Transcript Schizophrenia and the prefrontal cortex

Transcriptome changes in the
temporal cortex of subjects with autism
April 1, 2009
Károly Mirnics
Dept Psychiatry and Kennedy Center for Human Development
Vanderbilt University School of Medicine
DEVELOPMENT
INFECTIONS
NUTRITION
CHEMICALS
ENVIRONMENTAL INSULTS
GENE
EXPRESSION
IMPAIRMENTS
NEUROCHEMICAL &
METABOLIC
CHANGES
GENETIC SUSCEPTIBILITY
MET, GABRB3,
NRXN, NLGN,
RELN, CNVs…
ALTERED
CONNECTIVITY
IMPAIRED
INFORMATION
PROCESSSING
Courchesne Lab
BEHAVIOR
COGNITION
EMOTION
Watson-Crick Complementarity
• Four nucleotide types: A,C,G,T
• A’s paired with T’s (2 hydrogen bonds)
• C’s paired with G’s (3 hydrogen bonds)
The Central Dogma of Molecular Biology
Different methods,
different applications
Number of samples
1,000
RT qPCR
Expression
microarrays
100
10
Northern
ISH
SAGE
MPSS
TOGA
1
1
10
100
1,000
10,000
Number of
genes
queried
Mirnics Lab
THE BASIC PRINCIPLE OF DNA MICROARRAYS IS
COMPLEMETARY HYBRIDIZATION
Hybridization
Wash
Solid support (glass, membrane, metal)
http://www.affymetrix.com
http://www.affymetrix.com
Affymetrix GeneChips
HIGH-TROUGHPUT TRANSCRIPTOME
PROFILING METHODS ALLOW, BUT
DO NOT REQUIRE AN A PRIORY HYPOTHESIS
“HYPOTHESIS-FREE APPROACH”
“DATA-DRIVEN APPROACH”
“IGNORANCE-BASED APPROACH” (Dr. Eric Lander)
“FISHING EXPEDITION” (malicious reviewers)
… and what is wrong with fishing?!
a.k.a. Lina’s Rules
 If you don’t go fishing, you won’t catch a fish.
 To catch fish, you must wisely choose your
equipment, bait and a fishing spot.
 You are never certain what will you catch.
 Once you start catching fish, everyone will want to
fish at your fishing spot.
 Poor fisherman greatly outnumber skilled fisherman,
but they usually catch only Pomoxis annularis (crappies).
 Once you start fishing around a “taken spot”, the other
fishermen will try anything and everything to chase you
away.
 If you want to impress the tribe elders, you will have
to catch, clean, cook and properly serve the fish.
 When you talk to non-fishermen about your
adventures, they will find them boring and they will
rarely believe your stories.
 Not everyone likes fish, fishing or fishermen.
http://www.gofisherie.com/
EXPERIMENTAL DESIGN
 6 matched pairs of ASD-control subjects
(from >25 pairs surveyed)
 HG_U133plusV2 Affymetrix DNA microarrays
(RMA normalization, pairwise analysis)
 qPCR verification (SybrGreen method, ddCt method)
RESULTS
•
•
•
•
MET pathway
PKC
OAS family
Immune changes
I. Expression of MET signaling pathway
genes is impaired in ASD
Campbell et al, Ann Neurol. Vol.62, 3 Pages: 243-250
MET pathway: DNA microarray – qPCR data
Notice the decreased expression of MET and significantly increased expression
of messenger RNA for proteins involved in the activation of MET signaling.
Campbell et al, Ann Neurol. Vol.62, 3 Pages: 243-250
HEPATOCYTE GROWTH FACTOR
(HGF)
• neurotrophic factor in the developing brain
• both hepatocyte growth factor and its
receptor (MET) are present in third trimester
placentas, suggesting that HGF serves as a
paracrine mediator to control placental
development and growth
Dysregulation of MET/HGF ratio in autism
Notice that MET and HGF transcript levels are positively correlated in controls,
and negatively correlated in ASD.
Campbell et al, Ann Neurol. Vol.62, 3 Pages: 243-250
II. In addition to genetic susceptibility,
PKCB1 expression is reduced in the
temporal cortex of subjects with ASD
Lintas, C., Sacco, R., Gaita, L., D’Oronzio, R., Garbett, K., Mirnics, K., Militerni, R.,
Bravaccio, C., Curatolo, P., Manzi, P., Schneider, C., Melmed, R., Elia, M. & Persico, A.M.
