Shadi Khalil, PhD, 2017

Mechanisms of Erythroid Iron Sensing and Uptake: Roles for TfR2 in the Iron Deprivation Response and Mitochondrial Iron Delivery

Abstract:

Erythroid progenitors are the major consumers of iron in the human body. These cells must ultimately deliver iron to the mitochondrial matrix, where it is used in heme synthesis to support hemoglobinization. Iron homeostasis is tightly coupled with erythroid iron utilization: declining levels of circulating iron result in suppressed erythropoiesis, thus sparing the body’s supply of iron when threatened by erythroid consumption. This response, termed the erythroid iron deprivation response, underlies the pathogenic mechanism of various human anemias. Such iron-restricted human anemias are associated with resistance to erythropoietin (Epo), the major cytokine regulating survival, proliferation, and differentiation of erythroid progenitors. The mechanism of the erythroid iron deprivation response has remained unknown, but aconitase enzymes and transferrin receptor 2 (TfR2) have been identified as erythroid iron-sensing components. The results described herein define a pathway of erythroid iron sensing that culminates with regulation of Epo receptor (EpoR) surface presentation and signaling. Briefly, TfR2 undergoes accelerated lysosomal trafficking and degradation upon iron deprivation, resulting in the co-catabolism of the essential TfR2 binding partner: Scribble. Scribble is a key orchestrator of receptor trafficking and signaling that is required for erythroid surface presentation of EpoR and normal Epo-dependent Stat signaling. We identify the coordination of a TfR2-driven nutrient-sensing pathway with altered receptor trafficking and cytokine responsiveness, thus limiting progenitor expansion while maintaining survival. Interestingly, under iron replete conditions, TfR2 was found to traffic to the lysosome and undergo degradation, although the rate of degradation was reduced. The mechanism by which erythroid iron is ultimately trafficked from endosome to mitochondria has remained unclear, as cytosolic transit of iron is not observed. The existence of an alternative erythroid iron uptake pathway is suggested by the demonstration of residual erythropoiesis in mice lacking TfR1 and DMT1, proteins involved in canonical iron uptake. Further, mice with marrow-selective TfR2 deficiency have been found to exhibit microcytosis, suggesting that TfR2 may also contribute to erythroid hemoglobinization. Assessing whether TfR2 contributes to iron uptake identified an alternative pathway of iron uptake initiated by TfR2 lysosomal transferrin delivery. Imaging studies reveal an erythroid lineage-specific organelle arrangement consisting of a focal lysosomal cluster surrounded by a nest of mitochondria, with direct contacts between these two organelles. Erythroid TfR2 deficiency yields aberrant mitochondrial morphology and impaired heme synthesis, implicating TfR2-dependent transferrin trafficking in iron uptake and mitochondrial maintenance. Human TFR2 shares a lineage- and stage- specific expression pattern with MCOLN1, encoding a lysosomal iron channel, and MFN2, encoding a mitochondrial protein mediating organelle contacts. Functional studies reveal these latter factors to be involved in mitochondrial regulation and erythroid differentiation: Mcoln1 -/- animals exhibit anemia and erythroid mitochondrial perturbation, while erythroid Mfn2 knockdown reduces the frequency of mitochondrial-lysosomal contacts and blocks erythropoiesis. These findings identify an alternative role for TfR2, driving lysosomal delivery of transferrin and mitochondrial iron uptake. Thus, TfR2 is identified as a driver of both erythroid iron sensing and iron uptake.

Full Dissertation

Monique Anderson, PhD, 2017

The Role of HTLV-1 in Alteration of Immune Regulation Through Regulatory T Cell Dysfunction and Exosomal Manipulation

Abstract:

HTLV-1 is a human retrovirus that is associated with the neuroinflammatory disorder HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP). Much work has been undertaken to understand the mechanisms that lead to the development of this chronic progressive neuroinflammatory disorder in subsets of infected individuals. Similar to other inflammatory disorders, HAM/TSP appears to involve interaction between the immune system, viral/environmental triggers, and host genetics. When focusing on those individuals who develop HAM/TSP, certain factors are prominent. In particular, HTLV-1 is primarily found in the CD4+CD25+ T cell subset (Regulatory T cells: Tregs), which is responsible for peripheral immune tolerance and is known to be dysfunctional in HAM/TSP. This can be assessed in a number of ways. For example, recent evidence suggests that FoxP3, a lineage marker and master regulator of Tregs, expression and function is determined epigenetically through DNA demethylation in the Treg-specific demethylated region (TSDR). Additionally, Tregs have been shown to suppress activation through the production of microvesicles termed exosomes, which measure 30-150nm in size. Therefore, we analyzed the methylation state of the FoxP3 TSDR in peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and CD4+CD25+ T cells from normal healthy donors (NDs) and HAM/TSP patients. We demonstrated that there is decreased demethylation in analyzed PBMCs and CD4+CD25+ T cells from HAM/TSP patients as compared to NDs. Furthermore, decreased TSDR demethylation was associated with decreased functional suppression by Tregs and increased HTLV-1 Tax expression in HAM/TSP PBMC culture correlated with a concomitant decline in FoxP3 TSDR demethylation. Additionally, we assessed the content of exosomes produced by ND and HAM/TSP patient PBMCs and found that HAM/TSP exosomes contained HTLV-1 Tax protein, tax mRNA, and HTLV-1 bZIP (HBZ) protein. Furthermore, Tax containing exosomes were present in cell-free, virus-free HAM/TSP patient cerebrospinal fluid (CSF) supernatant. Importantly, HAM/TSP patient derived exosomes were able to sensitize targets for Tax-specific cytotoxic T lymphocyte (CTL) lysis. We extended our studies into the potential consequences of Treg dysfunction into other immune subsets and found preliminary evidence to suggest that B cell receptors (BCRs) sequenced from HAM/TSP patient CSF recognize HTLV-1 specific antigens, which may potentially be present in isolated exosomes. Overall, we suggest that HTLV-1 infection leads to neuroinflammation in HAM/TSP through HTLV-1 mediated epigenetic modification leading to Treg dysfunction and through incorporation of HTLV-1 products in secreted exosomes, leading to antigen sensing and subsequent destruction of target cells by HTLV-1 specific CTLs and potentially HTLV-1 specific antibodies in the CNS. All of these factors may therefore contribute to HAM/TSP disease pathogenesis and warrant further investigation with the goal of developing clinical strategies that target these mechanisms.

Full Dissertation

Eric Swanson, PhD, 2017

Extracellular Tau Oligomers Induce Disruption of Endogenous Tau Distribution, Invasion of Tau Into the Somatodendritic Compartment and Axonal Transport Dysfunction

Abstract:

Insoluble hyperphosphylated aggregates of the microtubule-associated protein tau define a subset of neurodegenerative disorders known as tauopathies, of which Alzheimer’s Disease is the most prevalent. Extracellular tau can induce the accumulation and aggregation of intracellular tau, and tau pathology can be transmitted along neural networks, in a manner that recapitulates the temporal spread of pathology observed upon post-mortem analysis of diseased tissue. There are six splice variants of central nervous system tau, and various oligomeric and fibrillar forms are associated with neurodegeneration in vivo. The particular extracellular forms of tau capable of transferring tau pathology from neuron to neuron remain ill-defined as do the consequences of intracellular tau aggregation on neuronal physiology. The work in the dissertation presented here was undertaken to compare the effects of extracellular tau monomers, oligomers and filaments comprising various tau isoforms on the behavior of cultured neurons. This work demonstrates that 2N4R or 2N3R tau oligomers provoked aggregation of endogenous intracellular tau much more effectively than monomers or fibrils, or of oligomers made from other tau isoforms, and that a mixture of all 6 isoforms most potently provoked focal, intracellular tau accumulation. These effects were associated with invasion of tau into the somatodendritic compartment. Preliminary data indicate that this somatodendritic tau accumulation may be due to disruption of ankyrin G and βIV spectrin, key components of the axon initial segment. Finally, this work shows that 2N4R oligomers perturbed fast axonal transport of membranous organelles along microtubules. Intracellular tau accumulation was often accompanied by increases in the run length, run time and instantaneous velocity of membranous cargo, and these alterations in fast axonal transport were diminished in neurons not expressing tau. This work provides a more physiological model of tau uptake in neurons and indicates that extracellular tau oligomers can disrupt normal neuronal homeostasis by triggering focal tau accumulation and loss of the polarized distribution of tau, and by impairing fast axonal transport. Additionally, by identifying species involved in cellular dysfunction, it provides a target for much needed future therapies in AD and non-AD tauopathies.

