• The Chemosensory-­Related Consequences of Fetal Ethanol or Fetal Nicotine Exposure: Their Contribution to Postnatal Nicotine Acceptance

      Youngentob, Steven; MANTELLA, NICOLE (2015)
      Human studies demonstrate a predictive association between gestational exposure to alcohol or nicotine and the probabilityoflater nicotine dependence.The flavor qualitiesof both drugsare known to influencetheir earlyacceptance and they share the perceptual attributesof an aversive odor, bitter taste and oral irritation.This dissertationexamined whether there are chemosensory-­‐related consequences offetal: (1) alcohol exposurethat contribute toenhanced nicotine acceptance; or (2)nicotine exposure that also enhances acceptance. The study rationale was drivenby overlappingliteraturesrelated to: (1) the relationship between gestational exposurewith chemosensory stimuli and their postnatal acceptance; (2) lessons learned from prenatal alcohol exposure and its postnatal consequences; and (3) perceptual commonalities between the flavor of alcohol and nicotine.Alcohol studies: rats were alcohol-­‐exposed during gestationvia the dams’ liquid diet. Control damsreceived ad libaccessto an iso-­‐caloric, iso-­‐nutritive diet. Nicotine studies: dams’ were implanted with a mini-­‐osmotic pump containing nicotine.Control animals received either vehicle only or no pump. Behaviorally, we found that fetal alcohol exposed adolescent rats showed anenhanced nicotine odor
    • Comorbidity of ADHD and adult bipolar disorder: A systematic review and meta-analysis

      Schiweck, Carmen; Arteaga-Henriquez, Gara; Aichholzer, Mareike; Edwin Thanarajah, Sharmili; Vargas-Cáceres, Sebastian; Matura, Silke; Grimm, Oliver; Haavik, Jan; Kittel-Schneider, Sarah; Ramos-Quiroga, Josep Antoni; et al. (Elsevier BV, 2021-05)
      Attention-deficit / hyperactivity disorder (ADHD) and Bipolar Disorder (BD) are common mental disorders with a high degree of comorbidity. However, no systematic review with meta-analysis has aimed to quantify the degree of comorbidity between both disorders. To this end we performed a systematic search of the literature in October 2020. In a meta-analysis of 71 studies with 646,766 participants from 18 countries, it was found that about one in thirteen adults with ADHD was also diagnosed with BD (7.95 %; 95 % CI: 5.31-11.06), and nearly one in six adults with BD had ADHD (17.11 %; 95 % CI: 13.05-21.59 %). Substantial heterogeneity of comorbidity rates was present, highlighting the importance of contextual factors: Heterogeneity could partially be explained by diagnostic system, sample size and geographical location. Age of BD onset occurred earlier in patients with comorbid ADHD (3.96 years; 95 % CI: 2.65-5.26, p < 0.001). Cultural and methodological differences deserve attention for evaluating diagnostic criteria and clinicians should be aware of the high comorbidity rates to prevent misdiagnosis and provide optimal care for both disorders.
    • The concept of target features in schizophrenia research

      Tsuang, M. T.; Faraone, Stephen V. (Wiley, 1999-05)
      Target features are clinical or neurobiological characteristics that arc expressions of the underlying predisposition to an illness. They comprise a wide range of phenomena, from thc classic signs and symptoms of psychopathology to sophisticated measures of brain structure and function. For schizophrenia, many target features have been identified. These include eye tracking dysfunction, attentional impairment, allusive thinking, neurological signs, thought disorder, characteristic auditory evoked potentials, neuropsychological impairment, structural brain abnormalities and functional brain abnormalities. In their most pathological forms, thcse features are present among many schizophrenic patients, yet it is their presence among their non-psychotic relatives that shows them to be target features. We discuss the theoretical background for target features, present examples and describe how the discovery of target features has implications I for schizophrenia research.
    • Connexin43 and immunity : macrophage phagocytosis, cardiac calcinosis and autoimmune myocarditis

