103 research outputs found
Transcriptional Regulation of Autophagy.
No full textMacroautophagy (hereafter autophagy) is a cellular recycling process through which cytoplasmic contents are delivered into the lysosome/vacuole for degradation by double-membrane organelles, autophagosomes. Autophagy is essential for cell survival under stress; however, too much autophagy can also be detrimental. Autophagy activity can be regulated by modulating the size or the number of autophagosomes. Although there are more than 40 autophagy-related (ATG) genes that have been identified, it is not fully understood how most of these genes contribute to these aspects of regulation. Autophagy is highly conserved among eukaryotic cells, and its molecular machinery has been best characterized in the budding yeast Saccharomyces cerevisiae. In my thesis studies, I use budding yeast as the model organism, taking advantage of its utility in genetic/genomic screening; in addition, high-throughput sequencing and powerful autophagy assays have been developed uniquely in the yeast system, to explore how autophagy is modulated through transcriptional regulation.
When I joined the Klionsky lab, I became involved in the study of a negative transcriptional regulator of autophagy, Ume6. Deletion of the UME6 gene results in an increase in the size, but not the number, of autophagosomes by increasing the expression of Atg8. From a subsequent genetic screen for autophagy modulators, I identified another transcription repressor of autophagy, Pho23. Intriguingly, Pho23 ended up being characterized as a specific regulator of the number, but not the size, of autophagosomes, or it can be viewed as controlling the rate of autophagosome formation by regulating Atg9 expression. These studies support a model whereby the size and numbers of autophagosomes are independently regulated through precise transcriptional regulation of different ATG genes.
Collaborating with Amélie Bernard, a postdoc in the lab, to further explore the transcriptional regulation network of autophagy, we analyzed 139 yeast strains each deleted for a single gene encoding a transcription factor; we profiled the transcription of several ATG genes in each strain. Through this screen we identified Gcn4, Slf1, Gat1 and Gln3 as transcriptional activators, and Spt10, Fyv5, and Rph1 as transcriptional repressors of autophagy. We also further investigated the detailed molecular mechanisms of the regulation of autophagy by Rph1.PhDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133444/1/jmeiyan_1.pd
The Role of Down Syndrome Cell Adhesion Molecule in Development and Diseases
No full textHow neurons elaborate dendrites and axons and how they form synapses with targeted cells are fundamental questions in neurodevelopment. Although a number of genes have been identified to play a role in these processes, the following two aspects are largely unknown and interesting to study. First, while most genes go through alternative splicing to generate different protein isoforms, how each protein isoform contributes to the function of this gene in neurodevelopment remains a challenging question, especially at the cell-type specific and endogenous levels. Second, in neurodevelopmental diseases, such as those in Down syndrome, the expression levels of hundreds or thousands of genes are altered. The dysregulation of which gene cause the neurite and synaptic defects in these brain disorders remains largely unknown. My dissertation aims to answer these two questions with the focus of one gene, Down syndrome cell adhesion molecule (Dscam).
Dscam is an evolutionarily conserved single-pass transmembrane protein that plays critical roles in multiple aspects of neuronal wiring, including dendritic/axonal growth and synapse formation. In Drosophila, alternative splicing of Dscam produces two mutually exclusive isoforms, Dscam[TM1] and [TM2], which differ in their transmembrane and juxtamembrane region. By developing a novel genetic method, termed isoTarget, which enables the investigation of isoform-specific function and endogenous localization in specific cells, I report differential function and localization of Dscam isoforms in axons versus dendrites. In addition, I uncovered an isoform-specific signaling pathway that involves DLK/Wallenda, Dock, and Dscam[TM2], but not Dscam[TM1], at the axon terminals of Drosophila sensory neurons. I provide evidence showing that Dscam[TM2]-specific function and signaling in axon terminals are caused by its isoform-specific localization, rather than by biochemical differences between Dscam[TM1] and [TM2]. These findings not only demonstrate the specific function, localization and signaling of Dscam isoforms but also demonstrate the critical role of subcellular localization in expanding isoform diversity.
