Transcript No Slide Title

Environmental Risks Factors in ASD: What
We Know / What We Need to Know
Isaac N. Pessah, PhD
Department of VM: Molecular Biosciences
Center for Children’s Environmental Health
The M.I.N.D. Institute
University of California, Davis
Scope of the Problem
• At present, the causes of the majority of developmental
defects are not understood.
• ~ 3% of all developmental defects are attributable to
exposure to toxic chemicals
• ~ 25% of all developmental defects may be due to a
combination of genetic and environmental factors.
• Underestimate: most neurological and behavioral problems
are not diagnosed until early childhood or young adulthood
National Res. Council Report (2000);
http://www.nap.edu/catalog.php?record_id=9871#toc
What we don’t know about environmental triggers
of developmental disorders
Thousands of commercially important chemicals
• industrial chemicals
• pesticides
• ingredients
• food additives
The vast majority of commercially important chemicals
lack adequate toxicity testing, especially for developmental
neurotoxicity
% with adverse effects
In any population, there are individual susceptible to
environmental toxicants
100
100
hypersensitive
individuals
Typical
resistant
individuals
50
50
0
TD50
Log Toxicant Concentration
mg/kg
0
% with adverse effects
Are children genetically predisposed to autism’s
deficits especially susceptible to environmental toxicants
100
100
Typical
50
0
Autism
TD50 TD50
Log Toxicant Concentration
mg/kg
50
0
% with adverse effects
Are children genetically predisposed to autism’s
deficits especially susceptible to environmental toxicants
100
50
0
100
Autism s
TD50
Typical
50
TD50
Log Toxicant Concentration
(mg/kg)
0
Autism is a Complex Disorder
•
•
•
•
Very high heritability
Multiple genes confer risk (estimates of 5-100)
Epigenetic mechanisms (DNA methylation)
Other mechanisms (de novo copy number variations)
Given that autism risk is contributed by multiple
“defective” genes….many expressed both within and
outside the central nervous system….
Could autism represent a multi-system disorder whose
outcome is likely to be more profoundly impacted by
environment than other disorders and diseases.
What are the possible mechanisms involved?
Strategy for generating hypotheses,
and testing them
Environmental
“Factors”
Genetic susceptibly
Repair
‘Normal’ phenotype
Autism
phenotype
A common mechanism for autism susceptibility may stem
from abnormal ratios of excitatory/inhibitory neurons and the
networks they form:
From Belmont and Bourgeron (2006) Nature Neuroscience 9(10):1221- 1225
Framework for understanding gene-environment
interactions impacting autism risk
Many of the most heavily used pesticides alter the
balance of excitation/inhibition within the developing
nervous system
Cholinergic Neurotransmission
• Organophosphate Insecticides (e.g. chlorpyrifos, diazinon)
Acetylcholinesterase inhibition- cholinergic synapses
Lower exposures (<10-9M)pCREB ; fgf expression
• Neonicotinoid Insecticides (e.g. imidacloprid, thiacloprid)
Target nicotinic cholinergic receptors (insect selective?)
Neuronal nAChR subunits:
a2
a3
a4
a5
a6
2
3
4
a7
a8
a9
a10
nAChR receptor differences in Autism
[3H]epibatidine
binding
1, 31
Parietal cortex
Cerebellum
Thalamus
[125I] aBgtx
binding
2Cortex
Level of heteromeric
subunit
cortex 2
Cerebellum
a4
Thalamus
2
(no difference) 1, 3Parietal
Cerebellum
1
2
3
Perry EK, Lee ML, Martin-Ruiz CM, et al (2001) Cholinergic activity in autism:
abnormalities in the cerebral cortex and basal forebrain. Am J Psychiatry. 158:1058-66.
PDD association with levels of OP metabolites
PON1 Polymorphisms associated with autism
Paraoxonase gene variants are associated with autism in North America,
but not in Italy: possible regional specificity in gene-environment interactions.
