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Pathogenic Characterization and Therapeutic Development for Fiblrolamellar Hepatocellular Carcinoma
Get PDFFibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that primarily affects adolescents and young adults. There are no known successful systemic chemotherapies for this disease, and thus, surgery is the only potential path to a cure in patients with FLC. Once the disease has grown or metastasized to a point where surgery is no longer an option, a patient\u27s chance for survival approaches zero. There is a recurrent genetic deletion in FLC cells, which has been found in almost all FLC tumor samples sequenced to date, but not in normal liver tissue from the same patients. The deletion encompasses ~400kb on chromosome 19 beginning after the first exon of DNAJB1, which codes for a member of the heat shock protein 40 (HSP40/DNAj) family, and ends before the second exon of PRKACA, which codes for the catalytic subunit of protein kinase A (PKAc). The deletion results in a functioning chimeric kinase with exon 1 of DNAJB1 and exons two through ten of PRKACA. In this thesis, I will present my work in two areas with regards to this disease. First, I will present my research working on understanding the pathogenesis of FLC. While we know the oncogene that is responsible for transformation, we do not have a good understanding of how this mutation leads to cancer. I will present proteomic data that shows a unique proteomic and phosphoproteomic signature in FLC and will show that quantification of the PKA system can provide insight into the pathogenesis. In the following chapter, I will present my research on developing the first systemic therapeutic for FLC, by targeting this mutation. I will show how I began by targeting the protein, which proved difficult given the similarities of the mutated protein to the wild-type protein. Then, I will discuss my work on developing an RNA-targeting therapy with antisense oligonucleotides and small interfering RNAs. I will close with a discussion on my thesis work and how I envision future research to continue from what I present here
Unraveling the Interaction Between Beige Adipocytes and the Sympathetic Nervous System
Get PDFObesity affects more than one in three adults in the United States and is a significant risk factor for a constellation of chronic diseases. The crucial role of adipose tissue in energy balance has driven great interest in investigating this tissue as a target for treatment of obesity and its sequelae. While white adipocytes store excess energy, thermogenic brown and beige adipocytes convert lipids and glucose into heat, thereby increasing energy expenditure. Unlike classical brown adipocytes which are thermogenic under basal conditions, inducible brown adipocytes, commonly known as beige adipocytes, reside in white adipose depots and need to be activated by external stimuli such as the sympathetic nervous system to drive thermogenesis. Recent studies have shown that active beige adipocytes can increase energy expenditure and are associated with anti-obesity and anti-diabetes effects in mice and humans. However, the origin of beige adipocytes and how they interact with other adipose cell types remains unclear, creating critical hurdles to manipulating these cells for therapeutic ends. To seek a comprehensive understanding of beige adipocyte formation, we developed a novel technique that enables whole-tissue immunostaining, clearing, and imaging in adipose tissue. Using this new method, we profiled various murine white adipose depots and observed pronounced depot to depot variability in tissue organization. Analysis of cold-induced beige adipocyte formation in whole adipose depots uncovered prominent regional variation in beige adipocyte distribution in subcutaneous fat. Through morphological characterization of the sympathetic nerve projections in subcutaneous fat, we found a dense network of sympathetic parenchymal neurites localizing to the same region where beige adipocytes readily arise. To understand how the dense sympathetic network is established, we used an adipocyte-specific Prdm16 knockout mouse model to ablate beige adipocyte function and demonstrated that the density of sympathetic parenchymal innervation depends on the presence of functional beige adipocytes. These results suggest that communication between beige adipocytes and the sympathetic neurites is important for the establishment of sympathetic innervation. To address whether the regulation by beige adipocytes occurs during early tissue morphogenesis, we applied whole-tissue imaging to examine the development of sympathetic innervation in subcutaneous fat. We found that parenchymal neurites actively grow between postnatal day 6 (P6) and P28, overlapping with early postnatal beige adipogenesis. Constitutive deletion of Prdm16 in adipocytes led to a significant reduction in early postnatal beige adipocytes and sympathetic density within this window. Using an inducible, adipocyte-specific Prdm16 knockout model, we ablated the function of early postnatal beige adipocytes and found strongly impaired sympathetic growth. These data suggest that sympathetic growth in subcutaneous fat depend on a PRDM16- mediated mechanism. However, deleting Prdm16 in adult animals, did not affect sympathetic structure. Together, these findings highlight that beige adipocyte-sympathetic neurite communication is crucial to establish sympathetic structure during the early postnatal period, but may be dispensable for its maintenance in mature animals. These studies unravel the complex interaction between beige adipocytes and the sympathetic nervous system, providing a framework for further investigation of the molecular mechanisms underlying this interaction. Lastly, investigation of the early postnatal beige adipocytes allowed us to appreciate an unprecedented link between early postnatal and adult beige adipocytes. By fate mapping beige adipocytes through development, we found that the majority of cold-induced beige adipocytes in adult subcutaneous fat arise from existing mature adipocytes that were once early postnatal beige adipocytes. These studies provide fundamental insights into beige adipocyte formation and will guide future investigation of the origin and fate of beige adipocytes
William H. Welch, Simon Flexner, and John D. Rockefeller, Jr.
