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1
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Herbst R, Huijbers MG, Oury J, Burden SJ. Building, Breaking, and Repairing Neuromuscular Synapses. Cold Spring Harb Perspect Biol 2024; 16:a041490. [PMID: 38697654 PMCID: PMC11065174 DOI: 10.1101/cshperspect.a041490] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/05/2024]
Abstract
A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.
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Affiliation(s)
- Ruth Herbst
- Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
| | - Maartje G Huijbers
- Department of Human Genetics, Leiden University Medical Centre LUMC, 2300 RC Leiden, the Netherlands
- Department of Neurology, Leiden University Medical Centre LUMC, 2333 ZA Leiden, the Netherlands
| | - Julien Oury
- Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, NYU School of Medicine, New York, New York 10016, USA
| | - Steven J Burden
- Neurology Department, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
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2
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Feng X, Liu S, Li K, Bu F, Yuan H. NCAD v1.0: a database for non-coding variant annotation and interpretation. J Genet Genomics 2024; 51:230-242. [PMID: 38142743 DOI: 10.1016/j.jgg.2023.12.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Revised: 12/15/2023] [Accepted: 12/18/2023] [Indexed: 12/26/2023]
Abstract
The application of whole genome sequencing is expanding in clinical diagnostics across various genetic disorders, and the significance of non-coding variants in penetrant diseases is increasingly being demonstrated. Therefore, it is urgent to improve the diagnostic yield by exploring the pathogenic mechanisms of variants in non-coding regions. However, the interpretation of non-coding variants remains a significant challenge, due to the complex functional regulatory mechanisms of non-coding regions and the current limitations of available databases and tools. Hence, we develop the non-coding variant annotation database (NCAD, http://www.ncawdb.net/), encompassing comprehensive insights into 665,679,194 variants, regulatory elements, and element interaction details. Integrating data from 96 sources, spanning both GRCh37 and GRCh38 versions, NCAD v1.0 provides vital information to support the genetic diagnosis of non-coding variants, including allele frequencies of 12 diverse populations, with a particular focus on the population frequency information for 230,235,698 variants in 20,964 Chinese individuals. Moreover, it offers prediction scores for variant functionality, five categories of regulatory elements, and four types of non-coding RNAs. With its rich data and comprehensive coverage, NCAD serves as a valuable platform, empowering researchers and clinicians with profound insights into non-coding regulatory mechanisms while facilitating the interpretation of non-coding variants.
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Affiliation(s)
- Xiaoshu Feng
- Institute of Rare Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610044, China
| | - Sihan Liu
- Institute of Rare Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610044, China
| | - Ke Li
- Institute of Rare Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610044, China
| | - Fengxiao Bu
- Institute of Rare Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610044, China.
| | - Huijun Yuan
- Institute of Rare Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610044, China.
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3
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Gessler L, Huraskin D, Jian Y, Eiber N, Hu Z, Prószyński T, Hashemolhosseini S. The YAP1/TAZ-TEAD transcriptional network regulates gene expression at neuromuscular junctions in skeletal muscle fibers. Nucleic Acids Res 2024; 52:600-624. [PMID: 38048326 PMCID: PMC10810223 DOI: 10.1093/nar/gkad1124] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Revised: 11/06/2023] [Accepted: 11/09/2023] [Indexed: 12/06/2023] Open
Abstract
We examined YAP1/TAZ-TEAD signaling pathway activity at neuromuscular junctions (NMJs) of skeletal muscle fibers in adult mice. Our investigations revealed that muscle-specific knockouts of Yap1 or Taz, or both, demonstrate that these transcriptional coactivators regulate synaptic gene expression, the number and morphology of NMJs, and synaptic nuclei. Yap1 or Taz single knockout mice display reduced grip strength, fragmentation of NMJs, and accumulation of synaptic nuclei. Yap1/Taz muscle-specific double knockout mice do not survive beyond birth and possess almost no NMJs, the few detectable show severely impaired morphology and are organized in widened endplate bands; and with motor nerve endings being mostly absent. Myogenic gene expression is significantly impaired in the denervated muscles of knockout mice. We found that Tead1 and Tead4 transcription rates were increased upon incubation of control primary myotubes with AGRN-conditioned medium. Reduced AGRN-dependent acetylcholine receptor clustering and synaptic gene transcription were observed in differentiated primary Tead1 and Tead4 knockout myotubes. In silico analysis of previously reported genomic occupancy sites of TEAD1/4 revealed evolutionary conserved regions of potential TEAD binding motifs in key synaptic genes, the relevance of which was functionally confirmed by reporter assays. Collectively, our data suggest a role for YAP1/TAZ-TEAD1/TEAD4 signaling, particularly through TAZ-TEAD4, in regulating synaptic gene expression and acetylcholine receptor clustering at NMJs.
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Affiliation(s)
- Lea Gessler
- Institute of Biochemistry, Medical Faculty, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany
| | - Danyil Huraskin
- Institute of Biochemistry, Medical Faculty, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany
| | - Yongzhi Jian
- Institute of Biochemistry, Medical Faculty, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany
| | - Nane Eiber
- Institute of Biochemistry, Medical Faculty, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany
| | - Zhaoyong Hu
- Nephrology Division, Department of Medicine, Baylor College of Medicine, Houston, TX, USA
| | - Tomasz J Prószyński
- Łukasiewicz Research Network-PORT Polish Center for Technology Development, Wrocław, Poland
| | - Said Hashemolhosseini
- Institute of Biochemistry, Medical Faculty, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany
- Muscle Research Center, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany
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4
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Qi Z, Wang S, Xuan A, Gu X, Deng J, Huang C, Zhang L, Yin X. MiR-142a-3p: A novel ACh receptor transcriptional regulator in association with peripheral nerve injury. MOLECULAR THERAPY. NUCLEIC ACIDS 2022; 30:325-336. [PMID: 36381585 PMCID: PMC9633872 DOI: 10.1016/j.omtn.2022.10.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Accepted: 10/12/2022] [Indexed: 12/15/2022]
Abstract
Long-term denervation leads to the disintegration of nicotinic acetylcholine receptor (nAChR) located at the endplate structure, which translates to deficits in functional activation despite nerve repair. Because of a lack of effective measures to protect AChR expression, we explored the effect of alterations in muscular miR-142a-3p on nAChR. In this study, we constructed a model of miR-142a-3p knockdown by transfecting a miR-142a-3p inhibitor short hairpin RNA (shRNA) into C2C12 myotubes, and we injected this miR-142a-3p inhibitor shRNA into the tibialis anterior (TA) muscle in uninjured mice and in denervated mice by transecting the sciatic nerve. Our results showed that miR-142a-3p knockdown led to an increased number and area of AChR clusters in myotubes in vitro and larger neuromuscular endplates in adult mice. Furthermore, miR-142a-3p knockdown delayed the disintegration of motor endplates after denervation. Last, upon miR-142a-3p knockdown in uninjured and denervated mice, we observed an increase in the mRNA levels of five AChR subunits as well as mRNAs of genes implicated in AChR transcription and AChR clustering. Together, these results suggest that miR-142a-3p may be a potential target for therapeutic intervention to prevent motor endplate degradation following peripheral nerve injury.
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Affiliation(s)
- Zhidan Qi
- Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing, China
| | - Shen Wang
- Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing, China
| | - Ang Xuan
- Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, China,MoE Key Laboratory for Biomedical Photonics, School of Engineering Sciences, Huazhong University of Science and Technology, Wuhan, China
| | - Xinyi Gu
- Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing, China
| | - Jin Deng
- Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing, China
| | - Chen Huang
- Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing, China
| | - Lei Zhang
- Electron Microscopy Analysis Laboratory, Medical and Health Analysis Center, Peking University, Beijing, China,Department of Biophysics, School of Basic Medical Sciences, Peking University, Beijing, China
| | - Xiaofeng Yin
- Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing, China,Pizhou People’s Hospital, Jiangsu, China,Corresponding author Xiaofeng Yin, Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing, China.
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5
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Planat M, Amaral MM, Fang F, Chester D, Aschheim R, Irwin K. Group Theory of Syntactical Freedom in DNA Transcription and Genome Decoding. Curr Issues Mol Biol 2022; 44:1417-1433. [PMID: 35723353 PMCID: PMC9164029 DOI: 10.3390/cimb44040095] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2022] [Revised: 03/19/2022] [Accepted: 03/20/2022] [Indexed: 12/24/2022] Open
Abstract
Transcription factors (TFs) are proteins that recognize specific DNA fragments in order to decode the genome and ensure its optimal functioning. TFs work at the local and global scales by specifying cell type, cell growth and death, cell migration, organization and timely tasks. We investigate the structure of DNA-binding motifs with the theory of finitely generated groups. The DNA ‘word’ in the binding domain—the motif—may be seen as the generator of a finitely generated group Fdna on four letters, the bases A, T, G and C. It is shown that, most of the time, the DNA-binding motifs have subgroup structures close to free groups of rank three or less, a property that we call ‘syntactical freedom’. Such a property is associated with the aperiodicity of the motif when it is seen as a substitution sequence. Examples are provided for the major families of TFs, such as leucine zipper factors, zinc finger factors, homeo-domain factors, etc. We also discuss the exceptions to the existence of such DNA syntactical rules and their functional roles. This includes the TATA box in the promoter region of some genes, the single-nucleotide markers (SNP) and the motifs of some genes of ubiquitous roles in transcription and regulation.
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Affiliation(s)
- Michel Planat
- Institut FEMTO-ST CNRS UMR 6174, Université de Bourgogne-Franche-Comté, F-25044 Besançon, France
- Correspondence:
| | - Marcelo M. Amaral
- Quantum Gravity Research, Los Angeles, CA 90290, USA; (M.M.A.); (F.F.); (D.C.); (R.A.); (K.I.)
| | - Fang Fang
- Quantum Gravity Research, Los Angeles, CA 90290, USA; (M.M.A.); (F.F.); (D.C.); (R.A.); (K.I.)
| | - David Chester
- Quantum Gravity Research, Los Angeles, CA 90290, USA; (M.M.A.); (F.F.); (D.C.); (R.A.); (K.I.)
| | - Raymond Aschheim
- Quantum Gravity Research, Los Angeles, CA 90290, USA; (M.M.A.); (F.F.); (D.C.); (R.A.); (K.I.)
| | - Klee Irwin
- Quantum Gravity Research, Los Angeles, CA 90290, USA; (M.M.A.); (F.F.); (D.C.); (R.A.); (K.I.)
