Livio Pellizzoni
Columbia University Health Sciences
Project start date: 2012-02-01
Project end date: 2014-01-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Livio Pellizzoni
A FUNCTIONAL CELL-BASED SCREEN FOR POTENTIAL SMA THERAPEUTIC COMPOUNDS
Livio Pellizzoni, Phd
Columbia University Health Sciences, Columbia University Medical Center, New York, Ny 10032-3702
Grant 1R21NS071092-01 from National Institute Of Neurological Disorders And Stroke
Abstract: Spinal muscular atrophy (SMA) is the most common genetic cause of death in infancy, but no effective treatment is available. SMA is caused by reduced levels of the survival motor neuron (SMN) protein - reflecting homozygous loss of the SMN1 gene but preservation of the nearly-identical SMN2 gene. Low SMN levels lead to motor neuron degeneration and loss of muscle strength. There is therefore an urgent need to identify treatments that can restore SMN levels or can correct the deficits downstream of SMN depletion. Since SMA patients and mouse models with higher copy numbers of the SMN2 gene show reduced disease severity, most drug screening efforts have focused on enhancing expression of full-length SMN from the SMN2 gene. This may occur either by increasing SMN2 transcription or by correcting the splicing defect, which leads it to be hypomorphic. However, most of the existing screening assays do not directly assess SMN function but instead measure reporter gene activity and/or SMN protein accumulation. Therefore, compounds that have a small effect on overall levels but a significant effect on SMN function may go undetected. Moreover, compounds acting on the disease pathway rather than on the disease trigger are not screened for. The current proposal aims to address each of these shortcomings. We have generated mouse fibroblast lines with regulated knockdown of endogenous SMN. Reducing mSmn expression to a level similar to that found in tissues of type I patients triggers growth arrest in these cells - a phenotype that can be corrected by ectopic expression of RNAi-resistant human SMN. In support of the idea that this assay mirrors the mechanisms at play in the disease, the ability of different point mutant forms of SMN to correct the growth defect is proportional to their potency in rescuing the motor phenotype in mouse models. We will develop a modified version of this cell line in which cell proliferation is dependent on SMN levels produced by the human SMN2 gene. The untreated cell line will show reduced growth, while agents that enhance SMN2 transcription or splicing, stabilize SMN protein, or correct downstream defects will promote increased proliferation. This new model will therefore provide a functional readout for multiple potential therapeutic targets in SMA. Following initial testing of the assay for reproducibility and responsiveness to known benchmarking compounds, we will perform a pilot screen of 4,000 chemical compounds known to show biological activity. Secondary screens have been devised to eliminate compounds acting through mechanisms that are not disease-related and to distinguish between those acting at the level of SMN2 and others acting downstream. The screen is designed to provide a first insight into mechanism(s) of action and priority ranking of compounds for follow-up studies. These will be of three types i) partnerships with academic and pharmaceutical screening centers to pursue a large-scale high-throughput screen on the validated assay, ii) analysis of potential disease mechanisms indicated by the hit compounds, and iii) investigation of therapeutic potential of the hit compounds in mouse models of SMA. Spinal muscular atrophy (SMA) is the most common genetic cause of death in infancy, but no effective treatment is available. We are developing new cell models which allow us to mimic the defect in the culture dish. As a first step toward new therapeutic strategies, we will test thousands of chemical compounds for their ability to correct the SMA-related defect in these cells
Keywords: Address; Affect; Aran-Duchenne disease; Assay; Benchmarking; Best Practice Analysis; Bioassay; Biologic Assays; Biological; Biological Assay; Biological Preservation; Biology; Blood Serum; Cause of Death; Cell Density; Cell Growth in Number; Cell Line; Cell Lines, Strains; Cell Multiplication; Cell Proliferation; Cell model; CellLine; Cells; Cellular Assay; Cellular Expansion; Cellular Growth; Cellular Proliferation; Cellular model; Chemicals; Collaborations; Crossmatching, Tissue; Cruveilhier disease; Cultured Cells; Data; Defect; Development; Disease; Disease Pathway; Disorder; Dose; Drug Evaluation, Preclinical; Drug Screening; ES cell; Ectopic Expression; Evaluation; Evaluation Studies, Drug, Pre-Clinical; Evaluation Studies, Drug, Preclinical; Exons; Fibroblasts; Follow-Up Studies; Followup