Faculty Bibliography

Cyclin D-CDK4/6 complexes drive cell-cycle entry through an RB-E2F-dependent transcriptional program, but how they coordinate proliferation with the membrane and organelle protein supply required for growth is unclear. We identify DeSI1 as a cyclin D-CDK4/6 substrate whose phosphorylation at S25 converts a latent homodimer into an active monomeric deubiquitylase that recognizes hydrophobic proteins such as those bearing transmembrane domains and mitochondrial targeting sequences. Phosphorylated, active DeSI1 extends the lifetime of newly synthesized hydrophobic proteins that support membrane and mitochondrial capacity. Constitutive DeSI1 activation uncouples this proteostatic program from metabolic supply, creating a cytidine-nucleotide supply-demand imbalance associated with impaired CTP-dependent phospholipid homeostasis, cardiolipin depletion, and mitochondrial decompensation -defects that cytidine reverses. In mice, constitutive DeSI1 activation causes progressive cerebellar degeneration with membrane-protein accumulation, respiratory-chain loss, and phospholipid depletion. These findings define a post-translational mechanism coupling cell-cycle entry to membrane and mitochondrial capacity, and reveal the cost of uncoupling this program from its metabolic support.

Immunization strategies are central to pathogen control, where efficacy relies on antigen distribution and inflammatory context. Lymphatic vessels are necessary conduits for antigen transport that actively remodel in peripheral tissues and draining lymph nodes (LNs) in response to their inflammatory context. In this study, we found that inhibiting viral-induced lymphatic remodeling in skin and LNs prompted germinal center (GC) expansion but, paradoxically, decreased the protective antibody response. While GCs scaled proportionately, larger GCs failed to support proliferative bursts consistent with clonal selection due to impaired T-B cell interactions. Accordingly, follicle and GC size are evolutionarily conserved across diverse mammals despite increased LN size. Lymphatic vessels controlled GC size through two coordinated processes: lymphatic remodeling in infected skin limited viral transport to the LN, while the resulting permissive environment in the LN supported perifollicular lymphangiogenesis that constrained GC expansion.

Age is the greatest risk factor for mortality and morbidity. We previously reported premature ageing in PKP2 arrhythmogenic cardiomyopathy (PKP2-ACM). However, depiction of cardiomyocyte molecular anatomy in premature ageing remains incomplete. Furthermore, the relationship between myocyte premature ageing and the molecular components of electrical homeostasis in PKP2-deficient hearts remains understudied. Therefore, we aimed to identify molecular anatomical changes of premature ageing in PKP2-deficient adult cardiomyocytes, the corresponding gene cohort and its imprint in the human electrocardiogram (ECG). We used a murine model of cardiomyocyte-specific PKP2 knockout. Molecular anatomy was resolved by expansion and structured illumination microscopy, allowing 3D-nanometric visualization, and by serial block-face scanning electron microscopy. We used omics databases to distill a subset of genes related to ageing and PKP2 deficiency containing single nucleotide polymorphisms (SNPs) with an ECG-relevant genomic signature. We found increased DNA damage and reduced abundance of transcriptionally repressed heterochromatin at the lamin-associated domain (LAD), and disrupted mitochondria ultrastructure at the intercalated disc. Genes dysregulated in ageing hearts and PKP2-deficient cells were distilled to identify those in the PKP2 human left ventricular gene network (by GTEx) that contain SNPs with an imprint in the human ECG (based on ECG GWAS). These findings indicate that in PKP2 deficiency, transcriptionally repressed heterochromatin in the LAD is vulnerable to damage, and erasing of otherwise transcriptionally inaccessible genomic regions, consistent with premature cellular ageing and transcriptional reprogramming. Multi-omics analysis indicates that reduced PKP2 expression accelerates cardiomyocyte ageing, and the age-related gene network contributes to dysfunction of cell metabolism and arrhythmia risk in PKP2-ACM. KEY POINTS: We used advanced imaging technology to unveil DNA damage and loss of heterochromatin architecture in the lamin-associated domain (LAD), and mitochondrial remodelling at the intercalated disc, as features of premature myocyte ageing in adult PKP2-deficient murine cardiomyocytes. Multi-omics analysis identified a network of genes dysregulated by ageing and by PKP2 deficiency. Cross-correlation with the ECG GWAS database found a subset of single nucleotide polymorphisms with an imprint in the human electrocardiogram. These results provide novel insight into the mechanisms of transcriptional remodelling and take an initial step toward a better understanding of the oligogenic bases of arrhythmia risk in PKP2-deficient hearts.

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.

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.

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.

