carlin, Virginie Carmignac, Leticia A M Carneiro, Serena Carra, Rosario A Caruso, Giorgio Casari, Caty Casas, Roberta Castino, Eduardo Cebollero, Francesco Cecconi, Jean Celli, Hassan Chaachouay, Han-Jung Chae, Chee-Yin Chai, David C Chan, Edmond Y Chan, Raymond Chuen-Chung Chang, Chi-Ming Che, Ching-Chow Chen, Guang-Chao Chen, Guo-Qiang Chen, Min Chen, Quan Chen, Steve S-L Chen, WenLi Chen, Xi Chen, Xiangmei Chen, Xiequn Chen, Ye-Guang Chen, Yingyu Chen, Yongqiang Chen, Yu-Jen Chen, Zhixiang Chen, Alan Cheng, Christopher H K Cheng, Yan Cheng, Heesun Cheong, Jae-Ho Cheong, Sara Cherry, Russ Chess-Williams, Zelda H Cheung, Eric Chevet, Hui-Ling Chiang, Roberto Chiarelli, Tomoki Chiba, Lih-Shen Chin, Shih-Hwa Chiou, Francis V Chisari, Chi Hin Cho, Dong-Hyung Cho, Augustine M K Choi, DooSeok Choi, Kyeong Sook Choi, Mary E Choi, Salem Chouaib, Divaker Choubey, Vinay Choubey, Charleen T Chu, Tsung-Hsien Chuang, Sheau-Huei Chueh, Taehoon Chun, Yong-Joon Chwae, Mee-Len Chye, Roberto Ciarcia, Maria R Ciriolo, Michael J Clague, Robert S B Clark, Peter G H Clarke, Robert Clarke, Patrice Codogno, Hilary A Coller, María I Colombo, Sergio Comincini, Maria Condello, Fabrizio Condorelli, Mark R Cookson, Graham H Coombs, Isabelle Coppens, Ramón Corbalán, Pascale Cossart, Paola Costelli, Safia Costes, Ana Coto-Montes, Eduardo Couve, Fraser P Coxon, James M Cregg, José L Crespo, Marianne J Cronjé, Ana Maria Cuervo, Joseph J Cullen, Mark J Czaja, Marcello D'Amelio, Arlette Darfeuille-Michaud, Lester M Davids, Faith E Davies, Massimo De Felici, John F de Groot, Cornelis A M de Haan, Luisa De Martino, Angelo De Milito, Vincenzo De Tata, Jayanta Debnath, Alexei Degterev, Benjamin Dehay, Lea M D Delbridge, Francesca Demarchi, Yi Zhen Deng, Jörn Dengjel, Paul Dent, Donna Denton, Vojo Deretic, Shyamal D Desai, Rodney J Devenish, Mario Di Gioacchino, Gilbert Di Paolo, Chiara Di Pietro, Guillermo Díaz-Araya, Inés Díaz-Laviada, Maria T Diaz-Meco, Javier Diaz-Nido, Ivan Dikic, Savithramma P Dinesh-Kumar, Wen-Xing Ding, Clark W Distelhorst, Abhinav Diwan, Mojgan Djavaheri-Mergny, Svetlana Dokudovskaya, Zheng Dong, Frank C Dorsey, Victor Dosenko, James J Dowling, Stephen Doxsey, Marlène Dreux, Mark E Drew, Qiuhong Duan, Michel A Duchosal, Karen Duff, Isabelle Dugail, Madeleine Durbeej, Michael Duszenko, Charles L Edelstein, Aimee L Edinger, Gustavo Egea, Ludwig Eichinger, N Tony Eissa, Suhendan Ekmekcioglu, Wafik S El-Deiry, Zvulun Elazar, Mohamed Elgendy, Lisa M Ellerby, Kai Er Eng, Anna-Mart Engelbrecht, Simone Engelender, Jekaterina Erenpreisa, Ricardo Escalante, Audrey Esclatine, Eeva-Liisa Eskelinen, Lucile Espert, Virginia Espina, Huizhou Fan, Jia Fan, Qi-Wen Fan, Zhen Fan, Shengyun Fang, Yongqi Fang, Manolis Fanto, Alessandro Fanzani, Thomas Farkas, Jean-Claude Farré, Mathias Faure, Marcus Fechheimer, Carl G Feng, Jian Feng, Qili Feng, Youji Feng, László Fésüs, Ralph Feuer, Maria E Figueiredo-Pereira, Gian Maria Fimia, Diane C Fingar, Steven Finkbeiner, Toren Finkel, Kim D Finley, Filomena Fiorito, Edward A Fisher, Paul B Fisher, Marc Flajolet, Maria L Florez-McClure, Salvatore