5430 research outputs found
Sort by
Marjorie Russel Oral History
No full textInterview recorded on February 13th, 2020. Part of The Rita and Frits Markus Library Oral History project.https://digitalcommons.rockefeller.edu/marjorie-russel/1000/thumbnail.jp
Neural Mechanisms of Homeostatic Need and Reward
No full textFeeding, drinking, sleep, mating, fighting, and parenting are instinct behaviors essential for animal survival. Brain has evolved sophisticated cell types and circuits that encode or fulfill the need states for food, water, sleep, sex, aggression and parenting. These need-sensing neurons monitor both environmental cues and interoceptive conditions to elicit appropriate and specific behavioral programs towards restoration of these needs. The homeostatic control of these need states further enables animals to maintain their survival strength and adapt to the environment. In contrast,dysregulation of these need-sensing neurons causes diseases such as obesity and sleep disorders,both of which are key risk factors for a shorter lifespan across animal species including human. This thus raises two key questions in neurobiology of need and homeostasis: 1. How do need-sensing neurons maintain physiological homeostasis? How does dysregulation of need-sensing neurons cause diseases? The present thesis aims to study these two questions, exemplified by the hypothalamic control of energy homeostasis and mesolimbic processing of motivation, as well as their roles in the pathogenesis of obesity and addiction, respectively. The first study aims to elucidate the mechanism of how the feeding center develops resistance to leptin, a satiety hormone, and means to reverse it. Diet-induced obese (DIO) mice and obese humans have high circulating levels of leptin and do not respond to the exogenous leptin suggesting that they develop leptin resistance. However, the underlying cellular and molecular mechanisms that reduce leptin signaling are unknown. As part of a metabolomic screen for biomarkers of a leptin effect, we found that leptin reduced the level of leucine and methionine, mTOR ligands, only in leptin-sensitive animals, raising the possibility that mTOR activation might contribute to leptin resistance. We tested this by first treating DIO, chow-fed, ob/ob and db/db mice with rapamycin, an mTOR inhibitor. Rapamycin reduced food intake and adiposity in DIO mice but not in ob/ob or db/db animals. Whole-brain mapping revealed that the levels of phosphoS6, a marker of mTOR activity, were increased in the arcuate nucleus (ARC) and other hypothalamic nuclei in DIO mice. Subsequent multi-modal single-nucleus RNA sequencing of the ARC in DIO, chow-fed and ob/ob mice revealed that rapamycin altered gene expression exclusively in POMC neurons of DIO mice which also showed normalization of pSTAT3 levels after rapamycin treatment. Consistent with an effect on POMC neurons, rapamycin did not alter food intake or adiposity in mice after an ablation of POMC neurons or in MC4R knockout mice. In contrast, a POMC-specific deletion of Tsc1, which leads to a cell specific increase of mTOR activity, resulted in leptin resistance in chow-fed animals and reduced leptin sensitivity in these and ob/ob mice. In ob/ob-POMCtsc1-/- mice, rapamycin did not reduce food intake or adiposity in the absence of leptin. Finally, POMC-specific deletion of mTOR activators decreased the weight gain in mice fed a HFD. These data suggest that leptin resistance in DIO mice is the result of increased mTOR activity in POMC neurons and that inhibition of mTOR reduces obesity by reversing leptin resistance. Feeding and energy homeostasis are regulated by a pair of cell types: POMC and Agouti-related- peptide (AgRP) neurons in the hypothalamus. Activation of POMC neurons decreases food intake and resembles the state of food satiation, while activation of AgRP neurons drives food seeking and consumption and resembles the state of hunger. However, neuronal activity of AgRP neurons decreases upon sensory detection of food in mice. Similarly, neuronal activity of nNOS neurons in the subfornical organ, the activation of which resembles thirst, also decreases upon sensory detection of water. The similar neural dynamics observed between hunger- and thirst-coding neurons raise the question: How are the need states for food and water fulfilled by neural substrates downstream of the feeding and drinking centers? In the second study, we examined nucleus accumbens (NAc), a canonical reward center that regulates feeding and drinking, but it is not known whether these behaviors are mediated by same or different neurons. We employed two-photon calcium imaging in awake, behaving mice and found that during the appetitive phase, both hunger and thirst are sensed by a nearly identical population of individual D1 and D2 neurons in the NAc that respond monophasically to food cues in fasted animals and water cues in dehydrated animals. During the consummatory phase, we identified three distinct neuronal clusters that are temporally correlated with action initiation, consumption, and