Individual Predoctoral Fellowship Awards to Current MSTP Students

Ashley Pandolfi, NIH NRSA F30 Individual Predoctoral Fellowship Award for a project entitled "Unraveling crucial roles of homeobox gene Hlx in hematopoiesis and leukemogenesis" (Sponsor, Uli Steidl, Cell Biology)

Corey Keller, NIH NRSA F31 Individual Predoctoral Fellowship Award for a project entitled "Localizing Functional And Pathological Networks In Epilepsy" (Sponsor, Fred Lado, Neuroscience)

Robert Tamayev, NIH NRSA F30 Individual Predoctoral Fellowship Award for a project entitled "Dementia due to mutations in BRI2; "Link to Alzheimer's Disease" (Sponsor, Luciano D'Adamio, Microbiology & Immunology)

Ujunwa Cynthia Okoye, NIH NRSA F31 Individual Predoctoral Fellowship Award for a project entitled "The Role Of c-MAF In Stem Cells In Leukemia" (Sponsor, Uli Steidl, Cell Biology)

Matthew Klein, NIH NRSA F31 Individual Predoctoral Fellowship Award for a project entitled "The Role of Sam68 in Synaptic Transmission And Plasticity" (Sponsor, Pablo Castillo and Bryen Jordan, Neuroscience)

Esther Berko, Autism Speaks Predoctoral Fellowship Award for a project entitled "Advanced Parental Age and Autism: The role of aneuploidy and uniparental disomy in ASD pathogenesis." (Sponsor, John Greally, Genetics)

Carlos Diaz-Balzac, NIH NRSA F31 Individual Predoctoral Fellowhip Award for a project entitled "Identification of Novel Loci Interacting with the Kallmann Syndrome Gene Kal-1." (Sponsor, Hannes Buelow, Genetics)

Rachel Fremont, NIH NRSA F30 Individual Predoctoral Fellowhip Award for a project entitled "The Role of a Cerebello-Thalamo-Basal Ganglia Pathway in Dystonia." (Sponsor, Kamran Khodakhah, Neuroscience)

Edward Manning, American Heart Association Individual Predoctoral Fellowhip Award for a project entitled "Modeling Cardiac Troponin." (Sponsor, Steve Schwartz, Physiology & Biophysics)

Jerri Chen, NIH Individual F30 Predoctoral Fellowhip Award for a project entitled "Structure-function analysis of cardiac KCNQ1 K+ channel interactions with KCNE1." (Sponsor, Thomas McDonald, Molecular Pharmacology)

Yamini Krishnan, NIH Individual F30 Predoctoral Fellowhip Award for a project entitled "GPCR-Based Regulation of the hERG Potassium Channel Biosynthesis and Function." (Sponsor, Thomas McDonald, Molecular Pharmacology)