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6
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Karmouch J, Delers P, Semprez F, Soyed N, Areias J, Bélanger G, Ravel-Chapuis A, Dobbertin A, Jasmin BJ, Legay C. AChR β-Subunit mRNAs Are Stabilized by HuR in a Mouse Model of Congenital Myasthenic Syndrome With Acetylcholinesterase Deficiency. Front Mol Neurosci 2020; 13:568171. [PMID: 33362463 PMCID: PMC7757417 DOI: 10.3389/fnmol.2020.568171] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2020] [Accepted: 08/13/2020] [Indexed: 11/13/2022] Open
Abstract
Collagen Q (COLQ) is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction. So far, no mutation has been identified in the ACHE human gene but over 50 different mutations in the COLQ gene are causative for a congenital myasthenic syndrome (CMS) with AChE deficiency. Mice deficient for COLQ mimic most of the functional deficit observed in CMS patients. At the molecular level, a striking consequence of the absence of COLQ is an increase in the levels of acetylcholine receptor (AChR) mRNAs and proteins in vivo and in vitro in murine skeletal muscle cells. Here, we decipher the mechanisms that drive AChR mRNA upregulation in cultured muscle cells deficient for COLQ. We show that the levels of AChR β-subunit mRNAs are post-transcriptionally regulated by an increase in their stability. We demonstrate that this process results from an activation of p38 MAPK and the cytoplasmic translocation of the nuclear RNA-binding protein human antigen R (HuR) that interacts with the AU-rich element located within AChR β-subunit transcripts. This HuR/AChR transcript interaction induces AChR β-subunit mRNA stabilization and occurs at a specific stage of myogenic differentiation. In addition, pharmacological drugs that modulate p38 activity cause parallel modifications of HuR protein and AChR β-subunit levels. Thus, our study provides new insights into the signaling pathways that are regulated by ColQ-deficiency and highlights for the first time a role for HuR and p38 in mRNA stability in a model of congenital myasthenic syndrome.
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Affiliation(s)
- Jennifer Karmouch
- CNRS UMR 8003, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Perrine Delers
- CNRS UMR 8003, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Fannie Semprez
- CNRS UMR 8003, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Nouha Soyed
- CNRS UMR 8003, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Julie Areias
- CNRS UMR 8003, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Guy Bélanger
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Aymeric Ravel-Chapuis
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
| | | | - Bernard J Jasmin
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Claire Legay
- CNRS UMR 8003, Université de Paris, Sorbonne Paris Cité, Paris, France
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Rodríguez Cruz PM, Cossins J, Beeson D, Vincent A. The Neuromuscular Junction in Health and Disease: Molecular Mechanisms Governing Synaptic Formation and Homeostasis. Front Mol Neurosci 2020; 13:610964. [PMID: 33343299 PMCID: PMC7744297 DOI: 10.3389/fnmol.2020.610964] [Citation(s) in RCA: 121] [Impact Index Per Article: 24.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Accepted: 10/30/2020] [Indexed: 12/28/2022] Open
Abstract
The neuromuscular junction (NMJ) is a highly specialized synapse between a motor neuron nerve terminal and its muscle fiber that are responsible for converting electrical impulses generated by the motor neuron into electrical activity in the muscle fibers. On arrival of the motor nerve action potential, calcium enters the presynaptic terminal, which leads to the release of the neurotransmitter acetylcholine (ACh). ACh crosses the synaptic gap and binds to ACh receptors (AChRs) tightly clustered on the surface of the muscle fiber; this leads to the endplate potential which initiates the muscle action potential that results in muscle contraction. This is a simplified version of the events in neuromuscular transmission that take place within milliseconds, and are dependent on a tiny but highly structured NMJ. Much of this review is devoted to describing in more detail the development, maturation, maintenance and regeneration of the NMJ, but first we describe briefly the most important molecules involved and the conditions that affect their numbers and function. Most important clinically worldwide, are myasthenia gravis (MG), the Lambert-Eaton myasthenic syndrome (LEMS) and congenital myasthenic syndromes (CMS), each of which causes specific molecular defects. In addition, we mention the neurotoxins from bacteria, snakes and many other species that interfere with neuromuscular transmission and cause potentially fatal diseases, but have also provided useful probes for investigating neuromuscular transmission. There are also changes in NMJ structure and function in motor neuron disease, spinal muscle atrophy and sarcopenia that are likely to be secondary but might provide treatment targets. The NMJ is one of the best studied and most disease-prone synapses in the nervous system and it is amenable to in vivo and ex vivo investigation and to systemic therapies that can help restore normal function.
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Affiliation(s)
- Pedro M. Rodríguez Cruz
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
- Neurosciences Group, Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Oxford, United Kingdom
| | - Judith Cossins
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
- Neurosciences Group, Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Oxford, United Kingdom
| | - David Beeson
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
- Neurosciences Group, Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Oxford, United Kingdom
| | - Angela Vincent
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
- Neurosciences Group, Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Oxford, United Kingdom
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8
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Cetin H, Beeson D, Vincent A, Webster R. The Structure, Function, and Physiology of the Fetal and Adult Acetylcholine Receptor in Muscle. Front Mol Neurosci 2020; 13:581097. [PMID: 33013323 PMCID: PMC7506097 DOI: 10.3389/fnmol.2020.581097] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Accepted: 08/13/2020] [Indexed: 12/31/2022] Open
Abstract
The neuromuscular junction (NMJ) is a highly developed synapse linking motor neuron activity with muscle contraction. A complex of molecular cascades together with the specialized NMJ architecture ensures that each action potential arriving at the motor nerve terminal is translated into an action potential in the muscle fiber. The muscle-type nicotinic acetylcholine receptor (AChR) is a key molecular component located at the postsynaptic muscle membrane responsible for the generation of the endplate potential (EPP), which usually exceeds the threshold potential necessary to activate voltage-gated sodium channels and triggers a muscle action potential. Two AChR isoforms are found in mammalian muscle. The fetal isoform is present in prenatal stages and is involved in the development of the neuromuscular system whereas the adult isoform prevails thereafter, except after denervation when the fetal form is re-expressed throughout the muscle. This review will summarize the structural and functional differences between the two isoforms and outline congenital and autoimmune myasthenic syndromes that involve the isoform specific AChR subunits.
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Affiliation(s)
- Hakan Cetin
- Department of Neurology, Medical University of Vienna, Vienna, Austria.,Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
| | - David Beeson
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
| | - Angela Vincent
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
| | - Richard Webster
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
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Belotti E, Schaeffer L. Regulation of Gene expression at the neuromuscular Junction. Neurosci Lett 2020; 735:135163. [PMID: 32553805 DOI: 10.1016/j.neulet.2020.135163] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2020] [Revised: 06/11/2020] [Accepted: 06/14/2020] [Indexed: 01/08/2023]
Abstract
Gene expression in skeletal muscle is profoundly changed upon innervation. 50 years of research on the neuromuscular system have greatly increased our understanding of the mechanisms underlying these changes. By controlling the expression and the activity of key transcription factors, nerve-evoked electrical activity in the muscle fiber positively and negatively regulates the expression of hundreds of genes. Innervation also compartmentalizes gene expression into synaptic and extra-synaptic regions of muscle fibers. In addition, electrically-evoked, release of several factors (e.g. Agrin, Neuregulin, Wnt ligands) induce the clustering of synaptic proteins and of a few muscle nuclei. The sub-synaptic nuclei acquire a particular chromatin organization and develop a specific gene expression program dedicated to building and maintaining a functional neuromuscular synapse. Deciphering synapse-specific, transcriptional regulation started with the identification of the N-box, a six base pair element present in the promoters of the acetylcholine δ and ε subunits. Most genes with synapse-specific expression turned out to contain at least one N-box in their promoters. The N-box is a response element for the synaptic signals Agrin and Neuregulins as well as a binding site for transcription factors of the Ets family. The Ets transcription factors GABP and Erm are implicated in the activation of post-synaptic genes via the N-box. In muscle fibers, Erm expression is restricted to the NMJ whereas GABP is expressed in all muscle nuclei but phosphorylated and activated by the JNK and ERK signaling pathways in response to Agrin and Neuregulins. Post-synaptic gene expression also correlates with chromatin modifications at the genomic level as evidenced by the strong enrichment of decondensed chromatin and acetylated histones in sub-synaptic nuclei. Here we discuss these transcriptional pathways for synaptic specialization at NMJs.
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Affiliation(s)
- Edwige Belotti
- INMG, Inserm U1217, CNRS UMR5310, Université Lyon 1, Université De Lyon, Lyon, France
| | - Laurent Schaeffer
- INMG, Inserm U1217, CNRS UMR5310, Université Lyon 1, Université De Lyon, Lyon, France; Centre De Biotechnologie Cellulaire, Hospices Civils De Lyon, Lyon, France.
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10
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Rudolf R, Khan MM, Witzemann V. Motor Endplate-Anatomical, Functional, and Molecular Concepts in the Historical Perspective. Cells 2019; 8:E387. [PMID: 31035624 PMCID: PMC6562597 DOI: 10.3390/cells8050387] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2019] [Revised: 04/20/2019] [Accepted: 04/25/2019] [Indexed: 11/17/2022] Open
Abstract
By mediating voluntary muscle movement, vertebrate neuromuscular junctions (NMJ) play an extraordinarily important role in physiology. While the significance of the nerve-muscle connectivity was already conceived almost 2000 years back, the precise cell and molecular biology of the NMJ have been revealed in a series of fascinating research activities that started around 180 years ago and that continues. In all this time, NMJ research has led to fundamentally new concepts of cell biology, and has triggered groundbreaking advancements in technologies. This review tries to sketch major lines of thought and concepts on NMJ in their historical perspective, in particular with respect to anatomy, function, and molecular components. Furthermore, along these lines, it emphasizes the mutual benefit between science and technology, where one drives the other. Finally, we speculate on potential major future directions for studies on NMJ in these fields.
