Faculty Bibliography

Neuronal axons have traditionally been considered to be the primary mediators of functional connectivity among brain regions. However, the role of astrocyte-mediated communication has been largely underappreciated. Astrocytes communicate with one another through gap junctions, but the extent and specificity of this communication remain poorly understood. Astrocyte gap junctions are necessary for memory formation^1,2, synaptic plasticity^3-5, coordination of neuronal signalling⁶, and closing the visual and motor critical periods^7,8. These findings indicate that this form of communication is essential for proper central nervous system development and function. Despite the importance of astrocyte gap junctional networks, studying them has been challenging. Current methods such as slice electrophysiology disrupt network connectivity and introduce artefacts due to tissue damage. Here, we developed a vector-based approach that labels molecules as they are fluxed by astrocyte gap junctions in awake, behaving animals to overcome these limitations. We then used whole-brain tissue clearing^9,10 to image these intact, three-dimensional astrocyte networks. We show that multiple astrocyte networks traverse the mouse brain. These networks selectively connect specific regions, rather than diffusing indiscriminately, and vary in size and organization. We observe local networks that are confined to single brain regions and long-range networks that robustly interconnect multiple regions across hemispheres, often exhibiting patterns distinct from known neuronal networks. We also demonstrate that astrocyte networks undergo structural reorganization in the adult brain after sensory deprivation. These findings reveal a mode of communication between distant brain regions that is mediated by plastic networks of gap junction-coupled astrocytes.

UNLABELLED:LKB1 mutations in lung cancer promote an immunosuppressive tumor microenvironment, but the underlying mechanisms remain unknown. Using genetically engineered mouse models and human tumor samples, we demonstrate that LKB1 loss leads to high expression of the cytokine leukemia-inhibitory factor (LIF), which through a cancer cell-autonomous autocrine loop, orchestrates the infiltration of immunosuppressive SiglecFHi neutrophils and Arg1+ interstitial macrophages. Genetic deletion of Lifr, the receptor for LIF, on Lkb1-mutant lung tumors revealed that autocrine LIF signaling induces tumor plasticity and the emergence of a Sox17+ dedifferentiated inflammatory cell state. Antibody-mediated LIF neutralization selectively eliminates the Sox17+ tumor cell state, reduces immunosuppressive myeloid cells, and enhances antitumor T-cell responses. Our study uncovers a novel LKB1-LIF axis driving immune evasion and identifies LIF as a potential therapeutic target in LKB1-mutant lung cancer. This work highlights the interplay between tumor genetics, cellular plasticity, and immune regulation in lung cancer progression. SIGNIFICANCE/UNASSIGNED:LKB1-mutant lung cancers express LIF, which induces an immunosuppressive Sox17+ tumor state. Anti-LIF therapy eliminates this state and restores antitumor immunity, revealing a novel vulnerability in this aggressive cancer subtype lacking effective targeted therapies.

Reactivation of quiescent neural stem cells (NSCs) in the central nervous system (CNS) is a tightly controlled process that generates new neurons and glia to maintain homeostasis or enable repair post-injury, but it remains unclear if reactivation of distinct NSC populations is coupled. Here, we discovered that NSC quiescence exit in Drosophila follows a hierarchical sequence, whereby activation of anterior stem cells in the brain lobes precedes and is required for the timely state-transition of more posterior NSCs in the ventral nerve cord. To achieve this, quiescent NSCs transiently activate neuronal genes. This transient neuronal state is temporary and specific to NSC dormancy, as neuronal genes are switched off after stem cells resume proliferation. Blocking neuronal firing in brain lobe neurons delays the onset of posterior NSC reactivation. Our results reveal long-range communication between quiescent NSCs to coordinate reactivation across the CNS, enabled by a transient, plastic neuron-like state that allows direct interaction with neuronal axons.

