scholarly journals Automated in vivo drug screen in zebrafish identifies synapse-stabilising drugs with relevance to spinal muscular atrophy

2021 ◽  
Vol 14 (4) ◽  
Author(s):  
Ana-Maria Oprişoreanu ◽  
Hannah L. Smith ◽  
Sophia Krix ◽  
Helena Chaytow ◽  
Neil O. Carragher ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Small Molecule ◽  
Synapse Formation ◽  
Muscular Atrophy ◽  
Neuromuscular Synapse ◽  
Axon Growth ◽  
Zebrafish Model ◽  
Synaptic Site ◽  
Automated Screening

ABSTRACT Synapses are particularly vulnerable in many neurodegenerative diseases and often the first to degenerate, for example in the motor neuron disease spinal muscular atrophy (SMA). Compounds that can counteract synaptic destabilisation are rare. Here, we describe an automated screening paradigm in zebrafish for small-molecule compounds that stabilize the neuromuscular synapse in vivo. We make use of a mutant for the axonal C-type lectin chondrolectin (chodl), one of the main genes dysregulated in SMA. In chodl−/− mutants, neuromuscular synapses that are formed at the first synaptic site by growing axons are not fully mature, causing axons to stall, thereby impeding further axon growth beyond that synaptic site. This makes axon length a convenient read-out for synapse stability. We screened 982 small-molecule compounds in chodl chodl−/− mutants and found four that strongly rescued motor axon length. Aberrant presynaptic neuromuscular synapse morphology was also corrected. The most-effective compound, the adenosine uptake inhibitor drug dipyridamole, also rescued axon growth defects in the UBA1-dependent zebrafish model of SMA. Hence, we describe an automated screening pipeline that can detect compounds with relevance to SMA. This versatile platform can be used for drug and genetic screens, with wider relevance to synapse formation and stabilisation.

scholarly journals Small Molecule Suppressors of Drosophila Kinesin Deficiency Rescue Motor Axon Development in a Zebrafish Model of Spinal Muscular Atrophy

PLoS ONE ◽  
2013 ◽  
Vol 8 (9) ◽  
pp. e74325 ◽  
Author(s):  
Andrew Gassman ◽  
Le T. Hao ◽  
Leena Bhoite ◽  
Chad L. Bradford ◽  
Chi-Bin Chien ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Small Molecule ◽  
Muscular Atrophy ◽  
Motor Axon ◽  
Zebrafish Model ◽  
Axon Development

scholarly journals Discovery of a CNS penetrant small molecule SMN2 splicing modulator with improved tolerability for spinal muscular atrophy

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shiori Ando ◽  
Shunya Suzuki ◽  
Shoichi Okubo ◽  
Kazuki Ohuchi ◽  
Kei Takahashi ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Small Molecule ◽  
Therapeutic Potential ◽  
Muscular Atrophy ◽  
Survival Motor Neuron ◽  
Tolerability Profile ◽  
Loss Of Function ◽  
Cryptic Exon

Abstract Spinal muscular atrophy (SMA) is a motor neuron disease, typically resulting from loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Nusinersen/SPINRAZA, a splice-switching oligonucleotide that modulates SMN2 (a paralog of SMN1) splicing and consequently increases SMN protein levels, has a therapeutic effect for SMA. Previously reported small-molecule SMN2 splicing modulators such as risdiplam/EVRYSDI and its analog SMN-C3 modulate not only the splicing of SMN2 but also that of secondary splice targets, including forkhead box protein M1 (FOXM1). Through screening SMA patient-derived fibroblasts, a novel small molecule, designated TEC-1, was identified that selectively modulates SMN2 splicing over three secondary splice targets. TEC-1 did not strongly affect the splicing of FOXM1, and unlike risdiplam, did not induce micronucleus formation. In addition, TEC-1 showed higher selectively on galactosylceramidase and huntingtin gene expression compared to previously reported compounds (e.g., SMN-C3) due to off-target effects on cryptic exon inclusion and nonsense-mediated mRNA decay. Moreover, TEC-1 significantly ameliorated the disease phenotype in an SMA murine model in vivo. Thus, TEC-1 may have promising therapeutic potential for SMA, and our study demonstrates the feasibility of RNA-targeting small-molecule drug development with an improved tolerability profile.

