Jamie Russell

NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1β (IL-1β) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.

A Dynamic Notch Injury Response Activates Epicardium and Contributes to Fibrosis Repair

Rationale: Transgenic Notch reporter mice express enhanced green fluorescent protein in cells with C-promoter binding factor-1 response element transcriptional activity (CBF1-REx4-EGFP), providing a unique and powerful tool for identifying and isolating “Notch-activated” progenitors. Objective: We asked whether, as in other tissues of this mouse, EGFP localized and functionally tagged adult cardiac tissue progenitors, and, if so, whether this cell-based signal could serve as a quantitative and qualitative biosensor of the injury repair response of the heart. Methods and Results: In addition to scattered endothelial and interstitial cells, Notch-activated (EGFP+) cells unexpectedly richly populated the adult epicardium. We used fluorescence-activated cell sorting to isolate EGFP+ cells and excluded hematopoietic (CD45+) and endothelial (CD31+) subsets. We analyzed EGFP+/CD45−/CD31− cells, a small (<2%) but distinct subpopulation, by gene expression profiling and functional analyses. We called this mixed cell pool, which had dual multipotent stromal cell and epicardial lineage signatures, Notch-activated epicardial-derived cells (NECs). Myocardial infarction and thoracic aortic banding amplified the NEC pool, increasing fibroblast differentiation. Validating the functional vitality of clonal NEC lines, serum growth factors triggered epithelial–mesenchymal transition and the immobilized Notch ligand Delta-like 1–activated downstream target genes. Moreover, cardiomyocyte coculture and engraftment in NOD-SCID (nonobese diabetic–severe combined immunodeficiency) mouse myocardium increased cardiac gene expression in NECs. Conclusions: A dynamic Notch injury response activates adult epicardium, producing a multipotent cell population that contributes to fibrosis repair.

Targeting native adult heart progenitors with cardiogenic small molecules

Targeting native progenitors with small molecule pharmaceuticals that direct cell fate decisions is an attractive approach for regenerative medicine. Here, we show that 3,5-disubstituted isoxazoles (Isx), stem cell-modulator small molecules originally recovered in a P19 embryonal carcinoma cell-based screen, directed cardiac muscle gene expression in vivo in target tissues of adult transgenic reporter mice. Isx also stimulated adult mouse myocardial cell cycle activity. Narrowing our focus onto one target cardiac-resident progenitor population, Isx directed muscle transcriptional programs in vivo in multipotent Notch-activated epicardium-derived cells (NECs), generating Notch-activated adult cardiomyocyte-like precursors. Myocardial infarction (MI) preemptively differentiated NECs toward fibroblast lineages, overriding Isxs cardiogenic influence in this cell population. Isx dysregulated gene expression in vivo in Notch-activated repair fibroblasts, driving distinctive (pro-angiogenesis) gene programs, but failed to mitigate fibrosis or avert ventricular functional decline after MI. In NECs in vitro, Isx directed partial muscle differentiation, which included biosynthesis and assembly of sarcomeric α-actinin premyofibrils, beaded structures pathognomonic of early developing cardiomyocytes. Thus, although Isx small molecules have promising in vivo efficacy at the level of cardiac muscle gene expression in native multipotent progenitors and are first in class in this regard, a greater understanding of the dynamic interplay between fibrosis and cardiogenic small molecule signals will be required to pharmacologically enable regenerative repair of the heart.

A small molecule differentiation inducer increases insulin production by pancreatic β cells

New drugs for preserving and restoring pancreatic β-cell function are critically needed for the worldwide epidemic of type 2 diabetes and the cure for type 1 diabetes. We previously identified a family of neurogenic 3,5-disubstituted isoxazoles (Isx) that increased expression of neurogenic differentiation 1 (NeuroD1, also known as BETA2); this transcription factor functions in neuronal and pancreatic β-cell differentiation and is essential for insulin gene transcription. Here, we probed effects of Isx on human cadaveric islets and MIN6 pancreatic β cells. Isx increased the expression and secretion of insulin in islets that made little insulin after prolonged ex vivo culture and increased expression of neurogenic differentiation 1 and other regulators of islet differentiation and insulin gene transcription. Within the first few hours of exposure, Isx caused biphasic activation of ERK1/2 and increased bulk histone acetylation. Although there was little effect on histone deacetylase activity, Isx increased histone acetyl transferase activity in nuclear extracts. Reconstitution assays indicated that Isx increased the activity of the histone acetyl transferase p300 through an ERK1/2-dependent mechanism. In summary, we have identified a small molecule with antidiabetic activity, providing a tool for exploring islet function and a possible lead for therapeutic intervention in diabetes.

Real-time resolution of point mutations that cause phenovariance in mice

Significance In forward genetics, a mutagen is used to randomly induce germline mutations that cause variant phenotypes. Forward genetics permits discovery of genes necessary for biological phenomena, but identifying the mutations that cause variant phenotypes is time-consuming and in the past usually occurred long after the phenotype was first recognized. Here we introduce a method and software tool, Linkage Analyzer, for identifying causative mutations present in the germline of mutant mice concurrent with recognition of variant phenotypes. It requires knowledge of genotype at all mutation sites in members of a pedigree prior to phenotypic assessment. Using this method and software, forward genetic studies in mice are limited only by the rates of mutant production and screening. With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled “superpedigrees” are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.