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Affiliation(s)
- Rüdiger Rudolf
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany.
- Interdisciplinary Center for Neuroscience, Heidelberg University, 69120 Heidelberg, Germany.
- Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen, Germany.
| | - Muzamil Majid Khan
- Cell Biology and Biophysics, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
| | - Veit Witzemann
- Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
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11
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Getz AM, Wijdenes P, Riaz S, Syed NI. Uncovering the Cellular and Molecular Mechanisms of Synapse Formation and Functional Specificity Using Central Neurons of Lymnaea stagnalis. ACS Chem Neurosci 2018. [PMID: 29528213 DOI: 10.1021/acschemneuro.7b00448] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
All functions of the nervous system are contingent upon the precise organization of neuronal connections that are initially patterned during development, and then continually modified throughout life. Determining the mechanisms that specify the formation and functional modulation of synaptic circuitry are critical to advancing both our fundamental understanding of the nervous system as well as the various neurodevelopmental, neurological, neuropsychiatric, and neurodegenerative disorders that are met in clinical practice when these processes go awry. Defining the cellular and molecular mechanisms underlying nervous system development, function, and pathology has proven challenging, due mainly to the complexity of the vertebrate brain. Simple model system approaches with invertebrate preparations, on the other hand, have played pivotal roles in elucidating the fundamental mechanisms underlying the formation and plasticity of individual synapses, and the contributions of individual neurons and their synaptic connections that underlie a variety of behaviors, and learning and memory. In this Review, we discuss the experimental utility of the invertebrate mollusc Lymnaea stagnalis, with a particular emphasis on in vitro cell culture, semi-intact and in vivo preparations, which enable molecular and electrophysiological identification of the cellular and molecular mechanisms governing the formation, plasticity, and specificity of individual synapses at a single-neuron or single-synapse resolution.
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Affiliation(s)
- Angela M. Getz
- Department of Cell Biology & Anatomy, Hotchkiss Brain Institute and Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 1N4, Canada
- Department of Neuroscience, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 1N4, Canada
| | - Pierre Wijdenes
- Department of Cell Biology & Anatomy, Hotchkiss Brain Institute and Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 1N4, Canada
- Biomedical Engineering Graduate Program, University of Calgary, Calgary, Alberta T2N 1N4, Canada
| | - Saba Riaz
- Department of Cell Biology & Anatomy, Hotchkiss Brain Institute and Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 1N4, Canada
| | - Naweed I. Syed
- Department of Cell Biology & Anatomy, Hotchkiss Brain Institute and Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 1N4, Canada
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12
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Engel AG. Genetic basis and phenotypic features of congenital myasthenic syndromes. HANDBOOK OF CLINICAL NEUROLOGY 2018; 148:565-589. [PMID: 29478601 DOI: 10.1016/b978-0-444-64076-5.00037-5] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/06/2022]
Abstract
The congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. The disease proteins reside in the nerve terminal, the synaptic basal lamina, or in the postsynaptic region, or at multiple sites at the neuromuscular junction as well as in other tissues. Targeted mutation analysis by Sanger or exome sequencing has been facilitated by characteristic phenotypic features of some CMS. No fewer than 20 disease genes have been recognized to date. In one-half of the currently identified probands, the disease stems from mutations in genes encoding subunits of the muscle form of the acetylcholine receptor (CHRNA1, CHRNB, CHRNAD1, and CHRNE). In 10-14% of the probands the disease is caused by mutations in RAPSN, DOK 7, or COLQ, and in 5% by mutations in CHAT. Other less frequently identified disease genes include LAMB2, AGRN, LRP4, MUSK, GFPT1, DPAGT1, ALG2, and ALG 14 as well as SCN4A, PREPL, PLEC1, DNM2, and MTM1. Identification of the genetic basis of each CMS is important not only for genetic counseling and disease prevention but also for therapy, because therapeutic agents that benefit one type of CMS can be harmful in another.
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Affiliation(s)
- Andrew G Engel
- Department of Neurology, Mayo Clinic College of Medicine, Rochester, MN, United States.
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HuR Mediates Changes in the Stability of AChR β-Subunit mRNAs after Skeletal Muscle Denervation. J Neurosci 2015; 35:10949-62. [PMID: 26245959 DOI: 10.1523/jneurosci.1043-15.2015] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
UNLABELLED Acetylcholine receptors (AChRs) are heteromeric membrane proteins essential for neurotransmission at the neuromuscular junction. Previous work showed that muscle denervation increases expression of AChR mRNAs due to transcriptional activation of AChR subunit genes. However, it remains possible that post-transcriptional mechanisms are also involved in controlling the levels of AChR mRNAs following denervation. We examined whether post-transcriptional events indeed regulate AChR β-subunit mRNAs in response to denervation. First, in vitro stability assays revealed that the half-life of AChR β-subunit mRNAs was increased in the presence of denervated muscle protein extracts. A bioinformatics analysis revealed the existence of a conserved AU-rich element (ARE) in the 3'-untranslated region (UTR) of AChR β-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR β-subunit 3'UTR, caused an increase in luciferase activity. By contrast, mutation of this ARE prevented this increase. We also observed that denervation increased expression of the RNA-binding protein human antigen R (HuR) and induced its translocation to the cytoplasm. Importantly, HuR binds to endogenous AChR β-subunit transcripts in cultured myotubes and in vivo, and this binding is increased in denervated versus innervated muscles. Finally, p38 MAPK, a pathway known to activate HuR, was induced following denervation as a result of MKK3/6 activation and a decrease in MKP-1 expression, thereby leading to an increase in the stability of AChR β-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR β-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression. SIGNIFICANCE STATEMENT Muscle denervation is a convenient model to examine expression of genes encoding proteins of the neuromuscular junction, especially acetylcholine receptors (AChRs). Despite the accepted model of AChR regulation, which implicates transcriptional mechanisms, it remains plausible that such events cannot fully account for changes in AChR expression following denervation. We show that denervation increases expression of the RNA-binding protein HuR, which in turn, causes an increase in the stability of AChR β-subunit mRNAs in denervated muscle. Our findings demonstrate for the first time the contribution of post-transcriptional events in controlling AChR expression in skeletal muscle, and points toward a central role for HuR in mediating synaptic development while also paving the way for developing RNA-based therapeutics for neuromuscular diseases.
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Tintignac LA, Brenner HR, Rüegg MA. Mechanisms Regulating Neuromuscular Junction Development and Function and Causes of Muscle Wasting. Physiol Rev 2015; 95:809-52. [DOI: 10.1152/physrev.00033.2014] [Citation(s) in RCA: 298] [Impact Index Per Article: 29.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
The neuromuscular junction is the chemical synapse between motor neurons and skeletal muscle fibers. It is designed to reliably convert the action potential from the presynaptic motor neuron into the contraction of the postsynaptic muscle fiber. Diseases that affect the neuromuscular junction may cause failure of this conversion and result in loss of ambulation and respiration. The loss of motor input also causes muscle wasting as muscle mass is constantly adapted to contractile needs by the balancing of protein synthesis and protein degradation. Finally, neuromuscular activity and muscle mass have a major impact on metabolic properties of the organisms. This review discusses the mechanisms involved in the development and maintenance of the neuromuscular junction, the consequences of and the mechanisms involved in its dysfunction, and its role in maintaining muscle mass during aging. As life expectancy is increasing, loss of muscle mass during aging, called sarcopenia, has emerged as a field of high medical need. Interestingly, aging is also accompanied by structural changes at the neuromuscular junction, suggesting that the mechanisms involved in neuromuscular junction maintenance might be disturbed during aging. In addition, there is now evidence that behavioral paradigms and signaling pathways that are involved in longevity also affect neuromuscular junction stability and sarcopenia.
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Affiliation(s)
- Lionel A. Tintignac
- Biozentrum, University of Basel, Basel, Switzerland; Department of Biomedicine, University of Basel, Basel, Switzerland; and INRA, UMR866 Dynamique Musculaire et Métabolisme, Montpellier, France
| | - Hans-Rudolf Brenner
- Biozentrum, University of Basel, Basel, Switzerland; Department of Biomedicine, University of Basel, Basel, Switzerland; and INRA, UMR866 Dynamique Musculaire et Métabolisme, Montpellier, France
| | - Markus A. Rüegg
- Biozentrum, University of Basel, Basel, Switzerland; Department of Biomedicine, University of Basel, Basel, Switzerland; and INRA, UMR866 Dynamique Musculaire et Métabolisme, Montpellier, France
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Ratti F, Ramond F, Moncollin V, Simonet T, Milan G, Méjat A, Thomas JL, Streichenberger N, Gilquin B, Matthias P, Khochbin S, Sandri M, Schaeffer L. Histone deacetylase 6 is a FoxO transcription factor-dependent effector in skeletal muscle atrophy. J Biol Chem 2014; 290:4215-24. [PMID: 25516595 DOI: 10.1074/jbc.m114.600916] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Skeletal muscle atrophy is a severe condition of muscle mass loss. Muscle atrophy is caused by a down-regulation of protein synthesis and by an increase of protein breakdown due to the ubiquitin-proteasome system and autophagy activation. Up-regulation of specific genes, such as the muscle-specific E3 ubiquitin ligase MAFbx, by FoxO transcription factors is essential to initiate muscle protein ubiquitination and degradation during atrophy. HDAC6 is a particular HDAC, which is functionally related to the ubiquitin proteasome system via its ubiquitin binding domain. We show that HDAC6 is up-regulated during muscle atrophy. HDAC6 activation is dependent on the transcription factor FoxO3a, and the inactivation of HDAC6 in mice protects against muscle wasting. HDAC6 is able to interact with MAFbx, a key ubiquitin ligase involved in muscle atrophy. Our findings demonstrate the implication of HDAC6 in skeletal muscle wasting and identify HDAC6 as a new downstream target of FoxO3a in stress response. This work provides new insights in skeletal muscle atrophy development and opens interesting perspectives on HDAC6 as a valuable marker of muscle atrophy and a potential target for pharmacological treatments.