Other Students Holding Individual Predoctoral Fellowships

See Below for Abstracts of Recent Projects

Ashley Pandolfi - ABSTRACT: While isolation of stem cells has advanced dramatically in the last few decades, understanding of the precise mechanisms that regulate the self-renewal and lineage commitment of a stem cell is still limited. During hematopoiesis, progeny of hematopoietic stem cells (HSC) become committed to differentiate into specific cell lineages to ultimately generate terminally differentiated cells. Transcription factors have been recognized for their ability to drive expression of a characteristic set of lineage-specific target genes, instructing a precursor cell to adopt a certain differentiation program. Dysregulation of transcription factor activity has an important role in leukemia, implicating these genes as potential targets for therapeutic intervention in blood, and other forms of cancer. When we analyzed purified pre-leukemic hematopoietic stem and progenitor cells (HSPC) in a murine acute myeloid leukemia (AML) model, we found 4-fold upregulation of a novel non-clustered homeobox gene, H2.0-like homeobox (Hlx), suggesting that Hlx may be involved in healthy hematopoiesis and malignant transformation. Our preliminary studies indicate that overexpression of Hlx disrupts healthy myeloid differentiation and confers unlimited serial clonogenicity and a myelomonocytic differentiation block to hematopoietic stem and progenitor cells in vitro. Furthermore, overexpression of Hlx causes loss of phenotypic HSC and persistence of an expanded, aberrant myeloid progenitor population in vivo. We also find that Hlx regulates a network of genes important for lineage commitment and myeloid differentiation of HSPC. Strikingly, we find that Hlx is overexpressed in the majority of patients with AML, and that Hlx expression is one of the strongest predictors of AML patient survival. We also find that Hlx downregulation inhibits growth of murine and human AML cells in vitro. This project aims to understand how Hlx is regulating these critical functions in HSC and during myeloid differentiation. To characterize the roles of Hlx in lineage commitment of stem and progenitor cells, as well as in myeloid differentiation and acute myeloid leukemia cells, we will utilize genetic murine models, stem cell transplantation assays and targeted reduction of Hlx levels in vivo and in vitro. To elucidate the mechanism of action of Hlx, we will study downstream pathways we have identified by transcriptional profiling and perform chromatin-immunoprecipitation to establish Hlx as a transcription factor capable of directly regulating its target genes.

Corey Keller - ABSTRACT: Recent advances in epilepsy stem from the notion that a network of brain areas, as opposed to a single onset zone, may be responsible for the onset and maintenance of seizures. This is critical as identifying and completely resecting the epileptogenic zone while preserving healthy eloquent cortex is considered to be the basis of the successful surgical treatment of epilepsy. However, methods to localize and quantitatively study the properties of these networks need to be improved. Recent advances in neuroscience include the development of tools for the analysis of cortical networks using graph theory and the identification of high frequency activity on EEG that correlates with neuronal population firing. Quantifying the connectivity between brain areas with graph theory allows comparison of normal and abnormal brain networks. Availability of a marker of highly localized neuronal population activity that can be recorded by conventional clinical electrodes offers a means to track brain activation rapidly as it propagates through cortex. Here, we propose to bridge the gap between current clinical practice that results in suboptimal surgical outcomes and a more detailed understanding of cortical networks and the spread of epileptic To determine the relationship between networks derived from neuroimaging and electrophysiology techniques, and 2) To localize functional and pathological networks using complementary methods from multiple recording techniques. The long term goal of this research is the application of network analysis to multimodal imaging to 1) better understand the relationship between non-invasive and invasive imaging and 2) develop more accurate techniques to localize epileptic networks to improve surgical outcome, both of which will be addressed in this proposal. This proposed study has two fundamental goals. PUBLIC HEALTH RELEVANCE: The cure rate of surgery for patients with epilepsy originating outside the temporal lobe continues to be less than 50% despite improvements in surgery and brain imaging. This low rate of success likely stems from the fact that our techniques to define epileptic regions of the brain are limited and cannot be identified at the time of surgery. This project proposes to develop novel methods for identifying the areas in the human brain which may be safely removed to control seizures not stopped by medications. This research has important significance in improving efficacy of localizing the seizure zone and developing tools to improve surgical outcome.