      Steven Taffet; Aaron Glass (2013)
      Connexin43 (Cx43) is a gap junction protein best known for coupling the cytoplasms of cardiac myocytes and allowing the efficient conduction of action potentials throughout the heart. In addition to the heart, Cx43 is also highly expressed in many immune cells and it has been attributed numerous roles in immunity. One such reported role was in macrophage phagocytosis. The first chapter in this dissertation explored the phagocytic activity of cultured and primary murine macrophages from wild type (WT) and Cx43-deleted (Cx43-/-) macrophages. No difference in phagocytic uptake was observed between the two groups using a series of target particles, indicating that Cx43 is dispensable for phagocytosis in macrophages. Given the spectrum of immune functions in which Cx43 has been ascribed a role, we set out to characterize its effect on a model of autoimmune myocarditis (EAM). Using the area of cardiac inflammatory infiltrate as a correlate of disease severity, we observed the progression of the disease to be independent of Cx43 status utilizing WT and Cx43-heterozygous (Cx43+/-) animals as well as radiation chimeric mice reconstituted with cells from donor WT, Cx43+/- and Cx43-/- mice. Although the severity of EAM did not measurably change when induced in animals with differing levels of Cx43 expression, substantial changes to ventricular Cx43 were noted in diseased hearts. Large foci were observed that completely lacked Cx43 immunofluorescence signal. Areas surrounding these foci exhibited disrupted Cx43 patterns such as internalization and lateralization. Similar alterations to Cx43 were also observed in the BALB/cByJ strain of laboratory mice that develop a spontaneous myocarditic disease. To investigate the electrophysiological ramifications of EAM, especially in the context of Cx43+/- mice, ECGs were recorded from animals over the course of EAM. Significant changes to the QRS interval were noted, including prolongation that was only observed in Cx43+/- animals.
    • Consortium neuroscience of attention deficit/hyperactivity disorder and autism spectrum disorder: The ENIGMA adventure

      Hoogman, Martine; Rooij, Daan; Klein, Marieke; Boedhoe, Premika; Ilioska, Iva; Li, Ting; Patel, Yash; Postema, Merel C.; Zhang‐James, Yanli; Anagnostou, Evdokia; et al. (Wiley, 2020-05-18)
      Neuroimaging has been extensively used to study brain structure and function in individuals with attention deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) over the past decades. Two of the main shortcomings of the neuroimaging literature of these disorders are the small sample sizes employed and the heterogeneity of methods used. In 2013 and 2014, the ENIGMA-ADHD and ENIGMA-ASD working groups were respectively, founded with a common goal to address these limitations. Here, we provide a narrative review of the thus far completed and still ongoing projects of these working groups. Due to an implicitly hierarchical psychiatric diagnostic classification system, the fields of ADHD and ASD have developed largely in isolation, despite the considerable overlap in the occurrence of the disorders. The collaboration between the ENIGMA-ADHD and -ASD working groups seeks to bring the neuroimaging efforts of the two disorders closer together. The outcomes of case–control studies of subcortical and cortical structures showed that subcortical volumes are similarly affected in ASD and ADHD, albeit with small effect sizes. Cortical analyses identified unique differences in each disorder, but also considerable overlap between the two, specifically in cortical thickness. Ongoing work is examining alternative research questions, such as brain laterality, prediction of case–control status, and anatomical heterogeneity. In brief, great strides have been made toward fulfilling the aims of the ENIGMA collaborations, while new ideas and follow-up analyses continue that include more imaging modalities (diffusion MRI and resting-state functional MRI), collaborations with other large databases, and samples with dual diagnoses.
    • A Controlled Study of Behavioral Inhibition in Children of Parents With Panic Disorder and Depression