Studies; Future; Gene Expression; Gene Transcription; Generalized Growth; Genes; Genetic; Genetic Transcription; Goals; Growth; High Throughput Assay; Histocompatibility Testing; Human; Human, General; Immunologic, Luciferase; Investigation; Knowledge; Lead; Length; Libraries; Luciferases; Mammals, Mice; Man (Taxonomy); Man, Modern; Measures; Mice; Modeling; Molecular; Molecular Bank; Motor; Motor Cell; Motor Neuron Disease; Motor Neurons; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Patients; Pb element; Pharmaceutical Agent; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Phenotype; Play; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Preclinical Drug Evaluation; Preservation, Biologic; Preservation, Biological; Production; Quelling; RNA Expression; RNA Interference; RNA Silencing; RNA Silencings; RNA Splicing; RNAi; Reading; Reporter Genes; Reproducibility; Resistance; SMN gene product (SMA); SMN protein (spinal muscular atrophy); SMN1; SMN1 gene; SMN2; SMN2 gene; Screening procedure; Sequence-Specific Posttranscriptional Gene Silencing; Serum; Severity of illness; Spinal Muscular Atrophy; Splicing; System; System, LOINC Axis 4; Testing; Therapeutic; Therapeutic Agents; Time; Tissue Crossmatchings; Tissue Growth; Tissue Typing; Transcription; Transcription, Genetic; Translational Research; Translational Research Enterprise; Translational Science; United States National Institutes of Health; Validation; Work; assay development; base; cell growth; cultured cell line; design; designing; disease severity; disease/disorder; effective therapy; embryonic stem cell; heavy metal Pb; heavy metal lead; high throughput analysis; high throughput screening; histocompatibility typing; infancy; infantile; insight; interest; motoneuron; motor neuron degeneration; motor neuron function; mouse model; muscle strength; mutant; new therapeutics; next generation therapeutics; novel therapeutics; ontogeny; pre-clinical; preclinical; preservation; public health relevance; resistant; response; screening; screenings; small molecule libraries; stem cell of embryonic origin; survival motor neuron gene product; survival motor neuron protein; survival of motor neuron 1, telomeric; survival of motor neuron 2, centromeric; therapeutic target; translation research enterprise
Relevance: Spinal muscular atrophy (SMA) is the most common genetic cause of death in infancy, but no effective treatment is available. We are developing new cell models which allow us to mimic the defect in the culture dish. As a first step toward new therapeutic strategies, we will test thousands of chemical compounds for their ability to correct the SMA-related defect in these cells
Project start date: 2010-07-15
Project end date: 2012-06-30
Budget start date: 15-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PAR-08-228
1R21NS071092-01 (2010): $201250
NONCODING RNA TARGETS OF THE SPINAL MUSCULAR ATROPHY PROTEIN
Livio Pellizzoni
Columbia University Health Sciences, Columbia University Medical Center, New York, Ny 10032-3702
Grant 5R21NS067448-02 from National Institute Of Neurological Disorders And Stroke
Abstract: SMA is an autosomal recessive disorder characterized by degeneration of motor neurons in the spinal cord and skeletal muscle atrophy. SMA is the leading genetic cause of death in infancy for which no effective treatment is currently available. Although decreased levels of the survival motor neuron (SMN) protein cause SMA and inversely correlate with disease severity in both human patients and mouse model, the molecular mechanisms of SMA are unknown. SMN interacts directly with Sm and LSm proteins-an evolutionarily conserved family of proteins with diverse roles in RNA metabolism-and mediates their specific association with small nuclear RNAs into ribonucleoprotein complexes (snRNPs) that function in pre-mRNA processing. However, SMN activity in snRNP assembly does not explain the selective degeneration of motor neurons observed in SMA. Our hypothesis is that SMN role in RNP assembly may extend to other cellular noncoding RNAs (ncRNAs), possibly with a motor neuron-specific expression profile, and that some of these RNAs are additional yet-to-be-identified targets of Sm/LSm proteins. This scenario is suggested by the versatility of Sm/LSm proteins in forming different heteromeric complexes with distinct RNA binding characteristics. Defects in the biogenesis and function of these novel ncRNA targets of SMN may represent the disease trigger of SMA. We will take advantage of the capacity to generate large numbers of motor neurons differentiated from mouse ES cells to explore this possibility in the cell type that is affected in SMA. We will characterize the repertoire of ncRNAs that associate with SMN and Sm/LSm proteins as well as carry out an initial assessment of the potential involvement