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Affiliation(s)
- Francesca Ratti
- From the Ecole Normale Supérieure de Lyon; CNRS UMR 5239; Equipe Différenciation Neuromusculaire, Université de Lyon, 46 allée d'Italie 69364 Lyon cedex 07, France, Université Lyon 1; Hospices civils de Lyon
| | - Francis Ramond
- From the Ecole Normale Supérieure de Lyon; CNRS UMR 5239; Equipe Différenciation Neuromusculaire, Université de Lyon, 46 allée d'Italie 69364 Lyon cedex 07, France, Université Lyon 1; Hospices civils de Lyon
| | - Vincent Moncollin
- From the Ecole Normale Supérieure de Lyon; CNRS UMR 5239; Equipe Différenciation Neuromusculaire, Université de Lyon, 46 allée d'Italie 69364 Lyon cedex 07, France, Université Lyon 1; Hospices civils de Lyon
| | - Thomas Simonet
- From the Ecole Normale Supérieure de Lyon; CNRS UMR 5239; Equipe Différenciation Neuromusculaire, Université de Lyon, 46 allée d'Italie 69364 Lyon cedex 07, France, Université Lyon 1; Hospices civils de Lyon
| | - Giulia Milan
- Department of Biomedical Sciences, University of Padova, 35131 Padova, Italy, and Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, 35129 Padova, Italy
| | - Alexandre Méjat
- From the Ecole Normale Supérieure de Lyon; CNRS UMR 5239; Equipe Différenciation Neuromusculaire, Université de Lyon, 46 allée d'Italie 69364 Lyon cedex 07, France, Université Lyon 1; Hospices civils de Lyon
| | - Jean-Luc Thomas
- From the Ecole Normale Supérieure de Lyon; CNRS UMR 5239; Equipe Différenciation Neuromusculaire, Université de Lyon, 46 allée d'Italie 69364 Lyon cedex 07, France, Université Lyon 1; Hospices civils de Lyon
| | | | - Benoit Gilquin
- INSERM U309, Institut Albert Bonniot, 38706 La Tronche Cedex, France
| | - Patrick Matthias
- Friedrich Miescher Institute, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Saadi Khochbin
- INSERM U309, Institut Albert Bonniot, 38706 La Tronche Cedex, France
| | - Marco Sandri
- Department of Biomedical Sciences, University of Padova, 35131 Padova, Italy, and Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, 35129 Padova, Italy
| | - Laurent Schaeffer
- From the Ecole Normale Supérieure de Lyon; CNRS UMR 5239; Equipe Différenciation Neuromusculaire, Université de Lyon, 46 allée d'Italie 69364 Lyon cedex 07, France, Université Lyon 1; Hospices civils de Lyon,
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Changeux JP. The concept of allosteric interaction and its consequences for the chemistry of the brain. J Biol Chem 2013; 288:26969-26986. [PMID: 23878193 DOI: 10.1074/jbc.x113.503375] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Throughout this Reflections article, I have tried to follow up on the genesis in the 1960s and subsequent evolution of the concept of allosteric interaction and to examine its consequences within the past decades, essentially in the field of the neuroscience. The main conclusion is that allosteric mechanisms built on similar structural principles operate in bacterial regulatory enzymes, gene repressors (and the related nuclear receptors), rhodopsin, G-protein-coupled receptors, neurotransmitter receptors, ion channels, and so on from prokaryotes up to the human brain yet with important features of their own. Thus, future research on these basic cybernetic sensors is expected to develop in two major directions: at the elementary level, toward the atomic structure and molecular dynamics of the conformational changes involved in signal recognition and transduction, but also at a higher level of organization, the contribution of allosteric mechanisms to the modulation of brain functions.
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Affiliation(s)
- Jean-Pierre Changeux
- Collège de France, 75005 Paris and the Institut Pasteur, 75724 Paris Cedex 15, France.
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Abstract
Muscle-specific kinase (MuSK) is essential for each step in neuromuscular synapse formation. Before innervation, MuSK initiates postsynaptic differentiation, priming the muscle for synapse formation. Approaching motor axons recognize the primed, or prepatterned, region of muscle, causing motor axons to stop growing and differentiate into specialized nerve terminals. MuSK controls presynaptic differentiation by causing the clustering of Lrp4, which functions as a direct retrograde signal for presynaptic differentiation. Developing synapses are stabilized by neuronal Agrin, which is released by motor nerve terminals and binds to Lrp4, a member of the low-density lipoprotein receptor family, stimulating further association between Lrp4 and MuSK and increasing MuSK kinase activity. In addition, MuSK phosphorylation is stimulated by an inside-out ligand, docking protein-7 (Dok-7), which is recruited to tyrosine-phosphorylated MuSK and increases MuSK kinase activity. Mutations in MuSK and in genes that function in the MuSK signaling pathway, including Dok-7, cause congenital myasthenia, and autoantibodies to MuSK, Lrp4, and acetylcholine receptors are responsible for myasthenia gravis.
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Yu S, Jing X, Colgan JD, Zhao DM, Xue HH. Targeting tetramer-forming GABPβ isoforms impairs self-renewal of hematopoietic and leukemic stem cells. Cell Stem Cell 2013; 11:207-19. [PMID: 22862946 DOI: 10.1016/j.stem.2012.05.021] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2011] [Revised: 02/26/2012] [Accepted: 05/03/2012] [Indexed: 11/27/2022]
Abstract
Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) are both capable of self-renewal, with HSCs sustaining multiple blood lineage differentiation and LSCs indefinitely propagating leukemia. The GABP complex, consisting of DNA binding GABPα subunit and transactivation GABPβ subunit, critically regulates HSC multipotency and self-renewal via controlling an essential gene regulatory module. Two GABPβ isoforms, GABPβ1L and GABPβ2, contribute to assembly of GABPα(2)β(2) tetramer. We demonstrate that GABPβ1L/β2 deficiency specifically impairs HSC quiescence and survival, with little impact on cell cycle or apoptosis in differentiated blood cells. The HSC-specific effect is mechanistically ascribed to perturbed integrity of the GABP-controlled gene regulatory module in HSCs. Targeting GABPβ1L/β2 also impairs LSC self-renewal in p210(BCR-ABL)-induced chronic myelogenous leukemia (CML) and exhibits synergistic effects with tyrosine kinase inhibitor imatinib therapy in inhibiting CML propagation. These findings identify the tetramer-forming GABPβ isoforms as specific HSC regulators and potential therapeutic targets in treating LSC-based hematological malignancy.
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Affiliation(s)
- Shuyang Yu
- Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
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Odrowaz Z, Sharrocks AD. The ETS transcription factors ELK1 and GABPA regulate different gene networks to control MCF10A breast epithelial cell migration. PLoS One 2012; 7:e49892. [PMID: 23284628 PMCID: PMC3527487 DOI: 10.1371/journal.pone.0049892] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2012] [Accepted: 10/17/2012] [Indexed: 12/29/2022] Open
Abstract
Members of the ETS transcription factor family often target the same binding regions and hence have the potential to regulate the same genes and downstream biological processes. However, individual family members also preferentially bind to other genomic regions, thus providing the potential for controlling distinct transcriptional programmes and generating specific biological effects. The ETS transcription factor ELK1 controls cell migration in breast epithelial cells through targeting a cohort of genes, independently from another family member GABPA, and therefore achieves biological specificity. Here, we demonstrate that GABPA also controls cell migration in breast epithelial cells. However, GABPA controls the expression of a different network of target genes to ELK1. Both direct and indirect target genes for GABPA are identified and amongst the direct targets we confirm the importance of RAC1 and KIF20A for cell migration. Therefore, although ELK1 and GABPA ultimately control the same biological process, they do so by regulating different cohorts of target genes associated with cytoskeletal functions and cell migration control.
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Affiliation(s)
- Zaneta Odrowaz
- Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
| | - Andrew D. Sharrocks
- Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
- * E-mail:
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20
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Abstract
Skeletal muscle innervation is a multi-step process leading to the neuromuscular junction (NMJ) apparatus formation. The transmission of the signal from nerve to muscle occurs at the NMJ level. The molecular mechanism that orchestrates the organization and functioning of synapses is highly complex, and it has not been completely elucidated so far. Neuromuscular junctions are assembled on the muscle fibers at very precise locations called end plates (EP). Acetylcholine receptor (AChR) clusterization at the end plates is required for an accurate synaptic transmission. This review will focus on some mechanisms responsible for accomplishing the correct distribution of AChRs at the synapses. Recent evidences support the concept that a dual transcriptional control of AChR genes in subsynaptic and extrasynaptic nuclei is crucial for AChR clusterization. Moreover, new players have been discovered in the agrin-MuSK pathway, the master organizer of postsynaptical differentiation. Mutations in this pathway cause neuromuscular congenital disorders. Alterations of the postynaptic apparatus are also present in physiological conditions characterized by skeletal muscle wasting. Indeed, recent evidences demonstrate how NMJ misfunctioning has a crucial role at the onset of age-associated sarcopenia.