No full textWilliam H. Welch (center), Simon Flexner (left), John D. Rockefeller, Jr. (right), ca. 1931
Courtesy of The Alan Mason Chesney Medical Archives
Simon Flexner, who succeeded Welch in 1902 as editor of The Journal of Experimental Medicine, told in his admirable biography William Welch and the Heroic Age of American Medicine that the new venture was itself a radical experiment; it was generally believed that America did not produce enough scientific work to fill its pages. Later, however, Dr. Flexner continued, the Journal succeeded beyond all expectations, since American laboratories proved able to produce far more articles worthy of its pages than anyone foreseen.https://digitalcommons.rockefeller.edu/jem-the-beginnings/1019/thumbnail.jp
The Identification of a Leptin Dependent Neural Pathway Regulating Adipose Tissue Innervation
Get PDFLeptin, secreted by the adipose tissue, is an afferent signal of a negative feedback loop that regulates body weight balance through its effects on feeding and energy expenditure. Mutations in the leptin gene or its cognate receptor result in severe obesity in both human and mice. My thesis work revealed a leptin-dependent, plastic pathway spanning the central to peripheral nervous system that is responsible for regulating energy homeostasis in mice. Leptin deficient (ob/ob) mice accumulate excessive fat mass due to increased food intake, and decreased energy expenditure partially as a result of defective fat utilization. Chronic leptin delivery to ob/ob mice reverses both phenotypes and leads to drastic fat loss. In contrast, dietary restriction of ob/ob mice fails to increase energy expenditure and results in reduced lean body mass rather than fat mass. These findings indicate that leptin is necessary for mice to efficiently utilize fat as energy source, however, the underlying mechanism is not known. The sympathetic nervous system (SNS) is the major regulator of several critical steps involved in fat utilization, we therefore hypothesized that leptin might regulate the plasticity of SNS innervation of adipose tissue to promote fat usage. We first visualized SNS innervation, using a whole-mount tissue clearing method (Adipo- Clear) paired with light sheet microscopy imaging, in both brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) of wild-type (WT) and ob/ob mice. Surprisingly, we found that ob/ob mice have a profound, around six-fold, reduction of SNS innervation density in both BAT and iWAT compared to age matched WT mice. The same phenotype was also observed in db/db mice which carry a mutation in leptin receptor. Furthermore, we showed that exogenous leptin delivery to adult ob/ob mice for 14 days through a subcutaneous osmotic pump normalizes their SNS innervation levels in both BAT and iWAT to WT level. This effect is independent of leptin-induced anorexia because ob/ob mice pair-fed to their leptin treated littermates fail to show any innervation increase in adipose tissues. These findings demonstrate that leptin regulates SNS innervation plasticity in adipose tissue. We then tested whether restoring adipose tissue innervation structure in ob/ob mice is sufficient to correct their fat utilization defects, such as thermogenesis and lipolysis defects. Thermogenesis is the process that dissipates energy as heat from BAT in cold conditions; and lipolysis is the process that breaks down lipid storage from iWAT to meet energy demand of other organs in times of privation. We first exposed mice to cold challenge and found that ob/ob mice, having BAT innervation density restored but having no leptin in serum, can still activate BAT thermogenesis similarly as WT mice. In contrast, ob/ob mice, not having innervation restored but having high leptin level in serum, fail to activate thermogenesis and succumb to cold just like their ob/ob littermates given no leptin treatment. This experiment led us to the surprising finding that SNS structure, rather than active leptin signaling, is critical for thermogenesis. Additionally, we observed similar trends when we fasted ob/ob mice to induce lipolysis from iWAT. In aggregate, these findings confirm our hypothesis that leptin regulates structural plasticity of SNS in adipose tissue, which in turn promotes fat utilization and energy expenditure. We next uncovered the neural mechanism underlying leptin dependent innervation regulation. We identified LepR expressing neurons in the arcuate nucleus of hypothalamus (ARC) as regulators of SNS innervation. In WT mice, either genetic deletion of LepR in ARC or diet induced leptin signaling loss in ARC leads to dramatic SNS innervation reduction in both BAT and iWAT. There are two major LepR expressing neuron populations in the ARC, agouti related peptide (AGRP) neurons and pro-opiomelanocortin (POMC) neurons. We employed a CRISPRbased gene editing strategy to selectively delete LepR in either AGRP or POMC neurons and found that leptin signaling loss in either population leads to same level of SNS innervation reduction in adipose tissue. Moreover, the magnitude of SNS innervation reduction resulted from ablating LepR in either AGRP or POMC is halved comparing to that from ablating LepR in the entire ARC region. These data suggest that AGRP and POMC neurons act synergistically in regulating leptin dependent SNS innervation. In order to regulate SNS plasticity in adipose tissue, leptin signaling needs to reach sympathetic preganglionic neurons in the spinal cord which act as the conduit between the brain and target-innervating sympathetic neurons in the periphery. However, AGRP and POMC neurons send efferents mostly within the brain, indicating the existence of downstream central populations that mediate leptin\u27s effects on innervation. We identified a group of brain-derived neurotropic factor (BDNF) expressing neurons in the paraventricular nucleus of hypothalamus (PVH) that 1) project directly to the sympathetic preganglionic neurons in the spinal cord, 2) are activated by leptin signaling and receive inputs from both AGPR and POMC neurons, and 3) are necessary for leptin to regulate SNS innervation. In conclusion, leptin requires downstream BDNF signaling to regulate sympathetic plasticity in adipose tissue. These downstream neurons may present therapeutic potentials for treating obesity associated with leptin resistance. The work mentioned above revealed a novel role of leptin in bidirectionally regulating sympathetic neural plasticity in adipose tissues and its underlying neural mechanism. Large-scale sympathetic neural plasticity is normally only observed during organ development or tissue injuries; therefore, it is important to understand how leptin turns on this process in adult animals. Since little is known about the connectivity between the brain and adipose tissue, we first used retrograde circuit tracing approaches to anatomically characterize the locations of the sympathetic pre- and postganglionic neurons innervating both iWAT and BAT. Interestingly, we found little overlap between iWAT and BAT innervating pre-ganglionic neurons, and zero overlap between post-ganglionic neurons, suggesting that the sympathetic neurons are highly specific to target organs. Furthermore, we revealed that there are similar number of fat innervating postganglionic neurons between WT and ob/ob mice, despite the drastic differences in adipose tissue SNS innervation density between these two mouse lines. These results suggest that the gene expression profiles of the fat innervating postganglionic neurons in WT and ob/ob mice are distinct. Therefore, we are currently conducting single cell sequencing experiments to uncover the molecular identities of the sympathetic postganglionic neurons in WT and ob/ob mice; we also hope to reveal the molecular mechanisms underlying leptin dependent plasticity in these neurons. We believe that targeting fat innervating postganglionic neurons might be a good strategy to treat metabolic diseases, and these experiments might help identify potential therapeutic targets
Quantifying the Release of Protein Substrates from AAA+ ATPase ClpX by Single Molecule Total Internal Reflection Fluorescence Microscopy