In human, the Dscam gene is localized in the Down syndrome critical region on chromosome 21. By taking advantage of sparse neuron labeling for morphological analysis and whole-cell patch-clamp for functional assay, I found that Dscam regulates inhibitory neuron development in the neocortex in a dose-dependent fashion. Loss of Dscam impairs the presynaptic growth in chandelier and basket cells, two major types of inhibitory neurons in the neocortex. In the Ts65Dn mouse model for Down syndrome, where Dscam is overexpressed, GABAergic inhibition of pyramidal neurons (PyNs), the main excitatory neurons in the cortex, is increased. Genetic normalization of Dscam expression rescues the excessive GABAergic synapse formation and inhibition of PyNs. These findings reveal abnormal GABAergic innervation in the neocortex of Down syndrome mouse model and identify Dscam overexpression as the cause. They also implicate dysregulated Dscam levels as a potential pathogenic driver in related neurological disorders.
In summary, by using the novel genetic method isoTarget, this dissertation uncovers Dscam isoform-specific subcellular signaling cascade in neurodevelopment; using genetic normalization in Down syndrome mouse model, it establishes a causal effect between dysregulated Dscam expression levels and cortical defects in Down syndrome pathology. These findings are expected to advance our understanding of the role of Dscam in neurodevelopment and neurological disorders. PhDCell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163118/1/falleave_1.pd
Dissecting the Mechanisms Shaping Liver Macrophage Heterogeneity and Function in Metabolic Liver Disease
No full textMacrophages play an integral role in host defense, tissue homeostasis, and disease progression. Altered macrophage polarization, characterized by changes in its transcriptional and functional states, has been causally linked to metabolic disease. Metabolic dysfunction-associated steatohepatitis (MASH) represents a severe stage of metabolic liver disease characterized by hepatocyte injury, inflammation, and liver fibrosis. Single-cell transcriptome analysis on non-parenchymal cells (NPC) isolated from healthy and MASH livers revealed unprecedented insights into the nature of intercellular crosstalk and reprogramming of diverse liver cell types during MASH pathogenesis. We observed a marked change of liver macrophage composition upon diet-induced MASH, characterized by a depletion of resident macrophages, and expansion of infiltrated macrophages. TREM2+ macrophages represent a unique population of monocyte-derived macrophages induced in both mouse and human MASH liver. These findings illustrate a new dimension of macrophage biology in MASH progression and raise several important questions regarding the mechanisms and pathophysiological role of liver macrophages during MASH progression and the development of MASH-associated liver cancer.
Macrophages play an important role in tissue homeostasis and disease pathogenesis; however, the nature of macrophage heterogeneity, disease-associated reprogramming, and contribution to MASH remains incompletely understood. In my thesis, we performed bulk and single-cell RNA sequencing analysis to delineate the landscape of macrophage transcriptomes in healthy and MASH liver. Our analysis uncovered cell type-specific transcriptomic signatures of liver cells upon diet-induced MASH. Gene ontology analysis indicated strongly activated inflammatory pathways in MASH. We identified brain abundant membrane attached signal protein 1 (Basp1) as a myeloid-enriched gene that is markedly induced in mouse and human MASH liver. Myeloid-specific inactivation of BASP1 attenuates the severity of diet-induced MASH pathologies as shown by reduced hepatocyte injury and liver fibrosis in mice. Mechanistically, cultured macrophages lacking BASP1 exhibited a diminished response to pro-inflammatory stimuli, impaired NLRP3 inflammasome activation, and reduced cytokine secretion. These findings uncover BASP1 as a critical regulator of myeloid inflammatory signaling that underlies MASH pathogenesis.
Cellular heterogeneity of macrophages in the liver is a hallmark of MASH pathogenesis. We identified TGF-beta signaling as a crucial regulator of disease-associated macrophages in the MASH liver. Myeloid-specific inactivation of Tgfbr1 in mice exacerbated diet-induced MASH. Mechanistically, ablation of TGF-beta signaling in myeloid cells altered liver macrophage composition characterized by a reduction of TREM2+ macrophages and a corresponding expansion of FCRL5+ macrophages in the liver. Additionally, macrophages lacking Tgfbr1 exhibited gene signatures associated with inflammasome activation, cytokine signaling, cellular senescence, and immunosuppression. These changes in macrophage composition and function promoted effector T cell exhaustion and the development of MASH-associated hepatocellular carcinoma in Tgfbr1-deficient mice. These studies uncover myeloid TGF-beta signaling as a driver that governs liver macrophage heterogeneity and polarization within the liver microenvironment during the development of MASH and MASH-associated liver cancer. Together, my thesis work has revealed disease-associated liver macrophage reprogramming, the molecular nature of inflammatory responses, and intrahepatic signaling pathways that shape macrophage heterogeneity in metabolic liver disease.PhDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/197280/1/ziyimeng_1.pd
Microphthalmia-associated transcription factors activate mTORC1 through RagD GTPase gene expression
No full textNovel Regulation of mTOR Complex 1 Signaling by Site-Specific mTOR Phosphorylation.