Robert Tamayev - ABSTRACT: Alzheimer’s disease (AD) pathogenesis is firmly associated with the processing of the amyloid precursor protein (APP), since mutations in APP or in the enzymes responsible for its processing cause Familial Alzheimer’s disease. Because of its biological and pathological importance, understanding how APP cleavage is controlled is of great relevance. Inhibition of APP processing may possibly hold the solution for therapeutic intervention in AD. We postulated the existence of membrane proteins that bind APP and regulate its processing and have identified BRI2 as an APP ligand. Of note, BRI2 mutations cause Familial British and Danish Dementia (FBD and FDD), two AD-like neurodegenerative disorders. Mature BRI2 inhibits APP processing and Aβ production and, interestingly, BRI2 mutants that cause FDD have a defect in maturation. These findings prompted us to hypothesize that BRI2 regulates and/or modifies AD pathogenesis and that BRI2 FDD mutants are ineffective inhibitors of Aβ generation in vivo. This last point hints to the possibility that dis-regulation of APP processing may participate in the pathogenesis of FDD and FBD. To test these hypothesis, we have created Bri2-null mice, and a mouse knock-in (KI) models of FDD (FDDKI), which is genetically congruous, carrying one wild-type and one mutant Bri2 allele, to the human cases. Our data shows that FDDKI mice have an impairment in synaptic plasticity and severe hippocampal memory deficits. Recovery from these defects is seen in FDDKI mice haplodeficient for APP. Bri2 heterozygous mice show similar memory and synaptic plasticity defects as FDDKI mice, leading us to believe this is a loss of function mutation, with the function lost being the inhibition of processing of APP by BRI2. We have also found that a BRI2-drived peptide binds and inhibits APP processing specifically, and can rescue the synaptic deficits found in the FDDKI and a popular transgenic mouse model of AD (APPtg2575). Here, we hope to further characterize the synaptic and hippocampal memory deficits in FDDKI and Bri2 heterozygous mice to shed light on the pathogenesis of AD, as well as test the Bri2-derived peptide in vivo as a possible therapeutic intervention for AD.

Ujunwa Cynthia Okoye - ABSTRACT: The transcription factor c-Maf is upregulated in hematopoietic stem cells of PU.1 knockdown mice which develop acute myeloid leukemia (AML). In another hematologic malignancy, multiple myeloma, c-Maf functions as an oncogene, contributing to the accumulation of malignant cells. Here we hypothesize that c-Maf is an important regulator of normal stem cells and a critical oncogenic target in AML induced by knockdown of the transcriptional master regulator PU.1. To test this hypothesis we will explore the consequences of c-Maf loss- of-function and gain-of-function in normal and malignant hematopoietic stem (HSC) and progenitor cells, as well as the mechanism through which c-Maf contributes to the pathogenesis and maintenance of leukemia stem cells. Strategies employed will include both biochemical and functional experiments. Biochemical assays such as Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, promoter-reporter assays and targeted mutagenesis, will be used to address the relationship between PU.1 knockdown and the upregulation of c-Maf, and to confirm whether c-Maf is a direct transcriptional target of PU.1. Meanwhile, in vitro and in vivo functional assays will help examine the importance of c-Maf in normal stem cell and leukemic stem cell development. Understanding the role c-Maf plays in AML can provide the basis for novel therapeutic approaches that aim at directly targeting c-Maf in the leukemia stem cell population. Relapses, which are very frequent in AML patients, are believed to occur as a consequence of the survival of these leukemia initiating cells; hence, by specifically targeting this population of malignant cells, we believe that this approach can lead to lasting cures of the disease and overall improvement in patient outcome. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the mechanism and role of c-Maf deregulation in hematopoietic stem and progenitor cells and in leukemia. In order to characterize the function of c-Maf in normal hematopoietic stem and progenitor cells, as well as in the pathogenesis of Acute Myeloid Leukemia (AML), we will conduct biochemical and functional assays. Through these experiments and analysis we can confirm whether c-Maf is a direct transcriptional target of the myeloid master regulator and stem cell tumor suppressor PU.1 and possibly elucidate a novel therapeutic approach at targeting the leukemia stem cell population in the hopes of providing a lasting cure.

