Mimicking of splicing-related retinitis pigmentosa mutations in C. elegans allow drug screens and identification of disease modifiers

2020 ◽  
Vol 29 (5) ◽  
pp. 756-765 ◽  
Author(s):  
Dmytro Kukhtar ◽  
Karinna Rubio-Peña ◽  
Xènia Serrat ◽  
Julián Cerón
Keyword(s):  
Retinitis Pigmentosa ◽  
Retinal Disease ◽  
Model Organisms ◽  
Splicing Factors ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Dominant Form ◽  
C Elegans ◽  
Autosomal Dominant Form ◽  
Approved Drugs

Abstract CRISPR/Cas and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease retinitis pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in Caenorhabditis elegans, we generated mutant strains to mimic s-adRP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations as homozygotes, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including reduced fertility. However, snrp-200(cer24[S1080L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens for identifying genetic interactions, which could uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors: isy-1/ISY1, cyn-15/PPWD1 and mog-2/SNRPA1, whose partial inactivation may modify the course of the disease. Interestingly, these three genes act as modifiers of prp-8(cer22) but not of snrp-200(cer24). Finally, a screen of the strong allele prp-8(cer14) with FDA-approved drugs did not identify molecules capable of alleviating the temperature-sensitive sterility. Instead, we detected drugs, such as dequalinium chloride, which exacerbated the phenotype, and therefore, are potentially harmful to s-adRP patients since they may accelerate the progression of the disease.

scholarly journals Splicing-related Retinitis Pigmentosa mutations mimicked in C. elegans allow the identification of disease modifiers and drug screens

2019 ◽  
Author(s):  
Dmytro Kukhtar ◽  
Karinna Rubio-Peña ◽  
Xènia Serrat ◽  
Julián Cerón
Keyword(s):  
Retinitis Pigmentosa ◽  
Retinal Disease ◽  
Model Organisms ◽  
Splicing Factors ◽  
Missense Mutations ◽  
Dominant Form ◽  
C Elegans ◽  
Autosomal Dominant Form ◽  
High Conservation ◽  
Approved Drugs

ABSTRACTCRISPR and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease Retinitis Pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in C. elegans, we generated mutant strains to mimic RP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations in homozygosis, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including a reduced fertility. However, snrp-200(cer24[S1080L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens to identify genetic interactions, which would uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors, isy-1/ISY1, cyn-15/PPWD1 and mog-2/SNRPA1 whose partial inactivation may modify the course of the disease. Interestingly, these three genes were acting as modifiers of prp-8(cer22) but no snrp-200(cer24).Finally, the strong allele prp-8(cer14) was used in a screen with FDA-approved drugs to find molecules capable of alleviating the phenotype. Instead, we detected drugs, as Dequalinium Chloride, which exacerbated the phenotype and therefore are potentially harmful for s-adRP patients since they may accelerate the progression of the disease.

The familial hemiplegic migraine type 1 mutation K1336E affects direct G protein-mediated regulation of neuronal P/Q-type Ca2+ channels

Cephalalgia ◽  
2013 ◽  
Vol 33 (6) ◽  
pp. 398-407 ◽  
Author(s):  
Edgar Garza-López ◽  
Ricardo González-Ramírez ◽  
María A Gandini ◽  
Alejandro Sandoval ◽  
Ricardo Felix
Keyword(s):  
G Protein ◽  
Familial Hemiplegic Migraine ◽  
Inactivation Kinetics ◽  
Missense Mutations ◽  
Hemiplegic Migraine ◽  
Gene Encoding ◽  
Dominant Form ◽  
Hek 293 ◽  
Autosomal Dominant Form