Florio, Edward A Fon, Francesco Fornai, Franco Fortunato, Rati Fotedar, Daniel H Fowler, Howard S Fox, Rodrigo Franco, Lisa B Frankel, Marc Fransen, José M Fuentes, Juan Fueyo, Jun Fujii, Kozo Fujisaki, Eriko Fujita, Mitsunori Fukuda, Ruth H Furukawa, Matthias Gaestel, Philippe Gailly, Malgorzata Gajewska, Brigitte Galliot, Vincent Galy, Subramaniam Ganesh, Barry Ganetzky, Ian G Ganley, Fen-Biao Gao, George F Gao, Jinming Gao, Lorena Garcia, Guillermo Garcia-Manero, Mikel Garcia-Marcos, Marjan Garmyn, Andrei L Gartel, Evelina Gatti, Mathias Gautel, Thomas R Gawriluk, Matthew E Gegg, Jiefei Geng, Marc Germain, Jason E Gestwicki, David A Gewirtz, Saeid Ghavami, Pradipta Ghosh, Anna M Giammarioli, Alexandra N Giatromanolaki, Spencer B Gibson, Robert W Gilkerson, Michael L Ginger, Henry N Ginsberg, Jakub Golab, Michael S Goligorsky, Pierre Golstein, Candelaria Gomez-Manzano, Ebru Goncu, Céline Gongora, Claudio D Gonzalez, Ramon Gonzalez, Cristina González-Estévez, Rosa Ana González-Polo, Elena Gonzalez-Rey, Nikolai V Gorbunov, Sharon Gorski, Sandro Goruppi, Roberta A Gottlieb, Devrim Gozuacik, Giovanna Elvira Granato, Gary D Grant, Kim N Green, Aleš Gregorc, Frédéric Gros, Charles Grose, Thomas W Grunt, Philippe Gual, Jun-Lin Guan, Kun-Liang Guan, Sylvie M Guichard, Anna S Gukovskaya, Ilya Gukovsky, Jan Gunst, Asa B Gustafsson, Andrew J Halayko, Amber N Hale, Sandra K Halonen, Maho Hamasaki, Feng Han, Ting Han, Michael K Hancock, Malene Hansen, Hisashi Harada, Masaru Harada, Stefan E Hardt, J Wade Harper, Adrian L Harris, James Harris, Steven D Harris, Makoto Hashimoto, Jeffrey A Haspel, Shin-Ichiro Hayashi, Lori A Hazelhurst, Congcong He, You-Wen He, Marie-Josee Hebert, Kim A Heidenreich, Miep H Helfrich, Gudmundur V Helgason, Elizabeth P Henske, Brian Herman, Paul K Herman, Claudio Hetz, Sabine Hilfiker, Joseph A Hill, Lynne J Hocking, Paul Hofman, Thomas G Hofmann, Jörg Höhfeld, Tessa L Holyoake, Ming-Huang Hong, David A Hood, Gökhan S Hotamisligil, Ewout J Houwerzijl, Maria Høyer-Hansen, Bingren Hu, Chien-An A Hu, Hong-Ming Hu, Ya Hua, Canhua Huang, Ju Huang, Shengbing Huang, Wei-Pang Huang, Tobias B Huber, Won-Ki Huh, Tai-Ho Hung, Ted R Hupp, Gang Min Hur, James B Hurley, Sabah N A Hussain, Patrick J Hussey, Jung Jin Hwang, Seungmin Hwang, Atsuhiro Ichihara, Shirin Ilkhanizadeh, Ken Inoki, Takeshi Into, Valentina Iovane, Juan L Iovanna, Nancy Y Ip, Yoshitaka Isaka, Hiroyuki Ishida, Ciro Isidoro, Ken-Ichi Isobe, Akiko Iwasaki, Marta Izquierdo, Yotaro Izumi, Panu M Jaakkola, Marja Jäättelä, George R Jackson, William T Jackson, Bassam Janji, Marina Jendrach, Ju-Hong Jeon, Eui-Bae Jeung, Hong Jiang, Hongchi Jiang, Jean X Jiang, Ming Jiang, Qing Jiang, Xuejun Jiang, Alberto Jiménez, Meiyan Jin, Shengkan Jin, Cheol O Joe, Terje Johansen, Daniel E Johnson, Gail V W Johnson, Nicola L Jones, Bertrand Joseph, Suresh K Joseph, Annie M Joubert, Gábor Juhász, Lucienne Juillerat-Jeanneret, Chang Hwa Jung, Yong-Keun Jung, Kai Kaarniranta, Allen Kaasik, Tomohiro Kabuta, Motoni Kadowaki, Katarina Kågedal, Yoshiaki Kamada, Vitaliy O Kaminskyy, Harm H Kampinga, Hiromitsu