cessation shared by feeding and drinking. These dynamic clusters also show a nearly complete overlap of individual D1 neurons and extensive overlap among D2 neurons. Modulating D1 and D2 neural activities revealed analogous effects on feeding versus drinking behaviors. In aggregate, these data show that a highly overlapping set of D1 and D2 neurons in NAc detect food and water reward and elicit concordant responses to hunger and thirst. These studies establish a general role of this mesolimbic pathway in fulfilling need states and refining ongoing behaviors by controlling motivation-associated variables. Food and water are also naturally rewarding. In the third study, we set out to study the mechanism of how drugs of abuse hijack the mesolimbic circuit that directs motivation. Using whole-brain FOS mapping and in vivo single-neuron calcium imaging, we found that drugs of abuse augment ensemble activity in the NAc and disorganize overlapping ensemble responses to natural rewards in a cell-type-specific manner. Combining FOS-Seq, CRISPR-perturbations, and snRNA-seq, we identified Rheb as a shared molecular substrate that regulates cell-type-specific signal transductions in NAc while enabling drugs to suppress natural reward responses. Retrograde circuit mapping pinpointed orbitofrontal cortex which, upon activation, mirrored drug effects on innate needs. These findings characterize the dynamic, molecular, and circuit basis of a common reward pathway, wherein drug exposure suppresses fulfillment of innate needs. In summary, the current work unravels the molecular and dynamic basis of neural substrates underpinning homeostatic needs, thereby elucidating fundamental mechanisms driving the pathogenesis of obesity and addiction
Converting an Allocentric Goal Direction into an Egocentric Steering Signal in Drosophila
No full textNeuronal signals relevant for spatial navigation have been described in many species, however, a circuit-level understanding of how such signals interact to guide behaviour is lacking. In this thesis, I characterize a neuronal circuit in the Drosophila central complex that compares internally generated estimates of the fly\u27s heading and goal angles––both encoded in world-centred, or allocentric, coordinates––to generate a body-centred, or egocentric, steering signal. Past work has argued that the activity of EPG cells, or compass neurons , represents the fly\u27s moment-to-moment angular orientation, or heading angle, during navigation. An animal\u27s heading angle, however, is not always aligned with its goal angle, i.e., the direction in which it wishes to progress forward. I discovered that a second set of neurons in the Drosophila brain, FC2 cells, express an activity pattern that correlates with the fly\u27s goal angle. Furthermore, focal optogenetic activation of FC2 neurons induces flies to orient along experimenter-defined directions as they walk forward. EPG and FC2 cells connect to a third neuronal class, PFL3 cells. I found that individual PFL3 cells show conjunctive spike-rate tuning to both heading and goal angles during navigation. Informed by the anatomy and physiology of these three cell classes, a formal model is proposed for how this circuit can compare allocentric heading- and goal-angles to build an egocentric steering signal in the PFL3 output terminals. Quantitative analyses and optogenetic manipulations of PFL3 activity support the model. The biological circuit described in this thesis reveals how two, population-level, allocentric signals are compared in the brain to produce an egocentric output signal appropriate for the motor system
Sex Differences and the Evolution of Gene Expression
Full text linkPhenotypic sex differences are essential byproducts and drivers of evolution in sexually reproducing species. Knowing how sex differences arise and how they influence evolution is central to our understanding of evolution. Although many facets of sex differences have been explored, many remain unknown. Here I focus on sex differences and the evolution of gene expression in three parts. First, I examine the evolutionary properties of genes with sex differences in mean expression levels in the Drosophila brain – a surprisingly understudied organ in this context. Second, I identify and characterize genes with sex differences in gene expression variability in human tissues – one of the first analyses of its kind. In both parts of the thesis, I focus on how the expression of genes with sex differences evolves. Finally, I step away from sex differences to investigate how chromatin accessibility evolves. Given the tight relationship between chromatin accessibility and gene expression regulation, this provides us with another window into the evolution of gene expression. In the first part, I show that sex-biased genes in the Drosophila brain are highly enriched on the X Chromosome. I show that X-linked male-biased genes, and to a lesser extent female biased genes, are enriched for signatures of directional selection at the gene expression level. By examining the evolutionary properties of gene-flanking regions on the X Chromosome, I find evidence that adaptive cis-regulatory changes are more likely to drive the expression evolution of X-linked male-biased genes than other X-linked genes. Finally, I examine how the shared genome between the sexes and expression breadth constrain the evolution of gene expression
