• 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.

      Sirotkin; Plante, Kyle (2015)
      Actin assembly into structures called endocytic actin patches is directly responsible for driving endocytic invagination and internalization. We tested the predictions made from the mathematical modeling of the Dendritic Nucleation Model of actin assembly at sites of clathrin-mediated endocytosis in fission yeast (Berro et al.; Sirotkin et al., 2010). The model predicts that increasing the concentration of cytoplasmic actin or deleting capping protein will cause an increase in the extent and the rate of actin assembly in actin patches. Conversely, the model predicts that increasing the concentration of capping protein or decreasing the actin concentration will cause a decrease in the extent and the rate of actin assembly in actin patches. To test these predictions, we used the actin cross-linking protein, fimbrin Fim1 tagged with a fluorescent protein to measure actin patch dynamics in strains that over- or under-express actin, over-express capping protein, or have deletions of the two capping protein genes. In contrast to model predictions, we found that manipulating capping protein concentrations did not have a significant effect on the extent of actin patch assembly and affected the rates of assembly to a lesser degree than expected from the model, suggesting that capping protein is not the only factor that limits actin patch assembly. Surprisingly, changes in the concentration of actin resulted in changes in the number of patches in a cell, suggesting that the concentration of actin is more important in controlling the initiation of new patches rather than in patch assembly. Through studying the biochemical pathway of actin assembly directly in living cells, we were able to gain insights into previously under-appreciated aspects of the mechanism of actin assembly at the sites of clathrin-mediated endocytosis.
    • Environmental risk factors for attention‐deficit hyperactivity disorder

      Banerjee, Tania Das; Middleton, Frank; Faraone, Stephen V. (Wiley, 2007-06-15)
      Attention-deficit hyperactivity disorder (ADHD) is the most common cognitive and behavioural disorder diagnosed among school children. It is characterized by deficient attention and problem solving, along with hyperactivity and difficulty withholding incorrect responses. This highly prevalent disorder is estimated to affect 5–10% of children and in many cases, persists into adulthood, leading to 4% prevalence among adults. Converging evidence from epidemiologic, neuropsychology, neuroimaging, genetic and treatment studies shows that ADHD is a valid medical disorder. The majority of studies performed to assess genetic risk factors in ADHD have supported a strong familial nature of this disorder. Family studies have identified a 2- to 8-fold increase in the risk for ADHD in parents and siblings of children with ADHD. Various twin and adoption studies have also highlighted the highly genetic nature of ADHD. In fact the mean heritability of ADHD was shown to be 0.77, which is comparable to other neuropsychiatric disorders such as schizophrenia or bipolar disorder. However, several biological and environmental factors have also been proposed as risk factors for ADHD, including food additives/diet, lead contamination, cigarette and alcohol exposure, maternal smoking during pregnancy, and low birth weight. Many recent studies have specifically examined the relationships between ADHD and these extraneous factors. This review describes some of these possible risk factors.
    • Ethanol-Induced Effects of the Microtranscriptome on Natural Gene Expression

      Middleton, Frank; Ignacio, Cherry Mae Gonzalez
      Reliable, minimally invasive biomarkers that predict the extent of alcoholism-induced CNS damage are currently lacking. This limits the selection of rational interventions and hampers the ability to gauge therapeutic effects. Developing biomarkers that indicate early CNS damage may prove useful in deterring the emergence of alcohol use disorders (AUDs). Extracellular microRNAs (miRNAs) can be informative molecular indicators of neuronal gene expression alterations. They repress large fractions of protein-coding genes and are highly-involved in intercellular signaling between both proximal and distal neurons. This work has focused on (1) examining whether extracellular miRNAs in the serum of individuals diagnosed with AUDs can be used as biomarkers of alcohol-induced brain damage, (2) determining in vivo the ethanol-inducedeffects imparted by miRNAs and their targets in the brain, (3) evaluating their role ininterventions that can reverse behavioral impairment and (4) testing the ability of extracellular miRNAs to transfer ethanol-induced pathologies to ethanol-naive cells. There are five major findings from this work. First, two independent quantification technologies demonstrated comparable differences in miRNA expression levels betweenAUDs and controls and revealed significant correlations between candidate miRNA biomarkers and medical, neuroimaging and drinking parameters. Second, in rats manymiRNAs significantly altered by ethanol in the hippocampus following maternal or postnatal exposure were also changed in the serum. Moreover, postnatal consumption activated cell-cycle pathways in the hippocampus while maternal exposure affected unfolded protein response pathways in adolescent offspring. Third, the lack of social motivation seen following fetal exposure was reversed as a result of social enrichment. Analysis of the integrated data in the amygdala and ventral striatum revealed several functional gene networks whose activation patterns following fetal ethanol exposure were reversed by social enrichment. Fourth, transfer of purified exosomes from ethanol-exposed to ethanol-naive cells conferred many gene expression changes consistent with ethanol exposure. Lastly, examination of all the data revealed consistent changes in miRNAs that independently converged on cell death, cell proliferation and cell cycle regulatory processes, regardless of the species, paradigm and source. The findings in this work illustrate the utility of miRNAs as peripheral biomarkers of AUDs and suggest novel epigenetic mechanisms affected by alcohol.