Background Familial hemiplegic migraine type 1 (FHM-1) is an autosomal dominant form of migraine with aura characterized by recurrent migraine, hemiparesis and ataxia. FHM-1 has been linked to missense mutations in the CACNA1A gene encoding the pore-forming subunit of the neuronal voltage-gated P/Q-type Ca2+ channel (CaV2.1α1). Methods Here, we explored the effects of the FHM-1 K1336E mutation on G protein-dependent modulation of the recombinant P/Q-type channel. The mutation was introduced into the human CaV2.1α1 subunit and its functional consequences investigated after heterologous expression in HEK-293 cells using patch-clamp recordings. Results Functional analysis of the K1336E mutation revealed a reduction of Ca2+ current densities, a ∼10 mV left-shift in the current-voltage relationship, and the slowing of current inactivation kinetics. When co-expressed along with the human μ-opioid receptor, application of the agonist DAMGO inhibited whole-cell currents through both the wild-type and the mutant channels. Prepulse facilitation was also reduced by the K1336E mutation. Likewise, the kinetic analysis of the onset and decay of facilitation showed that the mutation affects the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex. Conclusions These results suggest that the extent of G-protein-mediated inhibition is significantly reduced in the K1336E mutant CaV2.1 Ca2+ channels. This alteration would contribute to render the neuronal network hyperexcitable, possibly as a consequence of reduced presynaptic inhibition, and may help to explain some aspects of the FHM-1 pathophysiology.

scholarly journals The FDA-approved drugs ticlopidine, sertaconazole, and dexlansoprazole can cause morphological changes in C. elegans

2020 ◽  
Author(s):  
Kyle F Galford ◽  
Antony M Jose
Keyword(s):  
Proton Pump Inhibitor ◽  
Genetic Model ◽  
Morphological Changes ◽  
Model System ◽  
Model Organisms ◽  
Regulatory Agencies ◽  
C Elegans ◽  
Therapeutic Drugs ◽  
Approved Drugs ◽  
Fda Approved Drugs

AbstractUrgent need for treatments limit studies of therapeutic drugs before approval by regulatory agencies. Analyses of drugs after approval can therefore improve our understanding of their mechanism of action and enable better therapies. We screened a library of 1443 Food and Drug Administration (FDA)-approved drugs using a simple assay in the nematode C. elegans and found three compounds that caused morphological changes. While the anticoagulant ticlopidine and the antifungal sertaconazole caused morphologically distinct pharyngeal defects upon acute exposure, the proton-pump inhibitor dexlansoprazole caused molting defects and required exposure during larval development. Such easily detectable defects in a powerful genetic model system advocate the continued exploration of current medicines using a variety of model organisms to better understand drugs already prescribed to millions of patients.

scholarly journals Mutation analysis of CACNA1A and ATP1A2 genes in Brazilian FHM families

2006 ◽  
Vol 64 (3a) ◽  
pp. 549-552
Author(s):  
Luciana R. Lopes ◽  
Mario Fernando Prieto Peres ◽  
Kaate R.J. Vanmolkot ◽  
Patrícia R. Tobo ◽  
Eliova Zukerman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Autosomal Dominant ◽  
Familial Hemiplegic Migraine ◽  
Missense Mutations ◽  
Asymptomatic Individual ◽  
Hemiplegic Migraine ◽  
Dominant Form ◽  
The Third ◽  
Autosomal Dominant Form

Familial hemiplegic migraine (FHM) is a rare autosomal dominant form of migraine with aura. This disease has been associated with missense mutations in the CACNA1A and ATP1A2 genes. The aim of this study was to identify whether CACNA1A and ATP1A2 are or not related to Brazilian FHM. Here we screened four Brazilian FHM families (total of 26 individuals - 13 affected and 13 asymptomatic or normal) for mutations in both genes. We found an amino acid change in a member of family FHM-D (Arg2206Gly). However since this alteration is not present in all affected individuals and is present in one asymptomatic individual it should be considered a polymorphism. Further studies with additional families will be necessary to reveal the importance of both CACNA1A and ATP1A2 genes on the pathogeneses of FHM in Brazil and to test the third gene (SCN1A) in these FHM families.

scholarly journals ER network formation and membrane fusion by atlastin1/SPG3A disease variants

2015 ◽  
Vol 26 (9) ◽  
pp. 1616-1628 ◽  
Author(s):  
Idil Ulengin ◽  
John J. Park ◽  
Tina H. Lee
Keyword(s):  
Membrane Fusion ◽  
Motor Neurons ◽  
Network Formation ◽  
Missense Mutations ◽  
Fusion Activity ◽  
Limb Weakness ◽  
Dominant Form ◽  
Biochemical Assays ◽  
Autosomal Dominant Form ◽  
Membrane Morphogenesis

