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Cerebral Amyloid Angiopathy-Causing Beta-Amyloid Variants Have Altered Effects on the Clotting System
Get PDFCerebral amyloid angiopathy (CAA), where beta-amyloid (Aβ) deposits around cerebral blood vessels, is a major contributor of vascular dysfunction in Alzheimer\u27s disease (AD) patients. However, the molecular mechanism underlying CAA formation and CAA-induced cerebrovascular pathologies, including intracerebral hemorrhage (ICH) is unclear. Hereditary cerebral amyloid angiopathy (HCAA) is a rare familial form of CAA in which mutations within the Aβ peptide cause an increase in vascular deposits. Since the interaction between normal wild-type (WT) Aβ and fibrinogen increases CAA and plays an important role in cerebrovascular damage in AD, I investigated the role of the Aβ-fibrinogen interaction in HCAA pathology. My work revealed the most common forms of HCAA-linked mutations resulted in up to a 50-fold stronger binding affinity of Aβ for fibrinogen, increased perturbation of clot structure and fibrinolysis, and increased cortical fibrin(ogen)/Aβ co-deposition. Consistent with these in vitro results, HCAA patients have increased fibrin deposits compared to early-onset AD patients and non-demented individuals. In addition, because of the known WT Aβ enhancements to proteolytic/fibrinolytic system activation, I examined the effects of HCAA Aβ on the fibrinolytic system. I found that the majority of HCAA Aβ variants led to increased tPA-mediated plasminogen activation. The HCAA Arctic variant (E22G) did not, which may partially explain the unique clinical features of this mutation. To expand our understanding of the continuum of Aβs that can interact with fibrin(ogen) and the fibrinolytic system, I also explore the possible role of longer length Aβs in facets of AD-related vascular pathologies. My results suggest the HCAA-type mutations, with special emphasis on the Dutch and Iowa mutations, increase the interaction between Aβ, fibrinogen, and the fibrinolytic enzyme system. These findings provide a novel molecular mechanism for how CAA-linked mutations may lead to severe cerebrovascular pathology in HCAA patients by enhancing the Aβ-fibrin interaction and fibrinolytic activity
The Role of Nutrient Availability in Therapeutic Response of Leukemia
Get PDFTumor environment influences the response to anti-cancer therapy, but which extracellular nutrients impact drug sensitivity is largely unknown. In this work, we used functional genomics to identify metabolic modifiers of the response to L-asparaginase (ASNase), a therapy that depletes plasma asparagine and targets leukemic cells with insufficient asparagine synthesis. Our approach revealed thiamine pyrophosphate kinase 1 (TPK1), which converts vitamin B1 (thiamine) into the cofactor thiamine pyrophosphate (TPP), as a metabolic dependency under ASNase treatment. In glutamine-anaplerotic leukemia cells, we found that TPP availability enables asparagine synthesis from extracellular glutamine. Mechanistically, TPP is critical for the activity of alpha-ketoglutarate dehydrogenase (AKGDH), a TCA cycle enzyme that catalyzes a step in the overall conversion of glutamine to asparagine. When TPP availability is limiting for cell proliferation of TPK1 KOs, ASNase sensitivity is significantly increased. Standard cell culture media formulations provide thiamine at a concentration that is ~100-fold higher than that observed in human plasma. While thiamine is generally not limiting for cell proliferation under standard culture conditions, a DNA-barcode competition assay identified a subset of leukemia cell lines that grow sub-optimally under lower, more physiological thiamine levels. These cell lines are characterized by low expression of SLC19A2, a high affinity thiamine transporter. Intriguingly, SLC19A2 expression was necessary for not only optimal growth, but also for maintaining ASNase resistance, when standard media thiamine was lowered to the concentration of human plasma. Importantly, analyzing RNAseq data of pediatric acute lymphoblastic leukemia (ALL) tumor samples revealed that SLC19A2 is the primary thiamine transporter expressed in these cancers, and that SLC19A2-low tumors exist among patients. To model such tumors, we used a SLC19A2-low cell line to generate orthotopic tumors in NSG mice. Remarkably, humanizing blood thiamine content of mice through diet sensitized these leukemia cells to ASNase in vivo. Altogether, our work reveals that utilization of thiamine is a determinant of ASNase response for some cancer cells, and that over-supplying vitamins may impact therapeutic response in leukemia. Additionally, our work adds to the recent literature that demonstrates how physiological levels of certain nutrients in cell culture can affect therapy. Specifically, our work provides the first proof of principle that humanizing the vitamin levels of both in vitro and in vivo models can affect drug sensitivity. This has broad implications for the screening and validation of new therapeutic candidates
Details of the Exhibit
No full textDetails of the exhibit JEM: The Beginnings
Idea, design - Olga Nilova, Special Collections Librarian
Photograph - Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/jem-the-beginnings/1004/thumbnail.jp
The Journal of Experimental Medicine, Vol. 7, 1905
No full textThe Journal of Experimental Medicine, Vol. 7, 1905
In February 1905, Dr. Flexner and Dr. Eugene Opie, editors of the Journal, got out the final numbers of volume 6 of the JEM, which had been held up two years; numbers 4,5, and 6 appearing in a single issue composed, as Simon Flexner put it, of the most important manuscripts rescued from Welch\u27s study. This was the last issue to appear with Welch\u27s name on the Journal cover, and it contained 40 papers, all but two of which were by his Baltimore pupils. Ever since that time JEM has appeared regularly and has continued to be one of the world\u27s most respected medical research journals.
