Post on 31-Jan-2016
description
Nikolay N. Dygalo
Institute of Cytology & Genetics, RAS, Novosibirsk
International WorkshopEC-Russia: Cooperation in Medicine and Public Health
St.-Petersburg 11-12 October, 2007
International WorkshopEC-Russia: Cooperation in Medicine and Public Health
St.-Petersburg 11-12 October, 2007
Functional neurogenomics: brain neurochemical systems in
regulation of psychoneuroendocrine functions
Functional neurogenomics: brain neurochemical systems in
regulation of psychoneuroendocrine functions
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Cortex
Hippocampus
Cerebellum
Brainstem
DN
A f
rag
me
nta
tio
n in
de
x
p20 caspase 3 level (rel.units)Brainstem
Hippocamp.
Cerebellum
Cortex
Experimental Model of Neurodegeneration (natural apoptotic cell death in the week-old rat brain)
Caspase-3 proteinDNA fragmentation
Menshanov, Bannova, Dygalo, Neurochem. Res. 2006Bannova et al., Developmental Brain Res. 2004
Alpha2-adrenergic receptors (α2-ARs) regulate apoptosis in the cortex
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expressioncaspase-3Basal
compensation
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Cas
ppas
e-3
mR
NA
, % to
bas
al
Clonidine, mkg
Solvent Scrambled oligonucleotide Antisense to alpha2A-AR
Antisense knock-down of α2A-ARs increased caspase-3 expression in the week-old rat cortex and α2-AR agonist clonidine decreased it.
Data evidence for neuroprotective potency of α2-AR agonists in the cortex.
Control Yohimbine Clonidine Clonidine + Yohimbine
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casp
ase
-3 m
RN
A,%
,
caspase-3 mRNA
β-actin mRNA
Contro
l
Yohimbine
Clonidine
Clonidine +
Yohimbine
Stimulation of 2-AR increases caspase-3 mRNA in the week old rat brainstem
Stimulation of 2-AR increases caspase-3 mRNA in the week old rat brainstem
Cl
Cl
N
NH
NH
NH
N
H
H
H
OH
CO
OCH3
Cl
Cl
N
NH
NH
NH
N
H
H
H
OH
CO
OCH3
- Antagonist- Antagonist- Agonist- Agonist
Cl
Cl
N
NH
NH
NH
N
H
H
H
OH
CO
OCH3
Dygalo N.N. et al., Developmental Brain Res. 2004.Menshanov PN, Bannova AV, Dygalo NN. Neurochem Res. 2006.
Oligonucleotides (ОDN) enter brain cells
Oligonucleotides (ОDN) enter brain cells
5-day-old rat brain
Hematoxylin & Eosin Green fluorescence - ОDN
Site of injection
Sequence-specific inhibition of the gene expression in the brain in vivo
RNA interference:
5-day-old rat brain
Site of injection of siRNA targeted α2A-adrenergic receptor (α2A-AR) gene
siRNA inhibited targeted-gene expression in the brain in vivo
siRNA inhibited targeted-gene expression in the brain in vivo
-actin mRNA
α2А-АR mRNA
Substances injected into the brain
Shishkina G.T., Kalinina T.S., Dygalo N.N. Neuroscience, 2004.
dcRNAdcRNA siRNAsiRNASalineSaline
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Tim
e t
o t
he fi
rst
paw
movem
en
t, s
ec
Clonidine, mkgClonidine, mkgClonidine, mkgClonidine, mkg
SalineSaline dsRNA dsRNA siRNAsiRNA
*
F2,55=15.82; p<0.001F2,55=15.82; p<0.001
RNA interference (targeting 2A-AR mRNA) blocks behavioral response of the animal to the receptor stimulation
RNA interference (targeting 2A-AR mRNA) blocks behavioral response of the animal to the receptor stimulation
r = -0.485, p<0.05
Up-regulation of tryptophan hydroxylase-2 mRNA in the rat brain by chronic fluoxetine treatment correlates with its antidepressant effect
TPH2 mRNA
Real-time PCR
Tissue sample
Immobility in the forced-swim test
Correlation immobility - TPH2 mRNA
Shishkina, Kalinina, Dygalo, Neuroscience, 2007
Antidepressant Sensitive Catalepsy (ASC) mouse strain with depressive-like features
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Dis
tan
ce r
un
, cm
Imm
ob
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y t
ime,
s
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AKR CBA ASC
*
Catalepsy in ASC mice is sensitive to chronic, but not acute, treatment with classic
antidepressant drugs, imipramine and fluoxetine.
