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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 3, 2005 as doi:10.1096/fj.04-2106fje. |
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* The Life Sciences Institute, The Hebrew University of Jerusalem, Israel;
Departments of Neurology and Neurosurgery,
Zlotowsky Center of Neuroscience, Ben Gurion University and Soroka Medical Center, Beer Sheva, Israel;
Herzog Hospital and
|| Mount Scopus Hadassah University Hospital, Jerusalem, Israel
1Correspondence: E-mail: soreq{at}cc.huji.ac.il
SPECIFIC AIMS
To decipher gene/environment interactions that increase the risk of anticholinesterase exposure-induced Parkinson’s disease, we studied interacting polymorphisms in the ACHE/PON1 locus and corresponding expression variations in Israeli patients from agriculturally exposed areas. At a biochemical level, serum AChE and PON1 activities were compared between patients and healthy individuals and between risky polymorphism carriers to other parkinsonians.
PRINCIPAL FINDINGS
1. Apparent association with exposure-induced PD risk
Allelic composition of the ACHE/PON1 locus from insecticide-exposed and nonexposed PD patients was similar to that of healthy individuals, allowing comparison between patients and controls despite age differences. Three polymorphisms showed a nonsignificant trend for association with exposure-induced PD: –162PON1, –108PON1, 
ACHE, P < 0.1. The –108PON1 SNP and ACHE reflect functionally effective changes, with their rare alleles impairing production of PON1 and insult-induced overproduction of AChE, respectively. Using the PM program, –108PON1 in PON1 and the ACHE promoter deletion, but not the –162PON1 polymorphism, appeared to contribute to the formation of a core haplotype associated with significantly increased risk for exposure-induced parkinsonism. We analyzed distribution of the –108PON1 and ACHE haplotypes. Haplotype frequencies were estimated for these SNPs by the EM algorithm, tested by a likelihood ratio test, and empirically validated by permutations. Haplotypes containing the weaker T–108 promoter allelle in PON1 or the insult-nonresponsive ACHE appeared to be highly over-represented in subjects with exposure-induced parkinsonism (Table 1
). Conversely, the C-108-WTACHE haplotype ensuring robust PON1 and ACHE expression was significantly under-represented in exposed-PD patients vs. healthy subjects [odds ratio (OR)=2.4, 95% confidence interval: 1.3–4.4, P<0.0004] and nonexposed PD patients (OR=2.6, 95% confidence interval: 1.3–5.2, P<0.03). The highest statistical threshold (P<0.0004) remained significant even under the most conservative correction for multiple testing and assuming all tests were independent.
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Distribution of the combined genotypes of –108PON1 and ACHE was significantly different in insecticide-exposed vs. nonexposed PD patients (P=0.017) or healthy individuals (P=0.0037). Statistical analysis showed no deviation from independence for –108PON1 and ACHE genotypes among controls or nonexposed PD, but a significant deviation was found among insecticide-exposed PD patients (P=0.031). A logistic regression model showed a significant interaction effect of –108PON1 and ACHE genotypes on disease status (P=0.0035).
We reanalyzed a subsample of subjects of Ashkenazi Jewish origin (236 apparently healthy individuals, including 51 subjects from the original group+185 additional subjects, 22 nonexposed PD, and 30 exposed PD patients). Exposed PD patients from this subsample showed significant interaction effect between –108PON1 and ACHE (P=0.0057), a deviation from independence at the –108PON1 and ACHE genotypes (P=0.020), and an association between the exposed PD phenotype and combined genotypes of –108PON1 and ACHE (P=0.0067). Exposed PD patients from the Ashkenazi subsample showed a significant association with ACHE/PON1 haplotypes (P=0.0032).
2. Genetic variations and expression differences
Constitutive AChE overproduction such as occurs in carriers of the ACHE mutation prevents sufficient overproduction of the scavenging AChE molecules upon exposure to anti-AChEs. Reduced PON1 activities could be expected to subject carriers of this ACHE polymorphism to additional risk because of the insufficient PON1 capacity to hydrolyze organophosphate (OP) AChE inhibitors. To test the involvement of each enzyme in the OP-induced risk of PD, we measured serum cholinesterases, paraoxonase, and aryl esterase activities in exposed and nonexposed PD patients and in subjects with or without the potentially risk-inducing polymorphisms (Fig. 1
). In control subjects, serum AChE activities were found to increase by 
20% from 20 to 55 years of age, from 466 ± 186 to 657 ± 181 nmol substrate hydrolyzed/mL serum/min (r2=0.57, n=91), compatible with findings in the U.S. population. PON, but not aryl esterase, activity was found to decrease insignificantly with age both in U.S. and Israeli populations between 20 and 55 years of age (n=91, r2=0.018). In contrast, serum AChE and PON1 activity were significantly lower in all PD patients than in younger controls, but the AChE homologous enzyme butyrylcholinesterase (BChE) was not (Fig. 1A
, P=0–17; P=10–11, respectively). Aryl esterase activity of the PON1 protein was significantly higher in PD vs. healthy subjects (Fig. 1B
, P=0.0013), suggesting that paraoxonase, but not aryl esterase, activity of PON1 is causally involved in the progression of PD.
