Molecular mechanism underlying Depression: The relationship between serotonin system and glutamate system in mouse cortical neurons and HT22 cells

Adiba SHABNAM !2 September 2014!!

Overview

• Depression: Definition !

• Causes!

• Affected Brain areas!

• Antidepressant: Mechanism !

• Serotonin Theory!

• Glutamate Theory !

• Objective !

• Experimental Method!

• Results!

• Conclusion

DEPRESSION

A common psychiatric disorder characterized by low mood, loss of pleasure, sleep and appetite changes, poor concentration and suicidal tendency

Depression

Why to study depression?• Most common psychiatric

disorder • All age group affected (max.

40-59 yrs) • Women are more at risk • Lifetime prevalence 16.2 %

(USA) • Only 20 % people experience

the symptoms of depression. • Maximum global burden of

disease in 2030 (WHO, 2008)

Causes of depression

• Genetic or hereditary • Biochemical - monoamine • Endocrine factors • psychological factors

Involved Brain Parts

Involvement of Cortex in depression

Abnormal metabolism in the prefrontal cortex in various mood disorders

Involvement of Hippocampus in depression

Reduced Hippocampal volume (Bremner et al, 2000,Lange et al, 2004; Macmaster et al, 2007;Brien et al 2004, Sheline et al 1995 )

Chronic stress selectively reduces hippocampal volume in rats: a longitudinal MRI study (Lee, 2009)

Antidepressant SEROTONIN

Sero

toni

n Th

eory

of De

pres

sion

SSRI blocks 5-HT reuptake

Serotonin theory• Most widely accepted hypothesis • Depletion of 5-HT in depressed patients • Antidepressants like SSRI inhibits its re-uptake and

increases its concentration in the synaptic cleft. !

LIMITATIONS • Not all depressed patients (50%) responds to 5-HT

agonists and SSRI. (Mahar et al, 2014) • Delayed onset of effect (2 weeks) • A reduction in 5-HT level does not precipitate in

depressive phenotype in healthy individuals

What is the reason for the lag?

Glut

amat

e Th

eory

of De

pres

sion

Involvement of glutamate system in depression

The rapid response of ketamine (Berman et al, 2000)

Glutamatergic theoryMechanism of action of

Ketamine! AMPA-R activation

mTOR signalling pathway activated in prefrontal cortex

GluR1, Synapsin, spine density Synaptogenesis BDNF translation

Kavalali, 2012

Glutamate system also has antidepressant effect

Group&I&and&II&mGluR&antagonists&NMDAR&antagonists&

An6depressant�

5HT1A&agonist&5HT2A/2C&antagonist&

Ac6va6on&of&AMPAR�

BDNF&�&mTOR&signaling&�&Synapse&forma6on��

5HT&&&&�&BDNF&��(hippo,&cortex)&

Glutamate Receptor: classification

Glutamate system is related to serotonin system

• Dosing of fluoxetine (SSRI) changed AMPA mRNA expression and AMPA receptor phosphorylation in frontal cortex and hippocampus (Barbon et al, 2006)

• AMPA knockout mice displayed decreased serotonin levels.

• Serotonin may mediate the antidepressant effects of ketamine.

5HT1A & 5HT2A/2C receptors in depression

5HT1A R • 5HT-1a R agonists (8-OH-DPAT) produce antidepressant

like effect (Cryan, 2005). • 5HT-1A auto-receptor desensitize with antidepressant drugs. • Presynaptic 5HT1A R (autoreceptor): risk for depression • Postsynaptic 5HT1A R : produce antidepressant effect • 5HT-1A R mediates ketamine effect (Fukumoto et al, 2014) 5HT2A R • Antidepressant drugs block 5-HT2A R mediated responses

(Celada et al, 2004)

Objective of the experiment

Investigation of the effect of 5-HT1A agonist and 5-HT 2A/2C agonist on m-RNA expression of AMPA-R (GluR1, GluR2, GluR3, GluR4), and BDNF

5HT 2A/2C agonist

5HT,BDNF

Group II mGluR antagonist

NMDA-R antagonist

AMPA-R activation mTOR signaling BDNF, Synaptogenesis

Antidepression

5HT1A agonist

Mechanism of Depression

Experiment

m-RNA expression of GluR1, GluR2, GluR3, GluR4, BDNF

5-HT 1A agonist !(8-OH-DPAT)

5-HT 2A/2C agonist!(DOI)

Experimental methodology

Cortical cell & HT-22 cells culture

5-HT 1A & 5-HT 2A/2C agonists addition

Total RNA Isolation

Real time PCR Analysis

Cell culture and TrypsinizationCells +S-DMEM

HBSS + 0.05% trypsin-EDTA

Drug Addition: Serotonin Agonist

8-OH-DPAT and DOI at 4 different concentrations

!8-OH DPAT

(0 nM) 8-OH DPAT

(1nM)8-OH DPAT

(10nM)8-OH DPAT

(100nM)

DOI (0 nM)

DOI (1 nM)

DOI (10 nM)

DOI (100 nM)

After 4 hours (HT22 cells)

RNA IsolationSample!

+ RNAiso Plus!

+ Chloroform!

+ Isopropanol!

+ RNase free water

Absorption Spectrometry

Total RNA concentration measurement

DNA Removal

Thermal cycler

Isolated RNA!+!

RNase free water !+!

7 * gDNA wipeout buffer

Reverse Transcription

Template RNA !+!

RT Primer mix !+!

Quantiscript Reverse Transcriptase!+!

Quantiscript RT Buffer!+!

Placed in thermal cycler!

REAL TIME PCR

Amplification Plot

Melting curve

RESULTS

Relative expression: 8-OH-DPAT on cortical cells

GluR2

Rela

tive

Expl

essio

n0.0

15.0

30.0

45.0

60.0

8OH_0nM 8OH_1nM 8OH_10nM 8OH_100nM

GluR1

Rela

tive

Expr

essio

n

0.0

0.4

0.8

1.2

1.6

8OH_0nM 8OH_1nM 8OH_10nM 8OH_100nM

Decreased m-RNA expression of GluR1!&!

Increased m-RNA expression of GluR2

Relative expression: 8-OH-DPAT on HT-22 cells

GluR3

Rel

ativ

e Ex

pres

sion

0.00E+00

2.50E-03

5.00E-03

7.50E-03

1.00E-02

8OH 0nM 8OH 1nM 8OH 10nM 8OH 100nM

BDNF

Rel

ativ

e Ex

pres

sion

0.00E+00

1.25E-02

2.50E-02

3.75E-02

5.00E-02

8OH 0nM 8OH 1nM 8OH 10nM 8OH 100nM

Decreased m-RNA expression of GluR3 & BDNF

Rel

ativ

e Ex

pres

sion

0.00E+00

2.50E+00

5.00E+00

7.50E+00

1.00E+01

DOI 0nM DOI 1nM DOI 10nM DOI 100nM

Relative expression: DOI on HT-22 cells

Increased mRNA expression of BDNF

Conclusion • Decreased mRNA expression of GluR1 and increased mRNA expression of GluR2 by 8-OH-DPAT (Cortex)!

• Decreased mRNA expression of GluR3 and BDNF by 8-OH-DPAT(HT22 cells)!

• Increased mRNA expression of GluR3 and BDNF by DOI (HT22 cells)!

• No amplification of GluR1, GluR2, GluR4,(HT22 Cells)!!SHORTFALLS!

• Manual error ? • defective primers ? (>1 year) • cDNA of HT-22 cells got

modified.Interaction between GluR and cDNA of HT22 cells