of the novel ncRNAs in SMA pathology. First, ncRNAs will be isolated from whole cell extracts by immunoprecipitation with specific monoclonal antibodies. Then, cDNA libraries will be generated from each immunoprecipitate and comprehensive identification of ncRNAs will be performed using high-throughput sequencing technologies. Following bioinformatic analysis with stringent filter criteria, selected ncRNAs will be analyzed for expression and association with SMN and Sm/LSm proteins by independent validation methods including quantitative RT-PCR. We will employ the same approach to investigate whether SMN deficiency-the disease trigger of SMA-affects the metabolism of validated ncRNAs. To do so, we will compare ncRNA expression and assembly into Sm/LSm-containing RNPs in cells with normal and reduced SMN. By carrying out qualitative and quantitative assessment of potential changes in the metabolism of ncRNAs in two distinct cell types (ES cells and ES cell-derived motor neurons) and two different physiological conditions (normal and low SMN levels), our study will reveal both ubiquitous and cell type-specific ncRNA targets of SMN and highlight their possible connection to SMA pathogenesis. Spinal muscular atrophy (SMA) is a devastating motor neuron disease caused by decreased levels of the SMN protein. SMN plays a critical role in multiple aspects of RNA metabolism. We will carry out a comprehensive analysis of the repertoire of RNA targets of SMN with the goal of identifying novel RNA pathways that are regulated by SMN and whose disruption may contribute to motor neuron degeneration. These studies have the potential to provide unanticipated insights into the molecular mechanisms of SMA and to suggest new avenues of therapeutic intervention
Keywords: Affect; Animal Model; Animal Models and Related Studies; Aran-Duchenne disease; Atrophy, Muscle; Bio-Informatics; Biogenesis; Bioinformatics; Biological Models; Biology; Causality; Cause of Death; Cell Extracts; Cells; Characteristics; Co-Immunoprecipitations; Complex; Computing Methodologies; Cruveilhier disease; Data; Defect; Deficiency Diseases; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Disease; Disorder; Dysfunction; ES Cell Line; ES cell; Embryonic Stem Cell Line; Equipment; Etiology; Expression Profiling; Expression Signature; Functional disorder; Gene Products, RNA; Gene Transcription; Generations; Genes; Genetic; Genetic Transcription; Goals; Hereditary; Human; Human, General; Immune Precipitation; Immunoprecipitation; Inherited; Intermediary Metabolism; Link; Low Molecular Weight Nuclear RNA; METBL; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Medulla Spinalis; Metabolic Processes; Metabolism; Method LOINC Axis 6; Methodology; Methods; Mice; Moab, Clinical Treatment; Model System; Models, Biologic; Molecular; Molecular Fingerprinting; Molecular Profiling; Monoclonal Antibodies; Motor Cell; Motor Neuron Disease; Motor Neurons; Murine; Mus; Muscle, Skeletal; Muscle, Voluntary; Muscular Atrophy; Nerve Degeneration; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neuron Degeneration; Origin of Life; Outcome; Pathogenesis; Pathology; Pathway interactions; Patients; Physiologic; Physiological; Physiopathology; Play; Pre-mRNA; Process; Protein Family; Proteins; R01 Mechanism; R01 Program; RNA; RNA Binding; RNA Expression; RNA, Messenger, Precursors; RNA, Non-Polyadenylated; RNA, Noncoding; RNA, Nontranslated; RNA, Untranslated; RNP; RPG; RT-PCR; RTPCR; Reagent; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic Acid; Ribonucleoproteins; Role; SMN gene product (SMA); SMN protein (spinal muscular atrophy); Secure; Severity of illness; Skeletal Muscle Tissue; Skeletal muscle structure; Small Molecular Weight RNA; Small Nuclear RNA; Small Nuclear RNP; Small Nuclear Ribonucleoprotein Particle; Small Nuclear Ribonucleoproteins; Sorting - Cell Movement; Spinal Cord; Spinal Muscular Atrophy; Technology; Therapeutic Intervention; Transcription; Transcription, Genetic; Untranslated RNA; Validation; cDNA Library; cell type; computational methodology; computational methods; computer methods; design; designing; disease causation; disease etiology; disease severity; disease/disorder; disease/disorder etiology; disorder etiology; effective therapy; embryonic stem cell; experiment; experimental research; experimental study; gene product; human disease; infancy; infantile; insight; intervention therapy; mRNA Precursor; model organism; molecuar profile; molecular signature; motoneuron; motor neuron degeneration; motor neuron function; mouse model; neural degeneration; neurodegeneration; neurodegenerative illness; neuron cell death; neuron loss; neuronal cell death; neuronal degeneration; neuronal loss; next generation; novel; pathophysiology; pathway; premRNA; public health relevance; research study; reverse transcriptase PCR; snRNA; snRNP; social role; sorting; stem cell of embryonic origin; survival motor neuron gene product; survival motor neuron protein; therapeutic development; uRNA