Matthew Klein - ABSTRACT: The goal of this project is to describe a novel role for the RNA-binding protein Sam68 in synaptic transmission and plasticity. Sam68 is a member of the KH-domain containing family of proteins, which have been linked to the pathogenesis of several neurological disorders via their effects on synaptic transmission. For example, mutations in the KH-family member FMRP result in Fragile X Mental Retardation Syndrome, while mutations in the KH-family member QUAKING have been implicated in Schizophrenia. Two recent studies suggest that Sam68 is involved in the pathogenesis of Fragile X Tremor/Ataxia Syndrome, and Spinal Muscular Atrophy. Elucidation of Sam68's role in maintaining efficient synaptic transmission will provide valuable insights into how dysfunction of the family of KH-domain containing proteins, and Sam68 in particular, leads to neurological disease. Sam68 may serve dual roles at the synapse, first as a modulator of synaptic transmission through NMDA receptor trafficking, and second as a regulator of local protein synthesis at dendritic spines. This proposal includes training in a number of advanced laboratory skills in the fields of electrophysiology and molecular biology in order to conduct a detailed investigation of Sam68's role at the synapse. To study the effects of loss of Sam68 we will use in vivo molecular manipulations (e.g. viral injections), in addition to a complimentary set of experiments using a line of Sam68 null mice. We will examine the effect of loss of Sam68 on NMDA receptor trafficking using whole cell patch clamp recording and quantitative immunohistochemistry. We will examine the ability of Sam68 to regulate local protein synthesis at dendritic spines using fluorescence in situ hybridization, and the photoconvertable fluorescent protein Dendra2. Sam68 may represent a novel regulator of synaptic plasticity by participating in activity-dependent changes involving both receptor trafficking, and local protein synthesis. PUBLIC HEALTH RELEVANCE: Neurological disorders may arise from inefficiencies in the mechanisms that ensure proper communication between neurons in the central nervous system. This project aims to understand the role of the RNA-binding protein Sam68 in ensuring proper neuronal transmission. Understanding this role will be helpful for guiding the development of therapies for neurological disorders resulting from dysfunction of Sam68 and related proteins.

Esther Berko - ABSTRACT: Prominent experimental reports have repeatedly shown the correlation between increasing rates of autism prevalence and advanced parental age, suggesting that mechanisms involved in pathology of the aging germline may contribute to the etiology of Autism Spectrum Disorders (ASDs). While the effects of paternal age, namely higher rates of mutation and copy number variation in offspring, have indeed been linked to ASDs, no study has determined the potential role of maternal age. We propose that maternal non-disjunction and resulting aneuploidy could cause ASDs and remain undetected. Since most aneuploidies are lethal embryonically, surviving offspring often undergo a “rescue” event that restores normal chromosome number. Depending on when an aneuploidy rescue occurs and which chromosome is lost, offspring exhibit either covert mosaic aneuploidy in sub-populations of cells or heterodisomic uniparental disomy (UPD). These defects have been implicated in other genetic disorders and may contribute to the molecular basis of ASDs, but are, surprisingly, unlikely to have been detected by current approaches that utilize cultured blood, a tissue that demonstrates low or absent levels of aneuploidy in mosaic individuals. In this study, we will perform comprehensive analysis of the genomes of children with ASD born to parents of advanced age, employing DNA isolated from buccal epithelium. By comparing children’s genotypes with their parents and applying computational analysis on SNP array signal intensities, we will identify the prevalence of covert mosaicism and heterodisomic UPD in children with ASDs.

Carlos Diaz-Balzac - ABSTRACT: Kallmann Syndrome is a hereditary condition characterized by anosmia (the inability to smell) and hypogonadotropic hypogonadism resulting in infertility. This disorder affects 1 in 10,000 males and 1 in 40,000 females, but may be underdiagnosed due to mild cases of hypogonadism or hyposmia. To date, five genes associated with KS have been identified, namely, KAL1, FGFR1, FGF8, PROKR2, and PROK2; though these only account for approximately 30% of all KS cases. The goal of this project is to identify and characterize novel genes that genetically interact with kal-1, a gene that codes for a cell adhesion protein of the extracellular matrix. We propose to accomplish this using a modifier screen of a kal-1 gain of function axon branching phenotype in C. elegans. A pilot screen of this phenotype has been used successfully by our group to identify several novel loci that genetically interact with KAL-1, both in worms and humans. The newly isolated mutations will be molecularly identified through single nucleotide polymorphism mapping and whole genome sequencing approaches. RNAi mediated knock down experiments and transgenic rescue experiments will be performed to further corroborate the identity of the mutation. The identified genes will then be molecularly and genetically characterized by three complementary approaches to gain a deeper understanding of how kal-1 acts in concert with these genes on the development of the nervous system and the role of the extracellular matrix in this process. First, I will perform a detailed neuroanatomical and phenotypic analysis of the modifier mutants with a focus on the nervous system. Second, their site of expression and the subcellular localization will be determined, which will give important clues to the function of the protein. Third, double mutant and epistasis analyses will be performed in order to place the new mutations within a known genetic context. In the end, as more genes that interact with KAL-1 are identified, we will have a better understanding of their function and how their disruption in humans results in Kallmann Syndrome.

