Transcript Reelin and Autism

GABAA and GABAB
Receptor
Abnormalities
in Autism
S. Hossein Fatemi, M.D., Ph.D.
Professor of Psychiatry,
Pharmacology and Neuroscience
University of Minnesota
Medical School
Minneapolis, MN, USA
Autism
Neurodevelopmental disorder
– Brain pathology involving parietal, frontal, cerebellar,
hippocampal, and amygdalar areas
– Early onset of disease
Genetic etiologies
– MZ concordance 36-95%
– Chromosomes 1, 5, 6, 8, 13, 15, 16, 18, 21, x, y
– HOX A, Reelin, Wnt
Environmental etiologies
–
–
–
–
Rubella, borna, syphilis, CMV, mumps
Thalidomide
Valproic acid
Mercury
Brain Pathology in Autism
Selective cortical atrophy
Purkinje cell loss and atrophy (Piven; Fatemi)
Smaller cingulate gyrus
Higher packing density and smaller neuron
size in hippocampus, amygdala and septal
nucleus (Bauman and Kemper)
Macrocephaly (Courchesne)
Abnormal brain stem
Microcolumnar pathology (Casanova et al.)
Pathological Findings
Cell migration – Reelin
GABA transmission – GAD
Apoptosis – Bcl2 and P53
Cell injury – GFAP
cAMP/cGMP modulation - PDEs
Methods
SDS-PAGE
Western Blotting
qRT-PCR
HPLC
Table 1. Demographic data for subjects with autism and controls
Case
Dx
Sex
Age
PMI
(Hours)
Ethnicity
Medication History
Cause of Death
Seizure
Brain Areas
B1078
Autistic*
M
22
14.3
Caucasian
Dilantin, Tegretol, Phenobarbital,
Theodure
Asphyxia
Yes
BA40
B1045
Autistic*
M
28
16.3
Caucasian
Cefobid, Urecholine, Duracef
Cardiac arrest
Yes
Cer, BA40
B5000
Autistic*
M
27
8.3
Caucasian
Synthroid
Drowning
No
Cer
B1401
Autistic*
F
21
20.6
Caucasian
Tetracycline
Pneumonia, sepsis
Yes
Cer, BA9, BA40
B1664
Autistic*
M
20
15
Caucasian
Vitamins B, C
Perforation of ulcer; asphyxia
Yes
Cer, BA9, BA40
B2825
Autistic*
M
19
9.5
Caucasian
None
Seizure
Yes
Cer, BA9, BA40
B3511
Autistic*
M
29
15
Caucasian
None
Hit by train
Yes
Cer, BA9, BA40
B3845
Autistic*
M
30
28.4
Caucasian
Mellaril, Phenobarbital, Dilantin
Shock; acute pancreatitis
Yes
BA9, BA40
B1484
Autistic*
M
19
15
Caucasian
None
Burns
No
BA9, BA40
B3829
Control
M
22
24.3
Caucasian
None
MVA
No
Cer
B4267
Control
M
26
20
African-American
None
MVA
No
Cer
B4268
Control
M
30
22
African-American
None
Cardiomyopathy
No
Cer, BA40
B4269
Control
M
28
24
Caucasian
Lidocaine 12.0 mg/L found in blood
Arteriosclerotic cardiovascular
disease
No
Cer, BA9, BA40
B4272
Control
M
19
17
Caucasian
None
Accident; chest injuries
No
Cer
B4275
Control
M
20
16
Caucasian
None
Accident
No
Cer, BA9, BA40
B4279
Control
F
20
21
Caucasian
None
MVA
No
Cer
B4362
Control
M
30
20
African-American
None
MVA
No
Cer, BA9
B4101
Control
M
24
5
Unknown
None
Gun shot wound
No
Cer, BA40
B4271
Control
M
19
21
African-American
Ethyl alcohol, Advil, Amoxapine
Epiglottitis
No
BA40
B4756
Control
M
56
23
Unknown
None
Cardiac arrest
No
Cer
B4363
Control
M
21
9
Caucasian
None
MVA
No
Cer, BA40
Dx, diagnosis; PMI, postmortem interval; M, male; F, female; Autistic*, all autistic subjects were also mentally retarded[2]; MVA, motor vehicle accident
Figure 1. Representative samples of GABRA1 (51 kDa), GABRA2 (51 kDa),
GABRA3 (55 kDa), GABRA5 (52 kDa), GABRA6 (50 kDa), GABRG2 (45 kDa),
GABRG3 (51 kDa), GABRB3 (56 kDa), GABBR1 (108 kDa), GABBR2 (105 kDa),
and β-Actin (42 kDa) in BA9, BA40, and cerebellum of subjects with autism (A)
and matched controls (C).