Relevance: Spinal muscular atrophy (SMA) is a devastating motor neuron disease caused by decreased levels of the SMN protein. SMN plays a critical role in multiple aspects of RNA metabolism. We will carry out a comprehensive analysis of the repertoire of RNA targets of SMN with the goal of identifying novel RNA pathways that are regulated by SMN and whose disruption may contribute to motor neuron degeneration. These studies have the potential to provide unanticipated insights into the molecular mechanisms of SMA and to suggest new avenues of therapeutic intervention
Project start date: 2009-09-01
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-06-181
5R21NS067448-02 (2010): $199238
1R21NS067448-01 (2009): $240625
SMN CONTROL OF SNRNP BIOGENESIS: ROLE IN RNA SPLICING AND MOTOR NEURON SURVIVAL
Livio Pellizzoni, Assistant Professor
Columbia University Health Sciences, Columbia University Medical Center, New York, Ny 10032-3702
Grant 1R01NS069601-01 from National Institute Of Neurological Disorders And Stroke
Abstract: Project Summary Regulation of RNA splicing is the primary mechanism responsible for generating the proteome diversity and phenotypic complexity of humans. This post-transcriptional regulatory mechanism is particularly prominent in neuronal cells and the increasing number of neurodegenerative disorders that are associated with splicing dysfunction underscores its biological relevance. The study of the survival motor neuron (SMN) protein provides a unique opportunity to address the basic biology of splicing regulation and the role of RNA dysfunction in human disease. Reduced SMN levels cause spinal muscular atrophy (SMA)-a common inherited neuromuscular disorder characterized by motor neuron degeneration. SMN has a well-established function in the assembly of small nuclear ribonucleoproteins (snRNPs), which are the essential components of the splicing machinery. In SMA mice, the degree of snRNP assembly impairment correlates with disease severity and causes an uneven rather than uniform decrease in the levels of snRNPs, resulting in the alteration of the snRNP profile of tissues. Moreover, restoration of normal snRNP levels coincides with phenotypic correction in animal models of disease. Despite these advances, how defective SMN function in snRNP biogenesis selectively affects motor neurons is unknown. This project will investigate our hypothesis that SMN functions to endow distinct cell types with unique snRNP profiles for the purpose of splicing regulation and that alterations in this process triggered by SMN deficiency cause splicing defects in mRNAs critical for motor neuron biology. Building on the results of our preliminary studies, in Aim 1 we will analyze SMN role in establishing distinct snRNP profiles in different cell types as well as mouse tissues during development. The relevance for splicing regulation of these cell type-specific snRNP profiles will be studied in Aims 2 and 3. In Aim 2, we will investigate the consequences of SMN depletion on RNA splicing using microarray analyses and cellular model systems with regulated knockdown of SMN. Based on our ability to generate large numbers of motor neurons differentiated from mouse embryonic stem (ES) cells with normal and reduced levels of SMN, we will identify mRNAs affected by SMN deficiency in the cell type relevant to SMA. Through comparative analyses using other types of post-mitotic neurons as well as primary motor neurons from SMA mice, we will define the set of mRNAs whose expression or alternative splicing is selectively affected in motor neurons. In order to establish a mechanistic link between SMN control of snRNP biogenesis and splicing regulation, the cause-effect relationship between SMN-dependent alterations in the snRNP profile and splicing changes will be analyzed in Aim 3. Finally, in Aim 4, the functional role of selected genes and alternative splicing isoforms identified above will be studied using knockdown and over-expression experiments in both normal and SMN- deficient ES cell-derived motor neurons. This approach should identify mRNAs whose SMN-dependent expression or alternative splicing is critical for motor neuron survival and function. The biomedical relevance of understanding the mechanisms and regulation of RNA processing is highlighted by the growing list of human genetic disorders associated with defects in RNA metabolism. This project is designed to define the normal role of the spinal muscular atrophy (SMA) protein in the post-transcriptional control of gene expression as well as to identify genes whose altered expression may contribute to degeneration of SMN-deficient motor neurons, the neuronal cells selectively affected in SMA. These studies should provide insights into the basic mechanisms of RNA regulation and the molecular defects underlying SMA pathogenesis with the potential of identifying new candidate targets for therapeutic development
