Genetic disorders: Bipolar disorder

Research approach: Pharmacogenetics

System: Model (social amoeba)

Outcome: Identification of

mechanism of drug action

Bipolar disorderBipolar disorder•Bipolar disorder or manic depression affects

around 2% of the population• It causes a 30-50 fold increase risk of suicide • It is genetically inherited disorder with children

of a bipolar parent having between 6 and 25% chance of being bipolar

•There are three main treatments for this disorder, which must be taken throughout life, and how these drugs function is not known.

Bipolar disorder

Treatments for bipolar disorder

Treatments for bipolar disorder

The three main treatments for bipolar disorder are: • lithium

– shown to affect wnt and InP3 signaling– used for over 100 years– highly toxic and requires blood level monitoring

• valproic acid (2 propyl pentanoic acid)– originally identified as an anti-epileptic treatment– now used as first treatment in AUSTRALIA and

USA due to ease of use, non-toxicity and wide spectrum activity

• carbamazepine – thought to effect ion channel function

N

CNH2O

Defining the cause of bipolar disorder

Defining the cause of bipolar disorder

• Common biochemical changes in patient populations– Difficult due to patient population heterogeneity, access, treatment,

site of action

• Biochemical effects of drugs – Phenotypic/structural/signalling events altered by drug– Can be done in variety of cell types– Commonality of drug targets

» Structurally discrete drugs effecting common targets

• Family studies– Identify inherited chromosomal regions causing the disorder and

define genes within these regions– Complicated by multiple loci and large regions identified

• Pharmacogenetics– Genes controlling resistance/sensitivity to drug

Pharmacogenetics: Pharmacogenetics: ‘The study of the effect of genetic

factors on reactions to drugs’

• Approach complicated by the inability to identify these genetic factors

– Can you knock out every gene in a mouse and test every mutant for drug response? NO!

• Pharmacogenetic screen in simple system (model) overcomes this problem

1. Can you knock out every gene and isolate clonal lines – Thus, providing loci are non-lethal, every gene in the organism can

be analysed for the ability to cause drug resistance or sensitivity

2. Can you screen each mutant– enables the identification of loci causing drug resistance/sensitivity – drug must cause some phenotypic change and the screen must be

sufficiently large to ensure every gene KO is examined

3. Are there putative signalling cascades or orthologous target genes within the genome of the model system

4. Can you analyse the biochemical processes giving rise to resistance– requiring sufficient quantities of cellular material from each

resistant clone

Is a model system a suitable for a pharmacogenetics

approach:

Is a model system a suitable for a pharmacogenetics

approach:

Dictyostelium discoideum: A pharmacogenetic model

system

Dictyostelium discoideum: A pharmacogenetic model

system• Haploid • contains 11000 genes• genetically more closely

related to animals than plants, fungi and yeast

• develops over 24 hours• unicellular part of life cycle allows

gene knockouts and isolation of isogenic lines

• Can be grown in bulk (>gram weight) for biochemical analysis

Dictyostelium discoideum

(in time lapse)

Growth & division

Chemotaxis

Multicellularity

Culmination

QuickTime™ and aVideo decompressor

are needed to see this picture.

Mostly from Rick Firtel & Rob Kay

Dictyostelium discoideum

From Rick Firtel & Rex Chisholm

Pharmacogenetics: An example

Pharmacogenetics: An example

Process1. Screen for lithium resistant mutants in

Dictyostelium2. Characterise the biochemistry leading to this

resistance3. Transfer of this knowledge to primary

mammalian neurons

A common mechanism of action for three mood–stabilizing drugs Williams, Cheng, Mudge & Harwood (2002) Nature

REMI mutagenesis and drug screening

REMI mutagenesis and drug screening

Lithium target 1: wnt signalling (GSK3)

Lithium target 1: wnt signalling (GSK3)

• In Dicytostelium, GSK functions to regulate fruiting body shape

• In mammalian systems, GSK phosphorylates downstream targets causing both structural and transcriptional changes (eg -catenin)

