Floria Pancetti

Noncholinesterase effects induced by organophosphate pesticides and their relationship to cognitive processes: implication for the action of acylpeptide hydrolase.

Organophosphate pesticides have been classically described as inhibitors of acetylcholinesterase (AChE) activity in insects and invertebrates. However, there is now more evidence supporting the hypothesis that these compounds also act through noncholinergic pathways, especially those related to cognitive processes. The enzyme acylpeptide hydrolase was identified as a new target for organophosphate pesticides. This enzyme is more sensitive than AChE to some organophosphates (OP), including dichlorvos, which is the parent compound for metrifonate, a therapeutic agent used in the treatment of cognitive impairment associated to Alzheimer´s disease. Therefore, there is some doubt as to whether the mechanism of action of this drug is mediated by a potentiation of cholinergic transmission. However, the direct action of acylpeptide hydrolase in cognitive processes and the physiological and molecular mechanisms underlying subacute exposure to OP have yet to be demonstrated. This review deals with evidence demonstrating the existence of mechanisms of actions of OP, which are independent of cholinergic pathway potentiation and which have an effect on cognitive processes. In addition, the possible participation of the enzyme acylpeptide hydrolase in these processes is also discussed. Finally, the possibility of using this enzyme activity as a new biomarker for exposure to OP is considered.

Differential effects of GABAergic ligands in mouse and rat hippocampal neurons

Previous electrophysiological studies suggested that GABAA receptors in rat hippocampal neurons might be less sensitive to ethanol than mouse neurons. Therefore, we examined the effects of ethanol (0.5-850 mM) in cultured mouse (C57BL/6) and rat (Sprague-Dawley) neurons. In 35% of the mouse neurons, the Cl- current was potentiated by ethanol starting at 0.5 mM. In all of the rat neurons examined, on the other hand, the current was potentiated by concentrations starting at 200 mM. We also studied the effects of GABA and other GABAergic ligands. GABAA receptors in rat and mouse neurons displayed EC50s for GABA of 9 +/- 0.3 and 17 +/- 0.8 microM, respectively and ethanol did not significantly change these values. The EC50 for diazepam was 92 +/- 3 and 120 +/- 8 nM in rat and mouse, respectively. Pentobarbital enhanced the current with EC50s of 84 +/- 3 and 106 +/- 6 microM in rat and mouse, respectively. The sensitivity for Cl-218,872, which binds preferentially to the Type I benzodiazepine receptor, was similar in all the neurons. RO 15-4513, an inverse partial agonist to the benzodiazepine receptor, was not effective in reversing the potentiation of the Cl- current in rat neurons and only slightly reduced the potentiation in mouse neurons. The receptors in rat neurons were more sensitive to external Zn2+; the current was inhibited by 50% with a concentration of 93 +/- 3 and 244 +/- 9 microM in rat and mouse, respectively. Analysis of mRNA encoding for the gamma 2L receptor subunit showed similar levels in rat and mouse neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term fluoxetine treatment induces input-specific LTP and LTD impairment and structural plasticity in the CA1 hippocampal subfield.

Antidepressant drugs are usually administered for several weeks for the treatment of major depressive disorder. However, they are also prescribed in several additional psychiatric conditions as well as during long-term maintenance treatments. Antidepressants induce adaptive changes in several forebrain structures which include modifications at glutamatergic synapses. We recently found that repetitive administration of the selective serotonin reuptake inhibitor (SSRI) fluoxetine to naïve adult male rats induced an increase of mature, mushroom-type dendritic spines in several forebrain regions. This was associated with an increase of GluA2-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPA-Rs) in telencephalic postsynaptic densities. To unravel the functional significance of such a synaptic re-arrangement, we focused on glutamate neurotransmission in the hippocampus. We evaluated the effect of four weeks of 0.7 mg/kg fluoxetine on long-term potentiation (LTP) and long-term depression (LTD) in the CA1 hippocampal subfield. Recordings in hippocampal slices revealed profound deficits in LTP and LTD at Schaffer collateral-CA1 synapses associated to increased spine density and enhanced presence of mushroom-type spines, as revealed by Golgi staining. However, the same treatment had neither an effect on spine morphology, nor on LTP and LTD at perforant path-CA1 synapses. Cobalt staining and immunohistochemical experiments revealed decreased AMPA-R Ca2+ permeability in the stratum radiatum (s.r.) together with increased GluA2-containing Ca2+ impermeable AMPA-Rs. Therefore, 4 weeks of fluoxetine treatment promoted structural and functional adaptations in CA1 neurons in a pathway-specific manner that were selectively associated with impairment of activity-dependent plasticity at Schaffer collateral-CA1 synapses.

