• SELF-ASSOCIATION IS A COMMON PROPERTY IN BOVINE, MOUSE AND XENOPUSARRESTIN

      Calvert, Peter; Qiu, Shuang (2014)
      The light-driven translocation of arrestin from rod inner segment to outer segment was indicated to involve free diffusion with binding affinityto light-activated phosphorhodopsin, however, it is still debatable how arrestin is excluded from the dark-adapted outer segment. Previousstudies demonstrated that bovine visual arrestin had the property of self-association. The self-association propertyof both wild-type and mutated purified visual arrestin of several species (bovine, mouse and Xenopus laevis) was studiedby performing analytical ultracentrifugation experimentswhich providedthe oligomer formation information and association constants.Theself-association parameters of purified bovine and mousevisual arrestinwere investigated and compared with other studies. Results showed that arrestin of both species could self-associate, forming dimersin a concentration-dependent manner, but tetramerswere not detected at the highest concentrations examined.Xenopus arrestin was shown to self-associateas well, existing in a monomer-dimer equilibrium with the dimer dissociation constantKD,dim=80.8μM, which suggestedthat self-association was also a feature of Xenopus visual arrestin. Interestingly, homologous mutations (F78A/Y84A/F193A)of Xenopusarrestin, which were supposed to beconstitutive monomers, failed to completely disrupt the oligomerizationof Xenopusarrestinwith the dimer dissociation constantKD,dim=200.7μM , indicating that these regions were not conserved in amphibian visual arrestin, includingXenopuslaevis. The percentage of the dimer was higher than that of monomer at physiological concentrations in all species of arrestins tested.
    • Separating Attention Deficit Hyperactivity Disorder and Learning Disabilities in Girls: A Familial Risk Analysis

      Doyle, Alysa E.; Faraone, Stephen V.; DuPre, Emily P.; Biederman, Joseph (American Psychiatric Association Publishing, 2001-10)
      Objective: Familial risk analysis was used to clarify the relationship in girls between attention deficit hyperactivity disorder (ADHD) and learning disabilities in either mathematics or reading. Method: The authors assessed the presence of ADHD and learning disabilities in 679 first-degree relatives of three groups of index children: girls with ADHD and a comorbid learning disability, girls with ADHD but no learning disabilities, and a comparison group of girls without ADHD. Results: The risk for ADHD was similarly higher in families of ADHD probands with and without learning disabilities; both groups had significantly higher rates of ADHD than did families of the comparison girls. In contrast, only among relatives of ADHD probands with a learning disability was there a higher risk for learning disabilities. A strong (although statistically nonsignificant) difference emerged that suggested at least some degree of cosegregation of ADHD and learning disabilities in family members. There was no evidence of nonrandom mating between spouses with ADHD and learning disabilities. Conclusions: These results extend previously reported findings regarding the relationship of ADHD and learning disabilities to female subjects and raise the possibility that, in girls, the relationship between ADHD and learning disabilities is due to shared familial risk factors.
    • A seq2seq model to forecast the COVID-19 cases, deaths and reproductive R numbers in US counties

      Zhang-James, Yanli; Hess, Jonathan; Salekin, Asif; Wang, Dongliang; Chen, Samuel; Winkelstein, Peter; Morley, Christopher P; Faraone, Stephen V. (Cold Spring Harbor Laboratory, 2021-04-20)
      The global pandemic of coronavirus disease 2019 (COVID-19) has killed almost two million people worldwide and over 400 thousand in the United States (US). As the pandemic evolves, informed policy-making and strategic resource allocation relies on accurate forecasts. To predict the spread of the virus within US counties, we curated an array of county-level demographic and COVID-19-relevant health risk factors. In combination with the county-level case and death numbers curated by John Hopkins university, we developed a forecasting model using deep learning (DL). We implemented an autoencoder-based Seq2Seq model with gated recurrent units (GRUs) in the deep recurrent layers. We trained the model to predict future incident cases, deaths and the reproductive number, R. For most counties, it makes accurate predictions of new incident cases, deaths and R values, up to 30 days in the future. Our framework can also be used to predict other targets that are useful indices for policymaking, for example hospitalization or the occupancy of intensive care units. Our DL framework is publicly available on GitHub and can be adapted for other indices of the COVID-19 spread. We hope that our forecasts and model can help local governments in the continued fight against COVID-19.
    • Shared polygenic risk for ADHD, executive dysfunction and other psychiatric disorders