Kanamori, Chanhee Kang, Khong Bee Kang, Kwang Il Kang, Rui Kang, Yoon-A Kang, Tomotake Kanki, Thirumala-Devi Kanneganti, Haruo Kanno, Anumantha G Kanthasamy, Arthi Kanthasamy, Vassiliki Karantza, Gur P Kaushal, Susmita Kaushik, Yoshinori Kawazoe, Po-Yuan Ke, John H Kehrl, Ameeta Kelekar, Claus Kerkhoff, David H Kessel, Hany Khalil, Jan A K W Kiel, Amy A Kiger, Akio Kihara, Deok Ryong Kim, Do-Hyung Kim, Dong-Hou Kim, Eun-Kyoung Kim, Hyung-Ryong Kim, Jae-Sung Kim, Jeong Hun Kim, Jin Cheon Kim, John K Kim, Peter K Kim, Seong Who Kim, Yong-Sun Kim, Yonghyun Kim, Adi Kimchi, Alec C Kimmelman, Jason S King, Timothy J Kinsella, Vladimir Kirkin, Lorrie A Kirshenbaum, Katsuhiko Kitamoto, Kaio Kitazato, Ludger Klein, Walter T Klimecki, Jochen Klucken, Erwin Knecht, Ben C B Ko, Jan C Koch, Hiroshi Koga, Jae-Young Koh, Young Ho Koh, Masato Koike, Masaaki Komatsu, Eiki Kominami, Hee Jeong Kong, Wei-jia Kong, Viktor I Korolchuk, Yaichiro Kotake, Michael I Koukourakis, Juan B Kouri Flores, Attila L Kovács, Claudine Kraft, Dimitri Krainc, Helmut Krämer, Carole Kretz-Remy, Anna M Krichevsky, Guido Kroemer, Rejko Krüger, Oleg Krut, Nicholas T Ktistakis, Chia-Yi Kuan, Róza Kucharczyk, Ashok Kumar, Raj Kumar, Sharad Kumar, Mondira Kundu, Hsing-Jien Kung, Tino Kurz, Ho Jeong Kwon, Albert R La Spada, Frank Lafont, Trond Lamark, Jacques Landry, Jon D Lane, Pierre Lapaquette, Jocelyn F Laporte, Lajos László, Sergio Lavandero, Josée N Lavoie, Robert Layfield, Pedro A Lazo, Weidong Le, Laurent Le Cam, Daniel J Ledbetter, Alvin J X Lee, Byung-Wan Lee, Gyun Min Lee, Jongdae Lee, Ju-Hyun Lee, Michael Lee, Myung-Shik Lee, Sug Hyung Lee, Christiaan Leeuwenburgh, Patrick Legembre, Renaud Legouis, Michael Lehmann, Huan-Yao Lei, Qun-Ying Lei, David A Leib, José Leiro, John J Lemasters, Antoinette Lemoine, Maciej S Lesniak, Dina Lev, Victor V Levenson, Beth Levine, Efrat Levy, Faqiang Li, Jun-lin Li, Lian Li, Sheng Li, Weijie Li, Xue-Jun Li, Yan-Bo Li, Yi-Ping Li, Chengyu Liang, Qiangrong Liang, Yung-Feng Liao, Pawel P Liberski, Andrew Lieberman, Hyunjung J Lim, Kah-Leong Lim, Kyu Lim, Chiou-Feng Lin, Fu-Cheng Lin, Jian Lin, Jiandie D Lin, Kui Lin, Wan-Wan Lin, Weei-Chin Lin, Yi-Ling Lin, Rafael Linden, Paul Lingor, Jennifer Lippincott-Schwartz, Michael P Lisanti, Paloma B Liton, Bo Liu, Chun-Feng Liu, Kaiyu Liu, Leyuan Liu, Qiong A Liu, Wei Liu, Young-Chau Liu, Yule Liu, Richard A Lockshin, Chun-Nam Lok, Sagar Lonial, Benjamin Loos, Gabriel Lopez-Berestein, Carlos Lopez-Otin, Laura Lossi, Michael T Lotze, Péter Low, Binfeng Lu, Bingwei Lu, Bo Lu, Zhen Lu, Fredéric Luciano, Nicholas W Lukacs, Anders H Lund, Melinda A Lynch-Day, Yong Ma, Fernando Macian, Jeff P MacKeigan, Kay F Macleod, Frank Madeo, Luigi Maiuri, Maria Chiara Maiuri, Davide Malagoli, May Christine V Malicdan, Walter Malorni, Na Man, Eva-Maria Mandelkow, Stéphen Manon, Irena Manov, Kai Mao, Xiang Mao, Zixu Mao, Philippe Marambaud, Daniela Marazziti, Yves L Marcel, Katie Marchbank, Piero Marchetti, Stefan J Marciniak, Mateus Marcondes, Mohsen Mardi, Gabriella Marfè, Guillermo Mariño, Maria Markaki, Mark R Marten, Seamus J