For fifteen years Simon Flexner was chief editor, assisted by Opie until 1910 and by Benjamin D. Terry for the next two years. He carried on alone thereafter until 1921 when Peyton Rous became editor with him.https://digitalcommons.rockefeller.edu/jem-the-beginnings/1034/thumbnail.jp
Not Black and White: BMP Signaling Drives Melanocyte Differentiation Downstream of Stem Cell Activation
Get PDFTissue stem cells (SCs) maintain, regenerate, and repair the body over the course of an organism\u27s lifetime. To preserve their long-term function, SCs must exert precise control over their cell state dynamics as they move from quiescence to activation and commit to full differentiation. My graduate research has been centered on investigating the molecular mechanisms that fuel these transitions in melanocyte stem cells (McSCs), a unique neural crest-derived SC population located in the hair follicle (HF). Through periodic bouts synchronous with HF cycling, quiescent McSCs become activated to proliferate, giving rise to committed proliferative progeny (McCP) that differentiate into mature pigment-producing melanocytes. The signaling factors and gene expression programs that orchestrate these cellular changes are still incompletely understood. To elucidate new insights into this process, I developed fluorescence-activated cell sorting strategies to isolate quiescent, activated, and differentiating McSC lineage cells from the mouse skin at discrete stages of the hair cycle. I then performed single cell RNA-sequencing (scRNA-seq) to reveal the evolving transcriptional signatures of the lineage with high resolution. Comparative bioinformatic analyses suggested that BMP and WNT signaling increase concomitantly throughout differentiation. I then sought to understand the role of BMP signaling in McSC lineage progression and whether this pathway engages in crosstalk with WNT signaling. To do so, I performed conditional lineage-specific genetic ablation of Bmpr1a to extinguish BMP signaling, which resulted in gray hair. However, McSCs remained intact and functional in these mutant animals, indicating dysfunction in their differentiating progeny. ScRNA-seq and pseudotime analysis of Bmpr1a null cells indicated a block in the differentiation program just downstream of the early McCP stage, and I detected further signs of melanocyte immaturity using immunofluorescence and electron microscopy analyses. Seeking mechanistic understanding, I interrogated changes in transcription factor expression at this blockage point. I found reduced nuclear levels of the master regulator MITF and WNT mediator LEF1. Using WNT mutant mouse models, cell culture systems, and chromatin profiling, I provide evidence demonstrating cooperation of BMP and WNT signaling to trigger complete differentiation of McCP into melanocytes through MITF and LEF1 activity. Altogether, I have generated a thorough characterization of the transcriptional and chromatin landscape changes that temporally define McSC lineage commitment in vivo. My findings underscore a critical role for signaling through BMPR1a to achieve full melanocyte differentiation in the HF. These findings raise intriguing questions about the role of BMP in hair and coat color variation, age-related hair graying, and melanoma initiation and progression
A Translational Approach to Modeling Unique Aspects of Germ Cell Development During Self-Organization of the Primate Embryo
Get PDFThe specification of germ cells during embryonic development is vital not only for the development of an organism, but quite literally for the survival and propagation of its entire species. Recent work has demonstrated that several aspects of human primordial germ cell (hPGC) development are specific to primates, necessitating model systems and in vivo validation that is also species specific. In this work, a synthesis of in vitro and in vivo techniques is used to investigate hPGC specification within a human embryonic stem cell model of a gastrulating embryo known as a gastruloid. An hPGC transcriptomic signature that indicates migratory potential via canonical and novel mechanisms is indicated, raising several potential candidates for further investigation into the under-studied migratory phase of germ cell development. We seek to generate validation assays of hPGC function in an embryonic context by investigating migratory potential in grafts to the chick embryo, and demonstrate that despite significant differences between chick and human routes of migration in vivo, human in vitro-derived PGCs are in fact able to demonstrate migratory behavior in the chick, following chick migratory patterns and demonstrating a specific homing towards the chick mesonephros and gonad. These experiments not only provide a functional validation for in vitro-derived hPGCs that is complementary to molecular and