Full Dissertation

Brian Reon, PhD, 2017

Role of Long Noncoding RNAs in Aggressive Brain Tumors

Abstract:

Long noncoding RNAs (lncRNAs) are emerging as key regulators of cellular processes and are commonly found to be misregulated in many human pathologies, including cancer. Glioblastomas (GBM) and lower-grade gliomas (Grade II and III) are aggressive, difficult to treat brain tumors that have high mortality rates. Although much of the research into glioma biology has focused on identifying the molecular drivers of oncogenesis, these efforts have primarily focused on alterations in protein coding genes and the role of lncRNAs has not been sufficiently characterized. This dissertation focuses on expanding our knowledge of lncRNAs in gliomas by analyzing their expression globally and more targeted studies examining the role of two lncRNAs.
In our initial study, we analyzed the expression data from over 750 RNA-seq datasets from GBMs, Grade III and II gliomas and normal brain tissue. We found that hundreds of lncRNAs are differentially expressed in gliomas compared to normal brain tissue. Furthermore, many lncRNAs were found to be preferentially expressed within certain GBM and LGG subtypes. Using these subtype specific lncRNAs we identified similarities between the highly aggressive IDH1/2 wt LGG subtype and mesenchymal GBMs. We also used Cox regression to create a survival algorithm that is capable of separating LGG patients into two distinct prognostic groups. Lastly, we identified all lncRNAs that are associated with GBM patient survival, to aid in identifying which lncRNAs might play critical roles in brain tumors.
Using our brain tumor lncRNA expression and survival association data, we identified two oncogenic lncRNAs, GS1-124K5.4 and LINC00152, for further study. Both LINC00152 and GS1-124K5.4 are upregulated in GBMs and aggressive gliomas, and high expression of either lncRNAs is associated with negative patient outcomes in GBMs. High levels of LINC00152 has no affect on cell growth but lead to an increase invasion in U87 cells. In contrast, expression of GS1-124K5.4 does not affect invasion but high expression of GS1-124K5.4 leads to increased cell growth. Secondary structure analysis of LINC00152 suggests that a protein-bound stem-loop in the 3’ end of LINC00152 is partially involved in LINC00152’s proinvasive function. Furthermore, LINC00152 is upregulated in 10 other tumors types and high expression of is associated with a poor prognosis in 7 other tumors. These results suggest that LINC00152 potentially functions as an oncogene in many cancers.

Full Dissertation

Tzu-Ying Chuang, PhD, 2016

Myeloid LRP1 and Neuroinflammation

Abstract:

Forthcoming

Full Dissertation

Forthcoming

Sydney Strickland, PhD, 2016

HPV-16 E7 Attenuates AKT Signaling

Abstract:

While the role of high-risk HPV E6 and E7 in targeting p53 and Rb has been intensively studied, how E6 and E7 manipulate cellular signaling cascades to promote the viral life cycle and cancer development is less understood. We have shown that HPV-16, specifically 16E7, attenuates pAKT, enhances IRES-dependent translation of several cellular proteins, inhibits keratinocyte differentiation and translocates YAP to the nucleus. Attenuation of pAKT was ablated by a missense mutation in the E7 carboxy-terminus, H73E, thereby defining a novel structure-function phenotype for E7. Coinciding with the pAKT attenuation, phosphorylation of S6K and 4E-BP1 was also attenuated and correlated with an increase in IRES-dependent translation of cellular proteins, including cMYC. This shift in cellular translation could lead to the enhanced expression of other cellular proteins that are essential for the viral life cycle or a novel mechanism to express other HPV proteins, like E5. The attenuation of pAKT could also represent another mechanism by which E7 induces dysplastic phenotypes typically associated with cervical cancer, as Rb degradation alone does not induce invasive cervical cancers. Keratinocytes containing the HPV-16 genome showed severe dysplasia and impaired differentiation in organotypic raft cultures, while HPV-16 genomes harboring the 16E7 H73E mutation had enhanced differentiation and markedly reduced cellular dysplasia. This demonstrates that the ability of E7 to reduce AKT activation correlates with increased dysplasia and reduced keratinocyte differentiation. The block in keratinocyte differentiation could also promote the viral life cycle, as viral amplification is dependent upon terminally differentiated keratinocytes re-entering the cell cycle. Inhibition or delay of keratinocyte differentiation through attenuation of pAKT could promote a more “basal-like” phenotype, which is ideal for the amplification of the viral genome. We have also shown that 16E7 promotes the nuclear accumulation of YAP, where YAP acts as a transcriptional co-activator to promote the expression of genes controlling cell growth. Nuclear localization and activation of YAP have been associated with many cancers and thus, YAP has been termed an oncogene. We hypothesize that E7 could be mediating both the attenuation of pAKT and the nuclear localization of YAP through similar mechanisms involving PP2A or PDK1, both of which are involved in AKT and Hippo signaling. The attenuation of pAKT by 16E7 could be to promote a cellular environment that is ideal for viral amplification, by promoting cell cycle re-entry through inhibition of keratinocyte differentiation and activating YAP to enhance the expression of genes involved in cell proliferation.

Full Dissertation

Jennifer Kaplan, PhD, 2015

A Role for Adipocyte Progenitor Cells in Obesity-Induced MCP-1 and M1 Macrophage Accumulation in Visceral Adipose Tissue

Abstract:

Obesity is one of the top preventable causes of death in the United States, as it can lead to cardiovascular disease, type II diabetes, and cancer. Visceral adipose tissue (VAT) accumulation and inflammation directly link to metabolic dysfunction and obesity-associated disease, and act as predictors of obesity-associated mortality. As such, identification of novel targets to limit diet-induced VAT accumulation has the potential to impact morbidity and mortality.

Inflammatory cells, as well as the cytokines they produce, play a large role in obesity-related diseases. High-fat diet (HFD) results in the induction of expression of proinflammatory genes in both mice and humans, such as monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a potent chemotactic factor for monocytes. Once infiltrated into the adipose tissue in the advanced stages of obesity, macrophages participate in the inflammatory pathways that are activated in obese adipose tissue, including insulin signaling, toll-like receptor (TLR) activation, and the nuclear factor kappa B (NFκB) pathway. While it is evident that macrophages are the main producers of MCP-1 during later stages of obesity, it is less clear where the initial obesity-induced increases in MCP-1 are derived.

The helix-loop-helix (HLH) family of transcription factors is a highly conserved group that plays a role in the differentiation and growth of a variety of cell types. Inhibitor of Differentiation 3 (Id3) belongs to the HLH transcription factor family, and protein levels increase in the stromavascular fraction (SVF) of visceral adipose tissue during obesity. Global deletion of Id3 attenuates HFD-induced obesity, seen by decreased body weight and attenuated expansion of VAT. However, it is yet to be determined whether Id3 plays a role in obesity-induced metabolic perturbations or inflammation within VAT.

MCP-1 intracellular staining determined that CD45-CD31-Ter119-CD34+CD29+Sca-1+ adipocyte progenitor cells (AdPCs) within VAT are the first cells to produce MCP-1 after initiation of HFD. Production of MCP-1 was limited to the CD24- subpopulation of AdPCs, those that have been previously demonstrated to be further committed to the adipocyte lineage. 1 week of HFD results in an increase in the number of CD24- AdPCs, primarily via proliferation. Human AdPCs, identified as CD45-CD31-CD34+CD44+CD90+ cells, also expressed MCP-1, with higher expression in omental adipose tissue compared to subcutaneous adipose tissue. Additionally, high surface levels of CD44 on AdPCs marked the most abundant producers of MCP-1, in both murine and human VAT.