D'Agruma L, Muscarella LA, Guarnieri V, et al (2005) Mol Psychiatry. 10(11):1006-16.
Laboratory of Molecular Psychiatry and Neurogenetics, University Campus Bio-Medico, Rome, Italy.
Assessed linkage/association between autism and variants of the paraoxonase gene
(PON1) encoding paraoxonase, the enzyme responsible for OP detoxification.
Three functional (less active OP metabolizing activity) single nucleotide polymorphisms
PON1 C-108T, L55M, and Q192R.
Found significant association between autism and PON1 variants in
Caucasian-American, but not Italian families
PON1 Polymorphisms associated with autism
Pasca SP, Nemes B, Vlase L, Gagyi CE, Dronca E, Miu AC, Dronca M.
High levels of homocysteine and low serum paraoxonase 1 arylesterase activity
in children with autism. Life Sci. 2006 Apr 4;78(19):2244-8.
Environmental
OPÒFactorsÓ
insecticides
Genetic susceptibly
Repair
PON1
Activity
Ô
Normal Õphenotype
Autism
phenotype
Framework for understanding gene-environment
interactions impacting autism risk
Many of the most heavily used insecticides alter the
balance of excitation/inhibition within the developing
nervous system
GABA-mediated Neurotransmission
• Chlorinated hydrocarbon insecticides (e.g. lindane, heptachlor)
Block GABA activated Cl- movementspostnatal exposure causes hyper-excitability
gestational exposure may cause depression
• 4-alkyl-1-phenylpyrazole insecticides (e.g. Fipronil)
Non-competitive inhibitor of GABAA receptors (insect selective?)
Framework for understanding gene-environment
interactions impacting autism risk
Many of the most heavily used insecticides alter the
balance of excitation/inhibition within the developing
nervous system
GABAergic Neurotransmission
• 4-alkyl-1-phenylpyrazole insecticides (e.g. Fipronil)
Non-competitive inhibitor of GABAA receptors (insect selective?)
six types of α subunits (GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6)
three β's (GABRB1, GABRB2, GABRB3)
three γ's (GABRG1, GABRG2, GABRG3)
a δ (GABRD), an ε (GABRE), a π (GABRP), and a θ (GABRQ)
Pesticides that antagonize GABAA Receptors
Schematic illustration of a GABAA receptor
with its binding sites
Chlorinated hydrocarbon pore blockers
Lindane (head lice, scabies)
Endosulfan (agricultural crops)
Hepatchlor (1988)
Chlordane (1988)
Diledrin (1987)
Kepone (1978)
Toxaphene (1990)
Non-Competitive GABA antagonist
4-alkyl-1-phenylpyrazoles(e.g. Fipronil))
>800 tons applied in US 2000
4-alkyl-1-phenylpyrazoles may not be as selective for
Some GABAA receptors as we once thought
GABA receptor antagonists and insecticides: common structural features of 4-alkyl-1phenylpyrazoles and 4-alkyl-1-phenyltrioxabicyclooctanes
Sammelson RE, Caboni P, Durkin KA, Casida JE
Bioorg Med Chem. 2004 12(12):3345-55.
“Structure-activity studies described here reveal that fipronil retains its very high binding
potency at the human beta3 and house fly gamma-aminobutyric acid (GABA) receptors”
Control
1M
fipronil
Stehr et al, Tox Sci 92, 270 (2006)
Defective/deficient GABAA Receptors in Autisms
• Epigenetic mechanisms
MeCP2-deficiency associated with down regulation of GABR3
(Samaco et al 2005, Hum Mol Genet 14(4), 483-92)
• Complex gene-gene interactions
Polymorphisms at GABRa4 are involved in the etiology of autism,
interaction with GABR1 increases autism risk
(Ma et al, 2005, Am J Hum Gen 77, 377)
• Polymorphisms
GABR1, GABR3 genes…
(Vincent et al, 2006 J Med Gen 43, 429; Ashley-Koch et al, 2006 Ann Hum Gen 70, 281
Open Question
Could children with heritable deficiencies in GABA-mediated
neurotransmission be particularly susceptible to chemicals
that interfere with GABA neurotransmission? (Chlorinated
hydrocarbons, 4-alkyl-1-phenylpyrazoles, avermectins, etc)
Defective/deficient GABAA Receptors in Autisms
Open Question
Could children with heritable deficiencies in GABA-mediated
neurotransmission be particularly susceptible to chemicals that
interfere with GABA neurotransmission? (Organochlorine, 4-alkyl-1phenylpyrazoles, avermectins, etc)
Organochlorine insecticides - odds ratio for ASD of 6.1.
