44 results on '"Butchbach, Matthew E. R."'
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2. Detection of SMN1 to SMN2 gene conversion events and partial SMN1 gene deletions using array digital PCR
- Author
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Stabley, Deborah L., Holbrook, Jennifer, Scavina, Mena, Crawford, Thomas O., Swoboda, Kathryn J., Robbins, Katherine M., and Butchbach, Matthew E. R.
- Published
- 2021
- Full Text
- View/download PDF
3. Using Systems Biology and Mathematical Modeling Approaches in the Discovery of Therapeutic Targets for Spinal Muscular Atrophy
- Author
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Butchbach, Matthew E. R., Schousboe, Arne, Series Editor, Cheung-Hoi Yu, Albert, editor, and Li, Lina, editor
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- 2018
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4. Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument
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Jiang, Lingxia, Lin, Robert, Gallagher, Steve, Zayac, Andrew, Butchbach, Matthew E. R., and Hung, Paul
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- 2020
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5. Biological networks and complexity in early-onset motor neuron diseases
- Author
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Butchbach, Matthew E. R., primary and Scott, Rod C., additional
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- 2022
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6. Effects of inhibitors of SLC9A-type sodium-protein exchangers on Survival Motor Neuron2 (SMN2) mRNA splicing and expression
- Author
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Kanda, Sambee, primary, Moulton, Emily, additional, and Butchbach, Matthew E. R., additional
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- 2022
- Full Text
- View/download PDF
7. Comprehensive In Silico Analysis of Retrotransposon Insertions within the Survival Motor Neuron Genes Involved in Spinal Muscular Atrophy
- Author
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Pinto, Albano, primary, Cunha, Catarina, additional, Chaves, Raquel, additional, Butchbach, Matthew E. R., additional, and Adega, Filomena, additional
- Published
- 2022
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- View/download PDF
8. Genomic Variability in the Survival Motor Neuron Genes (SMN1 and SMN2): Implications for Spinal Muscular Atrophy Phenotype and Therapeutics Development
- Author
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Butchbach, Matthew E. R., primary
- Published
- 2021
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- View/download PDF
9. SMNΔ7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN
- Author
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Le, Thanh T., Pham, Lan T., Butchbach, Matthew E. R., Zhang, Honglai L., Monani, Umrao R., Coovert, Daniel D., Gavrilina, Tatiana O., Xing, Lei, Bassell, Gary J., and Burghes, Arthur H. M.
- Published
- 2005
10. Methyl-β-cyclodextrin but not retinoic acid reduces EAAT3-mediated glutamate uptake and increases GTRAP3-18 expression
- Author
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Butchbach, Matthew E. R., Guo, Hong, and Lin, Chien-liang Glenn
- Published
- 2003
11. The effects of C5-substituted 2,4-diaminoquinazolines on selected transcript expression in spinal muscular atrophy cells
- Author
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Gentillon, Cinsley, primary, Connell, Andrew J., additional, Kirk, Ryan W., additional, and Butchbach, Matthew E. R., additional
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- 2017
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12. Effect of the Butyrate Prodrug Pivaloyloxymethyl Butyrate (AN9) on a Mouse Model for Spinal Muscular Atrophy
- Author
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Edwards, Jonathan D., primary and Butchbach, Matthew E. R., additional
- Published
- 2016
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13. Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases
- Author
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Butchbach, Matthew E. R., primary
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- 2016
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- View/download PDF
14. SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR
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Stabley, Deborah L., primary, Harris, Ashlee W., additional, Holbrook, Jennifer, additional, Chubbs, Nicholas J., additional, Lozo, Kevin W., additional, Crawford, Thomas O., additional, Swoboda, Kathryn J., additional, Funanage, Vicky L., additional, Wang, Wenlan, additional, Mackenzie, William, additional, Scavina, Mena, additional, Sol‐Church, Katia, additional, and Butchbach, Matthew E. R., additional
- Published
- 2015
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15. Systems Biology Investigation of cAMP Modulation to Increase SMN Levels for the Treatment of Spinal Muscular Atrophy
- Author
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Mack, Sean G., primary, Cook, Daniel J., additional, Dhurjati, Prasad, additional, and Butchbach, Matthew E. R., additional
- Published
- 2014
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16. Transcriptome Profiling of Spinal Muscular Atrophy Motor Neurons Derived from Mouse Embryonic Stem Cells
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Maeda, Miho, primary, Harris, Ashlee W., additional, Kingham, Brewster F., additional, Lumpkin, Casey J., additional, Opdenaker, Lynn M., additional, McCahan, Suzanne M., additional, Wang, Wenlan, additional, and Butchbach, Matthew E. R., additional
- Published
- 2014
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17. Synthesis and Biological Evaluation of Novel 2,4-Diaminoquinazoline Derivatives as SMN2 Promoter Activators for the Potential Treatment of Spinal Muscular Atrophy
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Thurmond, John, primary, Butchbach, Matthew E. R., additional, Palomo, Marty, additional, Pease, Brian, additional, Rao, Munagala, additional, Bedell, Louis, additional, Keyvan, Monica, additional, Pai, Grace, additional, Mishra, Rama, additional, Haraldsson, Magnus, additional, Andresson, Thorkell, additional, Bragason, Gisli, additional, Thosteinsdottir, Margret, additional, Bjornsson, Jon Mar, additional, Coovert, Daniel D., additional, Burghes, Arthur H. M., additional, Gurney, Mark E., additional, and Singh, Jasbir, additional
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- 2008
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18. Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs
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Gabanella, Francesca, primary, Butchbach, Matthew E. R., additional, Saieva, Luciano, additional, Carissimi, Claudia, additional, Burghes, Arthur H. M., additional, and Pellizzoni, Livio, additional
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- 2007
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19. Association of Excitatory Amino Acid Transporters, Especially EAAT2, with Cholesterol-rich Lipid Raft Microdomains.