      Chang, Suhua; Yang, Li; Wang, Yufeng; Faraone, Stephen V. (Springer Science and Business Media LLC, 2020-06-09)
      Many psychiatric disorders are associated with impaired executive functioning (EF). The associated EF component varies by psychiatric disorders, and this variation might be due to genetic liability. We explored the genetic association between five psychiatric disorders and EF in clinically-recruited attention deficit hyperactivity disorder (ADHD) children using polygenic risk score (PRS) methodology. Genome-wide association study (GWAS) summary data for ADHD, major depressive disorder (MDD), schizophrenia (SZ), bipolar disorder (BIP) and autism were used to calculate the PRSs. EF was evaluated by the Stroop test for inhibitory control, the trail-making test for cognitive flexibility, and the digital span test for working memory in a Chinese ADHD cohort (n = 1147). Exploratory factor analysis of the three measures identified one principal component for EF (EF-PC). Linear regression models were used to analyze the association between each PRS and the EF measures. The role of EF measures in mediating the effects of the PRSs on ADHD symptoms was also analyzed. The result showed the PRSs for MDD, ADHD and BIP were all significantly associated with the EF-PC. For each EF component, the association results were different for the PRSs of the five psychiatric disorders: the PRSs for ADHD and MDD were associated with inhibitory control (adjusted P = 0.0183 and 0.0313, respectively), the PRS for BIP was associated with working memory (adjusted P = 0.0416), and the PRS for SZ was associated with cognitive flexibility (adjusted P = 0.0335). All three EF measures were significantly correlated with ADHD symptoms. In mediation analyses, the ADHD and MDD PRSs, which were associated with inhibitory control, had significant indirect effects on ADHD symptoms through the mediation of inhibitory control. These findings indicate that the polygenic risks for several psychiatric disorders influence specific executive dysfunction in children with ADHD. The results helped to clarify the relationship between risk genes of each mental disorder and the intermediate cognitive domain, which may further help elucidate the risk genes and motivate efforts to develop EF measures as a diagnostic marker and future treatment target.
    • SHP-1-dependent macrophage responses mediate virus-induced myositis and demyelinating disease.

      Massa, Paul; Watson, Neva (2015)
      Regulation of inflammatory immune responses to pathogenic microbial infections is critical for protecting against extensive tissue damage and chronic inflammation. Correspondingly, genes associated with inflammatory immune responses have been identified as potential genetic risk factors for chronic inflammatory diseases including multiple sclerosis (MS) and idiopathic inflammatory myopathies (IIMs). This dissertation will focus on characterizing how the key immune regulator, Src-homology 2 containing protein tyrosine phosphatase-1 (SHP-1), controls virus-induced inflammatory diseases in the central nervous system (CNS) and skeletal muscle. We previously reported that SHP-1 inhibits proinflammatory macrophage-mediated CNS demyelinating disease during Theiler’s murine encephalomyelitis (TMEV) infection in mice. Presently, we describe that SHP-1 controls TMEV-induced dystrophic calcification of skeletal muscle. Muscle-infiltrating macrophages displayed a proinflammatory M1-like phenotype and promoted muscle calcification in WT mice, whereas an increased infiltration of macrophages with a reduced M1 signature corresponded with absence of muscle disease in SHP-1-/-mice. These studies reveal SHP-1 as a key regulatory gene mediating CNS and skeletal muscle disease in response to a virus trigger.Proinflammatory macrophages promoted tissue damage in either skeletal muscle of WT mice or CNS of SHP-1-deficient mice following TMEV infection. We thus attempted to determine if SHP-1 activity within macrophages was sufficient to control the outcome of TMEV infection using multiple genetic approaches. However, since these cells have a high turnover rate, and SHP-1 was strongly induced in macrophages by TMEV infection, these approaches were not sufficient to address whether SHP-1 activity specifically within macrophages mediates tissue-specific disease outcomes following TMEV infection. The studies described here suggest that SHP-1 affects macrophage maturation in peripheral (muscle) and immune-privileged (CNS) tissues in opposite ways. However, SHP-1 inhibited inflammatory monocyte CCR2 expression and subsequent infiltration into both of these major sites of infection, indicating that additional environmental cues mediated by SHP-1 are needed to drive tissue-specific maturation of pathogenic M1-like macrophages in either the CNS or muscle, to explain tissue-specific disease outcomes in SHP-1-deficient mice. Thus, this dissertation characterized unique mechanisms by which SHP-1 mediates inflammatory responses to virus infections, and has revealed SHP-1 and proinflammatory M1-like macrophages as essential mediators of myositis and demyelinating CNS disease.
    • Six-week, double-blind, placebo-controlled study of desipramine for adult attention deficit hyperactivity disorder