ASD risk increased with poundage of organochlorine
applied and decreased with distance from field sites.
From Roberts et al Environ. Health Perspect.
Aromatic Chlorinated Hydrocarbons
Non-dioxin-like
Dioxin-like
O
Clx
O
Cly
Polychlorinated dibenzo-p-dioxin (PCDD)
O
Clx
Cly
Clx
Polychlorinated biphenyl
(non coplanar PCB)
Cly
Polychlorinated dibenzofuran (PCDF)
Brx
O
Bry
Polybrominated diphenylether
(non coplanar PBDE)
ClX
ClY
Polychlorinated biphenyl
(coplanar PCB)
Triclosan
X and Y represent the number of chlorine
on the respective rings
Triclosan
Non-coplanar Triclosan - Concerns for Human Health
F494/F0
1 min
2
Levels of Triclosan in the U.S. Population
100 nM
1
CDC scientists tested the urine of 2,517 people ages six years and
older who took part in nationwide survey, called the National Health and
Nutrition Examination Survey (NHANES), during 2003-2004.
250
• Triclosan was detected in the urine of nearly 75 percent of the
people tested.
• Triclosan levels were greater among people in the highest income
bracket than among those in either the middle or lower income
750
brackets.
1000
Triclosan
Table 1. Examples of Ca2+ regulating and Ca 2+ regulated genes linked to autism
Gene (map)
CACNA1C (12p13.3)
CACNA1H (16p13.3)
SLC25A12 (2q24)
KCNMA1 (10q22.3)
PTEN (10q23.3)
MECP2 (Xq28)
MET (7q21.1)
CADPS2 (7q31-q32)
NL-1; NL-3
3q26.31; Xq13.1
Function
Mutation (Dysfunction)
2+
L-ty pe voltage-dependent Ca
channel (CaV1.2)
2+
T-ty pe v oltage-dependent Ca
channel (CaV3.2)
2+
Ca -dependent mitochondrial
aspartate/glutamate carrier
2+
+
Ca -activ ated K channel
2+
(BKCa )
2+
Ca -regulated PI-3phosphatase; regulates CaV1.2
2+
DNA methy a
l tion (Ca dependent phosphoryation)
l
Ty rosine receptor kinase f or
hepatocyte growth f actor
2+
Ca -dependent activator protein
f or secretion
Neuroligin- sy napse formation
/f unction EF-hands
G406R-delay ed inactiv ation
(Timothy Syndrome)
R212C; R902W,W962C, A1874Valtered activ ation (autism)
SNPs (autism)
Balanced 9q23/10q22 translocation
H93R, D252G, F241S(macrocephaly ; autism
Down regulated/mutations-altered DNA
methy lation (autism, RETT sy ndrome)
Polymorphis m-down regulation (autism)
aberrant alternative splicing lacks exon
3 (autism)
NL-1 R476C (autism)
NL-3 R471C (autism)
Framework for understanding gene-environment
interactions impacting autism risk
Hypothesis generating concept:
Genetic susceptibilities x environmental exposures x timing =
Autism risk, severity, and treatment outcomes
How can autism susceptibility genes inform us about
Environmental modifiers of risk, severity, treatment