Martin, Camille Martinand-Mari, Wim Martinet, Marta Martinez-Vicente, Matilde Masini, Paola Matarrese, Saburo Matsuo, Raffaele Matteoni, Andreas Mayer, Nathalie M Mazure, David J McConkey, Melanie J McConnell, Catherine McDermott, Christine McDonald, Gerald M McInerney, Sharon L McKenna, BethAnn McLaughlin, Pamela J McLean, Christopher R McMaster, G Angus McQuibban, Alfred J Meijer, Miriam H Meisler, Alicia Meléndez, Thomas J Melia, Gerry Melino, Maria A Mena, Javier A Menendez, Rubem F S Menna-Barreto, Manoj B Menon, Fiona M Menzies, Carol A Mercer, Adalberto Merighi, Diane E Merry, Stefania Meschini, Christian G Meyer, Thomas F Meyer, Chao-Yu Miao, Jun-Ying Miao, Paul A M Michels, Carine Michiels, Dalibor Mijaljica, Ana Milojkovic, Saverio Minucci, Clelia Miracco, Cindy K Miranti, Ioannis Mitroulis, Keisuke Miyazawa, Noboru Mizushima, Baharia Mograbi, Simin Mohseni, Xavier Molero, Bertrand Mollereau, Faustino Mollinedo, Takashi Momoi, Iryna Monastyrska, Martha M Monick, Mervyn J Monteiro, Michael N Moore, Rodrigo Mora, Kevin Moreau, Paula I Moreira, Yuji Moriyasu, Jorge Moscat, Serge Mostowy, Jeremy C Mottram, Tomasz Motyl, Charbel E-H Moussa, Sylke Müller, Sylviane Muller, Karl Münger, Christian Münz, Leon O Murphy, Maureen E Murphy, Antonio Musarò, Indira Mysorekar, Eiichiro Nagata, Kazuhiro Nagata, Aimable Nahimana, Usha Nair, Toshiyuki Nakagawa, Kiichi Nakahira, Hiroyasu Nakano, Hitoshi Nakatogawa, Meera Nanjundan, Naweed I Naqvi, Derek P Narendra, Masashi Narita, Miguel Navarro, Steffan T Nawrocki, Taras Y Nazarko, Andriy Nemchenko, Mihai G Netea, Thomas P Neufeld, Paul A Ney, Ioannis P Nezis, Huu Phuc Nguyen, Daotai Nie, Ichizo Nishino, Corey Nislow, Ralph A Nixon, Takeshi Noda, Angelika A Noegel, Anna Nogalska, Satoru Noguchi, Lucia Notterpek, Ivana Novak, Tomoyoshi Nozaki, Nobuyuki Nukina, Thorsten Nürnberger, Beat Nyfeler, Keisuke Obara, Terry D Oberley, Salvatore Oddo, Michinaga Ogawa, Toya Ohashi, Koji Okamoto, Nancy L Oleinick, F Javier Oliver, Laura J Olsen, Stefan Olsson, Onya Opota, Timothy F Osborne, Gary K Ostrander, Kinya Otsu, Jing-hsiung James Ou, Mireille Ouimet, Michael Overholtzer, Bulent Ozpolat, Paolo Paganetti, Ugo Pagnini, Nicolas Pallet, Glen E Palmer, Camilla Palumbo, Tianhong Pan, Theocharis Panaretakis, Udai Bhan Pandey, Zuzana Papackova, Issidora Papassideri, Irmgard Paris, Junsoo Park, Ohkmae K Park, Jan B Parys, Katherine R Parzych, Susann Patschan, Cam Patterson, Sophie Pattingre, John M Pawelek, Jianxin Peng, David H Perlmutter, Ida Perrotta, George Perry, Shazib Pervaiz, Matthias Peter, Godefridus J Peters, Morten Petersen, Goran Petrovski, James M Phang, Mauro Piacentini, Philippe Pierre, Valérie Pierrefite-Carle, Gérard Pierron, Ronit Pinkas-Kramarski, Antonio Piras, Natik Piri, Leonidas C Platanias, Stefanie Pöggeler, Marc Poirot, Angelo Poletti, Christian Poüs, Mercedes Pozuelo-Rubio, Mette Prætorius-Ibba, Anil Prasad, Mark Prescott, Muriel Priault, Nathalie Produit-Zengaffinen, Ann Progulske-Fox, Tassula Proikas-Cezanne, Serge Przedborski, Karin Przyklenk, Rosa Puertollano, Julien Puyal, Shu-Bing Qian, Liang