      Wilen, T E; Biederman, J; Prince, J; spencer, T J; Faraone, Stephen V.; Warburton, R; Schleifer, D; Harding, M; Linehan, C; Geller, D (American Psychiatric Association Publishing, 1996-09)
      O bjective: The factor structures of individual positive and negative symptoms as well as global ratings were examined in a diagnostically heterogeneous group of subjects. Method: Subjects were identified through a clinical and family study of patients with major psychoses at a VA medical center and evaluated with the Scale for the Assessment of N egative Symptoms and the Scale for the Assessment of Positive Symptoms. For the examination of global-level factor structures (N =630), both principal-component analysis and factor analysis with orthogonal rotation were used. Factor analysis was used for the examination of item-level factor structures as well (N =549). Results: The principal-component analysis of global ratings revealed three factors: negative symptoms, positive symptoms, and disorganization. The factor analysis of global ratings revealed a negative symptom factor and a positive symptom factor. The itemlevel factor analysis revealed two negative symptom factors (diminished expression and disordered relating), two positive symptom factors (bizarre delusions and auditory hallucinations), and a disorganization factor. Conclusions: The generation of additional meaningful factors at the item level suggests that important information about symptoms is lost when only global ratings are viewed. Future work should explore clinical and pathological correlates of the more differentiated item-level symptom dimensions
    • Sodium hydrogen exchanger 9 NHE9 ( SLC9A9 ) and its emerging roles in neuropsychiatric comorbidity

      Patak, Jameson; Faraone, Stephen V.; Zhang‐James, Yanli (Wiley, 2020-05-13)
      Variations in SLC9A9 gene expression and protein function are associated with multiple human diseases, which range from Attention-deficit/hyperactivity disorder (ADHD) to glioblastoma multiforme. In an effort to determine the full spectrum of human disease associations with SLC9A9, we performed a systematic review of the literature. We also review SLC9A9's biochemistry, protein structure, and function, as well as its interacting partners with the goal of identifying mechanisms of disease and druggable targets. We report gaps in the literature regarding the genes function along with consistent trends in disease associations that can be used to further research into treating the respective diseases. We report that SLC9A9 has strong associations with neuropsychiatric diseases and various cancers. Interestingly, we find strong overlap in SLC9A9 disease associations and propose a novel role for SLC9A9 in neuropsychiatric comorbidity. In conclusion, SLC9A9 is a multifunctional protein that, through both its endosome regulatory function and its protein–protein interaction network, has the ability to modulate signaling axes, such as the PI3K pathway, among others.
    • Specific mutations in the α and ß subunits of the Kluyveromyces lactis F1-ATPase enhance ATP hydrolysis in the absence of the central γ-rotor

      Xin Jie Chen; Thuy La (2013)
      In eukaryotic cells, the mitochondria are vital organelles which are required for cell viability. Mitochondrial stresses such as oxidative stress, loss of membrane potential or loss of mitochondrial DNA are considered extreme and are associated with many neurodegenerative diseases and aging. The mitochondrial FoF1-synthase, where the majority of cellular ATP is synthesized, is composed of one inner membrane bound Fo domain and a water soluble F1 domain in the mitochondrial matrix. F1 contains the hexameric α3β3core and the centrally located γ subunit. The γ subunit is believed to play a key role in inducing conformational changes while rotating within the α3β3 core during ATP hydrolysis/synthesis. Previous studies have shown that the α3β3 core alone from the Thermophilic bacterium PS3 has a detectable hydrolyzing activity. In recent years, evidence of the rotary catalysis of Thermophilic Bacillus sp. PS3 F1-ATPase without its rotor - subunit γ - was shown using high-speed atomic force microscopy[1]. Moreover, previous study undertaken in our lab had utilized a unique genetic screen that allowed the identification of two specific mutations in the α and β subunits in the aerobic yeast Kluyveromyces lactis that stimulate ATP hydrolysis by the mitochondrial F1-ATPase in the absence of γ. This allows cells to survive upon the loss of mitochondrial DNA. In current work, we confirmed that the αF446I and βG419D mutations on the DELSEED loop are sufficient to allow ρ0 cells to survive in the absence of γ. Biochemical experiments showed that the γ -less F1-ATPase can be assembled to actively hydrolyze iv ATP in vivo, but this activity becomes extremely labile in vitro. These studies give insights into the catalytic mechanism of the α3β3 subcomplex and help to better understand the evolutionary origin of the mitochondrial F1-ATPase.
    • Stability of executive function deficits into young adult years: a prospective longitudinal follow-up study of grown up males with ADHD

