Mitochondrial Diseases: Hope for the Future.

Mitochondrial diseases are clinically heterogeneous disorders caused by a wide spectrum of mutations in genes encoded by either the nuclear or the mitochondrial genome. Treatments for mitochondrial diseases are currently focused on symptomatic management rather than improving the biochemical defect caused by a particular mutation. This review focuses on the latest advances in the development of treatments for mitochondrial disease, both small molecules and gene therapies, as well as methods to prevent transmission of mitochondrial disease through the germline.

A germline point mutation in the MYC-FBW7 phosphodegron initiates hematopoietic malignancies.

Oncogenic activation of MYC in cancers predominantly involves increased transcription rather than coding region mutations. However, MYC-dependent lymphomas frequently acquire point mutations in the MYC phosphodegron, including at threonine 58 (T58), where phosphorylation permits binding via the FBW7 ubiquitin ligase triggering MYC degradation. To understand how T58 phosphorylation functions in normal cell physiology, we introduced an alanine mutation at T58 (T58A) into the endogenous c-Myc locus in the mouse germline. While MYC-T58A mice develop normally, lymphomas and myeloid leukemias emerge in ∼60% of adult homozygous T58A mice. We found that primitive hematopoietic progenitor cells from MYC-T58A mice exhibit aberrant self-renewal normally associated with hematopoietic stem cells (HSCs) and up-regulate a subset of MYC target genes important in maintaining stem/progenitor cell balance. In lymphocytes, genomic occupancy by MYC-T58A was increased at all promoters compared with WT MYC, while genes differentially expressed in a T58A-dependent manner were significantly more proximal to MYC-bound enhancers. MYC-T58A lymphocyte progenitors exhibited metabolic alterations and decreased activation of inflammatory and apoptotic pathways. Our data demonstrate that a single point mutation stabilizing MYC is sufficient to skew target gene expression, producing a profound gain of function in multipotential hematopoietic progenitors associated with self-renewal and initiation of lymphomas and leukemias.

A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy.

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause.

Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state.

Growing old with Myc.

The Myc proto-oncogene has been intensively studied in tumorigenesis and development. A new paper in Cell reports the role of Myc as a determinant of mammalian longevity. Myc heterozygous mice exhibit extended lifespans resulting from alterations in multiple cellular processes distinct from those observed in other longevity models.

APC7 mediates ubiquitin signaling in constitutive heterochromatin in the developing mammalian brain.

Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons in vivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence.

Quiescence is a stress-resistant state in which cells reversibly exit the cell cycle and suspend most processes. Quiescence is essential for stem cell maintenance, and its misregulation is implicated in tumor formation. One of the hallmarks of quiescent cells is highly condensed chromatin. Because condensed chromatin often correlates with transcriptional silencing, it has been hypothesized that chromatin compaction represses transcription during quiescence. However, the technology to test this model by determining chromatin structure within cells at gene resolution has not previously been available. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide in quiescent Saccharomyces cerevisiae cells. We describe chromatin domains on the order of 10-60 kilobases that, only in quiescent cells, are formed by condensin-mediated loops. Condensin depletion prevents the compaction of chromatin within domains and leads to widespread transcriptional de-repression. Finally, we demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts.

Antigen-specific age-related memory CD8 T cells induce and track Alzheimer's-like neurodegeneration.

Cerebral (Aß) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aß/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aß/pTau, however, appears to vary depending on the animal model. Our prior work suggested that antigen-specific memory CD8 T ("hiT") cells act upstream of Aß/pTau after brain injury. Here, we examine whether hiT cells influence sporadic AD-like pathophysiology upstream of Aß/pTau. Examining neuropathology, gene expression, and behavior in our hiT mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. We identify an age-related factor acting upstream of Aß/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD.

Max deletion destabilizes MYC protein and abrogates Eµ-Myc lymphomagenesis.

Although MAX is regarded as an obligate dimerization partner for MYC, its function in normal development and neoplasia is poorly defined. We show that B-cell-specific deletion of Max has a modest effect on B-cell development but completely abrogates Eµ-Myc-driven lymphomagenesis. While Max loss affects only a few hundred genes in normal B cells, it leads to the global down-regulation of Myc-activated genes in premalignant Eµ-Myc cells. We show that the balance between MYC-MAX and MNT-MAX interactions in B cells shifts in premalignant B cells toward a MYC-driven transcriptional program. Moreover, we found that MAX loss leads to a significant reduction in MYC protein levels and down-regulation of direct transcriptional targets, including regulators of MYC stability. This phenomenon is also observed in multiple cell lines treated with MYC-MAX dimerization inhibitors. Our work uncovers a layer of Myc autoregulation critical for lymphomagenesis yet partly dispensable for normal development.