Ext

Int

GSK

-catenin

nucleus

signal

transcription

Li+

Lithium target 2: IP3 signalling and the inositol

depletion theory

Lithium target 2: IP3 signalling and the inositol

depletion theory

IMPase

signal

IP2

PIP2 IP3

IP1

Inositol

LiLi++

Gb

PLC

Lithium can phenocopy GSKA Null

Lithium can phenocopy GSKA Null

GSK null and wild-type cells (developed on 7mM lithium) show increased basal disk and reduced spore head, partially phenocopying the gskA null mutant

wt

gskA null

wt

+ 7mM Li

Lithium also inhibits aggregation in Dictyostelium

Lithium also inhibits aggregation in Dictyostelium

• Lithium severely retards aggregation at 10mM

• Lithium affects fruiting body shape at 7mM 10mM LiCl10mM NaCl

7mM LiCl

lisAlisC-E

Pharmacogenetics of Lithium action

Pharmacogenetics of Lithium action

• 30 000 Dictyostelium REMI mutants were screened for the ability to aggregate on media containing 10 mM lithium

• Thirteen mutants were isolated (Lis mutants: Lithium suppressor)

• These mutants were divided into two categories based upon altered gskA signalling pathway.

• Four of these mutants appear to have a wild-type gskA sigalling pathway

LisA NaCl

10mM LiCl

LisA

Continuing the analysis of LisA

LisA can aggregate in 10 mM lithium

LisA can aggregate in 10 mM lithium

LisA has a wild-type gsk signalling pathway

LisA has increased basal IP3 levels

LisA has increased basal IP3 levels

allowing it to aggregate in the presence of lithium

IP3 (

pm

ol/

10

7 c

ells)

0

5

10

15

30

25

20

gro

wt

h Na

Li

gro

wt

h Na

Li

These results suggest that high IP3 levels are necessary for aggregation

Cells can aggregate

Cells cannot aggregate

Wild type LisAIP3

IP1

Inositol

LiLi++

IP5/6

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

What is LisA? What is LisA?

•REMI insertion occurs at the N-terminus of the prolyl oligopeptidase (PO) gene

•LisA lacks activity (PO) activity

• Lithium (20mM) does not inhibit PO activity

PO

act

ivit

y

(O

D 4

05 n

m)

LisAWildtype

PO PO inhibitorinhibitor

20mM Li20mM Li

PO inhibitors also increase IP3 levels in

Dictyostelium

PO inhibitors also increase IP3 levels in

Dictyostelium30

25

20

15

10

5

0

Gro

wth

Na

Lithium+ino+PO inh

IP3 (

nm

ol/

10

7 c

ells)

•Addition of 10 mM myo-inositol or a specific prolyl oligopeptidase inhibitor to developing Dictyostelium cells overcomes the reduction in IP3 levels caused by lithium

Measuring IP3 in Dictyostelium

PO is associated with bipolar disorder

PO is associated with bipolar disorder

• Maes et al (1994/5) found bipolar patients have elevated plasma PO levels

• Successful treatment returns PO to normal

• This offers an possible molecular mechanism for depression.

• Suggests that IP3 signalling, not GSK is lithium target for affective disorders

IPIP33Li

DPO

AGGREGATION

cAMPcAMP

Li

Bipolar disorder

IPIP33

POInputInput

Does VPA and lithium share the same targetDoes VPA and lithium share the same target

Li+

GSKA

IP3 signalling

VPA?

Are the effects of VPA and lithium similar?

Are the effects of VPA and lithium similar?

