The field of stem cell research has been growing more because of the interest in using stem cells to cure diseases and heal injuries. Human embryonic stem cells, because of the controversy surrounding them—and subsequently the difficulties in acquiring samples of the existing aging cell lines—can only be used in limited capacities. While the development of induced pluripotent stem cells in the last decade has allowed the field to progress closer to medical treatments, the low efficiency of reprogramming a somatic cell to a pluripotent state, and the vast molecular and genomic differences between human embryonic stem cells and human induced pluripotent stem cells is still an issue. Therefore, the goal is to discover methods, chemicals, and factors that can reduce these differences and increase the efficiency of inducing pluripotency. This proposal aims to look at the effects of the protein ECAT1 in inducing pluripotency in human somatic cells. Little is known about ECAT1, otherwise known as Embryonic Stem Cell-Associated Transcript 1, beyond its presence in human embryonic stem cells and oocytes and its absence in differentiated cells. While originally considered by scientists during the development of the reprogramming technique, ECAT1's effects have not been tested in humans. Therefore, a series of experiments will be performed in which ECAT1 will be used in conjunction with OSKM to induce pluripotency in adult human dermal fibroblasts, which will then be differentiated into spinal motor neurons. The three stages of this proposal--inducing pluripotency, comparing pluripotencies in the reprogrammed cells and embryonic stem cells, and differentiating the stem cells--should answer questions about ECAT1 and the reprogramming process. It is predicted that ECAT1 should reduce the genomic and molecular differences between embryonic stem cells and induced pluripotent stem cells. ECAT1's presence should also increase the efficiency of reprogramming as well as successful differentiation to other cell types.

mRNA degradation is a fundamental process that controls both the level and the fidelity of gene expression. Using a combination of bioinformatic, genomic, genetic, and molecular biology approaches, we have shown that Edc3p, a yeast mRNA decay factor, controls the stability of the intron-containing YRA1 pre-mRNA. We found that Edc3p-mediated degradation of YRA1 pre-mRNA: 1) is a component of a negative feedback loop involved in the autoregulation of YRA1, 2) takes place in the cytoplasm, 3) is independent of translation, 4) occurs through a deadenylation-independent decapping and 5΄ to 3΄ exonucleotic decay mechanism, and 5) is controlled by specific cis-acting elements and trans-regulatory factors. Cis-regulation of YRA1 pre-mRNA degradation is complicated and precise. Sequences in exon1 inhibit YRA1 pre-mRNA splicing and/or promote pre-mRNA export in a size-dependent but sequence-independent manner. Sequences in the intron dictate the substrate specificity for Edc3p-mediated decay. Five structurally different but functionally interdependent modules were identified in the YRA1 intron. Two modules, designated Edc3p-responsive elements (EREs), are required for triggering an Edc3p-response. Three other modules, designated translational repression elements (TREs), are required for repressing translation of YRA1 pre-mRNA. TREs enhance the efficiency of the response of the EREs to Edc3p by inhibiting translation-dependent nonsense-mediated mRNA decay (NMD). Trans-regulation of YRA1 pre-mRNA is governed by Yra1p, which inhibits YRA1 pre-mRNA splicing and commits the pre-mRNA to nuclear export, and the RNP export factors, Mex67p and Crm1p, which jointly promote YRA1 pre-mRNA export. Mex67p also appears to interact with sequences in the YRA1 intron to promote translational repression and to enhance the Edc3p response of YRA1 pre-mRNA. These results illustrate how common steps in the nuclear processing, export, and degradation of a transcript can be uniquely combined to control the expression of a specific gene and suggest that Edc3p-mediated decay may have additional regulatory functions in eukaryotic cells.

Mutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.

Extracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data. We established that cholesterol-conjugated siRNAs passively associate to EV membrane and can be productively delivered to target neurons. We extensively characterized this loading process and optimized exosome-to-siRNA ratios for loading. We found that chemical stabilization of 5'-phosphate with 5'-E-vinylphosphonate and chemical stabilization of all nucleotides with 2'-O-methyl and 2'-fluoro increases the accumulation of siRNA and the level of mRNA silencing in target cells. Therefore, we recommend using fully modified siRNAs for lipid-mediated loading to EVs. Later, we identified that α-tocopherol-succinate (vitamin E) conjugation to siRNA increases productive loading to exosomes compared to originally described cholesterol. Low EV yield has been a rate-limiting factor in preclinical development of the EV technology. We developed a scalable EV manufacturing process based on three-dimensional, xenofree culture of mesenchymal stem cells and concentration of EVs from conditioned media using tangential flow filtration. This process yields exosomes more efficient at siRNA delivery than exosomes isolated via differential ultracentrifugation from two-dimensional cultures of the same cells. In-depth characterization of EV content is required for quality control of EV preparations as well as understanding composition–activity relationship of EVs. We have generated mass-spectrometry data on more than 3000 proteins and more than 2000 lipid species detected in exosomes (small EVs) and microvesicles (large EVs) isolated from five different producer cells: two cell lines (U87 and Huh7) and three mesenchymal stem cell types (derived from bone marrow, adipose tissue and umbilical cord Wharton’s jelly). These data represent an indispensable resource for the community. Furthermore, relating composition change to activity change of EVs isolated from cells upon serum deprivation allowed us to identify essential components of siRNA-delivering exosomes. Based on these data we reverse engineered natural exosomes into artificial exosomes consisting of dioleoyl-phosphatidylcholine, cholesterol, dilysocardiolipin, Rab7, AHSG and Desmoplakin. These artificial exosomes reproduced efficient siRNA delivery of natural exosomes both in vitro and in vivo. Artificial exosomes may facilitate manufacturing, quality control and cargo loading challenge that currently impede the therapeutic EV field.

The lifespan of an organism is determined by a complex array of genetic, environmental and nutritional factors. Yet single gene manipulations have been shown to significantly extend lifespan in several model organisms. Of all the genes that have been studied thus far, components of the insulin/IGF-1 signaling (IIS) pathway have emerged as the most robust regulators of longevity. In addition, IIS also regulates development, energy metabolism and the response to stress in a conserved manner. In Caenorhabditis elegans, signaling through this pathway is initiated by activation of the insulin/IGF-1 receptor tyrosine kinase DAF-2, which then activates a PI3-kinase signaling pathway involving additional downstream serine/threonine kinases such as PDK-1, AKT-1, AKT-2 and SGK-1. The concerted action of these kinases results in the negative regulation of the single FOXO transcription factor homolog DAF-16. Under reduced signaling conditions, active DAF-16 is able to translocate into the nucleus and regulate the expression of hundreds of genes regulating longevity, stress resistance, metabolism and development. The PTEN phosphatase homolog DAF-18, which antagonizes IIS at the level of PI3-kinase, is a major negative regulator of the pathway. However, not much was known about additional phosphatases that negatively regulated the kinases in the pathway. Dephosphorylation is a critical regulatory mechanism by which cellular signaling homeostasis is maintained. Aberrant hyper-activation of growth factor signaling pathways, including IIS, has been implicated in several cancers. In addition, deregulation of IIS is also closely linked to Type II diabetes. Therefore, the identification phosphatases that balance kinase activity will provide a better understanding of the regulation of the IIS pathway under normal as well as disease conditions. A directed RNAi screen using dauer diapause was conducted in our lab to identify serine/threonine phosphatases that modulated IIS. My work in the Tissenbaum Lab has primarily focused on characterization of the top three candidates from this screen, the genes pptr-1, pdp-1 and fem-2. From these studies, we have also uncovered novel crosstalk between the IIS and TGF-β signaling pathways. In Chapter 2, we demonstrate that PPTR-1, a PP2A phosphatase regulatory subunit negatively regulates the IIS pathway by modulating AKT-1 dephosphorylation. PPTR-1 modulates several outputs of IIS similar to DAF-18. In addition, PPTR-1 co-localizes and physically interacts with its