MDMA ("ecstasy") impairs learning in the Morris Water Maze and reduces hippocampal LTP in young rats.

3,4-Methylenedioxymethamphetamine (MDMA), an important recreational psychostimulant drug, was examined for its ability to alter visuo-spatial learning and synaptic plasticity. Young rats received MDMA (0.2 and 2mg/kg s.c.) twice per day for 6 days while their visuo-spatial learning was tested using the Morris Water Maze. After this, animals were sacrificed and LTP induced in hippocampal slices. Visuo-spatial learning was impaired and LTP reduced, both dose-dependently, without changes in serotonin levels or paired-pulse facilitation. We conclude that low, nontoxic doses of MDMA, applied during several days, slow learning by impairing postsynaptic plasticity.

Effect of short-term exposure to dichlorvos on synaptic plasticity of rat hippocampal slices: Involvement of acylpeptide hydrolase and α7 nicotinic receptors

Dichlorvos is the active molecule of the pro-drug metrifonate used to revert the cognitive deficits associated with Alzheimers disease. A few years ago it was reported that dichlorvos inhibits the enzyme acylpeptide hydrolase at lower doses than those necessary to inhibit acetylcholinesterase to the same extent. Therefore, the aim of our investigation was to test the hypothesis that dichlorvos can enhance synaptic efficacy through a mechanism that involves acylpeptide hydrolase instead of acetylcholinesterase inhibition. We used long-term potentiation induced in rat hippocampal slices as a model of synaptic plasticity. Our results indicate that short-term exposures (20 min) to 50 microM dichlorvos enhance long-term potentiation in about 200% compared to the control condition. This effect is correlated with approximately 60% inhibition of acylpeptide hydrolase activity, whereas acetylcholinesterase activity remains unaffected. Paired-pulse facilitation and inhibition experiments indicate that dichlorvos does not have any presynaptic effect in the CA3-->CA1 pathway nor affect gabaergic interneurons. Interestingly, the application of 100 nM methyllicaconitine, an alpha(7) nicotinic receptor antagonist, blocked the enhancing effect of dichlorvos on long-term potentiation. These results indicate that under the exposure conditions described above, dichlorvos enhances long-term potentiation through a postsynaptic mechanism that involves (a) the inhibition of the enzyme acylpeptide hydrolase and (b) the modulation of alpha(7) nicotinic receptors.

Homeostatic NMDA receptor down‐regulation via brain derived neurotrophic factor and nitric oxide‐dependent signalling in cortical but not in hippocampal neurons

J. Neurochem. (2011) 118, 760–772.

Neurological and neuropsychological deterioration in artisanal gold miners from the town of Andacollo, Chile

It is widely known that human exposure to mercury vapor can cause neurological and neuropsychological deterioration. We have investigated if a population of Chilean artisanal gold-mining workers heavily exposed to elemental mercury (Hg0) display neurological and neuropsychological impairment. Male volunteers occupationally exposed to Hg0 (“gold miners”, n = 35) were recruited and compared with a group of unexposed workers (n = 40). Blood specimens were obtained from both groups for total mercury quantification. Upon neurological examination, 71% of the “gold miners” group showed abnormalities expressed as frontal impairment, tremor, or simultaneously frontal impairment, parkinsonism, and pyramidal syndrome. In contrast, only 16% of the individuals in the control group displayed neurological abnormalities. The “gold miners” group also showed impairment of the neuropsychological performance and the distribution of abnormal scores for almost all the neuropsychological tests applied was significantly higher in this group compared to the control group. Educational level, alcohol intake, and smoking habits did not influence the outcome of the neurological or neuropsychological examinations. Finally, despite that the higher blood mercury concentrations were found in the “gold miners” group, they were not significantly different from the values obtained in the control group.

Assessing biomarkers and neuropsychological outcomes in rural populations exposed to organophosphate pesticides in Chile--study design and protocol.