Neural Control of Sickness Behavior
Full text linkInfections are an omnipresent threat to all species. Thus, effective adaptive responses to resist and tolerate infections are essential for survival. Beyond the immune response, animals also induce a set of pleiotropic responses including anorexia, adipsia, lethargy, and changes in temperature, collectively termed sickness, during infections. While these responses have been shown to be adaptive for animal survival during infection and have been attributed to the central nervous system, the underlying control mechanisms for this response have not been elucidated. The goal of this work was to quantitatively characterize the many phenotypes associated with infection-induced sickness and to identify neural substrates responsible for the induction and maintenance of these sickness phenotypes. To accomplish this, we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to bacterial endotoxin (lipopolysaccharide, LPS) which potently induces sickness. We first thoroughly characterized the multimodal sickness response using high resolution measures of behavior, autonomic function, and metabolism. We then performed whole brain activity mapping to reveal that subsets of neurons in the nucleus of the solitary tract (NTS) and area postrema (AP) acutely express FOS after LPS treatment. To causally show that these activated neurons were inducers of the sickness, we showed that subsequent reactivation of these specific neurons in Fos2A-iCreERT2 (TRAP2) mice replicates the behavioral and thermal component of sickness. In addition, inhibition of these same LPS-activated neurons diminished all of the behavioral responses to LPS. For further mechanistic delineation of the neurons involved in this response, we utilized singlenuclei RNA-sequencing of the NTS/AP to identify the LPS activated neural populations. Molecular profiling identified multiple neuronal types activated during LPS-induced sickness, with the greatest enrichment of Adcyap1+ neurons. We found that activation of these Adcyap1+ neurons in the NTS/AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia, and locomotor cessation seen after LPS injection. In aggregate, these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection
Structural and Functional Analysis of Substrate Recognition and Inhibition of the Multidrug Transporters MRP1 and MRP2
Full text linkNeuroblastoma is the most common extracranial tumor of young children, and the five-year overall survival of children with high-risk disease is less than 50%. Overexpression of multidrug resistance protein 1 (MRP1), a plasma membrane ATP-binding cassette (ABC) transporter, is associated with high-risk neuroblastoma. While it is known that MRP1 transports multiple neuroblastoma chemotherapeutic agents out of the cell in a glutathione (GSH)-dependent manner, the mechanism of GSH-dependent MRP1 substrate transport has previously not been understood. Additionally, over the past three decades, multiple small-molecule inhibitors of MRP1 transport have also been identified, with the activity of several inhibitors also determined to be GSH-dependent. The mechanism of GSH-dependent MRP1 inhibition, however, has also previously not been understood. This thesis consists of two parts. In part one, which covers GSH-dependent MRP1 substrate transport and inhibition (Chapter 2), I determine the mechanism of GSH-dependent MRP1 substrate transport by solving the structure of MRP1 bound to GSH and the chemotherapeutic vincristine. The cysteine thiol of GSH, which is bound in the relatively polar P-pocket of the MRP1 substrate-binding site, forms a key intermolecular interaction with vincristine, which is bound in the relatively hydrophobic H-pocket. I also determine the mechanism of GSH-dependent MRP1 inhibition by solving the structure of MRP1 bound to GSH and the small molecule Reversan, a lead compound with therapeutic potential. As with vincristine, the cysteine thiol of GSH forms a key intermolecular interaction with Reversan across the substrate-binding site, and Reversan competes directly with vincristine binding in the H-pocket. In part two, I investigate the mechanism and selectivity of a novel MRP1 cyclic peptide inhibitor, CPI1, developed with a collaborator (Chapter 3). The cross-inhibition of other ABC transporters beyond the intended target – MRP1 – may alter the pharmacokinetics of clinically-relevant substrates of other ABC transporters, thus resulting in toxicity. CPI1 binding arrests MRP1 in a conformation incompatible with substrate transport or ATP hydrolysis, but also inhibits the activity of MRP2 (Chapter 4). To begin to understand the mechanism of CPI1 cross-inhibition of MRP2, I solve the structure of MRP2 in a ligand-free state. Unlike previously resolved structures of MRP1, the ligand-free structure of MRP2 features a segment of its own cytoplasmic linker sequence bound in the substrate-binding site. This linker sequence likely functions as an affinity gate for MRP2 substrate transport
