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

0,00 0,01 0,02 0,03 0,040

5

10

15

20

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

60

80

100

120

140

160

expressioncaspase-3Basal

compensation

0 1 4

*

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

0

20

40

60

80

100

120

140

160

180

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

0 5 100

120

240

360

480

600

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

0

50

100

150

200

250

0

20

40

6080

100

120

140

Dis

tan

ce r

un

, cm

Imm

ob

ilit

y t

ime,

s

*

*

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

0

5

10

15

20

25

30

35

40

GFR,

ml/h

-1.

100g

b.w

t-1 ** ***

###

-base level-water loading -water loading + 5-HTP injection

Glomerular filtration rate

0

5

10

15

20

25

30

35

40

45

WISTAR BRATTLEBORO

TPH

ACTI

VITY

MIDDLE HYPOTHALAMUS

*****

++++++

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