GABAA and GABAB Receptor Abnormalities in Autism

To enlarge this document for easy viewing please click Fullscreen below.

Embedded Scribd iPaper - Requires Javascript and Flash Player
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) 14.3 16.3 8.3 20.6 15 9.5 15 28.4 15 24.3 20 22 24 17 16 21 20 5 21 23 9 Ethnicity Medication History Dilantin, Tegretol, Phenobarbital, Theodure Cefobid, Urecholine, Duracef Synthroid Tetracycline Vitamins B, C None None Mellaril, Phenobarbital, Dilantin None None None None Lidocaine 12.0 mg/L found in blood None None None None None Ethyl alcohol, Advil, Amoxapine None None Cause of Death Seizure Brain Areas
B1078 B1045 B5000 B1401 B1664 B2825 B3511 B3845 B1484 B3829 B4267 B4268 B4269 B4272 B4275 B4279 B4362 B4101 B4271 B4756 B4363
Autistic* Autistic* Autistic* Autistic* Autistic* Autistic* Autistic* Autistic* Autistic* Control Control Control Control Control Control Control Control Control Control Control Control
M M M F M M M M M M M M M M M F M M M M M
22 28 27 21 20 19 29 30 19 22 26 30 28 19 20 20 30 24 19 56 21
Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian African-American African-American Caucasian Caucasian Caucasian Caucasian African-American Unknown African-American Unknown Caucasian
Asphyxia Cardiac arrest Drowning Pneumonia, sepsis Perforation of ulcer; asphyxia Seizure Hit by train Shock; acute pancreatitis Burns MVA MVA Cardiomyopathy Arteriosclerotic cardiovascular disease Accident; chest injuries Accident MVA MVA Gun shot wound Epiglottitis Cardiac arrest MVA
Yes Yes No Yes Yes Yes Yes Yes No No No No No No No No No No No No No
BA40 Cer, BA40 Cer Cer, BA9, BA40 Cer, BA9, BA40 Cer, BA9, BA40 Cer, BA9, BA40 BA9, BA40 BA9, BA40 Cer Cer Cer, BA40 Cer, BA9, BA40 Cer Cer, BA9, BA40 Cer Cer, BA9 Cer, BA40 BA40 Cer 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 GABRA1 / β-Actin GABRA2 / β-Actin GABRA3 / β-Actin GABRB3 / β-Actin GABBR1 / β-Actin GABBR2 / β-Actin BA40 GABRA1 / β-Actin GABRA2 / β-Actin GABRA3 / β-Actin GABRB3 / β-Actin GABBR1 / β-Actin GABBR2 / β-Actin BA9 GABRA1 / β-Actin GABRA2 / β-Actin GABRA3 / β-Actin GABRB3 / β-Actin GABBR1 / β-Actin GABBR2 / β-Actin Control 1.350 ± 0.470 0.073 ± 0.028 0.216 ± 0.100 0.061 ± 0.022 0.051 ± 0.018 0.068 ± 0.028 Control 0.955 ± 0.305 0.214 ± 0.054 0.166 ± 0.039 0.039 ± 0.011 0.078 ± 0.049 0.118 ± 0.064 Control 0.260 ± 0.050 0.159 ± 0.041 0.127 ± 0.006 0.040 ± 0.002 0.076 ± 0.023 0.115 ± 0.016 Autistic 0.504 ± 0.356 0.053 ± 0.025 0.142 ± 0.073 0.030 ± 0.013 0.017 ± 0.006 0.037 ± 0.012 Autistic 0.458 ± 0.266 0.131 ± 0.060 0.072 ± 0.049 0.024 ± 0.006 0.023 ± 0.026 0.061 ± 0.039 Autistic 0.090 ± 0.062 0.092 ± 0.042 0.193 ± 0.083 0.041 ± 0.009 0.023 ± 0.024 0.053 ± 0.042 Change ↓ 63% ↓ 27% ↓ 34% ↓ 51% ↓ 67% ↓ 46% Change ↓ 52% ↓ 39% ↓ 57% ↓ 38% ↓ 71% ↓ 49% Change ↓ 65% ↓ 42% ↑ 52% ↑ 2.5% ↓ 70% ↓ 54% p* 0.007 0.21 0.17 0.008 0.005 0.026 p 0.018 0.033 0.005 0.006 0.019 0.104 