- Author
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Butchbach, Matthew E. R., Tian, Guilian, Hong Guo, and Lin, Chien-liang Glenn
- Subjects
- *
EXCITATORY amino acids , *AMINO acid neurotransmitters , *CHOLESTEROL , *CELL membranes , *GLUTAMIC acid esters , *LIPIDS - Abstract
In the present study, we investigated the role of membrane cholesterol in the function of glutamate transporters. Depletion of membrane cholesterol by methylf-β-cyclodextrin resulted in reduced Na+-dependent glutamate uptake in primary cortical cultures. Glial glutamate transporter EAAT2-mediated uptake was more sensitive to this effect. Cell surface biotinylation and immunostaining experiments revealed that the loss of cholesterol significantly altered the trafficking of EAAT2 to the plasma membrane as well as their membrane distribution. These effects were also observed in neuronal glutamate transporter EAAT3 but to a lesser extent. Furthermore, the treatment of mouse brain plasma membrane vesicles with methyl-β-cyclodextrin resulted in a significant reduction in glutamate uptake, suggesting that cholesterol depletion has a direct effect on the function of the glutamate transporters. Plasma membrane cholesterol is localized within discreet microdomains known as lipid rafts. Analyses of purified lipid raft microdomains revealed that a large portion of total EAAT2 and a minor portion of total EAAT1, EAAT3, and EAAT4 were associated with lipid rafts. Artificial aggregation of lipid rafts in vivo resulted in the formation of larger EAAT2-immunoreactive clusters on the cell surface. The purified lipid raft-associated fractions were capable of Na+-dependent glutamate uptake. Our data suggest that the glutamate transporters, especially EAAT2, are associated with cholesterol-rich lipid raft microdomains of the plasma membrane and that the association with these cholesterol-rich microdomains is important for excitatory amino acid transporter localization and function. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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20. Regulation of glutamate transport by GTRAP3-18 and by lipid rafts
- Author
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Butchbach, Matthew E. R.
- Subjects
- glutamate uptake, excitatory amino acid transporter, GTRAP3-18, cyclodextrin, lipid raft, cholesterol, neuron, astrocyte
- Abstract
Glutamate plays pivotal roles in maintaining cellular homeostasis. In the nervous system, glutamate can function as an excitatory neurotransmitter. Excessive stimulation of glutamatergic receptors can lead to neuronal cell death, a process known as excitotoxicity. The rapid removal of glutamate from the synaptic cleft is accomplished by the Na+/K+-coupled excitatory amino acid transporters (EAATs). The expression and function of the EAATs can be modulated by intracellular signaling cascades and by protein-protein interactions. Novel mechanisms by which glutamate transporters are regulated in neurons were examined in this dissertation. Isolation and characterization of the murine orthologue to GTRAP3-18 revealed that it is a highly conserved gene since its amino acid sequence and genomic organization are highly conserved amongst the vertebrates. GTRAP3-18 mRNA and protein are expressed in neuron-rich regions of the central nervous system as well as in peripheral tissues such as the kidney and liver. The expression profile closely matches the published expression profile for EAAT3, its interacting partner. Mouse GTRAP3-18 negatively modulates mouse EAAT3-mediated glutamate uptake into transfected cells. Methyl-β-cyclodextrin (MeβCD) reduces Na+-dependent, EAAT3-mediated glutamate uptake into cells and increases GTRAP3-18 protein expression. Chronic treatment of HEK293 cells and primary neuron cultures with MeβCD results in the intracellular accumulation of EAAT3 but does not affect the subcellular distribution of GTRAP3-18. Intracerebroventricular administration of MeβCD induces spontaneous, recurrent seizures and neuronal cell death in the adult mouse. These studies demonstrate a novel means of regulating glutamate transporter function and the expression of glutamate transporter regulatory proteins using the macrocyclic polysaccharide MeβCD. Transient depletion of membrane cholesterol with MeβCD significantly reduces EAAT2- as well as EAAT1/EAAT3-mediated glutamate transport function. This decrease in glutamate uptake is due, in part, to MeβCD-induced reduction in the trafficking of EAATs to the plasma membrane from the trans-Golgi network. Cholesterol depletion by MeβCD also has a direct effect on the glutamate transporters expressed on the plasma membrane by disrupting the clustered localization of EAATs on the plasma membrane. Biochemical, immunohistochemical and pharmacological approaches were used to show that the EAATs are partially localized onto cholesterol-rich microdomains on the plasma membrane. EAAT2 was more strongly associated with these cholesterol-rich microdomains than the other EAATs expressed in the brain. Finally, the functional importance of association of the EAATs with these cholesterol-rich microdomains was demonstrated by measuring glutamate transport activity in vesicles derived from these cholesterol-rich microdomains. Taken together, these studies demonstrate novel mechanisms by the expression, localization and function of the excitatory amino acid neurotransmitters can be regulated. Modulation of the membrane localization of glutamate transporters can dramatically affect the clearance of glutamate from the synapse which could result in neuronal cell death. Glutamate plays pivotal roles in maintaining cellular homeostasis. In the nervous system, glutamate can function as an excitatory neurotransmitter. Excessive stimulation of glutamatergic receptors can lead to neuronal cell death, a process known as excitotoxicity. The rapid removal of glutamate from the synaptic cleft is accomplished by the Na+/K+-coupled excitatory amino acid transporters (EAATs). The expression and function of the EAATs can be modulated by intracellular signaling cascades and by protein-protein interactions. Novel mechanisms by which glutamate transporters are regulated in neurons were examined in this dissertation. Isolation and characterization of the murine orthologue to GTRAP3-18 revealed that it is a highly conserved gene since its amino acid sequence and genomic organization are highly conserved amongst the vertebrates. GTRAP3-18 mRNA and protein are expressed in neuron-rich regions of the central nervous system as well as in peripheral tissues such as the kidney and liver. The expression profile closely matches the published expression profile for EAAT3, its interacting partner. Mouse GTRAP3-18 negatively modulates mouse EAAT3-mediated glutamate uptake into transfected cells. Methyl-β-cyclodextrin (MeβCD) reduces Na+-dependent, EAAT3-mediated glutamate uptake into cells and increases GTRAP3-18 protein expression. Chronic treatment of HEK293 cells and primary neuron cultures with MeβCD results in the intracellular accumulation of EAAT3 but does not affect the subcellular distribution of GTRAP3-18. Intracerebroventricular administration of MeβCD induces spontaneous, recurrent seizures and neuronal cell death in the adult mouse. These studies demonstrate a novel means of regulating glutamate transporter function and the expression of glutamate transporter regulatory proteins using the macrocyclic polysaccharide MeβCD. Transient depletion of membrane cholesterol with MeβCD significantly reduces EAAT2- as well as EAAT1/EAAT3-mediated glutamate transport function. This decrease in glutamate uptake is due, in part, to MeβCD-induced reduction in the trafficking of EAATs to the plasma membrane from the trans-Golgi network. Cholesterol depletion by MeβCD also has a direct effect on the glutamate transporters expressed on the plasma membrane by disrupting the clustered localization of EAATs on the plasma membrane. Biochemical, immunohistochemical and pharmacological approaches were used to show that the EAATs are partially localized onto cholesterol-rich microdomains on the plasma membrane. EAAT2 was more strongly associated with these cholesterol-rich microdomains than the other EAATs expressed in the brain. Finally, the functional importance of association of the EAATs with these cholesterol-rich microdomains was demonstrated by measuring glutamate transport activity in vesicles derived from these cholesterol-rich microdomains. Taken together, these studies demonstrate novel mechanisms by the expression, localization and function of the excitatory amino acid neurotransmitters can be regulated. Modulation of the membrane localization of glutamate transporters can dramatically affect the clearance of glutamate from the synapse which could result in neuronal cell death.