substrate, AKT-1. PPTR-1 modulates dephosphorylation of AKT-1 at a conserved threonine site and we show the molecular conservation of this interaction in mammalian adipocytes. Ultimately, this negative regulation by PPTR-1 results in increased DAF-16 nuclear localization and transcriptional activity. Next, in Chapter 3, we show how PDP-1 is a novel link between the IIS and TGF-β signaling pathways. Similar to DAF-18 and PPTR-1, PDP-1 regulates multiple outputs of the IIS pathway and promotes DAF-16 activity. Interestingly, PDP-1 acts at the level of DAF-8 and DAF-14, two R-SMAD proteins that function in a TGF-β pathway. Our data suggests that PDP-1 may negatively regulate TGF-β signaling to downregulate the expression of several insulin(s). Without the insulin ligands, there is less activation of the IIS pathway, and DAF-16 is more active, thereby promoting transcription of genes that act to enhance longevity and stress resistance. In Chapter 4, we investigate possible crosstalk between IIS and the TGF-β signaling pathways, as the latter was previously considered as a parallel independent pathway. From our studies on PDP-1, we knew that this phosphatase, despite acting in the TGF-β pathway, was a robust modulator of multiple outputs of IIS. Using double mutant combinations as well as RNAi we unravel complex and extensive crosstalk between the two pathways. Importantly, our results suggest that DAF-16 is likely to be the most downstream component of the two pathways. In Chapter 5, we describe genetic characterization of fem-2, and its regulation of the IIS pathway. RNAi of fem-2 results in robust suppression of dauer formation, similar to pptr-1 and pdp-1 RNAi but this phenotype is only observed in the e1370 allele of daf-2. While knockdown of pptr-1 and pdp-1 suppress dauer formation of additional alleles of daf-2, fem-2 RNAi has no effect. These results reveal a complex genetic interaction between fem-2 and the daf-2 receptor. Taken together, our results identify several novel regulators of IIS that modulate this pathway by distinct mechanisms.

The alarming rate of increase in incidence and prevalence of the type 2 diabetes mellitus has prompted intense research on understanding the pathogenesis of the type 2 diabetes. It is observed that the development of type 2 diabetes is preceded by a state of insulin resistance and obesity. Previous studies have suggested that the obesity induced insulin resistance may be mediated by elevated levels of circulating free fatty acids (FFAs). The increase in circulating levels of FFAs may be contributed by the release of FFAs from stored triglycerides (TG) in adipocytes via lipolysis. It is hypothesized that the decrease in levels of circulating FFAs by sequestration and storage of FFAs in adipocytes may prevent deleterious effects of FFAs on insulin sensitivity. Recently our lab and others have shown that the storage of TG in adipocytes is promoted by a novel protein, Fat Specific Protein 27 (FSP27). Although, these studies also revealed FSP27 to be a lipid droplet associated protein that suppresses lipolysis to enhance TG accumulation in adipocytes, the role of FSP27 in lipolysis remains largely undetermined. Therefore, this study investigates the role and regulation of FSP27 in adipocytes in both the basal state, as well as during lipolysis. The studies presented here show FSP27 to be a remarkably short-lived protein (half-life=15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, I tested the hypothesis that lipolytic agents like the cytokine, TNF-α and the catecholamine isoproterenol modulate FSP27 protein levels to regulate FFA release. Consistent with this concept, TNF-α markedly decreased FSP27 mRNA and protein along with lipid droplet size as it increased lipolysis in cultured adipocytes. Similarly, FSP27 depletion using siRNA mimicked the effect of TNF-α to enhance lipolysis, while maintaining stable FSP27 protein levels by expression of HA epitope-tagged FSP27 blocked TNF-α mediated lipolysis. In contrast, the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 protein and a delayed degradation rate that corresponds to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism to restrain excessive lipolysis by catecholamines, is mimicked by forskolin or 8-Bromo-cAMP treatment, and prevented by Protein Kinase A (PKA) inhibitor KT5720 or PKA depletion using siRNA. These results show that isoproterenol stabililizes FSP27 via the canonical PKA pathway and increased cAMP levels. However, the work presented here also suggests that FSP27 does not get phosphorylated in response to isoproterenol treatment, and the stabilization of FSP27 is independent of isoproterenol mediated lipolysis. The data presented in this thesis not only identifies the regulation of FSP27 as an important intermediate in mechanism of lipolysis in adipocytes in response to TNF-α and isoproterenol, but also suggests that FSP27 may be a possible therapeutic target to modulate lipolysis in adipocytes.

