Adjuvants influence the maturation of VRC01-like antibodies during immunization.

Once naive B cells expressing germline VRC01-class B cell receptors become activated by germline-targeting immunogens, they enter germinal centers and undergo affinity maturation. Booster immunizations with heterologous Envs are required for the full maturation of VRC01-class antibodies. Here, we examined whether and how three adjuvants, Poly(I:C), GLA-LSQ, or Rehydragel, that activate different pathways of the innate immune system, influence the rate and type of somatic mutations accumulated by VRC01-class BCRs that become activated by the germline-targeting 426c.Mod.Core immunogen and the heterologous HxB2.WT.Core booster immunogen. We report that although the adjuvant used had no influence on the durability of plasma antibody responses after the prime, it influenced the plasma VRC01 antibody titers after the boost and the accumulation of somatic mutations on the elicited VRC01 antibodies.

HIV-1 VRC01 Germline-Targeting Immunogens Select Distinct Epitope-Specific B Cell Receptors.

Activating precursor B cell receptors of HIV-1 broadly neutralizing antibodies requires specifically designed immunogens. Here, we compared the abilities of three such germline-targeting immunogens against the VRC01-class receptors to activate the targeted B cells in transgenic mice expressing the germline VH of the VRC01 antibody but diverse mouse light chains. Immunogen-specific VRC01-like B cells were isolated at different time points after immunization, their VH and VL genes were sequenced, and the corresponding antibodies characterized. VRC01 B cell sub-populations with distinct cross-reactivity properties were activated by each immunogen, and these differences correlated with distinct biophysical and biochemical features of the germline-targeting immunogens. Our study indicates that the design of effective immunogens to activate B cell receptors leading to protective HIV-1 antibodies will require a better understanding of how the biophysical properties of the epitope and its surrounding surface on the germline-targeting immunogen influence its interaction with the available receptor variants in vivo.

Epigenetic signatures of attachment insecurity and childhood adversity provide evidence for role transition in the pathogenesis of perinatal depression.

Early life adversity and insecure attachment style are known risk factors for perinatal depression. The biological pathways linking these experiences, however, have not yet been elucidated. We hypothesized that overlap in patterns of DNA methylation in association with each of these phenomena could identify genes and pathways of importance. Specifically, we wished to distinguish between allostatic-load and role-transition hypotheses of perinatal depression. We conducted a large-scale analysis of methylation patterns across 5 × 106 individual CG dinucleotides in 54 women participating in a longitudinal prospective study of perinatal depression, using clustering-based criteria for significance to control for multiple comparisons. We identified 1580 regions in which methylation density was associated with childhood adversity, 3 in which methylation density was associated with insecure attachment style, and 6 in which methylation density was associated with perinatal depression. Shorter telomeres were observed in association with childhood trauma but not with perinatal depression or attachment insecurity. A detailed analysis of methylation density in the oxytocin receptor gene revealed similar patterns of DNA methylation in association with perinatal depression and with insecure attachment style, while childhood trauma was associated with a distinct methylation pattern in this gene. Clinically, attachment style was strongly associated with depression only in pregnancy and the early postpartum, whereas the association of childhood adversity with depression was time-invariant. We concluded that the broad DNA methylation signature and reduced telomere length associated with childhood adversity could indicate increased allostatic load across multiple body systems, whereas perinatal depression and attachment insecurity may be narrower phenotypes with more limited DNA methylation signatures outside the CNS, and no apparent association with telomere length or, by extension, allostatic load. In contrast, the finding of matching DNA methylation patterns within the oxytocin receptor gene for perinatal depression and attachment insecurity is consistent with the theory that the perinatal period is a time of activation of existing attachment schemas for the purpose of structuring the mother-child relationship, and that such activation may occur in part through specific patterns of methylation of the oxytocin receptor gene.

Overcoming Steric Restrictions of VRC01 HIV-1 Neutralizing Antibodies through Immunization.

Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2∗02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies.

Local and global chromatin interactions are altered by large genomic deletions associated with human brain development.