      Biederman, J.; Petty, C. R.; Fried, R.; Doyle, A. E.; Spencer, T.; Seidman, L. J.; Gross, L.; Poetzl, K.; Faraone, Stephen V. (Wiley, 2007-08)
      Objective: Although individuals with attention deficit‐hyperactivity disorder (ADHD) commonly exhibit deficits in executive functions that greatly increase the morbidity of the disorder, all available information on the subject is cross sectional. Method: Males (n = 85) 9–22 years with ADHD followed over 7 years into young adulthood were assessed on measures of sustained attention/vigilance, planning and organization, response inhibition, set shifting and categorization, selective attention and visual scanning, verbal and visual learning, and memory. A binary definition of executive function deficits (EFDs) was defined based on a subject manifesting at least two abnormal tests 1.5 standard deviations from controls. Results: The majority of subjects maintained EFDs over time (kappa: 0.41, P < 0.001; sensitivity: 55%, specificity: 85%, positive predictive value: 69%, and negative predictive value: 75%). Conclusion: Considering the morbidity of EFDs, these findings stress the importance of their early recognition for prevention and early intervention strategies. EFDs are stable over time.
    • A STRING OF LIGHTS: MATURIN EXPRESSION AND POTENTIAL ROLE DURING MOUSE RETINOGENESIS

      Ly, Christine (2020)
      During retinal development, a pool of progenitor cells divides to generate daughter cells that eventually differentiate into the seven retinal cell types, including horizontal cells (HCs) and retinal ganglion cells (RGCs). Much about how cells exit the cell cycle and maintain a differentiated state remain unknown. Dysregulation of this process can alter the cellular composition and function of the retina.Thus, by studying this developmental process, we can better understand the mechanisms by which progenitor cells become functional, differentiated cells. Our previous work determined that Maturin (Mturn)is highly conserved in its expression pattern and protein sequence across various vertebrate species. Furthermore, we concluded that it is required for differentiation of primary neurons in Xenopuslaevis. Preliminary work in mice revealed that in the absence of Mturn, extensive folds occur in the retina. I used this model to characterize the expression of Mturnin the mouse retina and ask if Mturn is required for normal mice retinogenesis. By immunostaining retinal sections with various cell type-specific antibodies, I found that Mturn is expressed in differentiating cells and not in proliferating cells. In addition to determining that its expression is maintained in mature HCs, I concluded that Mturn is not required for generating the proper number HCs. Our results from studies on Mturn in both frogs and mice have led us to hypothesize that Mturn may function to maintain HCs in differentiated state and prevent their reentry into the cell cycle. Although preliminary experiments testing this hypothesis were inconclusive, future work should continue to investigate the role of Mturn in retinogenesis.
    • Structural and functional characterization of the V-ATPase membrane sector

      Couoh-Carde, Sergio J. (2017)
      The vacuolar ATPase (V1VO-ATPase, V-ATPase) is a H+-pump involved in the acidification of organelles in eukaryotes. Under certain physiological conditions, the VATPase disassociates into an inactive soluble ATPase sector (V1) and a membrane sector (VO) that is impermeable to protons. Due to the lack of detailed structural and functional information, the auto-inhibition mechanism of VO is not well understood. Although the V-ATPase shares a similar structure and rotary catalysis mechanism with the F- and AATPases, V-ATPase’s increased structural complexity and unique mode of regulation suggest other functions beyond its canonical proton pumping. We purified Vo and Vo sub-complexes for structural and functional characterization. First, our ~18 Å cryo-EM model of Vo suggests that c-ring (c8c’c’’) is partially surrounded by the C-terminal membrane integral portion of subunit a (aCT). On the other hand, the soluble N-terminal portion of subunit a (aNT) interacts with subunit d that sits atop of the c-ring. Selective removal of subunit d (VoΔd) did not allow passive proton translocation. Second, the c-ring was isolated and its X-ray crystal structure was solved at ~4 Å resolution. Two c-rings interact to form a gap-junction like structure. The presence of c’’ disrupts the intrinsic and global symmetry of the c8c’ sub-complex, constituting a kinetic barrier during c-ring axial rotation. Third, we discovered that c-ring can act as a large-conductance ion-channel independently from its canonical function in proton pumping. Our biophysical, biochemical, and functional data suggest that exquisite kinetic barriers play a primary role in the auto-inhibition of Vo, and that Vo may have noncanonical functions in intercellular communication.
    • STRUCTURAL BASIS OF THE FUNCTIONAL ROLES OF HUMAN AROMATASE

