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Long term survival of breast cancer patients is limited due to recurrence from metastatic dormant cancer cells. However, the mechanisms by which these dormant breast cancer cells survive and awaken remain poorly understood. Our unbiased genome-scale genetic screen in mice identified Med4 as a novel cancer-cell intrinsic gatekeeper in metastatic reactivation. MED4 haploinsufficiency is prevalent in metastatic breast cancer patients and correlates with poorer prognosis. Syngeneic xenograft models revealed that Med4 enforces breast cancer dormancy. Contrary to the canonical function of the Mediator complex in activating gene expression, Med4 maintains 3D chromatin compaction and enhancer landscape, by preventing enhancer priming or activation through the suppression of H3K4me1 deposition. Med4 haploinsufficiency disrupts enhancer poise and reprograms the enhancer dynamics to facilitate extracellular matrix (ECM) gene expression and integrin-mediated mechano-transduction, driving metastatic growth. Our findings establish Med4 as a key regulator of cellular dormancy and a potential biomarker for high-risk metastatic relapse.

For over a century, an explanation for how concentrated ions denature proteins has proven elusive. Here, we report a novel mechanism of protein denaturation driven by entropy changes in water networks. Experiments and simulations show that ion pairs like LiBr and LiCl localize water molecules and disrupt the water network's structure, while others exert a more global effect without compromising network integrity. This disruption reduces the entropy penalty when proteins sequester water molecules during unfolding, resulting in a peculiar yet universal "inverse hydrophobic effect" that potentiates protein denaturation. Through successful isolation and systematic study of indirect solute effects, our findings offer a universal approach to salt induced protein denaturation and provide a unified framework for the decoding of the protein-water-solute nexus.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury. Recently, it has been demonstrated that ablation of astrocytic transglutaminase 2 (TG2) modulates the phenotype of reactive astrocytes in a way that improves neuronal injury outcomes both in vitro and in vivo. In an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopies the neurosupportive effects of TG2 deletion in astrocytes. In this study, we provide insights into the mechanisms by which TG2 deletion or inhibition result in a more neurosupportive astrocytic phenotype. Using a neuron-astrocyte co-culture model, we show that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix. To better understand how pharmacologically altering TG2 affects its ability to regulate reactive astrocyte phenotypes, we assessed how VA4 inhibition impacts TG2's interaction with Zbtb7a, a transcription factor we have previously identified as a functionally relevant TG2 nuclear interactor. The results of these studies demonstrate that VA4 significantly decreases the interaction of TG2 and Zbtb7a. TG2's interactions with Zbtb7a, as well as a wide range of other transcription factors and chromatin regulatory proteins, suggest that TG2 may act as an epigenetic regulator to modulate gene expression. To begin to understand if TG2-mediated epigenetic modification may impact astrocytic phenotypes in our models, we interrogated the effect of TG2 deletion and VA4 treatment on histone acetylation and found significantly greater acetylation in both experimental groups. Consistent with these findings, previous RNA-sequencing and our present proteomic analysis also supported a predominant transcriptionally suppressive role of TG2 in astrocytes. Our proteomic data additionally unveiled pronounced changes in lipid and antioxidant metabolism in astrocytes with TG2 deletion or inhibition, which likely contribute to the enhanced neurosupportive function of these astrocytes.

In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In Drosophila, Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. Here, we demonstrate that the conserved PY motifs in the cytoplasmic domain of Comm are required allow the ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1 positive late endosomes and to promote axon growth across the midline in vivo. In addition, we conclusively demonstrate a role for Nedd4 in midline crossing. Genetic analysis shows that nedd4 mutations result in midline crossing defects in the Drosophila embryonic nerve cord, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a three-member complex with Comm and Robo1 in a PY motif-dependent manner. Finally, we present genetic evidence that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo1 ubiquitination by Nedd4, ultimately leading to its degradation.

Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders such as addiction and Parkinson's disease. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter (VMAT2), vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44%), VGAT+ (37%) and VGLUT2+ (41%) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54%), fewer were VGAT+ (42%), and VGLUT2+ neurons were least abundant (16%). Moreover, 20% of VTA neurons and 10% of SNc neurons expressed more than one vesicular transporter, including 45% of VGLUT2+ neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.

