Disease-relevant Phenotypes Research Articles

Interaction between smoking and ATG16L1T300A triggers Paneth cell defects in Crohn's disease.

It is suggested that subtyping of complex inflammatory diseases can be based on genetic susceptibility and relevant environmental exposure (G+E). We propose that using matched cellular phenotypes in human subjects and corresponding preclinical models with the same G+E combinations is useful to this end. As an example, defective Paneth cells can subtype Crohn's disease (CD) subjects; Paneth cell defects have been linked to multiple CD susceptibility genes and are associated with poor outcome. We hypothesized that CD susceptibility genes interact with cigarette smoking, a major CD environmental risk factor, to trigger Paneth cell defects. We found that both CD subjects and mice with ATG16L1T300A (T300A; a prevalent CD susceptibility allele) developed Paneth cell defects triggered by tobacco smoke. Transcriptional analysis of full-thickness ileum and Paneth cell-enriched crypt base cells showed the T300A-smoking combination altered distinct pathways, including proapoptosis, metabolic dysregulation, and selective downregulation of the PPARγ pathway. Pharmacologic intervention by either apoptosis inhibitor or PPARγ agonist rosiglitazone prevented smoking-induced crypt apoptosis and Paneth cell defects in T300A mice and mice with conditional Paneth cell-specific knockout of Atg16l1. This study demonstrates how explicit G+E can drive disease-relevant phenotype and provides rational strategies for identifying actionable targets.

Crybb2 Mutations Consistently Affect Schizophrenia Endophenotypes in Mice.

As part of the βγ-superfamily, βB2-crystallin (CRYBB2) is an ocular structural protein in the lens, and mutation of the corresponding gene can cause cataracts. CRYBB2 also is expressed in non-lens tissue such as the adult mouse brain and is associated with neuropsychiatric disorders such as schizophrenia. Nevertheless, the robustness of this association as well as how CRYBB2 may contribute to disease-relevant phenotypes is unknown. To add further clarity to this issue, we performed a comprehensive analysis of behavioral and neurohistological alterations in mice with an allelic series of mutations in the C-terminal end of the Crybb2 gene. Behavioral phenotyping of these three βB2-mutant lines Crybb2O377, Crybb2Philly, and Crybb2Aey2 included assessment of exploratory activity and anxiety-related behavior in the open field, sensorimotor gating measured by prepulse inhibition (PPI) of the acoustic startle reflex, cognitive performance measured by social discrimination, and spontaneous alternation in the Y-maze. In each mutant line, we also quantified the number of parvalbumin-positive (PV+) GABAergic interneurons in selected brain regions that express CRYBB2. While there were allele-specific differences in individual behaviors and affected brain areas, all three mutant lines exhibited consistent alterations in PPI that paralleled alterations in the PV+ cell number in the thalamic reticular nucleus (TRN). The direction of the PPI change mirrored that of the TRN PV+ cell number thereby suggesting a role for TRN PV+ cell number in modulating PPI. Moreover, as both altered PPI and PV+ cell number are schizophrenia-associated endophenotypes, our result implicates mutated Crybb2 in the development of this neuropsychiatric disorder.

Using Patient-Derived Induced Pluripotent Stem Cells to Identify Parkinson's Disease-Relevant Phenotypes.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting older individuals. The specific cause underlying dopaminergic (DA) neuron loss in the substantia nigra, a pathological hallmark of PD, remains elusive. Here, we highlight peer-reviewed reports using induced pluripotent stem cells (iPSCs) to model PD in vitro and discuss the potential disease-relevant phenotypes that may lead to a better understanding of PD etiology. Benefits of iPSCs are that they retain the genetic background of the donor individual and can be differentiated into specialized neurons to facilitate disease modeling. Mitochondrial dysfunction, oxidative stress, ER stress, and alpha-synuclein accumulation are common phenotypes observed in PD iPSC-derived neurons. New culturing technologies, such as directed reprogramming and midbrain organoids, offer innovative ways of investigating intraneuronal mechanisms of PD pathology. PD patient-derived iPSCs are an evolving resource to understand PD pathology and identify therapeutic targets.