Large copy number variants (CNVs) in the human genome are strongly associated with common neurodevelopmental, neuropsychiatric disorders such as schizophrenia and autism. Here we report on the epigenomic effects of the prominent large deletion CNVs on chromosome 22q11.2 and on chromosome 1q21.1. We use Hi-C analysis of long-range chromosome interactions, including haplotype-specific Hi-C analysis, ChIP-Seq analysis of regulatory histone marks, and RNA-Seq analysis of gene expression patterns. We observe changes on all the levels of analysis, within the deletion boundaries, in the deletion flanking regions, along chromosome 22q, and genome wide. We detect gene expression changes as well as pronounced and multilayered effects on chromatin states, chromosome folding and on the topological domains of the chromatin, that emanate from the large CNV locus. These findings suggest basic principles of how such large genomic deletions can alter nuclear organization and affect genomic molecular activity.

Multiplex assessment of protein variant abundance by massively parallel sequencing.

Determining the pathogenicity of genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes requires generalizable, scalable assays. We describe variant abundance by massively parallel sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance simultaneously. We apply VAMP-seq to quantify the abundance of 7,801 single-amino-acid variants of PTEN and TPMT, proteins in which functional variants are clinically actionable. We identify 1,138 PTEN and 777 TPMT variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and show that p.Pro38Ser, which accounts for ~10% of PTEN missense variants in melanoma, functions via a dominant-negative mechanism. Finally, we demonstrate that VAMP-seq is applicable to other genes, highlighting its generalizability.

Corrigendum: Snf1-related kinase improves cardiac mitochondrial efficiency and decreases mitochondrial uncoupling.

This corrects the article DOI: 10.1038/ncomms14095.

Snf1-related kinase improves cardiac mitochondrial efficiency and decreases mitochondrial uncoupling.

Ischaemic heart disease limits oxygen and metabolic substrate availability to the heart, resulting in tissue death. Here, we demonstrate that the AMP-activated protein kinase (AMPK)-related protein Snf1-related kinase (SNRK) decreases cardiac metabolic substrate usage and mitochondrial uncoupling, and protects against ischaemia/reperfusion. Hearts from transgenic mice overexpressing SNRK have decreased glucose and palmitate metabolism and oxygen consumption, but maintained power and function. They also exhibit decreased uncoupling protein 3 (UCP3) and mitochondrial uncoupling. Conversely, Snrk knockout mouse hearts have increased glucose and palmitate oxidation and UCP3. SNRK knockdown in cardiac cells decreases mitochondrial efficiency, which is abolished with UCP3 knockdown. We show that Tribbles homologue 3 (Trib3) binds to SNRK, and downregulates UCP3 through PPARα. Finally, SNRK is increased in cardiomyopathy patients, and SNRK reduces infarct size after ischaemia/reperfusion. SNRK also decreases cardiac cell death in a UCP3-dependent manner. Our results suggest that SNRK improves cardiac mitochondrial efficiency and ischaemic protection.

Increased Heme Levels in the Heart Lead to Exacerbated Ischemic Injury.

BACKGROUND: Heme is an essential iron-containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid synthase 2 (ALAS2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart. METHODS AND RESULTS: We first showed that ALAS2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac-specific overexpression of ALAS2. While ALAS2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with ALAS2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS2-overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to nonischemic cardiomyopathy. CONCLUSIONS: Heme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a novel approach for protection against the development of heart failure.

Cardiac-specific ablation of ARNT leads to lipotoxicity and cardiomyopathy.