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21
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Herndon CA, Snell J, Fromm L. Chromatin modifications that support acetylcholine receptor gene activation are established during muscle cell determination and differentiation. Mol Biol Rep 2011; 38:1277-85. [PMID: 20574709 PMCID: PMC3008550 DOI: 10.1007/s11033-010-0227-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2010] [Accepted: 06/11/2010] [Indexed: 10/19/2022]
Abstract
Localization of acetylcholine receptors (AChRs) to the postsynaptic region of muscle is mediated in part by transcriptional mechanisms. An important way of regulating transcription is through targeting histone modifications on chromatin to distinct gene loci. Using chromatin immunoprecipitation, we examined the developmental regulation of certain histone modifications at the AChR epsilon subunit locus, including methylations at lysine residues K4 and K27 and acetylations at K9 and K14. We modeled various stages of muscle development in cell culture, including pre-determined cells, committed but undifferentiated myoblasts, and differentiated myotubes, and modeled synaptic myotube nuclei by stimulating myotubes with neuregulin (NRG) 1. We found that a pattern of histone modifications associated with transcriptional activation is targeted to the AChR epsilon subunit locus in myotubes prior to stimulation with NRG1 and does not change upon addition of NRG1. Instead, we found that during muscle cell determination and differentiation, specific histone modifications are targeted to the AChR epsilon subunit locus. Within the gene, at K4, dimethylation is induced during muscle cell determination, while trimethylation is induced during differentiation. At K27, loss of trimethylation and appearance of monomethylation occurs during determination and differentiation. In addition, in a region upstream of the gene, K4 di- and trimethylation, and K9/14 acetylation are induced in a distinct developmental pattern, which may reflect a functional regulatory element. These results suggest synaptic signaling does not directly target histone modifications but rather the histone modification pattern necessary for transcriptional activation is previously established in a series of steps during muscle development.
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Affiliation(s)
- Carter A. Herndon
- Indiana University School of Medicine-Muncie and Ball State University, 2000 University Avenue, Muncie, IN 47306, USA
| | - Jeff Snell
- Indiana University School of Medicine-Muncie and Ball State University, 2000 University Avenue, Muncie, IN 47306, USA
| | - Larry Fromm
- Indiana University School of Medicine-Muncie and Ball State University, 2000 University Avenue, Muncie, IN 47306, USA,
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22
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23
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Scarpulla RC. Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev 2008; 88:611-38. [PMID: 18391175 DOI: 10.1152/physrev.00025.2007] [Citation(s) in RCA: 1226] [Impact Index Per Article: 72.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Mitochondria contain their own genetic system and undergo a unique mode of cytoplasmic inheritance. Each organelle has multiple copies of a covalently closed circular DNA genome (mtDNA). The entire protein coding capacity of mtDNA is devoted to the synthesis of 13 essential subunits of the inner membrane complexes of the respiratory apparatus. Thus the majority of respiratory proteins and all of the other gene products necessary for the myriad mitochondrial functions are derived from nuclear genes. Transcription of mtDNA requires a small number of nucleus-encoded proteins including a single RNA polymerase (POLRMT), auxiliary factors necessary for promoter recognition (TFB1M, TFB2M) and activation (Tfam), and a termination factor (mTERF). This relatively simple system can account for the bidirectional transcription of mtDNA from divergent promoters and key termination events controlling the rRNA/mRNA ratio. Nucleomitochondrial interactions depend on the interplay between transcription factors (NRF-1, NRF-2, PPARalpha, ERRalpha, Sp1, and others) and members of the PGC-1 family of regulated coactivators (PGC-1alpha, PGC-1beta, and PRC). The transcription factors target genes that specify the respiratory chain, the mitochondrial transcription, translation and replication machinery, and protein import and assembly apparatus among others. These factors are in turn activated directly or indirectly by PGC-1 family coactivators whose differential expression is controlled by an array of environmental signals including temperature, energy deprivation, and availability of nutrients and growth factors. These transcriptional paradigms provide a basic framework for understanding the integration of mitochondrial biogenesis and function with signaling events that dictate cell- and tissue-specific energetic properties.
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Affiliation(s)
- Richard C Scarpulla
- Department of Cell and Molecular Biology, Northwestern Medical School, Chicago, Illinois 60611, USA
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Herndon CA, Fromm L. Neuregulin-1 induces acetylcholine receptor transcription in the absence of GABPα phosphorylation. J Neurosci Res 2008; 86:982-91. [DOI: 10.1002/jnr.21563] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Jin TE, Wernig A, Witzemann V. Changes in acetylcholine receptor function induce shifts in muscle fiber type composition. FEBS J 2008; 275:2042-54. [DOI: 10.1111/j.1742-4658.2008.06359.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Affiliation(s)
- Andrew G Engel
- Department of Neurology, Mayo Clinic, Rochester, MN, USA.
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Hippenmeyer S, Huber RM, Ladle DR, Murphy K, Arber S. ETS Transcription Factor Erm Controls Subsynaptic Gene Expression in Skeletal Muscles. Neuron 2007; 55:726-40. [PMID: 17785180 DOI: 10.1016/j.neuron.2007.07.028] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2007] [Revised: 06/22/2007] [Accepted: 07/24/2007] [Indexed: 11/23/2022]
Abstract
Accumulation of specific proteins at synaptic structures is essential for synapse assembly and function, but mechanisms regulating local protein enrichment remain poorly understood. At the neuromuscular junction (NMJ), subsynaptic nuclei underlie motor axon terminals within extrafusal muscle fibers and are transcriptionally distinct from neighboring nuclei. In this study, we show that expression of the ETS transcription factor Erm is highly concentrated at subsynaptic nuclei, and its mutation in mice leads to severe downregulation of many genes with normally enriched subsynaptic expression. Erm mutant mice display an expansion of the muscle central domain in which acetylcholine receptor (AChR) clusters accumulate, show gradual fragmentation of AChR clusters, and exhibit symptoms of muscle weakness mimicking congenital myasthenic syndrome (CMS). Together, our findings define Erm as an upstream regulator of a transcriptional program selective to subsynaptic nuclei at the NMJ and underscore the importance of transcriptional control of local synaptic protein accumulation.
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Affiliation(s)
- Simon Hippenmeyer
- Biozentrum, Department of Cell Biology, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland
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Jaworski A, Smith CL, Burden SJ. GA-binding protein is dispensable for neuromuscular synapse formation and synapse-specific gene expression. Mol Cell Biol 2007; 27:5040-6. [PMID: 17485447 PMCID: PMC1951497 DOI: 10.1128/mcb.02228-06] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2006] [Revised: 01/17/2007] [Accepted: 04/24/2007] [Indexed: 01/04/2023] Open
Abstract
The mRNAs encoding postsynaptic components at the neuromuscular junction are concentrated in the synaptic region of muscle fibers. Accumulation of these RNAs in the synaptic region is mediated, at least in part, by selective transcription of the corresponding genes in synaptic myofiber nuclei. The transcriptional mechanisms that are responsible for synapse-specific gene expression are largely unknown, but an Ets site in the promoter regions of acetylcholine receptor (AChR) subunit genes and other "synaptic" genes is required for synapse-specific transcription. The Ets domain transcription factor GA-binding protein (GABP) has been implicated to mediate synapse-specific gene expression. Inactivation of GABPalpha, the DNA-binding subunit of GABP, leads to early embryonic lethality, preventing analysis of synapse formation in gabpalpha mutant mice. To study the role of GABP at neuromuscular synapses, we conditionally inactivated gabpalpha in skeletal muscle and studied synaptic differentiation and muscle gene expression. Although expression of rb, a target of GABP, is elevated in muscle tissue deficient in GABPalpha, clustering of synaptic AChRs at synapses and synapse-specific gene expression are normal in these mice. These data indicate that GABP is dispensable for synapse-specific transcription and maintenance of normal AChR expression at synapses.
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Affiliation(s)
- Alexander Jaworski
- The Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomoledular Medicine, NYU School of Medicine, New York, NY 10016, USA
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Perkins KJ, Basu U, Budak MT, Ketterer C, Baby SM, Lozynska O, Lunde JA, Jasmin BJ, Rubinstein NA, Khurana TS. Ets-2 repressor factor silences extrasynaptic utrophin by N-box mediated repression in skeletal muscle. Mol Biol Cell 2007; 18:2864-72. [PMID: 17507653 PMCID: PMC1949368 DOI: 10.1091/mbc.e06-12-1069] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPalpha/beta to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest "repressing repressors" as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.
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Affiliation(s)
- Kelly J Perkins
- Department of Physiology and Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085, USA
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O'Leary DA, Noakes PG, Lavidis NA, Kola I, Hertzog PJ, Ristevski S. Targeting of the ETS factor GABPalpha disrupts neuromuscular junction synaptic function. Mol Cell Biol 2007; 27:3470-80. [PMID: 17325042 PMCID: PMC1899955 DOI: 10.1128/mcb.00659-06] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
The GA-binding protein (GABP) transcription factor has been shown in vitro to regulate the expression of the neuromuscular proteins utrophin, acetylcholine esterase, and acetylcholine receptor subunits delta and epsilon through the N-box promoter motif (5'-CCGGAA-3'), but its in vivo function remains unknown. A single point mutation within the N-box of the gene encoding the acetylcholine receptor epsilon subunit has been identified in several patients suffering from postsynaptic congenital myasthenic syndrome, implicating the GA-binding protein in neuromuscular function and disease. Since conventional gene targeting results in an embryonic-lethal phenotype, we used conditional targeting to investigate the role of GABPalpha in neuromuscular junction and skeletal muscle development. The diaphragm and soleus muscles from mutant mice display alterations in morphology and distribution of acetylcholine receptor clusters at the neuromuscular junction and neurotransmission properties consistent with reduced receptor function. Furthermore, we confirmed decreased expression of the acetylcholine receptor epsilon subunit and increased expression of the gamma subunit in skeletal muscle tissues. Therefore, the GABP transcription factor aids in the structural formation and function of neuromuscular junctions by regulating the expression of postsynaptic genes.