Rachel Fremont - ABSTRACT: Dystonia is a movement disorder characterized by simultaneous and prolonged co-contraction of agonist and antagonist muscles, causing patients to adopt painful twisting postures. The dystonias are heterogeneous in their etiologies and few of these are well understood. Dystonia can be generated by lesions in the basal ganglia and many genetic and secondary dystonias are associated with altered basal ganglia activity suggesting the basal ganglia plays a central role in this disorder. Certain dystonias present after cerebellar insult and others are relieved by lesion of the cerebellar output nuclei suggesting that the cerebellum is involved in certain cases of dystonia. In rodents, perfusing a selective Na/K ATPase (sodium pump) blocker ouabain into the cerebellum results in dystonia. Studies in our lab suggest this mechanism of dystonia generation may be important in a particular genetic dystonia, DYT12 or Rapid-Onset Dystonia-Parkinsonism. We hypothesize that in this model, aberrant activity in the cerebellum is transferred to the basal ganglia and mediates the observed dystonia. A disynaptic connection between the cerebellar output nuclei and basal ganglia input nuclei has been identified in rodents and primates. The physiologic role of this pathway is currently unknown. We evaluate the role of this disynaptic pathway and specifically one of its intermediate nuclei, the centrolateral nucleus of the thalamus (CL), in dystonia induced by ouabain administration to the cerebellum. Our data suggest that lesioning of CL prior to ouabain perfusion attenuates the generation of dystonia. We will test if this attenuation is long term and if it is accompanied by other motor abnormalities. We will also explore CL as a potential site of intervention after animals already have dystonia. Finally we will determine how CL neuron firing changes in response to ouabain perfusion onto the cerebellum. This will give us insight into the role of the disynaptic pathway through CL in diseased animals and facilitate development of new interventions to test on dystonic ouabain perfused mice including pharmacologic treatments and deep brain stimulation. These experiments will help define one mechanism of dystonia induction and suggest new methods to alleviate dystonia in certain human cases.

Edward Manning - ABSTRACT: The goal of my proposal is to build theoretical models of cardiac troponin to elucidate mechanisms by which mutations in cardiac troponin T (cTnT) result in Familial Hypertrophic Cardiomyopathy (FHC).

My proposal outlines three separate aims. Aim 1 involves correlating results from MD simulations of a segment of cTnT, residues 70-170, with functional results of respective ternary complex cTnT in vitro motility assays. We believe that a correlation is possible between the dynamics of the N-tail of cTnT, where nearly three-quarter of all cTnT FHC mutations are located, and functional aspects of the complex. A proper correlation study between the two will result in a useful tool to investigate mutations in this region of cTnT.

My second aim is to build a complete atomistic model of cardiac troponin interacting with the overlapping region of tropomyosins. We recognize from physiological experiments that calcium-binding is affected by cTnT N-tail domain mutations; therefore, changes that result from mutations in the N-tail domain of cTnT would otherwise be undetermined without a complete model of cTn which includes cTnC (the calcium binding domain) and cTnI. We expect that our model will capture these changes and be validated by our collaborator's results. We hope that in the future this model will expand into a tool for anyone investigating mutations throughout the cardiac troponin complex.