Qin, Zheng-Hong Qin, Susan E Quaggin, Nina Raben, Hannah Rabinowich, Simon W Rabkin, Irfan Rahman, Abdelhaq Rami, Georg Ramm, Glenn Randall, Felix Randow, V Ashutosh Rao, Jeffrey C Rathmell, Brinda Ravikumar, Swapan K Ray, Bruce H Reed, John C Reed, Fulvio Reggiori, Anne Regnier-Vigouroux, Andreas S Reichert, John J Reiners, Russel J Reiter, Jun Ren, Jose L Revuelta, Christopher J Rhodes, Konstantinos Ritis, Elizete Rizzo, Jeffrey Robbins, Michel Roberge, Hernan Roca, Maria C Roccheri, Stéphane Rocchi, H Peter Rodemann, Santiago Rodríguez de Córdoba, Bärbel Rohrer, Igor B Roninson, Kirill Rosen, Magdalena M Rost-Roszkowska, Mustapha Rouis, Kasper M A Rouschop, Francesca Rovetta, Brian P Rubin, David C Rubinsztein, Klaus Ruckdeschel, Edmund B Rucker, Assaf Rudich, Emil Rudolf, Nelson Ruiz-Opazo, Rossella Russo, Tor Erik Rusten, Kevin M Ryan, Stefan W Ryter, David M Sabatini, Junichi Sadoshima, Tapas Saha, Tatsuya Saitoh, Hiroshi Sakagami, Yasuyoshi Sakai, Ghasem Hoseini Salekdeh, Paolo Salomoni, Paul M Salvaterra, Guy Salvesen, Rosa Salvioli, Anthony M J Sanchez, José A Sánchez-Alcázar, Ricardo Sánchez-Prieto, Marco Sandri, Uma Sankar, Poonam Sansanwal, Laura Santambrogio, Shweta Saran, Sovan Sarkar, Minnie Sarwal, Chihiro Sasakawa, Ausra Sasnauskiene, Miklós Sass, Ken Sato, Miyuki Sato, Anthony H V Schapira, Michael Scharl, Hermann M Schätzl, Wiep Scheper, Stefano Schiaffino, Claudio Schneider, Marion E Schneider, Regine Schneider-Stock, Patricia V Schoenlein, Daniel F Schorderet, Christoph Schüller, Gary K Schwartz, Luca Scorrano, Linda Sealy, Per O Seglen, Juan Segura-Aguilar, Iban Seiliez, Oleksandr Seleverstov, Christian Sell, Jong Bok Seo, Duska Separovic, Vijayasaradhi Setaluri, Takao Setoguchi, Carmine Settembre, John J Shacka, Mala Shanmugam, Irving M Shapiro, Eitan Shaulian, Reuben J Shaw, James H Shelhamer, Han-Ming Shen, Wei-Chiang Shen, Zu-Hang Sheng, Yang Shi, Kenichi Shibuya, Yoshihiro Shidoji, Jeng-Jer Shieh, Chwen-Ming Shih, Yohta Shimada, Shigeomi Shimizu, Takahiro Shintani, Orian S Shirihai, Gordon C Shore, Andriy A Sibirny, Stan B Sidhu, Beata Sikorska, Elaine C M Silva-Zacarin, Alison Simmons, Anna Katharina Simon, Hans-Uwe Simon, Cristiano Simone, Anne Simonsen, David A Sinclair, Rajat Singh, Debasish Sinha, Frank A Sinicrope, Agnieszka Sirko, Parco M Siu, Efthimios Sivridis, Vojtech Skop, Vladimir P Skulachev, Ruth S Slack, Soraya S Smaili, Duncan R Smith, María S Soengas, Thierry Soldati, Xueqin Song, Anil K Sood, Tuck Wah Soong, Federica Sotgia, Stephen A Spector, Claudia D Spies, Wolfdieter Springer, Srinivasa M Srinivasula, Leonidas Stefanis, Joan S Steffan, Ruediger Stendel, Harald Stenmark, Anastasis Stephanou, Stephan T Stern, Cinthya Sternberg, Björn Stork, Peter Stralfors, Carlos S Subauste, Xinbing Sui, David Sulzer, Jiaren Sun, Shi-Yong Sun, Zhi-Jun Sun, Joseph J Y Sung, Kuninori Suzuki, Toshihiko Suzuki, Michele S Swanson, Charles Swanton, Sean T Sweeney, Lai-King Sy, Gyorgy Szabadkai, Ira Tabas, Heinrich Taegtmeyer, Marco Tafani, Krisztina Takács-Vellai, Yoshitaka Takano, Kaoru Takegawa, Genzou Takemura, Fumihiko