• 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.
    • Dopamine D4 Gene 7-Repeat Allele and Attention Deficit Hyperactivity Disorder

      Faraone, Stephen V.; Biederman, Joseph; Weiffenbach, Barbara; Keith, Tim; Chu, Monica P.; Weaver, Alix; Spencer, Thomas J.; Wilens, Timothy E.; Frazier, Jean; Cleves, Mario; et al. (American Journal of Psychiatry, 1999-05)
      Objective: Family, twin, and adoption studies show attention deficit hyperactivity disorder (ADHD) to have a substantial genetic component, and some studies have reported an association between ADHD and the dopamine D4 (DRD4) gene. Method: The authors recruited 27 triads that comprised an ADHD adult, his or her spouse, and their ADHD child. These triads were assessed for ADHD, and their DNA was genotyped for DRD4 alleles. Results: A multiallelic transmission disequilibrium test suggested an association between ADHD and the DRD4 7-repeat allele. Among family members, the number of 7-repeat alleles predicted the diagnosis of ADHD. Conclusions: Prior reports of an association between ADHD and DRD4 generalize to families recruited through clinically referred ADHD adults. However, because there are some conflicting studies, further work is needed to clarify the role of DRD4 in the etiology of the disorder.
    • The dopamine receptor D4 7-repeat allele influences neurocognitive functioning, but this effect is moderated by age and ADHD status: An exploratory study

      Altink, Marieke E.; Rommelse, Nanda N.J.; Slaats-Willemse, Dorine I.E.; Väsquez, Alejandro Arias; Franke, Barbara; Buschgens, Cathelijne J.M.; Fliers, Ellen A.; Faraone, Stephen V.; Sergeant, Joseph A.; Oosterlaan, Jaap; et al. (Informa UK Limited, 2011-11-23)
      Objectives. Evidence suggests the involvement of the dopamine D4 receptor gene ( DRD4 ) in the pathogenesis of ADHD, but the exact mechanism is not well understood. Earlier reports on the effects of DRD4 polymorphisms on neurocognitive and neuroimaging measures are inconsistent. This study investigated the functional consequences of the 7-repeat allele of DRD4 on neurocognitive endophenotypes of ADHD in the Dutch subsample of the International Multicenter ADHD Genetics study. Methods. Participants were 350 children (5 – 11.5 years) and adolescents (11.6 – 19 years) with ADHD and their 195 non-affected siblings. An overall measure of neuropsychological functioning was derived by principal component analysis from five neurocognitive and five motor tasks. The effects of DRD4 and age were examined using Linear Mixed Model analyses. Results. The analyses were stratified for affected and non-affected participants after finding a significant three-way interaction between ADHD status, age and the 7-repeat allele. Apart from a main effect of age, a significant interaction effect of age and DRD4 was found in non-affected but not in affected participants, with non-affected adolescent carriers of the 7-repeat allele showing worse neuropsychological performance. In addition, carrying the 7-repeat allele of DRD4 was related to a significantly worse performance on verbal working memory in non-affected siblings, independent of age. Conclusions. These results might indicate that the effect of the DRD4 7-repeat allele on neuropsychological functioning is dependent on age and ADHD status.
    • Dr. Glatt and Colleagues Reply

      Glatt, Stephen J.; Faraone, Stephen V.; Tsuang, Ming T. (American Psychiatric Association Publishing, 2004-06)
    • DRAMS: A tool to detect and re-align mixed-up samples for integrative studies of multi-omics data

      Jiang, Yi; Giase, Gina; Grennan, Kay; Shieh, Annie W.; Xia, Yan; Han, Lide; Wang, Quan; Wei, Qiang; Chen, Rui; Liu, Sihan; et al. (Public Library of Science (PLoS), 2020-04-13)
    • Effectiveness and Tolerability of Tomoxetine in Adults With Attention Deficit Hyperactivity Disorder

      Spencer, Thomas; Biederman, Joseph; Wilens, Timothy; Prince, Jeffry; Hatch, Mary; Jones, Janice; Harding, Margaret; Faraone, Stephen V.; Seidman, Larry (American Psychiatric Association Publishing, 1998-05)
      Objective: The authors assessed the experimental noradrenergic compound tomoxetine as an alternative treatment for adult attention deficit hyperactivity disorder (ADHD). Method: They conducted a double-blind, placebo-controlled, crossover study of tomoxetine in 22 adults with well-characterized ADHD. Results: Treatment with tomoxetine at an average oral dose of 76 mg/day was well tolerated. Drug-specific improvement in ADHD symptoms was highly significant overall and sufficiently robust to be detectable in a parallel-groups comparison restricted to the first 3 weeks of the protocol. Eleven of 21 patients showed improvement after receiving tomoxetine, compared with only two of 21 patients who improved after receiving placebo. Significant tomoxetine-associated improvement was noted on neuropsychological measures of inhibitory capacity from Stroop tests. Conclusions: This preliminary study showed that tomoxetine was effective in treating adult ADHD and was well tolerated. These promising results provide support for further studies of tomoxetine over an extended period of treatment.
    • EFFECTS OF FOCAL SEGMENTAL GLOMERULOSCLEROSIS-ASSOCIATED MUTATIONS ON MYOSIN 1E LOCALIZATION AND ACTIVITY