No full textPhDCell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/94087/1/bekim_1.pd
Novel Roles for mTORC1-dependent Translational Control during Synaptic Homeostasis.
No full textThe mechanistic target of rapamycin complex 1 (mTORC1), a kinase involved in regulating translation initiation, has recently emerged as a critical player responsible for orchestrating dynamic changes in local protein synthesis in response to altered synaptic activity. Here we identify a novel mode of synaptic regulation conferred by mTORC1-dependent signaling in dendrites, wherein mTORC1 activation gates a local retrograde signaling mechanism that drives changes in presynaptic function from apposed postsynaptic terminals. This unique role for dendritic mTORC1 signaling is critically dependent on BDNF release, which couples loss of excitatory synaptic drive with retrograde compensation of presynaptic function. Acute activation of mTORC1 signaling using the lipid second messenger phosphatidic acid (PA) or overexpression of the endogenous mTOR activator RhebGTPase exerts a powerful influence on network function, which is also dependent on dendritic synthesis of BDNF as a retrograde signal. We identify an additional feature of the putative postsynaptic homeostatic sensor mechanism, showing that phospholipase D (PLD)-mediated hydrolysis of the lipid second messenger phosphatidic acid (PA) is a crucial component of the signaling pathway which relays homeostatic signals to postsynaptic mTORC1 after loss of excitatory input. Lastly, we find that mTORC1-dependent retrograde signaling acts in coordination with dynamic relocalization of the ubiquitin proteasome system to and from axonal boutons to regulate presynaptic function in the expression of synaptic homeostasis. Increasing neuronal firing rates enriches proteasome accumulation at synaptic terminals, whereas inhibiting neuronal firing results in a dramatic redistribution away from synaptic terminals. This altered localization is due, at least in part, to an activity-dependent sequestration mechanism at presynaptic terminals. Moreover, activity-dependent phosphorylation of the Rpt6 subunit of the 19S proteasome is necessary and sufficient for axonal proteasome redistribution, and this altered localization plays a critical role in establishing mTORC1-dependent retrograde homeostatic changes in presynaptic function after loss of postsynaptic drive. Several monogenic neurodevelopmental conditions related to Autism Spectrum Disorder and Intellectual Disability share the common molecular phenotype of increased mTORC1 signaling. As such, a more thorough understanding of how mTORC1 regulates synaptic function may provide insights for targeting this signaling pathway as a therapeutic option for cognitive dysfunction.PhDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107207/1/fehenry_1.pd
Single-Cell Profiling of Paracrine Network and Immune Landscape in Non-Alcoholic Steatohepatitis
No full textThe liver is a heterogeneous organ comprising a diverse array of cells including parenchymal hepatocytes, and non-parenchymal cells (NPCs) including endothelial cells, hepatic stellate cells, cholangiocytes, and various immune cells. These different cell types function in concert to regulate hepatic metabolism and maintain tissue homeostasis. Non-alcoholic fatty liver disease (NAFLD) is a major hepatic comorbidity of metabolic syndrome that is characterized by pathogenic fat accumulation. Chronic NAFLD may progress to non-alcoholic steatohepatitis (NASH), an inflammatory condition that is associated with liver injury, immune cell infiltration, and liver fibrosis, and increases the risk for end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. Despite the prominent roles of NPCs in NASH pathogenesis, the molecular nature of intercellular crosstalk among different liver cell types and their reprogramming in disease remains poorly understood.