At least 38 distinct missense mutations in the neuronal atlastin1/SPG3A GTPase are implicated in an autosomal dominant form of hereditary spastic paraplegia (HSP), a motor-neurological disorder manifested by lower limb weakness and spasticity and length-dependent axonopathy of corticospinal motor neurons. Because the atlastin GTPase is sufficient to catalyze membrane fusion and required to form the ER network, at least in nonneuronal cells, it is logically assumed that defects in ER membrane morphogenesis due to impaired fusion activity are the primary drivers of SPG3A-associated HSP. Here we analyzed a subset of established atlastin1/SPG3A disease variants using cell-based assays for atlastin-mediated ER network formation and biochemical assays for atlastin-catalyzed GTP hydrolysis, dimer formation, and membrane fusion. As anticipated, some variants exhibited clear deficits. Surprisingly however, at least two disease variants, one of which represents that most frequently identified in SPG3A HSP patients, displayed wild-type levels of activity in all assays. The same variants were also capable of co-redistributing ER-localized REEP1, a recently identified function of atlastins that requires its catalytic activity. Taken together, these findings indicate that a deficit in the membrane fusion activity of atlastin1 may be a key contributor, but is not required, for HSP causation.

scholarly journals Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing

2005 ◽  
Vol 168 (4) ◽  
pp. 599-606 ◽  
Author(s):  
Katia J. Evans ◽  
Edgar R. Gomes ◽  
Steven M. Reisenweber ◽  
Gregg G. Gundersen ◽  
Brett P. Lauring
Keyword(s):  
Axonal Degeneration ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Missense Mutations ◽  
Spinal Neurons ◽  
Dominant Form ◽  
Microtubule Severing ◽  
Autosomal Dominant Form ◽  
Dorsal Columns ◽  
Mutant Forms

Mutations in the AAA adenosine triphosphatase (ATPase) Spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia, which is a retrograde axonopathy primarily characterized pathologically by the degeneration of long spinal neurons in the corticospinal tracts and the dorsal columns. Using recombinant Spastin, we find that six mutant forms of Spastin, including three disease-associated forms, are severely impaired in ATPase activity. In contrast to a mutation designed to prevent adenosine triphosphate (ATP) binding, an ATP hydrolysis–deficient Spastin mutant predicted to remain kinetically trapped on target proteins decorates microtubules in transfected cells. Analysis of disease-associated missense mutations shows that some more closely resemble the canonical hydrolysis mutant, whereas others resemble the ATP-binding mutant. Using real-time imaging, we show that Spastin severs microtubules when added to permeabilized, cytosol-depleted cells stably expressing GFP-tubulin. Using purified components, we also show that Spastin interacts directly with microtubules and is sufficient for severing. These studies suggest that defects in microtubule severing are a cause of axonal degeneration in human disease.

scholarly journals RBM-5 modulates U2AF large subunit-dependent alternative splicing in C. elegans

2018 ◽  
Author(s):  
Chuanman Zhou ◽  
Xiaoyang Gao ◽  
Surong Hu ◽  
Wenjing Gan ◽  
Xu Jing ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Rna Splicing ◽  
Large Subunit ◽  
Splicing Factors ◽  
Temperature Sensitive ◽  
Subunit Gene ◽  
C Elegans ◽  
Expression Of Genes ◽  
Specific Manner