Small molecule inhibition of RAS/MAPK signaling ameliorates developmental pathologies of Kabuki Syndrome

Kabuki Syndrome (KS) is a rare disorder characterized by distinctive facial features, short stature, skeletal abnormalities, and neurodevelopmental deficits. Previously, we showed that loss of function of RAP1A, a RAF1 regulator, can activate the RAS/MAPK pathway and cause KS, an observation recapitulated in other genetic models of the disorder. These data suggested that suppression of this signaling cascade might be of therapeutic benefit for some features of KS. To pursue this possibility, we performed a focused small molecule screen of a series of RAS/MAPK pathway inhibitors, where we tested their ability to rescue disease-relevant phenotypes in a zebrafish model of the most common KS locus, kmt2d. Consistent with a pathway-driven screening paradigm, two of 27 compounds showed reproducible rescue of early developmental pathologies. Further analyses showed that one compound, desmethyl-Dabrafenib (dmDf), induced no overt pathologies in zebrafish embryos but could rescue MEK hyperactivation in vivo and, concomitantly, structural KS-relevant phenotypes in all KS zebrafish models (kmt2d, kmd6a and rap1). Mass spectrometry quantitation suggested that a 100 nM dose resulted in sub-nanomolar exposure of this inhibitor and was sufficient to rescue both mandibular and neurodevelopmental defects. Crucially, germline kmt2d mutants recapitulated the gastrulation movement defects, micrognathia and neurogenesis phenotypes of transient models; treatment with dmDf ameliorated all of them significantly. Taken together, our data reinforce a causal link between MEK hyperactivation and KS and suggest that chemical suppression of BRAF might be of potential clinical utility for some features of this disorder.

Discovery of a drug candidate for GLIS3-associated diabetes

GLIS3 mutations are associated with type 1, type 2, and neonatal diabetes, reflecting a key function for this gene in pancreatic β-cell biology. Previous attempts to recapitulate disease-relevant phenotypes in GLIS3−/− β-like cells have been unsuccessful. Here, we develop a “minimal component” protocol to generate late-stage pancreatic progenitors (PP2) that differentiate to mono-hormonal glucose-responding β-like (PP2-β) cells. Using this differentiation platform, we discover that GLIS3−/− hESCs show impaired differentiation, with significant death of PP2 and PP2-β cells, without impacting the total endocrine pool. Furthermore, we perform a high-content chemical screen and identify a drug candidate that rescues mutant GLIS3-associated β-cell death both in vitro and in vivo. Finally, we discovered that loss of GLIS3 causes β-cell death, by activating the TGFβ pathway. This study establishes an optimized directed differentiation protocol for modeling human β-cell disease and identifies a drug candidate for treating a broad range of GLIS3-associated diabetic patients.

Plate-Based Phenotypic Screening for Pain Using Human iPSC-Derived Sensory Neurons

Screening against a disease-relevant phenotype to identify compounds that change the outcome of biological pathways, rather than just the activity of specific targets, offers an alternative approach to find modulators of disease characteristics. However, in pain research, use of in vitro phenotypic screens has been impeded by the challenge of sourcing relevant neuronal cell types in sufficient quantity and developing functional end-point measurements with a direct disease link. To overcome these hurdles, we have generated human induced pluripotent stem cell (hiPSC)–derived sensory neurons at a robust production scale using the concept of cryopreserved “near-assay-ready” cells to decouple complex cell production from assay development and screening. hiPSC sensory neurons have then been used for development of a 384-well veratridine-evoked calcium flux assay. This functional assay of neuronal excitability was validated for phenotypic relevance to pain and other hyperexcitability disorders through screening a small targeted validation compound subset. A 2700-compound chemogenomics screen was then conducted to profile the range of target-based mechanisms able to inhibit veratridine-evoked excitability. This report presents the assay development, validation, and screening data. We conclude that high-throughput-compatible pain-relevant phenotypic screening with hiPSC sensory neurons is feasible and ready for application for the identification of new targets, pathways, mechanisms of action, and compounds for modulating neuronal excitability.

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration.

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

Microhomology-assisted scarless genome editing in human iPSCs

Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.

Neural Stem Cells and Human Induced Pluripotent Stem Cells to Model Rare CNS Diseases.