      Mitchell, David; Smith, Brandon (2013)
      Cilia and flagella are essential for the function of nearly all eukaryotes. This organelle is made up of nine outer doublet microtubules and two central singlet microtubules to form the canonical (9+2) ciliary structure. Cilia and flagella use this structure, as well as several protein complexes, such as the outer and inner dynein arms, the radial spokes, and the proteins that decorate the central pair to propagate the bending that produces motion. Flagellar motion is highly regulated, and each of these structures is necessary to regulate the dynein arms that generate the motile force. The central pair is one of the least understood of these structures. To date there are two major impediments hindering our understanding of the central pair: a lack of understanding as to how distinct central pair structures work in concert, and a general lack of available central pair mutant strains in the model organism Chlamydomonas reinhardtii. In order to further our understanding of how the central pair functions I have used multiple strategies. Firstly I have used previously characterized central pair mutants to study both structural interactions within the central pair and how the double mutant affects motility regulation. Secondly I provide evidence that a potential central pair mutant, H2, is indeed a central pair mutant and affects the C2b projection. Lastly I will attempt to characterize a new Chlamydomonas mutant, 10B5. Together these analyses will demonstrate that double mutants can have an additive effect on the structure of the central pair, and that double central pair mutants do not appear to suppress one another, but are at least ivepistatic to the most severe phenotype. I will also show evidence that 10B5 is not a central pair mutant, but with further study it may offer new insight into motilityregulation.
    • Evidence for Similar Structural Brain Anomalies in Youth and Adult Attention-Deficit/Hyperactivity Disorder: A Machine Learning Analysis

      Zhang-James, Yanli; Helminen, Emily C; Liu, Jinru; Franke, Barbara; Hoogman, Martine; Faraone, Stephen V. (Cold Spring Harbor Laboratory, 2019-02-11)
      Attention-deficit/hyperactivity disorder (ADHD) affects 5% of children world-wide. Of these, two-thirds continue to have impairing symptoms of ADHD into adulthood. Although a large literature implicates structural brain differences of the disorder, it is not clear if adults with ADHD have similar neuroanatomical differences as those seen in children with recent reports from the large ENIGMA-ADHD consortium finding structural differences for children but not for adults. This paper uses deep learning neural network classification models to determine if there are neuroanatomical changes in the brains of children with ADHD that are also observed for adult ADHD, and vice versa. We found that structural MRI data can significantly separate ADHD from control participants for both children and adults. Consistent with the prior reports from ENIGMA-ADHD, prediction performance and effect sizes were better for the child than the adult samples. The model trained on adult samples significantly predicted ADHD in the child sample, suggesting that our model learned anatomical features that are common to ADHD in childhood and adulthood. These results support the continuity of ADHD’s brain differences from childhood to adulthood. In addition, our work demonstrates a novel use of neural network classification models to test hypotheses about developmental continuity.
    • Evidence for the multigenic inheritance of schizophrenia