Genome-Scale Quantification of Target Vulnerability in Mycobacterium Tuberculosis
No full textEssential bacterial genes orchestrate core biological processes and represent the targets of nearly all antibacterial drugs. Traditional genetic approaches such as knockout studies and transposon mutagenesis have cataloged hundreds of essential bacterial genes but provide no basis on which to identify the most attractive targets for antibiotics. In contrast to the binary definition of gene essentiality, the gene vulnerability is an expression that relates the magnitude of gene inhibition with the resulting impact on bacterial fitness. Partial gene inhibition results in stronger fitness costs for vulnerable genes than invulnerable genes. Understanding vulnerability is critical for prioritizing targets for drug development and in understanding the growth rate limiting steps in bacterial physiology. To quantify gene vulnerability, we developed a CRISPR interference-based functional genomics method to systematically titrate the expression of nearly all essential genes and monitor fitness outcomes in the global pathogen, Mycobacterium tuberculosis (Mtb). Additionally we created an approach to study differential vulnerability in clinical Mtb strains and under host-relevant growth conditions. We find that different biological pathways, while all essential for Mtb viability, vary widely in their vulnerability. We define highly vulnerable steps in Mtb central dogma processes, protein secretion and cell wall synthesis, including targets currently unexplored in the drug discovery pipeline. Likewise, we identify invulnerable processes that can withstand substantial inhibition with little impact on bacterial fitness, including targets of failed efforts in drug discovery. Comparison of vulnerability in Mtb and the model bacterium M. smegmatis resulted in substantial overlap in vulnerability predictions but with notable exceptions, suggesting considerable but incomplete conservation of expression-fitness relationships within mycobacteria. Bioinformatic analysis of 10,000 Mtb clinical isolates showed that vulnerable Mtb genes are under higher purifying selection than invulnerable genes, indicating that these gene classes are under distinct evolutionary pressures. Evolutionary analysis further identified that vulnerable genes are more likely to be conserved and essential across \u3e2 billion years of evolutionary separation in bacteria. Differential vulnerability analyses between the reference H37Rv Mtb strain and a hypervirulent Mtb isolate HN878 as well as between standard culture conditions and different iron and carbon source conditions, revealed clinically relevant differences in vulnerability. Lastly, we find that differential genetic vulnerability can predict differential susceptibility to chemical inhibitors. This work provides a quantitative redefinition of essential bacterial processes and serves as a roadmap for developing more effective drugs against Mtb and other bacterial pathogens
Neotelomere Formation by Telomerase in Human Cells
Full text linkTelomeres define and protect the ends of linear chromosomes. Genome stability requires that cells differentiate telomeres from DNA double-strand breaks (DSBs), which humans accomplish with the shelterin complex. The reverse transcriptase telomerase counteracts the shortening of telomeres that occurs with cell division due to incomplete DNA replication and nucleolytic resection. During human development, the expression of the reverse transcriptase component of telomerase (TERT) is restricted to the germ line and select stem cell pools, causing telomeres to shorten in most somatic cells. After a certain number of divisions, one or a few telomeres become too short to adequately suppress DNA damage response signaling, driving cells into senescence or apoptosis. Cells deficient in the p53 and Rb pathways, however, disregard these alarms, continue to divide, and ultimately enter telomere crisis, marked by restricted cell viability due to severe genome instability. During crisis, dysfunctional telomeres undergo repeated breakage-fusion-bridge cycles, forming dicentric chromosomes that snap during mitosis. Ultimately, clinically detectable tumors emerge from this process with shuffled, aneuploid genomes after acquiring a telomere maintenance mechanism, usually by reactivating TERT expression. Canonically, telomerase is believed to function in cancer by rapidly restoring critically short telomeres to a tolerable length and then maintaining replicative immortality thereafter. Nevertheless, this dogma does not explain how cells restore end protection to deeply damaged chromosomes, which likely require a mechanism for reestablishing their telomeres. We suspected that telomerase might aid incipient tumor cells in their escape from telomere