Despite the great effort spent over recent decades to unravel the pathological mechanisms underpinning the development of central nervous system disorders, most of them still remain unclear. In particular, the study of rare CNS diseases is hampered by the lack of postmortem samples and of reliable epidemiological studies, thus the setting of in vitro modeling systems appears essential to dissect the puzzle of genetic and environmental alterations affecting neural cells viability and functionality. The isolation and expansion in vitro of embryonic (ESC) and fetal neural stem cells (NSC) from human tissue have allowed the modeling of several neurological diseases "in a dish" and have also provided a novel platform to test potential therapeutic strategies in a pre-clinical setting. In recent years, the development of induced pluripotent stem cell (iPS) technology has added enormous value to the aforementioned approach, thanks to their capability for generating diseaserelevant cell phenotypes in vitro and to their perspective use in autologous transplantation. However, while the potentiality of ESC, NSC and iPS has been widely sponsored, the pitfalls related to the available protocols for differentiation and the heterogeneity of lines deriving from different individuals have been poorly discussed. Here we present pro and contra of using ESC, NSC or iPS for modeling rare diseases like Lysosomal Storage disorders and Motor Neuron Diseases. In this view, the advent of gene editing technologies is a unique opportunity to standardize the data analysis in preclinical studies and to tailor clinical protocols for stem cell-mediated therapy.

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models

Most neurodegenerative disorders are associated with accumulation of disease-relevant proteins. Among them, Huntington disease (HD) is of particular interest because of its monogenetic nature. HD is mainly caused by cytotoxicity of the defective protein encoded by the mutant Huntingtin gene (HTT). Thus, lowering mutant HTT protein (mHTT) levels would be a promising treatment strategy for HD. Here we report two kinases HIPK3 and MAPK11 as positive modulators of mHTT levels both in cells and in vivo. Both kinases regulate mHTT via their kinase activities, suggesting that inhibiting these kinases may have therapeutic values. Interestingly, their effects on HTT levels are mHTT-dependent, providing a feedback mechanism in which mHTT enhances its own level thus contributing to mHTT accumulation and disease progression. Importantly, knockout of MAPK11 significantly rescues disease-relevant behavioral phenotypes in a knockin HD mouse model. Collectively, our data reveal new therapeutic entry points for HD and target-discovery approaches for similar diseases.

Foxp1 in Forebrain Pyramidal Neurons Controls Gene Expression Required for Spatial Learning and Synaptic Plasticity.

Genetic perturbations of the transcription factor Forkhead Box P1 (FOXP1) are causative for severe forms of autism spectrum disorder that are often comorbid with intellectual disability. Recent work has begun to reveal an important role for FoxP1 in brain development, but the brain-region-specific contributions of Foxp1 to autism and intellectual disability phenotypes have yet to be determined fully. Here, we describe Foxp1 conditional knock-out (Foxp1cKO) male and female mice with loss of Foxp1 in the pyramidal neurons of the neocortex and the CA1/CA2 subfields of the hippocampus. Foxp1cKO mice exhibit behavioral phenotypes that are of potential relevance to autism spectrum disorder, including hyperactivity, increased anxiety, communication impairments, and decreased sociability. In addition, Foxp1cKO mice have gross deficits in learning and memory tasks of relevance to intellectual disability. Using a genome-wide approach, we identified differentially expressed genes in the hippocampus of Foxp1cKO mice associated with synaptic function and development. Furthermore, using magnetic resonance imaging, we uncovered a significant reduction in the volumes of both the entire hippocampus as well as individual hippocampal subfields of Foxp1cKO mice. Finally, we observed reduced maintenance of LTP in area CA1 of the hippocampus in these mutant mice. Together, these data suggest that proper expression of Foxp1 in the pyramidal neurons of the forebrain is important for regulating gene expression pathways that contribute to specific behaviors reminiscent of those seen in autism and intellectual disability. In particular, Foxp1 regulation of gene expression appears to be crucial for normal hippocampal development, CA1 plasticity, and spatial learning.SIGNIFICANCE STATEMENT Loss-of-function mutations in the transcription factor Forkhead Box P1 (FOXP1) lead to autism spectrum disorder and intellectual disability. Understanding the potential brain-region-specific contributions of FOXP1 to disease-relevant phenotypes could be a critical first step in the management of patients with these mutations. Here, we report that Foxp1 conditional knock-out (Foxp1cKO) mice with loss of Foxp1 in the neocortex and hippocampus display autism and intellectual-disability-relevant behaviors. We also show that these phenotypes correlate with changes in both the genomic and physiological profiles of the hippocampus in Foxp1cKO mice. Our work demonstrates that brain-region-specific FOXP1 expression may relate to distinct, clinically relevant phenotypes.