(2007) Involvement of the PRKCB1 gene in autistic disorder: significant genetic
association and reduced neocortical gene expression. Mol Psychiatry, in press.
Protein Kinase C-beta (PRKCB1)
• located on chromosome 16 p
• a family of serine- and threonine-specific protein
kinases that can be activated by calcium and second
messenger diacylglycerol
• plays an important role in signal transduction,
regulation of gene expression and control of cell
division and differentiation in the brain and epithelium
• Previous reports of association with autism (Philippi
et al, 2005)
Both PRKCB1 probesets show a downregulation
(DNA microarrays and qPCR data)
III. The OAS gene expression is increased in autism
(2’-5’- OLIGOADENYLATE SYNTHETASE)
• 3 genes, OAS1, OAS2, OAS3
• Bind to and activate RNase L, which degrades viral
and cellular RNAs, leading to inhibition of cellular
protein synthesis and impairment of viral replication
• mediates resistance to virus infection
• implicated in the control of cell growth, differentiation,
and apoptosis.
Postmortem expression data lead to identification of the
OAS (2',5'-oligoadenylate synthetase) gene cluster as
an ASD susceptibility locus
12q24.2
Confidential information, collaboration between the Levitt, Sutcliffe, Persico and Mirnics
laboratories, to be submitted.
OAS gene cluster is an ASD
susceptibility locus
 629 pedigrees including 1,228 individuals with ASD plus 312 unrelated
controls
 Association of the OAS1 marker rs3741981 allele G (P = 0.032) and a
four-marker haplotype spanning the OAS1, OAS3 and OAS2 genes (P =
0.008).
 292 individuals with narrowly-defined autism revealed significant
association of the OAS3 marker rs2285932 allele C by both family-based
association test (P = 0.008) and case-control analyses (P = 0.019).
 The relative risk of autism diagnosis was 2.745 (95% CI: 1.169-6.449)
for the rs2285932 C/C genotype and 3.277 (95% CI: 1.386-7.747) for the C/T
genotype compared to the T/T genotype.
IV. Profound dysregulation of immune
system genes in the temporal cortex
with ASD
Garbett et al, Neurobiol Dis, 30 (3):303-11, 2008
PROBESET
GENE
SYMBOL
prPval
grPval
Fold
244699_at
heat shock 70kDa protein 6 (HSP70B')
HSPA6
0.03223
0.03902
12.9
242138_at
serpin peptidase inhibitor, clade H (heat shock protein 47), mem 1
SERPINH1
0.02988
0.01320
11.1
241758_at
BCL2-associated athanogene 3
BAG3
0.02634
0.01613
8.4
240282_at
adrenomedullin
ADM
0.01214
0.00691
7.7
239001_at
chitinase 3-like 1 (cartilage glycoprotein-39)
CHI3L1
0.04795
0.03390
7.3
238320_at
GTP binding protein overexpressed in skeletal muscle
GEM
0.03275
0.02341
6.8
237584_at
growth arrest and DNA-damage-inducible, beta
GADD45B
0.03507
0.02532
5.6
236106_at
heat shock 27kDa protein 1 /// Meis1, homolog 3 (mouse)
HSPB1
0.01708
0.01592
5.5
236034_at
trophoblast-derived noncoding RNA
TncRNA
0.01877
0.01900
5.4
235420_at
growth arrest and DNA-damage-inducible, beta
GADD45B
0.01990
0.02254
5.1
234989_at
nucleoside phosphorylase
NP
0.04096
0.02375
5.0
234024_at
tubulin, beta 6
TUBB6
0.01193
0.01366
4.8
233471_at
TIMP metallopeptidase inhibitor 1
