Faculty Bibliography

Coupling of stem/progenitor cell proliferation and differentiation to organismal physiological demands ensures the proper growth and homeostasis of tissues. However, in vivo mechanisms underlying this control are poorly characterized. We investigated the role of ribosomal protein S6 kinase (S6K) at the intersection of nutrition and the establishment of a stem/progenitor cell population using the C. elegans germ line as a model. We find that rsks-1 (which encodes the worm homolog of mammalian p70S6K) is required germline-autonomously for proper establishment of the germline progenitor pool. In the germ line, rsks-1 promotes cell cycle progression and inhibits larval progenitor differentiation, promotes growth of adult tumors and requires a conserved TOR phosphorylation site. Loss of rsks-1 and ife-1 (eIF4E) together reduces the germline progenitor pool more severely than either single mutant and similarly to reducing the activity of let-363 (TOR) or daf-15 (RAPTOR). Moreover, rsks-1 acts in parallel with the glp-1 (Notch) and daf-2 (insulin-IGF receptor) pathways, and does not share the same genetic dependencies with its role in lifespan control. We show that overall dietary restriction and amino acid deprivation cause germline defects similar to a subset of rsks-1 mutant phenotypes. Consistent with a link between diet and germline proliferation via rsks-1, loss of rsks-1 renders the germ line largely insensitive to the effects of dietary restriction. Our studies establish the C. elegans germ line as an in vivo model to understand TOR-S6K signaling in proliferation and differentiation and suggest that this pathway is a key nutrient-responsive regulator of germline progenitors.

Introduction: Atherosclerosis is the leading cause of death in the Western world. Despite huge advances in understanding its pathophysiological mechanisms, current treatment is mostly based on 'traditional' risk factors. The introduction of statins more than 20 years ago reduced morbidity and mortality of atherosclerosis by 30%, leaving a residual cardiovascular risk. Therefore, efforts continue toward the development of novel therapies that can be added to established treatments. Besides targeting dyslipidemia, recent focus has been put on preventing or resolving inflammatory processes involved in atherosclerosis. Areas covered: The article discusses therapeutic and diagnostic targets in atherosclerosis and how they can be discovered and studied in preclinical animal models. The roles of immune cells, specifically macrophages and monocytes, in plaque inflammation are discussed. The article also describes current preclinical models of atherosclerosis, specifically the mouse, study designs (for progression and regression studies), basic and advanced methods of analysis of atherosclerotic lesions, and discusses the challenges of translating the findings to humans. Expert opinion: Advances in genomics, proteomics, lipidomics and the development of high-throughput screening techniques help to improve our understanding of atherosclerosis disease mechanisms immensely and facilitate the discovery of new diagnostic and therapeutic targets. Preclinical studies in animals are still indispensable to uncover pathways involved in atherosclerotic disease and to evaluate novel drug targets. The translation of these targets, however, from animal studies to humans remains challenging. There is a strong need for novel biomarkers that can be used to prove the concept of a new target in humans.

Since ion channels move electrical charge during their activity, they have traditionally been studied using electrophysiological approaches. This was sometimes combined with mathematical models, for example with the description of the ionic mechanisms underlying the initiation and propagation of action potentials in the squid giant axon by Hodgkin and Huxley. The methods for studying ion channels also have strong roots in protein chemistry (limited proteolysis, the use of antibodies, etc.). The advent of the molecular cloning and the identification of genes coding for specific ion channel subunits in the late 1980s introduced a multitude of new techniques with which to study ion channels and the field has been rapidly expanding ever since (e.g. antibody development against specific peptide sequences, mutagenesis, the use of gene targeting in animal models, determination of their protein structures) and new methods are still in development. This review focuses on techniques commonly employed to examine ion channel function in an electrophysiological laboratory. The focus is on the K(ATP) channel, but many of the techniques described are also used to study other ion channels.

Toll-like receptors (TLRs) are innate sentinels required for clearance of bacterial and fungal infections of the cornea, but their role in viral immunity is currently unknown. We report that TLR signaling is expendable in herpes simplex virus (HSV)-1 containment as depicted by plaque assays of knockout mice (MyD88(-/-), Trif(-/-) and MyD88(-/-) Trif(-/-) double knockout) resembling wild-type controls. To identify the key sentinel in viral recognition of the cornea, in vivo knockdown of the DNA sensor IFI-16/p204 in the corneal epithelium was performed and resulted in a loss of IFN-regulatory factor-3 (IRF-3) nuclear translocation, interferon-alpha production, and viral containment. The sensor seems to have a similar function in other HSV clinically relevant sites such as the vaginal mucosa in which a loss of p204/IFI-16 results in significantly more HSV-2 shedding. Thus, we have identified an IRF-3-dependent, IRF-7- and TLR-independent innate sensor responsible for HSV containment at the site of acute infection.

BACKGROUND: C/EBP homologous protein-10 (CHOP-10) is a novel developmentally regulated nuclear protein that emerges as a critical transcriptional integrator among pathways regulating differentiation, proliferation, and survival. In the present study, we analyzed the role of CHOP-10 in postnatal neovascularization. METHODS AND RESULTS: Ischemia was induced by right femoral artery ligation in wild-type and CHOP-10(-/-) mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes mellitus. Angiographic score, capillary density, and foot perfusion were increased in CHOP-10(-/-) mice compared with wild-type mice. This effect was associated with a reduction in apoptosis and an upregulation of endothelial nitric oxide synthase (eNOS) levels in ischemic legs of CHOP-10(-/-) mice compared with wild-type mice. In agreement with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 short interfering RNA-transfected human endothelial cells, whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using a chromatin immunoprecipitation assay, we also showed that CHOP-10 was bound to the eNOS promoter. Interestingly, enhanced postischemic neovascularization in CHOP-10(-/-) mice was fully blunted in CHOP-10/eNOS double-knockout animals. Finally, we showed that induction of diabetes mellitus is associated with a marked upregulation of CHOP-10 that substantially inhibited postischemic neovascularization. CONCLUSIONS: This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.

Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a approximately 1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism is a common human single nucleotide polymorphism (SNP) that affects the regulated release of BDNF, and has been implicated in affective disorders and cognitive dysfunction. A decreased activation of the infralimbic medial prefrontal cortex (IL-mPFC), a brain region critical for the regulation of affective behaviors, has been described in BDNF(Met) carriers. However, it is unclear whether and how the Val66Met polymorphism affects the IL-mPFC synapses. Here, we report that spike timing-dependent plasticity (STDP) was absent in the IL-mPFC pyramidal neurons from BDNF(Met/Met) mice, a mouse that recapitulates the specific phenotypic properties of the human BDNF Val66Met polymorphism. Also, we observed a decrease in NMDA and GABA receptor-mediated synaptic transmission in the pyramidal neurons of BDNF(Met/Met) mice. While BDNF enhanced non-NMDA receptor transmission and depressed GABA receptor transmission in the wild-type mice, both effects were absent in BDNF(Met/Met) mice after BDNF treatment. Indeed, exogenous BDNF reversed the deficits in STDP and NMDA receptor transmission in BDNF(Met/Met) neurons. BDNF-mediated selective reversal of the deficit in plasticity and NMDA receptor transmission, but its lack of effect on GABA and non-NMDA receptor transmission in BDNF(Met/Met) mice, suggests separate mechanisms of Val66Met polymorphism upon synaptic transmission. The effect of the Val66Met polymorphism on synaptic transmission and plasticity in the IL-mPFC represents a mechanism to account for this impact of SNP on affective disorders and cognitive dysfunction.

Myelination is a highly regulated developmental process whereby oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system ensheathe axons with a multilayered concentric membrane. Axonal myelination increases the velocity of nerve impulse propagation. In this work, we present a novel in vitro system for coculturing primary dorsal root ganglia neurons along with myelinating cells on a highly restrictive and micropatterned substrate. In this new coculture system, neurons survive for several weeks, extending long axons on defined Matrigel tracks. On these axons, myelinating cells can achieve robust myelination, as demonstrated by the distribution of compact myelin and nodal markers. Under these conditions, neurites and associated myelinating cells are easily accessible for studies on the mechanisms of myelin formation and on the effects of axonal damage on the myelin sheath.

Cardiomyocyte hypertrophy is a complex cellular behavior involving coordination of cell size expansion and myofibril content increase. Here, we investigate the contribution of cardiomyocyte hypertrophy to cardiac chamber emergence, the process during which the primitive heart tube transforms into morphologically distinct chambers and increases its contractile strength. Focusing on the emergence of the zebrafish ventricle, we observed trends toward increased cell surface area and myofibril content. To examine the extent to which these trends reflect coordinated hypertrophy of individual ventricular cardiomyocytes, we developed a method for tracking cell surface area changes and myofibril dynamics in live embryos. Our data reveal a previously unappreciated heterogeneity of ventricular cardiomyocyte behavior during chamber emergence: although cardiomyocyte hypertrophy was prevalent, many cells did not increase their surface area or myofibril content during the observed timeframe. Despite the heterogeneity of cell behavior, we often found hypertrophic cells neighboring each other. Next, we examined the impact of blood flow on the regulation of cardiomyocyte behavior during this phase of development. When blood flow through the ventricle was reduced, cell surface area expansion and myofibril content increase were both dampened, and the behavior of neighboring cells did not seem coordinated. Together, our studies suggest a model in which hemodynamic forces have multiple influences on cardiac chamber emergence: promoting both cardiomyocyte enlargement and myofibril maturation, enhancing the extent of cardiomyocyte hypertrophy, and facilitating the coordination of neighboring cell behaviors

INrf2 (Keap1) serves as a negative regulator of the cytoprotective transcription factor Nrf2. At basal levels, INrf2 functions as a substrate adaptor to sequester Nrf2 into the Cul3-Rbx1 E3 ligase complex for ubiquitylation and proteasomal degradation. In response to antioxidants, Nrf2 is released from the INrf2-Cul3-Rbx1 complex and translocates into the nucleus, where it activates ARE-mediated cytoprotective gene expression. The present studies demonstrate that INrf2, Cul3 and Rbx1 export out of the nucleus and are degraded during the early or pre-induction response to antioxidants. Mutation of Tyr85 in INrf2 stymied the nuclear export of INrf2, suggesting that tyrosine phosphorylation controls the pre-induction nuclear export and degradation in response to antioxidants. The nuclear export of Cul3-Rbx1 were also blocked when INrf2Tyr85 was mutated, suggesting that INrf2-Cul3-Rbx1 undergo nuclear export as a complex. INrf2 siRNA also inhibited the nuclear export of Cul3-Rbx1, confirming that Cul3-Rbx1 requires INrf2 for nuclear export. Newly synthesized INrf2-Cul3-Rbx1 is imported back into the nucleus during the post-induction period to ubiquitylate and degrade Nrf2. Mutation of INrf2Tyr85 had no effect on activation of Nrf2 but led to nuclear accumulation of Nrf2 during the post-induction period owing to reduced export and degradation of Nrf2. Our results also showed that nuclear export and degradation followed by the new synthesis of INrf2-Cul3-Rbx1 controls the cellular abundance of the proteins during different phases of antioxidant responses. In conclusion, the early or pre-induction nuclear export of INrf2 in response to antioxidants is controlled by tyrosine phosphorylation, whereas the nuclear export of Cul3 and Rbx1 is controlled by INrf2, allowing normal activation or repression of Nrf2.