Bazovkina et al., Genetika, 2005Tikhonova et al., Bull. Exp. Biol. Med., 2006
ASC mice have decreased open field locomotion
AKR CBA ASC
ASC mice have increased immobility in the forced swim
test
ASC – originated from backcrosses between CBA и AKR mouse strains
Linkage between the main gene of catalepsy and 5-HT1A serotonin receptor
in mice
QTL analysis mapped the main gene of catalepsy on the 50-75 cM
fragment of mouse chromosome 13 near the 5-HT1A receptor gene
Selective breeding of mice for catalepsy removes the AKR-
derived marker linked to 5-HT1A receptor and fixed the CBA-like.
Kulikov et al., Dokl. Acad. Sci. 2003, Genetika, 2003 Kondaurova et al., Genes Brain Behav., 2006
C1473G polymorphism in mouse tph2 gene (tryptophan hydroxylase-2) determines the activity of this key enzyme of
serotonin synthesis in the brain and intermale aggressiveness in mice
Strains: 1-CBA, 2-PT, 3-C57BL/6, 4-C3H/He, 5-AKR, 6-YT, 7-DD, 8-BALB/c, 9-A/He, 10-CC57BR, 11-DBA/2
Kulikov et al., Genes Brain Behav., 2005
Number of attacks
C1473G polymorphism in mouse tph2 gene Tph2 activity
Sequencing of the 5-HT1A serotonin receptor gene of hibernating ground squirrel revealed insertion of three
nucleotides (marked in red) in the third intracellular loop that is lacking in the 5-HT1A receptor gene of nonhibernators
Naumenko et al., Genes Brain and Behavior, 2007
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GFR,
ml/h
-1.
100g
b.w
t-1 ** ***
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-base level-water loading -water loading + 5-HTP injection
Glomerular filtration rate
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WISTAR BRATTLEBORO
TPH
ACTI
VITY
MIDDLE HYPOTHALAMUS
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Activity of TPH2 (pmol/mg/min)
-control-water deprivation -hydration
WISTAR BRATTLBORO
Ivanova et al. Neuroendocrinology, 2007Popova et al. Pflugers Arch., 2002
Vasopressin deficiency affects brain 5-HT system reactions to hydration and water deprivation.
Serotonin has a role in regulation of renal function.
Vasopressin deficient Brattboro rats were used to analyze interrelations between serotonin and vasopressin in regulation of renal function
Models:Experimental Model of NeurodegenerationGenetic Models of Psychopathology Genetic Model of Vasopressin DeficiencyNatural Model of specific form of behavior (hibernation)Depression-like behavior
Methods:Gene silencing: RNA interference, Antisense knockdown Gene expression: Real-Time PCR, RT-PCR, WesternblotGene sequencingQuantitative trait linkage analysisNeurochemistry: Enzymes activities, Receptors densities, Neurotransmitters concentrations (HPLC)Behavioral testsMeasurements of physiological functions
Applications:NeurodegenerationPsychopathologyTarget validation for new drugsSearch for new targets of the existing drugs
Participants of the work:
Institute of Cytology & Genetics, RAS, Novosibirsk Laboratory of Functional Neurogenomics Laboratory of Behavioral Neurogenomics Laboratory of Physiological Genetics
Institute of Chemical Biology & Fundamental Medicine, RAS, Novosibirsk
Thank you for attention