Table 2. Expression of GABRA1-GABRA3, GABRB3, GABBR1, and GABBR2 in cerebellum, BA40, and
BA9 in subjects with autism vs. controls
Cerebellum
Control
Autistic
Change
p*
mRNA
GABRA1 / β-Actin
1.350 ± 0.470
0.504 ± 0.356
↓ 63%
0.007
nc
GABRA2 / β-Actin
0.073 ± 0.028
0.053 ± 0.025
↓ 27%
0.21
nc
GABRA3 / β-Actin
0.216 ± 0.100
0.142 ± 0.073
↓ 34%
0.17
nc
GABRB3 / β-Actin
0.061 ± 0.022
0.030 ± 0.013
↓ 51%
0.008
↓**
GABBR1 / β-Actin
0.051 ± 0.018
0.017 ± 0.006
↓ 67%
0.005
↓**
GABBR2 / β-Actin
0.068 ± 0.028
0.037 ± 0.012
↓ 46%
0.026
nc
BA40
Control
Autistic
Change
p
mRNA
GABRA1 / β-Actin
0.955 ± 0.305
0.458 ± 0.266
↓ 52%
0.018
nc
GABRA2 / β-Actin
0.214 ± 0.054
0.131 ± 0.060
↓ 39%
0.033
nc
GABRA3 / β-Actin
0.166 ± 0.039
0.072 ± 0.049
↓ 57%
0.005
nc
GABRB3 / β-Actin
0.039 ± 0.011
0.024 ± 0.006
↓ 38%
0.006
nc
GABBR1 / β-Actin
0.078 ± 0.049
0.023 ± 0.026
↓ 71%
0.019
nc
GABBR2 / β-Actin
0.118 ± 0.064
0.061 ± 0.039
↓ 49%
0.104
nc
BA9
Control
Autistic
Change
p
mRNA
GABRA1 / β-Actin
0.260 ± 0.050
0.090 ± 0.062
↓ 65%
0.012
nc
GABRA2 / β-Actin
0.159 ± 0.041
0.092 ± 0.042
↓ 42%
0.057
nc
GABRA3 / β-Actin
0.127 ± 0.006
0.193 ± 0.083
↑ 52%
0.236
nc
GABRB3 / β-Actin
0.040 ± 0.002
0.041 ± 0.009
↑ 2.5%
0.853
nc
GABBR1 / β-Actin
0.076 ± 0.023
0.023 ± 0.024
↓ 70%
0.021
nc
GABBR2 / β-Actin
0.115 ± 0.016
0.053 ± 0.042
↓ 54%
0.064
nc
*, Two-tailed independent group t-tests; **, p<0.05; nc, no change
Table 3. Expression of GABRA5, GABRA6, GABRG2, and GABRG3 in cerebellum, BA40, and BA9 in
subjects with autism vs. controls
Cerebellum
Control
Autistic
Change
p*
mRNA
GABRA5 / β-Actin
0.387 ± 0.194
0.436 ± 0.152
↑ 13%
0. 52
nc
GABRA6 / β-Actin
0.417 ± 0.134
0.442 ± 0.195
↑ 6%
0.75
nc
GABRG2 / β-Actin
0.153 ± 0.084
0.122 ± 0.057
↓ 20%
0.41
nc
GABRG3 / β-Actin
0.313 ± 0.089
0.331 ± 0.070
↑ 6%
0.65
nc
BA40
Control
Autistic
Change
p
mRNA
GABRA5 / β-Actin
0.340 ± 0.155
0.272 ± 0.038
↓ 20%
0.42
nc
GABRA6 / β-Actin
0.198 ± 0.071
0.286 ± 0.092
↑ 44%
0.13
nc
GABRG2 / β-Actin
0.255 ± 0.137
0.198 ± 0.050
↓ 22%
0.46
nc
GABRG3 / β-Actin
0.229 ± 0.075
0.246 ± 0.061
↑ 7%
0.72
nc
BA9
Control
Autistic
Change
p
mRNA
GABRA5 / β-Actin
0.576 ± 0.040
0.288 ± 0.176
↓ 50%
0.035
nc
GABRA6 / β-Actin
0.153 ± 0.040
0.085 ± 0.035
↓ 44%
0.046
nc
GABRG2 / β-Actin
0.216 ± 0.048
0.111 ± 0.058
↓ 49%
0.039
nc
GABRG3 / β-Actin
0.109 ± 0.032
0.069 ± 0.013
↓ 37%
0.067
nc
*, Two-tailed independent group t-tests; **, p<0.05; nc, no change
Figure 2. Neurochemical analysis of brain regions of control and autistic subjects in BA9 (A),
BA40 (B), and cerebellum (C). Shown are mean values ± SEM of the mean for dopamine
(DA), serotonin (5-HT), glutamate (Glu), glutamine (Gln), taurine (tau), and GABA. Statistical
significance of difference analyzed by one way analysis of variance followed by Holm Sidak
post hoc test. **p<0.05
Protein Levels
 GABAA a1 protein by 63% in cerebellum
 GABAA b3 protein by 51% in cerebellum
 GABAB R1 protein by 67% in cerebellum
 GABAB R2 protein by 46% in cerebellum
 GABAA a1 protein by 52% in BA40
 GABAA a2 protein by 39% in BA40
 GABAA a3 protein by 57% in BA40
 GABAA b3 protein by 38% in BA40
 GABAB R1 protein by 71% in BA40
 GABAA a1 protein by 65% in BA9
 GABAB R1 protein by 70% in BA9
HPLC Studies
 cerebellar serotonin (p<0.05) in autism
No difference in levels of GABA,
glutamate, dopamine, glycine, or taurine
between two group
Supported by central serotonergic
