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 A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging

2001 ◽  
Vol 6 (4) ◽  
pp. 219-223 ◽  
Author(s):  
John M. Joern ◽  
Takeshi Sakamoto ◽  
Akira Arisawa ◽  
Frances H. Arnold
Keyword(s):  
Image Analysis ◽  
Directed Evolution ◽  
High Throughput ◽  
Digital Imaging ◽  
Solid Phase ◽  
High Throughput Screen ◽  
Microplate Reader ◽  
Dihydrodiol Dehydrogenase ◽  
Dioxygenase Activity

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-di- hydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30,uM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.

scholarly journals Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Melissa B. Walsh ◽  
Eva Janzen ◽  
Emily Wingrove ◽  
Seyyedmohsen Hosseinibarkooie ◽  
Natalia Rodriguez Muela ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Muscular Atrophy ◽  
Protein Complexes ◽  
Genetic Modifiers ◽  
Functional Connection ◽  
C Elegans ◽  
Hnrnp F ◽  
Smn Protein ◽  
Survival Of Motor Neuron

Abstract Background Understanding the genetic modifiers of neurodegenerative diseases can provide insight into the mechanisms underlying these disorders. Here, we examine the relationship between the motor neuron disease spinal muscular atrophy (SMA), which is caused by reduced levels of the survival of motor neuron (SMN) protein, and the actin-bundling protein Plastin 3 (PLS3). Increased PLS3 levels suppress symptoms in a subset of SMA patients and ameliorate defects in SMA disease models, but the functional connection between PLS3 and SMN is poorly understood. Results We provide immunohistochemical and biochemical evidence for large protein complexes localized in vertebrate motor neuron processes that contain PLS3, SMN, and members of the hnRNP F/H family of proteins. Using a Caenorhabditis elegans (C. elegans) SMA model, we determine that overexpression of PLS3 or loss of the C. elegans hnRNP F/H ortholog SYM-2 enhances endocytic function and ameliorates neuromuscular defects caused by decreased SMN-1 levels. Furthermore, either increasing PLS3 or decreasing SYM-2 levels suppresses defects in a C. elegans ALS model. Conclusions We propose that hnRNP F/H act in the same protein complex as PLS3 and SMN and that the function of this complex is critical for endocytic pathways, suggesting that hnRNP F/H proteins could be potential targets for therapy development.

scholarly journals High-throughput screening reveals novel mutations in spinal muscular atrophy patients

2019 ◽  
Author(s):  
Jianbo Shu ◽  
Jingrui Wang ◽  
Yulian Fang ◽  
Zanmei Xu ◽  
Xiaowei Wang ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Throughput Sequencing ◽  
Muscular Atrophy ◽  
Point Mutations ◽  
Control Group ◽  
Compound Heterozygous ◽  
Single Nucleotide Variants ◽  
Illumina Hiseq

Abstract Background Some spinal muscular atrophy (SMA) cases are caused by either compound heterozygosity with a point mutation in one allele and a deletion in the other or compound heterozygous point mutations in SMN1 or other genes. Methods To explore more genes and mutations in the onset of SMA, 83 whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was firstly performed with a SALSA MLPA Kit P021 for preliminary diagnosis. Afterwards, the complete gene sequence of SMN1 gene was detected with the high-throughput sequencing platform of Illumina HiSeq-2500 to find more mutations in the 28 core families. Furthermore, 20 SMA patients were selected from the 28 prodands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results MLPA results showed that 22 probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. 6 SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C>T], c.[271C>T], c.[-39A>G] and g.[70240639G>C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[-41_-40insCTCT], FUT5 SNV c.[1001A>G], and MCCC2 SNV c.[-117A>G] were the 3 most frequent mutations in SMA group (95%, 85% and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C>T], c.[-41_-40insCTCT], c.[1001A>G] and c.[-117A>G] might be associated with the onset of SMA.

scholarly journals Impairment of the neurotrophic signaling hub B-Raf contributes to motoneuron degeneration in spinal muscular atrophy

2021 ◽  
Vol 118 (18) ◽  
pp. e2007785118
Author(s):  
Niko Hensel ◽  
Federica Cieri ◽  
Pamela Santonicola ◽  
Ines Tapken ◽  
Tobias Schüning ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Signaling Pathways ◽  
Muscular Atrophy ◽  
Direct Interaction ◽  
Erk Pathway ◽  
Motoneuron Survival ◽  
C Elegans ◽  
Protein Levels ◽  
Approved Drugs ◽  
Motoneuron Degeneration

Spinal muscular atrophy (SMA) is a motoneuron disease caused by deletions of the Survival of Motoneuron 1 gene (SMN1) and low SMN protein levels. SMN restoration is the concept behind a number of recently approved drugs which result in impressive yet limited effects. Since SMN has already been enhanced in treated patients, complementary SMN-independent approaches are needed. Previously, a number of altered signaling pathways which regulate motoneuron degeneration have been identified as candidate targets. However, signaling pathways form networks, and their connectivity is still unknown in SMA. Here, we used presymptomatic SMA mice to elucidate the network of altered signaling in SMA. The SMA network is structured in two clusters with AKT and 14-3-3 ζ/δ in their centers. Both clusters are connected by B-Raf as a major signaling hub. The direct interaction of B-Raf with 14-3-3 ζ/δ is important for an efficient neurotrophic activation of the MEK/ERK pathway and crucial for motoneuron survival. Further analyses in SMA mice revealed that both proteins were down-regulated in motoneurons and the spinal cord with B-Raf being reduced at presymptomatic stages. Primary fibroblasts and iPSC-derived motoneurons from SMA patients both showed the same pattern of down-regulation. This mechanism is conserved across species since a Caenorhabditis elegans SMA model showed less expression of the B-Raf homolog lin-45. Accordingly, motoneuron survival was rescued by a cell autonomous lin-45 expression in a C. elegans SMA model resulting in improved motor functions. This rescue was effective even after the onset of motoneuron degeneration and mediated by the MEK/ERK pathway.