TIMP1
0.04213
0.02646
4.8
233364_s_at
TGFB-induced factor (TALE family homeobox)
TGIF
0.00522
0.00125
4.5
232882_at
cold shock domain protein A
CSDA
0.00614
0.01571
4.5
232693_s_at
C-type lectin domain family 2, member B
CLEC2B
0.01113
0.00592
4.3
232668_at
cysteine-rich, angiogenic inducer, 61
CYR61
0.01124
0.03898
4.2
230482_at
growth arrest and DNA-damage-inducible, beta
GADD45B
0.04512
0.03109
4.2
230304_at
collagen, type IV, alpha 1
COL4A1
0.02879
0.03452
4.1
230109_at
phospholipase A1 member A
PLA1A
0.03479
0.01116
4.0
229435_at
Yes-associated protein 1, 65kDa
YAP1
0.03955
0.03993
4.0
228758_at
CDNA FLJ26120 fis, clone SYN00419
---
0.00445
0.00372
4.0
~110 genes separate the ASD and CNT phenotypes
NEURONAL DEVELOPMENT IS DISRUPTED
IN THE BRAINS OF SUBJECTS WITH ASD
Cell communication and motility
Cell fate and differentiation
EMP1
CSDA
SPP1
CHI3L1
ADM
DTNA
CYR61
TIMP1
TGIF
COL4A1
tncRNA
IFITM2
BCL6
CRYAB
BAG3
PLSCR1
HSPB1
INHBA
BAG2
FOXO1A
S100A10
AQP4
RFX4
ITGAV
F3
HSPA6
IFI16
PTDSR
IFITM3
CDS2
GADD45G
SERPINH1
IL1R1
CLEC2B
GADD45B
GEM
EMP3
SCARA3
TNFRSF1A
DNAJB1
FAS
TPD52L1
ITGA5
TJP2
ALCAM
ADAMTS9
TNFRSF10B
DNAJB6
NFKBIZ
HSPB8
ELAVL1
CMKOR1
FGF2
CHRNA7
TNPO1
HSPA1A
MKNK2
ZAK
EFNA1
RAB13
TP53BP2
ZFP36L1
DNMBP
HMG20B
TNFRSF1B
RIT1
DLX1
TNFAIP3
TAP1
FGF12
>3 ALR
>2 ALR
>1 ALR
<-1 ALR
qPCR confirmed
Gene Set
SIZE
ES
NES
p-val
q-val
RANK
NFKBPATHWAY
22
0.83
2.10
0.0000
0.0000
2082
IL1RPATHWAY
30
0.77
2.05
0.0000
0.0000
2338
TOLLPATHWAY
32
0.75
2.05
0.0000
0.0000
2338
NKTPATHWAY
25
0.76
2.00
0.0000
0.0000
1019
INFLAMPATHWAY
28
0.73
1.96
0.0000
0.0000
2966
GSK3PATHWAY
25
0.73
1.91
0.0000
0.0020
2082
TOB1PATHWAY
16
0.79
1.87
0.0000
0.0040
2231
TNFR2PATHWAY
17
0.77
1.86
0.0000
0.0040
2207
EGFPATHWAY
16
0.79
1.86
0.0000
0.0040
2405
P38MAPKPATHWAY
37
0.66
1.84
0.0030
0.0050
2216
TIDPATHWAY
17
0.75
1.79
0.0000
0.0120
2600
G1PATHWAY
24
0.68
1.76
0.0000
0.0200
2697
HIVNEFPATHWAY
54
0.59
1.73
0.0010
0.0290
3323
41BBPATHWAY
16
0.73
1.73
0.0020
0.0270
2338
MCALPAINPATHWAY
23
0.66
1.70
0.0050
0.0340
1653
P53HYPOXIAPATHWAY
18
0.70
1.69
0.0050
0.0350
2740
METPATHWAY
36
0.61
1.68
0.0030
0.0360
1753
DEATHPATHWAY
32
0.60
1.67
0.0060
0.0370
3084
ATMPATHWAY
18
0.68
1.66
0.0160
0.0390
2338
IL6PATHWAY
20
0.67
1.66
0.0130
0.0400
2724
ASD SUBJECTS SHOW HIGH VARIABILITY IN GENE EXPRESSION
GENE NAME
SYMBOL
AUT Sd
CNT Sd
Sd
1
serpin peptidase inhibitor A3 (alpha-1 antiproteinase)
SERPINA3
4.21
0.88
3.33
2
hypothetical protein FLJ10847
FLJ10847
3.33
0.04
3.29
3
stratifin*
SFN
3.25
0.06
3.19
4
secreted frizzled-related protein 2*
SFRP2
3.44
0.29
3.15
5
secretory leukocyte peptidase inhibitor
SLPI
3.13
0.07
3.06
6
stratifin*
SFN
3.37
0.33
3.04
7
scavenger receptor class A, member 5*
SCARA5
3.70
0.66
3.03
8
heat shock 70kDa protein 6*
HSPA6
3.27
0.38
2.89
9
secreted frizzled-related protein 2*
SFRP2
3.60
0.71
2.88
10
CD44 molecule*
CD44
2.88
0.01
2.87
11
heat shock 70kDa protein 6*
HSPA6
3.69
0.94
2.75
12
scavenger receptor class A, member 5*
SCARA5
3.16
0.47
2.69
13
CD44 molecule*
CD44
3.32
0.67
2.65
14
neuronal PAS domain protein 4
NPAS4
3.75