Cerebral malaria is a major complication of Plasmodium falciparum infection that occurs upon the sequestration of infected red blood cells (iRBCs) in brain capillaries, resulting in the loss of endothelial barrier integrity, brain swelling, and frequently long-term sequelae or death. P. falciparum-iRBCs cause the disruption of human brain microvascular endothelial cell barrier integrity in vitro, mimicking the microenvironment of cerebral malaria, yet the specific mechanisms mediating this process remain unknown. Upon infection of the host RBCs, P. falciparum produces hemozoin, a crystal form of heme generated following the degradation of hemoglobin by the parasite. Here, we show that the endothelial barrier-disrupting activity is found entirely in the hemozoin fraction of P. falciparum-iRBCs. This activity is not caused by the hemozoin crystal itself, which is not able to induce barrier disruption, but by the biomolecules that are associated with it. Treatment of purified P. falciparum hemozoin with proteases inhibits the disruption of endothelial barrier integrity caused by the hemozoin, indicating an important role for proteins in the disruption of the barrier. Conversely, treatment with nucleases did not affect hemozoin barrier-disrupting activity. These results identify a key molecular mechanism in the P. falciparum-mediated brain endothelial barrier disruption during cerebral malaria and may open new avenues for the treatment of this complication.IMPORTANCEWhile several specific biomolecules have been proposed to contribute to the disruption of endothelial barrier integrity in cerebral malaria, no single Plasmodium falciparum- or host-derived factor has been definitively identified as the primary driver of this disruption. Here, we identify the brain endothelial barrier-disruptive P. falciparum-infected red blood cell (iRBC)-derived activity to be caused by biomolecules bound to hemozoin, identifying a key, novel mechanism in the pathogenesis of cerebral malaria. The finding that P. falciparum hemozoin also disrupts a pulmonary endothelial cell barrier opens the possibility that this mechanism underlies other severe malaria complications. The implication of P. falciparum-iRBC-derived proteins in this mechanism is in line with previous reports, providing a novel interpretation of these findings in the context of hemozoin-binding. This knowledge provides a new perspective in the search for specific molecules and mechanisms involved in barrier disruption, which may lead to the development of much-needed specific treatments for cerebral malaria.

RAS proteins control signals required for cell growth and survival and, when constitutively activated by mutation, can drive oncogenesis. RAS proteins are primarily regulated by their GTP or GDP binding state, which is controlled by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). RAS proteins are also substrates for dozens of posttranslational modifications (PTMs) that target them to membranes and serve as a secondary means of regulation. Because the newly developed direct RAS inhibitors do not produce durable responses in RAS-dependent cancer, there is renewed interest in targeting the PTMs of RAS. These modifications are the subject of this review.

Over the past decade, the field of human embryo editing has witnessed remarkable advancements and triggered significant ethical debates. The groundbreaking tool, CRISPR/Cas9, has revolutionized the landscape of genetic engineering by enabling modifications at the genomic level in germ cells. Since the first case of human embryo gene editing in 2015, the field has rapidly progressed, presenting promising avenues for therapeutic interventions. However, it still grapples with safety concerns, including off-target effects, mosaicism, and the long-term impacts of genetic alterations, as well as ongoing ethical controversies. In this review, we will systematically overview the significant research in this field and provide insights into the potential applications of basic research in early embryonic development and the treatment of genetic diseases.

LRP2 (Megalin or low-density lipoprotein-related receptor 2), together with Cubilin and Amnionless, is responsible for binding and internalizing a wide range of nutrients and toxins from the kidney's glomerular filtrate by endocytosis. Accordingly, Lrp2 deletion or mutation results in the loss of these ligands into the urine. Yet Lrp2 is essential not only for receptor-mediated but also for fluid-phase endocytosis, implicating a broader role beyond ligand binding. To identify the linkage between Lrp2 and endocytosis, we engineered Lrp2-APEX2-expressing mice and performed biotinylation in vivo to label Lrp2's cytoplasmic partners. We demonstrated the specificity and sensitivity of this technique by mass spectrometric identification of biotinylated proteins from kidney lysate and immunostaining kidney sections. We identified critical endocytic regulators interacting with Lrp2, but also many proteins functionally associated with endocytosis that are not already known to interact with Lrp2. These data suggest that Lrp2 plays a central role in organizing apical membranes through PDZ domain proteins and engages with regulators and molecular motors during endocytosis. These interactions are abolished in the absence of Lrp2.