Get PDFThe safe disposal of proteins is an essential process for maintaining proteostasis in cells. AAA+ proteases, such as proteasomes, play a major role in selective degradation of proteins. Degradation by AAA+ proteases typically requires the substrate to be physically unfolded before proteolysis. The efficiency of the unfolding process is hence a critical parameter for determining the turnover rate of the substrate. However, the mechanism by which force is utilized to unfold the protein substrate is not fully characterized. One parameter regulating the efficiency of the unfolding process is the rate by which a substrate is prematurely released before unfolded. Importantly, a propensity to be prematurely released by AAA+ proteases has been proposed to be an important mechanism allowing some substrates to escape proteolysis, but factors that influence the substrate release rate have not been well characterized. In this thesis, we investigate the parameters that influence the premature release of the substrate using the AAA+ ATPase ClpXP. We present a new assay based on Total-Internal-Reflection Fluorescence (TIRF) microscopy that measures the lifetime of the ClpX-substrate complex. We demonstrate that the technique has the potential to identify factors that affect the mean lifetime of the ClpX-substrate complex. Using this method, we show that the substrate release rate can be reduced by simply increasing the length of the unstructured region where ClpX grips the substrate. Next, we demonstrate that reducing the frequency of the ClpX mechanical cycle prolongs the lifetime of ClpX-substrate interaction. We also offer evidence that the association of the protease particle ClpP to the AAA+ particle stabilizes the interaction between ClpX and the protein substrate, even when the substrate does not directly interact with ClpP. Finally, we show that the glycine-alanine repeats, which are associated with proteins that cannot be fully degraded by the proteasomes, do not increase the release rate of the substrate from ClpX. The results indicate that the force ClpX exerts on the substrate can lead to the release of the substrate when it cannot be unfolded. The results support the hypothesis that the transient loss of grip between ClpX and its substrate could be a frequent side-effect of the substrate unfolding process, but ClpX is often able to recover its grip before the substrate dissociates
Genome-Wide Human-Specific RNA Regulatory Elements in the Brain
Get PDFAdvancements in DNA sequencing technology and the implementation of clinical genome and exome sequencing have allowed for the identification of candidate variants and genes essential in brain development and pathogenesis of neurological diseases, yet there is still much to be learned. It is estimated that the molecular diagnostic rate of whole exome sequencing (WES) of constitutional diseases is between 9-41%; however, it is thought that diagnostic yield could be much improved by gaining a better understanding of individual variation and regulation at the transcript level. Identifying the root cause of diversity between humans and specifically in the human brain has been a long-standing question for the scientific field. At the turn of the 20thcentury, Santiago Ramón y Cajal illustrated thousands of neurons spanning across brain regions to irrefutably demonstrate the morphological complexity and diversity of individual neurons (Levine and Marcillo, 2008). Discoveries spanning centuries continue to drive investigators to question not only why neuronal diversity exists, but how a single genome gives rise to the remarkable heterogeneity observed between single cells, tissue structures, and circuit connections in the brain. Post-transcriptional gene regulation contributes to organism complexity in eukaryotes. RNA regulation is strictly controlled by RNA-binding proteins that are important for the coordination and regulation of gene expression and eukaryotic cells have evolved intricate posttranscriptional mechanisms that permit for precise spatial and temporal control of RNA steady state levels in any given cell. Alternative RNA processing via RNA binding proteins underlies distinct phenotypic and functional diversity specifically observed among mammalian cells. Alternative splicing (AS) and alternative polyadenylation (APA) are widely utilized to expand and diversify both transcriptome and proteome. Alternative RNA processing allows one gene to produce multiple transcripts with distinct coding and regulatory sequences giving rise to multifaceted protein function. RNA binding proteins dictate alternative RNA processing which is a key player in most, if not all, biological