Incomplete reporting of microscopy methods undermines transparency, reproducibility, and data reuse. Despite recent initiatives, comprehensive, broadly endorsed, and accessible reporting guidelines are still lacking. Here, we present a bare minimal microscopy reporting requirements checklist that integrates human- and machine-readable input to provide clear, actionable guidance for researchers, reviewers, and publishers and to advance community standards in microscopy.

Excessive accumulation of lipids within cardiomyocytes can sometimes initiate cardiomyopathy, while in other situations excess lipids do not cause harm. To understand how pathologic and non-pathologic lipid accumulation differ, we isolated lipid droplets (LDs) from two genetically altered mouse lines and from wild-type (WT) mice after an overnight fast. The LDs from MHC-peroxisomal proliferator-activated receptor γ1(MHC-Pparg1) transgenic mice were threefold larger than those from either fasted WT or non-cardiomyopathy MHC-diacylglycerol acyl transferase 1 (MHC-Dgat1) transgenic mice. Proteomic analysis of the LD-associated membrane proteins (LDAMPs) showed that MHC-Pparg1 LDs had less perilipin (PLIN). Proteins associated with lipolysis and LD formation (CIDEs and MTP), lipid synthesis, and Pparg signaling pathways were increased in MHC-Pparg1 LDAMPs. Unlike in MHC-Pparg1, MHC-Dgat1 LDAMPs exhibited increased mitochondrial peroxidative proteins with reduced adipose triglyceride lipase (Pnpla2), and Pparg coactivator 1 alpha (Pgc1A). Cardiomyocytes from MHC-Pparg1 hearts had transmission electron microscopy (TEM) images of ongoing lipolysis and greater amounts of lipolytic proteins. In contrast, images from MHC-Dgat1 cardiomyocytes showed more lipophagy. Consistent with the proteomic study and EM images, cardiac immunofluorescence staining showed that PLIN5 protein, thought to block LD lipolysis, was markedly reduced with MHC-Pparg1 overexpression, while hormone-sensitive lipase was increased. The autophagosome marker protein LC3B was increased in MHC-Dgat1 but not in MHC-Pparg1 hearts. Potentially toxic lipids like diacylglycerols and ceramides were increased in hearts but not LDs from MHC-Pparg1 mice. Our data indicate that cardiomyocyte LDs vary in size, composition, and metabolism. Cardiotoxicity was associated with greater LD lipolysis, which we postulate leads to intracellular release of toxic lipids.

The capsule of Streptococcus pneumoniae (Spn) is highly heterogeneous based on the expression of distinct polysaccharides. Spn transformation, controlled by the Com regulon, has been predominantly studied using unencapsulated laboratory strains. However, genomic studies revealed different rates of recombination events in clinical isolates of different serotypes. As these isolates were genetically distinct beyond capsule-encoding genes, the exact relationship between transformation and capsule remains unclear. Herein, we compared the transformability of a collection of isogenic capsule-switch strains. Strains with different capsule types and amounts significantly differed in their transformation frequency, with the unencapsulated strain having a higher frequency compared to encapsulated strains. A GFP-reporter of each strain monitoring the expression of a Com regulon-controlled gene showed similar kinetics, indicating differences in transformability were due to processes downstream of competence activation. The Com pilus, induced by competence, binds and takes in the donor DNA and is the central component of the transformation apparatus. The surface exposure of Com pilus significantly differed among serotypes with highly transformable strains having more cells binding ComGC antibody. Furthermore, electron microscopy demonstrated that transformability correlated with the proportion of cells bearing a Com pilus, which was affected by both the presence of capsule and serotype. Additionally, the unencapsulated strain displayed longer pili than encapsulated strains. Examination of capsule porosity revealed that serotypes with higher transformation frequencies had more porous capsules. Together, these results indicate that the capsule interferes with the assembly of Com pilus, thereby inhibiting the natural transformation of Spn.IMPORTANCEThe capsule is a major virulence factor of Streptococcus pneumoniae (Spn), providing a physical shield and exhibiting extensive diversity across at least 100 serotypes. Although natural transformation of Spn has predominantly been characterized in unencapsulated laboratory strains, clinical encapsulated isolates also exhibit transformability and demonstrate varied recombination rates during host carriage. We utilized otherwise genetically identical capsule-switch strains to isolate the effect of capsule on transformation. We demonstrate serotype- and quantity-dependent inhibition of transformation by the capsule, mediated through hindrance with the transformation pilus assembly and function. This study challenges the paradigm that unencapsulated laboratory strains fully recapitulate natural transformation dynamics. By redefining the capsule as a multifunctional modulator of Spn biology, balancing virulence and adaptability, our findings advance our understanding of pneumococcal evolution.