hypofunction in autism (Croonenbergh et al.,
2007)
mRNA Levels
 mRNA for GABAA b3 levels by 29% in
cerebella of autistics vs. controls
 mRNA for GABAB R1 levels by 15% in
cerebella of autistics vs. controls
GABAA b3
Implicated by multiple association studies in autism
(Buxbaum JD et al., 2002; Cook EH et al., 1998; McCauley JL
et al., 2004; Shao Y et al., 2003)
 expression in autism, Angelman syndrome and
Rett’s disease (Samaco RC et al., 2005)
Pentameric chloride channels
Crucial site of action for intravenous anesthetics,
ethanol, and developmental processes (i.e.,
ventromedial nucleus of hypothalamus)
Localized to 15q11-q13, a site for b3, a5, and g3
subunits of GABAA receptor
Our results are the first to show significant decreases
in b3 proteins in cerebellum and BA40 of autistics, as
well as b3 mRNA and protein decreases in cerebellum
of autistics
GABAB R1
Signal transduction/G protein activation (Jones KA et al.,
2000)
Downregulation in kainic acid-induced seizures in rats
signify neurodegeneration (Furtingers et al., 2003)
Impairment of attentional processing (Prosser HM et al.,
2001)
Ca-dependent receptor activation; tubulin-dependent
receptor trafficking
Insulin resistance, hyperlocomotion, and atypical
absence seizure
Our results are the first to demonstrate R1 deficiency
in 3 important brain areas in autism
GABAB R2
Gene locus at 9q22.1
G-protein coupled receptor 51
Associated with nicotine dependence
Increased with absence seizure in rat
somatosensory cortex and thalamus (Principalle
et al., 2003)
GABAA a1
Gene locus 5q34-q35
Associated with juvenile myoclonic epilepsy
Associated with depression in probands (Murray
et al., 1994)
 5HT1A receptor in brains of subjects with
JME (Meschaks et al., 2005). Serotonin is
reduced in cerebellum of autistics (Fatemi et al.,
2008)
No previous publication indicating its reduction
in autism
GABAA a2
Gene locus at 4p12
Modulates anxiety and stress response
May be involved in plasticity of ventrobasal
complex and posterior nucleus of thalamus in
chronic inflammatory pain (Ferreria-Gomes et al.,
2006)
Expression in medial amygdala (Byrnes et al.,
2007)
Involved in hippocampal dentate granule cell
during development (Brooks-Kayal et al., 2001)
Associated with alcohol dependence (Soyka et
al., 2008)
GABAA a3
Gene locus at Xq28
Localized to substantia nigra parvalbuminpositive nonpigmented cells (Waldvogel et al.,
2008)
Involved in unipolar major depression (Henket et
al., 2004)
GABAA a5
Gene locus at 15q11.2-q12
Component of a pentameric receptor mediating
inhibitory neurotransmission
Involved in the susceptibility to schizophrenia
(Papadimitriou et al., 2001a) and bipolar affective
disorder (Papadimitriou et al., 2001b; Otani et al.,
2005)
Implicated in autism (Menold et al., 2001; AshleyKoch et al., 2005; Tochigi et al., 2007), Angelman
syndrome (Nurmi et al., 2001), and childhood
absence epilepsy (CAE) (Lu et al., 2004)
GABAA a6
Gene locus at 5q34
Component of a pentameric receptor mediating
inhibitory neurotransmission, expressed in the
cerebellar granule cells and the related cells of
cochlear nucleus
Implicated in schizophrenia (Petryshen et al.,
2005), alcoholism (Chang et al., 2002; Dick et al.,
2003; Sen et al., 2004), and heroin abuse (Loh et
al., 2007)
GABAA g2
Gene locus at 5q31.1-q33.1
This subunit carries the benzodiazepine binding site
Component of a pentameric receptor mediating inhibitory