      Rosenbaum, Jerrold F.; Biederman, Joseph; Hirshfeld-Becker, Dina R.; Kagan, Jerome; Snidman, Nancy; Friedman, Deborah; Nineberg, Allan; Gallery, Daniel J.; Faraone, Stephen V. (American Psychiatric Association Publishing, 2000-12)
      Objective: “Behavioral inhibition to the unfamiliar” has been proposed as a precursor to anxiety disorders. Children with behavioral inhibition are cautious, quiet, introverted, and shy in unfamiliar situations. Several lines of evidence suggest that behavioral inhibition is an index of anxiety proneness. The authors sought to replicate prior findings and examine the specificity of the association between behavioral inhibition and anxiety. Method: Laboratory-based behavioral observations were used to assess behavioral inhibition in 129 young children of parents with panic disorder and major depression, 22 children of parents with panic disorder without major depression, 49 children of parents with major depression without panic disorder, and 84 children of parents without anxiety disorders or major depression (comparison group). A standard definition of behavioral inhibition based on previous research (“dichotomous behavioral inhibition”) was compared with two other definitions. Results: Dichotomous behavioral inhibition was most frequent among the children of parents with panic disorder plus major depression (29% versus 12% in comparison subjects). For all definitions, the univariate effects of parental major depression were significant (conferring a twofold risk for behavioral inhibition), and for most definitions the effects of parental panic disorder conferred a twofold risk as well. Conclusions: These results suggest that the comorbidity of panic disorder and major depression accounts for much of the observed familial link between parental panic disorder and childhood behavioral inhibition. Further work is needed to elucidate the role of parental major depression in conferring risk for behavioral inhibition in children.
    • Deciphering a hippocampus to hypothalamus feeding circuit via the septal nucleus.

      Yang, Yunlei; Sweeney, Patrick (2017)
      The neural circuits controlling feeding are concentrated in the hypothalamus and hindbrain. These circuits primarily control homeostatic feeding behavior, which can be broadly defined as increasing feeding in response to hunger or decreasing feeding in response to satiety. However, non-homeostatic factors, such as the emotional state of an animal, can also profoundly affect feeding behavior. Therefore, the current thesis project sought to determine how primary emotion centers in the brain influence the known homeostatic feeding circuitry in the hypothalamus. In particular, given that ventral hippocampus (vHPC) and septum are involved in emotional processes, influence feeding behavior, and are anatomically connected to hypothalamic feeding circuitry, this dissertation aimed to determine the cell-types in vHPC and septum that control feeding and to functionally connect these cell-types to the primary feeding circuitry located in the hypothalamus. To accomplish these central aims, chemogenetic and optogenetic approaches were utilized to selectively manipulate neural activity within distinct ventral hippocampal and septal cell types and neural circuits. These approaches were complemented by traditional anterograde and retrograde tracing techniques and chemo/optogenetic circuit mapping approaches to define the neural circuits responsible for vHPC and septal control of feeding behavior. We find that chemogenetic activation of ventral hippocampal glutamate neurons reduces feeding, while inhibition facilitates feeding. We further dissect a functional neural circuit pathway from ventral hippocampus to lateral septum that is sufficient to suppress feeding behavior. Within the septum, both chemo/optogenetic activation of septal GABAergic neurons reduces feeding, while inhibition of these neurons increases food intake. Utilizing optogenetic circuit manipulation approaches, we demonstrate that septal GABAergic neurons reduce feeding, at least in part, by projecting to hyperphagia-inducing GABAergic neurons located within the lateral hypothalamus. Taken together, our findings expand upon the known roles for ventral hippocampus and septum in energy homeostasis by providing the specific cell-types and neural circuits governing vHPC and septal control of feeding behavior. Given the role for ventral hippocampus and septum in emotional processes and energy homeostasis, we propose that the described vHPC and septal circuits represent promising neural circuits for investigating interactions between feeding, emotional state, and motivated behavior.
    • DECIPHERINGGLUTAMATERGIC NEUROTRANSMITTER SPECIFICATION IN THE ZEBRAFISH SPINAL CORD