- Published
- 2003
21. Human Glioma Cells and Undifferentiated Primary Astrocytes That Express Aberrant EAAT2 mRNA Inhibit Normal EAAT2 Protein Expression and Prevent Cell Death
- Author
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Guo, Hong, Lai, Liching, Butchbach, Matthew E. R., and Lin, Chien-liang Glenn
- Subjects
- *
GLIOMAS , *AMYOTROPHIC lateral sclerosis , *ALZHEIMER'S disease - Abstract
Abnormal splicing of astroglial glutamate transporter EAAT2 mRNA has been suggested to account for the loss of EAAT2 protein in amyotrophic lateral sclerosis (ALS) and Alzheimer''s disease (AD). We have identified several clones of human U251 glioma cells which express varying amounts of aberrantly spliced EAAT2 mRNA; these clones do not express any detectable EAAT2 protein. When the wild-type EAAT2 cDNA was expressed in each of these clones, we found that the amount of EAAT2 protein inversely correlated with the levels of endogenous aberrant EAAT2 mRNA. We also observed that ectopic expression of normal EAAT2 protein is toxic to U251 cells as well as to undifferentiated primary astrocytes. We conclude that expression of aberrant EAAT2 mRNA may be one possible mechanism to repress normal EAAT2 protein expression. The implication of this study for the mechanisms of EAAT2 protein loss in ALS and AD is discussed. [Copyright &y& Elsevier]
- Published
- 2002
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22. NF-κB-mediated Pax7 dysregulation in the muscle microenvironment promotes cancer cachexia.
- Author
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He, Wei A, Berardi, Emanuele, Cardillo, Veronica M, Acharyya, Swarnali, Aulino, Paola, Thomas-Ahner, Jennifer, Wang, Jingxin, Bloomston, Mark, Muscarella, Peter, Nau, Peter, Shah, Nilay, Butchbach, Matthew E R, Ladner, Katherine, Adamo, Sergio, Rudnicki, Michael A, Keller, Charles, Coletti, Dario, Montanaro, Federica, and Guttridge, Denis C
- Abstract
Cachexia is a debilitating condition characterized by extreme skeletal muscle wasting that contributes significantly to morbidity and mortality. Efforts to elucidate the underlying mechanisms of muscle loss have predominantly focused on events intrinsic to the myofiber. In contrast, less regard has been given to potential contributory factors outside the fiber within the muscle microenvironment. In tumor-bearing mice and patients with pancreatic cancer, we found that cachexia was associated with a type of muscle damage resulting in activation of both satellite and nonsatellite muscle progenitor cells. These muscle progenitors committed to a myogenic program, but were inhibited from completing differentiation by an event linked with persistent expression of the self-renewing factor Pax7. Overexpression of Pax7 was sufficient to induce atrophy in normal muscle, while under tumor conditions, the reduction of Pax7 or exogenous addition of its downstream target, MyoD, reversed wasting by restoring cell differentiation and fusion with injured fibers. Furthermore, Pax7 was induced by serum factors from cachectic mice and patients, in an NF-κB-dependent manner, both in vitro and in vivo. Together, these results suggest that Pax7 responds to NF-κB by impairing the regenerative capacity of myogenic cells in the muscle microenvironment to drive muscle wasting in cancer. [ABSTRACT FROM AUTHOR]
- Published
- 2013
23. Translational Control of Glial Glutamate Transporter EAAT2 Expression.
- Author
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Guilian Tian, Liching Lai, Hong Guo, Yuan Lin, Butchbach, Matthew E. R., Yueming Chang, and Chien-liang Glenn Lin
- Subjects
- *
NEUROTRANSMITTERS , *CENTRAL nervous system , *AMYOTROPHIC lateral sclerosis , *MESSENGER RNA , *ALZHEIMER'S disease , *CELL death - Abstract
Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5′-untranslated regions (5′-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5′-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5′-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
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24. Effects of Inhibitors of SLC9A-Type Sodium-Proton Exchangers on Survival Motor Neuron 2 ( SMN2 ) mRNA Splicing and Expression.
- Author
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Kanda S, Moulton E, and Butchbach MER
- Subjects
- Alternative Splicing, Humans, Motor Neurons metabolism, RNA, Messenger genetics, Muscular Atrophy, Spinal drug therapy, Muscular Atrophy, Spinal genetics, Sodium-Hydrogen Exchangers antagonists & inhibitors, Survival of Motor Neuron 2 Protein genetics
- Abstract
Spinal muscular atrophy (SMA) is an autosomal recessive, pediatric-onset disorder caused by the loss of spinal motor neurons, thereby leading to muscle atrophy. SMA is caused by the loss of or mutations in the survival motor neuron 1 ( SMN1 ) gene. SMN1 is duplicated in humans to give rise to the paralogous survival motor neuron 2 ( SMN2 ) gene. This paralog is nearly identical except for a cytosine to thymine transition within an exonic splicing enhancer element within exon 7. As a result, the majority of SMN2 transcripts lack exon 7 (SMNΔ7), which produces a truncated and unstable SMN protein. Since SMN2 copy number is inversely related to disease severity, it is a well established target for SMA therapeutics development. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs), has previously been shown to increase exon 7 inclusion and SMN protein levels in SMA cells. In this study, NHE inhibitors were evaluated for their ability to modulate SMN2 expression. EIPA as well as 5-(N,N-hexamethylene)amiloride (HMA) increase exon 7 inclusion in SMN2 splicing reporter lines as well as in SMA fibroblasts. The EIPA-induced exon 7 inclusion occurs via a unique mechanism that does not involve previously identified splicing factors. Transcriptome analysis identified novel targets, including TIA1 and FABP3 , for further characterization. EIPA and HMA are more selective at inhibiting the NHE5 isoform, which is expressed in fibroblasts as well as in neuronal cells. These results show that NHE5 inhibition increases SMN2 expression and may be a novel target for therapeutics development. SIGNIFICANCE STATEMENT: This study demonstrates a molecular mechanism by which inhibitors of the sodium-protein exchanger increase the alternative splicing of SMN2 in spinal muscular atrophy cells. NHE5 selective inhibitors increase the inclusion of full-length SMN2 mRNAs by targeting TIA1 and FABP3 expression, which is distinct from other small molecule regulators of SMN2 alternative splicing. This study provides a novel means to increase full-length SMN2 expression and a novel target for therapeutics development., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2022