scholarly journals Chondrolectin affects cell survival and neuronal outgrowth in in vitro and in vivo models of spinal muscular atrophy

2013 ◽  
Vol 23 (4) ◽  
pp. 855-869 ◽  
Author(s):  
James N. Sleigh ◽  
Antón Barreiro-Iglesias ◽  
Peter L. Oliver ◽  
Angeliki Biba ◽  
Thomas Becker ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Cell Survival ◽  
Muscular Atrophy ◽  
In Vivo Models ◽  
Neuronal Outgrowth

scholarly journals Synthesis and Characterization of Pseudocantharidins, Novel Phosphatase Modulators That Promote the Inclusion of Exon 7 into the SMN (Survival of Motoneuron) pre-mRNA

2011 ◽  
Vol 286 (12) ◽  
pp. 10126-10136 ◽  
Author(s):  
Zhaiyi Zhang ◽  
Olga Kelemen ◽  
Maria A. van Santen ◽  
Sharon M. Yelton ◽  
Alison E. Wendlandt ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Central Element ◽  
Detectable Effect ◽  
Splice Site Selection ◽  
Eukaryotic Gene ◽  
Constitutive Splicing ◽  
New Compounds

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.

scholarly journals Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy

PLoS Genetics ◽  
2017 ◽  
Vol 13 (4) ◽  
pp. e1006744 ◽  
Author(s):  
Penelope J. Boyd ◽  
Wen-Yo Tu ◽  
Hannah K. Shorrock ◽  
Ewout J. N. Groen ◽  
Roderick N. Carter ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Muscular Atrophy ◽  
Zebrafish Model

scholarly journals Automated screening ofC. elegansneurodegeneration mutants enabled by microfluidics and image analysis algorithms

2018 ◽  
Vol 10 (9) ◽  
pp. 539-548 ◽  
Author(s):  
Ivan de Carlos Cáceres ◽  
Daniel A. Porto ◽  
Ivan Gallotta ◽  
Pamela Santonicola ◽  
Josue Rodríguez-Cordero ◽  
...  
Keyword(s):  
Image Analysis ◽  
Spinal Muscular Atrophy ◽  
High Throughput ◽  
Muscular Atrophy ◽  
High Throughput Screen ◽  
C Elegans ◽  
Genetic Mechanisms ◽  
Automated Screening

A fully automated high-throughput screen usingC. elegansto investigate genetic mechanisms affecting spinal muscular atrophy (SMA).

scholarly journals Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1636 ◽  
Author(s):  
Chris D. Balak ◽  
Jesse M. Hunter ◽  
Mary E. Ahearn ◽  
David Wiley ◽  
Gennaro D'urso ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Diagnostic Assays ◽  
Missense Variants ◽  
Thioester Bond ◽  
In The Wild ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1’s enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners.  These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations’ biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

scholarly journals Negative Cooperativity between Gemin2 and RNA provides Insights into RNA Selection and the SMN Complex’s Release in snRNP Assembly

2018 ◽  
Author(s):  
Hongfei Yi ◽  
Li Mu ◽  
Congcong Shen ◽  
Xi Kong ◽  
Yingzhi Wang ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Basic Mechanism ◽  
Negative Cooperativity ◽  
Sm Proteins ◽  
Early Step ◽  
Smn Complex ◽  
Narrow State ◽  
Snrnp Core

ABSTRACTThe assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex’s release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

scholarly journals Negative cooperativity between Gemin2 and RNA provides insights into RNA selection and the SMN complex's release in snRNP assembly

2019 ◽  
Vol 48 (2) ◽  
pp. 895-911 ◽  
Author(s):  
Hongfei Yi ◽  
Li Mu ◽  
Congcong Shen ◽  
Xi Kong ◽  
Yingzhi Wang ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Basic Mechanism ◽  
Negative Cooperativity ◽  
Sm Proteins ◽  
Early Step ◽  
Smn Complex ◽  
Narrow State ◽  
Snrnp Core

Abstract The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex's release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

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