p 0.012 0.057 0.236 0.853 0.021 0.064 mRNA nc nc nc ↓** ↓** nc mRNA nc nc nc nc nc nc mRNA nc nc nc nc nc 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 GABRA5 / β-Actin GABRA6 / β-Actin GABRG2 / β-Actin GABRG3 / β-Actin BA40 GABRA5 / β-Actin GABRA6 / β-Actin GABRG2 / β-Actin GABRG3 / β-Actin BA9 GABRA5 / β-Actin GABRA6 / β-Actin GABRG2 / β-Actin GABRG3 / β-Actin Control 0.387 ± 0.194 0.417 ± 0.134 0.153 ± 0.084 0.313 ± 0.089 Control 0.340 ± 0.155 0.198 ± 0.071 0.255 ± 0.137 0.229 ± 0.075 Control 0.576 ± 0.040 0.153 ± 0.040 0.216 ± 0.048 0.109 ± 0.032 Autistic 0.436 ± 0.152 0.442 ± 0.195 0.122 ± 0.057 0.331 ± 0.070 Autistic 0.272 ± 0.038 0.286 ± 0.092 0.198 ± 0.050 0.246 ± 0.061 Autistic 0.288 ± 0.176 0.085 ± 0.035 0.111 ± 0.058 0.069 ± 0.013 Change ↑ 13% ↑ 6% ↓ 20% ↑ 6% Change ↓ 20% ↑ 44% ↓ 22% ↑ 7% Change ↓ 50% ↓ 44% ↓ 49% ↓ 37% p* 0. 52 0.75 0.41 0.65 p 0.42 0.13 0.46 0.72 p 0.035 0.046 0.039 0.067 mRNA nc nc nc nc mRNA nc nc nc nc mRNA nc nc nc 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
↓ GABAA α1 protein by 63% in cerebellum ↓ GABAA β3 protein by 51% in cerebellum ↓ GABAB R1 protein by 67% in cerebellum ↓ GABAB R2 protein by 46% in cerebellum ↓ GABAA α1 protein by 52% in BA40 ↓ GABAA α2 protein by 39% in BA40 ↓ GABAA α3 protein by 57% in BA40 ↓ GABAA β3 protein by 38% in BA40 ↓ GABAB R1 protein by 71% in BA40 ↓ GABAA α1 protein by 65% in BA9
Protein Levels
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 β3 levels by 29% in cerebella of autistics vs. controls ↓ mRNA for GABAB R1 levels by 15% in cerebella of autistics vs. controls
Implicated by multiple association studies in autism
GABAA β 3
(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 β3, α5, and γ3 subunits of GABAA receptor Our results are the first to show significant decreases in β3 proteins in cerebellum and BA40 of autistics, as well as β3 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 α 1
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
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) 2007)
GABAA α 2
Expression in medial amygdala (Byrnes et al., Involved in hippocampal dentate granule cell during development (Brooks-Kayal et al., 2001) Associated with alcohol dependence (Soyka et
al., 2008)
GABAA α 3
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 α 5
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 α 6
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)
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 γ 2
GABAA γ 3
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
GABAA α1 and β3, GABAB R1 and R2 proteins decreased in cerebellum of autistics (p<0.05) GABAA α1, α2, α3, and β3 and GABAB R1 proteins decreased in BA40 of autistics (p<0.05) GABAA α1, α5, α6, γ2, and GABAB R1 proteins decreased in BA9 of autistics (p<0.05) GABAA β3 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
Conclusions
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