Harish C Prasad

Vanderbilt University, Nashville, MI, United States

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Publications (19)102.99 Total impact

  • 2013 International Meeting for Autism Research; 05/2013
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    ABSTRACT: The Na(+)/Cl(-)-dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear. In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIalpha specifically associates with hSERT. Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIalpha that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIalpha supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.
    Molecular Brain 09/2009; 2:26. · 4.20 Impact Factor
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    ABSTRACT: Alterations in peripheral and central indices of serotonin (5-hydroxytryptamine, 5-HT) production, storage and signaling have long been associated with autism. The 5-HT transporter gene (HTT, SERT, SLC6A4) has received considerable attention as a potential risk locus for autism-spectrum disorders, as well as disorders with overlapping symptoms, including obsessive-compulsive disorder (OCD). Here, we review our efforts to characterize rare, nonsynonymous polymorphisms in SERT derived from multiplex pedigrees carrying diagnoses of autism and OCD and present the initial stages of our effort to model one of these variants, Gly56Ala, in vivo. We generated a targeting vector to produce the Gly56Ala substitution in the Slc6a4 locus by homologous recombination. Following removal of a neomycin resistance selection cassette, animals exhibiting germline transmission of the Ala56 variant were bred to establish a breeding colony on a 129S6 background, suitable for initial evaluation of biochemical, physiological and behavioral alterations relative to SERT Gly56 (wild-type) animals. SERT Ala56 mice were achieved and exhibit a normal pattern of transmission. The initial growth and gross morphology of these animals is comparable to wildtype littermate controls. The SERT Ala56 variant can be propagated in 129S6 mice without apparent disruption of fertility and growth. We discuss both the opportunities and challenges that await the physiological/behavioral analysis of Gly56Ala transgenic mice, with particular reference to modeling autism-associated traits.
    Journal of Neurodevelopmental Disorders 06/2009; 1(2):158-71. · 3.45 Impact Factor
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    ABSTRACT: Background: Elevated platelet serotonin (5-HT), termed hyperserotonemia, is present in about one-quarter of children with autism. Linkage studies in families containing two or more male children with autism have implicated the chromosome 17q11.2 region containing the serotonin transporter gene (SERT, SLC6A4). Common functional variants in SERT fail to explain the strong linkage signal observed at this locus. In families with male-only probands, rare SERT amino acid variants show an association with autism. The most common of these variants, Gly56Ala, is associated with rigid-compulsive behaviors and sensory aversion. Objectives: In vitro studies (Prasad et al., 2005; Prasad et al., 2009) demonstrated that the 56Ala variant (1) displays increased basal 5-HT transport, (2) exhibits elevated basal phosphorylation, and (3) is refractory to regulation through PKG and p38 MAPK pathways. To pursue the physiological significance of these findings in vivo, we developed a SERT Gly56Ala knock-in mouse line. Methods: Serotonin levels were measured in whole blood and in brain by high-performance liquid chromatography. Serotonin uptake was measured in platelets and midbrain synaptosomes. Mouse behavior was assessed using standard techniques to measure activity level, anxiety-like behavior, social behavior, and acoustic startle / prepulse inhibition. Results: SERT 56Ala variant mice exhibit elevated whole blood 5-HT. Initial behavioral studies suggest social and sensory alterations in the SERT 56Ala variant mice. Conclusions: The SERT 56Ala mouse is the first mouse model targeting an autism-associated gene to manifest hyperserotonemia. Initial studies provide preliminary support for altered behavioral traits with face validity for autism spectrum disorder. Ongoing efforts seek to connect the intermediate steps between the SERT 56Ala variant and the observed neurochemical and behavioral phenotype.
    International Meeting for Autism Research 2009; 05/2009
  • 55th Meeting of American Academy of Child and Adolescent Psychiatry; 11/2008
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    ABSTRACT: Rare, functional, non-synonymous variants in the human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT) gene (SLC6A4) have been identified in both autism and obsessive-compulsive disorder (OCD). Within autism, rare hSERT coding variants associate with rigid-compulsive traits, suggesting both phenotypic overlap with OCD and a shared relationship with disrupted 5-HT signalling. Here, we document functional perturbations of three of these variants: Ile425Leu; Phe465Leu; and Leu550Val. In transiently transfected HeLa cells, the three variants confer a gain of 5-HT transport phenotype. Specifically, enhanced SERT activity was also observed in lymphoblastoid lines derived from mutation carriers. In contrast to previously characterized Gly56Ala, where increased transport activity derives from catalytic activation, the three novel variants exhibit elevated surface density as revealed through both surface antagonist-binding and biotinylation studies. Unlike Gly56Ala, mutants Ile425Leu, Phe465Leu and Leu550Val retain a capacity for acute PKG and p38 MAPK regulation. However, both Gly56Ala and Ile425Leu demonstrate markedly reduced sensitivity to PP2A antagonists, suggesting that deficits in trafficking and catalytic modulation may derive from a common basis in perturbed phosphatase regulation. When expressed stably from the same genomic locus in CHO cells, both Gly56Ala and Ile425Leu display catalytic activation, accompanied by a striking loss of SERT protein.