Keywords: Address; Affect; Alternate Splicing; Alternative Splicing; Animal Disease Models; Aran-Duchenne disease; Assay; Atrophy, Muscle; Axon; Bioassay; Biologic Assays; Biological; Biological Assay; Biological Models; Biology; Body Tissues; Causality; Cell Line; Cell Lines, Strains; Cell model; CellLine; Cells; Cellular model; Connector Neuron; Cruveilhier disease; Defect; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Development; Disease; Disorder; Dysfunction; ES cell; Embryo; Embryonic; Etiology; Exons; Fibroblasts; Functional disorder; Gene Expression; Gene Expression Monitoring; Gene Expression Pattern Analysis; Gene Expression Profiling; Gene Products, RNA; Genes; Genetic Condition; Genetic Diseases; Genetics, Human; Goals; Hereditary; Hereditary Disease; Human; Human Genetics; Human, General; Impairment; Individual; Inherited; Intercalary Neuron; Intercalated Neurons; Intermediary Metabolism; Interneurons; Internuncial Cell; Internuncial Neuron; Isoforms; Lead; Link; Low Molecular Weight Nuclear RNA; METBL; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Medulla Spinalis; Metabolic Processes; Metabolism; Mice; Mitotic; Model System; Models, Biologic; Molecular; Molecular Disease; Motor Cell; Motor Neurons; Murine; Mus; Muscle, Skeletal; Muscle, Voluntary; Muscular Atrophy; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neuromuscular Diseases; Neurons; Pathogenesis; Pathway interactions; Pb element; Phenotype; Physiopathology; Post-Transcriptional Control; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Post-Transcriptional Regulation; Post-Transcriptional Regulation Process; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Process; Profilings, Gene Expression; Protein Isoforms; Proteins; Proteome; Quelling; RNA; RNA Interference; RNA Metabolism[{..}] Processing and Transport; RNA Processing; RNA Silencing; RNA Silencings; RNA Splicing; RNA Splicing, Alternative; RNA, Non-Polyadenylated; RNAi; RT-PCR; RTPCR; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic Acid; Role; SMN gene product (SMA); SMN protein (spinal muscular atrophy); Sequence-Specific Posttranscriptional Gene Silencing; Severity of illness; Skeletal Muscle Tissue; Skeletal muscle structure; Small Molecular Weight RNA; Small Nuclear RNA; Small Nuclear RNP; Small Nuclear Ribonucleoprotein Particle; Small Nuclear Ribonucleoproteins; Spinal; Spinal Cord; Spinal Muscular Atrophy; Splicing; Staging; Survival Analyses; Survival Analysis; Therapeutic Intervention; Time; Tissue Extracts; Tissues; Transcript Expression Analyses; Transcript Expression Analysis; Validation; base; cell type; comparative; cultured cell line; design; designing; disease causation; disease etiology; disease severity; disease/disorder; disease/disorder etiology; disorder etiology; embryonic stem cell; experiment; experimental research; experimental study; gene product; genetic disorder; genome-wide; heavy metal Pb; heavy metal lead; hereditary disorder; human disease; in vivo; insight; intervention therapy; lentiviral-mediated; mRNA Expression; motoneuron; motor neuron degeneration; motor neuron function; myoneural disorder; neurodegenerative illness; neuromuscular disorder; neuronal; new therapeutic target; novel; pathophysiology; pathway; public health relevance; research study; restoration; reverse transcriptase PCR; snRNA; snRNP; snRNP Biogenesis; social role; stem; stem cell of embryonic origin; survival motor neuron gene product; survival motor neuron protein; therapeutic development; uRNA
Relevance: The biomedical relevance of understanding the mechanisms and regulation of RNA processing is highlighted by the growing list of human genetic disorders associated with defects in RNA metabolism. This project is designed to define the normal role of the spinal muscular atrophy (SMA) protein in the post-transcriptional control of gene expression as well as to identify genes whose altered expression may contribute to degeneration of SMN-deficient motor neurons, the neuronal cells selectively affected in SMA. These studies should provide insights into the basic mechanisms of RNA regulation and the molecular defects underlying SMA pathogenesis with the potential of identifying new candidate targets for therapeutic development
Project start date: 2010-04-15
Project end date: 2015-03-31
Budget start date: 15-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: PA-07-070
1R01NS069601-01 (2010): $343874
Livio Pellizzoni
Columbia University Health Sciences
Project start date: 2010-04-15
Project end date: 2015-03-31