      Ghosh, Debashis; Lo, Jessica (2015)
      Humanaromatase(AROM) catalyzes theconversion of androgens to estrogensand is a major breast cancer drug target. Structural investigation has provided insights intothe active siteandaromatization mechanism.Utilization of the structural data has permitted rational design of a series of novel steroidal inhibitors. Investigation ofthe roles of key amino acids is facilitated by a recombinant AROM identical in crystal structureto the placental AROM.We use mutagenesis, chromatography, ultracentrifugation, spectrophotometry, enzyme kinetics, and X-ray crystallography to probe the roles of critical residues and the molecular basis of oligomerization. Furthermore, weevaluate the potencies of novel inhibitors and determine the structural basis of inhibition andselectivity. A critical active site residue D309 withan elevated pKa remainsprotonated at neutral pHand facilitatessubstrate binding and catalysis.The “gatekeeper” R192, linked to D309 via a watermolecule, is postulated to have a role in proton relay and substrate selectivity. D309N and R192Q mutants are virtually inactive supporting thehypothesis that both play keyrolesin aromatization.AROM oligomerization is driven bytheD-E loop of one moleculeand heme-proximal region of another via hydrogen bonding, electrostatic interactions between E181 and K440, and shape complementarity.Del7, generated by deletionof 7 residues in the D-E loop, experiences 65% reductionin activitydue to the loss of oligomer formation. Mutants Del4, E181A, and E181K exhibit normal enzymatic activity,and maintain some oligomeric interactions. The heme-proximal interface is also the putative coupling site of the reductasethatsupplieselectronsfor aromatization. The siteis larger than the active site, and at least twice aslarge asother P450s.MutantsK440Qand Y361Fof this region are virtuallyinactive.Collectivelythe results suggestfunctional significanceof oligomerization. Several newly designedAIs are superiortoexemestane, the steroidal AI currently used as a drug, in inhibition and anti-proliferation assays. The C6β-(pent-2-yn-1-yloxy) side chains ofthe most potent compoundspenetrate the access channelunique to AROM and havethe sameconformation asin the enzyme-free state.Astructural-based approachcan improve drug efficacy by improving specificity and selectivity, and reducing sideeffects.
    • Structural Brain Imaging Studies Offer Clues about the Effects of the Shared Genetic Etiology among Neuropsychiatric Disorders

      Radonjić, Nevena V.; Hess, Jonathan L.; Rovira, Paula; Andreassen, Ole; Buitelaar, Jan K.; Ching, Christopher R. K.; Franke, Barbara; Hoogman, Martine; Jahanshad, Neda; McDonald, Carrie; et al. (Cold Spring Harbor Laboratory, 2019-10-17)
      Genomewide association studies have found significant genetic correlations among many neuropsychiatric disorders. In contrast, we know much less about the degree to which structural brain alterations are similar among disorders and, if so, the degree to which such similarities have a genetic etiology. From the Enhancing Neuroimaging Genetics through Meta- Analysis (ENIGMA) consortium, we acquired standardized mean differences (SMDs) in regional brain volume and cortical thickness between cases and controls. We had data on 41 brain regions for: attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), bipolar disorder (BD), epilepsy, major depressive disorder (MDD), obsessive compulsive disorder (OCD), and schizophrenia (SCZ). These data had been derived from 24,360 patients and 37,425 controls. The SMDs were significantly correlated between SCZ and BD, OCD, MDD, and ASD. MDD was positively correlated with BD and OCD. BD was positively correlated with OCD and negatively correlated with ADHD. These pairwise correlations among disorders were correlated with the corresponding pairwise correlations among disorders derived from genomewide association studies (r = 0.494). Our results show substantial similarities in sMRI phenotypes among neuropsychiatric disorders and suggest that these similarities are accounted for, in part, by corresponding similarities in common genetic variant architectures.
    • STRUCTURE AND FORMATION OF PERINEURONAL NETS: A UNIQUE FORM OF EXTRACELLULAR MATRIX IN THE CENTRAL NERVOUS SYSTEM