      Freedman, Robert; Leonard, Sherry; Olincy, Ann; Kaufmann, Charles A.; Malaspina, Dolores; Cloninger, C. Robert; Svrakic, Dragan; Faraone, Stephen V.; Tsuang, Ming T. (Wiley, 2002-08-21)
      Schizophrenia is assumed to have complex inheritance because of its high prevalence and sporadic familial transmission. Findings of linkage on different chromosomes in various studies corroborate this assumption. It is not known whether these ®endings represent heterogeneous inheritance, in which various ethnic groups inherit illness through different major gene effects, or multigenic inheritance, in which affected individuals inherit several common genetic abnormalities. This study therefore examined inheritance of schizophrenia at different genetic loci in a nationally collected European American and African American sample. Seventy-seven families were previously genotyped at 458 markers for the NIMH Schizophrenia Genetics Initiative. Initial genetic analysis tested a dominant model, with schizophrenia and schizoaffective disorder, depressed type, as the affected phenotype. The families showed one genome-wide significant linkage (Z ¼ 3.97) at chromosome 15q14, which maps within 1 cM of a previous linkage at the a7-nicotinic receptor gene. Chromosome 10p13 showed suggestive linkage (Z ¼ 2.40). Six others (6q21, 9q32, 13q32, 15q24, 17p12, 20q13) were positive, with few differences between the two ethnic groups. The probability of each family transmitting schizophrenia through two genes is greater than expected from the combination of the independent segregation of each gene. Two trait-locus linkage analysis supports a model in which genetic alleles associated with schizophrenia are relatively common in the general population and affected individuals inherit risk for illness through at least two different loci.
    • Examining the Comorbidity Between Attention Deficit Hyperactivity Disorder and Bipolar I Disorder: A Meta-Analysis of Family Genetic Studies

      Faraone, Stephen V.; Biederman, Joseph; Wozniak, Janet (American Psychiatric Association Publishing, 2012-12)
      Objective: The existence of comorbidity between attention deficit hyperactivity disorder (ADHD) and bipolar I disorder has been documented in clinical and epidemiological studies, in studies of children and adults, and in diagnosed ADHD and bipolar I patient samples. Yet questions remain about the validity of diagnosing bipolar I disorder in ADHD youth. The authors aim to clarify these issues by reviewing family genetic studies of ADHD and bipolar I disorder. Method: The authors applied randomeffects meta-analysis to family genetic studies of ADHD and bipolar I disorder. Twenty bipolar proband studies provided 37 estimates of the prevalence of ADHD in 4,301 relatives of bipolar probands and 1,937 relatives of comparison probands. Seven ADHD proband studies provided 12 estimates of the prevalence of bipolar I disorder in 1,877 relatives of ADHD probands and 1,601 relatives of comparison probands. Results: These studies found a significantly higher prevalence of ADHD among relatives of bipolar probands and a significantly higher prevalence of bipolar I disorder among relatives of ADHD probands. These results could not be accounted for by publication biases, unusual results from any one observation, sample characteristics, or study design features. The authors found no evidence of heterogeneity in the ADHD or bipolar family studies. Conclusions: The results suggest that ADHD plus bipolar comorbidity cannot be accounted for by misdiagnoses, but additional research is needed to rule out artifactual sources of comorbidity. More research is also needed to determine whether comorbidity of ADHD and bipolar I disorder constitutes a familial subtype distinct from its constituent disorders, which if confirmed would have implications for diagnostic nosology and genetic studies.
    • Expression and Function of Paxillin Genes in Zebrafish: A Role in Skeletal Muscle Development