    Philosophical Transactions of The Royal Society B Biological Sciences 11/2008; 364(1514):163-73. · 6.23 Impact Factor
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    ABSTRACT: Unipolar major depressive disorder (MDD) is a prevalent, disabling condition with multiple genetic and environmental factors impacting disease risk. The diagnosis of MDD relies on a cumulative measure derived from multiple trait dimensions and alone is limited in elucidating MDD genetic determinants. We and others have proposed that MDD may be better dissected using paradigms that assess how specific genes associate with component features of MDD. This within-disease design requires both a well-phenotyped cohort and a robust statistical approach that retains power with multiple tests of genetic association. In the present study, common polymorphic variants of genes related to central monoaminergic and cholinergic pathways that previous studies align with functional change in vitro or depression associations in vivo were genotyped in 110 individuals with unipolar MDD. Subphenotypic characteristics were examined using responses to individual items assessed with the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders (DSM IV), the 17-item Hamilton Rating Scale for Depression (HAM-D) and the NEO Five Factor Inventory. Multivariate Permutation Testing (MPT) was used to infer genotype-phenotype relationships underlying dimensional findings within clinical categories. MPT analyses show significant associations of the norepinephrine transporter (NET, SLC6A2) -182 T/C (rs2242446) with recurrent depression [odds ratio, OR = 4.15 (1.91-9.02)], NET -3081 A/T (rs28386840) with increase in appetite [OR = 3.58 (1.53-8.39)] and the presynaptic choline transporter (CHT, SLC5A7) Ile89Val (rs1013940) with HAM-D-17 total score {i.e. overall depression severity [OR = 2.74 (1.05-7.18)]}. These relationships illustrate an approach to the elucidation of gene influences on trait components of MDD and with replication, may help identify MDD subpopulations that can benefit from more targeted pharmacotherapy.
    Genes Brain and Behavior 07/2008; 7(4):487-95. · 3.60 Impact Factor
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    ABSTRACT: Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V(max)). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.
    Proceedings of the National Academy of Sciences 09/2005; 102(32):11545-50. · 9.81 Impact Factor
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    ABSTRACT: Autism is a spectrum of neurodevelopmental disorders with a primarily genetic etiology exhibiting deficits in (1) development of language and (2) social relationships and (3) patterns of repetitive, restricted behaviors or interests and resistance to change. Elevated platelet serotonin (5-HT) in 20%-25% of cases and efficacy of selective 5-HT reuptake inhibitors (SSRIs) in treating anxiety, depression, and repetitive behaviors points to the 5-HT transporter (5-HTT; SERT) as a strong candidate gene. Association studies involving the functional insertion/deletion polymorphism in the promoter (5-HTTLPR) and a polymorphism in intron 2 are inconclusive, possibly because of phenotypic heterogeneity. Nonetheless, mounting evidence for genetic linkage of autism to the chromosome 17q11.2 region that harbors the SERT locus (SLC6A4) supports a genetic effect at or near this gene. We confirm recent reports of sex-biased genetic effects in 17q by showing highly significant linkage driven by families with only affected males. Association with common alleles fails to explain observed linkage; therefore, we hypothesized that preferential transmission of multiple alleles does explain it. From 120 families, most contributing to linkage at 17q11.2, we found four coding substitutions at highly conserved positions and 15 other variants in 5' noncoding and other intronic regions transmitted in families exhibiting increased rigid-compulsive behaviors. In the aggregate, these variants show significant linkage to and association with autism. Our data provide strong support for a collection of multiple, often rare, alleles at SLC6A4 as imposing risk of autism.
    The American Journal of Human Genetics 09/2005; 77(2):265-79. · 11.20 Impact Factor
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    ABSTRACT: The alpha(1A)-adrenergic receptor is highly expressed in human vasculature including resistance arteries and veins, and its stimulation is primarily responsible for adrenergically mediated smooth muscle contraction. Variability in sensitivity to phenylephrine, an alpha(1A) adrenergic agonist, has a large genetic component. We examined the hypothesis that a common polymorphism of alpha(1A)-adrenergic receptor (Arg347Cys) affects in vivo response. We measured vascular sensitivity to phenylephrine using the dorsal hand vein linear variable differential transformer technique and determined alpha(1A)-adrenergic receptor genotype in 74 healthy, nonsmoking adults (28 Arg/Arg, 30 Arg/Cys, and 16 Cys/Cys). Sensitivity to phenylephrine, expressed as the dose of phenylephrine resulting in 50% venoconstriction (Phe(50)), was not significantly different in subjects with the 3 alpha(1A) adrenergic receptor genotypes: Phe(50) geometric mean (95% confidence interval) was 513 ng/min (287-918 ng/min) for Arg/Arg, 431 ng/min (274-680 ng/min) for Arg/Cys, and 471 ng/min (197-1124 ng/min) for Cys/Cys (P =.90). We conclude that the Arg347Cys receptor polymorphism does not alter agonist-mediated venoconstriction in vivo.
    Clinical Pharmacology &#38 Therapeutics 07/2004; 75(6):539-45. · 6.85 Impact Factor
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    ABSTRACT: To examine the hypothesis that sildenafil, a phosphodiesterase type 5 inhibitor that inhibits cGMP breakdown, could enhance nitric oxide-mediated vasodilation and reverse endothelial dysfunction in chronic smokers. Flow-mediated dilation of the brachial artery and forearm postischemic reactive hyperemia (both nitric oxide-mediated responses) were measured before and after sildenafil 50 mg and placebo in a double-blind, randomized, crossover study in 9 men who were chronic smokers (21 +/- 3 pack years). There was no significant change in flow-mediated dilation after either sildenafil (0.18%, 95%CI -1.7-2%) or placebo (0.24%, 95%CI -2.8-3.3%) (P = 0.88 and 0.8, respectively). Sildenafil had no significant effect on resting forearm blood flow or postischemic reactive hyperemia (P = 0.39 and 0.7, respectively). Resting heart rate and blood pressure were unaffected by sildenafil. Acute sildenafil administration did not improve endothelial function in chronic smoking men.
    British Journal of Clinical Pharmacology 03/2004; 57(2):209-12. · 3.69 Impact Factor
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    ABSTRACT: The genetic basis for salt-sensitive hypertension is not known. Altered regulatory mechanisms such as vascular reactivity and/or sodium excretion are thought to contribute to salt sensitivity. Linkage analysis indicates that the blood pressure response to sodium loading is associated with the beta-2 adrenergic receptor (BAR2) locus suggesting that this gene may be an important regulator of blood pressure responses to salt. The common Arg16Gly and Gln27Glu BAR2 polymorphisms have been associated with enhanced agonist mediated desensitization and increased agonist-mediated responsiveness, respectively but their effects on salt sensitivity are not known. Healthy subjects (n = 23) received a high (400meq) and a low (10meq) salt diet for 5 days in random order. Automated blood pressure monitoring was performed on the last day of each diet. The change in blood pressure between low and high salt days was calculated and compared among genotypes. There was no significant genotypic difference in salt induced changes in systolic (Arg16Gln27[n = 8] 2.3 ± 3.8 mmHg, Gly16Gln27 [n = 7] 3.1 ± 3.3 mmHg and Gly16Glu27 [n = 8] 6.1 ± 2.8 mmHg: P = 0.66) or diastolic (Arg16Gln27 −2.4 ± 1.7 mmHg, Gly16Gln27 1.1 ± 1.6 mmHg and Gly16Glu27 −1.5 ± 1.2 mmHg: P = 0.26) blood pressure. These findings suggest that the BAR2 Arg16Gly and Gln27Glu polymorphisms are not a significant determinant of blood pressure responses to salt.
    Clinical Pharmacology &#38 Therapeutics 01/2004; · 6.85 Impact Factor
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    ABSTRACT: A common polymorphism of the beta(1)-adrenergic receptor Arg389Gly markedly affects function in vitro, but little is known about its in vivo significance. Resting and exercise hemodynamic responses were measured in subjects homozygous for Arg389 (n = 21) or Gly389 (n = 13) alleles before and 3 hours after administration of a beta-blocker, atenolol. Demographic characteristics and atenolol concentrations were similar in the two genotypic groups. Genotype had a marked effect on resting hemodynamic responses to atenolol, with Arg389-homozygous subjects having a larger decrease in resting systolic blood pressure (8.7 +/- 1.3 mm Hg versus 0.2 +/- 1.7 mm Hg, P < .001) and mean arterial blood pressure (7.2 +/- 1.0 mm Hg versus 2.0 +/- 1.7 mm Hg, P = .009). Attenuation of exercise-induced hemodynamic responses by atenolol was not affected by genotype. There is reduced sensitivity of Gly389 homozygotes to a beta-adrenergic receptor antagonist, and this polymorphism may be an important determinant of variability in response to beta-blockade.
    Clinical Pharmacology &#38 Therapeutics 04/2003; 73(4):366-71. · 6.85 Impact Factor
  • Clinical Pharmacology &#38 Therapeutics 02/2003; 73(2). · 6.85 Impact Factor
  • Clinical Pharmacology &#38 Therapeutics 02/2003; 73(2):P98-P98. · 6.85 Impact Factor
  • Clinical Pharmacology &#38 Therapeutics 01/2003; · 6.85 Impact Factor
  • Clinical Pharmacology &#38 Therapeutics 01/2003; 73(2). · 6.85 Impact Factor
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    ABSTRACT: Cytochrome P-450 (CYP) 2C9 CYP2C9 is a polymorphically expressed enzyme responsible for the metabolism of several clinically important drugs, some with a low therapeutic index. This review summarizes the structure-function relationship of the CYP2C9 promoter and coding regions, known polymorphisms, the functional significance of various CYP2C9 alleles in vitro and in vivo, and their population frequencies. In addition, possible molecular mechanisms underlying ethnic variability in the metabolism of CYP2C9 substrate drugs are discussed.
    Advanced Drug Delivery Reviews 12/2002; 54(10):1257-70. · 12.89 Impact Factor
  • 56th Meeting of American Academy of Child and Adolescent Psychiatry;