A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I.

Myotonic dystrophy Type 1 (DM1) is a rare genetic disease caused by the expansion of CTG trinucleotide repeats ((CTG)exp) in the 3' untranslated region of the DMPK gene. The repeat transcripts sequester the RNA binding protein Muscleblind-like protein 1 (MBNL1) and hamper its normal function in pre-mRNA splicing. Overexpressing exogenous MBNL1 in the DM1 mouse model has been shown to rescue the splicing defects and reverse myotonia. Although a viable therapeutic strategy, pharmacological modulators of MBNL1 expression have not been identified. Here, we engineered a ZsGreen tag into the endogenous MBNL1 locus in HeLa cells and established a flow cytometry-based screening system to identify compounds that increase MBNL1 level. The initial screen of small molecule compound libraries identified more than thirty hits that increased MBNL1 expression greater than double the baseline levels. Further characterization of two hits revealed that the small molecule HDAC inhibitors, ISOX and vorinostat, increased MBNL1 expression in DM1 patient-derived fibroblasts and partially rescued the splicing defect caused by (CUG)exp repeats in these cells. These findings demonstrate the feasibility of this flow-based cytometry screen to identify both small molecule compounds and druggable targets for MBNL1 upregulation.

Transcriptional Profiling of a Selective CREB Binding Protein Bromodomain Inhibitor Highlights Therapeutic Opportunities.

Bromodomains are involved in transcriptional regulation through the recognition of acetyl lysine modifications on diverse proteins. Selective pharmacological modulators of bromodomains are lacking, although the largely hydrophobic nature of the pocket makes these modules attractive targets for small-molecule inhibitors. This work describes the structure-based design of a highly selective inhibitor of the CREB binding protein (CBP) bromodomain and its use in cell-based transcriptional profiling experiments. The inhibitor downregulated a number of inflammatory genes in macrophages that were not affected by a selective BET bromodomain inhibitor. In addition, the CBP bromodomain inhibitor modulated the mRNA level of the regulator of G-protein signaling 4 (RGS4) gene in neurons, suggesting a potential therapeutic opportunity for CBP inhibitors in the treatment of neurological disorders.

Identification of a Novel Mucin Gene HCG22 Associated With Steroid-Induced Ocular Hypertension.

PURPOSE: The pathophysiology of ocular hypertension (OH) leading to primary open-angle glaucoma shares many features with a secondary form of OH caused by treatment with glucocorticoids, but also exhibits distinct differences. In this study, a pharmacogenomics approach was taken to discover candidate genes for this disorder. METHODS: A genome-wide association study was performed, followed by an independent candidate gene study, using a cohort enrolled from patients treated with off-label intravitreal triamcinolone, and handling change in IOP as a quantitative trait. RESULTS: An intergenic quantitative trait locus (QTL) was identified at chromosome 6p21.33 near the 5' end of HCG22 that attained the accepted statistical threshold for genome-level significance. The HCG22 transcript, encoding a novel mucin protein, was expressed in trabecular meshwork cells, and expression was stimulated by IL-1, and inhibited by triamcinolone acetate and TGF-ß. Bioinformatic analysis defined the QTL as an approximately 4 kilobase (kb) linkage disequilibrium block containing 10 common single nucleotide polymorphisms (SNPs). Four of these SNPs were identified in the National Center for Biotechnology Information (NCBI) GTEx eQTL browser as modifiers of HCG22 expression. Most are predicted to disrupt or improve motifs for transcription factor binding, the most relevant being disruption of the glucocorticoid receptor binding motif. A second QTL was identified within the predicted signal peptide of the HCG22 encoded protein that could affect its secretion. Translation, O-glycosylation, and secretion of the predicted HCG22 protein was verified in cultured trabecular meshwork cells. CONCLUSIONS: Identification of two independent QTLs that could affect expression of the HCG22 mucin gene product via two different mechanisms (transcription or secretion) is highly suggestive of a role in steroid-induced OH.

A systems biology approach utilizing a mouse diversity panel identifies genetic differences influencing isoniazid-induced microvesicular steatosis.