Coupling hippocampal neurogenesis to brain pH through proneurogenic small molecules that regulate proton sensing G protein-coupled receptors.

Acidosis, a critical aspect of central nervous system (CNS) pathophysiology and a metabolic corollary of the hypoxic stem cell niche, could be an expedient trigger for hippocampal neurogenesis and brain repair. We recently tracked the function of our isoxazole stem cell-modulator small molecules (Isx) through a chemical biology-target discovery strategy to GPR68, a proton (pH) sensing G protein-coupled receptor with no known function in brain. Isx and GPR68 coregulated neuronal target genes such as Bex1 (brain-enriched X-linked protein-1) in hippocampal neural progenitors (HCN cells), which further amplified GPR68 signaling by producing metabolic acid in response to Isx. To evaluate this proneurogenic small molecule/proton signaling circuit in vivo, we explored GPR68 and BEX1 expression in brain and probed brain function with Isx. We localized proton-sensing GPR68 to radial processes of hippocampal type 1 neural stem cells (NSCs) and, conversely, localized BEX1 to neurons. At the transcriptome level, Isx demonstrated unrivaled proneurogenic activity in primary hippocampal NSC cultures. In vivo, Isx pharmacologically targeted type 1 NSCs, promoting neurogenesis in young mice, depleting the progenitor pool without adversely affecting hippocampal learning and memory function. After traumatic brain injury, cerebral cortical astrocytes abundantly expressed GPR68, suggesting an additional role for proton-GPCR signaling in reactive astrogliosis. Thus, probing a novel proneurogenic synthetic small molecules mechanism-of-action, candidate target, and pharmacological activity, we identified a new GPR68 regulatory pathway for integrating neural stem and astroglial cell functions with brain pH.

Molecular signatures define myogenic stem cell populations.

Developmental and regenerative mechanisms are directed by stem cell populations. Skeletal muscle is a dynamic tissue that is capable of adapting to stress and severe injury due to a resident somatic stem cell population. In response to a severe injury that destroys upward of 90% of the tissue, skeletal muscle efficiently and reproducibly regenerates damaged tissue and restores the cellular architecture within a 2-wk period. Recent studies have localized and examined the molecular regulation of skeletal muscle stem cell populations using emerging molecular biological technologies. These studies enhance the understanding of the regulatory mechanisms that direct the somatic stem cell populations and the role they play in development and regeneration. Furthermore, these basic science studies will serve as a platform for future therapies directed toward patients with myopathic diseases.

Probability of phenotypically detectable protein damage by ENU-induced mutations in the Mutagenetix database

Computational inference of mutation effects is necessary for genetic studies in which many mutations must be considered as etiologic candidates. Programs such as PolyPhen-2 predict the relative severity of damage caused by missense mutations, but not the actual probability that a mutation will reduce/eliminate protein function. Based on genotype and phenotype data for 116,330 ENU-induced mutations in the Mutagenetix database, we calculate that putative null mutations, and PolyPhen-2-classified “probably damaging”, “possibly damaging”, or “probably benign” mutations have, respectively, 61%, 17%, 9.8%, and 4.5% probabilities of causing phenotypically detectable damage in the homozygous state. We use these probabilities in the estimation of genome saturation and the probability that individual proteins have been adequately tested for function in specific genetic screens. We estimate the proportion of essential autosomal genes in Mus musculus (C57BL/6J) and show that viable mutations in essential genes are more likely to induce phenotype than mutations in non-essential genes.Programs such as PolyPhen-2 predict the relative severity of damage by missense mutations. Here, Wang et al estimate probabilities that putative null or missense alleles would reduce protein function to cause detectable phenotype by analyzing data from ENU-induced mouse mutations.

Large-scale forward genetics screening identifies Trpa1 as a chemosensor for predator odor-evoked innate fear behaviors

Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly, Trpa1−/− mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism.TMT is a chemical that evokes innate defensive behaviors yet the molecular mechanisms are not well understood. Here the authors perform a large-scale forward genetics screen in mice and identify Trpa1, a pungency/irritancy receptor, as a chemosensor for predator odor-evoked innate fear and defensive behaviors.

The class I myosin MYO1D binds to lipid and protects against colitis

ABSTRACT Myosin ID (MYO1D) is a member of the class I myosin family. We screened 48,649 third generation (G3) germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). We found and validated mutations in Myo1d as a cause of increased susceptibility to DSS-induced colitis. MYO1D is produced in the intestinal epithelium, and the colitis phenotype is dependent on the nonhematopoietic compartment of the mouse. Moreover, MYO1D appears to couple cytoskeletal elements to lipid in an ATP-dependent manner. These findings demonstrate that MYO1D is needed to maintain epithelial integrity and protect against DSS-induced colitis. Summary: Using random germline mutagenesis and screening of mice, we determined that loss of MYO1D function in nonhematopoietic tissues renders mice susceptible to colitis induced by dextran sodium sulfate challenge.