A gene regulatory network combining Pax3/7, Sox10 and Mitf generates diverse pigment cell types in medaka and zebrafish.

Neural crest cells generate numerous derivatives, including pigment cells, and are a model for studying how fate specification from multipotent progenitors is controlled. In mammals, the core gene regulatory network for melanocytes (their only pigment cell type) contains three transcription factors, Sox10, Pax3 and Mitf, with the latter considered a master regulator of melanocyte development. In teleosts, which have three to four pigment cell types (melanophores, iridophores and xanthophores, plus leucophores e.g. in medaka), gene regulatory networks governing fate specification are poorly understood, although Mitf function is considered conserved. Here, we show that the regulatory relationships between Sox10, Pax3 and Mitf are conserved in zebrafish, but the role for Mitf is more complex than previously emphasized, affecting xanthophore development too. Similarly, medaka Mitf is necessary for melanophore, xanthophore and leucophore formation. Furthermore, expression patterns and mutant phenotypes of pax3 and pax7 suggest that Pax3 and Pax7 act sequentially, activating mitf expression. Pax7 modulates Mitf function, driving co-expressing cells to differentiate as xanthophores and leucophores rather than melanophores. We propose that pigment cell fate specification should be considered to result from the combinatorial activity of Mitf with other transcription factors.

Current and upcoming treatment approaches to common subtypes of PTCL (PTCL, NOS; ALCL; and TFHs).

ABSTRACT: The treatment of common nodal peripheral T-cell lymphomas (PTCLs), including PTCL, not otherwise specified (PTCL, NOS), anaplastic large-cell lymphomas, and T-follicular helper lymphomas, is evolving. These entities are currently treated similarly with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisone (CHOEP) for CD30-negative diseases, or brentuximab vedotin plus cyclophosphamide, doxorubicin, and prednisone (CHP) for CD30-positive diseases, followed by consolidation with autologous stem cell transplantation in the first remission. Ongoing improvements in PTCL classification, identification of predictive biomarkers, and development of new targeted agents will lead to more specific therapies that address the unique biologic and clinical properties of each entity. For example, widespread efforts focused on molecular profiling of PTCL, NOS is likely to identify distinct subtypes that warrant different treatment approaches. New agents, such as EZH1/2 and JAK/STAT pathway inhibitors, have broadened treatment options for relapsed or refractory diseases. Furthermore, promising strategies for optimizing immune therapy for PTCL are currently under investigation and have the potential to significantly alter the therapeutic landscape. Ongoing frontline study designs incorporate an understanding of disease biology and drug sensitivities and are poised to evaluate whether newer-targeted agents should be incorporated into frontline settings for various disease entities. Although current treatment strategies lump most disease entities together, future treatments will include distinct strategies for each disease subtype that optimize therapy for individuals. This movement toward individualized therapy will ultimately lead to dramatic improvements in the prognosis of patients with PTCL.

Molecular Interactions Required for Activation of Complement Component C2 Include Exosites Located on the Serine Protease Domain of C1s and Mannose-Binding Lectin Associated Protease-2.

The activation of the CP/LP C3 proconvertase complex is a key event in complement activation and involves cleavage of C4 and C2 by the C1s protease (classical pathway) or the mannose-binding lectin-associated serine protease (MASP)-2 (lectin pathway). Efficient cleavage of C4 by C1s and MASP-2 involves exosites on the complement control protein and serine protease (SP) domains of the proteases. The complement control protein domain exosite is not involved in cleavage of C2 by the proteases, but the role of an anion-binding exosite (ABE) on the SP domains of the proteases has (to our knowledge) never been investigated. In this study, we have shown that the ABE on the SP of both C1s and MASP-2 is crucial for efficient cleavage of C2, with mutant forms of the proteases greatly impaired in their rate of cleavage of C2. We have additionally shown that the site of binding for the ABE of the proteases is very likely to be located on the von Willebrand factor domain of C2, with the precise area differing between the enzymes: whereas C1s requires two anionic clusters on the von Willebrand factor domain to enact efficient cleavage of C2, MASP-2 apparently only requires one. These data provide (to our knowledge) new information about the molecular determinants for efficient activation of C2 by C1s and MASP-2. The enhanced view of the molecular events underlying the early stages of complement activation provides further possible intervention points for control of this activation that is involved in a number of inflammatory diseases.

Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1.

The pyruvate kinase isoforms PKM1 and PKM2 are alternatively spliced products of the PKM2 gene. PKM2, but not PKM1, alters glucose metabolism in cancer cells and contributes to tumorigenesis by mechanisms that are not explained by its known biochemical activity. We show that PKM2 gene transcription is activated by hypoxia-inducible factor 1 (HIF-1). PKM2 interacts directly with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements, whereas PKM1 fails to regulate HIF-1 activity. Interaction of PKM2 with prolyl hydroxylase 3 (PHD3) enhances PKM2 binding to HIF-1α and PKM2 coactivator function. Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O(2) consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells.