Id3 was identified as a critical regulator of AdPC proliferation, which may be dependent on Id3-induced repression of the p21Cip1 promoter. Id3-deficient mice had fewer CD24- AdPCs, as well as reduced MCP-1 levels and attenuated accumulation of M1 macrophages within the VAT. Id3-/- mice also had improved glucose uptake during glucose tolerance tests (GTTs) as well as enhanced insulin-stimulated phosphorylation of AKT. Adoptive transfer of Id3+/+ AdPCs to Id3-/- mice resulted in increased MCP-1 secretion and significantly enhanced M1/M2 macrophage ratio in VAT. Adoptive transfer also resulted in increased weight gain and worsened glucose tolerance. However, adoptive transfer of Id3-/- AdPCs to Id3-/- mice had no effect on these parameters, leading us to believe that AdPCs must express Id3 to have MCP-1-mediated increases in M1 macrophages and glucose intolerance.

Full Dissertation

Samuel Rosenfeld, PhD, 2015

B-1B Cells are a Novel Atheroprotective B Cell Subset and are Negatively Regulated by ID3

Abstract:

Vascular disease remains the leading cause of death globally through its sequelae myocardial infarction and stroke. The primary contributor to vascular disease, atherosclerosis, is defined by intimal plaque formation in response to lipid deposition. Atherosclerosis is a chronic, progressive disease of the medium to large size arteries and has been well described as a product of low grade inflammation.

B cells are highly involved in the development and progression of atherosclerosis, responding to atherogenic antigen and aggregating in the vascular adventitia. The contributions of B cells to atherosclerosis are subset dependent. Adaptive B-2 cells are considered atherogenic though they can also have atheroprotective function based on the context and model used to study them. Innate B-1a cells are atheroprotective through the secretion of IgM natural antibodies reactive to oxidized LDL. B-1b cells, a unique subset of B-1 cells that are able to provide T cell-independent immune memory, are also atheroprotective and secrete IgM reactive to oxidation-specific epitopes on oxidized LDL. Inhibitor of differentiation 3 (Id3) is a helix-loop-helix transcription factor known to regulate B cell development and homeostasis as well as atherosclerosis. Studies in a B cell specific Id3 knockout model (Id3BKO) revealed that these mice have a significantly expanded B-1b cell population in all compartments without a difference in B-1a or B-2 numbers. Additional studies demonstrated that Id3BKO mice developed attenuated atherosclerosis and increased titers of atheroprotective IgM supporting B-1b cells as atheroprotective and suggesting Id3 selectively negatively regulates their numbers. Attempts to define a mechanism by which Id3 regulates B-1b cells have been inconclusive.

Given their contributions to T cell independent memory, B-1b cells are a novel target for immunization strategies against atherosclerosis and Id3 could be an important target for B-1 directed therapies

Full Dissertations

Han Dong, PhD, 2015

The Function of CD27 Costimulation in the Activation and Fate Decisions of CD8+ T Cells

Abstract:

CD8+ cytotoxic T lymphocytes are critical components of adaptive immunity against a variety of intracellular pathogens, and can play a key role in the control of tumors. Effective vaccination strategies against viral infections and tumors will likely require the development of potent CD8+ T cell responses, which are constituted by the expansion of robust primary CD8+ T cell populations and the establishment of long-lasting memory. Fully functional CD8+ T cell responses are highly dependent upon CD4+ helper T cells and Signal 3 inflammatory cytokine pathways. CD4+ T cells have been demonstrated to play a critical role in inducing the expression of CD70, the ligand for CD27, on dendritic cells. However, it is not clear to what extent the ‘help’ provided by CD4+ T cells is manifest via CD70, or how CD70-mediated stimulation of CD8+ T cells is integrated with signals that emanate from Signal 3 pathways, such as type-1 interferon (IFN-1) and IL-12. In this work, by enforcing or abrogating CD27 function by genetic or protein intervention in murine models, we sought to identify the function of CD27 costimulation in the activation and fate decisions of CD8+ T cells, to determine the extent it resembles CD4+ T cell help, and how inflammation impacts the relative importance of CD70-CD27 interactions in CD8+ T cell primary responses and CD8+ T cell memory. Both subunit vaccine and replicating/non-replicating viral infection settings have been used to facilitate our comprehensive understanding of the role of CD27 costimulation in CD8+ T cell responses, which has been previously complicated by its variable requirement during different stages of CD8+ T cell responses, depending on the nature of the immunogen. We have demonstrated that in the low inflammatory setting of subunit vaccines, CD27 costimulation synergizes with IFN-1 at the level of CD8+ T cells to achieve robust primary CD8+ T cell responses; while in the high inflammatory setting of replicating viral infection, CD27 costimulation antagonizes with impact of IL-12 to promote CD8+ T cell memory. In addition, we identify CD70-CD27 interactions as one main downstream functional consequence of CD4+ T cell help. Mechanistically, we demonstrate that CD27 costimulation supports CD8+ T cell responses in part by modulating the expression of receptors for ‘fate-decision’ cytokines, including IL-7, IL-12 and likely IL-2. Particularly, IL-7Rα has been identified as a functional marker for memory precursor effector cells, and our data indicate that CD27 signals promote the re-expression of IL-7Rα on effector CD8+ T cells via mRNA regulation. Furthermore, CD27 costimulation strongly induces transcription factor Eomesodermin (Eomes), which is a main contributor of effector function and memory differentiation. Based on this new information, we propose that the interplay between CD27 costimulatory and inflammatory cytokine pathways leads to delicate regulation of transcription factor pair Eomes and T-bet, a critical axis for the activation and fate decisions of CD8+ T cells. In summary, our work has 1) identified complex interactions between Signal 3 and costimulatory pathways, 2) provided insights into the mechanistic basis by which CD27 costimulation influences CD8+ T cell activation and fate decisions, 3) demonstrated a previously unappreciated role of CD27 costimulation as a positive regulator of IL-7Rα during CD8 T cell responses, and 4) highlighted the potential of targeting CD27-CD70 axis for antiviral/antitumor immunotherapy.

Full Dissertation

Thomas Mendel, PhD, 2014

Treatment of Diabetic Retinopathy With Adipose-Derived Stem Cells

Abstract:

Diabetic retinopathy threatens the vision of over 100 million people in the world. Retinal pericytes are critical cells that enshealth and protect the delicate retinal microvasculature via direct contact and paracrine mechanisms. After diabetes causes pericyte dysfunction over decades with the disease, a cascade of microvascular pathology ensues that results in blindness. Current therapies aid patients with their symptoms, but do not address the fundamental cellular biology of diabetic retinopathy.

Mesenchymal stem cells, found throughout the body, are capable of acting as pericytes. This suggests that they may be useful as a protective and regenerative cellular therapy for retinal vascular disease, applied in a way that is mindful of the underlying cellular pathology. Easily harvested from adipose, these stem cells may serve as a regenerative cure for diabetic retinopathy.

Our work demonstrated, for the first time, that adipose-derived stem cells are capable of engraftment into multiple pre-clinical models of diabetic retinopathy, where they appear and function like pericytes to stabilize retinopathy, sometimes dramatically. These models included short experimental times of only a few days as well as longer experiments, which revealed transplanted stem cells adopting pericyte morphology along vessels.

Over the course of our experiments, we came to appreciate the impact that the transplanted stem cells could have via paracrine signaling, even without full engraftment and microvascular contact. We also discovered that the diabetic status of the stem cell donor impacted the signaling and disease-modulating efficacy of the cells transplanted into the eye.

While clarity is difficult to find in the field of mesenchymal stem cell research, largely due to its relative youth as a field of study, these experiments confirm the bright promise that stem cell therapy holds for degenerative diseases. Further work should be directed towards understanding the relationship between mesenchymal stem cells and pericytes, as well as investigating precisely how they control their own differentiation and interaction with their surrounding microenvironment.

Full Dissertation