Isoniazid (INH), the mainstay therapeutic for tuberculosis infection, has been associated with rare but serious hepatotoxicity in the clinic. However, the mechanisms underlying inter-individual variability in the response to this drug have remained elusive. A genetically diverse mouse population model in combination with a systems biology approach was utilized to identify transcriptional changes, INH-responsive metabolites, and gene variants that contribute to the liver response in genetically sensitive individuals. Sensitive mouse strains developed severe microvesicular steatosis compared with corresponding vehicle control mice following 3 days of oral treatment with INH. Genes involved in mitochondrial dysfunction were enriched among liver transcripts altered with INH treatment. Those associated with INH treatment and susceptibility to INH-induced steatosis in the liver included apolipoprotein A-IV, lysosomal-associated membrane protein 1, and choline phosphotransferase 1. These alterations were accompanied by metabolomic changes including reduced levels of glutathione and the choline metabolites betaine and phosphocholine, suggesting that oxidative stress and reduced lipid export may additionally contribute to INH-induced steatosis. Finally, genome-wide association mapping revealed that polymorphisms in perilipin 2 were linked to increased triglyceride levels following INH treatment, implicating a role for inter-individual differences in lipid packaging in the susceptibility to INH-induced steatosis. Taken together, our data suggest that INH-induced steatosis is caused by not one, but multiple events involving lipid retention in the livers of genetically sensitive individuals. This work also highlights the value of using a mouse diversity panel to investigate drug-induced responses across a diverse population.

More than just hormones: H295R cells as predictors of reproductive toxicity.

Many of the commonly observed reproductive toxicities associated with therapeutic compounds can be traced to a disruption of the steroidogenic pathway. We sought to develop an in vitro assay that would predict reproductive toxicity and be high throughput in nature. H295R cells, previously validated as having an intact and functional steroidogenic pathway, were treated with 83 known-positive and 79 known-negative proprietary and public-domain compounds. The assay measured the expression of the key enzymes STAR, 3ßHSD2, CYP17A1, CYP11B2, CYP19A1, CYP21A2, and CYP11A1 and the hormones DHEA, progesterone, testosterone, and cortisol. We found that a Random Forest model yielded a receiver operating characteristic area under the curve (ROC AUC) of 0.845, with sensitivity of 0.724 and specificity of 0.758 for predicting in vivo reproductive toxicity with this in vitro assay system.

Dystrophic muscle improvement in zebrafish via increased heme oxygenase signaling.

Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx(5cv) mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications.

Transcriptomic analysis of genetically defined autism candidate genes reveals common mechanisms of action.

BACKGROUND: Austism spectrum disorder (ASD) is a heterogeneous behavioral disorder or condition characterized by severe impairment of social engagement and the presence of repetitive activities. The molecular etiology of ASD is still largely unknown despite a strong genetic component. Part of the difficulty in turning genetics into disease mechanisms and potentially new therapeutics is the sheer number and diversity of the genes that have been associated with ASD and ASD symptoms. The goal of this work is to use shRNA-generated models of genetic defects proposed as causative for ASD to identify the common pathways that might explain how they produce a core clinical disability. METHODS: Transcript levels of Mecp2, Mef2a, Mef2d, Fmr1, Nlgn1, Nlgn3, Pten, and Shank3 were knocked-down in mouse primary neuron cultures using shRNA constructs. Whole genome expression analysis was conducted for each of the knockdown cultures as well as a mock-transduced culture and a culture exposed to a lentivirus expressing an anti-luciferase shRNA. Gene set enrichment and a causal reasoning engine was employed to identify pathway level perturbations generated by the transcript knockdown. RESULTS: Quantification of the shRNA targets confirmed the successful knockdown at the transcript and protein levels of at least 75% for each of the genes. After subtracting out potential artifacts caused by viral infection, gene set enrichment and causal reasoning engine analysis showed that a significant number of gene expression changes mapped to pathways associated with neurogenesis, long-term potentiation, and synaptic activity. CONCLUSIONS: This work demonstrates that despite the complex genetic nature of ASD, there are common molecular mechanisms that connect many of the best established autism candidate genes. By identifying the key regulatory checkpoints in the interlinking transcriptional networks underlying autism, we are better able to discover the ideal points of intervention that provide the broadest efficacy across the diverse population of autism patients.

Assessing the translatability of in vivo cardiotoxicity mechanisms to in vitro models using causal reasoning.