VPA does not target GSK-3VPA does not target GSK-3

In vitro, VPA produces 50% inactivation of mammalian GSK3 and Dictyostelium GSKA at around 200mM (more than 300 fold plasma levels)Work by Jonathan Ryves

0

25

50

75

100

125

0.01 0.1 1

VPA (mM)

Gsk a

cti

vit

y (

% t

ota

l) Dicty gsk

Effect of VPA on GSK

gsk3

10 100 1000

125

100

75

50

25

0

Cont 10 0.1 0.5 1.0 (mM) LiCl VPA

VPAIP

3 (

pm

ol/

10

7 c

ells)

40

30

0

20

10

Li+

VPA, like lithium, lowers IP3 levels in DictyosteliumVPA, like lithium, lowers

IP3 levels in Dictyostelium

LisA is resistant to VPA during aggregation

LisA is resistant to VPA during aggregation

• LisA, shows resistance to VPA during early aggregation (8h)

• LisA has elevated IP3 levels giving it resistance to lithium during aggregation

• This suggests VPA is inhibiting an increase in IP3 during aggregation

wild typewild type LisALisA

control

1 mM VPA

( :

Thus we have defined a gene which controls resistance to both lithium and VPA by the modification of IP3 signalling………..

..using a social amoeba

Does the relationship between PO activity and anti-manic drug

action hold in mammals ?

Does the relationship between PO activity and anti-manic drug

action hold in mammals ?

Examine the effects of lithium, VPA and CBZ on primary rat dorsal root

ganglia neurons

Rat Dorsal Root Ganglion Neurons

Rat Dorsal Root Ganglion Neurons

• Assess drug effects on dorsal root ganglia neurons:

– Changes in axon structure and growth cone structure

muscle

Input(touch/pain etc)

Dorsal

Ventral

Sensory neuron

Motor neuron

Spinal cord

Dorsal root ganglion neurons

+ NGF

Neuron growth cone

Anti-manic drugs alter DRG growth cone morphology

Anti-manic drugs alter DRG growth cone morphology

Double stained for acetylated-tubulin (green) and calcein (blue - cytosol)

Simple Giantlarge

Mean growth cone size (um2)

0 30 40 50 60 70 80

control5

10

15

20

25

30

35

All three drugs reduce collapse and increase growth cone size

Li+

3 mM10 mM

VPA1 mM

3 mM

CBZ50 mM

0

GSKGSK

-catenin

P

VPA and CBZ do not inhibit phosphorylation of downstream GSK3

targets

VPA and CBZ do not inhibit phosphorylation of downstream GSK3

targets

Li+

-catenin

Cont CBZ TSACont VPA Li

Western analysis using rat DRG neurons treated for 24 hours with 10 mM Li+, 3mM VPA or 50µM CBZ

-catenin

gsk3

Mean growth cone size (um2)

0 30 40 50 60 70 80

PO inhibitor

and PO inhibitors

control Li+

VPA

CBZ

3 mM

1 mM

50 mM

Growth cone size is reversed by addition of inositol

Inositol

SummarySummary• Lithium, VPA and CBZ are used for the

treatment of manic depression• Screening a Dictyostelium mutant bank

identified prolyl oligopeptidase as controlling lithium and VPA sensitivity, via IP3 signalling

• Ablation of PO activity negates the common effect of all three anti-manic drugs in primary rat neurons

• Thus, we have linked a marker for manic depression with a common action for all three major anti-manic treatments using Dictyostelium as a model system

Reading Reading • Williams (2005) Pharmacogenetics in model systems:

defining a common mechanism of action for mood stabilizers. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 29(6), 1029-1037.

• Eickholt, Towers, Ryves, Eikel D, Adley, Ylinen, Chadborn Harwood, Nau and Williams (2005) Effects of valproic acid derivatives on inositol trisphosphate depletion, teratogenicity, GSK-3 inhibition and viral replication - A screening approach for new bipolar disorder drugs based on the valproic acid core structure. Molecular Pharmacology, 67, 1-8.

• Williams (2004) Prolyl oligopeptidase and bipolar disorder in ‘Lithium and Mood Stabilizers: Mechanisms of Action’, Clinical Neuroscience Research. Vol 4/3-4 pp 233-242.

• Williams, Cheng, Mudge, and Harwood (2002) A common mechanism of action for three mood-stabilizing drugs. Nature 417, 292-95.