In search of a small molecule agonist of the relaxin receptor RXFP1 for the treatment of liver fibrosis

The peptide hormone human relaxin-2 (H2-RLX) has emerged as a potential therapy for cardiovascular and fibrotic diseases, but its short in vivo half-life is an obstacle to long-term administration. The discovery of ML290 demonstrated that it is possible to identify small molecule agonists of the cognate G-protein coupled receptor for H2-RLX (relaxin family peptide receptor-1 (RXFP1)). In our efforts to generate a new medicine for liver fibrosis, we sought to identify improved small molecule functional mimetics of H2-RLX with selective, full agonist or positive allosteric modulator activity against RXFP1. First, we confirmed expression of RXFP1 in human diseased liver. We developed a robust cellular cAMP reporter assay of RXFP1 signaling in HEK293 cells transiently expressing RXFP1. A high-throughput screen did not identify further specific agonists or positive allosteric modulators of RXFP1, affirming the low druggability of this receptor. As an alternative approach, we generated novel ML290 analogues and tested their activity in the HEK293-RXFP1 cAMP assay and the human hepatic cell line LX-2. Differences in activity of compounds on cAMP activation compared with changes in expression of fibrotic markers indicate the need to better understand cell- and tissue-specific signaling mechanisms and their disease-relevant phenotypes in order to enable drug discovery.

In silico prediction of novel therapeutic targets using gene\u2013disease association data

BackgroundTarget identification and validation is a pressing challenge in the pharmaceutical industry, with many of the programmes that fail for efficacy reasons showing poor association between the drug target and the disease. Computational prediction of successful targets could have a considerable impact on attrition rates in the drug discovery pipeline by significantly reducing the initial search space. Here, we explore whether gene–disease association data from the Open Targets platform is sufficient to predict therapeutic targets that are actively being pursued by pharmaceutical companies or are already on the market.MethodsTo test our hypothesis, we train four different classifiers (a random forest, a support vector machine, a neural network and a gradient boosting machine) on partially labelled data and evaluate their performance using nested cross-validation and testing on an independent set. We then select the best performing model and use it to make predictions on more than 15,000 genes. Finally, we validate our predictions by mining the scientific literature for proposed therapeutic targets.ResultsWe observe that the data types with the best predictive power are animal models showing a disease-relevant phenotype, differential expression in diseased tissue and genetic association with the disease under investigation. On a test set, the neural network classifier achieves over 71% accuracy with an AUC of 0.76 when predicting therapeutic targets in a semi-supervised learning setting. We use this model to gain insights into current and failed programmes and to predict 1431 novel targets, of which a highly significant proportion has been independently proposed in the literature.ConclusionsOur in silico approach shows that data linking genes and diseases is sufficient to predict novel therapeutic targets effectively and confirms that this type of evidence is essential for formulating or strengthening hypotheses in the target discovery process. Ultimately, more rapid and automated target prioritisation holds the potential to reduce both the costs and the development times associated with bringing new medicines to patients.

Serotonin in psychiatry: in vitro disease modeling using patient-derived neurons.

Several lines of evidence implicate serotonin in the etiology of multiple psychiatric disorders, especially mood disorders, such as major depressive disorder (MDD) and bipolar disorder (BD). Much of our current understanding of biological mechanisms underlying serotonergic alterations in mood disorders comes from animal studies. Innovation in induced pluripotent stem cell and transdifferentiation technologies for deriving neurons from adult humans has enabled the study of disease-relevant cellular phenotypes in vitro. In this context, human serotonergic neurons can now be generated using three recently published methodologies. In this mini-review, we broadly discuss evidence linking altered serotonergic neurotransmission in MDD and BD and focus on recently published methods for generating human serotonergic neurons in vitro.

Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation

Three-prime repair exonuclease 1 (TREX1) is an anti-viral enzyme that cleaves nucleic acids in the cytosol, preventing accumulation and a subsequent type I interferon-associated inflammatory response. Autoimmune diseases, including Aicardi-Goutières syndrome (AGS) and systemic lupus erythematosus, can arise when TREX1 function is compromised. AGS is a neuroinflammatory disorder with severe and persistent intellectual and physical problems. Here we generated a human AGS model that recapitulates disease-relevant phenotypes using pluripotent stem cells lacking TREX1. We observed abundant extrachromosomal DNA in TREX1-deficient neural cells, of which endogenous Long Interspersed Element-1 retrotransposons were a major source. TREX1-deficient neurons also exhibited increased apoptosis and formed three-dimensional cortical organoids of reduced size. TREX1-deficient astrocytes further contributed to the observed neurotoxicity through increased type I interferon secretion. In this model, reverse-transcriptase inhibitors rescued the neurotoxicity of AGS neurons and organoids, highlighting their potential utility in therapeutic regimens for AGS and related disorders.