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Affiliation(s)
- Debra A O'Leary
- Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia
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33
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Ravel-Chapuis A, Vandromme M, Thomas JL, Schaeffer L. Postsynaptic chromatin is under neural control at the neuromuscular junction. EMBO J 2007; 26:1117-28. [PMID: 17304221 PMCID: PMC1852850 DOI: 10.1038/sj.emboj.7601572] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2006] [Accepted: 01/04/2007] [Indexed: 12/25/2022] Open
Abstract
In adult skeletal muscle, the nicotinic acetylcholine receptor (AChR) specifically accumulates at the neuromuscular junction, to allow neurotransmission. This clustering is paralleled by a compartmentalization of AChR genes expression to subsynaptic nuclei, which acquire a unique gene expression program and a specific morphology in response to neural cues. Our results demonstrate that neural agrin-dependent reprogramming of myonuclei involves chromatin remodelling, histone hyperacetylation and histone hyperphosphorylation. Activation of AChR genes in subsynaptic nuclei is mediated by the transcription factor GABP. Here we demonstrate that upon activation, GABP recruits the histone acetyl transferase (HAT) p300 on the AChR epsilon subunit promoter, whereas it rather recruits the histone deacetylase HDAC1 when the promoter is not activated. Moreover, the HAT activity of p300 is required in vivo for AChR expression. GABP therefore couples chromatin hyperacetylation and AChR activation by neural factors in subsynaptic nuclei.
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Affiliation(s)
- Aymeric Ravel-Chapuis
- Equipe Différenciation Neuromusculaire; IFR128; UMR5161; ENS Lyon; CNRS; INRA; Université de Lyon; Lyon Cedex, France
| | - Marie Vandromme
- Equipe Différenciation Neuromusculaire; IFR128; UMR5161; ENS Lyon; CNRS; INRA; Université de Lyon; Lyon Cedex, France
| | - Jean-Luc Thomas
- Equipe Différenciation Neuromusculaire; IFR128; UMR5161; ENS Lyon; CNRS; INRA; Université de Lyon; Lyon Cedex, France
| | - Laurent Schaeffer
- Equipe Différenciation Neuromusculaire; IFR128; UMR5161; ENS Lyon; CNRS; INRA; Université de Lyon; Lyon Cedex, France
- Equipe Différenciation Neuromusculaire; IFR128; UMR5161; ENS Lyon; CNRS; INRA; Université de Lyon; 46 allée d'Italie, 69364 Lyon Cedex 07, France. Tel.: +33 4 72 72 85 73; Fax: +33 4 72 72 80 80; E-mail:
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34
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Herndon CA, Fromm L. Directing RNA interference specifically to differentiated muscle cells. J Muscle Res Cell Motil 2006; 28:11-7. [PMID: 17187237 DOI: 10.1007/s10974-006-9098-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2006] [Accepted: 09/19/2006] [Indexed: 10/23/2022]
Abstract
A common approach for mediating RNA interference (RNAi) is to introduce DNA that encodes short hairpin RNA (shRNA), which is often contained in a plasmid that can express a shRNA in a wide variety of cell types. Muscle cells and certain other cell types grown in culture can exist in both a dividing state and in a post-mitotic, differentiated state, and it is sometimes useful to induce RNAi selectively in terminally differentiated cells to study the function of a gene, particularly when the gene is also required for propagation of dividing cells. We describe two methods for studying gene function by RNAi specifically in terminally differentiated skeletal muscle cells in culture. We developed a shRNA expression vector, based on myosin light chain 1f gene regulatory sequences, which is designed to induce shRNA expression specifically after differentiation has been initiated. We show that this vector can mediate RNAi and is only active in differentiated muscle cells. Also, we developed an adenoviral vector that is designed to be able to deliver shRNAs directly to post-mitotic muscle cells. We show that adenoviruses produced using this vector mediate RNAi in differentiated muscle cells. These methods add to the repertoire of RNAi tools that can be used for identifying genes involved in any event of interest that occurs in differentiated muscle cells.
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Affiliation(s)
- Carter A Herndon
- Indiana University School of Medicine-Muncie, 2000 University Avenue, Muncie, IN 47306, USA
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35
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Abstract
Members of the receptor tyrosine kinase family, that include EGFR, ErbB-2/HER-2, ErbB-3/HER-3 and ErbB-4/HER-4, are frequently implicated in experimental models of epithelial cell neoplasia as well as in human cancers. Therefore, interference with the activation of these growth factor receptors represents a promising strategy for development of novel and selective anticancer therapies. Indeed, a number of inhibitors that target either EGFR or HER-2, with the exception of a few that target both; have been developed for treatment of epithelial cancers. Since most solid tumors express different ErbB receptors and/or their ligands, identification of inhibitor(s), targeting multiple EGFR family members may provide a therapeutic benefit to a broader patient population. Here we describe the significance of an ErbB family of receptors in epithelial cancers, and summarize different available therapeutics targeting these receptors. It also emphasizes the need to develop pan-ErbB inhibitors and discusses EGF-Receptor Related Protein, a recently isolated negative regulator of EGFR as a potential pan-ErbB therapeutic for a wide variety of epithelial cancers.
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Affiliation(s)
- Jyoti Nautiyal
- Karmanos Cancer Institute, Detroit, MI 48201, United States
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36
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Cohen TV, Randall WR. The regulation of acetylcholinesterase by cis-elements within intron I in cultured contracting myotubes. J Neurochem 2006; 98:723-34. [PMID: 16787423 DOI: 10.1111/j.1471-4159.2006.03897.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
The onset of spontaneous contraction in rat primary muscle cultures coincides with an increase in acetylcholinesterase (AChE) activity. In order to establish whether contractile activity modulates the rate of AChE transcript synthesis, and what elements of the gene are determinant, we examined the promoter and intron I in contracting muscle cultures. Ache genomic fragments attached to a luciferase reporter were transfected into muscle cultures that were either electrically stimulated or paralyzed with tetrodotoxin to enhance or inhibit contractions, respectively. Cultures transfected with intron I-containing constructs showed a 2-fold increase in luciferase activity following electrical stimulation, compared to tetrodotoxin treatment, suggesting that this region contains elements responding to contractile activity. Deleting a 780 bp distal region within intron I, containing an N-box element at +890 bp, or introducing a 2-bp mutation within its core sequence, eliminated the contraction-induced response. In contrast, mutating an N-box element at +822 bp had no effect on the response. Furthermore, co-transfecting a dominant negative GA-binding protein (GABP), a transcription factor known to selectively bind N-box elements, reduced the stimulation-mediated increase. Our results suggest that the N-box within intron I at +890 bp is a regulatory element important in the transcriptional response of Ache to contractile activity in muscle.
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Affiliation(s)
- Tatiana V Cohen
- Department of Pharmacology and Experimental Therapeutics, School of Medicine University of Maryland, Baltimore, MD 21201-1559, USA
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37
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Jaworski A, Burden SJ. Neuromuscular synapse formation in mice lacking motor neuron- and skeletal muscle-derived Neuregulin-1. J Neurosci 2006; 26:655-61. [PMID: 16407563 PMCID: PMC6674415 DOI: 10.1523/jneurosci.4506-05.2006] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2005] [Revised: 11/21/2005] [Accepted: 11/23/2005] [Indexed: 11/21/2022] Open
Abstract
The localization of acetylcholine receptors (AChRs) to the vertebrate neuromuscular junction is mediated, in part, through selective transcription of AChR subunit genes in myofiber subsynaptic nuclei. Agrin and the muscle-specific receptor tyrosine kinase, MuSK, have critical roles in synapse-specific transcription, because AChR genes are expressed uniformly in mice lacking either agrin or MuSK. Several lines of evidence suggest that agrin and MuSK stimulate synapse-specific transcription indirectly by regulating the distribution of other cell surface ligands, which stimulate a pathway for synapse-specific gene expression. This putative secondary signal for directing AChR gene expression to synapses is not known, but Neuregulin-1 (Nrg-1), primarily based on its presence at synapses and its ability to induce AChR gene expression in vitro, has been considered a good candidate. To study the role of Nrg-1 at neuromuscular synapses, we inactivated nrg-1 in motor neurons, skeletal muscle, or both cell types, using mice that express Cre recombinase selectively in developing motor neurons or in developing skeletal myofibers. We find that AChRs are clustered at synapses and that synapse-specific transcription is normal in mice lacking Nrg-1 in motor neurons, myofibers, or both cell types. These data indicate that Nrg-1 is dispensable for clustering AChRs and activating AChR genes in subsynaptic nuclei during development and suggest that these aspects of postsynaptic differentiation are dependent on Agrin/MuSK signaling without a requirement for a secondary signal.
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MESH Headings
- Agrin/physiology
- Animals
- Cell Differentiation
- Diaphragm/embryology
- Diaphragm/innervation
- ErbB Receptors/metabolism
- Genes, Reporter
- Integrases/genetics
- Integrases/metabolism
- Intercostal Muscles/embryology
- Intercostal Muscles/innervation
- Mice
- Mice, Knockout
- Motor Neurons/metabolism
- Motor Neurons/ultrastructure
- Muscle Fibers, Skeletal/metabolism
- Muscle Fibers, Skeletal/ultrastructure
- Muscle, Skeletal/embryology
- Muscle, Skeletal/innervation
- Nerve Tissue Proteins/deficiency
- Nerve Tissue Proteins/genetics
- Nerve Tissue Proteins/physiology
- Neuregulin-1
- Neuromuscular Junction/embryology
- Neuromuscular Junction/physiology
- Neuromuscular Junction/ultrastructure
- RNA, Messenger/biosynthesis
- RNA, Messenger/genetics
- Receptor Protein-Tyrosine Kinases/biosynthesis
- Receptor Protein-Tyrosine Kinases/genetics
- Receptor Protein-Tyrosine Kinases/physiology
- Receptor, ErbB-2/metabolism
- Receptor, ErbB-4
- Receptors, Cholinergic/biosynthesis
- Receptors, Cholinergic/genetics
- Receptors, Cholinergic/physiology
- Reverse Transcriptase Polymerase Chain Reaction
- Sequence Deletion
- Viral Proteins/genetics
- Viral Proteins/metabolism
- beta-Galactosidase/analysis
- beta-Galactosidase/genetics
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Affiliation(s)
- Alexander Jaworski
- Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York 10016, USA
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38
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Abstract
The neuromuscular junction (NMJ) is a complex structure that serves to efficiently communicate the electrical impulse from the motor neuron to the skeletal muscle to signal contraction. Over the last 200 years, technological advances in microscopy allowed visualization of the existence of a gap between the motor neuron and skeletal muscle that necessitated the existence of a messenger, which proved to be acetylcholine. Ultrastructural analysis identified vesicles in the presynaptic nerve terminal, which provided a beautiful structural correlate for the quantal nature of neuromuscular transmission, and the imaging of synaptic folds on the muscle surface demonstrated that specializations of the underlying protein scaffold were required. Molecular analysis in the last 20 years has confirmed the preferential expression of synaptic proteins, which is guided by a precise developmental program and maintained by signals from nerve. Although often overlooked, the Schwann cell that caps the NMJ and the basal lamina is proving to be critical in maintenance of the junction. Genetic and autoimmune disorders are known that compromise neuromuscular transmission and provide further insights into the complexities of NMJ function as well as the subtle differences that exist among NMJ that may underlie the differential susceptibility of muscle groups to neuromuscular transmission diseases. In this review we summarize the synaptic physiology, architecture, and variations in synaptic structure among muscle types. The important roles of specific signaling pathways involved in NMJ development and acetylcholine receptor (AChR) clustering are reviewed. Finally, genetic and autoimmune disorders and their effects on NMJ architecture and neuromuscular transmission are examined.