      Turner, Chris; Amack, Jeffrey; Jacob, Andrew (2017)
      Paxillin is a key component of the Integrin adhesion complex, which regulates cellular signaling events in response to extracellular matrix interactions. Although the roles for Paxillin in cell migration have been extensively studied, less is understood about its role in vertebrate development. Depletion of Paxillin from mouse embryos results in early lethality due to impaired cardiovascular development and function, necessitating the development of alternative vertebrate genetic models for examining the role of Paxillin during embryogenesis. Zebrafish have emerged as an experimental vertebrate model amenable to genetic manipulation. The work compiled herein first characterizes the expression profiles for Paxillin genes in zebrafish, and then describes the embryonic phenotypes observed upon mutation of these genes. The identification of two Paxillin genes in zebrafish, pxnaand pxnb, provided new insight into the evolution of this gene family in the Teleost lineage. Both overlapping and unique expression profiles for these genes during zebrafish embryogenesis were uncovered. While both genes are expressed in developing skeletal muscle, pxnawas restricted to the notochord during earlier stages of embryogenesis and pxnbwas expressed in the developing heart. Targeted mutation of either gene alone did not impair embryonic development, suggesting partial functional redundancy between each gene during embryogenesis. Accordingly, combined mutations in pxnaand pxnbrevealed defects during the development ofseveral embryonic tissues. In particular, skeletal muscle morphogenesis iiiwas perturbed in these double mutant embryos. Further characterization revealed that Paxillin genes in zebrafish serve to regulate embryonic myotome shape and proper extracellular matrix composition during muscle development. The amount of Laminin was reduced, while the abundance of Fibronectin persisted, during myotome morphogenesis in Paxillin double mutant embryos. In addition, a role for cytoskeletal contractility in regulatingsubcellular localization of Paxillin in developing skeletal muscle was established. Defects in the development of the cardiovascular system were also apparent in Paxillin double mutant embryos, and future work will focus on characterizing these in further detail. Altogether, this work provides a new vertebrate model to use for understanding the role of Paxillin during embryonic development, and uncovers an unrecognized role for Paxillin in establishing the extracellular matrix of skeletal muscle.
    • Familial Risk Analyses of Attention Deficit Hyperactivity Disorder and Substance Use Disorders

      Biederman, Joseph; Petty, Carter R.; Wilens, Timothy E.; Fraire, Maria G.; Purcell, Caitlin A.; Mick, Eric; Monuteaux, Michael C.; Faraone, Stephen V. (American Psychiatric Association Publishing, 2008-01)
      Objective: A robust and bidirectional comorbidity between attention deficit hyperactivity disorder (ADHD) and psychoactive substance use disorder (alcohol or drug abuse or dependence) has been consistently reported in the extant literature. Method: First-degree relatives from a large group of pediatrically and psychiatrically referred boys with (112 probands, 385 relatives) and without (105 probands, 358 relatives) ADHD were comprehensively assessed by blind raters with structured diagnostic interviews. Familial risk analysis examined the risks in first-degree relatives for ADHD, psychoactive substance use disorder, alcohol dependence, and drug dependence after stratifying probands by the presence and absence of these disorders. Results: ADHD in the proband was consistently associated with a significant risk for ADHD in relatives. Drug dependence in probands increased the risk for drug dependence in relatives irrespective of ADHD status, whereas alcohol dependence in relatives was predicted only by ADHD probands with comorbid alcohol dependence. In addition, ADHD in the proband predicted drug dependence in relatives, and drug dependence in comparison probands increased the risk for ADHD in relatives. Both alcohol dependence and drug dependence bred true in families without evidence for a common risk between these disorders. Conclusions: Patterns of familial risk analysis suggest that the association between ADHD and drug dependence is most consistent with the hypothesis of variable expressivity of a common risk between these disorders, whereas the association between ADHD and alcohol dependence is most consistent with the hypothesis of independent transmission of these disorders. Findings also suggest specificity for the transmission of alcohol and drug dependence.
    • Family based association analysis of statistically derived quantitative traits for drug use in ADHD and the dopamine transporter gene

      Lasky-Su, Jessica; Biederman, Joseph; Doyle, Alysa E.; Wilens, Timothy; Monuteaux, Michael; Smoller, Jordan W.; Faraone, Stephen V. (Elsevier BV, 2006-06)
      Objective To determine whether SNPs within the dopamine transporter gene (DAT) are associated with quantitative phenotypes generated from drug frequency variables in an ADHD sample. Method 35 SNPs were genotyped in and around DAT. We developed a quantitative phenotype at each SNP by weighting the drug frequency variables. The weights were selected to maximize the heritability at each SNP. Once a quantitative phenotype was generated at each SNP, a screening procedure was used to select and test the SNPs with the greatest power to detect an association in DAT. Results No SNPs in DAT were associated with the quantitative phenotypes generated from the drug frequency variables after the multiple comparisons adjustment; however, some SNPs achieved nominal significance. A sliding window of analysis of 3 SNPs also resulted in only nominal associations. Conclusions SNPs in DAT do not appear to be associated with the phenotypes generated from drug frequency variables among individuals with ADHD.
    • Family based association study of pediatric bipolar disorder and the dopamine transporter gene (SLC6A3)