neurotransmission, complexing with DRD5 and promoting
mutually inhibitory functional interactions between these
receptor systems
Implicated in schizophrenia (Petryshen et al., 2005) and mood
disorders (Yamada, 2003)
Involved in the physiological dependence on alcohol (Sander
et al., 1999; Chang et al., 2002; Dick et al., 2003), heroin
abuse (Loh et al., 2007), and methamphetamine abuse
(Nishiyama et al., 2005)
Cause of childhood absence epilepsy type 2 (ECA2) (Olsen et
al., 1999; Wallace et al., 2001) generalized epilepsy with
febrile seizures plus type 3 (GEFS+3) (Baulac et al., 2001)
severe myoclonic epilepsy in infancy (SMEI)(Jansen et al.,
2006)
GABAA g3
Gene locus at 15q12
This subunit also carries the benzodiazepine
binding site
Component of a pentameric receptor
mediating inhibitory neurotransmission
Associated with alcohol dependence (Dick et
al., 2004)
Associated with risk for autism (Ma et al., 2005;
Ashley-Koch et al., 2006)
Glutamic Acid Decarboxylase 65kDa
Localized to axon terminals
Membrane bound and in vesicles
Involved in vesicular release of GABA
Involved in synthesis of GABA by phasically
firing neurons
Can exist as either amphiphillic GAD65
homodimer or GAD65/67 heterodimer
Glutamic Acid Decarboxylase 67kDa
Concentrated in interneurons
Cytosolic
Involved in non-vesicular synthesis of GABA
Involved in continuous synthesis of GABA in
tonically firing neurons
Used for synthesis of GABA for general
metabolic activity
Can exist as either hydrophillic GAD67
homodimer or amphiphillic GAD65/67
homodimer
 Glutamate & aspartate in plasma of autistic
children (Moreno et al., 1992; Moreno-Fuenmayer et
al., 1996)
 CSF glutamate in 4 patients with Rett’s syndrome
(Hamberger et al., 1992)
 Glutamate & GABA in platelets of drug-naïve
autistic patients (Rolf et al., 1993)
 Glutamate to N-acetylaspartate ratio in gray
matter in Rett’s syndrome (Pan et al., 1999)
 mRNA of excitatory amino acid transporter 1 and
AMPA1 receptor with  density of AMPA1 receptor in
autistic cerebellum (Purcell et al., 2001)
GAD 65/67 in Parietal Cortex
GAD 65/67 in Cerebellum
Conclusions
GABAA a1 and b3, GABAB R1 and R2 proteins
decreased in cerebellum of autistics (p<0.05)
GABAA a1, a2, a3, and b3 and GABAB R1 proteins
decreased in BA40 of autistics (p<0.05)
GABAA a1, a5, a6, g2, and GABAB R1 proteins
decreased in BA9 of autistics (p<0.05)
GABAA b3 and GABAB R1 mRNAs decreased in
cerebellum of autistics (p<0.05)
Serotonin levels reduced in cerebellum of autistics
without any change in levels of glutamate or GABA
Results support ↑ rates of seizure disorder and mental
retardation in autistic subjects
Acknowledgements
Human tissue was obtained from the NICHD
Brain and Tissue Bank for Developmental
Disorders; the Harvard Brain Tissue Resource
Center, which is supported in part by PHS grant
number R24 MH068855; the Brain Endowment
Bank, which is funded in part by the National
Parkinson Foundation, Inc., Miami, Florida; and
the Autism Tissue Program and is gratefully
acknowledged. Grant support by National
Institute of Child Health and Human
Development (#5R01HD052074-01A2) to SHF
is also gratefully acknowledged.
Collaborators
University of Minnesota, Minneapolis, MN
USA
- TJ Reutiman, TD Folsom, PD Thuras
Charité Campus Mitte, University Medicine,
Berlin, Germany
- C Winter, R Sohr, J Klein
University of Rochester, Rochester, NY USA
- DA Pearce, M Zanche