Drug-induced cardiac toxicity has been implicated in 31% of drug withdrawals in the USA. The fact that the risk for cardiac-related adverse events goes undetected in preclinical studies for so many drugs underscores the need for better, more predictive in vitro safety screens to be deployed early in the drug discovery process. Unfortunately, many questions remain about the ability to accurately translate findings from simple cellular systems to the mechanisms that drive toxicity in the complex in vivo environment. In this study, we analyzed translatability of cardiotoxic effects for a diverse set of drugs from rodents to two different cell systems (rat heart tissue-derived cells (H9C2) and primary rat cardiomyocytes (RCM)) based on their transcriptional response. To unravel the altered pathway, we applied a novel computational systems biology approach, the Causal Reasoning Engine (CRE), to infer upstream molecular events causing the observed gene expression changes. By cross-referencing the cardiotoxicity annotations with the pathway analysis, we found evidence of mechanistic convergence towards common molecular mechanisms regardless of the cardiotoxic phenotype. We also experimentally verified two specific molecular hypotheses that translated well from in vivo to in vitro (Kruppel-like factor 4, KLF4 and Transforming growth factor beta 1, TGFB1) supporting the validity of the predictions of the computational pathway analysis. In conclusion, this work demonstrates the use of a novel systems biology approach to predict mechanisms of toxicity such as KLF4 and TGFB1 that translate from in vivo to in vitro. We also show that more complex in vitro models such as primary rat cardiomyocytes may not offer any advantage over simpler models such as immortalized H9C2 cells in terms of translatability to in vivo effects if we consider the right endpoints for the model. Further assessment and validation of the generated molecular hypotheses would greatly enhance our ability to design predictive in vitro cardiotoxicity assays.

Usf1, a suppressor of the circadian Clock mutant, reveals the nature of the DNA-binding of the CLOCK:BMAL1 complex in mice.

Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a ∼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals. DOI:http://dx.doi.org/10.7554/eLife.00426.001.

Evaluating genetic markers and neurobiochemical analytes for fluoxetine response using a panel of mouse inbred strains.

RATIONALE: Identification of biomarkers that establish diagnosis or treatment response is critical to the advancement of research and management of patients with depression. OBJECTIVE: Our goal was to identify biomarkers that can potentially assess fluoxetine response and risk to poor treatment outcome. METHODS: We measured behavior, gene expression, and the levels of 36 neurobiochemical analytes across a panel of genetically diverse mouse inbred lines after chronic treatment with water or fluoxetine. RESULTS: Glyoxylase 1 (GLO1) and guanine nucleotide-binding protein 1 (GNB1) mostly account for baseline anxiety-like and depressive-like behavior, indicating a common biological link between depression and anxiety. Fluoxetine-induced biochemical alterations discriminated positive responders, while baseline neurobiochemical differences differentiated negative responders (p < 0.006). Results show that glial fibrillary acidic protein, S100 beta protein, GLO1, and histone deacetylase 5 contributed most to fluoxetine response. These proteins are linked within a cellular growth/proliferation pathway, suggesting the involvement of cellular genesis in fluoxetine response. Furthermore, a candidate genetic locus that associates with baseline depressive-like behavior contains a gene that encodes for cellular proliferation/adhesion molecule (Cadm1), supporting a genetic basis for the role of neuro/gliogenesis in depression. CONCLUSION: We provided a comprehensive analysis of behavioral, neurobiochemical, and transcriptome data across 30 mouse inbred strains that has not been accomplished before. We identified biomarkers that influence fluoxetine response, which, altogether, implicate the importance of cellular genesis in fluoxetine treatment. More broadly, this approach can be used to assess a wide range of drug response phenotypes that are challenging to address in human samples.

Integrative analysis of low- and high-resolution eQTL.

The study of expression quantitative trait loci (eQTL) is a powerful way of detecting transcriptional regulators at a genomic scale and for elucidating how natural genetic variation impacts gene expression. Power and genetic resolution are heavily affected by the study population: whereas recombinant inbred (RI) strains yield greater statistical power with low genetic resolution, using diverse inbred or outbred strains improves genetic resolution at the cost of lower power. In order to overcome the limitations of both individual approaches, we combine data from RI strains with genetically more diverse strains and analyze hippocampus eQTL data obtained from mouse RI strains (BXD) and from a panel of diverse inbred strains (Mouse Diversity Panel, MDP). We perform a systematic analysis of the consistency of eQTL independently obtained from these two populations and demonstrate that a significant fraction of eQTL can be replicated. Based on existing knowledge from pathway databases we assess different approaches for using the high-resolution MDP data for fine mapping BXD eQTL. Finally, we apply this framework to an eQTL hotspot on chromosome 1 (Qrr1), which has been implicated in a range of neurological traits. Here we present the first systematic examination of the consistency between eQTL obtained independently from the BXD and MDP populations. Our analysis of fine-mapping approaches is based on 'real life' data as opposed to simulated data and it allows us to propose a strategy for using MDP data to fine map BXD eQTL. Application of this framework to Qrr1 reveals that this eQTL hotspot is not caused by just one (or few) 'master regulators', but actually by a set of polymorphic genes specific to the central nervous system.

Quantitative trait locus analysis identifies Gabra3 as a regulator of behavioral despair in mice.