      Russell Matthews; Sinha, Ashis (2021)
      The complex organization and trajectory of development of the central nervous system (CNS) depends not only on cell-cell interactions but also on interactions of cells with the extracellular environment. The extracellular environment in the CNS contains an organized network of proteins, glycans and glycoproteins and comprises what is called the CNS extracellular matrix (ECM). Components of this ECM are dynamically regulated not just during development but are also involved in various neurobiological processes in the adult. They are implicated in normal nervous system physiology as well as in nervous system disease and pathology. A complete understanding of the CNS therefore, requires an understanding of the composition, structure and function of the neural ECM also. Our laboratory focuses on a specialized substructure of the neural ECM, called perineuronal nets (PNNs). PNNs are conspicuous structures within the neural ECM and are thought to be key regulators of neuronal plasticity. Traditionally their role has been demonstrated in regulating forms of plasticity seen during development, but recent work has implicated PNNs in other forms of learning and plasticity across various brain regions as well. Interest in these structures has been further peaked by studies linking their alterations to a variety of neurological and neuropsychiatric diseases such as Alzheimer's disease and schizophrenia. However, despite this growing interest in PNNs, the mechanisms by which they modulate neural functions are poorly understood. The limited mechanistic understanding of PNN function is derived primarily from the fact that there are no existing models, tools or techniques that specifically target them without also disrupting the surrounding neural ECM. Therefore, a rigorous investigation of PNN function to date has not been possible. Our inability to specifically target PNNs is driven by an incomplete of understanding of their molecular composition and structure. While PNNs comprise of many known ECM components which are broadly expressed in the CNS ECM, PNNs appear clearly distinct from their surroundings. They are highly ordered and stable and show a regular organization and geometry not present in the diffuse neural ECM. The exact molecular mechanisms by which various PNN components come together and aggregate on the cell surface to form these structures however, is not iii clearly known. The primary goal of our laboratory has therefore been to determine the molecular structure, composition and formation of PNNs. Through our work described in chapters 2 and 3 of this thesis, we were able to develop a new model of PNN structure. In our proposed model, PNN components are bound to the cell surface by two distinct types of interactions, one dependent on the classical HA scaffold of PNNs and the other mediated by a complex formed by the ECM glycoprotein tenascin-R (TNR) and the chondroitin sulfate proteoglycan receptor protein tyrosine phosphatase zeta (RPTPz). Our work also allowed us to provide evidence that PNN components are immobilized on the neuronal surface by a GPI-linked mechanism. We identify the GPI-linked protein contactin-1 (CNTN1) as a key receptor molecule for PNNs. To our knowledge, this is the first ever identified receptor for PNNs. We feel that our findings presented here give us important insight into PNN structure and represent the initial important steps in understanding the formation of this unique ECM subcompartment.
    • STUDIES ON PHOTORECEPTOR GENE EXPRESSION

      Knox, Barry; ZHUO, XINMING (2013)
      Rod photoreceptors are a group of specialized retinal neurons that convert light into a neuronal signal in low light condition. The phototransduction function of rodsrequires expression of a group of rod genes. The homeostasis of these genes is primarily regulated by a photoreceptor regulatory network, which contains two major retina-specific transcription factors, neural retina leucine zipper (Nrl) and cone-rod homeobox(Crx). Nrl and Crx synergistically activate the expression of several phototransduction genes, most notably rhodopsin. Base on the studies done in cell culture, the synergy is theconsequence of interaction of Nrl and Crx. However, the interaction of Nrl and Crx has not been studied in live rods. The goal of thesis is to develop methods that can be used for studying Nrl and Crx interaction in live rods.In order to study Nrl and Crx in live rods without altering cell fate and inducing cell degeneration, I developed a novel inducible system, G3U, which can regulate gene expression in live rods. In this chapter, we investigated the characters of G3U by using rhodopsin-mCherry as a reporter and monitoring the induction response in transgenic Xenopus. The results of live rod imaging suggest that the inducible system has negligible background expression before induction and significant fold increase of expression after induction. Moreover, the induction response of G3U is reproducible in rods. These findings suggest that G3U system is a good candidate for expressing Nrl and Crx temporally in rods. In the second part of my thesis, I developed a flow cytometry based FRET method, FC-FRET, to study Nrl and Crx interaction in live cells. This method allows non-invasively analysis of protein-protein interaction in large population of live cells in a very short time. Furthermore, researchers will be able to analyze the concentration effect of FRET conveniently. In this study, I investigated the orientation of Nrl-Nrl, Crx-Crx and Nrl-Crx interactions. Our studies revealed that the Nrl-Nrl homodimer has a head-to-head and tail-to-tai l conformation. Both the Crx-Crx and the Nrl-Crx dimers have a head-to-head conformation in interacting complex. I also performed structure-function studies on both Nrl and Crx and classified the role of their different domains in the interactions. In addition to the known Nrl basic leucine zipper domain (b-ZIP) and Crx homeobox domain (HD), we found that the Nrl extended homology domain (EHD) plays an important role in the Nrl-Crx interaction. Two methods presented in this thesis are my major achievement during my graduate studies. The G3U inducible can be used to generate transgenic animals carrying inducible Nrl and Crx in rods. These transgenic animals allow researchers to study the interaction of Nrl and Crx in live rods by FC-FRET assay.
    • STUDYING RETINA DEVELOPMENT USING SMALL MOLECULE BMP INHIBITORS AND MAKING STABLE MOUSE EMBRYONIC STEM CELL LINES