Takeshita, Nicholas J Talbot, Kevin S W Tan, Keiji Tanaka, Kozo Tanaka, Daolin Tang, Dingzhong Tang, Isei Tanida, Bakhos A Tannous, Nektarios Tavernarakis, Graham S Taylor, Gregory A Taylor, J Paul Taylor, Lance S Terada, Alexei Terman, Gianluca Tettamanti, Karin Thevissen, Craig B Thompson, Andrew Thorburn, Michael Thumm, Fengfeng Tian, Yuan Tian, Glauco Tocchini-Valentini, Aviva M Tolkovsky, Yasuhiko Tomino, Lars Tönges, Sharon A Tooze, Cathy Tournier, John Tower, Roberto Towns, Vladimir Trajkovic, Leonardo H Travassos, Ting-Fen Tsai, Mario P Tschan, Takeshi Tsubata, Allan Tsung, Boris Turk, Lorianne S Turner, Suresh C Tyagi, Yasuo Uchiyama, Takashi Ueno, Midori Umekawa, Rika Umemiya-Shirafuji, Vivek K Unni, Maria I Vaccaro, Enza Maria Valente, Greet Van den Berghe, Ida J van der Klei, Wouter van Doorn, Linda F van Dyk, Marjolein van Egmond, Leo A van Grunsven, Peter Vandenabeele, Wim P Vandenberghe, Ilse Vanhorebeek, Eva C Vaquero, Guillermo Velasco, Tibor Vellai, Jose Miguel Vicencio, Richard D Vierstra, Miquel Vila, Cécile Vindis, Giampietro Viola, Maria Teresa Viscomi, Olga V Voitsekhovskaja, Clarissa von Haefen, Marcela Votruba, Keiji Wada, Richard Wade-Martins, Cheryl L Walker, Craig M Walsh, Jochen Walter, Xiang-Bo Wan, Aimin Wang, Chenguang Wang, Dawei Wang, Fan Wang, Fen Wang, Guanghui Wang, Haichao Wang, Hong-Gang Wang, Horng-Dar Wang, Jin Wang, Ke Wang, Mei Wang, Richard C Wang, Xinglong Wang, Xuejun Wang, Ying-Jan Wang, Yipeng Wang, Zhen Wang, Zhigang Charles Wang, Zhinong Wang, Derick G Wansink, Diane M Ward, Hirotaka Watada, Sarah L Waters, Paul Webster, Lixin Wei, Conrad C Weihl, William A Weiss, Scott M Welford, Long-Ping Wen, Caroline A Whitehouse, J Lindsay Whitton, Alexander J Whitworth, Tom Wileman, John W Wiley, Simon Wilkinson, Dieter Willbold, Roger L Williams, Peter R Williamson, Bradly G Wouters, Chenghan Wu, Dao-Cheng Wu, William K K Wu, Andreas Wyttenbach, Ramnik J Xavier, Zhijun Xi, Pu Xia, Gengfu Xiao, Zhiping Xie, Zhonglin Xie, Da-zhi Xu, Jianzhen Xu, Liang Xu, Xiaolei Xu, Ai Yamamoto, Akitsugu Yamamoto, Shunhei Yamashina, Michiaki Yamashita, Xianghua Yan, Mitsuhiro Yanagida, Dun-Sheng Yang, Elizabeth Yang, Jin-Ming Yang, Shi Yu Yang, Wannian Yang, Wei Yuan Yang, Zhifen Yang, Meng-Chao Yao, Tso-Pang Yao, Behzad Yeganeh, Wei-Lien Yen, Jia-Jing Yin, Xiao-Ming Yin, Ook-Joon Yoo, Gyesoon Yoon, Seung-Yong Yoon, Tomohiro Yorimitsu, Yuko Yoshikawa, Tamotsu Yoshimori, Kohki Yoshimoto, Ho Jin You, Richard J Youle, Anas Younes, Li Yu, Long Yu, Seong-Woon Yu, Wai Haung Yu, Zhi-Min Yuan, Zhenyu Yue, Cheol-Heui Yun, Michisuke Yuzaki, Olga Zabirnyk, Elaine Silva-Zacarin, David Zacks, Eldad Zacksenhaus, Nadia Zaffaroni, Zahra Zakeri, Herbert J Zeh, Scott O Zeitlin, Hong Zhang, Hui-Ling Zhang, Jianhua Zhang, Jing-Pu Zhang, Lin Zhang, Long Zhang, Ming-Yong Zhang, Xu Dong Zhang, Mantong Zhao, Yi-Fang Zhao, Ying Zhao, Zhizhuang J Zhao, Xiaoxiang Zheng, Boris Zhivotovsky, Qing Zhong, Cong-Zhao Zhou, Changlian Zhu, Wei-Guo Zhu, Xiao-feng Zhu, Xiongwei Zhu, Yuangang Zhu, Teresa Zoladek, Wei-Xing Zong, Antonio Zorzano, Jürgen Zschocke, Brian Zuckerbraun.