1.16
2.59
15
CD44 molecule*
CD44
2.72
0.15
2.58
16
CD44 molecule*
CD44
2.68
0.11
2.57
17
guanylate binding protein 2, interferon-inducible
GBP2
2.64
0.07
2.57
18
chemokine (C-C motif) ligand 19
CCL19
3.07
0.51
2.56
There are significant similarities between
immune system dysregulation in autism
and schizophrenia: a common gene
expression signature of developmental
origin?
Schizophrenia is characterized by a systemic increase of
immune system transcripts in the prefrontal cortex
Arion, D., Unger, T., Lewis, D.A., Levitt, P. & Mirnics, K. (2007) Molecular evidence for
increased expression of genes related to immune and chaperone function in the
prefrontal cortex in schizophrenia. Biol Psychiatry, 62, 711-721.
Can we recapitulate the human gene
expression changes observed in ASD in a
mouse model?
Poly (I:C) treated mice
• polyinosinic-polycytidylic is an immunostimulant used
to mimic viral infection
•
binds to Toll-like receptors, elevates a wide range of
cytokine, including IL1, IL6 and IFNgamma
• Pregnant mice exposed at E13 to poly(I:C) show
sickness behavior
• Offspring shows behavioral deficits in social
interactions.
Prediction: the offspring of poly(I:C)-treated
mice will show the same gene expression
deficit as the postmortem samples from
ASD and/or schizophrenia
Result: not really. There are gene
expression changes as a result of
intrauterine poly(I:C) exposure, but they
are not common with the postmortem
findings of ASD or schizophrenia.
Question: is the immune activation seen in
humans not an immune scar, but an active
inflammatory process?
CONCLUSIONS
• DNA microarray analysis of the postmortem tissue originating from autistic
subjects is feasible and yields high quality data.
• Subjects in autism show dysregulated gene expression of the MET
pathway and activated immune system transcripts
• Some of the genes showing altered expression (PKCB, OAS) also confer
genetic susceptibility to autism
• ASD shows significant inter-subject variability of gene expression profile,
stressing the heterogeneity of disorder
• Immune activation in the brain may be characteristic of multiple brain
disorders, including ASD and schizophrenia, and may be an ongoing,
active process
• We all need to join efforts and mechanistically follow up the data
generated in these transcriptome profiling studies.
COLLABORATORS on the AUTISM PROJECT
 Pat Levitt and Dan Campbell (Vanderbilt)
 James Sutcliffe (Vanderbilt)
 Tony Persico (Rome)
 Paul Patterson (Caltech)
 Eric Courchesne (UCSD)
Thank you, ATP.
MIRNICS LAB
 Phil Ebert
 Krassimira Garbett
 Dominique Arion
 Annie Bedison
We greatly appreciate the sacrifice that the
families made by donating us the brain tissue
of their loved ones. This amazing gift is the
ultimate display of humanity and love that
triumphs over the tragedy of their loss, offering
hope to those sill waiting for the cure.
 Kathie Douglass
 Melissa Macioce
THANK YOU !!!
 Travis Unger
 Amanda Mitchell
 Rodica Gal-Chis
THE AUTISM RESEARCH OF THE MIRNICS LAB HAVE
BEEN FULLY FUNDED BY THE VANDERBILT
UNIVERSITY KENNEDY CENTER STARTUP FUNDS.