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- View/download PDF
25. Spinal muscular atrophy diagnosis and carrier screening from genome sequencing data.
- Author
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Chen X, Sanchis-Juan A, French CE, Connell AJ, Delon I, Kingsbury Z, Chawla A, Halpern AL, Taft RJ, Bentley DR, Butchbach MER, Raymond FL, and Eberle MA
- Subjects
- Base Sequence, Child, Child, Preschool, Humans, Survival of Motor Neuron 1 Protein genetics, Muscular Atrophy, Spinal diagnosis, Muscular Atrophy, Spinal genetics
- Abstract
Purpose: Spinal muscular atrophy (SMA), caused by loss of the SMN1 gene, is a leading cause of early childhood death. Due to the near identical sequences of SMN1 and SMN2, analysis of this region is challenging. Population-wide SMA screening to quantify the SMN1 copy number (CN) is recommended by the American College of Medical Genetics and Genomics., Methods: We developed a method that accurately identifies the CN of SMN1 and SMN2 using genome sequencing (GS) data by analyzing read depth and eight informative reference genome differences between SMN1/2., Results: We characterized SMN1/2 in 12,747 genomes, identified 1568 samples with SMN1 gains or losses and 6615 samples with SMN2 gains or losses, and calculated a pan-ethnic carrier frequency of 2%, consistent with previous studies. Additionally, 99.8% of our SMN1 and 99.7% of SMN2 CN calls agreed with orthogonal methods, with a recall of 100% for SMA and 97.8% for carriers, and a precision of 100% for both SMA and carriers., Conclusion: This SMN copy-number caller can be used to identify both carrier and affected status of SMA, enabling SMA testing to be offered as a comprehensive test in neonatal care and an accurate carrier screening tool in GS sequencing projects.
- Published
- 2020
- Full Text
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26. Establishing a reference dataset for the authentication of spinal muscular atrophy cell lines using STR profiling and digital PCR.
- Author
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Stabley DL, Holbrook J, Harris AW, Swoboda KJ, Crawford TO, Sol-Church K, and Butchbach MER
- Subjects
- DNA Copy Number Variations, Family, Humans, Muscular Atrophy, Spinal genetics, Reference Values, Survival of Motor Neuron 1 Protein genetics, Survival of Motor Neuron 1 Protein metabolism, Survival of Motor Neuron 2 Protein genetics, Survival of Motor Neuron 2 Protein metabolism, Validation Studies as Topic, Cell Line, Fibroblasts cytology, Fibroblasts metabolism, Microsatellite Repeats, Muscular Atrophy, Spinal metabolism, Polymerase Chain Reaction
- Abstract
Fibroblasts and lymphoblastoid cell lines (LCLs) derived from individuals with spinal muscular atrophy (SMA) have been and continue to be essential for translational SMA research. Authentication of cell lines helps ensure reproducibility and rigor in biomedical research. This quality control measure identifies mislabeling or cross-contamination of cell lines and prevents misinterpretation of data. Unfortunately, authentication of SMA cell lines used in various studies has not been possible because of a lack of a reference. In this study, we provide said reference so that SMA cell lines can be subsequently authenticated. We use short tandem repeat (STR) profiling and digital PCR (dPCR), which quantifies SMN1 and SMN2 copy numbers, to generate molecular identity codes for fibroblasts and LCLs that are commonly used in SMA research. Using these molecular identity codes, we clarify the familial relationships within a set of fibroblasts commonly used in SMA research. This study presents the first cell line reference set for the SMA research community and demonstrates its usefulness for re-identification and authentication of lines commonly used as in vitro models for future studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
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27. Identification of early gene expression changes in primary cultured neurons treated with topoisomerase I poisons.
- Author
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Rossi SL, Lumpkin CJ, Harris AW, Holbrook J, Gentillon C, McCahan SM, Wang W, and Butchbach MER
- Subjects
- Animals, Antigens, Differentiation metabolism, Antineoplastic Agents chemistry, Camptothecin chemistry, Cells, Cultured, DNA Topoisomerases, Type I metabolism, Early Growth Response Protein 2 metabolism, Fibroblasts metabolism, Microscopy, Fluorescence, Neurons drug effects, Oligonucleotide Array Sequence Analysis, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins c-fos metabolism, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Gene Expression Regulation, Neurons metabolism, Topoisomerase I Inhibitors chemistry
- Abstract
Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1-CPT, actinomycin D (ActD) and β-lapachone (β-Lap)-on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify differentially regulated transcripts in MNs in response to a brief exposure to CPT. Pathway analysis of the differentially expressed transcripts revealed activation of ERK and JNK signaling cascades in CPT-treated MNs. Immediate-early genes like Fos, Egr-1 and Gadd45b were upregulated in CPT-treated MNs. Fos mRNA levels were elevated in all cell types treated with CPT; Egr-1, Gadd45b and Dyrk3 transcript levels, however, increased in CPT-treated MNs and CNs but decreased in CPT-treated fibroblasts. These transcripts may represent new targets for the development of therapeutic agents that mitigate the off-target effects of chemotherapy on the nervous system., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
28. Applicability of digital PCR to the investigation of pediatric-onset genetic disorders.
- Author
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Butchbach ME
- Abstract
Early-onset rare diseases have a strong impact on child healthcare even though the incidence of each of these diseases is relatively low. In order to better manage the care of these children, it is imperative to quickly diagnose the molecular bases for these disorders as well as to develop technologies with prognostic potential. Digital PCR (dPCR) is well suited for this role by providing an absolute quantification of the target DNA within a sample. This review illustrates how dPCR can be used to identify genes associated with pediatric-onset disorders, to identify copy number status of important disease-causing genes and variants and to quantify modifier genes. It is also a powerful technology to track changes in genomic biomarkers with disease progression. Based on its capability to accurately and reliably detect genomic alterations with high sensitivity and a large dynamic detection range, dPCR has the potential to become the tool of choice for the verification of pediatric disease-associated mutations identified by next generation sequencing, copy number determination and noninvasive prenatal screening.