      Lewis, Kate; Hilinski, William (2016)
      The correct specification of neurotransmitter phenotypes is crucial for properly functioning neuronal circuitry. Neurons specify their neurotransmitter phenotypes via transcription factors that they express as they differentiate. Often, transcription factors that specify neurotransmitter phenotypes are expressed in multiple populations with the same neurotransmitter phenotype. Since, the transcription factors that specify this characteristic are not yet known for all spinal cord glutamatergic populations, we identified additional transcription factors expressed at relatively higher levels in glutamatergic neurons compared to inhibitory neurons. We have functionally tested three of these (Lmx1bb, Skor1a and Skor1b) to determine if they are required for correct spinal, glutamatergic phenotypes. We demonstrate that Lmx1b likely maintains a subset of glutamatergicphenotypes in the spinal cord. In lmx1bb mutant embryos, the number of cells that initially express glutamatergic markers are unchanged but become reduced at 36 h and to a greater degree at 48 h. In contrast, we observe no changes in the total number of dI5 or V0v neurons, which express lmx1bb,nor do we detect elevated levels of apoptosis between 36 h and 48 h in lmx1bb mutants. Lastly, we show that at least some of the cells that lose their glutamatergic neurotransmitter phenotype are likely to be V0v cells.Additionally, we demonstrate that skor1a and skor1b are expressed predominantly by glutamatergic spinal interneurons, many of which are V0v neurons. When skor1a and skor1b are knocked-down, we observe a significant reduction in the number of glutamatergic neurons and no change in the number of inhibitory neurons suggesting that these genesmay be required to specify the glutamatergic neurotransmitter phenotype of a subset of spinal neurons. In parallel studies, it was shown that evx1 and evx2, genes expressed exclusively by V0v spinal neurons, are required to specify the V0v glutamatergic phenotype. Interestingly, we show that lmx1bb, skor1a and skor1b require evx1 and evx2 for their expression. In summary, these results suggest that skor1, skor1b and lmx1bb may function downstream of evx1 and evx2 to specify and/ormaintain the glutamatergic neurotransmitter phenotype ofat least a subset of V0v neurons.
    • Deficient Emotional Self-Regulation and Adult Attention Deficit Hyperactivity Disorder: A Family Risk Analysis

      Surman, Craig B.H.; Biederman, Joseph; Spencer, Thomas; Yorks, Dayna; Miller, Carolyn A.; Petty, Carter R.; Faraone, Stephen V. (American Psychiatric Association Publishing, 2011-06)
      Objective: A growing body of research suggests that deficient emotional self-regulation (DESR) is prevalent and morbid among patients with attention deficit hyperactivity disorder (ADHD). Family studies provide a method of clarifying the co-occurrence of clinical features, but no family studies have yet addressed ADHD and DESR. Method: Participants were 83 probands with and without ADHD and 128 siblings. All were assessed for axis I DSM-IV conditions with structured diagnostic interviews. The authors defined DESR in adult probands and siblings using items from the Barkley Current Behavior Scale. Analyses tested hypotheses about the familial relationship between ADHD and DESR. Results: Siblings of ADHD probands were at elevated risk of having ADHD, irrespective of the presence or absence of DESR in the proband. The risk for DESR was elevated in siblings of ADHD plus DESR probands but not in siblings of ADHD probands. ADHD and DESR cosegregated in siblings. The risk for other psychiatric disorders was similar in siblings of the ADHD proband groups. Conclusions: The pattern of inheritance of ADHD with DESR preliminarily suggests that DESR may be a familial subtype of ADHD. Our data suggest that DESR is not an expression of other axis I DSM-IV disorders or of nonfamilial environmental factors. The authors cannot exclude contribution of non-axis-I DSM-IV disorders to risk for DESR and cannot determine whether the cosegregation of ADHD in DESR within families is a result of genes or familial environmental risk factors. Further investigation of DESR and its correlates and treatment both in and outside the context of ADHD is warranted.
    • Defining the Determinants of the Bok-IP3R Interaction and the Bok Interactome