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Exposed PD patients presented lower AChE activity than nonexposed PD patients (by 12%, n=13, 32 patients). Carriers of the C-108 allele in PON1 presented significantly higher aryl esterase activities (P=1.2x10–5, r2=22.7%). AChE activity was significantly (P=0.02) lower in PD carriers of the risk-associated haplotypes C-108/ACHE and T-108/WTACHE than in PD patient carriers of the protective haplotype C-108/WTACHE. ACHE was found to be associated with impaired AChE overproduction upon exposure in vivo. ACHE gene expression is regulated by the same mechanism in blood and in brain. Therefore, our findings are compatible with the assumption that after exposure, low cholinesterase activities in ACHE carriers may increase the risk for premature death of substantia nigra neurons and PD.
CONCLUSIONS AND SIGNIFICANCE
Separate studies have indicated a potential association of AChE or PON1 with an environmentally induced risk of PD, but to the best of our knowledge, causal interaction in the induction of PD between these two proteins was never suspected.
Here, we report findings attributing the increased PD risk in rural Israel to interacting debilitating polymorphisms in the ACHE/PON1 locus and corresponding expression variations. Polymorphisms that debilitate PON1 activity constitutively and impair the overproduction of AChE under anticholinesterase exposure were overrepresented in patients from agricultural areas, predicting abnormal cholinergic-mediated neuronal activity. Serum AChE and PON activities were selectively and significantly lower in patients than in healthy individuals and in carriers of the risky polymorphisms vs. other parkinsonian patients. Control experiments demonstrated that for PON1 the decrease in PD patients is substantially greater than would be expected based on age alone; for AChE, this decrease is opposite of what would be expected.
Agricultural workers under chronic exposure to anti-AChEs suffer increased risk of developing PD with odds ration (OR) of 3.55 (confidence interval (CI) 95%, 1.75–7.18). Carriers of the M55 allele of PON1, with a deficient capacity to degrade paraoxon, present an OR of 2.19 (CI 95%, 1.52–3.14), suggesting this allele contributes at least part of the anti-AChE induced risk. The R192 variant of PON1 was reported to increase the risk of early-onset cardiovascular disease, where cholinergic neurotransmission plays an important role. Our study is the first to demonstrate that inherited ACHE/PON1 allelic variants exert interrelated effects on the risk for neurodegeneration due to a modified capacity to confront an environmental challenge.
Under exposure to organophosphates, increased levels of acetylcholine (ACh), due to blocked AChE activity by undegraded OPs, activate ACh receptors and induce AChE overproduction as a feedback response. PON1 protects from the consequences of such exposure by degrading the poisonous OPs and by protecting AChE from oxidative stress via its peroxidase activity. AChE is highly sensitive to oxidative damage, suggesting further reduction in AChE activity under inheritably low PON1 activity and a higher risk of PD. AChE overproduction and protection would retrieve balanced ACh levels, ensuring retrieval of the correct extent of activation of dopaminergic neurons [the so-called ACh-dopamine (DA) balance]. Figure 2
A presents this principle. However, carriers of the risk-associated polymorphism in ACHE fail to initiate AChE overexpression under acute or chronic exposure. Debilitated PON1 will leave even more undegraded OPs and free oxygen radicals, exacerbating AChE blockade. This would elevate ACh levels, impairing the ACh/DA balance and facilitating PD symptoms by contributing to the vulnerability of dopaminergic neurons. Such carriers will be vulnerable to consistently increased ACh levels and may fail to retrieve balanced ACh/DA status (Fig. 2B
). This proposed mechanism is compatible with the apparently damaging interaction between risk-associated ACHE and PON1 polymorphisms, and with the relation between AChE release, activation of dopaminergic receptors, and neuronal firing rate in the nigrostriatal pathway.
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The PD phenotype is evident only when 60–70% of substantia nigra neurons stop functioning. Exposure of susceptible subjects exerts a delayed effect but is not expected to cause evolutionary pressure. The high incidence in Middle East populations of a haplotype spanning 5.5 Mb the ACHE/PON1 locus likely reflects a founder effect; parallel tests in U.S. groups revealed 10-fold lower incidence of the ACHE promoter and coding polymorphisms. This indicates the emergence of this haplotype after the migration of the native population from the Middle East 6500 B.C. to the Caucasus.
Our findings suggest that environmental exposure may significantly increase the risk to those individuals who live close to exposed areas. Genomic interactions with the environment thus call for special attention.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2106fje;
This article has been cited by other articles:
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  B. A. Golomb Acetylcholinesterase inhibitors and Gulf War illnesses PNAS, March 18, 2008; 105(11): 4295 - 4300. [Abstract] [Full Text] [PDF]
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 R. O. Browne, L. B. Moyal-Segal, D. Zumsteg, Y. David, O. Kofman, A. Berger, H. Soreq, and A. Friedman Coding region paraoxonase polymorphisms dictate accentuated neuronal reactions in chronic, sub-threshold pesticide exposure FASEB J, August 1, 2006; 20(10): 1733 - 1735. [Abstract] [Full Text] [PDF]
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