Single-Cell Transcriptomic Analysis Links Nonmyelinating Schwann Cells to Proinflammatory Response in the Lung.

The lung is a barrier tissue with constant exposure to the inhaled environment. Therefore, innate immunity against particulates and pathogens is of critical importance to maintain tissue homeostasis. Although the lung harbors both myelinating and nonmyelinating Schwann cells (NMSCs), NMSCs represent the most abundant Schwann cell (SC) population in the lung. However, their contribution to lung physiology remains largely unknown. In this study, we used the human glial fibrillary acidic protein promoter driving tdTomato expression in mice to identify SCs in the peripheral nervous system and determine their location within the lung. Single-cell transcriptomic analysis revealed the existence of two NMSC populations (NMSC1 and NMSC2) that may participate in pathogen recognition. We demonstrated that these pulmonary SCs produce chemokines and cytokines upon LPS stimulation using in vitro conditions. Furthermore, we challenged mouse lungs with LPS and found that NMSC1 exhibits an enriched proinflammatory response among all SC subtypes. Collectively, these findings define the molecular profiles of lung SCs and suggest a potential role for NMSCs in lung inflammation.

Tetrahydrofuran-Based Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists: Ligand-Based Discovery, Activity in a Rodent Asthma Model, and Mechanism-of-Action via Cryogenic Electron Microscopy.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.

A TRPA1 inhibitor suppresses neurogenic inflammation and airway contraction for asthma treatment.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

Behavioral characterization of a CRISPR-generated TRPA1 knockout rat in models of pain, itch, and asthma.

The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1 KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.

A Damage-Independent Role for 53BP1 that Impacts Break Order and Igh Architecture during Class Switch Recombination.

During class switch recombination (CSR), B cells replace the Igh Cµ or δ exons with another downstream constant region exon (CH), altering the antibody isotype. CSR occurs through the introduction of AID-mediated double-strand breaks (DSBs) in switch regions and subsequent ligation of broken ends. Here, we developed an assay to investigate the dynamics of DSB formation in individual cells. We demonstrate that the upstream switch region Sµ is first targeted during recombination and that the mechanism underlying this control relies on 53BP1. Surprisingly, regulation of break order occurs through residual binding of 53BP1 to chromatin before the introduction of damage and independent of its established role in DNA repair. Using chromosome conformation capture, we show that 53BP1 mediates changes in chromatin architecture that affect break order. Finally, our results explain how changes in Igh architecture in the absence of 53BP1 could promote inversional rearrangements that compromise CSR.

Defining ATM-Independent Functions of the Mre11 Complex with a Novel Mouse Model.

UNLABELLED: The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATM(flox)) was crossed to hypomorphic NBS1 mutants (Nbs1(ΔB/ΔB) mice). Nbs1(ΔB/ΔB) Atm(-/-) hematopoietic cells derived by crossing to vav(cre) were viable in vivo. Nbs1(ΔB/ΔB) Atm(-/-) (VAV) mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. IMPLICATIONS: A novel mouse model was developed, by combining a vav(cre)-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis.

The Ku heterodimer and the metabolism of single-ended DNA double-strand breaks.

Single-ended double-strand breaks (DSBs) are a common form of spontaneous DNA break, generated when the replisome encounters a discontinuity in the DNA template. Given their prevalence, understanding the mechanisms governing the fate(s) of single-ended DSBs is important. We describe the influence of the Ku heterodimer and Mre11 nuclease activity on processing of single-ended DSBs. Separation-of-function alleles of yku70 were derived that phenocopy Ku deficiency with respect to single-ended DSBs but remain proficient for NHEJ. The Ku mutants fail to regulate Exo1 activity, and bypass the requirement for Mre11 nuclease activity in the repair of camptothecin-induced single-ended DSBs. Ku mutants exhibited reduced affinity for DNA ends, manifest as both reduced end engagement and enhanced probability of diffusing inward on linear DNA. This study reveals an interplay between Ku and Mre11 in the metabolism of single-ended DSBs that is distinct from repair pathway choice at double-ended DSBs.

Functional interplay of the Mre11 nuclease and Ku in the response to replication-associated DNA damage.

The Mre11 complex is a central component of the DNA damage response, with roles in damage sensing, molecular bridging, and end resection. We have previously shown that in Saccharomyces cerevisiae, Ku70 (yKu70) deficiency reduces the ionizing radiation sensitivity of mre11Δ mutants. In this study, we show that yKu70 deficiency suppressed the camptothecin (CPT) and methyl methanesulfonate (MMS) sensitivity of nuclease-deficient mre11-3 and sae2Δ mutants in an Exo1-dependent manner. CPT-induced G(2)/M arrest, γ-H2AX persistence, and chromosome breaks were elevated in mre11-3 mutants. These outcomes were reduced by yKu70 deficiency. Given that the genotoxic effects of CPT are manifest during DNA replication, these data suggest that Ku limits Exo1-dependent double-strand break (DSB) resection during DNA replication, inhibiting the initial processing steps required for homology-directed repair. We propose that Mre11 nuclease- and Sae2-dependent DNA end processing, which initiates DSB resection prevents Ku from engaging DSBs, thus promoting Exo1-dependent resection. In agreement with this idea, we show that Ku affinity for binding to short single-stranded overhangs is much lower than for blunt DNA ends. Collectively, the data define a nonhomologous end joining (NHEJ)-independent, S-phase-specific function of the Ku heterodimer.

GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63.

Activation of the protein kinases ATM and ATR following chromosomal breakage prevents initiation of DNA replication and entry into mitosis. However, the effects of ATM and ATR activation in cells already progressing through mitosis are poorly understood. Here we report that ATM and ATR activation induced by DNA double-strand breaks (DSBs) inhibits centrosome-driven spindle assembly in Xenopus laevis mitotic egg extract and somatic cells, delaying mitotic progression. Using a cDNA expression library to screen for ATM and ATR substrates, we identified centrosomal protein CEP63 as an ATM and ATR target required for normal spindle assembly. ATM and ATR phosphorylate Xenopus CEP63 (XCEP63) on Ser 560 and promote its delocalization from the centrosome. Suppression of ATM and ATR activity or mutation of XCEP63 Ser 560 to Ala prevented spindle assembly defects. Consistently, inactivation of the CEP63 gene in avian DT40 cells impaired spindle assembly and prevented ATM- and ATR-dependent effects on mitosis. These data indicate that ATM and ATR control mitotic events in vertebrate cells by targeting CEP63 and centrosome dependent spindle assembly.

Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity.

DNA double-strand breaks (DSBs) can be processed by the Mre11-Rad50-Nbs1 (MRN) complex, which is essential to promote ataxia telangiectasia-mutated (ATM) activation. However, the molecular mechanisms linking MRN activity to ATM are not fully understood. Here, using Xenopus laevis egg extract we show that MRN-dependent processing of DSBs leads to the accumulation of short single-stranded DNA oligonucleotides (ssDNA oligos). The MRN complex isolated from the extract containing DSBs is bound to ssDNA oligos and stimulates ATM activity. Elimination of ssDNA oligos results in rapid extinction of ATM activity. Significantly, ssDNA oligos can be isolated from human cells damaged with ionizing radiation and injection of small synthetic ssDNA oligos into undamaged cells also induces ATM activation. These results suggest that MRN-dependent generation of ssDNA oligos, which constitute a unique signal of ongoing DSB repair not encountered in normal DNA metabolism, stimulates ATM activity.

Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint.

Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G(1) phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Delta dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.