The stepwise assembly of the neonatal virome is modulated by breastfeeding.

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, which-in some cases-leads to gastrointestinal disorders1-4. Here we show that the assembly of the viral community in neonates takes place in distinct steps. Fluorescent staining of virus-like particles purified from infant meconium or early stool samples shows few or no particles, but by one month of life particle numbers increase to 109 per gram, and these numbers seem to persist throughout life5-7. We investigated the origin of these viral populations using shotgun metagenomic sequencing of virus-enriched preparations and whole microbial communities, followed by targeted microbiological analyses. Results indicate that, early after birth, pioneer bacteria colonize the infant gut and by one month prophages induced from these bacteria provide the predominant population of virus-like particles. By four months of life, identifiable viruses that replicate in human cells become more prominent. Multiple human viruses were more abundant in stool samples from babies who were exclusively fed on formula milk compared with those fed partially or fully on breast milk, paralleling reports that breast milk can be protective against viral infections8-10. Bacteriophage populations also differed depending on whether or not the infant was breastfed. We show that the colonization of the infant gut is stepwise, first mainly by temperate bacteriophages induced from pioneer bacteria, and later by viruses that replicate in human cells; this second phase is modulated by breastfeeding.

An alphacoronavirus polymerase structure reveals conserved replication factor functions.

Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine structures of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. One structure shows an unexpected nsp8 stoichiometry despite remaining bound to RNA. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha- and betacoronaviruses. Our work demonstrates the importance of studying diverse coronaviruses in revealing aspects of coronavirus replication and identifying areas of conservation to be targeted by antiviral drugs.

α-lipoic acid ameliorates consequences of copper overload by up-regulating selenoproteins and decreasing redox misbalance.

α-lipoic acid (LA) is an essential cofactor for mitochondrial dehydrogenases and is required for cell growth, metabolic fuel production, and antioxidant defense. In vitro, LA binds copper (Cu) with high affinity and as an endogenous membrane permeable metabolite could be advantageous in mitigating the consequences of Cu overload in human diseases. We tested this hypothesis in 3T3-L1 preadipocytes with inactivated Cu transporter Atp7a; these cells accumulate Cu and show morphologic changes and mitochondria impairment. Treatment with LA corrected the morphology of Atp7a-/- cells similar to the Cu chelator bathocuproinedisulfonate (BCS) and improved mitochondria function; however, the mechanisms of LA and BCS action were different. Unlike BCS, LA did not decrease intracellular Cu but instead increased selenium levels that were low in Atp7a-/- cells. Proteome analysis confirmed distinct cell responses to these compounds and identified upregulation of selenoproteins as the major effect of LA on preadipocytes. Upregulation of selenoproteins was associated with an improved GSH:GSSG ratio in cellular compartments, which was lowered by elevated Cu, and reversal of protein oxidation. Thus, LA diminishes toxic effects of elevated Cu by improving cellular redox environment. We also show that selenium levels are decreased in tissues of a Wilson disease animal model, especially in the liver, making LA an attractive candidate for supplemental treatment of this disease.

Atp7b-dependent choroid plexus dysfunction causes transient copper deficit and metabolic changes in the developing mouse brain.

Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl' cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-ß-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.

Reduced fire severity offers near-term buffer to climate-driven declines in conifer resilience across the western United States.

Increasing fire severity and warmer, drier postfire conditions are making forests in the western United States (West) vulnerable to ecological transformation. Yet, the relative importance of and interactions between these drivers of forest change remain unresolved, particularly over upcoming decades. Here, we assess how the interactive impacts of changing climate and wildfire activity influenced conifer regeneration after 334 wildfires, using a dataset of postfire conifer regeneration from 10,230 field plots. Our findings highlight declining regeneration capacity across the West over the past four decades for the eight dominant conifer species studied. Postfire regeneration is sensitive to high-severity fire, which limits seed availability, and postfire climate, which influences seedling establishment. In the near-term, projected differences in recruitment probability between low- and high-severity fire scenarios were larger than projected climate change impacts for most species, suggesting that reductions in fire severity, and resultant impacts on seed availability, could partially offset expected climate-driven declines in postfire regeneration. Across 40 to 42% of the study area, we project postfire conifer regeneration to be likely following low-severity but not high-severity fire under future climate scenarios (2031 to 2050). However, increasingly warm, dry climate conditions are projected to eventually outweigh the influence of fire severity and seed availability. The percent of the study area considered unlikely to experience conifer regeneration, regardless of fire severity, increased from 5% in 1981 to 2000 to 26 to 31% by mid-century, highlighting a limited time window over which management actions that reduce fire severity may effectively support postfire conifer regeneration.