scholarly journals Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy

2005 ◽  
Vol 14 (14) ◽  
pp. 2003-2018 ◽  
Author(s):  
Jill Jarecki ◽  
Xiaocun Chen ◽  
Alexandra Bernardino ◽  
Daniel D. Coovert ◽  
Michael Whitney ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Small Molecule ◽  
High Throughput ◽  
Muscular Atrophy ◽  
Compound Screening ◽  
Small Molecule Modulators

scholarly journals High-throughput screening reveals novel mutations in spinal muscular atrophy patients

2020 ◽  
Vol 46 (1) ◽  
Author(s):  
Ruiping Zhang ◽  
Chunyu Gu ◽  
Linjie Pu ◽  
Yingtao Meng ◽  
Jianbo Shu ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Throughput Sequencing ◽  
Muscular Atrophy ◽  
Hereditary Disease ◽  
Control Group ◽  
Single Nucleotide Variants ◽  
Frequent Mutations ◽  
Multiplex Ligation Probe Amplification

Abstract Background Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disease associated with severe muscle atrophy and weakness in the limbs and trunk. The discovery of mutated genes is helpful in diagnosis and treatment for SMA. Methods Eighty-three whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was performed. Afterwards, the complete gene sequence of SMN1 gene was detected. Furthermore, 20 SMA patients were selected from the 28 probands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results Twenty-two probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. Six SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C > T], c.[271C > T], c.[−39A > G] and g.[70240639G > C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[−41_-40insCTCT], FUT5 SNV c.[1001A > G], and MCCC2 SNV c.[−117A > G] were the 3 most frequent mutations in SMA group (95, 85 and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C > T], c.[−41_-40insCTCT], c.[1001A > G] and c.[−117A > G] might be associated with the onset of SMA.

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 Decreased microRNA levels lead to deleterious increases in neuronal M2 muscarinic receptors in Spinal Muscular Atrophy models

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Patrick J O'Hern ◽  
Inês do Carmo G. Gonçalves ◽  
Johanna Brecht ◽  
Eduardo Javier López Soto ◽  
Jonah Simon ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Muscular Atrophy ◽  
Spinal Motor Neuron ◽  
C Elegans ◽  
M2 Muscarinic Receptor ◽  
Smn Protein ◽  
Survival Of Motor Neuron ◽  
Microrna Function ◽  
M2 Muscarinic Receptors

Spinal Muscular Atrophy (SMA) is caused by diminished Survival of Motor Neuron (SMN) protein, leading to neuromuscular junction (NMJ) dysfunction and spinal motor neuron (MN) loss. Here, we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs. Loss of the C. elegans SMN ortholog, SMN-1, causes NMJ defects. We found that increased levels of the C. elegans Gemin3 ortholog, MEL-46, ameliorates these defects. Increased MEL-46 levels also restored perturbed microRNA (miR-2) function in smn-1(lf) animals. We determined that miR-2 regulates expression of the C. elegans M2 muscarinic receptor (m2R) ortholog, GAR-2. GAR-2 loss ameliorated smn-1(lf) and mel-46(lf) synaptic defects. In an SMA mouse model, m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects. Collectively, these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation, followed by increased m2R expression, and neuronal dysfunction in SMA.

scholarly journals Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

2017 ◽  
Author(s):  
Blanca Hernando-Rodríguez ◽  
Annmary Paul Erinjeri ◽  
María Jesús Rodríguez-Palero ◽  
Val Millar ◽  
Sara González-Hernández ◽  
...  
Keyword(s):  
Image Analysis ◽  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Rnai Screen ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Automated Image Analysis ◽  
C Elegans ◽  
Larval Stages ◽  
Lethal Mutations

ABSTRACTBackgroundThe advancement in automated image based microscopy platforms coupled with high throughput liquid workflows has facilitated the design of large scale screens utilizing multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high throughput approaches.ResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosis using chromosome balancers, commonly labelled with GFP in the pharynx. Moreover gene-expression is typically monitored by the use of fluorescent reporters marked with the same fluorophore. Therefore, the separation of the different populations of animals at early larval stages represents a challenge. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at early larval stages. Because sorting is not completely error-free, we develop an automated high-throughput image-analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image-analysis in a functional genomic RNAi screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while, homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both, known and new PHB genetic interactors affecting the UPRmt and growth.ConclusionsA systematic way to analyse genetic interactions of essential genes in multicellular organisms is lacking. The method presented here allows the study of balanced lethal mutations in a high-throughput manner and can be easily adapted depending on the user’s requirements. Therefore, it will serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

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