      Mick, Eric; Kim, Jang Woo; Biederman, Joseph; Wozniak, Janet; Wilens, Timothy; Spencer, Thomas; Smoller, Jordan W.; Faraone, Stephen V. (Wiley, 2008-10-05)
      Thedopaminetransportergene(SLC6A3) isacompelling candidate for pediatric bipolar disorder because (a) it has been associated with ADHD, (b) bipolar comorbidity with ADHD has been hypothesized to be an etiologically distinct familial subtype (c) blockade of the dopamine transporter with psychostimulants can induce mania in susceptible individualsand(d) previous studies have implicated the gene in bipolar disorder in adults. We conducted a family-based association study of SLC6A3 in 170 affected offspring trios defined by a child (12.9 5.3 years of age)with DSM-IV Bipolar-I disorder. Twenty-eight tag SNPs were chosen from the CEU (European) population of the International HapMap project (www.hapmap.org). Results indicated nominally positive association for 4 SNPs (rs40184, rs11133767, rs3776512, and rs464049), but only rs40184 survived correction formultiple statistical comparisons (P¼0.038). This is the first examination of the association with SLC6A3 and bipolar disorder in children and, like previous findings in adults with bipolar disorder, we found evidence of association with SNPs in the 30 region of the gene. These data provide suggestive evidence supporting a role for SLC6A3 in the etiology of pediatric bipolar disorder.
    • Family-based and case-control association studies of catechol-O-methyltransferase in attention deficit hyperactivity disorder suggest genetic sexual dimorphism

      Qian, Qiujin; Wang, Yufeng; Zhou, Rulun; Li, Jun; Wang, Bing; Glatt, Stephen; Faraone, Stephen V. (Wiley, 2003-03-04)
      Attention deficit hyperactivity disorder (ADHD) is the most common childhood-onset behavioral disorder. Boys are more often affected than girls. Family, twin, and adoption studies have supported a strong genetic basis. Some studies show that a catechol-O-methyltransferase (COMT) polymorphism affecting enzyme activity was associated with personality characteristics and diseases, such as novelty-seeking personality, substance abuse, and heroin addiction, whose features are similar to ADHD or are associated with ADHD. These findings suggest that the COMT gene may be a candidate gene for ADHD. TDT, HHRR, and case-control association studies were conducted within a sample of 202 nuclear ADHD families, 340 ADHD cases, and 226 controls in the Han Chinese population. Diagnoses and ADHD subtypes were ascertained according to DSM-IV criteria using American Clinical Diagnostic Interviewing Scales. The HHRR analysis suggested that the low enzyme-activity COMT Met allele was preferentially transmitted to ADHD boys (160 trios, χ2 = 3.858, P = 0.05, df = 1) but not girls. This association is particularly pronounced among male ADHD probands without any comorbidity (50 trios, HHRR: χ2 = 5.128, P = 0.024, df = 1; TDT: χ2 = 4.558, P = 0.033, df = 1), especially the ADHD-I subtype (32 trios, HHRR: χ2 = 5.792, P = 0.016, df = 1; TDT: χ2 = 5.333, P = 0.021, df = 1). The case-control study revealed that the Val allele was more frequent in females meeting ICD-10 or DSM-IV criteria for ADHD than in female controls (86 and 79.5%, respectively, χ2 = 4.059, P = 0.044, df = 1). Although these results suggest the COMT gene exerts some influence on the risk for ADHD in the Han Chinese population, given the potential for Type I error, these findings require replication before drawing definitive conclusions. © 2003 Wiley-Liss, Inc.
    • Family-based association study of markers on chromosome 22 in schizophrenia using African-American, European-American, and Chinese families

