Genetic disorders: Bipolar disorder Research approach ...
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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.
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