Patients with type 2 diabetes often present with cardiovascular complications; however, it is not clear how diabetes promotes cardiac dysfunction. In murine models, deletion of the gene encoding aryl hydrocarbon nuclear translocator (ARNT, also known as HIF1ß) in the liver or pancreas leads to a diabetic phenotype; however, the role of ARNT in cardiac metabolism is unknown. Here, we determined that cardiac-specific deletion of Arnt in adult mice results in rapid development of cardiomyopathy (CM) that is characterized by accumulation of lipid droplets. Compared with hearts from ARNT-expressing mice, ex vivo analysis of ARNT-deficient hearts revealed a 2-fold increase in fatty acid (FA) oxidation as well as a substantial increase in the expression of PPARα and its target genes. Furthermore, deletion of both Arnt and Ppara preserved cardiac function, improved survival, and completely reversed the FA accumulation phenotype, indicating that PPARα mediates the detrimental effects of Arnt deletion in the heart. Finally, we determined that ARNT directly regulates Ppara expression by binding to its promoter and forming a complex with HIF2α. Together, these findings suggest that ARNT is a critical regulator of myocardial FA metabolism and that its deletion leads to CM and an increase in triglyceride accumulation through PPARα.

Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation.

Doxorubicin is an effective anticancer drug with known cardiotoxic side effects. It has been hypothesized that doxorubicin-dependent cardiotoxicity occurs through ROS production and possibly cellular iron accumulation. Here, we found that cardiotoxicity develops through the preferential accumulation of iron inside the mitochondria following doxorubicin treatment. In isolated cardiomyocytes, doxorubicin became concentrated in the mitochondria and increased both mitochondrial iron and cellular ROS levels. Overexpression of ABCB8, a mitochondrial protein that facilitates iron export, in vitro and in the hearts of transgenic mice decreased mitochondrial iron and cellular ROS and protected against doxorubicin-induced cardiomyopathy. Dexrazoxane, a drug that attenuates doxorubicin-induced cardiotoxicity, decreased mitochondrial iron levels and reversed doxorubicin-induced cardiac damage. Finally, hearts from patients with doxorubicin-induced cardiomyopathy had markedly higher mitochondrial iron levels than hearts from patients with other types of cardiomyopathies or normal cardiac function. These results suggest that the cardiotoxic effects of doxorubicin develop from mitochondrial iron accumulation and that reducing mitochondrial iron levels protects against doxorubicin-induced cardiomyopathy.

ATP-binding cassette B10 regulates early steps of heme synthesis.

RATIONALE: Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia-reperfusion injury in the heart; however, its primary function has not been established. OBJECTIVE: The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells. METHODS AND RESULTS: Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with δ-aminolevulinic acid reversed these defects. Overexpression of mitochondrial δ-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of δ-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy. CONCLUSIONS: ABCB10 plays a critical role in heme synthesis pathway by facilitating δ-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.

MicroRNA-210 decreases heme levels by targeting ferrochelatase in cardiomyocytes.

BACKGROUND: MicroRNA-210 (miR-210) increases in hypoxia and regulates mitochondrial respiration through modulation of iron-sulfur cluster assembly proteins (ISCU1/2), a protein that is involved in Fe/S cluster synthesis. However, it is not known how miR-210 affects cellular iron levels or production of heme, another iron containing molecule that is also needed for cellular and mitochondrial function. METHODS AND RESULTS: To screen for micro-ribonucleic acids (miRNAs) regulated by iron, we performed a miRNA gene array in neonatal rat cardiomyocytes treated with iron chelators. Levels of miR-210 are significantly increased with iron chelation, however, this response was mediated entirely through the hypoxia-inducible factor (HIF) pathway. Furthermore, miR-210 reduced cellular heme levels and the activity of mitochondrial and cytosolic heme-containing proteins by modulating ferrochelatase (FECH), the last enzyme in heme biosynthesis. Mutation of the 2 miR-210 binding sites in the 3' untranslated region (UTR) of FECH reversed the miR-210 response, while mutation of either binding site in isolation did not exert any effects. Changes mediated by miR-210 in heme and FECH were independent of ISCU, as overexpression of an ISCU construct lacking the 3' UTR does not alter miR-210 regulation of heme and FECH. Finally, FECH levels increased in hypoxia, and this effect was not reversed by miR-210 knockdown, suggesting that the effects of miR-210 on heme are restricted to normoxic conditions, and that the pathway is overriden in hypoxia. CONCLUSIONS: Our results identify a role for miR-210 in the regulation of heme production by targeting and inhibiting FECH under normoxic conditions.