Aneuploidy is common in cancer and has been implicated in promoting tumor progression, yet the underlying mechanisms remain poorly understood. By generating models of aneuploidy, we found that aneuploidy confers resistance to reactive oxygen species (ROS)-mediated cell death, independent of the specific chromosomes gained or lost. Mechanistically, poly(ADP-ribose) polymerase 1 (PARP1) is suppressed in aneuploid cells, which inhibits PARP1-mediated cell death (parthanatos). We validated aneuploidy-associated PARP1 suppression across 15 cell models and human tumors, with pronounced effects in metastatic tumors. Importantly, PARP1 downregulation promotes tumor metastasis while PARP1 upregulation suppresses it. Through a genome-wide CRISPR screen and functional validation, we identified the transcription factor CCAAT/enhancer-binding protein beta (CEBPB) as a mediator of PARP1 downregulation and ROS resistance in aneuploid cells. Lysosomal dysfunction serves as the upstream activator of CEBPB in aneuploid cells. We propose that aneuploidy-driven CEBPB activation suppresses PARP1, fostering ROS resistance and cancer progression.

BACKGROUND:are the most common cause of familial arrhythmogenic right ventricular cardiomyopathy. This study tests whether plakophilin-2 (PKP2) deficiency only in cardiomyocytes is sufficient to provoke premature aging and proinflammatory senescence in nonmyocyte, cardiac resident cells. METHODS:We studied mice with cardiomyocyte-specific, tamoxifen-activated loss of PKP2 (cardiomyocyte-specific conditional knockout of plakophilin-2) using conventional and multiplex imaging, cytokine arrays, epigenetic clocks, spatial transcriptomics, expansion and structured illumination microscopy, and correlative data analysis. We examined nonmyocytes and cardiomyocytes for premature aging and senescence. RESULTS:We observed senescence-associated heterochromatin foci in nonmyocytes, predominantly in cells positive for α-smooth muscle actin staining. Cytokines in media of nonmyocyte cells were consistent with senescence-associated secretory phenotype. Epigenetic clocks identified premature aging. Multiplex immunohistochemistry showed nonmyocyte cells in niches, intermingled with cardiomyocytes. Spatial transcriptomics showed overrepresentation of senescence-associated secretory phenotype-related transcripts, predominantly in myocyte-rich areas of the left ventricle. Senescence-associated heterochromatin foci and increased epigenetic age were not found in cardiomyocytes from cardiomyocyte-specific conditional knockout of plakophilin-2 hearts, although we observed structural features associated with premature aging. Cross-reference analysis showed correlation between the cardiomyocyte-specific conditional knockout of plakophilin-2 cardiac proteome and that of mice 5 or 6 times their chronological age, as well as transcriptional signatures of neurodegenerative diseases. CONCLUSIONS:Loss of PKP2 expression only in adult cardiac myocytes is sufficient to induce proinflammatory senescence in nonmyocytes, and overall premature cardiac aging. This is the first study to intersect cellular senescence and premature aging with desmosomal arrhythmogenic cardiomyopathies. We speculate that cell-agnostic molecular signatures, biomarkers, and pharmacology of senescence and of neurodegenerative diseases may be relevant to diagnose or treat PKP2 arrhythmogenic right ventricular cardiomyopathy.

During development and homeostasis, tissues move and rearrange to form organs, seal wounds, or-in the case of cancer-spread in the body. To accomplish this, cells in tissues need to communicate with each other, generate force to push themselves forward, and know where to go to-all of this with little to no error. Here, we discuss how a migrating tissue-the zebrafish posterior lateral line primordium-solves these challenges. We focus on the strategies that ensure signaling within the tissue, enable the tissue to generate and transmit force to its substrate for propulsion, and allow robust directional sensing and migration by the tissue. These strategies include facilitated diffusion and ligand trapping for focal signaling, a self-generated attractant gradient for long-distance migration, clamping of the attractant concentration to the attractant receptor's K