      Viczian, Andrea; Keshvani, Caezaan (2013)
      Age related macular degeneration results in loss ofconephotoreceptors. Studies have not been able to efficientlytransplant cone cells, possiblydue to their limited numbersand lack of information about their development in the mammalian retina.Our lab has discoveredthat BMP signal inhibition is important for eye developmentin the frogas well as generating retinal cone photoreceptorsin mouse embryonic stem cell cultures. The frog, Xenopus laevis,can grow eyeswithin a few days; it is also amenable to transplantation and genetic alteration. In this study, two small molecule BMP inhibitors, LDN193189 and Dorsomorphin, causedshortened tails(dorsalized) phenotypein Xenopusembryos as well asexpansionofthe neural plate in neural stage embryos. This suggests that these chemical inhibitors can be used in place of the standard BMP antagonist, Noggin, in cell culture experiments. Mouse embryonic stem cell cultures treated with Noggin have been used to generate retinal progenitors that generate cone photoreceptorsin our lab. In order to further study cones indetail,we needed to enrich the population of cone photoreceptors from retinal progenitors by marking these cells in-­‐vitro. We generated stable mouse embryonic stem cells expressing a promoter that drives expression in rod/cone progenitor cells upstream from the mCherry fluorescent protein. This construct also contained aubiquitous promoterdriving an antibiotic resistance genefor antibiotic selection. In future studies, these stable ES celllines could be used to differentiate into rod/cone progenitors selected specifically by FAC sorting. Small molecule BMP inhibitors could be used in place of Noggin and analyzed in the generation of conecells.
    • Substance Use among ADHD Adults: Implications of Late Onset and Subthreshold Diagnoses

      Faraone, Stephen V.; Wilens, Timothy E.; Petty, Carter; Antshel, Kevin; Spencer, Thomas; Biederman, Joseph (Wiley, 2007-01)
      Diagnosing ADHD in adults is difficult when the diagnostician cannot establish an onset prior to the DSM-IV criterion of age seven or if the number of symptoms does not achieve the DSM threshold for diagnosis. These diagnostic issues are an even larger concern for clinicians faced with adults with substance use disorders (SUD). The present study compared four groups of adults: full ADHD subjects who met all DSM-IV criteria for childhood onset ADHD, late onset ADHD subjects who met all criteria except the age at onset criterion, subthreshold ADHD subjects who did not meet full symptom criteria, and nonADHD subjects who did not meet any of the above criteria. Diagnoses were by the Structured Clinical Interview for DSM-IV, and the Drug Use Severity Index (DUSI) was used for self-report of substance use. Cigarette and marijuana use was significantly greater in all ADHD groups relative to non-ADHD controls. Although usage rates of other drugs failed to reach significance, the ADHD groups were more likely to have used each drug (except alcohol) compared with the non-ADHD group. The late onset and full ADHD groups were more likely to have endorsed ever having a problem due to use of cigarettes, alcohol, or marijuana and reported more trouble resisting use of drugs or alcohol. The full ADHD group was more likely than the other groups to have reported ‘‘getting high’’ as their reason for using their preferred drug. Adults with ADHD have elevated rates of substance use and related impairment. Data about late onset ADHD provides further support for the idea that the DSM-IV age at onset criterion is too stringent. In contrast, subthreshold ADHD seems to be a milder form of the disorder, or perhaps a heterogeneous group of true ADHD cases and false positives.
    • Substance Use Disorders in High-Risk Adolescent Offspring

      Milberger, Stephen V. Faraone, Jose, Sharon (Wiley, 1999-01)
      Objective: To examine the risk for substance use disorders (SUD) in offspring of SUD parents who were not selected due to referral to SUD treatment centers. Method: The original sample was ascertained through two groups of index children: 140 ADHD probands and 120 non-ADHD comparison probands. These groups had 174 and 129 biological siblings and 279 and 240 parents, respectively. Results: We found that: 1) parental SUD was associated with SUD and all SUD subtypes in the offspring; 2) parental alcohol use disorders were associated with alcohol use disorders in the offspring as well as co-occurring alcohol and drug use disorders but not drug use disorders alone in the offspring; and 3) drug use disorders in the parents were associated with drug use disorders but not alcohol use disorders in the offspring. Conclusions: These findings suggest that alcoholism and drug abuse may breed true from parents to their offspring, but further work with larger samples is needed to confirm this idea. Our findings also suggest a possible common diathesis that is expressed as comorbid alcohol and drug use in the offspring of alcoholic parents. If confirmed, these findings may be useful for the development of preventive and early intervention strategies for adolescents at high risk for SUD based on parental history of SUD.
    • T Cell Factor-1 (TCF-1) Regulates Mature Alloactivated T Cells to Separate GVHD From GVL