Human obesity is increasing globally at an impressive rate. The rise in obesity has led to an increase in diseases associated with obesity, such as type 2 diabetes. A major prerequisite for this disease is the development of insulin resistance in the muscle and adipose tissues. Interestingly, experiments in rodent models suggest that adipocytes and macrophages can profoundly influence the development of insulin resistance. Accordingly, the number of adipose tissue macrophages increases substantially during the development of obesity. Numerous research models have demonstrated that macrophages promote insulin resistance by secreting cytokines, like TNFα, which impair whole body insulin sensitivity and adipose tissue function. Additionally, enhancements of murine adipose function, particularly glucose disposal, prevent the development of insulin resistance in mice on a high fat diet. Thus, mechanisms which enhance adipose function or attenuate macrophage inflammation are of interest. Our lab previously identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potent negative regulator of adipocyte function. In these studies, TNFα treatment increased the expression of adipocyte MAP4K4. Furthermore, the use of small interfering RNAs (siRNA) to block the increase in MAP4K4 expression protected adipocytes from some of the adverse effects of TNFα. Because MAP4K4 is a potent negative regulator of adipocyte function, an understanding of the mechanisms by which TNFα regulates MAP4K4 expression is of interest. Thus, for the first part of this thesis, I characterized the signaling pathways utilized by TNFα to regulate MAP4K4 expression in cultured adipocytes. Here I show that TNFα increases MAP4K4 expression through a pathway requiring the transcription factors activating transcription factor 2 (ATF2) and the JUN oncogene (cJUN). Through TNFα receptor 1 (TNFR1), but not TNFR2, TNFα increases MAP4K4 expression. This increase is highly specific to TNFα, as the inflammatory agents IL-1β, IL-6 and LPS did not affect MAP4K4 expression. In agreement, the activation of cJUN and ATF2 by TNFα is sustained over a longer period of time than by IL-1β in adipocytes. Finally, MAP4K4 is unique as the expression of other MAP kinases tested fails to change substantially with TNFα treatment. For the second part of this thesis, I assessed the role of MAP4K4 in macrophage inflammation in vitro and in vivo. To accomplish this task, pure β1,3-D-glucan shells were used to encapsulate siRNA. Glucan shells were utilized because they are effectively taken up by macrophages which express the dectin-1 receptor and they survive oral delivery. I demonstrate that these β1,3-D-glucan encapsulated RNAi particles (GeRPs) are efficiently phagocytosed and capable of mediating the silencing of multiple macrophage genes in vitro and in vivo. Importantly, oral treatment of mice with GeRPs fails to increase plasma IFNγ and TNFα or alter serum AST and ALT levels. Orally administered GeRPs are found in macrophages isolated from the spleen, liver, lung and peritoneal cavity and mediate macrophage gene silencing in these tissues. Utilizing this technology, I reveal that MAP4K4 augments the expression of TNFα in macrophages following LPS treatment. Oral delivery of MAP4K4 siRNA in GeRPs silences MAP4K4 expression by 70% and reduces basal TNFα and IL-1β expression significantly. The depletion of MAP4K4 in macrophages protects 40% of mice from death in the LPS/D- galactosamine (D-GalN) model of septicemia, compared to less than 10% in the control groups. This protection associates with significant decreases in serum TNFα concentrations following LPS/D-GalN challenge. Consistent with reduced macrophage inflammation, hepatocytes from mice treated orally with GeRPs targeting MAP4K4 present less apoptosis following LPS/D-GalN treatment. Thus, MAP4K4 is an important regulator of macrophage TNFα production in response to LPS. The results presented here add to the knowledge of MAP4K4 action in adipocyte and macrophage inflammation substantially. Prior to these studies, the mechanism by which TNFα controlled MAP4K4 expression in adipocytes remained unknown. Considering that MAP4K4 is a negative regulator of adipocyte function, identifying the mechanisms that control MAP4K4 expression was of interest. Furthermore, the role of macrophage MAP4K4 in LPS stimulated TNFα production was also unknown. To address this question in vivo, new technology specifically targeting macrophages was needed. Thus, we developed a technology for non toxic and highly specific macrophage gene silencing in vivo. Considering that macrophages mediate numerous diseases, the application of GeRPs to these disease models is an exciting new possibility.