      Takahashi, Sakae; Cui, Yu-hu; Kojima, Takuya; Han, Yong-hua; Zhou, Ru-lum; Kamioka, Masashi; Yu, Shun-ying; Matsuura, Masato; Matsushima, Eisuyke; Wilcox, Marsha; et al. (Wiley, 2003-06-13)
      Several studies suggest that loci at chromosome 22q11.2-q13 might be linked to susceptibility to schizophrenia. Here we performed family-based association studies on chromosome 22q using 12 DNA microsatellite markers in African-American, European-American, and Chinese pedigrees. The marker D22S683 showed significant linkage and association with schizophrenia in not only the European-American sample but also in a combined sample (European-American and Chinese samples). Notably, D22S683 is located nearby and between D22S278 and D22S283, which have shown linkage and association to schizophrenia in prior reports. However, we found no significant association for the African-American sample. In conclusion, our data provide further support for the idea that the region around D22S683 contains a susceptibility gene for schizophrenia. © 2003 Wiley-Liss, Inc.
    • FH2-dependent Localization of FHOD Formins in the Sarcomere

      Blystone, Scott; Hamilton, Elisabeth (2016)
      Formins are a class of actin nucleating factors containing a highly-conserved FH2 domain, which binds actin. There are 15 mammalian formin proteins in seven sub-families that have been found to nucleate, cap, sever, bundle, and polymerize linear actin filaments. An expression analysis of all 15 human formins across 22 different cell and tissue types showed high levels of expression for one formin sub-family in striated muscle: FHOD. While FHOD1 is highly expressed across many cell and tissue types, FHOD3 is only found at comparatively high levels in striated muscle cells. The mature structure of skeletal muscle is a very recognizable periodic repetition of filaments that allows muscles to contract. The sarcomere is the smallest contractile unit of skeletal muscle, with the two main filaments, the thick filament containing myosin, and the thin filament containing actin moving back and forth via the interaction of myosin heads with the thin filament, which allows muscles to contract. While the molecular functioning and the mature structure of skeletal muscle is well understood, the exact mechanism by which the sarcomere is assembled, and specifically, how the actin-core of the thin filament is formed and incorporated into the thin filament remains unknown. The high levels of expression of FHOD formins in striated muscle, combined with their ability to interact with filamentous actin warranted a closer look at FHOD1 and FHOD3 in the sarcomere. In this study we found that FHOD1 and FHOD3 have distinct sarcomeric localizations in C2C12 cells. FHOD1 localizes to the barbed end of the actin filament at the Z-disk and FHOD3 localizes to the pointed end of the actin filament near the center of the sarcomere. Full-length cDNA constructs for FHOD1 and FHOD3 were introduced into skeletal muscle cells, and we were able to recreate the endogenous localization of FHOD3 with the exogenous cDNA. The FHOD1 cDNA localized not to the barbed end of the actin filament, as endogenous FHOD1 does, but instead to the pointed end of the actin filament, where endogenous and exogenous FHOD3 were found. We hypothesized that FHOD binding to actin is dependent upon two highly conserved actin-binding sites in the FH2 domain. Mutations of the actin-binding residues in the FH2 domain impaired the actin-binding ability of both exogenous FHOD1 and FHOD3 and showed that the localization of FHOD1 and FHOD3 in the sarcomere is actin-binding dependent.
    • The Formin FMNL1 Contributes to the Macrophage Inflammatory Response by Regulating Podosome-dependent Adhesion and Migration.