processes. Understanding how RNA binding proteins regulate RNA steady state levels and alternative RNA processing requires knowledge of the genomic positions to which these proteins function in vivo. Mouse models have facilitated genome-wide, unbiased discovery of RNA regulatory sites, and the link between causation and functional effect with extraordinary resolution and specificity. The inability to translate many RNA regulatory events from the mouse to the human genome emphasizes the need to perform biochemistry on the human brain directly to compare with the mouse. Identification of RNA regulatory regions via CLIP-sequencing provides valuable information as to how human RNA profiles are dictated and controlled in normal and disease states. Studying neuronal splicing factor NOVA elucidates previously unidentified non-coding RNA regulatory sites as functional and unique to human neurons. We show that human expansion in NOVA regulatory elements diversifies the RNA landscape specifically in human neurons and differentially across brain regions. Taken together, these findings demonstrate mouse to human conserved relationships between RNA binding proteins and targets, however the activity, specificity, and usage of RNA regulatory elements is largely species-specific
William H. Welch in 1884
No full textWilliam H. Welch after his appointment at Johns Hopkins, 1884
Courtesy of the Alan Mason Chesney Archives of The Johns Hopkins Medical Institutions
In early March of 1884, William Welch was interviewed by Daniel Gilman, President of The Johns Hopkins University, and offered the professorship of pathology. The offer included another year of travel and study in Europe and funding to set up a pathological laboratory at Johns Hopkins.
While in Europe, Welch studied the organization and instrumentation of the most renowned laboratories, including those of Koch, Bollinger, Kitt, von Pettenkofer, von Ziemssen, Weigert, Ludwig, Flugge, and Pasteur. There he collected cultures to take to Johns Hopkins and also purchased laboratory instruments and equipment.https://digitalcommons.rockefeller.edu/jem-the-beginnings/1008/thumbnail.jp
Louisville, Kentucky in 1850
No full textLouisville, Kentucky in 1850
Courtesy of the University of Louisville Photographic Archives
This is probably one of the first photographs taken in Louisville. It was found in the cornerstone of the Old Masonic Temple and shows North side of Main Street, form 3rd Street, about 1850 with two-horse tandem drays then used for hauling hogsheads of tobacco, etc. In the foreground is shown the omnibus driven from Louisville by Jim Porter, the famous Kentucky giant.
Simon Flexner, born in 1863 in Louisviell, was the fourth child of Morris Flexner, member of an educated Jewish family in Czechoslovakia, who immigrated to Kentucky.https://digitalcommons.rockefeller.edu/jem-the-beginnings/1021/thumbnail.jp
Dining room at Johns Hopkins Hospital
No full textLewellys F. Barker, Franklin P. Mall, F. R. Smith, Simon Flexner, William Sydney Thayer, seated, informal, in officer\u27s dining room at Johns Hopkins Hospital, circa 1895
Courtesy of the American Philosophical Societyhttps://digitalcommons.rockefeller.edu/jem-the-beginnings/1026/thumbnail.jp
Enroute to the Ming Tombs, China, 1915
No full textEnroute to the Ming Tombs, China, 1915. Photograph by Simon Flexner
Courtesy of Medical Archives of The Johns Hopkins Medical Institutions
In 1915 William Welch, then President of the Board of Scientific Directors of the Rockefeller Institute, and Simon Flexner went to China as the medical members of a commission to examine progress in establishing the Peking Union Medical College there under auspices of The Rockefeller Foundation. Like so many men of scientific genius Dr. Welch was interested in art and architecture, and he took any every occasion to explore the Imperial city. Thus, with Simon Flexner and Mrs.Flexner and Caroline Buttrick, wife of Wallace Buttrick, Director of the Commission, Welch set forth on a two-day expedition north to Nankou to see the Great Wall and the Ming Tombs.
At one point as the expedition paused to admire a monument, Simon Flexner snapped a photograph of William Welch on his pony.https://digitalcommons.rockefeller.edu/jem-the-beginnings/1017/thumbnail.jp