      Richard Wojcikiewicz; Szczesniak, Laura (2021)
      Bok is a Bcl-2 protein family member that is often grouped with the pro-apoptotic family members Bax and Bak due to high sequence homology and because exogenously overexpressed Bok induces apoptosis by causing mitochondrial outer membrane permeabilization. However, the cellular roles of Bok remain unclear, as Bok KO cell lines and mice have failed to demonstrate a significant phenotype under normal conditions. Our lab discovered that Bok interacts with inositol 1,4,5-trisphosphate receptors (IP3Rs), tetrameric Ca2+ channels found in the ER membrane of mammalian cells that play an integral role in cell signaling. While other Bcl-2 family members have been reported to weakly interact with IP3Rs, the Bok-IP3R interaction is much more efficient, with essentially all cellular Bok constitutively bound to IP3Rs. We have generated full-length IP3R1 mutants that resolve the Bok-binding region to a small, unstructured loop in the cytosolic region of IP3R1 between _ helices 72 and 73. Additional bioinformatic analysis has revealed that the Bok-IP3R interaction is likely dependent upon helical and dynamic determinants within this loop. Interestingly, Bok KO cell lines demonstrate mitochondrial fragmentation and only minor changes in mitochondrial bioenergetics. We have investigated the role of Bok in mitochondrial dynamics through an in vivo proximity labeling technique known as TurboID. A TurboID-Bok fusion protein expressed in Bok KO HeLas has identified several mitochondrial fission proteins through mass spectroscopy analysis, suggesting that Bok acts at mitochondria-ER contact sites to inhibit fission, and this function may be independent of the Bok-IP3R interaction. The results provided from binding studies and proximity labeling have furthered our knowledge of the Bok-IP3R interaction and of Bok itself to better define, or re-define, the role of Bok within the cell. Understanding how and why these interactions occur will help us further understand fundamental cellular processes in health and human disease.
    • Deletion at the SLC1A1 glutamate transporter gene co-segregates with schizophrenia and bipolar schizoaffective disorder in a 5-generation family

      Myles-Worsley, Marina; Tiobech, Josepha; Browning, Sharon R.; Korn, Jeremy; Goodman, Sarah; Gentile, Karen; Melhem, Nadine; Byerley, William; Faraone, Stephen V.; Middleton, Frank A. (Wiley, 2013-01-22)
      Growing evidence for genetic overlap between schizophrenia (SCZ) and bipolar disorder (BPD) suggests that causal variants of large effect on disease risk may cross traditional diagnostic boundaries. Extended multigenerational families with both SCZ and BPD cases can be a valuable resource for discovery of shared biological pathways because they can reveal the natural evolution of the underlying genetic disruptions and their phenotypic expression. We investigated a deletion at the SLC1A1 glutamate transporter gene originally identified as a copy number variant exclusively carried by members of a 5-generation Palauan family. Using an expanded sample of 21 family members, quantitative PCR confirmed the deletion in all seven individuals with psychosis, three “obligate-carrier” parents and one unaffected sibling, while four marry-in parents were non-carriers. Linkage analysis under an autosomal dominant model generated a LOD-score of 3.64, confirming co-segregation of the deletion with psychosis. For more precise localization, we determined the approximate deletion end points using alignment of next-generation sequencing data for one affected deletion-carrier and then designed PCR amplicons to span the entire deletion locus. These probes established that the deletion spans 84,298 bp, thus eliminating the entire promoter, the transcription start site, and the first 59 amino acids of the protein, including the first transmembrane Na2+/dicarboxylate symporter domain, one of the domains that perform the glutamate transport action. Discovery of this functionally relevant SLC1A1 mutation and its co-segregation with psychosis in an extended multigenerational pedigree provides further support for the important role played by glutamatergic transmission in the pathophysiology of psychotic disorders. © 2013 Wiley Periodicals, Inc.
    • Design and Characterization of Proteins Rationally Engineered to Domain Swap by Mutually Exclusive Folding

      Loh, Stewart; Karchin, Joshua Michael (2016)
      Domain swapping is a mechanism for proteins to form dimers and higher order oligomers through the exchange of a section of their 3D structures. The backbone peptides of domain swapped oligomers are intertwined, but their 3D structures remain identical to their monomeric state, except for where they cross-over, termed a hinged region. We have developed a technique to engineer domain swapping interfaces with mutually exclusive folding (MEF). MEF achieves this by inserting a ‘lever’ protein into the surface loops of a host ‘target’ protein to form a target-lever fusion. This target-leveris conformationally strained with the lever and the target in a thermodynamic tug-of-war. When the lever is folded, the long distance between its N- and C-termini stretches apart the target and splits it in half. Conversely, when the target is folded, the short length of the loop where the lever was inserted compresses the lever and unfolds it. Domain swapping provides an escape from this tug-of-war as it allows the split target to refold and bypass the conformational strain. Because the lever is external to the target, adjusting the stability of the lever, through well-established thermodynamic principles, allows the propensity for domain swapping to be modulated without affecting the binding interface. This enables the design of “triggerable” levers which can reversibly induce domain swapping in response to a signal. Further, we can use domain swapping to turn the function of a target domain on and off. Two target-lever constructs are created with functional mutants in the target domain, one N-terminal to the lever and the other one C-terminal to the lever. Individually, both of these mutants are inactive, however if they are mixed and allowed to domain swap, then up to half of the target domains can swap out the functional mutations into the native active form. This bimolecular system in combination with induced domain swapping enables the design of modular bioswitches and biosensors.
    • Development and Characterization of Transgenic Models for Studying Progressive Retinal Degeneration and Regeneration