- Published
- 2016
- Full Text
- View/download PDF
29. Protective effects of butyrate-based compounds on a mouse model for spinal muscular atrophy.
- Author
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Butchbach MER, Lumpkin CJ, Harris AW, Saieva L, Edwards JD, Workman E, Simard LR, Pellizzoni L, and Burghes AHM
- Subjects
- Animals, Behavior, Animal, Butyrates pharmacokinetics, Cell Survival drug effects, Female, Glycogen Synthase Kinase 3 metabolism, Glycogen Synthase Kinase 3 beta, Histone Deacetylase Inhibitors therapeutic use, Male, Mice, Mice, Knockout, Motor Neurons pathology, Muscular Atrophy, Spinal pathology, Muscular Atrophy, Spinal psychology, Neuroprotective Agents pharmacokinetics, Oncogene Protein v-akt metabolism, Phosphorylation, Prodrugs therapeutic use, Spinal Cord growth & development, Spinal Cord pathology, Butyrates therapeutic use, Muscular Atrophy, Spinal prevention & control, Neuroprotective Agents therapeutic use
- Abstract
Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analog (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs-glyceryl tributyrate (BA3G) and VX563-on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3β, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favorable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
30. The effect of the DcpS inhibitor D156844 on the protective action of follistatin in mice with spinal muscular atrophy.
- Author
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Harris AW and Butchbach ME
- Subjects
- Animals, Body Weight drug effects, Disease Models, Animal, Drug Therapy, Combination, Female, Follistatin therapeutic use, Kaplan-Meier Estimate, Male, Mice, Mice, Transgenic, Motor Activity drug effects, Muscular Atrophy, Spinal genetics, Muscular Atrophy, Spinal physiopathology, Quinazolines therapeutic use, Recombinant Proteins administration & dosage, Recombinant Proteins therapeutic use, Survival of Motor Neuron 1 Protein genetics, Survival of Motor Neuron 2 Protein genetics, Disease Progression, Endoribonucleases antagonists & inhibitors, Follistatin administration & dosage, Muscular Atrophy, Spinal prevention & control, Quinazolines administration & dosage
- Abstract
Spinal muscular atrophy (SMA), a leading genetic cause of pediatric death in the world, is an early-onset disease affecting the motor neurons in the anterior horn of the spinal cord. This degeneration of motor neurons leads to loss of muscle function. At the molecular level, SMA results from the loss of or mutation in the survival motor neuron 1 (SMN1) gene. The number of copies of the nearly duplicated gene SMN2 modulates the disease severity in humans as well as in transgenic mouse models for SMA. Most preclinical therapeutic trials focus on identifying ways to increase SMN2 expression and to alter its splicing. Other therapeutic strategies have investigated compounds which protect affected motor neurons and their target muscles in an SMN-independent manner. In the present study, the effect of a combination regimen of the SMN2 inducer D156844 and the protectant follistatin on the disease progression and survival was measured in the SMNΔ7 SMA mouse model. The D156844/follistatin combination treatment improved the survival of, delayed the end stage of disease in and ameliorated the growth rate of SMNΔ7 SMA mice better than follistatin treatment alone. The D156844/follistatin combination treatment, however, did not provide additional benefit over D156844 alone with respect to survival and disease end stage even though it provided some additional therapeutic benefit over D156844 alone with respect to motor phenotype., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
31. The effect of diet on the protective action of D156844 observed in spinal muscular atrophy mice.
- Author
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Butchbach ME, Singh J, Gurney ME, and Burghes AH
- Subjects
- Animals, Brain drug effects, Brain metabolism, Disease Models, Animal, Female, Male, Mice, Muscular Atrophy, Spinal drug therapy, Pregnancy, Prenatal Exposure Delayed Effects drug therapy, Quinazolines pharmacology, Severity of Illness Index, Spinal Cord drug effects, Spinal Cord metabolism, Treatment Outcome, Diet, Muscular Atrophy, Spinal genetics, Prenatal Exposure Delayed Effects genetics, Quinazolines therapeutic use, SMN Complex Proteins genetics
- Abstract
Spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of spinal motor neurons which leads to skeletal muscle atrophy. Proximal SMA results from the loss or mutation of the survival motor neuron (SMN) gene. In humans, the SMN gene is duplicated to produce two nearly identical genes, SMN1 and SMN2. SMN1 is lost in SMA but SMN2 is retained; in fact, the number of SMN2 copies correlates with disease severity. The SMN2 inducer D156844 increases the survival and improves phenotype of SMN∆7 SMA mice. Maternal diet also modifies the survival and phenotype of these mice. In this study, we show the effect of maternal diet on the protective effects of D156844 in SMN∆7 SMA mice. SMA mice maintained on the PicoLab20 Mouse diet survived longer when treated with D156844; the effect of diet was additive to the effect of D156844 on these mice. Brain levels of D156844 were higher in neonatal mice maintained on the PicoLab20 diet than those on the Harlan-Teklad 22/5 diet. SMN protein levels in the spinal cord were modestly elevated in D156844-treated, PicoLab20-maintained SMA mice. These data show that maternal diet does influence the responsiveness of D156844 in neonatal SMN∆7 SMA mice., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
32. Trans-splicing, more than meets the eye: multifaceted therapeutics for spinal muscular atrophy.
- Author
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Butchbach ME
- Subjects
- Animals, Gene Expression, Humans, Insulin-Like Growth Factor I metabolism, Mice, Mice, Transgenic, Survival of Motor Neuron 1 Protein genetics, Survival of Motor Neuron 2 Protein genetics, Genetic Therapy, Muscular Atrophy, Spinal genetics, Muscular Atrophy, Spinal therapy, Trans-Splicing
- Published
- 2011
- Full Text
- View/download PDF
33. Effects of 2,4-diaminoquinazoline derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy.
- Author
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Butchbach ME, Singh J, Thorsteinsdóttir M, Saieva L, Slominski E, Thurmond J, Andrésson T, Zhang J, Edwards JD, Simard LR, Pellizzoni L, Jarecki J, Burghes AH, and Gurney ME
- Subjects
- Animals, Cell Survival drug effects, Disease Models, Animal, Humans, Mice, Mice, Knockout, Mice, Transgenic, Motor Neurons drug effects, Motor Neurons metabolism, Muscular Atrophy, Spinal genetics, Muscular Atrophy, Spinal metabolism, Muscular Atrophy, Spinal physiopathology, Phenotype, Promoter Regions, Genetic drug effects, Survival of Motor Neuron 2 Protein metabolism, Gene Expression drug effects, Muscular Atrophy, Spinal drug therapy, Quinazolines administration & dosage, Quinazolines chemistry, Survival of Motor Neuron 2 Protein genetics
- Abstract
Proximal spinal muscular atrophy (SMA), one of the most common genetic causes of infant death, results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of survival motor neuron (SMN) protein. In humans, the SMN gene is duplicated; SMA results from the loss of SMN1 but SMN2 remains intact. SMA severity is related to the copy number of SMN2. Compounds which increase the expression of SMN2 could, therefore, be potential therapeutics for SMA. Ultrahigh-throughput screening recently identified substituted quinazolines as potent SMN2 inducers. A series of C5-quinazoline derivatives were tested for their ability to increase SMN expression in vivo. Oral administration of three compounds (D152344, D153249 and D156844) to neonatal mice resulted in a dose-dependent increase in Smn promoter activity in the central nervous system. We then examined the effect of these compounds on the progression of disease in SMN lacking exon 7 (SMNDelta7) SMA mice. Oral administration of D156844 significantly increased the mean lifespan of SMNDelta7 SMA mice by approximately 21-30% when given prior to motor neuron loss. In summary, the C5-quinazoline derivative D156844 increases SMN expression in neonatal mouse neural tissues, delays motor neuron loss at PND11 and ameliorates the motor phenotype of SMNDelta7 SMA mice.