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Affiliation(s)
- Benjamin W Hughes
- Department of Neurology, Case Western University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA
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39
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Costa E, Dong E, Grayson DR, Ruzicka WB, Simonini MV, Veldic M, Guidotti A. Epigenetic Targets in GABAergic Neurons to Treat Schizophrenia. GABA 2006; 54:95-117. [PMID: 17175812 DOI: 10.1016/s1054-3589(06)54005-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Affiliation(s)
- E Costa
- Department of Psychiatry, Psychiatric Institute, University of Illinois at Chicago, Chicago, Illinois 60612, USA
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40
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Tang H, Veldman MB, Goldman D. Characterization of a muscle-specific enhancer in human MuSK promoter reveals the essential role of myogenin in controlling activity-dependent gene regulation. J Biol Chem 2005; 281:3943-53. [PMID: 16361705 DOI: 10.1074/jbc.m511317200] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Neuromuscular synaptogenesis is initiated by the release of agrin from motor neurons and the activation of the receptor tyrosine kinase, MuSK, in the postsynaptic membrane. MuSK gene expression is regulated by nerve-derived agrin and muscle activity. Agrin stimulates synapse-specific MuSK gene expression by activating GABP(alphabeta) transcription factors in endplate-associated myonuclei. In contrast, the mechanism by which muscle activity regulates MuSK gene expression is not known. We report on a 60-bp MuSK enhancer that confers promoter regulation by muscle differentiation, changes in intracellular calcium, and muscle activity. Within this enhancer, we identified a single E-box that is essential for this regulation. This E-box binds myogenin, and we showed that myogenin is necessary for not only MuSK but also nAChR gene regulation by muscle activity. Surprisingly, the same E-box functions in vivo to mediate muscle-specific and differentiation-dependent gene induction in zebrafish, suggesting an evolutionary conserved mechanism of regulation of synaptic protein gene expression.
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Affiliation(s)
- Huibin Tang
- Molecular and Behavior Neuroscience Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, 48109, USA
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41
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Ongwijitwat S, Wong-Riley MTT. Is nuclear respiratory factor 2 a master transcriptional coordinator for all ten nuclear-encoded cytochrome c oxidase subunits in neurons? Gene 2005; 360:65-77. [PMID: 16126350 DOI: 10.1016/j.gene.2005.06.015] [Citation(s) in RCA: 82] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2005] [Revised: 05/22/2005] [Accepted: 06/03/2005] [Indexed: 11/23/2022]
Abstract
Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial electron transport chain, is a multi-subunit, bigenomically encoded inner mitochondrial membrane protein. Of the thirteen subunits, three are encoded in the mitochondrial genome and ten others are encoded in the nuclear genome. Transcriptional coordination of nuclear-encoded COX subunit genes is likely accomplished by transcription factors responding to upstream signals. Previous studies have found that nuclear-encoded COX subunit genes are under the control of specific transcription factors, such as nuclear respiratory factor 2 (NRF-2). However, it is not known if a single transcription factor binds to all ten of COX subunit promoters. In the current study, we identified in silico putative NRF-2 binding sites on all ten nuclear-encoded COX gene promoters in the rat genome. Chromatin immunoprecipitation assay showed that NRF-2 bound in vivo to six of the ten nuclear-encoded COX subunit promoters. Electrophoretic mobility supershift assays demonstrated binding of NRF-2 to the other four subunits, and promoter mutation study confirmed the functionality of these NRF-2 binding sites. Finally, transfection of dominant-negative constructs of NRF-2 proteins caused a significant reduction of COX expression. We conclude that NRF-2 is an important mediator of coordinated regulation of all ten nuclear-encoded COX subunit genes in neurons.
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Affiliation(s)
- Sakkapol Ongwijitwat
- Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
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42
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Shimokawa T, Ra C. C/EBPα functionally and physically interacts with GABP to activate the human myeloid IgA Fc receptor (FcαR, CD89) gene promoter. Blood 2005; 106:2534-42. [PMID: 15928042 DOI: 10.1182/blood-2004-06-2413] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractHuman Fcα receptor (FcαR; CD89), the receptor for the crystallizable fragment (Fc) of immunoglobulin A (IgA), is expressed exclusively in myeloid cells, including granulocytes and monocytes/macrophages, and is considered to define a crucial role of these cells in immune and inflammatory responses. A 259-base pair fragment of the FCAR promoter is sufficient to direct myeloid expression of a reporter gene and contains functionally important binding sites for CCAAT/enhancer-binding protein α (C/EBPα) (CE1, CE2, and CE3) and an unidentified Ets-like nuclear protein. Here, we show that the Ets-binding site is bound by a heterodimer composed of GA-binding protein α (GABPα), an Ets-related factor, and GABPβ, a Notch-related protein. Cotransfection of GABP increased FCAR promoter activity 3.7-fold through the Ets-binding site. GABP and C/EBPα synergistically activated the FCAR promoter 280-fold. Consistent with these observations, in vitro binding analyses revealed a physical interaction between the GABPα subunit and C/EBPα. This is the first report demonstrating both physical and functional interactions between GABP and C/EBPα and will provide new insights into the molecular basis of myeloid gene expression. (Blood. 2005;106:2534-2542)
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Affiliation(s)
- Toshibumi Shimokawa
- Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Sciences, Itabashi-ku, Tokyo, Japan
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43
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Angus LM, Chakkalakal JV, Méjat A, Eibl JK, Bélanger G, Megeney LA, Chin ER, Schaeffer L, Michel RN, Jasmin BJ. Calcineurin-NFAT signaling, together with GABP and peroxisome PGC-1α, drives utrophin gene expression at the neuromuscular junction. Am J Physiol Cell Physiol 2005; 289:C908-17. [PMID: 15930144 DOI: 10.1152/ajpcell.00196.2005] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1α alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1α with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1α.
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Affiliation(s)
- Lindsay M Angus
- Department of Cellular and Molecular Medicine, and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5
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44
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Tsuchimochi K, Yagishita N, Yamasaki S, Amano T, Kato Y, Kawahara KI, Aratani S, Fujita H, Ji F, Sugiura A, Izumi T, Sugamiya A, Maruyama I, Fukamizu A, Komiya S, Nishioka K, Nakajima T. Identification of a crucial site for synoviolin expression. Mol Cell Biol 2005; 25:7344-56. [PMID: 16055742 PMCID: PMC1190266 DOI: 10.1128/mcb.25.16.7344-7356.2005] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Synoviolin is an E3 ubiquitin ligase localized in the endoplasmic reticulum (ER) and serving as ER-associated degradation system. Analysis of transgenic mice suggested that synoviolin gene dosage is implicated in the pathogenesis of arthropathy. Complete deficiency of synoviolin is fatal embryonically. Thus, alternation of Synoviolin could cause breakdown of ER homeostasis and consequently lead to disturbance of cellular homeostasis. Hence, the expression level of Synoviolin appears to be important for its biological role in cellular homeostasis under physiological and pathological conditions. To examine the control of protein level, we performed promoter analysis to determine transcriptional regulation. Here we characterize the role of synoviolin transcription in cellular homeostasis. The Ets binding site (EBS), termed EBS-1, from position -76 to -69 of the proximal promoter, is responsible for synoviolin expression in vivo and in vitro. Interestingly, transfer of EBS-1 decoy into NIH 3T3 cells conferred not only the repression of synoviolin gene expression but also a decrease in cell number. Fluorescence-activated cell sorter analysis using annexin V staining confirmed the induction of apoptosis by EBS-1 decoy and demonstrated recovery of apoptosis by overexpression of Synoviolin. Our results suggest that transcriptional regulation of synoviolin via EBS-1 plays an important role in cellular homeostasis. Our study provides novel insight into the transcriptional regulation for cellular homeostasis.
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Affiliation(s)
- Kaneyuki Tsuchimochi
- Department of Genomic Science, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan
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45
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Kim CH, Xiong WC, Mei L. Inhibition of MuSK expression by CREB interacting with a CRE-like element and MyoD. Mol Cell Biol 2005; 25:5329-38. [PMID: 15964791 PMCID: PMC1156998 DOI: 10.1128/mcb.25.13.5329-5338.2005] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The type I receptor-like protein tyrosine kinase MuSK is essential for the neuromuscular junction formation. MuSK expression is tightly regulated during development, but the underlying mechanisms were unclear. Here we identified a novel mechanism by which MuSK expression may be regulated. A cyclic AMP response element (CRE)-like element in the 5'-flanking region of the MuSK gene binds to CREB1 (CRE-binding protein 1). Mutation of this element increases the MuSK promoter activity, suggesting a role for CREB1 in attenuation of MuSK expression. Interestingly, CREB mutants unable to bind to DNA also inhibit MuSK promoter activity, suggesting a CRE-independent inhibitory mechanism. In agreement, CREB1 could inhibit a mutant MuSK transgene reporter whose CRE site was mutated. We provide evidence that CREB interacts directly with MyoD, a myogenic factor essential for MuSK expression in muscle cells. Suppression of CREB expression by small interfering RNA increases MuSK promoter activity. These results demonstrate an important role for CREB1 in the regulation of MuSK expression.