      Zuber, Michael; Yueh Ku, Ray (2017)
      Vision is the sense that human beings rely on the most in the daily life. The irreversibility of retinal damage has been a great challenge to the modern medicine and remains an interest of research in hope of treating, even curing the loss of vision. The work presented in this dissertationutilizes transgenic Xenopus. laevisas an animal model to investigate the possibility of studying progressive retinal degeneration in a species that is known for robust regeneration of damaged retina. I investigated the expression of intermediate filament proteins during retinal gliosis, which has been suggested to be an inhibitory component that prevents effective treatment in degenerating retina. In order to understand the response of retinal bipolar cells in degenerating X. laevisretina, I characterized the expression pattern of metabotropoic glutamate receptor 6 (grm6) and the transgenic X. laevismodel that expresses eGFP under the control of mouse Grm6 promoter in the retina.As a follow up study of a previous study published by our lab,I optimized the condition of using F2 transgenic animals in preparation of a long term study of retinal degeneration in X. laevis. In conclusion, the work in my thesis includes development of tools to further the understanding of retinal degeneration and regeneration in transgenic X. laevis.
    • Differential Effect of Environmental Adversity by Gender: Rutter’s Index of Adversity in a Group of Boys and Girls With and Without ADHD

      Biederman, Joseph; Faraone, Stephen V.; Monuteaux, Michael C. (American Psychiatric Association Publishing, 2002-09)
      Objective: This study examined the effect of gender in mediating the association between environmental adversity and the risk of attention deficit hyperactivity disorder (ADHD) and associated impairments. Method: The authors studied 280 ADHD and 242 healthy comparison probands of both genders who were between the ages of 6 and 17 years. They tested the association between Rutter’s indicators of adversity (including family conflict, social class, family size, maternal psychopathology, and paternal criminality) and ADHD, comorbidity, and functioning. Results: Greater levels of environmental adversity were associated with a greater risk for ADHD and other comorbidity in both genders in a dose-dependent fashion. However, learning disability and global functioning were modified by gender, with more detrimental effects observed in boys than in girls. Low social class, maternal psychopathology, and family conflict were significantly associated with psychopathology and functional impairment in the probands, with control for gender, parental ADHD, proband ADHD status, and maternal smoking during pregnancy. Conclusions: Psychosocial adversity in general and low social class, maternal psychopathology, and family conflict in particular increased the risk for ADHD and associated morbidity independently of gender and other risk factors, but gender modified the risk for adverse cognitive and interpersonal outcomes; boys were more vulnerable to the disorder than girls. Because of the difficulties in separating the effects of genetics from environment, these results must be interpreted as provisional until confirmation from twin and adoption studies.
    • DIRECTING DENDRITOGENESIS: DEFINING THE ROLE OF REELIN AND CSPGS IN THE CONTROL OF CORTICAL DENDRITE FORMATION