Type 2 Nuclear Receptors (T2NRs) require heterodimerization with a common partner, the Retinoid X Receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and over-expression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged retinoid X receptor (RXR) and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

The coordination of forelimb and orofacial movements to compose an ethological reach-to-consume behavior likely involves neural communication across brain regions. Leveraging wide-field imaging and photo-inhibition to survey across the cortex, we identified a cortical network and a high-order motor area (MOs-c), which coordinate action progression in a mouse reach-and-withdraw-to-drink (RWD) behavior. Electrophysiology and photo-inhibition across multiple projection neuron types within the MOs-c revealed differential contributions of pyramidal tract and corticothalamic (CT^MOs) output channels to action progression and hand-mouth coordination. Notably, CT^MOs display sustained firing throughout RWD sequence and selectively enhance RWD-relevant activity in postsynaptic thalamus neurons, which also contribute to action coordination. CT^MOs receive converging monosynaptic inputs from forelimb and orofacial sensorimotor areas and are reciprocally connected to thalamic neurons, which project back to the cortical network. Therefore, motor cortex corticothalamic channel may selectively amplify the thalamic integration of cortical and subcortical sensorimotor streams to coordinate a skilled motor sequence.

Background: Modeling brain stimulation at the microscopic scale may reveal new paradigms for a variety of stimulation modalities.

Objective: We present the largest map of distributions of the extracellular electric field to date within a layer L2/L3 mouse primary visual cortex brain sample, which was enabled by automated analysis of serial section electron microscopy images with improved handling of image defects (250×140×90 μm ³ volume).

Methods: We used the map to identify microscopic perturbations of the extracellular electric field and their effect on the activating thresholds of individual neurons. Previous relevant studies modeled a macroscopically homogeneous cortical volume. Result: Our immediate result is a reduction of the predicted stimulation field strength necessary for neuronal activation by a factor of approximately 0.7 (or by 30%) on average, due to microscopic perturbations of the extracellular electric field-an electric field "spatial noise" with a mean value of zero.

Conclusion: Although this result is largely sample-specific, it aligns with experimental data indicating that existing macroscopic theories substantially overestimate the electric fields necessary for brain stimulation.

Significance statement: Currently, there is a discrepancy between macroscopic volumetric brain modeling for brain stimulation and experimental results: experiments typically reveal lower electric intensities required for brain stimulation. This study is arguably the first attempt to model brain stimulation at the microscopic scale, enabled by automated analysis of modern scanning electron microscopy images of the brain. The immediate result is a prediction of lower electric field intensities necessary for brain stimulation, with an average reduction factor of 0.7.

Both enhanced motion-induced nausea and increased static imbalance are observed symptoms in migraine and especially vestibular migraine (VM). Motion-induced nausea and static imbalance were investigated in a mouse model, nestin/hRAMP1, expressing elevated levels of human RAMP1 which enhances CGRP signaling in the nervous system, and compared to non-affected littermate controls. Behavioral surrogates such as the motion- induced thermoregulation and postural sway center of pressure (CoP) assays were used to assess motion sensitivity. Nausea readouts revealed that the nestin/hRAMP1 mouse exhibit an increased sensitivity to CGRP's effects at lower doses compared to unaffected controls. In addition, the nestin/hRAMP1 mice exhibit a higher dynamic range in postural sway than their wildtype counterparts, along with increased sway observed in nestin/hRAMP1 male mice that was not present in male unaffected controls. Results from migraine blocker experiments were challenging to interpret, but the data suggests that olcegepant is incapable of reversing CGRP-induced or endogenous alterations in the nestin/hRAMP1 mice, while rizatriptan was ineffective in both the nestin/hRAMP1 and control mice. The results indicate that overexpression of hRAMP1 leads to heightened endogenous CGRP signaling. Results also suggest that both olcegepant and rizatriptan are ineffective in reducing nausea and sway in this hypersensitive CGRP mouse model. This study suggests that the hypersensitive nestin/hRAMP1 mouse may serve as a model for difficult to treat cases of migraine that exhibit increased motion sensitivity.

Sleep disturbances are associated with poor long-term memory (LTM) formation, yet the underlying cell types and neural circuits involved have not been fully decoded. Dopamine neurons (DANs) are involved in memory processing at multiple stages. Here, using both male and female flies, Drosophila melanogaster , we show that, during the first few hours of memory consolidation, disruption of basal activity of a small subset of protocerebral anterior medial DANs (PAM-DANs), by either brief activation or inhibition of the two dorsal posterior medial (DPM) neurons, impairs 24 h LTM. Interestingly, these brief changes in activity using female flies result in sleep loss and fragmentation, especially at night. Pharmacological rescue of sleep after manipulation restores LTM. A specific subset of PAM-DANs (PAM-α1) that synapse onto DPM neurons specify the microcircuit that links sleep and memory. PAM-DANs, including PAM-α1, form functional synapses onto DPM mainly via multiple dopamine receptor subtypes. This PAM-α1 to DPM microcircuit exhibits a synchronized, transient, post-training increase in activity during the critical memory consolidation window, suggesting an effect of this microcircuit on maintaining the sleep necessary for LTM consolidation. Our results provide a new cellular and circuit basis for the complex relationship between sleep and memory.