Homeostatic functions of monocytes and interstitial lung macrophages are regulated via collagen domain-binding receptor LAIR1.

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.

Integration of innate immune signalling by caspase-8 cleavage of N4BP1.

Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.

The RIPK4-IRF6 signalling axis safeguards epidermal differentiation and barrier function.

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.

TEAD Proteins Associate With DNA Repair Proteins to Facilitate Cellular Recovery From DNA Damage.

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage-induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action.

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity. METHODS: This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products. RESULTS: A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response. CONCLUSIONS: pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

Assessment and comparison of viability assays for cellular products.

BACKGROUND AIMS: Accurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs and cultured chimeric antigen receptor and T-cell receptor-engineered T-cell products. METHODS: Viability assays, including manual Trypan Blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining or cell surface marker staining in conjunction with 7-AAD, Cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA) Acridine Orange/PI staining and Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter, Inc, Brea, CA, USA), were evaluated. A viability standard was established using live and dead cell mixtures to assess the accuracy of these assays. Furthermore, precision assessment was conducted to determine the reproducibility of the viability assays. Additionally, the viability of individual cell populations from cryopreserved PBSC and PBMC apheresis products was examined. RESULTS: All methods provided accurate viability measurements and generated consistent and reproducible viability data. The assessed viability assays were demonstrated to be reliable alternatives when evaluating the viability of fresh cellular products. However, cryopreserved products exhibited variability among the tested assays. Additionally, analyzing the viability of each subset of the cryopreserved PBSC and PBMC apheresis products revealed that T cells and granulocytes were more susceptible to the freeze-thaw process, showing decreased viability. CONCLUSIONS: The study demonstrates the importance of careful assay selection, validation and standardization, particularly for assessing the viability of cryopreserved products. Given the complexity of cellular products, choosing a fit-for-purpose viability assay is essential.

Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling.

Cell-surface death receptors such as DR4 and DR5 trigger apoptosis through a death-inducing signaling complex (DISC) that recruits the apical protease caspase-8. Apoptosis commitment requires efficient activation and autocatalytic release of caspase-8 into the cytoplasm to engage executioner caspases. While DISC recruitment initiates caspase-8 stimulation, full activation of the protease depends on further molecular aggregation events that are not fully understood. Here, we show that death receptor ligation induces polyubiquitination of caspase-8, through a previously unknown interaction of the DISC with a cullin3 (CUL3)-based E3 ligase. CUL3-mediated caspase-8 polyubiquitination required the RING box protein RBX1, whereas the deubiquitinase A20 reversed this modification. The ubiquitin-binding protein p62/sequestosome-1 promoted aggregation of CUL3-modified caspase-8 within p62-dependent foci, leading to full activation and processing of the enzyme and driving commitment to cell death. These results identify a mechanism that positively controls apoptosis signaling by polyubiquitination and aggregation of a key initiator caspase.

Targeting MCL-1 and BCL-2 with polatuzumab vedotin and venetoclax overcomes treatment resistance in R/R non-Hodgkin lymphoma: Results from preclinical models and a Phase Ib study.

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.

Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy.

Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.

MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1ß and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.

Correction: MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

[This corrects the article DOI: 10.1371/journal.pbio.3000354.].

RIPK1 inhibits ZBP1-driven necroptosis during development.

Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1RHIM/RHIMMlkl-/- macrophages, but not in wild-type, Mlkl-/- or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.

Proteomic Analyses Identify a Novel Role for EZH2 in the Initiation of Cancer Cell Drug Tolerance.

Acquisition of drug resistance remains a chief impediment to successful cancer therapy, and we previously described a transient drug-tolerant cancer cell population (DTPs) whose survival is in part dependent on the activities of the histone methyltransferases G9a/EHMT2 and EZH2, the latter being the catalytic component of the polycomb repressive complex 2 (PRC2). Here, we apply multiple proteomic techniques to better understand the role of these histone methyltransferases (HMTs) in the establishment of the DTP state. Proteome-wide comparisons of lysine methylation patterns reveal that DTPs display an increase in methylation on K116 of PRC member Jarid2, an event that helps stabilize and recruit PRC2 to chromatin. We also find that EZH2, in addition to methylating histone H3K27, also can methylate G9a at K185, and that methylated G9a better recruits repressive complexes to chromatin. These complexes are similar to complexes recruited by histone H3 methylated at K9. Finally, a detailed histone post-translational modification (PTM) analysis shows that EZH2, either directly or through its ability to methylate G9a, alters H3K9 methylation in the context of H3 serine 10 phosphorylation, primarily in a cancer cell subpopulation that serves as DTP precursors. We also show that combinations of histone PTMs recruit a different set of complexes to chromatin, shedding light on the temporal mechanisms that contribute to drug tolerance.