      Olson, Eric; Zluhan, Eric (2020)
      Appropriate dendritic development is essential for normal neuronal function throughout life. Disruptions in neuronal dendrite structure alter brain circuitry and are associated with debilitating neurological disorders.The Reelin signaling pathway is critical for proper cortical dendrite orientation and outgrowth. In Reelin null cortices (reeler), dendrites are unstable, retracting from and avoiding their normal target region called the marginal zone (MZ). This observation raises the possible existenceof a dendritic destabilizing cue in the MZ that can be counteracted by Reelin-signaling.The MZis cell sparse but highly enriched in chondroitin sulfate proteoglycans (CSPGs). While CSPGs are known to inhibit axonal outgrowth, their impact on dendritic growth is unclear.Here, we demonstrate that the growth of the apical dendrite is also inhibited by CSPGs. Soluble CSPGs and CSPG-patterned stripes are inhibitory to dendrite growth, butthis inhibitory effect can be reversed by CSPG ablation via chondroitinase treatment and by activation of the Reelin signaling pathway. In reeler explants, chondroitinase treatment rescues dendrite growth into MZ. Prior studies have shown that the serine threonine kinase Akt is essential for Reelin-dependent dendritic growthand also functions in CSPG-dependent neurite retraction. We find that CSPGs induce Akt dephosphorylation which isreversed by Reelin addition. CSPG presence had no effect on the cytoplasmic adaptor Dab1, which is rapidly phosphorylated in response to Reelin. Dab1-deficient neurons were sensitive to CSPG stimulation, but Reelin-dependent phosphorylation was blunted. This suggests that the extracellular signals imparted on dendrites by Reelin and CSPGs at the MZ converge intracellularly downstream of Dab1 atthe level of Akt to regulate dendritogenesis in the MZ.Disruptions in Reelin signaling cause intellectual disability and have been linked to autism. Thus, these findings identify a context in which Reelin signaling operates and provide insight into the underlying mechanism of neurodevelopmental disorders.
    • DISCOVERY OF A ROLE OF FMRP IN R-LOOP REGULATION AND GENOME MAINTENANCE THROUGH BREAK-SEQ ANALYSIS OF THE FRAGILE X GENOME

      Chakraborty, Arijita (2020)
      Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMRP translation regulator 1 (FMR1) gene and deficiency of its product, FMRP. FMRP is known as a translation repressor whose nuclear function is poorly understood. We investigated the global impact on genome stability due to FMRP loss. We applied Break-seq to a human cell line-based model for FXS and mapped genome-wide spontaneous and replication stress-induced DNA double strand breaks (DSBs) for the first time. We report that the genomes of FXS patient-derived cells are inherently unstable and accumulate more than twice as many DSBs as those fromnormal cells. The DSBs in FXS cells are enriched in neuron projection and synapse organization pathways. We further demonstrate that replication stress-induced DSBs in FXS cells correlate with R-loop forming sequences. FMRP, and not an RNA-binding mutant FMRP-I304N, abates R-loop-induced DSBs during programmed replication-transcription conflict.Moreover, exogenously expressed FMRP in FXS patient-derived cells reduces the replication stress-induced DSB formation. We conclude that the FXS cells are more susceptible to DNA replication stress. Furthermore, we identified chromatin binding sites of FMRP for the first time in human lymphoblastoid cells.Through mapping FMRP-bound chromatin loci in normal cells and correlating with FX-specific chromosome breaks, we identified novel FXS-susceptible genes. We show that FX cells have reduced expression of the uridine diphosphoglucuronosyl transferase 1 family enzymes, suggesting defective xenobioticmetabolism. In addition, using transcriptome analysis, we show that DNA repair genes are downregulated in FX cells under replication stress. Finally, we report a direct binding interaction between FMRP and R-loop and that the C-terminal domain is important for this interaction. Therefore, we proposethat FMRP is a novel genome maintenance protein required for preventing R-loop formation during replication stress. Our study provides newinsights into the etiological basis for FXS.
    • Disorder Versus Disability: The Challenge of ADHD in the Context of a High IQ

      Antshel, Kevin M.; Hendricks, Kaitlin; Faraone, Stephen V.; Gordon, Michael (Guilford Publications, 2011-04)
    • DISSECTING THE ROLE OF YEAST RIM8 IN THEINTERNALIZATION OF THE PLASMA MEMBRANE PROTON PUMPPMA1