      Blystone, Scott; Miller, Matthew (2015)
      Macrophages are indispensible white blood cells (leukocytes) that contribute to both the innate and adaptive immune response. They are crucial for resolving inflammatory events by clearing pathogens and cellular debris, in addition to promoting wound repair. Macrophages are derived from peripherally circulating monocytes, which after stimulation undergo diapedesis from the vasculature into the underlying complex extracellular matrix, where they can become fully differentiated macrophages and migrate to inflammatory loci. Tissues also contain residential populations of macrophages that aid in immediate immune responses and maintain tissue homeostasis. Conversely, unwarranted macrophage activation largely contributes to the onset and progression of inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, in addition to aiding cancer metastasis and facilitating organ transplant rejection. In order for macrophages to effectively resolve inflammatory events or contribute to disease pathology, they must be able to undergo directional migration, which is mediated by integrin-dependent adhesion complexes termed podosomes. Macrophage podosomes are the most prominent structure of the macrophage actin cytoskeleton, containing a pillar-like core of dense filamentous actin that is tethered to the cortical actincytoskeleton via radial actin filaments. Podosomes also contain a variety of proteins that are circumferentially arranged orassociated with the core, and thatare involved in signaling, linking, and scaffolding,as well as modulating the actin cytoskeleton.Historically, our lab has been interested in leukocyte integrin biology and understanding how these receptors mediate adhesion and migration through complex extracellular matrices. Previous studies in our lab demonstrated the novel podosomal association of an actin modulating protein with the ability to processively elongate unbranched linear actin filaments. Subsequent studies determined this protein to be the formin FMNL1, which is predominantly expressed in hematopoietic cells. Consequently, we further revealed that FMNL1 localizes to the apex of the dense actin core, and is required for podosome stability and macrophage adhesion.The work described in this dissertation has greatly expanded on these findings, demonstrating for the first time that primary macrophage migration is dependent on the formin FMNL1. Utilizing in vitro and in vivotechniques with aid of a novel conditional murine FMNL1 KO, we have observed that macrophage podosome formation, migration, and tissue distribution are dependent on FMNL1. Additionally, we have indicated that FMNL1 is required for embryonic development. Remarkably, our findings also suggest that FMNL1-dependent macrophage migration and podosome localization rely on the specific isoform FMNL1γ. Foremost, we have demonstrated that barbed end binding by the FMNL1γ FH2 domain is dispensable for its cellularfunction in macrophages, which has not been previously shown for any other cellular formin function. Thus, these findings, in addition to current formin knowledge, have allowed us develop a working model for FMNL1 function at macrophage podosomes. This work has distinguished FMNL1 as a unique therapeutic target to restrict macrophage migration that contributes to macrophage-mediated diseases. Furthermore, this could translate to treatment of certain cancers, since FMNL1 has been suggested to promote leukemic cell migration.
    • Functional alterations and rhythmic disturbances by pan-histone deacetylase inhibition in the heart.

      Veenstra, Richard; Patel, Dakshesh (2016)
      Histone acetyl transferases (HATs) and histone deacetylases (HDACs) maintain a dynamic balance of acetylation and deacetylation of histone and non-histone proteins. HDAC inhibitors are small molecules anti-cancer therapeutics that exhibited dose limiting cardiac toxicities during clinical and preclinical trials. Multiple instances of abnormal T-waves, ST segment depression, QT prolongation, grade 3 sinus bradycardia and non- circumstantial deaths have been observed in patients. The underlying electrophysiological and molecular mechanism of these cardiac side-effects are poorly understood. In our in vivo ECG monitoring using Data Science International® telemetry transmitters, mice injected with panobinostat showed ventricular tachychardic and atrial fibrillation episodes with significant prolongation of ST, QT and QTc intervals. In whole cell patch clamp studies, we observed no significant change in transient and steady state K currents in myocyte ventricular cultures suggesting no role of hERG currents in ventricular arrhythmias. The majority (>90%) of congenital and drug induced QT prolongation is caused by alterations of hERG (IKr, Kv11.1) current. Interestingly, we observed significant reductions of INa and gap junctional conductance along with reductions in protein expression of Nav1.5 & Cx43 in vivo and in vitro. We conclude that pan-HDAC inhibition reduced cardiac INa density and gap junctional coupling with unaltered late INa and K+ currents explaining the cardiac abnormalities exhibited by panHDAC inhibitors. Decreased gap junctional coupling can enhance triggered activity by limiting electrotonic inhibition, combined with reduced INa density which can lead to slow myocardial conduction. Both of them taken together, increases the vulnerability to reentrant arrhythmias.
    • Functional Studies of Tumor Suppressor ECRG2 in the Regulation of Cancer Cell Death and Drug Resistance