      Mobin Karimi; Harris, Rebecca (2021)
      Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment used for patients with cancer or other hematological malignancies. However, widespread use of this treatment is hindered by development of graft-versus-host disease (GVHD), a life-threatening complication of allo-HSCT. Mature donor T cells in the graft mediate GVHD, but also help kill residual malignant cells in the patient by the graft-versus-leukemia (GVL) effect. Depletion of mature T cells from the graft eliminates this beneficial anti-tumor response. Mature T cells are also needed for proper stem cell engraftment. Therefore, current work has focused on how to modulate T cell signaling and function to separate GVHD from GVL. T Cell Factor-1 (TCF-1) is a T cell developmental transcription factor that is also important in some contexts for T cell activation. The role of TCF-1 in alloactivated mature T cells is completely unknown. To examine the role of TCF-1 in this context, a mouse model of allo-HSCT leading to GVHD/GVL was used to study T cells from mice with a T cell-specific deletion of TCF-1. This work showed that loss of TCF-1 separates GVHD from GVL, with reduced disease severity and persistence yet maintained GVL effects. TCF-1 affects alloactivated T cell phenotypes and suppressive profiles, as well as the major T cell functions (proliferation, migration, and cytokine/cytotoxic mediator production). TCF-1 also controls alloactivated T cell survival, apoptosis, and gene expression programs. The regulation of these functions and programs by TCF-1 is distinct in CD4 versus CD8 T cells. TCF-1 also controls two unique T cell subsets - stem-like CXCR5+ CD8+ T cells, and CD25- noncanonical Tregs. Therefore, TCF-1, or these two unique T cell types, may be a therapeutic target to improve allo-HSCT outcomes by separating GVHD from GVL effects. Expansion of CD25- Tregs during TCF-1 deficiency may also be useful for treatment of other T cell-mediated disorders as well.
    • Targetingof PIM1 KinaseinMyeloproliferative NeoplasmsInduced by JAK2V617F

      Mohi, Golam; Stuver, Matthew (2017)
      Myeloproliferative neoplasms (MPNs) arestem cell-derivedblood disorders. The most common mutation found in MPN patients is the JAK2V617Fmutation. JAK2 is anon-receptor tyrosine kinase involved in STAT signaling. The JAK2V617F mutation is asingle amino acid substitution of a phenylalanine for valine, whichcauses JAK2 to be constitutively activated. This mutation can cause ahematopoietic transformation. Eventuallythis transformationcan lead to the development of one of thethree different Philadelphia-negative MPN diseases: Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF). The JAK2V617F mutationhas been identified in 95% PVpatients,and 50-60% ofETand PMF patients.A JAK1/2 inhibitor (ruxolitinib) has been approved for MF and PV patients and,though it provides initial benefits, it is not effective enough to causelong-termremission in patients. This creates a critical need to identify new therapeutic targets for MPN patients. We found that PIM1 levels were significantly increased inMPN patients, as well asour JAK2V617F mouse modelof MPN.We observedthatknockdown of PIM1 caused a significant decrease in proliferationof JAK2V617F expressing cells. We also found that PIM1 knockdownhad no effect on the proliferation of hematopoietic cells not expressing JAK2V617F, leading us to believe PIM1 is only required in JAK2V617F mediated proliferation. Pharmacological inhibition of PIM kinases,using TP-3654,(kindly provided by Tolero pharmaceuticals)also led to a significant decrease in proliferation of JAK2V617F-expressing cells, but had no effect on cellslacking the mutation. We also found thatthePIM inhibitor,TP-3654,workssynergistically with ruxolitinibto achieve an even greater decrease in proliferation. We found that using the combination of ruxolitiniband TP-3654,we could use both drugs at lower concentrations andachieve an even greater decrease in proliferation and an increase apoptosis. Furthermore,we found that inhibition of PIM kinasesusing TP-3654can resensitize ruxolitinib-resistant cells to ruxolitinibtreatment.These important findingsshow that PIM1 plays animportantrolein the proliferation of hematopoietic cells expressing the JAK2V617F mutation, but is dispensable for the maintenance of cells lacking the mutation. We also found that targeting PIM kinases with TP-3654,significantly decreasedthe proliferation, and increaseapoptosisactivationof JAK2V617Fexpressing cells. We also showedthat TP-3654 and ruxolitinibcan work synergistically. Lastly, we showed that inhibition of PIM kinases,using TP-3654,caused ruxolitinib-resistant cells tobecome resensitized toruxolitinib. These findings helpedus come to the conclusionthatPIM1 kinase, is an importanttherapeutictargetin JAK2V617F-induced MPNs.