crisis by healing broken chromosomes with neotelomeres. In a subset of human patients with terminal chromosome deletions, telomeric repeats directly abut reference genomic sequence at the breakpoint, suggesting that telomerase itself might have directly added to a DSB at that site. Consistent with this interpretation, the breakpoint sequence from one of these patients, known as TS, has been shown to be an excellent primer for telomerase in vitro. Here, we use this TS sequence to develop a sensitive and specific qPCR assay to detect and measure telomeric repeat addition at a programmed DSB in human cells. With this assay, we demonstrate that telomeric repeat addition occurs at a Cas9- induced DSB in a telomerase-dependent fashion, though such events are rare in cells with physiologic levels of telomerase activity. We identify long-range DSB resection and ATR kinase signaling as repressors of telomerase at DSBs in human cells. Finally, we provide preliminary evidence that telomerase can synthesize a fully functional telomere de novo at a DSB. Our findings unveil a new function for telomerase in carcinogenesis and provide a mechanistic explanation for the recent identification of neotelomeres in human tumors
Cortical Control of Naturalistic Facial Expression Production in Primates
No full textFacial expressions constitute a fundamental class of social communication signals in primates. Nonverbal expressions shared between individuals can instantly and effortlessly communicate endogenous affective state, the valence of external stimuli, as well as more complex concepts such as intentions, social rank, and receptivity to future social interactions. All of this information is transmitted through the fine, dynamic arrangement of dozens of small facial expression muscles. Commiserate with their importance, the muscles of facial expression are under direct cortical control from descending projections to the facial nucleus. These direct corticomotoneuron projections are unique to primates and originate in multiple distinct cortical face representations, including primary (M1), ventral premotor (PMv), and cingulate motor (M3) cortices. Much like their human counterparts, macaques use facial expressions to navigate both cooperative and competitive interactions with conspecifics. Yet despite their integral role in social communication, the neural bases for facial expression production are not well understood. Neither the relative contributions of each of these areas, nor the local codes supporting facial expression production, have been systematically explored. To this end, we first identified these three cortical face motor representations via fMRI, before targeting them for electrophysiological recording with chronic electrode arrays. Subsequently, we recorded from many single cells in each region simultaneously, while subjects produced naturalistic facial expressions. These expressions included socially-meaningful expressions elicited through a number of visuosocial stimuli, in addition to non-socially meaningful, volitional facial movements, as well as periods of rest. We found widespread electrophysiological activation of single cells in all cortical face motor regions during naturalistic facial expression production, during both volitional and socioemotional facial expressions. While individual neuron responses were highly heterogenous in terms of tuning, latency, and shape, neural populations within each cortical region supported linearly discriminable representations of expression type. Thus, all cortical face motor regions contained unique representations for facial expressions. However, this was achieved via regionally distinct coding regimes, with primary motor cortex enacting two temporally-distinct population codes, and premotor cortex, a single temporally-stable code. Furthermore, the dynamic structure of neural activities was not preserved across expressions, and this was not due to non-overlapping subpopulations of neurons, each specialized for one expression type. Instead, each expression was accompanied by a unique temporal evolution of population activity, formed by neurons which flexibly recombined their activities. At the level of the entire cortical face motor system, each expression engaged a unique combination of neural modes, which themselves contained a mixture of temporal and expression-specific features. Within motor cortex, expressions with similar kinematic features engaged a subset of shared neural modes, perhaps reflecting the direction of moment-by-moment movements -- a feature not observed in the dynamics of premotor or cingulate cortex. Indeed, we found that primary motor cortex contained substantial information regarding the continuous kinematics of the face during naturalistic facial expression production. Together, these results reveal the first cortical control mechanisms for naturalistic facial expression production in primates and generate new hypotheses for testing how motor programs are instantiated in a distributed system, which interfaces with various domains of social cognition