Natural variation of macrophage activation as disease-relevant phenotype predictive of inflammation and cancer survival

Although mouse models exist for many immune-based diseases, the clinical translation remains challenging. Most basic and translational studies utilize only a single inbred mouse strain. However, basal and diseased immune states in humans show vast inter-individual variability. Here, focusing on macrophage responses to lipopolysaccharide (LPS), we use the hybrid mouse diversity panel (HMDP) of 83 inbred strains as a surrogate for human natural immune variation. Since conventional bioinformatics fail to analyse a population spectrum, we highlight how gene signatures for LPS responsiveness can be derived based on an Interleukin-12β and arginase expression ratio. Compared to published signatures, these gene markers are more robust to identify susceptibility or resilience to several macrophage-related disorders in humans, including survival prediction across many tumours. This study highlights natural activation diversity as a disease-relevant dimension in macrophage biology, and suggests the HMDP as a viable tool to increase translatability of mouse data to clinical settings.

Parent-of-origin effects on schizophrenia-relevant behaviours of type III neuregulin 1 mutant mice.

A robust, disease-relevant phenotype is paramount to the validity of genetic mouse models, which are an important tool in understanding complex diseases. Recent evidence from genome-wide association studies suggests the genetic contribution of parents to offspring is not equivalent. Despite this, few studies to date have examined the potential impact of parent genotype (i.e. origin of mutation) on the offspring of disease-relevant genetic mouse models. To elucidate the potential impact of the sex of the mutant parent on offspring phenotype, we characterized male and female offspring of an established schizophrenia mouse model, which had been generated using two different breeding schemes, in a range of disease-relevant behaviours. We compared heterozygous type III neuregulin 1 mutant (type III Nrg1+/-) and wild type-like control (WT) offspring from mutant father x WT mother pairings with offspring from mutant mother x WT father pairings. Offspring were tested in schizophrenia-relevant paradigms including the elevated plus maze (EPM), fear conditioning (FC), prepulse inhibition (PPI), social interaction (SI), and open field (OF). We found type III Nrg1+/- males from mutant fathers, but not mutant mothers, showed deficits in contextual fear-associated memory and exhibited increased social interaction, compared to their WT littermates. Type III Nrg1+/- females across breeding colonies only exhibited a subtle change to their acoustic startle response and sensorimotor gating. These results suggest a paternal-dependent transmission of genetically induced behavioural characteristics. Though the mechanisms governing this phenomenon are unclear, our results show that parental origin of mutation can alter the behavioural phenotype of genetic mouse models. Thus, researchers should carefully consider their breeding scheme when dealing with genetic mouse models of diseases such as schizophrenia.

Defective synaptic connectivity and axonal neuropathology in a human iPSC-based model of familial Parkinson’s disease

α-Synuclein (αSyn) is the major gene linked to sporadic Parkinson's disease (PD), whereas the G209A (p.A53T) αSyn mutation causes a familial form of PD characterized by early onset and a generally severe phenotype, including nonmotor manifestations. Here we generated de novo induced pluripotent stem cells (iPSCs) from patients harboring the p.A53T mutation and developed a robust model that captures PD pathogenic processes under basal conditions. iPSC-derived mutant neurons displayed novel disease-relevant phenotypes, including protein aggregation, compromised neuritic outgrowth, and contorted or fragmented axons with swollen varicosities containing αSyn and Tau. The identified neuropathological features closely resembled those in brains of p.A53T patients. Small molecules targeting αSyn reverted the degenerative phenotype under both basal and induced stress conditions, indicating a treatment strategy for PD and other synucleinopathies. Furthermore, mutant neurons showed disrupted synaptic connectivity and widespread transcriptional alterations in genes involved in synaptic signaling, a number of which have been previously linked to mental disorders, raising intriguing implications for potentially converging disease mechanisms.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Target Discovery for Precision Medicine Using High-Throughput Genome Engineering.

Over the past few years, programmable RNA-guided nucleases such as the CRISPR/Cas9 system have ushered in a new era of precision genome editing in diverse model systems and in human cells. Functional screens using large libraries of RNA guides can interrogate a large hypothesis space to pinpoint particular genes and genetic elements involved in fundamental biological processes and disease-relevant phenotypes. Here, we review recent high-throughput CRISPR screens (e.g. loss-of-function, gain-of-function, and targeting noncoding elements) and highlight their potential for uncovering novel therapeutic targets, such as those involved in cancer resistance to small molecular drugs and immunotherapies, tumor evolution, infectious disease, inborn genetic disorders, and other therapeutic challenges.