- Published
- 2010
- Full Text
- View/download PDF
34. Effect of diet on the survival and phenotype of a mouse model for spinal muscular atrophy.
- Author
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Butchbach ME, Rose FF Jr, Rhoades S, Marston J, McCrone JT, Sinnott R, and Lorson CL
- Subjects
- 3-Hydroxybutyric Acid blood, Animals, Blood Glucose analysis, Diet, Disease Models, Animal, Female, Male, Mice, Mice, Transgenic, Muscular Atrophy, Spinal mortality, Survival of Motor Neuron 1 Protein genetics, Muscular Atrophy, Spinal diet therapy, Muscular Atrophy, Spinal physiopathology
- Abstract
Proximal spinal muscular atrophy (SMA) is a leading genetic cause of infant death. Patients with SMA lose alpha-motor neurons in the ventral horn of the spinal cord which leads to skeletal muscle weakness and atrophy. SMA is the result of reduction in Survival Motor Neuron (SMN) expression. Transgenic mouse models of SMA have been generated and are extremely useful in understanding the mechanisms of motor neuron degeneration in SMA and in developing new therapeutic candidates for SMA patients. Several research groups have reported varying average lifespans of SMNDelta7 SMA mice (SMN2(+/+);SMNDelta7(+/+);mSmn(-/-)), the most commonly used mouse model for preclinical therapeutic candidate testing. One environmental factor that varied between research groups was maternal diet. In this study, we compared the effects of two different commercially available rodent chows (PicoLab20 Mouse diet and Harlan-Teklad 22/5 diet) on the survival and motor phenotype of the SMNDelta7 mouse model of SMA. Specifically, the PicoLab20 diet significantly extends the average lifespan of the SMNDelta7 SMA mice by approximately 25% and improved the motor phenotype as compared to the Harlan diet. These findings indicate that maternal diet alone can have considerable impact on the SMA phenotype., (Copyright 2009 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
35. Detection of human survival motor neuron (SMN) protein in mice containing the SMN2 transgene: applicability to preclinical therapy development for spinal muscular atrophy.
- Author
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Mattis VB, Butchbach ME, and Lorson CL
- Subjects
- Alternative Splicing genetics, Amino Acid Sequence, Animals, Antibodies, Monoclonal metabolism, Cells, Cultured, Disease Models, Animal, Enzyme Inhibitors, Epitope Mapping methods, Fibroblasts drug effects, Fibroblasts metabolism, Gene Expression Regulation drug effects, Humans, Hydroxyurea therapeutic use, Indoles, Mice, Muscular Atrophy, Spinal drug therapy, Muscular Atrophy, Spinal pathology, SMN Complex Proteins genetics, SMN Complex Proteins immunology, Spinal Cord drug effects, Spinal Cord metabolism, Survival of Motor Neuron 2 Protein, Valproic Acid therapeutic use, Mice, Transgenic metabolism, Muscular Atrophy, Spinal metabolism, SMN Complex Proteins metabolism
- Abstract
Spinal muscular atrophy (SMA), the leading genetic cause of infant death results from loss of spinal motor neurons causing atrophy of skeletal muscle. SMA is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, however, an identically coding gene called SMN2 is retained, but is alternatively spliced to produce approximately 90% truncated protein. Most SMA translational and preclinical drug development has relied on the use of SMA mice to determine changes in SMN protein levels. However, the SMA mouse models are relatively severe and analysis of SMN-inducing compounds is confounded by the early mortality of these animals. An antibody that could detect SMN protein on a Smn background could circumvent this limitation and allow unaffected, heterozygous animals to be examined. Here we describe the generation and characterization of a monoclonal anti-SMN antibody, 4F11, which specifically recognizes human SMN protein. 4F11 detects SMN (human) but not native Smn (mouse) protein in SMN2 transgenic mice and in SMA cell lines. We demonstrate the feasibility of using 4F11 to detect changes in SMN2-derived SMN protein in SMA patient fibroblasts and in healthy SMN2 transgenic mice. This antibody is, therefore, an excellent tool for examining SMN2-inducing therapeutics in patient cells as well as in transgenic mice.
- Published
- 2008
- Full Text
- View/download PDF
36. Let all DNA vote: who are the amyotrophic lateral sclerosis candidates?
- Author
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Burghes AH and Butchbach ME
- Subjects
- Animals, Genetic Predisposition to Disease genetics, Humans, Mice, Mice, Neurologic Mutants, Polymorphism, Single Nucleotide genetics, Receptors, AMPA genetics, Receptors, N-Methyl-D-Aspartate genetics, Superoxide Dismutase genetics, Superoxide Dismutase-1, DNA Mutational Analysis, Genetic Markers genetics, Motor Neuron Disease genetics
- Published
- 2008
- Full Text
- View/download PDF
37. Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing.