Finally, I propose a third aim investigating the temporal nature of mutational mechanisms that will not be accessible in the above aims due to the computational expense of atomistic models. We propose to integrate the coarse-graining methodology with our cardiac troponin model. Our goal is to access physiologic timescales with respect to cardiac contraction, on the order of milliseconds. We expect the results from this aim to be more readily comparable to those from experiments thanks to their greater temporal perspective.

Jerri Chen - ABSTRACT: The IKs delayed rectifier potassium current is critical for cardiac action potential repolarization. The channel that carries the IKs current has pore-forming α subunits encoded by the KCNQ1 gene, and regulatory β subunits encoded by the KCNE1 gene. Interactions between these subunits regulate the current, allowing adaptation to changes in heart rate. Hundreds of mutations identified in the two genes have been associated with Long QT Syndrome (LQTS) and Familial Atrial Fibrillation (FAF), both of which increase the risk of potentially fatal arrhythmias in patients. The goal of this project is to determine the structure-function relationships between KCNQ1 and KCNE1, with particular attention to how the interactions between the C-termini of the subunits influence IKs kinetics and adaptation to heart rate changes (Aim #1), and how mutations alter these interactions resulting in disease phenotypes (Aim #2). Site-directed mutagenesis will target predicted areas of interaction, with functional studies carried out via whole-cell patch clamping to determine current densities, gating kinetics, and voltage dependence of gating. Specific sites of contact between the two C-termini will be determined by hydrogen/deuterium exchange mass spectrometry and thermodynamic double-mutant cycle analysis. The sites of contact will be biochemically characterized, with subtle changes in binding affinity quantified by surface plasmon resonance analysis. Mutagenesis will be used to introduce known LQTS mutations in the KCNQ1 and KCNE1 C-termni, to explore the structural basis of LQTS phenotypes.

Yamini Krishnan - ABSTRACT: Mutations in the hERG (human ether-a-go-go related gene) potassium channel are linked to the hereditary Long QT syndrome (LQTS, locus LQT2). Patients with the hereditary LQTS or drug-induced LQTS are susceptible to fatal tachyarrhythmias. HERG channels are regulated by several intracellular signaling pathways that together contribute to the overall modulation of the potassium current IKr in normal and disease states. Previous studies have established the acute regulation of HERG current and gating though the β-adrenergic system via direct phosphorylation of the HERG channel. Recent studies show that acute treatment of HERG-expressing Xenopus oocytes with phorbol esters, a broad PKC activator, leads to a shift in the voltage dependence of activation of HERG. Chronic effects of adrenergic stimulation on the HERG channel have not been studied. Our experiments show that 24-hour stimulation with increased intracellular cAMP levels (β-adrenergic pathway) or phorbol esters (α-adrenergic pathway) result in distinct increases in HERG protein abundance which are not transcriptionally mediated. The goal of the proposed research is to elucidate the mechanisms by which the β- and α-adrenergic pathways (via PKA and PKC) modulate changes in HERG protein abundance. We hypothesize that chronic β-and α-adrenergic receptor stimulation specifically enhances the rate of HERG synthesis through mechanisms involving PKA and the PKC isoforms which ultimately lead to translational up-regulation. We propose to determine the roles of PKA and PKC during translation of HERG using polysomal profiling with quantitative real-time PCR and in-vitro translation methods with radioisotope labeling. We also propose to dissect the upstream adrenergic signaling pathways leading to increases in HERG abundance by using pharmacological and biochemical methods in a heterologous expression system.
Long-term increases in the circulating levels of the hormones epinephrine and norepinephrine are consistently observed in patients with chronic heart disease and this may put them at an increased risk for abnormal heart rhythms. Our research is designed to examine the molecular pathways between the long-term hormonal stimulation and changes in the cardiac HERG ion-channel, which normally helps to maintain the heart rhythm. This work may identify potential targets for therapy in the prevention of rhythm disturbances accompanying chronic heart disease.