epigenetic analysis, but also hint at the elements of hPGC development that are conserved throughout evolution. The gastruloid system is then used for further investigation into the hPGC niche. The power of this model system relies upon self-organization due to endogenous signaling in response to an exogenous BMP4 initiating signal, in a manner analogous to that found in the human embryo. We harness this power, using single cell image quantification and genetic tools including CRISPR-Cas9 to probe further into the signaling environment of the hPGC niche in the gastruloid model. We elucidate the role of each leg of the BMP4 – WNT – ACTIVIN/NODAL signaling cascade in development of this niche, which determines gastrulation events, in hPGC specification. These experiments not only demonstrate how BMP4 acts independently in addition to this cascade to directly specify hPGCs, but also how all three pathways work in harmony to generate self-organization of hPGCs within their gastruloid niche. By probing further into the ontogeny of hPGC specification, we find that upregulation of BLIMP1 alone, a canonical regulator of PGCs, is sufficient to induce later markers of hPGC fate, and surprisingly is also sufficient to downregulate SOX2 (a marker of epiblast and early ectodermal fate) and upregulate SOX17, which has been previously placed upstream of BLIMP1. Finally, we propose the marmoset as a good model of primate reproduction and embryogenesis, in an attempt to describe a non-human primate (NHP) system for validation of hPGC characteristics observed in vitro. We systematically investigate marmoset embryonic development in vivo using serial, high temporal and spatial resolution ultrasound imaging. We describe the morphological characteristics of implantation, gastrulation, neurulation, and organogenesis, as well as the curious marmoset phenotype of an elongated peri-gastrulation window, incorporating slowed embryonic growth and rapid extraembryonic development. In doing so, we generate an annotated ultrasound atlas of marmoset embryogenesis, and train models to identify developmental stages and predict fertilization ages from a single frame. In addition, we suggest that the extended peri-gastrulation window in the marmoset will provide a unique opportunity to perform in utero genetic editing, lineage tracing, and even allogenic transplantation to complement in vitro studies of hPGC development. This body of work provides a synthesis of culture techniques, genetic and molecular tools, and imaging systems that will provide a foundation for the exploration of not only human PGC development, but also hopefully a generalizable roadmap for translation between in vitro and in vivo studies early primate development
Linker Histone Medicated Regulation of Mitotic Chromosome Compaction and Individualization
Get PDFMitotic chromosomes are scaled to the cell size to ensure effective chromosome segregation. Recent studies have shown how condensins and DNA topoisomerase II organize the mitotic chromosome. However, the regulation of these factors in maintaining proper chromosome size in different cell types remains a mystery. Here, I investigated the role of the linker histone variant H1.8 in regulating mitotic chromosome structure. I showed that H1.8 suppresses binding of condensins and topo II to mitotic chromatin in Xenopus egg extracts. Using an in vitro reconstitution system, I showed that H1.8 inhibits binding of purified condensins and topo II to nucleosome arrays. I also showed that condensin binding to nucleosome arrays is sensitive to magnesium dependent chromatin compaction. By using direct measurement of chromosome length, I then showed that H1.8 suppresses chromosome length solely through condensin I enrichment on chromatin. I then investigated the organization of Xenopus egg extract chromosomes using chromosome conformation capture technique Hi-C. Using Hi-C analysis, I showed that condensin I organizes both mitotic loops and loop layers of mitotic chromosomes and that H1.8 mediated suppression of condensin I increases both mitotic loop and layer sizes. This analysis also corroborates direct measurements of chromosome length. I also showed that nucleosome depletion results in further reduction in loop and layer sizes over H1.8 depletion. This suggests that chromosome length can be regulated by condensin I binding through competitive inhibition by both nucleosomes and linker histones. Mitotic chromosomes are organized in a rod to increase both physical rigidity of chromosomes and to ensure effective resolution. Using Hi-C data, I observed that both condensins play a role in maintaining chromosome rigidity and subsequently in maintaining chromosome individualization. I then showed that, like sister chromatid resolution, condensin activity drives topo II activity to continuously resolve