- Author
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Novoyatleva T, Heinrich B, Tang Y, Benderska N, Butchbach ME, Lorson CL, Lorson MA, Ben-Dov C, Fehlbaum P, Bracco L, Burghes AH, Bollen M, and Stamm S
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cell Line, Conserved Sequence, Cyclic AMP Response Element-Binding Protein chemistry, Cyclic AMP Response Element-Binding Protein genetics, Cyclic AMP Response Element-Binding Protein metabolism, DNA Primers genetics, Evolution, Molecular, Exons, Humans, Molecular Sequence Data, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Phylogeny, RNA-Binding Proteins chemistry, RNA-Binding Proteins genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, SMN Complex Proteins, Sequence Homology, Amino Acid, Serine-Arginine Splicing Factors, Alternative Splicing, Protein Phosphatase 1 metabolism, RNA Precursors metabolism, RNA-Binding Proteins metabolism
- Abstract
Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein phosphatase 1 (PP1) binds to the splicing factor transformer2-beta1 (tra2-beta1) via a phylogenetically conserved RVDF sequence located on the RNA recognition motif (RRM) of tra2-beta1. PP1 binds directly to tra2-beta1 and dephosphorylates it, which regulates the interaction between tra2-beta1 and other proteins. Eight other proteins, including SF2/ASF and SRp30c, contain an evolutionary conserved PP1 docking motif in the beta-4 strand of their RRMs indicating that binding to PP1 is a new function of some RRMs. Reducing PP1 activity promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 exon 7 in a mouse model expressing the human gene. This suggests that reducing PP1 activity could be a new therapeutic principle to treat spinal muscular atrophy and other diseases caused by missplicing events. Our data indicate that the binding of PP1 to evolutionary conserved motifs in several RRMs is the link between known signal transduction pathways regulating PP1 activity and pre-mRNA processing.
- Published
- 2008
- Full Text
- View/download PDF
38. Abnormal motor phenotype in the SMNDelta7 mouse model of spinal muscular atrophy.
- Author
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Butchbach ME, Edwards JD, and Burghes AH
- Subjects
- Animals, Genotype, Mice, Mice, Transgenic, Muscular Atrophy, Spinal genetics, SMN Complex Proteins, Cyclic AMP Response Element-Binding Protein genetics, Disease Models, Animal, Motor Activity physiology, Muscular Atrophy, Spinal physiopathology, Nerve Tissue Proteins genetics, Phenotype, RNA-Binding Proteins genetics
- Abstract
Spinal muscular atrophy (SMA) is a recessive motor neuron disease that affects motor neurons in the anterior horn of the spinal cord. SMA results from the reduction of SMN (survival motor neuron) protein. Even though SMN is ubiquitously expressed, motor neurons are more sensitive to the reduction in SMN than other cell types. We have previously generated mouse models of SMA with varying degrees of clinical severity. So as to more clearly understand the pathogenesis of motor neuron degeneration in SMA, we have characterized the phenotype of the SMNDelta7 SMA mouse which normally lives for 13.6+/-0.7 days. These mice are smaller than their non-SMA littermates and begin to lose body mass at 10.4+/-0.4 days. SMNDelta7 SMA mice exhibit impaired responses to surface righting, negative geotaxis and cliff aversion but not to tactile stimulation. Spontaneous motor activity and grip strength are also significantly impaired in SMNDelta7 SMA mice. In summary, we have demonstrated an impairment of neonatal motor responses in SMNDelta7 SMA mice. This phenotype characterization could be used to assess the effectiveness of potential therapies for SMA.
- Published
- 2007
- Full Text
- View/download PDF
39. A novel method for oral delivery of drug compounds to the neonatal SMNDelta7 mouse model of spinal muscular atrophy.
- Author
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Butchbach ME, Edwards JD, Schussler KR, and Burghes AH
- Subjects
- Administration, Oral, Animals, Animals, Newborn, Female, Humans, Male, Mice, Organ Specificity, Tissue Distribution, Brain metabolism, Bromodeoxyuridine administration & dosage, Bromodeoxyuridine pharmacokinetics, Disease Models, Animal, Drug Delivery Systems methods, Muscular Atrophy, Spinal metabolism, Spinal Cord metabolism
- Abstract
Spinal muscular atrophy (SMA) is a devastating motor neuron disease that is one of the leading genetic causes of infant mortality. Currently, there is no cure for SMA. Mouse models that genetically and phenotypically resemble SMA have been generated and have the potential to be used for the discovery of novel therapeutics. Oral administration is a commonly used mode of drug delivery in humans as well as in rodents. Unfortunately, there is no method of drug delivery that can accurately and reliably deliver drug compounds orally to neonatal mice. In this report, we describe a novel method to orally administer compounds to neonatal SMA mice. Oral delivery to neonatal mice, usually starting at postnatal day 4 (PND04), is both rapid and safe to the pup. Oral delivery of two different commonly used vehicle formulations, distilled water and 2-hydroxypropyl-beta-cyclodextrin, does not affect the survival of SMA mice. After oral delivery for 3 days, 5-bromo-2'-deoxyuridine could be detected in the kidneys, brains and spinal cords of treated non-SMA as well as SMA neonatal pups. In conclusion, we have developed a method by which drugs can be safely and reliably administered orally to neural targets of neonatal mice. This approach offers a simple and rapid means by which potential therapeutics for SMA can be identified.
- Published
- 2007
- Full Text
- View/download PDF
40. Translational control of glial glutamate transporter EAAT2 expression.
- Author
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Tian G, Lai L, Guo H, Lin Y, Butchbach ME, Chang Y, and Lin CL
- Subjects
- 5' Untranslated Regions, Animals, Astrocytes metabolism, Biological Transport, Corticosterone metabolism, Humans, Mice, Neurons metabolism, Rats, Vitamin A metabolism, Amino Acid Transport System X-AG physiology, Excitatory Amino Acid Transporter 2 biosynthesis, Neuroglia metabolism, Protein Biosynthesis
- Abstract
Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5'-untranslated regions (5'-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5'-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5'-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions.
- Published
- 2007
- Full Text
- View/download PDF
41. Dystrophin glycoprotein complex dysfunction: a regulatory link between muscular dystrophy and cancer cachexia.
- Author
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Acharyya S, Butchbach ME, Sahenk Z, Wang H, Saji M, Carathers M, Ringel MD, Skipworth RJ, Fearon KC, Hollingsworth MA, Muscarella P, Burghes AH, Rafael-Fortney JA, and Guttridge DC
- Subjects
- Adult, Aged, Animals, Cachexia complications, Dystrophin metabolism, Dystrophin-Associated Proteins metabolism, Female, Founder Effect, Humans, Male, Mice, Mice, Inbred BALB C, Mice, Inbred mdx, Mice, Transgenic, Middle Aged, Muscle, Skeletal metabolism, Muscle, Skeletal pathology, Muscular Atrophy metabolism, Muscular Atrophy pathology, Neoplasms complications, Neoplasms pathology, Ubiquitin-Protein Ligases metabolism, Cachexia metabolism, Dystrophin physiology, Dystrophin-Associated Proteins physiology, Glycoproteins physiology, Neoplasms metabolism
- Abstract
Cachexia contributes to nearly a third of all cancer deaths, yet the mechanisms underlying skeletal muscle wasting in this syndrome remain poorly defined. We report that tumor-induced alterations in the muscular dystrophy-associated dystrophin glycoprotein complex (DGC) represent a key early event in cachexia. Muscles from tumor-bearing mice exhibited membrane abnormalities accompanied by reduced levels of dystrophin and increased glycosylation on DGC proteins. Wasting was accentuated in tumor mdx mice lacking a DGC but spared in dystrophin transgenic mice that blocked induction of muscle E3 ubiquitin ligases. Furthermore, DGC deregulation correlated positively with cachexia in patients with gastrointestinal cancers. Based on these results, we propose that, similar to muscular dystrophy, DGC dysfunction plays a critical role in cancer-induced wasting.