      Krendel, Mira; Karchin, Jing Bi (2015)
      Our lab has discovered that an actin-dependent molecular motor called Myosin 1e (Myo1e) is required for maintaining normal morphology and function in vivo of podocytes, a specialized epithelial cell in the kidney. We have found that Myo1e-null mice develop proteinuria, and mutations in the MYO1E gene, including missense mutations A159P and T119I, and nonsense mutation Y695X, have been identified in focal segmental glomerulosclerosis (FSGS), a primary kidney disease that often leads to end stage renal disease (ESRD). Based on these findings, we have proposed that Myo1e and especially its motor domain, plays a key role in regulating actin cytoskeleton organization in kidney podocytes. To study Myo1e activity at the junctions, we have used cell culture systems. We confirmed that Myo1e is a component of the podocyte slit diaphragm using glomerular fractionation assay and immune-gold labeling electron microscopy. Disruption of Myo1e motor activity by point mutation (A159P) completely disrupted Myo1e cellular localization and led to defective actin assembly at nascent cell-cell contacts. Domain mapping experiments in MDCK cells have suggested that the Myo1e TH2 domain is necessary, but not sufficient for its localization, but addition of the TH1 domain restores its localization to junctions. We have also found that the Myo1e SH3 domain interacts with ZO-1, a slit diaphragm and tight junction protein, in invitro pulldown assays, which might contribute to ZO-1 exchange activity at the junctions. Another FSGS-associated Myo1e motor domain mutation (T119I) also caused mis-localization of Myo1e in the cultured mouse podocytes, suggesting loss-of-function of the motor domain mutants. We have also shown that ZO-1 is not recruited to the nascent cell-cell contacts at the same time with the Myo1e T119I mutants. Finally, by using fission yeast as a model system, we have demonstrated that human kidney disease-associated mutations in fission yeast caused defects in yeast growth and endocytosis processes. Interestingly, after analyzing the colocalization patterns between the FSGS-associated Myo1 mutants and Chaperone Rng3, we have proposed that these two kidney disease-associated mutants likely possess different disease-causing mechanisms.Above all, we have concluded that Myo1e motor domain plays an important role in its localization and activity in podocyte actin cytoskeleton, which might be the link to the disease mechanism of FSGS at the molecular level.
    • Effects of Rab4A Mutations on Mouse Behavior, mTORC1 Activity,and Surface 8Receptor/TransporterRecycling

      Perl, Andras; Winans, Thomas C (2020-10-03)
      Through studying endosomal regulation, I found that a single amino acid 47mutation (Q72L) in the Rab4A gene leads to neurological disorders in two separate 48mouse strains. In the C57Bl/6 (SLE(WT)) background,I found thatknock-in the 49Rab4A gene leads to hyperactivity, which resembles both autism spectrum 50disorders (ASD) and attention-deficit hyperactivity disorder (ADHD). On a lupus-51prone background (SLE(1.2.3)) I found thatthe same mutation led to hypoactivity, 52which indicates a more severe neuropsychiatric systemic lupus erythematosus 53(NPSLE) than SLE(1.2.3)mice with wild type Rab4A.54The same mice were studied in chapter two, where mTORC1 activity was 55confirmed to be elevated in CD4+ T cells when Rab4A was knocked-in (Rab4A(KI))56compared to Rab4A(WT) cells.In young mouse brains prior to disease onset, I found57increases of mTORC1 and oxidative stress in Rab4A(KI) brains relative to 58Rab4A(WT) brains. In the same brains, there was also a depletion of GLUT1 and 59IFNGR1.Many of these changes were absent in the adult mice, after disease had 60developed.61SLE(1.2.3) mice with the three Rab4A alleleswere treated withrapamycin or 62NAC,and brains were collected.In these brains, there wasevidence that the 63hypoactive Rab4A(KI) SLE(1.2.3) had lower mTORC1 activity than Rab4A(WT) and 64Rab4A(KO) mice.This finding indicatesdepression, which is a pattern seen in major 65depressive disorder(MDD). Depression is also a symptoms of NPSLE. Interestingly, 66rapamycin increased mTORC1 activity in theRab4A(KI)brains compared Rab4A(KI) 67mice treated with vehicle, indicating a positive effect from the drug.