Molecular Determinants of the Regulation of Development and Metabolism by Neuronal eIF2α Phosphorylation in

Cell-nonautonomous effects of signaling in the nervous system of animals can influence diverse aspects of organismal physiology. We previously showed that phosphorylation of Ser49 of the α-subunit of eukaryotic translation initiation factor 2 (eIF2α) in two chemosensory neurons by PEK-1/PERK promotes entry of Caenorhabditis elegans into dauer diapause. Here, we identified and characterized the molecular determinants that confer sensitivity to effects of neuronal eIF2α phosphorylation on development and physiology of C. elegans. Isolation and characterization of mutations in eif-2Ba encoding the α-subunit of eIF2B support a conserved role, previously established by studies in yeast, for eIF2Bα in providing a binding site for phosphorylated eIF2α to inhibit the exchange factor eIF2B catalytic activity that is required for translation initiation. We also identified a mutation in eif-2c, encoding the γ-subunit of eIF2, which confers insensitivity to the effects of phosphorylated eIF2α while also altering the requirement for eIF2Bγ. In addition, we show that constitutive expression of eIF2α carrying a phosphomimetic S49D mutation in the ASI pair of sensory neurons confers dramatic effects on growth, metabolism, and reproduction in adult transgenic animals, phenocopying systemic responses to starvation. Furthermore, we show that constitutive expression of eIF2α carrying a phosphomimetic S49D mutation in the ASI neurons enhances dauer entry through bypassing the requirement for nutritionally deficient conditions. Our data suggest that the state of eIF2α phosphorylation in the ASI sensory neuron pair may modulate internal nutrient sensing and signaling pathways, with corresponding organismal effects on development and metabolism. Keywords: Caenorhabditis elegans; Dauer; EIF2α; phosphorylation; sensory neurons; translational contro

Cell Type of Origin Influences the Molecular and Functional Properties of Mouse Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.Stem Cell and Regenerative Biolog

Genetic analysis of the neuronal integrated stress response in developmental plasticity and organismal physiology of C. elegans

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.Cataloged from PDF version of thesis.Includes bibliographical references.The genetic study of the C. elegans dauer developmental decision has served as an experimental paradigm for understanding how environmental cues influence organismal physiology through evolutionarily conserved neuroendocrine signaling mechanisms. My genetic characterization of the previously isolated daf-28(sa191) mutant that enters dauer constitutively has revealed cell-nonautonomous roles of conserved stress signaling pathways-the Unfolded Protein Response (UPR) and translational control mediated by eIF2[alpha] phosphorylation. While the cell-autonomous functions of these stress-responsive mechanisms in maintaining cellular homeostasis have been examined, their organismal effects on remodeling development and stress responses remain largely unexplored. Chapter II will highlight the hypotheses and approaches that led to identification of the PEK-1/PERK branch of the UPR, functioning in a pair of chemosensory neurons, as a novel regulator of the dauer developmental decision. Chapter III will examine the systemic effects of eIF2[alpha] phosphorylation, downstream of PERK/PEK-1 activation, in the sensory nervous system on larval development and stress responses. Specifically, the identification of the C. elegans translational regulatory factors that function as molecular determinants of cellular and systemic sensitivity to eIF2[alpha] phosphorylation will be described. Subsections of Chapter III and IV will also highlight genes whose functions can modify the organismal effects of the UPR and eIF2[alpha] phosphorylation: these genes are involved in modulation of ER proteostasis or function in the dauer neuroendocrine pathways that interact with the UPR or eIF2[alpha] phosphorylation. Finally, we proceed to show that alterations in the neuronal eIF2[alpha] phosphorylation status may modulate sensory processing to influence diverse physiological outputs, mimicking the effects of starvation or unfavorable microbial environment. Collectively, results from my study indicate that modulation of the UPR and eIF2[alpha]-mediated translational control in the sensory nervous system confers substantial cell-nonautonomous effects on animal physiology. These findings underscore how molecular events underlying cellular homeostasis, which can be perturbed by fluctuating environmental and developmental conditions, may be co-opted to systemically reprogram organismal stress responses in C. elegans.by Warakorn Kulalert.Ph. D

The Unfolded Protein Response in a Pair of Sensory Neurons Promotes Entry of C. elegans into Dauer Diapause

In response to unfavorable environmental conditions such as starvation, crowding, and elevated temperature, Caenorhabditis elegans larvae enter an alternative developmental stage known as dauer [ 1], which is characterized by adaptive changes in stress resistance and metabolism [ 2 ; 3]. The genetic dissection of the molecular mechanisms of the C. elegans dauer developmental decision has defined evolutionarily conserved signaling pathways of organismal neuroendocrine physiology [ 2; 3 ; 4]. Here, we have identified a mechanism by which a dominant mutation in a neuronal insulin gene, daf-28(sa191) [ 5; 6 ; 7], causes constitutive entry into dauer diapause. We demonstrate that expression of the mutant DAF-28 insulin peptide results in endoplasmic reticulum (ER) stress in the ASI pair of chemosensory neurons. The neuronal ER stress does not compromise cellular survival but activates PEK-1, the C. elegans ortholog of the mammalian eIF2α kinase PERK, which in turn phosphorylates Ser49 of eIF2α, specifically in the ASI neuron pair, to promote entry into dauer diapause. Our data establish a novel role for ER stress and the unfolded protein response (UPR) in promoting entry into dauer diapause and suggest that, in addition to cell-autonomous activities in the maintenance of ER homeostasis, the UPR may act in a non-cell-autonomous manner to promote organismal adaptation to stress during larval development.National Institutes of Health (U.S.) (grant GM084477)Lawrence Ellison Foundation (Ellison New Scholar in Aging Award

Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages