Download At the same time, thyroid hormone deficit markedly decreased Bmax

Functional neurogenomics:
brain neurochemical systems in regulation
of psychoneuroendocrine functions
Nikolay N. Dygalo
Institute of Cytology & Genetics, RAS, Novosibirsk
International Workshop
EC-Russia: Cooperation in Medicine and Public Health
St.-Petersburg 11-12 October, 2007
Experimental Model of Neurodegeneration
(natural apoptotic cell death in the week-old rat brain)
DNA fragmentation
DNA fragmentation index
Caspase-3 protein
20
Brainstem
15
Cortex
Hippocampus
10
5
Cerebellum
0
0,00
0,01
0,02
0,03
0,04
p20 caspase 3 level (rel.units)
Bannova et al., Developmental Brain Res. 2004
Menshanov, Bannova, Dygalo, Neurochem. Res. 2006
Solvent
Scrambled oligonucleotide
Antisense to alpha2A-AR
*
e
160
ns
140
ti
ic
log
An
co
120
ma
sе
ar
Ph
Casppase-3 mRNA, % to basal
Alpha2-adrenergic receptors (α2-ARs) regulate apoptosis in the cortex
compensation
100
80 Basal
60
caspase-3
expression
0
1
4
Clonidine, mkg
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.
Stimulation of 2-AR increases caspase-3 mRNA in the
week old rat brainstem
caspase-3 mRNA
caspase-3 mRNA,%
β-actin mRNA
180
Control
Yohimbine - Antagonist
Clonidine - Agonist
Clonidine + Yohimbine
160
140
120
100
80
60
Cl
N
N
H
H
O
N
H
,
N
H
Cl
C
OCH3
Cl
0
N
H
NH
H
O
N
H
H
H
C
OH
NOCH3
N
40
20
N
H
H
NH
Cl NH
OH
Cl NH
NH
Cl NH
H
O
C
OCH3
Dygalo N.N. et al., Developmental Brain Res. 2004.
Menshanov PN, Bannova AV, Dygalo NN. Neurochem Res. 2006.
OH
RNA interference: Sequence-specific inhibition of the gene
expression in the brain in vivo
5-day-old rat brain
Oligonucleotides (ОDN)
enter brain cells
Site of
injection
Hematoxylin & Eosin
Green fluorescence - ОDN
5-day-old rat brain
siRNA inhibited targeted-gene
expression in the brain in vivo
Site of injection of siRNA targeted
α2A-adrenergic receptor (α2A-AR) gene
α2А-АR mRNA
-actin mRNA
Saline
dcRNA
siRNA
Substances injected
into the brain
Shishkina G.T., Kalinina T.S., Dygalo N.N. Neuroscience, 2004.
Time to the first paw movement, sec
RNA interference (targeting 2A-AR mRNA) blocks behavioral
response of the animal to the receptor stimulation
Saline
dsRNA
siRNA
600
480
360
240
*
120
0
0
F2,55=15.82; p<0.001
5
10
Clonidine, mkg
Up-regulation of tryptophan hydroxylase-2 mRNA in the rat brain by
chronic fluoxetine treatment correlates with its antidepressant effect
Immobility in the forced-swim test
TPH2 mRNA
Correlation
immobility - TPH2 mRNA
Real-time PCR
Tissue
sample
r = -0.485, p<0.05
Shishkina, Kalinina, Dygalo, Neuroscience, 2007
Antidepressant Sensitive Catalepsy (ASC) mouse
strain with depressive-like features
250
200
150
*
*
CBA
ASC
100
50
Immobility time, s
0
140
120
100
80
60
40
20
0
AKR
ASC mice have decreased
open field locomotion
*
AKR
CBA
ASC
ASC mice have increased immobility
in the forced swim test
Catalepsy in ASC mice is sensitive to
chronic, but not acute, treatment
with classic antidepressant drugs,
imipramine and fluoxetine.
ASC – originated from backcrosses
between CBA и AKR mouse strains
Bazovkina et al., Genetika, 2005
Tikhonova et al., Bull. Exp. Biol. Med., 2006
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
Kulikov et al., Dokl. Acad. Sci. 2003, Genetika, 2003
Selective breeding of mice for
catalepsy removes the AKRderived marker linked to 5-HT1A
receptor and fixed the CBA-like.
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
C1473G polymorphism in mouse tph2 gene
Tph2 activity
Number of attacks
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
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
Vasopressin deficient Brattboro rats were used to analyze interrelations
between serotonin and vasopressin in regulation of renal function
Vasopressin deficiency affects brain 5-HT system
reactions to hydration and water deprivation.
Activity of TPH2 (pmol/mg/min)
45
40
**
Glomerular filtration rate
-control
-water deprivation
-hydration
40
MIDDLE HYPOTHALAMUS
***
-base level
-water loading
-water loading + 5-HTP injection
35
30
25
+++
20
+++
15
10
GFR, ml/h-1.100g b.wt-1
35
TPH ACTIVITY
Serotonin has a role in
regulation of renal function.
30
25
20
15
5
0
0
BRATTLEBORO
Popova et al. Pflugers Arch., 2002
###
10
5
WISTAR
***
**
WISTAR
BRATTLBORO
Ivanova et al. Neuroendocrinology, 2007
Models:
Experimental Model of Neurodegeneration
Genetic Models of Psychopathology
Genetic Model of Vasopressin Deficiency
Natural Model of specific form of behavior (hibernation)
Depression-like behavior
Methods:
Gene silencing: RNA interference, Antisense knockdown
Gene expression: Real-Time PCR, RT-PCR, Westernblot
Gene sequencing
Quantitative trait linkage analysis
Neurochemistry: Enzymes activities, Receptors densities,
Neurotransmitters concentrations (HPLC)
Behavioral tests
Measurements of physiological functions
Applications:
Neurodegeneration
Psychopathology
Target validation for new drugs
Search 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