This study investigated how genetic variation in the serotonin transporter gene (Slc6a4) and integrin beta 3 gene (Itgb3) interact to modulate serotonin uptake and transporter expression in mouse brain synapses. The researchers prepared synaptoneurosomes (preserved pre- and post-synaptic structures) from mouse midbrain, hippocampus and cortex with different genotypes of Slc6a4 and Itgb3. They found reduced serotonin transporter expression and uptake activity in midbrain synaptoneurosomes of mice with both genes heterozygous, revealing an interaction between the two genes. In contrast, changes were driven mostly by Slc6a4 in the hippocampus. The study provides evidence that
Activity-dependent transcriptional dynamics in mouse primary cortical and hum...Darya Vanichkina
Poster I presented at Lorne Genome 2012. Subsequently formed part of the paper
Barry G, Briggs JA, Vanichkina DP, Poth EM, Beveridge NJ, Ratnu VS, Nayler SP, Nones K, Hu J, Bredy TW, Nakagawa S, Rigo F, Taft RJ, Cairns MJ, Blackshaw S, Wolvetang EJ, Mattick JS (2013). The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing. Molecular psychiatry doi: 10.1038/mp.2013.45
Duplicate of http://figshare.com/articles/Activity_dependent_transcriptional_dynamics_in_mouse_primary_cortical_and_human_iPS_derived_neurons/978468
Activity-dependent transcriptional dynamics in mouse primary cortical and hum...Darya Vanichkina
Poster I presented at Lorne Genome 2012. Subsequently formed part of the paper
Barry G, Briggs JA, Vanichkina DP, Poth EM, Beveridge NJ, Ratnu VS, Nayler SP, Nones K, Hu J, Bredy TW, Nakagawa S, Rigo F, Taft RJ, Cairns MJ, Blackshaw S, Wolvetang EJ, Mattick JS (2013). The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing. Molecular psychiatry doi: 10.1038/mp.2013.45
Duplicate of http://figshare.com/articles/Activity_dependent_transcriptional_dynamics_in_mouse_primary_cortical_and_human_iPS_derived_neurons/978468
WEBWARE36 - Organisation und Know-how für Web-Agenturensoftenginegmbh
Mit ERP und einem ganzheitlichen Beratungsansatz die nächste Professionalisierungsstufe im E-Commerce meistern.
Fest steht jedenfalls, dass es künftig nicht mehr reichen wird, einen Shop aufzusetzen, ein ansehnliches Design zu entwickeln
und den Content suchmaschinenoptimiert (SEO) aufzubereiten. Gefragt sind stattdessen Geschäftspartner, die über ganzheitliches
betriebswirtschaftliches Know-how verfügen – und dabei Themen abdecken wie:
Eine Agentur mit ERP-Kompetenz bleibt im Boot. Das gilt selbst dann, wenn der Webshop eines Tages gewechselt wird.
Der Grund hierfür ist einfach: Nicht der Shop ist die zentrale Plattform, sondern das ERP-System.
■ Lager & Logistik
■ Warenbeschaffung
■ Kundenbeziehungsmanagement (CRM)
■ Multishop-Anbindung
■ Integration unterschiedlicher Plattformen
WEBWARE36 - Organisation und Know-how für Web-Agenturensoftenginegmbh
Mit ERP und einem ganzheitlichen Beratungsansatz die nächste Professionalisierungsstufe im E-Commerce meistern.
Fest steht jedenfalls, dass es künftig nicht mehr reichen wird, einen Shop aufzusetzen, ein ansehnliches Design zu entwickeln
und den Content suchmaschinenoptimiert (SEO) aufzubereiten. Gefragt sind stattdessen Geschäftspartner, die über ganzheitliches
betriebswirtschaftliches Know-how verfügen – und dabei Themen abdecken wie:
Eine Agentur mit ERP-Kompetenz bleibt im Boot. Das gilt selbst dann, wenn der Webshop eines Tages gewechselt wird.
Der Grund hierfür ist einfach: Nicht der Shop ist die zentrale Plattform, sondern das ERP-System.
■ Lager & Logistik
■ Warenbeschaffung
■ Kundenbeziehungsmanagement (CRM)
■ Multishop-Anbindung
■ Integration unterschiedlicher Plattformen
Presentación donde platicamos sobre cómo en Phyne Games buscando balancear el arte y la tecnología en nuestros juegos.
Esta fue presentada durante el evento "GameDev XP" en la facultad de Ingeniería de la UNAM.
Gracias a SODVI por el espacio y la invitación.
Los servicios de orientación y asistencia legal dirigidos a población en situ...EUROsociAL II
Los servicios de orientación y asistencia legal dirigidos a población en situación de vulnerabilidad en colaboración con la sociedad civil: criterios para considerar buenas prácticas en la materia y propuesta de planes de acción para su fortalecimiento / Olga Lucía Gaitán García
Getting Traction for (your) Open Source ProjectsMichael Boelen
So you got an idea, some code, and now you only need the users and contributors? A good idea is not enough to have a successful project. Open source software projects need also marketing, promotion, and optimization. We will look at the technical and non-technical level on how to enhance OSS projects. This with the goal to get more happy users and gain more traction. I will share from personal experience what works (and what not!), including examples. Subjects include simplicity, the website, dealing with social media, optimize for users and search engines, and the tiny details that matter.
This talk is useful for developers, contributors, and also users of open source software. No programming skills are required.
Proyecto ELECTIVA VI EMPRESA LAMIDECO, C.AZaibel Campos
Misión.
Ofrecer a nuestros clientes un producto de calidad cumpliendo con las exigencias y necesidades requeridas, permitiéndonos competir en el mercado estatal y regional, posicionándonos como la empresa número uno en ventas y distribución de láminas decorativas, apalancando nuestra rentabilidad.
Visión.
Constituirse como una de las mejores empresas en su ramo, así como expandir nuestros locales comerciales con la finalidad de abarcar todos los espacios del Estado Monagas, aplicando nuestros conocimientos en beneficio de la sociedad y el bienestar colectivo.
Objetivos.
Obtener y mantener un amplio crecimiento de clientes.
Ser una Empresa reconocida en el mercado nacional.
Lograr un crecimiento organizacional orientado a la alta rentabilidad de la empresa, así como al desarrollo y aumento de la calidad de los productos y servicios prestados.
Tener un personal altamente capacitado en la fuerza labor, apalancando su formación con adiestramientos y actualizaciones continuas en el ramo de producción del laminado decorativo.
Organización.
Somos una empresa preocupada por la necesidad y satisfacción de la clientela y que a través de brindar la mejor calidad en productos y servicios nos comprometemos y garantizamos una atención completa.
Nuestra prioridad son los clientes y todo lo que hacemos está orientado a satisfacer sus necesidades y superar continuamente sus expectativas. Nos esforzamos cada día en buscar mejoras que optimicen los procesos y tareas en todas las áreas de la organización.
Ubicación.
Láminas de Maderas Decorativas “LamiDeco, C.A.” está ubicada en Venezuela, Maturín estado Monagas. Específicamente en la parroquia La Cruz, Zona Industrial
Mega Madera CA.
Maderera Tumero, C.A.
HERMANOS PETRIGLIERI, C.A.
INSUMOSLos insumos son todos los recursos con los que cuenta la empresa para generar productos y servicios. El principal insumo de “LamiDeco, C.A.”, es La Madera; así como también cuenta con Insumos Humanos, Tecnológicos y Organizacionales.
Sin embargo, sin duda alguna la mayor oportunidad para aumentar la productividad se encuentra en el propio trabajo, en el conocimiento y, en especial, en la administración. La productividad implica eficacia y eficiencia en el desempeño individual y organizacional, Ambiente específicos y proveedores
Proceso de manufacturaSelección de la materia.
Sierra de cortar.
Cepillo automático.
Sierra cortes múltiples.
Cepillo automático secundario.
Maquina talladora.
Pegado ensamblado.
Lijado y limpiado.
Control de calidad.
Área de productos terminados.
Productos terminados
Esta empresa se encarga de fabricar láminas de madera decorativas que luego son usadas en principalmente a espacios que requieren un revestimiento con resistencia a los golpes, rayado, agua y calor así como también en muebles decorativos. Su uso es utilitario y no tiene uso específico, sino genérico. Sus texturas, colores, e
Entelios AG - Demand Response for Germany and EuropeAQAL Group
Entelios AG is Germany’s Demand Response Aggregator. We introduce proven and powerful cleantech technology to the European smart grid markets. Entelios is applying a leapfrog strategy, providing the next evolutionary step called Demand Response 2.0, doing our part in enabling renewable energy and reducing the need for more fossil fuel power plants.
Guia Municipal de servicios de Bermeo con los directorios y telefonos de comercios, restaurantes, servicios, instalaciones (culturales, deportivas, ocio, educación, ...), empresas, industria, adminsitración municipal, ....
cloning. Second, it is sensitive. Activities canbe detected WilheminaRossi174
cloning. Second, it is sensitive. Activities can
be detected in the purified GST-ORF pools
that simply cannot be detected in extracts or
cells, the starting point of both conventional
purification and expression cloning. Because
the GST-ORFs are individually expressed at
high levels and are largely free of extract
proteins after purification, activities can be
measured for hours without competing activ-
ities that destroy the substrate, the product, or
the enzymes.
In addition to the conventional use demon-
strated here, this array could be used in two
other ways: (i) to determine the range of poten-
tial substrate proteins for any protein-modifying
enzyme (such as a protein kinase) before genet-
ic or biochemical tests to establish authentic
substrates and (ii) to identify genes encoding
proteins that bind any particular macromole-
cule, ligand, or drug. Thus, one could rapidly
ascribe function to many presently unclassified
yeast proteins, complementing other genomic
approaches to deduce gene function from ex-
pression patterns, mutant phenotypes, localiza-
tion of gene products, and identification of in-
teracting partners.
References and Notes
1. H. Simonsen and H. F. Lodish, Trends Pharmacol. Sci.
15, 437 (1994).
2. Plasmid pYEX 4T-1 (Clontech, Palo Alto, CA) was
modified by the addition of a 140-nucleotide recom-
bination domain, 39 of its Eco RI site, linearized within
the recombination domain by restriction digestion,
and cotransformed with a genomic set of reamplified
ORFs that had the same ends as the linearized plas-
mid [ J. R. Hudson Jr. et al., Genome Res. 7, 1169
(1997)] into strain EJ 758 [MATa his3-D200, leu2-
3,112, ura3-52, pep4::URA3], a derivative of JHRY-
20-2Ca (5). Transformants obtained on synthetic
minimal (SD) 2 Ura drop-out plates [F. Sherman,
Methods Enzymol. 194, 3 (1991)] (.100 in all cases,
and more than five times the cut vector in 97% of the
cases) were eluted in batch and saved in 96-well
microtiter plates. The library contains 6080 ORF-
containing strains and 64 strains with vector only.
3. Cell patches were inoculated in SD 2 Ura liquid
medium, grown overnight, reinoculated, and grown
overnight in SD 2 Ura 2 Leu medium, and then
inoculated into 250 ml of SD 2 Ura 2 Leu medium,
grown to absorbance at 600 nm of 0.8, and induced
with 0.5 mM copper sulfate for 2 hours before har-
vest [I. G. Macreadie, O. Horaitis, A. J. Verkuylen,
K. W. Savin, Gene 104, 107 (1991)]. Cells were re-
suspended in 1 ml of buffer [50 mM tris-HCl (pH 7.5),
1 mM EDTA, 4 mM MgCl2, 5 mM dithiothreitol (DT T),
10% glycerol, and 1 M NaCl] containing leupeptin (2
mg/ml) and pepstatin (1 mg/ml), and extracts were
made with glass beads [S. M. McCraith and E. M.
Phizicky, Mol. Cell. Biol. 10, 1049 (1990)], followed
by supplementation with 1 mM phenylmethylsulfo-
nyl fluoride and centrifugation. GST-ORF fusion pro-
teins were purified by glutathione agarose chroma-
tography in buffer containing 0.5 M NaCl, essentially
as described [ J. ...
Genomic gene expression changes resulting from Trypanosomiasis: a horizontal study Examining expression changes elucidated by micro arrays in seminal tissues associated with the pathophysiology of Trypanosomiasis during disease progression
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Age Related Histomorphological and Transmission Electron Microscopic Studies ...iosrjce
IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) is a double blind peer reviewed International Journal edited by the International Organization of Scientific Research (IOSR). The journal provides a common forum where all aspects of Agricultural and Veterinary Sciences are presented. The journal invites original papers, review articles, technical reports and short communications containing new insight into any aspect Agricultural and Veterinary Sciences that are not published or not being considered for publication elsewhere.
Crimson publishers-5-MethylcytosineDNA Methylation Patterns among Gut Predomi...CrimsonpublishersMedical
5-MethylcytosineDNA Methylation Patterns among Gut Predominate Commensal Escherichia coli and Lactobacilli from the Balbas and Mazekh Domestic Sheep Breeds by Pepoyan AZ* in Research in Medical &Engineering Sciences
1. Serotonin transporter and integrin beta 3 genes interact to modulate
serotonin uptake in mouse brain
Alonzo Whyte a
, Tammy Jessen b
, Seth Varney b
, Ana M.D. Carneiro b,⇑
a
Neuroscience Graduate Program, Vanderbilt Brain Institute, U1205 Medical Research Building III, 465 21st Avenue South, Nashville, TN 37232, United States
b
Department of Pharmacology, Vanderbilt University School of Medicine, 215 Light Hall, Nashville, TN 37232, United States
a r t i c l e i n f o
Article history:
Available online xxxx
Keywords:
Serotonin transporter
Integrin
Genetic interaction
Neuropsychiatric disorders
Autism
a b s t r a c t
Dysfunctions in serotonin (5-hydroxytryptamine, 5-HT) systems have been associated with several psy-
chiatric illnesses, including anxiety, depression, obsessive–compulsive disorders and autism spectrum
disorders. Convergent evidence from genetic analyses of human subjects has implicated the integrin b3
subunit gene (ITGB3) as a modulator of serotonergic systems via genetic interactions with the 5-HT trans-
porter gene (SLC6A4, SERT). While genetic interactions may result from contributions of each gene at sev-
eral levels, we hypothesize that ITGB3 modulates the 5-HT system at the level of the synapse, through the
actions of integrin avb3. Here we utilized a genetic approach in mouse models to examine Itgb3 contribu-
tions to SERT function both in the context of normal and reduced SERT expression. As integrin avb3 is
expressed in postsynaptic membranes, we isolated synaptoneurosomes, which maintain intact pre- and
post-synaptic associations. Citalopram binding revealed significant Slc6a4-driven reductions in SERT
expression in midbrain synapses, whereas no significant changes were observed in hippocampal or corti-
cal projections. Expecting corresponding changes to SERT function, we also measured 5-HT uptake activity
in synaptoneurosomal preparations. Itgb3 single heterozygous mice displayed significant reductions in 5-
HT Vmax, with no changes in Km, in midbrain preparations. However, in the presence of both Itgb3 and
Slc6a4 heterozygozity, 5-HT uptake was similar to wild-type levels, revealing a significant Slc6a4 by Itgb3
genetic interaction in the midbrain. Similar findings were observed in cortical preparations, whereas in the
hippocampus, most Vmax changes were driven solely by Slc6a4. Our findings provide evidence that integrin
avb3 is involved in the regulation of serotonergic systems in some, but not all 5-HT synapses, revealing
novel contributions to synaptic specificity within the central nervous system.
Ó 2013 Published by Elsevier Ltd.
1. Introduction
Dysfunction in serotonin (5-hydroxytryptamine, 5-HT) neuro-
transmission has been implicated in the etiology of mood and
developmental disorders including anxiety, depression, and aut-
ism-spectrum disorders (ASD). Several genetic variants in the 5-
HT transporter gene (SERT, SLC6A4) have been associated with
behavioral phenotypes manifested in these disorders, especially
in the context of genetic interactions or under specific environ-
mental conditions (Caspi et al., 2003; Sutcliffe et al., 2005; Murphy
and Moya, 2011). Variations in whole blood 5-HT levels, found in
several neuropsychiatric disorders, including autism, bipolar disor-
der and seasonal affective disorder (Velayudhan et al., 1999; Wil-
leit et al., 2008), are associated with non-coding variation in
ITGB3 (Weiss et al., 2004). Genetic interaction of ITGB3, which
encodes the integrin b3 subunit (forming the integrin aIIbb3 in
platelets and integrin avb3 in brain), and SLC6A4, either in mRNA
expression or autism susceptibility, further reinforces the sugges-
tion that these two genes may interact to modify 5-HT homeostasis
(Weiss et al., 2004). Whereas genetic interactions do not typically
translate into functional or biochemical interactions, we have re-
ported a physical and functional association between integrin
aIIbb3 and SERT in platelets (Carneiro et al., 2008). Thus we
hypothesize that ITGB3 and SLC6A4 interact to modulate SERT
expression and function in the brain. Here we utilized a genetic ap-
proach to document unique and interactive contributions of these
genes to transporter expression and function in the mouse synaptic
preparations.
2. Materials and methods
2.1. Animals
Mouse studies were performed in accordance with humane
guidelines established by the Vanderbilt Institutional Animal Care
0197-0186/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.neuint.2013.09.014
⇑ Corresponding author. Address: 461 Preston Research Building, Vanderbilt
University Medical Center, 23rd Ave. South at Pierce, Nashville, TN 37232-6600,
United States. Tel.: +1 615 875 5635.
E-mail address: ana.carneiro@vanderbilt.edu (A.M.D. Carneiro).
Neurochemistry International xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Neurochemistry International
journal homepage: www.elsevier.com/locate/nci
Please cite this article in press as: Whyte, A., et al. Serotonin transporter and integrin beta 3 genes interact to modulate serotonin uptake in mouse brain.
Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.09.014
2. and Use Committee under approved protocol (M/09/198). Both
Itgb3À/À
(Hodivala-Dilke et al., 1999) and Slc6a4À/À
(Bengel et al.,
1998) mouse lines were previously backcrossed onto C57BL/6 for
more than 20 generations. Slc6a4+/À
, Itgb3+/À
mice were generated
by crossing C57BL/6 Itgb3À/À
males and C57BL/6 Slc6a4À/À
females.
Mice derived from this crossing were not used for experiments to
avoid rearing effects caused by Slc6a4À/À
dam phenotypes. Instead,
the Itgb3+/À
, Slc6a4+/À
male offspring were paired with wildtype
C57BL/6J females producing offspring of four genotypes: Itgb3+/+
,
Slc6a4+/+
(WT); Itgb3+/À
, Slc6a4+/+
(Itgb3+/À
); Itgb3+/+
, Slc6a4+/À
(Slc6a4+/À
); and Itgb3+/À
, Slc6a4+/À
. Male and female offspring were
housed by sex with mixed-genotype littermates in groups of 2–5
per cage. Mice were maintained on a 12-h light–dark cycle, and
provided with food and water ad libitum. Littermate males and fe-
males were utilized for all biochemical and neurochemical assays.
2.2. Synaptoneurosome preparation
Synaptoneurosomes were obtained as previously described
(Phillips et al., 2001). Briefly, mice were rapidly decapitated and
brain regions were dissected onto 0.32 M sucrose in HEPES con-
taining 0.1 mM CaCl2 and 1 mM MgCl2 at 4 °C. Samples were
homogenized in a piston-type TeflonÒ
pestle with stainless steel
shaft and replaceable grinding vessel and cell debris/nuclei sepa-
rated by centrifugation at 1000g. Supernatants were collected
and spun at 10,000g for isolation of crude synaptoneurosomes.
Immediately after preparation, synaptoneurosomal protein was
measured using a modified Lowry protocol with bicinchoninic acid
(BCA Protein Assay Kit, Pierce Chemical Company, Rockford, IL).
Approximately 1 mg was used immediately for 5-HT saturation ki-
netic studies of 5-HT uptake, and the remaining was frozen for
citalopram binding and Western blot studies.
2.3. Saturation kinetic studies of [3
H] 5-HT uptake
Synaptoneurosome pellets were resuspended in Krebs–Ringer’s
HEPES (KRH) buffer (130 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl2,
1.2 mM MgSO4, 1.2 mM KH2PO4, 1.8 g/L glucose, 10 mM HEPES,
pH 7.4 containing 100 lM ascorbic acid and 100 lM pargyline).
Synaptoneurosomes (100 lg for midbrain and 200 lg for hippo-
campus and cortex) were incubated for 10 min at 37 °C in test
tubes containing 100 ll of KRH buffer, and 50 ll [3
H] 5-HT (Con-
centrations ranging from 12.5–400 nM. Hydroxytryptamine Creat-
inine Sulfate, 5-[1,2-3H(N)]-(Serotonin). Perkin Elmer, Walthman,
MA). An identical set of tubes contained 50 ll of 1 lM paroxetine
(Sigma Aldrich, Saint Louis, MO) to define SERT-specific uptake.
Next, samples were harvested via Brandel tissue harvester and fil-
tered onto GF/B Whatman filters (Brandel, Gaithersburg, MD). Fil-
ters were dissolved overnight in scintillation fluid (Econo-Safe™,
Research Products International Corp., Mount Prospect, IL) then
radioactivity was quantified in a Packard counter by QuantaSmart
4.0 software.
2.4. [3
H]-Citalopram binding
Synaptoneurosomes (100 lg for midbrain and 250 lg for hippo-
campus and cortex) were incubated with 5 nM [3
H]-citalopram
(Racemic citalopram, [N-Methyl-3
H]. Perkin-Elmer, Walthman,
MA) on ice for 20 min then harvested using a Brandel tissue har-
vester onto GF/B Whatman filters. An identical set of tubes con-
tained 1 lM paroxetine (Sigma Aldrich, Saint Louis, MO) to
define SERT-specific binding. Filters were dissolved overnight in
scintillation fluid then radioactivity was quantified in a Packard
counter by QuantaSmart 4.0 software.
2.5. Western blotting
Midbrain synaptoneurosomes pellets were resuspended in 1%
sodium dodecyl sulfate in phosphate buffered saline pH 7.4 and
protein was measured using a modified Lowry protocol with
bicinchoninic acid (BCA Protein Assay Kit, Pierce Chemical Com-
pany, Rockford, IL). No hippocampal or cortical samples were avail-
able for Western blots. 50 lg of protein were loaded onto 17-well
Pierce Protein Gels (Thermo Scientific). Gel electrophoresis was
performed at 100 v for 3 h then proteins were transferred over-
night at 4 °C onto PVDF membranes (Immobilon, Millipore, Bille-
rica, MA). After transfer, membranes were blocked with 5% milk
in 1x tris-buffered saline pH 7.4 and incubated with antibodies at
1:250 or 1:1000 dilutions overnight at 4 °C. Secondary antibodies
were added at 1:2500 dilution and proteins detected with chemi-
luminescence. Amersham Hyperfilm ECL films were exposed at
1,5,10, and 30 min to address linearity of the data (GE Healthcare,
Pittsburgh, PA). Films were scanned in tagged image file format
(.tiff) and bands quantified by densitometry using Image J. Anti-
bodies included: rabbit anti-integrin av and rabbit anti-integrin
b3 (Cell Signaling Technology, Denvers, MA), mouse anti-syntaxin
(Millipore, Billerica, MA), and guinea pig anti-5-HT transporter
(Frontier Science Co., Ltd., Hokkaido, Japan).
2.6. Data analysis
All data was analyzed in Prism 4.0c (Graphpad Software, Inc.,
LaJolla, CA). Two-way ANOVA was used with Slc6a4 and Itgb3 as
variables to identify contributions of each gene. Dunnett’s multiple
comparison tests were used to compare each genotype to wild-
type (WT). Kruskal–Wallis test was used to analyze Western blot
samples as each group of samples was run in a different day and
normalized to each individual control (WT = 100%). In this particu-
lar case we used Dunn’s post-tests to identify statistical significant
genotype differences. Saturation data was fit to a one-site non-lin-
ear regression model. Scatchard plots were fit by linear regression
for calculation of Vmax and Km. A P value of less than 0.05 was con-
sidered statistically significant. All data are shown as mean ± stan-
dard error of the mean (SEM, represented by error bars).
3. Results and discussion
3.1. Synaptic SERT expression is reduced in the midbrains of double
heterozygous mice
To examine the influence of Itgb3 heterozygosity on SERT
expression and uptake activity, we studied Itgb3+/À
and Slc6a4+/À
,
Itgb3+/À
mice. Whereas SERT expression patterns in midbrain neu-
rons and in projection areas have been extensively studied (Bengel
et al., 1997; Tao-Cheng and Zhou, 1999), we have little information
on the expression of integrin avb3 in the intact brain. Few studies
have identified post-synaptic expression of integrin avb3 in hippo-
campal synapses (Cingolani et al., 2008); moreover, it is possible
that extracellular-matrix proteins, which bind integrins, maintain
synaptic structure and thus pre- and post-synaptic interactions
may be essential for proper synaptic function (Wang et al., 2008).
Therefore, to examine the influence of Slac6a4 and Itgb3 heterozy-
gozity in synaptic SERT expression and uptake activity, we isolated
synaptoneurosomes in the presence of CaCl2 and MgCl2, maintain-
ing N-cadherin, NCAM, and integrin-mediated interactions (Phil-
lips et al., 2001).
We prepared synaptoneurosomes from midbrain, hippocampus,
and cortices dissected from WT, Itgb3+/À
, Slc6a4+/À
; and Itgb3+/À
,
Slc6a4+/À
littermates and assessed [3
H]-citalopram binding.
The data revealed a significant a significant reduction in
2 A. Whyte et al. / Neurochemistry International xxx (2013) xxx–xxx
Please cite this article in press as: Whyte, A., et al. Serotonin transporter and integrin beta 3 genes interact to modulate serotonin uptake in mouse brain.
Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.09.014
3. [3
H]-citalopram binding in the context of Slc6a4 heterozygosity in
midbrain synaptoneurosomes (Fig. 1a). We used Western blot
analysis to determine whether these changes may correspond to
reductions in SERT expression in terminals. Our data indicates that
Slc6a4 modifies SERT expression in midbrain terminals (Fig. 1b and
c). Similar findings were found in previous studies of the Slc6a4+/À
mice (Bengel et al., 1998). As synapse number/size may be influ-
enced by 5-HT signaling (Udo et al., 2005) or integrin function
(Cingolani et al., 2008), we assessed syntaxin expression as a con-
trol for pre-synaptic terminal expression. No significant changes
were found in integrin av, integrin b3 or syntaxin expression
(Fig. 1b). We found no significant alterations in [3
H]-citalopram
binding in synaptic preparations from two terminal fields: hippo-
campus and cortex (Fig. 1d and e, respectively). These findings
indicate that, although SERT tissue expression may be influenced
by genotype, neither Slc6a4 nor Itgb3 modified synaptic SERT
expression in the two terminal fields examined. The discrepancy
between midbrain and cortical and hippocampal SERT synaptic
expression may be due to differences in the localization of SERT
in these brain regions. While SERT is strictly localized to axonal/
pre-synaptic terminals in cortex and hippocampus, both at the
perisynaptic plasma membrane and in intracellular vesicles, mid-
brain SERTs localize to both axonal/pre-synaptic and dendritic/
post-synaptic terminals (Tao-Cheng and Zhou, 1999). It is possible
that axonal SERT localization is tightly regulated by trafficking
mechanisms, independent of the total protein expressed in the cell
body, whereas dendritic expression, predominantly intracellular,
may be directly correlated with mRNA/protein expression at the
cell body. To determine whether these changes in expression are
correlated with changes in SERT function, we performed 5-HT
reuptake studies.
3.2. Itgb3 and Slc6a4 interact to modulate SERT uptake
Saturation kinetic analysis of SERT 5-HT uptake from midbrains
synaptoneurosomes revealed a significant interaction between
Slc6a4 and Itgb3 genes (Fig. 2a–c). Itgb3+/À
mice exhibited signifi-
cant reductions in midbrain Vmax (Fig. 2b) compared to WT, while
Itgb3+/À
, Slc6a4+/À
mice exhibited normal 5-HT uptake (Fig. 2b). No
significant changes were observed in Km (Fig. 2c). This result sup-
ports the hypothesis that axonal/pre-synaptic plasma membrane
SERT expression is not reduced in Slc6a4+/À
or Itgb3+/À
, Slc6a4+/À
mice and replicates the initial functional studies performed in
Slc6a4+/À
mice (Bengel et al., 1998). Our results reveal that hetero-
zygosity in the Itgb3+/À
line is sufficient to reduce SERT activity, by
a mechanism yet unidentified, as no changes in integrin avb3 were
observed in the same preparations. It is possible that pre-synaptic
levels of integrin avb3 are modified, but remain undetected in syn-
aptoneurosomal preparations. Importantly, no 5-HT uptake
changes were observed in Itgb3+/À
platelets (Carneiro et al.,
2008), suggesting a specific neuronal mechanism for integrin
avb3 modulation of SERT activity in the midbrain.
5-HT uptake was differentially modulated in the two terminal
fields studied. In the hippocampus (Fig. 2d–f), we found significant
Vmax increases driven by Slc6a4 (Fig. 2e). No changes in Km were
observed (Fig. 2f). In cortical preparations, Vmax reductions were
found in Itgb3+/À
mice compared to WT (Fig. 2h). These changes
in hippocampal uptake are not replicated in other studies; most
laboratories report reductions in hippocampal Vmax in Slc6a4+/À
mice (Murphy and Moya, 2011). These differences may result from
different fractionation methods used to isolate 5-HT synapses i.e.
synaptosomes vs. synaptoneurosomes. Thus, we have detected
changes in SERT transport activity, increases driven by genetic
Fig. 1. SERT expression levels are reduced in midbrain synapses of Itgb3+/+
, Slc6a4+/À
and Itgb3+/À
, Slc6a4+/À
mice. (a) Two-way ANOVA reveals significant contributions of
Slc6a4 to midbrain synaptoneurosomal [3
H]-citalopram binding. WT: 143.8 ± 14.46 fmol/mg, n = 12; Itgb3+/À
: 115.6 ± 19.47 fmol/mg, n = 12; Slc6a4+/À
: 98.26 ± 13.06 fmol/mg,
n = 12; Itgb3+/À
, Slc6a4+/À
: 97.18 ± 8.43 fmol/mg, n = 12; two-way ANOVA: Slc6a4 P < 0.05. (b) Representative Western blot showing SERT, integrin av, integrin b3 and syntaxin
(as a presynaptic control) expression in midbrain synaptoneurosomes. (c) Densitometry analysis of Western blots for SERT expression levels in midbrain synaptoneurosomes.
WT: 100 ± 0%, n = 9; Itgb3+/À
, Slc6a4+/+
: 89.88 ± 19.4%, n = 12; Itgb3+/+
, Slc6a4+/À
: 66.26 ± 18.4%, n = 10; Itgb3+/À
, Slc6a4+/À
: 33.68 ± 9.79%, n = 8; Kruskal–Wallis one-way ANOVA:
P < 0.05, Dunn’s post hoc WT vs. Itgb3+/À
, Slc6a4+/À
: ⁄
P < 0.05. Hippocampus (d) and cortex (e) synaptoneurosomal [3
H]-citalopram binding is not significantly different across
terminal fields examined. Data is shown as means ± SEM.
A. Whyte et al. / Neurochemistry International xxx (2013) xxx–xxx 3
Please cite this article in press as: Whyte, A., et al. Serotonin transporter and integrin beta 3 genes interact to modulate serotonin uptake in mouse brain.
Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.09.014
4. alterations in Slc6a4 in the hippocampus, and decreases driven by
Itgb3. Together, in Itgb3+/À
, Slc6a4+/À
preparations, we observe no
significant changes in SERT uptake activity.
How can we interpret alterations in SERT uptake vs. expression
levels in Itgb3+/À
, Slc6a4+/À
mice? One possibility is the presence of
two distinct SERT populations at the plasma membrane. One pop-
ulation has high capacity for 5-HT uptake, which is preferentially
expressed at the plasma membrane of Slc6a4+/À
mice. The second
population, perhaps with a low-capacity uptake, is absent in
Slc6a4+/À
mice. Therefore 100% of SERTs at the plasma membrane
of Slc6a4+/À
mice have high capacity uptake, independently of
Itgb3. As integrin avb3 may modulate SERT levels at the plasma
membrane (Carneiro et al., 2008), Itgb3+/À
mice may have reduced
plasma membrane SERT expression in both low- and high-capacity
uptake populations, and thus the resulting 5-HT reuptake observed
is significantly reduced. While highly speculative, this interpreta-
tion is supported by the data observed in hippocampal prepara-
tions, where Itgb3 heterozygozity has no effect in SERT uptake
and Slc6a4+/À
mice show elevated SERT activity. Interestingly, these
data suggest a protective effect of Slc6a4 heterozygozity in the con-
text of Itgb3 heterozygozity. As both genes are highly polymorphic
in humans, it is possible that this genetic interaction may play an
important role in conferring risk for neuropsychiatric disorders.
4. Conclusions
Our studies reveal a surprising genetic interaction between
Slc6a4 and Itgb3 in the modulation of 5-HT uptake in midbrain
Fig. 2. 3
[H] 5-HT uptake in synaptoneurosomes reveals differential changes in midbrain and cortices of Itgb3+/À
mice. (a) Saturation kinetic studies and Scatchard plot for
midbrain synaptoneurosomes. (b) Two-way ANOVA of Vmax for 5-HT uptake in the midbrain reveals a significant statistical interaction between Slc6a4 and Itgb3. WT:
144.4 ± 4.58 fmol/min/mg protein, n = 4; Itgb3+/À
: 91.19 ± 3.33 fmol/min/mg protein, n = 4; Slc6a4+/À
: 142.1 ± 5.3 fmol/min/mg protein, n = 4; Itgb3+/À
, Slc6a4+/À
:
156.7 ± 8.45 fmol/min/mg protein, n = 4; Two-way ANOVA: Interaction P = 0.0183; Dunnett’s post hoc WT vs. Itgb3+/À
: #
P < 0.05. (c) No changes in Km were observed in
the midbrain. (d) Saturation kinetics studies and Scatchard plots for SERT-mediated 5-HT uptake from hippocampal synaptoneurosomes. (e) Two-way ANOVA analysis of Vmax
values obtained in d reveal significant contributions of Slc6a4 to 5-HT Vmax in the hippocampus. WT: 160 ± 12 fmol/min/mg protein, n = 4; Itgb3+/À
: 119.1 ± 4.55 fmol/min/mg
protein, n = 4; Slc6a4+/À
: 150 ± 4.99 fmol/min/mg protein, n = 4; Itgb3+/À
, Slc6a4+/À
: 148.5 ± 3.5 fmol/min/mg protein, n = 4; two-way ANOVA: Slc6a4: P = 0.0330. (f) No
significant changes were observed in Km. (g) Saturation kinetics studies and Scatchard plots for SERT-mediated 5-HT uptake in cortical synaptoneurosomes. (h) Two-way
ANOVA analysis of cortical SERT Vmax reveals significant reductions in Itgb3+/À
samples. WT: 84.37 ± 5.79 fmol/min/mg protein, n = 4; Itgb3+/À
: 53 ± 2 fmol/min/mg protein,
n = 4; Slc6a4+/À
: 73.76 ± 3.1 fmol/min/mg protein, n = 4; Itgb3+/À
, Slc6a4+/À
: 73.76 ± 3.02 fmol/min/mg protein, n = 4; two-way ANOVA: Interaction P = 0.0731; Dunnett’s post
hoc Itgb3+/À
, Slc6a4+/À
vs. Itgb3+/À
: #
P < 0.05. (i) No significant changes were observed in SERT Km in cortical preparations. Data is shown as means ± SEM.
4 A. Whyte et al. / Neurochemistry International xxx (2013) xxx–xxx
Please cite this article in press as: Whyte, A., et al. Serotonin transporter and integrin beta 3 genes interact to modulate serotonin uptake in mouse brain.
Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.09.014
5. and cortical synapses. In accordance to previous studies, deletion
or loss of function in Itgb3 reduces SERT uptake capacity in some
brain areas, whereas Slc6a4 heterozygozity seems to increase or
has no effect in SERT uptake. These effects suggest an important
role of integrin avb3 in the midbrain. Future studies will reveal
whether these alterations are sufficient to modify 5-HT signaling.
Acknowledgements
We thank Dennis Murphy and Richard Hynes for generating the
Slc6a4 and Itgb3 lines used in this paper. We thank Jeremy Veen-
stra-Vanderweele and Randy D. Blakely for many helpful discus-
sions. This work was supported by NIMH Grant 090256-01A1.
References
Bengel, D., Johren, O., Andrews, A.M., Heils, A., Mossner, R., Sanvitto, G.L., Saavedra,
J.M., Lesch, K.P., Murphy, D.L., 1997. Cellular localization and expression of the
serotonin transporter in mouse brain. Brain Res. 778, 338–345.
Bengel, D., Murphy, D.L., Andrews, A.M., Wichems, C.H., Feltner, D., Heils, A.,
Mossner, R., Westphal, H., Lesch, K.P., 1998. Altered brain serotonin homeostasis
and locomotor insensitivity to 3,4-methylenedioxymethamphetamine
(‘‘Ecstasy’’) in serotonin transporter-deficient mice. Mol. Pharmacol. 53, 649–
655.
Carneiro, A.M., Cook, E.H., Murphy, D.L., Blakely, R.D., 2008. Interactions between
integrin alphaIIbbeta3 and the serotonin transporter regulate serotonin
transport and platelet aggregation in mice and humans. J. Clin. Invest. 118,
1544–1552.
Caspi, A., Sugden, K., Moffitt, T.E., Taylor, A., Craig, I.W., Harrington, H., McClay, J.,
Mill, J., Martin, J., Braithwaite, A., Poulton, R., 2003. Influence of life stress on
depression: moderation by a polymorphism in the 5-HTT gene. Science 301,
386–389.
Cingolani, L.A., Thalhammer, A., Yu, L.M., Catalano, M., Ramos, T., Colicos, M.A.,
Goda, Y., 2008. Activity-dependent regulation of synaptic AMPA receptor
composition and abundance by beta3 integrins. Neuron 58, 749–762.
Hodivala-Dilke, K.M., McHugh, K.P., Tsakiris, D.A., Rayburn, H., Crowley, D., Ullman-
Cullere, M., Ross, F.P., Coller, B.S., Teitelbaum, S., Hynes, R.O., 1999. Beta3-
integrin-deficient mice are a model for Glanzmann thrombasthenia showing
placental defects and reduced survival. J. Clin. Invest. 103, 229–238.
Murphy, D.L., Moya, P.R., 2011. Human serotonin transporter gene (SLC6A4)
variants: their contributions to understanding pharmacogenomic and other
functional GxG and GxE differences in health and disease. Curr. Opin.
Pharmacol. 11, 3–10.
Phillips, G.R., Huang, J.K., Wang, Y., Tanaka, H., Shapiro, L., Zhang, W., Shan, W.S.,
Arndt, K., Frank, M., Gordon, R.E., Gawinowicz, M.A., Zhao, Y., Colman, D.R.,
2001. The presynaptic particle web: ultrastructure, composition, dissolution
and reconstitution. Neuron 32, 63–77.
Sutcliffe, J.S., Delahanty, R.J., Prasad, H.C., McCauley, J.L., Han, Q., Jiang, L., Li, C.,
Folstein, S.E., Blakely, R.D., 2005. Allelic heterogeneity at the serotonin
transporter locus (SLC6A4) confers susceptibility to autism and rigid-
compulsive behaviors. Am. J. Hum. Genet. 77, 265–279.
Tao-Cheng, J.H., Zhou, F.C., 1999. Differential polarization of serotonin transporters
in axons versus soma-dendrites: an immunogold electron microscopy study.
Neuroscience 94, 821–830.
Udo, H., Jin, I., Kim, J.H., Li, H.L., Youn, T., Hawkins, R.D., Kandel, E.R., Bailey, C.H.,
2005. Serotonin-induced regulation of the actin network for learning-related
synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons.
Neuron 45, 887–901.
Velayudhan, A., Sunitha, T.A., Balachander, S., Reddy, J.Y., Khanna, S., 1999. A study
of platelet serotonin receptor in mania. Biol. Psychiatry 45, 1059–1062.
Wang, X.B., Bozdagi, O., Nikitczuk, J.S., Zhai, Z.W., Zhou, Q., Huntley, G.W., 2008.
Extracellular proteolysis by matrix metalloproteinase-9 drives dendritic spine
enlargement and long-term potentiation coordinately. Proc. Natl. Acad. Sci.
U.S.A. 105, 19520–19525.
Weiss, L.A., Veenstra-Vanderweele, J., Newman, D.L., Kim, S.J., Dytch, H., McPeek,
M.S., Cheng, S., Ober, C., Cook Jr., E.H., Abney, M., 2004. Genome-wide
association study identifies ITGB3 as a QTL for whole blood serotonin. Eur. J.
Hum. Genet. 12, 949–954.
Willeit, M., Sitte, H.H., Thierry, N., Michalek, K., Praschak-Rieder, N., Zill, P., Winkler,
D., Brannath, W., Fischer, M.B., Bondy, B., Kasper, S., Singer, E.A., 2008. Enhanced
serotonin transporter function during depression in seasonal affective disorder.
Neuropsychopharmacology 33, 1503–1513.
A. Whyte et al. / Neurochemistry International xxx (2013) xxx–xxx 5
Please cite this article in press as: Whyte, A., et al. Serotonin transporter and integrin beta 3 genes interact to modulate serotonin uptake in mouse brain.
Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.09.014