The Tail Suspension Test (TST), which measures behavioral despair, is widely used as an animal model of human depressive disorders and antidepressant efficacy. In order to identify novel genes involved in the regulation of TST performance, we crossed an inbred strain exhibiting low immobility in the TST (RIIIS/J) with two high-immobility strains (C57BL/6J and NZB/BlNJ) to create two distinct F2 hybrid populations. All F2 offspring (n = 655) were genotyped at high density with a panel of SNP markers. Whole-genome interval mapping of the F2 populations identified statistically significant quantitative trait loci (QTLs) on mouse chromosomes (MMU) 4, 6, and X. Microarray analysis of hippocampal gene expression in the three parental strains was used to identify potential candidate genes within the MMUX QTLs identified in the NZB/BlNJ x RIIIS/J cross. Expression of Gabra3, which encodes the GABA(A) receptor alpha3 subunit, was robust in the hippocampus of B6 and RIIIS mice but absent from NZB hippocampal tissue. To verify the role of Gabra3 in regulating TST behavior in vivo, mice were treated with SB-205384, a positive modulator of the alpha3 subunit. SB-205384 significantly reduced TST immobility in B6 mice without affecting general activity, but it had no effect on behavior in NZB mice. This work suggests that GABRA3 regulates a behavioral endophenotype of depression and establishes this gene as a viable new target for the study and treatment of human depression.

Phenotypic characterization of a genetically diverse panel of mice for behavioral despair and anxiety.

BACKGROUND: Animal models of human behavioral endophenotypes, such as the Tail Suspension Test (TST) and the Open Field assay (OF), have proven to be essential tools in revealing the genetics and mechanisms of psychiatric diseases. As in the human disorders they model, the measurements generated in these behavioral assays are significantly impacted by the genetic background of the animals tested. In order to better understand the strain-dependent phenotypic variability endemic to this type of work, and better inform future studies that rely on the data generated by these models, we phenotyped 33 inbred mouse strains for immobility in the TST, a mouse model of behavioral despair, and for activity in the OF, a model of general anxiety and locomotor activity. RESULTS: We identified significant strain-dependent differences in TST immobility, and in thigmotaxis and distance traveled in the OF. These results were replicable over multiple testing sessions and exhibited high heritability. We exploited the heritability of these behavioral traits by using in silico haplotype-based association mapping to identify candidate genes for regulating TST behavior. Two significant loci (-logp >7.0, gFWER adjusted p value <0.05) of approximately 300 kb each on MMU9 and MMU10 were identified. The MMU10 locus is syntenic to a major human depressive disorder QTL on human chromosome 12 and contains several genes that are expressed in brain regions associated with behavioral despair. CONCLUSIONS: We report the results of phenotyping a large panel of inbred mouse strains for depression and anxiety-associated behaviors. These results show significant, heritable strain-specific differences in behavior, and should prove to be a valuable resource for the behavioral and genetics communities. Additionally, we used haplotype mapping to identify several loci that may contain genes that regulate behavioral despair.

Expression quantitative trait loci mapping identifies new genetic models of glutathione S-transferase variation.

Expression quantitative trait loci (eQTL) mapping can be used to identify the genetic variations that underlie inherited differences in gene transcription. We performed eQTL mapping by combining whole genome transcriptional data from the hypothalami of 33 strains of inbred mice with a detailed haplotype map of those same strains, revealing 10,655 trans associations and 31 cis eQTLs. One of the cis associations was found to be driven by strain-specific variation in the expression of Glutathione S-transferase, mu 5 (Gstm5). Gstm5 is one of seven members of the glutathione S-transferase, Mu family of genes. The glutathione S-transferases are phase II metabolic enzymes and are key regulators of drug and toxin clearance. In mouse, all seven family members are tightly clustered on mouse chromosome 3. Investigation of the Gstm5 cis association in multiple tissues types revealed that an 84-kilobase region on MMU3 acts as a haplotype-specific locus control region for the glutathione S-transferase, Mu cluster. In the strains that share the minor haplotype, drastic reductions in mRNA levels in multiple members of the Gst Mu family were observed. The strain-specific differences in Gst Mu transcription characterized here accurately model the human population, in which extreme variations in expression of GST Mu family members have been observed. Furthermore, the reduction in Gst Mu levels has important relevance for pharmacology and toxicology studies conducted in these strains. For instance, the reduced levels of Gst Mu in general and Gstm5 in particular have implications in models of dopamine metabolism, Parkinson's disease, and chemical neurotoxicity.

Glucocorticoid receptor polymorphisms and intraocular pressure response to intravitreal triamcinolone acetonide.