AbstractA key step in pre-mRNA splicing is the recognition of 3’ splicing sites by the U2AF large and small subunits, a process regulated by numerous trans-acting splicing factors. How these trans-acting factors interact with U2AF in vivo is unclear. From a screen for suppressors of the temperature-sensitive (ts) lethality of the C. elegans U2AF large subunit gene uaf-1(n4588) mutants, we identified mutations in the RNA binding motif gene rbm-5, a homolog of the tumor suppressor RBM5. rbm-5 mutations can suppress uaf-1(n4588) ts-lethality by loss of function and neuronal expression of rbm-5 was sufficient to rescue the suppression. Transcriptome analyses indicate that uaf-1(n4588) affected the expression of numerous genes and rbm-5 mutations can partially reverse the abnormal gene expression to levels similar to that of wild type. Though rbm-5 mutations did not obviously affect alternative splicing per se, they can suppress or enhance, in a gene-specific manner, the altered splicing of genes in uaf-1(n4588) mutants. Specifically, the recognition of a weak 3’ splice site was more susceptible to the effect of rbm-5. Our findings provide novel in vivo evidence that RBM-5 can modulate UAF-1-dependent RNA splicing and suggest that RBM5 might interact with U2AF large subunit to affect tumor formation.Author summaryRNA splicing is a critical regulatory step for eukaryotic gene expression and has been involved in the pathogenesis of multiple diseases. How RNA splicing factors interact in vivo to affect the splicing and expression of genes is unclear. In studying the temperature-sensitive lethal phenotypes of a mutation affecting the splicing factor U2AF large subunit gene uaf-1 in the nematode Caenorhabditis elegans, we isolated suppressive mutations in the rbm-5 gene, a homolog of the human tumor suppressor gene RBM5. rbm-5 is broadly expressed in neurons to enhance the lethality of the uaf-1 mutants. We found that the uaf-1 mutation causes aberrant expression of genes in numerous biological pathways, a large portion of which can be corrected by rbm-5 mutations. The abnormal splicing of multiple genes caused by the uaf-1 mutation is either corrected or enhanced by rbm-5 mutations in a gene-specific manner. We propose that RBM-5 interacts with UAF-1 to affect RNA splicing and the tumor suppressor function of RBM5 might involve U2AF-dependent RNA splicing.

scholarly journals Drug screening with zebrafish visual behavior identifies carvedilol as a potential treatment for an autosomal dominant form of retinitis pigmentosa

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Logan Ganzen ◽  
Mee Jung Ko ◽  
Mengrui Zhang ◽  
Rui Xie ◽  
Yongkai Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Retinitis Pigmentosa ◽  
Motor Response ◽  
Autosomal Dominant ◽  
Potential Treatment ◽  
Visual Behavior ◽  
Dominant Form ◽  
Autosomal Dominant Form ◽  
Visual Motor ◽  
Inherited Retinal Degeneration

AbstractRetinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

scholarly journals Retinitis pigmentosa is associated with shifts in the gut microbiome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Oksana Kutsyr ◽  
Lucía Maestre-Carballa ◽  
Mónica Lluesma-Gomez ◽  
Manuel Martinez-Garcia ◽  
Nicolás Cuenca ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Gut Microbiome ◽  
Functional Decline ◽  
Retinal Disease ◽  
Photoreceptor Cells ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Alpha And Beta Diversity ◽  
Potential Biomarker

AbstractThe gut microbiome is known to influence the pathogenesis and progression of neurodegenerative diseases. However, there has been relatively little focus upon the implications of the gut microbiome in retinal diseases such as retinitis pigmentosa (RP). Here, we investigated changes in gut microbiome composition linked to RP, by assessing both retinal degeneration and gut microbiome in the rd10 mouse model of RP as compared to control C57BL/6J mice. In rd10 mice, retinal responsiveness to flashlight stimuli and visual acuity were deteriorated with respect to observed in age-matched control mice. This functional decline in dystrophic animals was accompanied by photoreceptor loss, morphologic anomalies in photoreceptor cells and retinal reactive gliosis. Furthermore, 16S rRNA gene amplicon sequencing data showed a microbial gut dysbiosis with differences in alpha and beta diversity at the genera, species and amplicon sequence variants (ASV) levels between dystrophic and control mice. Remarkably, four fairly common ASV in healthy gut microbiome belonging to Rikenella spp., Muribaculaceace spp., Prevotellaceae UCG-001 spp., and Bacilli spp. were absent in the gut microbiome of retinal disease mice, while Bacteroides caecimuris was significantly enriched in mice with RP. The results indicate that retinal degenerative changes in RP are linked to relevant gut microbiome changes. The findings suggest that microbiome shifting could be considered as potential biomarker and therapeutic target for retinal degenerative diseases.

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