Autophagy
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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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Genetic animal models for evaluating the role of autophagy in etiopathogenesis of Parkinson disease.
Autophagy
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Parkinson disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by the formation of ubiquitin and SNCA/?-synuclein-containing inclusions (Lewy bodies), dystrophic midbrain dopaminergic (DAergic) terminals, and degeneration of midbrain DAergic neurons. The vast majority of PD occurs sporadically, while approximately 5% of all PD cases are inherited. Genetic mutations of a few genes have been identified as causes of familiar PD, i.e., mutations in SNCA, PARK2/parkin, UCHL1, PARK7/DJ1, PINK1 and LRRK2, leading to DAergic cell death, but variable pathological changes. The evidence supports the hypothesis that several pathogenic mechanisms are likely involved at initial stages of the disease, and eventually they merge to cause parkinsonism. The current challenge facing PD research is to unravel the components in these pathways that contribute to the pathogenesis of PD. Accumulating evidence has implicated dysfunctional autophagy, a regulated lysosomal pathway with a capacity for clearing protein aggregates and cellular organelles, as one of the pathogenic systems contributing to the development of idiopathic PD.
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Animal models of Parkinsons disease: limits and relevance to neuroprotection studies.
Mov. Disord.
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Over the last two decades, significant strides has been made toward acquiring a better knowledge of both the etiology and pathogenesis of Parkinsons disease (PD). Experimental models are of paramount importance to obtain greater insights into the pathogenesis of the disease. Thus far, neurotoxin-based animal models have been the most popular tools employed to produce selective neuronal death in both in vitro and in vivo systems. These models have been commonly referred to as the pathogenic models. The current trend in modeling PD revolves around what can be called the disease gene-based models or etiologic models. The value of utilizing multiple models with a different mechanism of insult rests on the premise that dopamine-producing neurons die by stereotyped cascades that can be activated by a range of insults, from neurotoxins to downregulation and overexpression of disease-related genes. In this position article, we present the relevance of both pathogenic and etiologic models as well as the concept of clinically relevant designs that, we argue, should be utilized in the preclinical development phase of new neuroprotective therapies before embarking into clinical trials.
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Disrupted autophagy leads to dopaminergic axon and dendrite degeneration and promotes presynaptic accumulation of ?-synuclein and LRRK2 in the brain.
J. Neurosci.
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Parkinsons disease (PD) is characterized pathologically by the formation of ubiquitin and ?-synuclein (?-syn)-containing inclusions (Lewy bodies), dystrophic dopamine (DA) terminals, and degeneration of midbrain DA neurons. The precise molecular mechanisms underlying these pathological features remain elusive. Accumulating evidence has implicated dysfunctional autophagy, the cell self-digestion and neuroprotective pathway, as one of the pathogenic systems contributing to the development of idiopathic PD. Here we characterize autophagy-deficient mouse models and provide in vivo evidence for the potential role that impaired autophagy plays in pathogenesis associated with PD. Cell-specific deletion of essential autophagy gene Atg7 in midbrain DA neurons causes delayed neurodegeneration, accompanied by late-onset locomotor deficits. In contrast, Atg7-deficient DA neurons in the midbrain exhibit early dendritic and axonal dystrophy, reduced striatal dopamine content, and the formation of somatic and dendritic ubiquitinated inclusions in DA neurons. Furthermore, whole-brain-specific loss of Atg7 leads to presynaptic accumulation of ?-syn and LRRK2 proteins, which are encoded by two autosomal dominantly inherited PD-related genes. Our results suggest that disrupted autophagy may be associated with enhanced levels of endogenous ?-syn and LRRK2 proteins in vivo. Our findings implicate dysfunctional autophagy as one of the failing cellular mechanisms involved in the pathogenesis of idiopathic PD.