      Huang, Ying; Lucchesi, Chris (2015)
      Esophageal Cancer Related Gene 2 (ECRG2) is a newer tumor suppressor whose mRNAhas previously been shown to be decreased in multiple human malignanceswhencompared to normal/adjacent tissues.Of importance, ECRG2 has previously been revealedto possess tumor suppressive attributes,including the ability to induce cell death in cancer cells. However, how ECRG2 is able to activate the apoptotic machinery has yet to be elucidated. In the present study,we highlight multiple angles that ECRG2 leverages in order to sensitize cancer cells to apoptosis. Moreover, we report for the first time,that ECRG2 protein expression in lung cancer patientsamples is lost/decreased in upwards of 90% ofthecancer tissues evaluatedcompared to normal tissue. Additionally, a single somatic point mutant found in patient tumor samples, V30E, was shown to lose tumor suppressive abilities and acquired resistance against multiple anticancer drugs. Our results demonstrate that ECRG2is upregulated in response to DNA damage, andis capable of inducing the activation of both caspase cascades (intrinsic and extrinsic) leadingto cancer cell death. We further illustratedthat ECRG2-mediated cell deathwasattributed to a reduction in the levels of apoptosis inhibitor, X chromosome-linked inhibitor of apoptosis protein (XIAP). ECRG2 was revealedto regulate XIAP protein levels via RNA-binding protein,human antigen R(HuR).We further highlight that ECRG2 causes increased HuR ubiquitination,subsequently leading to its degradation. Thus, we demonstrate that ECRG2 sensitizescancer cells to apoptosis through the downregulation of HuR, and consequent downregulation of XIAP. Next, we have identified ECRG2 as a potent positive regulator of death receptor 5 (DR5) gene expression.ECRG2-mediated upregulation of DR5 was shown to be facilitated through the upregulation of tumor suppressor p53 and transcription factors ATF3 and NFⱪB. Together, in a cooperative fashion, increased levelsof p53, ATF3 and NFⱪB stimulateDR5 gene expression. Contrastingly, silencing of ECRG2 not only decreased the levels of NFⱪB and DR5, but also resulted in decreased cancer cell sensitivity to genotoxic stressandTRAIL treatment. Collectively,our work establishes that ECRG2 is capable of inducing apoptosis in cancer cells by increasing the expression of pro-apoptotic proteins, while also negatively influencing anti-apoptotic proteins.Further, the loss of ECRG2’stumor suppressive abilities, as wehaveshownby the loss of ECRG2in lung patient tumor samples,and through the somatic point mutantV30E, illuminates possible mechanismsin which cancer cells can acquiremultiple drug resistance.
    • Further evidence of an association between maternal smoking during pregnancy and attention deficit hyperactivity disorder: Findings from a high-risk sample of siblings

      Milberger, Sharon; Biederman, Joseph; Faraone, Stephen V.; Jones, Janice (Informa UK Limited, 1998-09)
      The authors investigated the relationship between attentiondeficit/byperactivity disorder (ADHD) and cigarette smoking in siblings of ADHD and non-ADHD probands. They conducted a 4-year follow-up of siblings from ADHD and control-group families. In the siblings of ADHD probands, ADHD was associated with higher rates and earlier onset of cigarette smoking. There was also a significant positive association between cigarette smoking and conduct disorder, major depression, and drug abuse in the siblings, even after adjusting for confounding variables. Moreover, smoking was found to be familial among ADHD families but not control-group families. Our findings indicate that ADHD is a risk factor for early initiation of cigarette smoking in the high-risk siblings of ADHD probands
    • Further Evidence of Association Between Behavioral Inhibition and Social Anxiety in Children

      Biederman, Joseph; Hirshfeld-Becker, Dina R.; Rosenbaum, Jerrold F.; Hérot, Christine; Friedman, Deborah; Snidman, Nancy; Kagan, Jerome; Faraone, Stephen V. (American Psychiatric Association Publishing, 2001-10)
      Objective: The authors sought to examine psychopathological correlates of behavioral inhibition in young offspring of parents with panic disorder and/or major depression. Method: Behavioral inhibition, determined by using standard laboratory observations, was assessed in four groups of children (age 2–6 years): 129 children of parents with both panic disorder and major depression, 22 children of parents with panic disorder alone, 49 children of parents with major depression alone, and 84 comparison children of parents with neither panic disorder nor major depression. Psychopathology in children ≥5 years was compared between children with behavioral inhibition (N=64) and without (N=152). Results: Social anxiety disorder (social phobia or avoidant disorder) was significantly more likely to be found in the children with behavioral inhibition (17%) than in those without (5%). Noninhibited children were significantly more likely than inhibited children to have disruptive behavior disorders (20% versus 6%, respectively) and had higher scores on the attention problems scale of the Child Behavior Checklist (mean=52.1 versus 50.8). Conclusions: This study adds to the growing literature suggesting an association between behavioral inhibition and social anxiety disorder and an inverse relationship between inhibition and disruptive behavior disorders.