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Affiliation(s)
- Chang-Hoon Kim
- Program of Developmental Neurobiology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, CB2803, 1120 15th Street, Augusta, Georgia 30912, USA
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46
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Escher P, Lacazette E, Courtet M, Blindenbacher A, Landmann L, Bezakova G, Lloyd KC, Mueller U, Brenner HR. Synapses form in skeletal muscles lacking neuregulin receptors. Science 2005; 308:1920-3. [PMID: 15976301 DOI: 10.1126/science.1108258] [Citation(s) in RCA: 99] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
The formation of the neuromuscular junction (NMJ) is directed by reciprocal interactions between motor neurons and muscle fibers. Neuregulin (NRG) and Agrin from motor nerve terminals are both implicated. Here, we demonstrate that NMJs can form in the absence of the NRG receptors ErbB2 and ErbB4 in mouse muscle. Postsynaptic differentiation is, however, induced by Agrin. We therefore conclude that NRG signaling to muscle is not required for NMJ formation. The effects of NRG signaling to muscle may be mediated indirectly through Schwann cells.
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MESH Headings
- Agrin/physiology
- Animals
- Animals, Newborn
- Cells, Cultured
- ErbB Receptors/genetics
- ErbB Receptors/physiology
- Genes, erbB
- Genes, erbB-2
- Membrane Potentials
- Mice
- Motor Endplate/metabolism
- Motor Endplate/physiology
- Motor Endplate/ultrastructure
- Muscle, Skeletal/innervation
- Muscle, Skeletal/ultrastructure
- Mutation
- Neuregulins/metabolism
- Neuromuscular Junction/embryology
- Neuromuscular Junction/metabolism
- Neuromuscular Junction/physiology
- Neuromuscular Junction/ultrastructure
- Presynaptic Terminals/physiology
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Receptor, ErbB-2/genetics
- Receptor, ErbB-2/physiology
- Receptor, ErbB-4
- Receptors, Cholinergic/chemistry
- Receptors, Cholinergic/genetics
- Receptors, Cholinergic/metabolism
- Recombination, Genetic
- Schwann Cells/physiology
- Signal Transduction
- Synaptic Transmission
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Affiliation(s)
- P Escher
- Institute of Physiology, Biozentrum, University of Basel, 4056 Basel, Switzerland
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47
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Kishi M, Kummer TT, Eglen SJ, Sanes JR. LL5beta: a regulator of postsynaptic differentiation identified in a screen for synaptically enriched transcripts at the neuromuscular junction. ACTA ACUST UNITED AC 2005; 169:355-66. [PMID: 15851520 PMCID: PMC2171857 DOI: 10.1083/jcb.200411012] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that encode soluble membrane and nuclear proteins. One gene product, LL5β, binds to both phosphoinositides and a cytoskeletal protein, filamin, one form of which is concentrated at synaptic sites. LL5β is itself associated with the cytoplasmic face of the postsynaptic membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in “corraling” AChRs. Consistent with this idea, perturbing LL5β expression in myotubes inhibits AChR aggregation. Thus, a strategy designed to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.
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Affiliation(s)
- Masashi Kishi
- Department of Anatomy and Neurobiology, Washington University Medical Center, St. Louis, MO 63110, USA
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48
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Bardet PL, Schubert M, Horard B, Holland LZ, Laudet V, Holland ND, Vanacker JM. Expression of estrogen-receptor related receptors in amphioxus and zebrafish: implications for the evolution of posterior brain segmentation at the invertebrate-to-vertebrate transition. Evol Dev 2005; 7:223-33. [PMID: 15876195 DOI: 10.1111/j.1525-142x.2005.05025.x] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
Summary The evolutionary origin of vertebrate hindbrain segmentation is unclear since the amphioxus, the closest living invertebrate relative to the vertebrates, possesses a hindbrain homolog that displays no gross morphological segmentation. Three of the estrogen-receptor related (ERR) receptors are segmentally expressed in the zebrafish hindbrain, suggesting that their common ancestor was expressed in a similar, reiterated manner. We have also cloned and determined the developmental expression of the single homolog of the vertebrate ERR genes in the amphioxus (AmphiERR). This gene is also expressed in a segmented manner in a region considered homologous to the vertebrate hindbrain. In contrast to the expression of amphioxus islet (a LIM-homeobox gene that also labels motoneurons), AmphiERR expression persists longer in the hindbrain homolog and does not later extend to additional posterior cells. In addition, AmphiERR and one of its vertebrate homologs (ERRalpha) are expressed in the developing somitic musculature of amphioxus and zebrafish, respectively. Altogether, our results are consistent with fine structural evidence suggesting that the amphioxus hindbrain is segmented, and indicate that chordate ERR gene expression is a marker for both hindbrain and muscle segmentation. Furthermore, our data support an evolution model of chordate brain segmentation: originally, the program for anterior segmentation in the protochordate ancestors of the vertebrates resided in the developing axial mesoderm which imposed reiterated patterning on the adjacent neural tube; during early vertebrate evolution, this segmentation program was transferred to and controlled by the neural tube.
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Affiliation(s)
- Pierre-Luc Bardet
- Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5161, IFR128 BioSciences Lyon-Gerland, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69007 Lyon, France
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O'Leary DA, Pritchard MA, Xu D, Kola I, Hertzog PJ, Ristevski S. Tissue-specific overexpression of the HSA21 gene GABPalpha: implications for DS. Biochim Biophys Acta Mol Basis Dis 2005; 1739:81-7. [PMID: 15607120 DOI: 10.1016/j.bbadis.2004.09.005] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2004] [Revised: 09/07/2004] [Accepted: 09/08/2004] [Indexed: 11/15/2022]
Abstract
The ETS transcription factor GABPalpha is encoded by a gene on HSA21 and interacts with an ankyrin repeat-containing beta subunit to form the GABP complex. GABP regulates expression of genes involved in mitochondrial respiration and neuromuscular signalling. When GABPalpha mRNA is overexpressed in human DS fibroblast cell lines, or by tranfection in NIH3T3 cells, no increase in protein level is detected. However, increased Gabpalpha gene dosage in the Ts65Dn segmental trisomy mouse model of DS (DS) results in elevated Gabpalpha protein levels in brain and skeletal muscle only. These findings suggest that GABPalpha protein levels are tightly regulated in a tissue-specific manner, and consequently GABP may play a role in DS pathologies in tissues where GABPalpha protein levels are elevated.
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Affiliation(s)
- Debra A O'Leary
- Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia
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50
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Méjat A, Ramond F, Bassel-Duby R, Khochbin S, Olson EN, Schaeffer L. Histone deacetylase 9 couples neuronal activity to muscle chromatin acetylation and gene expression. Nat Neurosci 2005; 8:313-21. [PMID: 15711539 DOI: 10.1038/nn1408] [Citation(s) in RCA: 137] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2004] [Accepted: 01/19/2005] [Indexed: 11/08/2022]
Abstract
Electrical activity arising from motor innervation influences skeletal muscle physiology by controlling the expression of many muscle genes, including those encoding acetylcholine receptor (AChR) subunits. How electrical activity is converted into a transcriptional response remains largely unknown. We show that motor innervation controls chromatin acetylation in skeletal muscle and that histone deacetylase 9 (HDAC9) is a signal-responsive transcriptional repressor which is downregulated upon denervation, with consequent upregulation of chromatin acetylation and AChR expression. Forced expression of Hdac9 in denervated muscle prevents upregulation of activity-dependent genes and chromatin acetylation by linking myocyte enhancer factor 2 (MEF2) and class I HDACs. By contrast, Hdac9-null mice are supersensitive to denervation-induced changes in gene expression and show chromatin hyperacetylation and delayed perinatal downregulation of myogenin, an activator of AChR genes. These findings show a molecular mechanism to account for the control of chromatin acetylation by presynaptic neurons and the activity-dependent regulation of skeletal muscle genes by motor innervation.
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MESH Headings
- Acetylation
- Age Factors
- Animals
- Animals, Newborn
- Blotting, Western/methods
- Chromatin/metabolism
- Cloning, Molecular
- DNA-Binding Proteins/metabolism
- DNA-Binding Proteins/physiology
- Electroporation/methods
- Embryo, Mammalian
- Fluorescent Antibody Technique/methods
- Gene Expression/physiology
- Gene Expression Regulation, Developmental/physiology
- Green Fluorescent Proteins/metabolism
- Histone Deacetylases/classification
- Histone Deacetylases/deficiency
- Histone Deacetylases/genetics
- Histone Deacetylases/metabolism
- Histones/metabolism
- Immunoprecipitation/methods
- MEF2 Transcription Factors
- Mice
- Mice, Knockout
- Muscle Denervation/methods
- Muscle, Skeletal/innervation
- Muscle, Skeletal/physiology
- Myogenic Regulatory Factors
- Myogenin/metabolism
- Neurons/physiology
- RNA, Messenger/biosynthesis
- Receptors, Cholinergic/genetics
- Receptors, Cholinergic/metabolism
- Repressor Proteins/genetics
- Repressor Proteins/metabolism
- Reverse Transcriptase Polymerase Chain Reaction/methods
- Time Factors
- Transcription Factors/metabolism
- Transcription Factors/physiology
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Affiliation(s)
- Alexandre Méjat
- Equipe Différenciation Neuromusculaire, Institut Fédératif de Recherche 128, Unité Mixte de Recherche 5161, Centre National de la Recherche Scientifique/Ecole Normale Supérieure, Ecole Normale Supérieure 46, allée d'Italie, 69364 Lyon Cedex 07, France
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