Heme levels are increased in human failing hearts.

OBJECTIVES: The goal of this study was to characterize the regulation of heme and non-heme iron in human failing hearts. BACKGROUND: Iron is an essential molecule for cellular physiology, but in excess it facilitates oxidative stress. Mitochondria are the key regulators of iron homeostasis through heme and iron-sulfur cluster synthesis. Because mitochondrial function is depressed in failing hearts and iron accumulation can lead to oxidative stress, we hypothesized that iron regulation may also be impaired in heart failure (HF). METHODS: We measured mitochondrial and cytosolic heme and non-heme iron levels in failing human hearts retrieved during cardiac transplantation surgery. In addition, we examined the expression of genes regulating cellular iron homeostasis, the heme biosynthetic pathway, and micro-RNAs that may potentially target iron regulatory networks. RESULTS: Although cytosolic non-heme iron levels were reduced in HF, mitochondrial iron content was maintained. Moreover, we observed a significant increase in heme levels in failing hearts, with corresponding feedback inhibition of the heme synthetic enzymes and no change in heme degradation. The rate-limiting enzyme in heme synthesis, delta-aminolevulinic acid synthase 2 (ALAS2), was significantly upregulated in HF. Overexpression of ALAS2 in H9c2 cardiac myoblasts resulted in increased heme levels, and hypoxia and erythropoietin treatment increased heme production through upregulation of ALAS2. Finally, increased heme levels in cardiac myoblasts were associated with excess production of reactive oxygen species and cell death, suggesting a maladaptive role for increased heme in HF. CONCLUSIONS: Despite global mitochondrial dysfunction, heme levels are maintained above baseline in human failing hearts.

mTOR regulates cellular iron homeostasis through tristetraprolin.

Iron is an essential cofactor with unique redox properties. Iron-regulatory proteins 1 and 2 (IRP1/2) have been established as important regulators of cellular iron homeostasis, but little is known about the role of other pathways in this process. Here we report that the mammalian target of rapamycin (mTOR) regulates iron homeostasis by modulating transferrin receptor 1 (TfR1) stability and altering cellular iron flux. Mechanistic studies identify tristetraprolin (TTP), a protein involved in anti-inflammatory response, as the downstream target of mTOR that binds to and enhances degradation of TfR1 mRNA. We also show that TTP is strongly induced by iron chelation, promotes downregulation of iron-requiring genes in both mammalian and yeast cells, and modulates survival in low-iron states. Taken together, our data uncover a link between metabolic, inflammatory, and iron-regulatory pathways, and point toward the existence of a yeast-like TTP-mediated iron conservation program in mammals.

Disruption of ATP-binding cassette B8 in mice leads to cardiomyopathy through a decrease in mitochondrial iron export.

Mitochondrial iron levels are tightly regulated, as iron is essential for the synthesis of Fe/S clusters and heme in the mitochondria, but high levels can cause oxidative stress. The ATP-binding cassette (ABC) transporter ABCB8 is a mitochondrial inner membrane protein with an unknown function. Here, we show that ABCB8 is involved in mitochondrial iron export and is essential for baseline cardiac function. Induced genetic deletion of ABCB8 in mouse hearts resulted in mitochondrial iron accumulation and cardiomyopathy, as assessed by echocardiography and invasive hemodynamics. Mice with ABCB8 deletion in the heart also displayed mitochondrial damage, and higher levels of reactive oxygen species and cell death. Down-regulation of ABCB8 in vitro resulted in decreased iron export from isolated mitochondria, whereas its overexpression had the opposite effect. Furthermore, ABCB8 is needed for the maturation of the cytosolic Fe/S proteins, as its deletion in vitro and in vivo led to decreased activity of cytosolic, but not mitochondrial, iron-sulfur-containing enzymes. These results indicate that ABCB8 is essential for normal cardiac function, maintenance of mitochondrial iron homeostasis and maturation of cytosolic Fe/S proteins. In summary, this report provides characterization of a protein involved in mitochondrial iron export.