Single-cell RNA sequencing (scRNA-seq) is a powerful technique for unraveling cellular heterogeneity in complex tissue through profiling transcriptomes of individual cells. In my thesis research, I performed scRNA-seq on NPCs isolated from healthy and diet-induced NASH mouse livers to dissect the paracrine signaling network and to uncover the immune cell landscape in NASH. We found that each cell type exhibited enriched expression of a unique subset of secreted ligands and membrane receptors, in a restricted pattern conserved from mouse to human. We confirmed expression for this paracrine network through quantitative proteomics and found macrophages and stellate cells to be major hubs of ligands and receptors which were upregulated in NASH. The hepatic stellate cells (HSCs) provide a source of stellakines which are predicted to act on endothelial and immune cells. Functionally, we showed that HSCs express a class of G-protein coupled receptors that regulate cellular contractility in response to vasoactive ligands.
ScRNA-seq revealed a highly disease-specific population of NASH-associated macrophages (NAMs) marked by abundant expression of marker genes including Trem2 and Gpnmb. NAMs were found at higher levels in both human and mouse NASH and decreased upon dietary and therapeutic interventions for NASH. Their transcriptional profile indicates a propensity for phagocytosis, antigen presentation, and extracellular matrix remodeling, illustrating a potentially important role for NAMs in NASH progression. Tracing hematopoietic cells through bone marrow transplantation, we demonstrated that NAMs originate from the bone marrow and not tissue-resident progenitors. We developed a Trem2 Cre knockin mouse strain to track the emergence of NAMs during NASH progression. Our mouse liver injury and fibrosis assays were unaffected by Trem2 knockout in NASH, indicating Trem2 itself may be unimportant in NASH, but the role of the NAMs they mark are not yet precluded from pathophysiology. Finally, we discovered that NASH is linked to liver CD8+ T cell exhaustion, which is characterized by high levels of PD1 and LAG3 expression and diminished IFNγ, IL2, and TNFα secretion upon T cell stimulation. NASH-associated T cell exhaustion is attenuated by the adipose hormone Neuregulin 4 (NRG4), which protects mice from diet-induced NASH and hepatocellular carcinoma. Taken together, my thesis work has revealed the transcriptomic nature of liver cell heterogeneity, the global landscape of cell-cell signaling in the liver, and NASH-associated NPC reprogramming at a single-cell resolution.PhDCellular & Molec Biology PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/172722/1/henrykg_1.pd
Cascading Samll GTPases in Insulin Action.
No full textInsulin stimulates glucose uptake into adipocytes and muscle cells by stimulating the translocation of the glucose transporter 4 Glut4 from intracellular storage vesicles to the plasma membrane. This process is tightly regulated by insulin signaling cascades in concert with vesicle transport machineries. Small GTPases such as Rab10 and RalA play important roles in insulin-stimulated Glut4 translocation by functioning at the intersection of insulin signaling and vesicle trafficking. A novel effector pull-down assay to evaluate the intracellular activity of Rab10 was developed and used to characterize Rab10 as a functional target of the Akt substrate protein AS160. AS160 contains a GAP (GTPase-activating protein) domain that directly stimulates guanosine triphosphate hydrolysis of Rab10, leading to inactivation of the small GTPase. We show that Akt-catalyzed phosphorylation of AS160 inhibits the GAP activity, as activated Akt relieves the inhibitory effect of AS160 on Rab10 activity.
Activated Rab10 promotes Glut4 translocation to the plasma membrane by recruiting downstream effector proteins. One potential downstream target of Rab10 is the RalA GTPase, which has been reported to mobilize the exocyst complex, targeting complex that guides the Glut4 vesicle to the plasma membrane. Overexpression of constitutively active Rab10 increases RalA activity, while depletion of Rab10 with siRNA-mediated knockdown significantly reduces RalA activation by insulin in adipocytes. Rab10 potentially regulates RalA activity through an as-yet-unidentified guanine-nucleotide exchange factor for RalA, which facilitates guanosine triphosphate binding to RalA, leading to activation of the small GTPase and mobilization of the exocyst complex. Taken together, these data suggest that a small GTPase cascade of Rab10 and RalA, together with their regulatory GAP and GEF proteins, connects the insulin/Akt signaling pathway with the transport machineries in Glut4 translocation.PhDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/93937/1/ttxiong_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/93937/2/ttxiong_2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/93937/3/ttxiong_3.pd
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