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The BECN1 coiled coil domain: an "imperfect" homodimer interface that facilitates ATG14 and UVRAG binding.
Autophagy
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The coiled-coil domain of BECN1 serves as a protein interaction platform to recruit two major autophagy regulators ATG14 and UVRAG. Our crystal structure of the BECN1 coiled-coil domain reveals a homodimer with an imperfect dimer interface. This "imperfect" feature favors the formation of a stable BECN1-ATG14 or BECN1-UVRAG heterodimer over a metastable BECN1 homodimer to promote autophagy and/or endocytic pathways.
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Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak.
J. Biol. Chem.
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Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.
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Generation of a novel rodent model for DYT1 dystonia.
Neurobiol. Dis.
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A mutation in the coding region of the Tor1A gene, resulting in a deletion of a glutamic acid residue in the torsinA protein (?ETorA), is the major cause of the inherited autosomal-dominant early onset torsion dystonia (DYT1). The pathophysiological consequences of this amino acid loss are still not understood. Currently available animal models for DYT1 dystonia provided important insights into the disease; however, they differ with respect to key features of torsinA associated pathology. We developed transgenic rat models harboring the full length human mutant and wildtype Tor1A gene. A complex phenotyping approach including classical behavioral tests, electrophysiology and neuropathology revealed a progressive neurological phenotype in ?ETorA expressing rats. Furthermore, we were able to replicate key pathological features of torsinA associated pathology in a second species, such as nuclear envelope pathology, behavioral abnormalities and plasticity changes. We therefore suggest that this rat model represents an appropriate new model suitable to further investigate the pathophysiology of ?ETorA and to test for therapeutic approaches.
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Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.
Cell Host Microbe
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Phagocytosis and autophagy are two important and related arms of the hosts first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicons actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.
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Involvement of Beclin 1 in engulfment of apoptotic cells.
J. Biol. Chem.
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Efficient apoptotic cell engulfment is important for both tissue homeostasis and immune response in mammals. In the present study, we report that Beclin 1 (a regulator of autophagy) is required for apoptotic cell engulfment. The engulfment process was largely abolished in Beclin 1 knock-out cells, and Beclin 1 knockdown significantly decreased apoptotic cell internalization in macrophage and fibroblast cell lines. Beclin 1 was recruited to the early phagocytic cup along with the generation of phosphatidylinositol 3-phosphate and Rac1, which regulates actin dynamics in lamellipodia. No lamellipodia were formed in Beclin 1 knock-out cells, and Beclin 1 knockdown completely inhibited the promotion of engulfment by ectopic expression of Rac1. Beclin 1 was co-immunoprecipitated with Rac1. These data indicate that Beclin 1 coordinates actin dynamics and membrane phospholipid synthesis to promote efficient apoptotic cell engulfment.
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Imperfect interface of Beclin1 coiled-coil domain regulates homodimer and heterodimer formation with Atg14L and UVRAG.
Nat Commun
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Beclin 1 is a core component of the Class III Phosphatidylinositol 3-Kinase VPS34 complex. The coiled coil domain of Beclin 1 serves as an interaction platform for assembly of distinct Atg14L- and UVRAG-containing complexes to modulate VPS34 activity. Here we report the crystal structure of the coiled coil domain that forms an antiparallel dimer and is rendered metastable by a series of imperfect a-d pairings at its coiled coil interface. Atg14L and UVRAG promote the transition of metastable homodimeric Beclin 1 to heterodimeric Beclin1-Atg14L/UVRAG assembly. Beclin 1 mutants with their imperfect a-d pairings modified to enhance self-interaction, show distinctively altered interactions with Atg14L or UVRAG. These results suggest that specific utilization of the dimer interface and modulation of the homodimer-heterodimer transition by Beclin 1-interacting partners may underlie the molecular mechanism that controls the formation of various Beclin1-VPS34 subcomplexes to exert their effect on an array of VPS34-related activities, including autophagy.
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Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.
Gastroenterology
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The pathogenesis of liver fibrosis involves activation of hepatic stellate cells, which is associated with depletion of intracellular lipid droplets. When hepatocytes undergo autophagy, intracellular lipids are degraded in lysosomes. We investigated whether autophagy also promotes loss of lipids in hepatic stellate cells to provide energy for their activation and extended these findings to other fibrogenic cells.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.