      Kane, Patricia; Shoniwa, Makandiwana (2013)
      The vacuolar A-TPase (V-ATPase) is a proton pump that is found ubiquitiously throughout the cells. It uses the hydrolysis of ATP to transport protons across membranes, thereby maintaining homeostatic pH. pH control in the cells of an organism is vital, a disturbance in cellular pH may be lethal. The maintenance of homeostatic pH within the cell appears to be a result of the interplay between V-ATPasesandproton exporters. In yeast and plants, the major proton exporter isthe plasma membrane proton exporter, Pma1. Pma1 is the transporter that is primarily involved in themaintenance of cytosolic pH. In cells in which the function of V-ATPase has been compromised (vma mutants) Pma1 is partially mislocalized.It is known thatmembrane transporterslacking the PY motifare endocytosed via the action of an Arrestin Related Trafficking (ART) protein, which translocates an E3 ligase into close proximity with the transporter, so as to allow for the ubiquitination of the transporter. Rim 8 is the ART protein (adaptor) that has been linked to the endocytosis of Pma1, along with E3 Ubiquitin ligase Rsp5. It is of interest to this project that Rim8 is well studied in its role as an adaptor in the alkaline ambient pH pathway. We thus propose that there may be crosstalk between the ambient pH pathway and the pathway that leads to the internalization of Pma1. Therefore, in this body of work we seek to find other players that may be involved in the Pma1 pathway, as well as to elucidate theareas of interaction between Rim8 and Pma1. Ourfinal goal isbringing a better understanding ofthe pathway that leads to the endocytosis of Pma1. To answer the question posed in this work we monitored the growth phenotype and the localization of Pma1 indouble mutants lacking both V-ATPase subunits and key players in the ambient pH pathway. In addition, we looked to see which cytosolically exposed terminal of Pma1 may be involved in theinteraction with Rim8. In yet another experiment, we mutatedRim8 so as to find which areas of the adaptor werevital for Pma1 internalization.Our results showed that other players tested in the Rim pathway(vma2∆rim20∆and vma2∆vps23∆)werenot required for Pma1 internalization. In addition we observed that mutations in Rim8 that compromise its function in the Rim pathway still allow Pma1 internalization, even though they show synthetic growth phenotypes with vma2∆ mutations. Two-hybrid assay could not detect thesites of interaction between Rim8 and Pma1 and newstrategies will be employed to determine these sites. Changes in electrophoretic mobility of Rim8 suggested that Rim8 undergoes posttranslational modifications, and showed differences in vma2∆mutants and WT mutants.
    • Domain swapping, fragment complementation, and self-assembly in engineered chimeric proteins.

      Loh, Stewart; Craft, Matthew (2014)
      Domain swapping is a form of protein oligomerization in which two or more identical proteins reciprocally exchange parts of their tertiary structure. The structure of the domain swapped proteins is identical to the structure formed by the monomer except for the hinge region linking the two domains. Domain swapping provides the cell with a way to control complex assembly, alter enzyme kinetics and specificity. Domain swapping can also be used to reconstitute enzyme function or as a form of molecular recognition. Only a small number of proteins are known domain swap, and the forces behind domain swapping are not well understood. Much more need to be understood abouthow and why proteins domain swap, before it would be possible to reliably engineer proteins to do so. In an effort to understand the thermodynamic forces that drive domain swapping, the goal of this project was to induce domain swapping and investigate the effects different hinge regions have on an otherwise identical domain swapped structure. To accomplish this we inserted ubiquitin (Ub) into five surface loops of ribose binding protein (RBP), a protein that does not naturally domain swap. The presenceof ubiquitin puts conformational strain on RBP and vice versa, where the folding of one causes the other to unfold. The entropic penalty for having unfolded domains can be relieved by domain swapping, allowing all protein domains to be folded. Using gelfiltration and circular dichroism we determined that our RBP-Ub (RU) fusion proteins domain swap. This domain swapping is dependent upon the conformational strain caused by a folded Ub, and can be reversed by the addition of a flexible glycine linker. Using our RU system we provide the first evidence that proteins with non-identical hinge regions can domain swap to form stable, functional oligomers. Finally we present a physical model that explains the ability of different RU’s to domain swap, which provides at least some of the criteria required of domain swapping proteins.