TRAF2 Sets a threshold for extrinsic apoptosis by tagging caspase-8 with a ubiquitin shutoff timer.

Apoptotic caspase activation mechanisms are well defined, yet inactivation modes remain unclear. The death receptors (DRs), DR4, DR5, and Fas, transduce cell-extrinsic apoptotic signals by recruiting caspase-8 into a death-inducing signaling complex (DISC). At the DISC, Cullin3-dependent polyubiquitination on the small catalytic subunit of caspase-8 augments stimulation. Here we report that tumor necrosis factor receptor-associated factor 2 (TRAF2) interacts with caspase-8 at the DISC, downstream of Cullin3. TRAF2 directly mediates RING-dependent, K48-linked polyubiquitination on the large catalytic domain of caspase-8. This modification destines activated caspase-8 molecules to rapid proteasomal degradation upon autoprocessing and cytoplasmic translocation. TRAF2 depletion lowers the signal threshold for DR-mediated apoptosis, altering cell life versus death decisions in vitro and in vivo. Thus, TRAF2 sets a critical barrier for cell-extrinsic apoptosis commitment by tagging activated caspase-8 with a K48-ubiquitin shutoff timer. These results may have important implications for caspase regulation mechanisms.

Deubiquitinase USP37 is activated by CDK2 to antagonize APC(CDH1) and promote S phase entry.

Cell cycle progression requires the E3 ubiquitin ligase anaphase-promoting complex (APC/C), which uses the substrate adaptors CDC20 and CDH1 to target proteins for proteasomal degradation. The APC(CDH1) substrate cyclin A is critical for the G1/S transition and, paradoxically, accumulates even when APC(CDH1) is active. We show that the deubiquitinase USP37 binds CDH1 and removes degradative polyubiquitin from cyclin A. USP37 was induced by E2F transcription factors in G1, peaked at G1/S, and was degraded in late mitosis. Phosphorylation of USP37 by CDK2 stimulated its full activity. USP37 overexpression caused premature cyclin A accumulation in G1 and accelerated S phase entry, whereas USP37 knockdown delayed these events. USP37 was inactive in mitosis because it was no longer phosphorylated by CDK2. Indeed, it switched from an antagonist to a substrate of APC(CDH1) and was modified with degradative K11-linked polyubiquitin.

Peptide level immunoaffinity enrichment enhances ubiquitination site identification on individual proteins.

Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.

Development of orthogonally protected hypusine for solid-phase peptide synthesis.

An orthogonally protected hypusine reagent was developed for solid-phase synthesis of hypusinated peptides using the Fmoc/t-Bu protection strategy. The reagent was synthesized in an overall yield of 27% after seven steps from Cbz-Lys-OBzl and (R)-3-hydroxypyrrolidin-2-one. The side-chain protecting groups (Boc and t-Bu) are fully compatible with standard Fmoc chemistry and can be readily removed during the peptide cleavage step. The utility of the reagent was demonstrated by solid-phase synthesis of hypusinated peptides.

Negative Space: An Alternative Framework for Archaeoacoustics.

Hearing the remote past seems impossible. Archaeoacoustics is a contemporary field intent on reconstructing the evolution of early communication systems, offering the possibility of developing methodologies relating to past sound signaling and music. Through a contribution of the emerging sensory field of archaeoacoustics and an example of acoustic assessments conducted at the site of Coves del Toll, can we understand signals of the past in order to investigate human behaviour and trace its cognitive evolution? This paper explores alternative methodological and theoretical approaches to understanding prehistoric sonic behaviours in early hominids and aims to set out a framework to theoretically and philosophically approach the "sound record" of the past. The theoretical proposition of this paper integrates the musical and sound art disciplines of spectralism and sound ecology to challenge the current limitations of listening to sound.

Relapse to cocaine seeking is regulated by medial habenula NR4A2/NURR1 in mice.

Drugs of abuse can persistently change the reward circuit in ways that contribute to relapse behavior, partly via mechanisms that regulate chromatin structure and function. Nuclear orphan receptor subfamily4 groupA member2 (NR4A2, also known as NURR1) is an important effector of histone deacetylase 3 (HDAC3)-dependent mechanisms in persistent memory processes and is highly expressed in the medial habenula (MHb), a region that regulates nicotine-associated behaviors. Here, expressing the Nr4a2 dominant negative (Nurr2c) in the MHb blocks reinstatement of cocaine seeking in mice. We use single-nucleus transcriptomics to characterize the molecular cascade following Nr4a2 manipulation, revealing changes in transcriptional networks related to addiction, neuroplasticity, and GABAergic and glutamatergic signaling. The network controlled by NR4A2 is characterized using a transcription factor regulatory network inference algorithm. These results identify the MHb as a pivotal regulator of relapse behavior and demonstrate the importance of NR4A2 as a key mechanism driving the MHb component of relapse.