BACKGROUND: Elevation of intraocular pressure (IOP) following injection of intravitreal triamcinolone acetonide (IVTA) is an important clinical problem. The etiology of the steroid response is poorly understood, although a genetic determinant has long been suspected. We performed a pharmacogenomic association study with glucocorticoid receptor polymorphisms. MATERIALS AND METHODS: Fifty-two patients (56 eyes) who underwent treatment with IVTA for various retinal diseases were genotyped for six well-studied glucocorticoid receptor polymorphisms (ER22/23EK, N363S, BclI, N766N, and single nucleotide polymorphisms (SNPs) within introns 3 and 4). RESULTS: Three polymorphisms (ER22/23EK, N363S, and the intron 3 SNP) were essentially nonpolymorphic within this population sample and excluded from further analysis. The remaining three polymorphisms (BclI, N766N, and within intron 4) passed the Hardy-Weinberg Equilibrium test, indicating good genotyping quality and normal population distribution of allelic frequency. No statistically significant correlations were found between these three polymorphisms and magnitude of IOP elevation following IVTA, using single point association and haplotype analyses. CONCLUSIONS: In this small, pilot study, we found no statistically significant relationship between glucocorticoid receptor polymorphisms and IOP elevation following IVTA. The precise etiology of the steroid response remains obscure. To our knowledge, this is the first published pharmacogenomic study of this common clinical entity.

Association between the nociceptin receptor gene (OPRL1) single nucleotide polymorphisms and alcohol dependence.

OPRL1 encodes the nociceptin receptor, which has been shown to be involved in alcohol dependence in previous studies. In the present study, we investigated the association between genetic polymorphisms of OPRL1 and alcohol dependence in a Scandinavian population. We genotyped 15 single nucleotide polymorphisms (SNPs) spanning the OPRL1 locus and found that SNP rs6010718 was significantly associated with both Type I and Type II alcoholics (P < 0.05). Linkage disequilibrium and haplotype analysis identified two haplotype blocks in this region. Furthermore, two haplotypes composed of five tag SNPs showed significant association with alcohol dependence. These findings suggest that genetic variants of the OPRL1 gene play a role in alcohol dependence in the Scandinavian population, warranting further investigation at the OPRL1 locus.

Genetic regulation of behavioral and neuronal responses to fluoxetine.

Despite widespread use of antidepressants, the factors underlying the behavioral response to antidepressants are unknown. It has been shown that antidepressant treatment promotes the proliferation and survival of neurons in the adult hippocampus via enhanced serotonergic signaling, but it is unclear whether hippocampal neurogenesis is responsible for the behavioral response to antidepressants. Furthermore, a large subpopulation of patients fails to respond to antidepressant treatment due to presumed underlying genetic factors. In the present study, we have used the phenotypic and genotypic variability of inbred mouse strains to show that there is a genetic component to both the behavioral and neuronal effects of chronic fluoxetine treatment, and that this antidepressant induces an increase in hippocampal cell proliferation only in the strains that also show a positive behavioral response to treatment. Furthermore, the behavioral and neuronal responses are associated with an upregulation of genes known to promote neuronal proliferation and survival. These results suggest that inherent genetic predisposition to increased serotonin-induced neurogenesis may be a determinant of antidepressant efficacy.

Generation, identification and functional characterization of the nob4 mutation of Grm6 in the mouse.

We performed genome-wide chemical mutagenesis of C57BL/6J mice using N-ethyl-N-nitrosourea (ENU). Electroretinographic screening of the third generation offspring revealed two G3 individuals from one G1 family with a normal a-wave but lacking the b-wave that we named nob4. The mutation was transmitted with a recessive mode of inheritance and mapped to chromosome 11 in a region containing the Grm6 gene, which encodes a metabotropic glutamate receptor protein, mGluR6. Sequencing confirmed a single nucleotide substitution from T to C in the Grm6 gene. The mutation is predicted to result in substitution of Pro for Ser at position 185 within the extracellular, ligand-binding domain and oocytes expressing the homologous mutation in mGluR6 did not display robust glutamate-induced currents. Retinal mRNA levels for Grm6 were not significantly reduced, but no immunoreactivity for mGluR6 protein was found. Histological and fundus evaluations of nob4 showed normal retinal morphology. In contrast, the mutation has severe consequences for visual function. In nob4 mice, fewer retinal ganglion cells (RGCs) responded to the onset (ON) of a bright full field stimulus. When ON responses could be evoked, their onset was significantly delayed. Visual acuity and contrast sensitivity, measured with optomotor responses, were reduced under both photopic and scotopic conditions. This mutant will be useful because its phenotype is similar to that of human patients with congenital stationary night blindness and will provide a tool for understanding retinal circuitry and the role of ganglion cell encoding of visual information.