- Published
- 2005
- Full Text
- View/download PDF
42. Increased expression of the glial glutamate transporter EAAT2 modulates excitotoxicity and delays the onset but not the outcome of ALS in mice.
- Author
-
Guo H, Lai L, Butchbach ME, Stockinger MP, Shan X, Bishop GA, and Lin CL
- Subjects
- Amyotrophic Lateral Sclerosis enzymology, Amyotrophic Lateral Sclerosis metabolism, Amyotrophic Lateral Sclerosis pathology, Animals, Brain metabolism, Brain pathology, Caspases drug effects, Caspases metabolism, Cell Membrane metabolism, Crosses, Genetic, Disease Models, Animal, Enzyme Activation drug effects, Excitatory Amino Acid Transporter 2 genetics, Glial Fibrillary Acidic Protein genetics, Glutamic Acid metabolism, Glutamic Acid pharmacokinetics, Mice, Mice, Transgenic, Motor Neurons enzymology, Motor Neurons pathology, Promoter Regions, Genetic, Spinal Cord metabolism, Spinal Cord pathology, Superoxide Dismutase metabolism, Superoxide Dismutase-1, Time Factors, Transgenes, Amyotrophic Lateral Sclerosis genetics, Cell Death genetics, Excitatory Amino Acid Transporter 2 metabolism, Gene Expression
- Abstract
The glial glutamate transporter EAAT2 is primarily responsible for clearance of glutamate from the synaptic cleft and loss of EAAT2 has been previously reported in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. The loss of functional EAAT2 could lead to the accumulation of extracellular glutamate, resulting in cell death known as excitotoxicity. However, it is still unknown whether it is a primary cause in the cascade leading to neuron degeneration or a secondary event to cell death. The goals of this study were to generate transgenic mice overexpressing EAAT2 and then to cross these mice with the ALS-associated mutant SOD1(G93A) mice to investigate whether supplementation of the loss of EAAT2 would delay or rescue the disease progression. We show that the amount of EAAT2 protein and the associated Na+-dependent glutamate uptake was increased about 2-fold in our EAAT2 transgenic mice. The transgenic EAAT2 protein was properly localized to the cell surface on the plasma membrane. Increased EAAT2 expression protects neurons from L-glutamate induced cytotoxicity and cell death in vitro. Furthermore, our EAAT2/G93A double transgenic mice showed a statistically significant (14 days) delay in grip strength decline but not in the onset of paralysis, body weight decline or life span when compared with G93A littermates. Moreover, a delay in the loss of motor neurons and their axonal morphologies as well as other events including caspase-3 activation and SOD1 aggregation were also observed. These results suggest that the loss of EAAT2 may contribute to, but does not cause, motor neuron degeneration in ALS.
- Published
- 2003
- Full Text
- View/download PDF
43. Methyl-beta-cyclodextrin but not retinoic acid reduces EAAT3-mediated glutamate uptake and increases GTRAP3-18 expression.
- Author
-
Butchbach ME, Guo H, and Lin CL
- Subjects
- Animals, Cell Line, Cells, Cultured, Cerebral Cortex drug effects, Cerebral Cortex metabolism, Cyclodextrins administration & dosage, Excitatory Amino Acid Transporter 3, Glutamate Plasma Membrane Transport Proteins, Heat-Shock Proteins, Hippocampus drug effects, Hippocampus metabolism, Humans, Hypothalamus cytology, Injections, Intraventricular, Kidney cytology, Kidney drug effects, Kidney metabolism, Male, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Neurons cytology, Neurons drug effects, Neurons metabolism, Sodium metabolism, Amino Acid Transport System X-AG metabolism, Carrier Proteins metabolism, Cyclodextrins pharmacology, Glutamic Acid pharmacokinetics, Symporters metabolism, Tretinoin pharmacology, beta-Cyclodextrins
- Abstract
The Na+-dependent glutamate transporter EAAT3 facilitates glutamate uptake into neurons as well as many other cell types. GTRAP3-18 (JWA, Arl6ip5) is a novel protein that interacts with EAAT3 and negatively modulates EAAT3-mediated glutamate uptake. Previous studies suggest that retinoic acid (RA) decreases Na+-dependent glutamate uptake and increases GTRAP3-18 protein expression. However, the RA used in those studies was complexed with methyl-beta-cyclodextrin (MebetaCD). In the present study we found that MebetaCD, but not RA, significantly reduced Na+-dependent EAAT3-mediated [3H]glutamate uptake in human embryonic kidney 293 (HEK293) cells. MebetaCD also significantly increased GTRAP3-18 protein expression in HEK293 cells as well as in rat hypothalamic neuron cultures. Intracerebroventricular administration of MebetaCD to the mouse brain resulted in a significant increase in GTRAP3-18 immunoreactivity in the hippocampus and cerebral cortex. In conclusion, we have shown that MebetaCD reduces EAAT3-mediated glutamate uptake and induces the expression of GTRAP3-18 protein.
- Published
- 2003
- Full Text
- View/download PDF
44. Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3-18.
- Author
-
Butchbach ME, Lai L, and Lin CL
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cell Line, Cloning, Molecular, DNA, Complementary chemistry, DNA, Complementary genetics, Excitatory Amino Acid Transporter 3, Genes genetics, Glutamate Plasma Membrane Transport Proteins, Glutamic Acid drug effects, Glutamic Acid pharmacokinetics, Heat-Shock Proteins, Immunoblotting, Immunohistochemistry, Male, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Molecular Sequence Data, RNA genetics, RNA metabolism, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Amino Acid Transport System X-AG, Carrier Proteins genetics, Carrier Proteins metabolism, Gene Expression Profiling, Symporters
- Abstract
Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na(+)-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3-interacting protein called GTRAP3-18 has been identified by a yeast two-hybrid screening. GTRAP3-18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3-18, we cloned the mouse orthologue to GTRAP3-18 and determined its gene structure and its expression pattern. GTRAP3-18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3-18 genes contain three exons separated by two introns. The GTRAP3-18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3-18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3-18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3-18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.
- Published
- 2002
- Full Text
- View/download PDF
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