interchromosomal links in mitosis. Since H1.8 suppresses both condensin and topo II, it suppresses chromosome individualization. I then go on to show that this suppression of chromosome individualization is necessary to maintain spindle integrity. Based on these data, I propose a model where mitotic chromosome length and individualization can be regulated by using linker histone stoichiometry on chromatin as a rheostat. As linker histones are a dynamic component of chromatin that have been shown to have extensive cell cycle dependent phosphorylation, I discuss the possibility that titrating linker histone stoichiometry on chromatin may be used as a mechanism to control the binding of DNA binding proteins in both interphase and mitosis and thus regulate cellular functions
Structural Study of Disease Relevant ABC Transporters-Cystic Fibrosis Transmembrane Conductance Regulator and ABCA4
Get PDFATP-binding cassette (ABC) transporters are primary transporters that utilize the energy from ATP binding and hydrolysis to transport substrates across membrane against their concentration gradients. Structurally, canonical ABC transporters consist of four subunits— two transmembrane domains (TMDs) which form the substrate transport pathway and two nucleotide binding domains (NBDs) which dimerize upon ATP binding to provide energy for substrate transport. Most mammalian ABC transporters are exporters, with three exceptions: SUR—a regulatory protein for the KATP channel, cystic fibrosis transmembrane conductance regulator (CFTR)—an chloride channel and ABCA4 (aka the Rim protein and ABCR)—an retinylidene-PE importer. In addition to their unique functional properties, both CFTR and ABCA4 are very important in human health. Mutations in CFTR cause cystic fibrosis, a lethal disease with a prevalence of 1 in 2,500 in Caucasian populations. Over 800 mutations have been identified in ABCA4 to associate with various types of retinal disease, including the Stargardt disease (also known as juvenile macular degeneration), the most common form of inherited macular degeneration. To understand how those two proteins function, I mainly took a structural approach to capture the structures of CFTR and ABCA4 in different functional states. In correlating the structures with functional data, we now have a deeper mechanistic understanding of these two important ABC transporters. The function of CFTR is regulated by ATP and phosphorylation. Once phosphorylated, ATP binding opens the CFTR channel and ATP hydrolysis closes it. First, we determined by cryo-electron microscopy (cryo-EM), the structure of dephosphorylated human CFTR in the absence of ATP (Chapter 2). With this structure, in conjunction with the functional studies performed by our collaborator (Prof. Laszlo Csanady and Prof. David C. Gadsby), we were able to propose a mechanism of how phosphorylation regulates CFTR. In addition, we identified a structural feature distinguishing CFTR from all other ABC transporters, which likely forms the structural basis for CFTR\u27s channel function. Next, we determined the structure of CFTR in the phosphorylated, ATP-bound state (Chapter 3 and 4). By comparing the ATP-free and -bound structures, we identified the nature of conformational changes that lead to channel opening. These structures also allow us to map many disease-causing mutants and explain how they lead to the malfunctioning of CFTR. To understand how small molecules, called potentiators, interact with CFTR and increase its open probability, we determined the structures of CFTR in complex with 2 potentiators— ivacaftor and GLPG1837 (Chapter 5). Interestingly, both small molecules bind to the same pocket inside the transmembrane region. These studies identified a hotspot on CFTR for rational drug design. Finally, I also studied ABCA4, the only known importer in mammalian ABC transporters. To understand how ABCA4 functions, I determined the structures of ABCA4 in the absence and presence of ATP (Chapter 6). Based on these structures, we propose a rudimentary transport mechanism for ABCA4. Future work will be carried out in the Chen lab to test this model
Details of the Exhibit
No full textDetails of the exhibit JEM: The Beginnings
Idea, design: Olga Nilova, Special Collections Librarian
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/jem-the-beginnings/1020/thumbnail.jp
The Cornerstone
No full textThe ground was broken in July 1904 for the first building, now called Founder\u27s Hall. This faded photograph survives to commemorate the occasion on December 3rd when without public ceremony Prudden, Holt, Biggs, and Flexner stood beside the masons to the cornerstone well and truly laid. - George W. Corner. A History of the Rockefeller Institute. The Rockefeller Press, 1964https://digitalcommons.rockefeller.edu/jem-the-beginnings/1028/thumbnail.jp