BackgroundHealth effects of pesticides are easily diagnosed when acute poisonings occurs, nevertheless, consequences from chronic exposure can only be observed when neuropsychiatric, neurodegenerative or oncologic pathologies appear. Therefore, early monitoring of this type of exposure is especially relevant to avoid the consequences of pathologies previously described; especially concerning workers exposed to pesticides on the job. For acute organophosphate pesticides (OPP) exposure, two biomarkers have been validated: plasma cholinesterase (ChE) and acetylcholinesterase (AChE) from erythrocytes. These enzymes become inhibited when people are exposed to high doses of organophosphate pesticides, along with clear signs and symptoms of acute poisoning; therefore, they do not serve to identify risk from chronic exposure. This study aims to assess a novel biomarker that could reflect neuropsychological deterioration associated with long-term exposure to organophosphate pesticides via the enzyme acylpeptide-hydrolase (ACPH), which has been recently identified as a direct target of action for some organophosphate compounds.Methods/DesignThree population groups were recruited during three years (2011–2013): Group I having no exposure to pesticides, which included people living in Chilean coastal areas far from farms (external control); Group II included those individuals living within the rural and farming area (internal control) but not occupationally exposed to pesticides; and Group III living in rural areas, employed in agricultural labour and having had direct contact with pesticides for more than five years. Blood samples to assess biomarkers were taken and neuropsychological evaluations carried out seasonally; in three time frames for the occupationally exposed group (before, during and after fumigation period); in two time frames for internal control group (before and during fumigation), and only once for the external controls. Neuropsychological evaluations considered cognitive functions, affectivity and psychomotor activity. The biomarkers measured included ChE, AChE and ACPH. Statistical analysis and mathematical modelling used both laboratory results and neuropsychological testing outcomes in order to assess whether ACPH would be acceptable as biomarker for chronic exposure to OPP.DiscussionThis study protocol has been implemented successfully during the time frames mentioned above for seasons 2011, 2012 and 2013–2014.

Synaptic localization of acylpeptide hydrolase in adult rat telencephalon.

Acylpeptide hydrolase (ACPH), a serine protease present in the central nervous system (CNS), is believed to have a function in modulating synaptic plasticity, cleavage of beta amyloid peptide and degradation of aggregated oxidized proteins. In this report, we demonstrate for the first time the presence of ACPH in the synapse and its preferential localization at the pre-synaptic side. We isolated subcellular fractions from the rat telencephalon enriched in pre- versus post-synaptic components by using differential centrifugation steps to evaluate ACPH catalytic activity and expression level. Relative ACPH levels were determined by Western blot techniques while antibodies against synaptophysin and PSD-95 were used as positive pre- and post-synaptic markers, respectively. Our results show that ACPH protein levels are significantly increased at the synapse, which correlates with a 56% increase in ACPH activity. Furthermore, Western blot experiments show that ACPH is preferentially located at the pre-synaptic side and this is consistent with the increase of its enzymatic activity in fractions enriched in pre-synaptic components. These results give new insights regarding the localization and a putative role of ACPH in the CNS.

Cognitive impairment in agricultural workers and nearby residents exposed to pesticides in the Coquimbo Region of Chile

Chronic exposure to organophosphate pesticides is a worldwide public health concern associated with several psychiatric disorders and dementia. Most existing studies on the effects of pesticides only evaluate agricultural workers. Therefore, this study sought to establish if individuals indirectly exposed to pesticides, such as residents in agricultural areas, also suffer cognitive impairments. Neuropsychological evaluations were carried out on three groups (n=102): agricultural workers directly exposed to pesticides (n=32), individuals living in agricultural areas indirectly (i.e. environmentally) exposed to pesticides (n=32), and an unexposed control group (n=38). The assessed cognitive processes included memory, executive functions, attention, language praxis, and visuoconstruction. The direct exposure group performed significantly lower in executive function, verbal fluency, and visual and auditory memory tests than the indirect exposure group, which, in turn, performed worse than the unexposed group. Even after adjusting for age, gender, and educational level, both exposure groups showed higher rates of cognitive deficit than control individuals. In conclusion, both direct and indirect chronic exposure to pesticides affects cognitive functioning in adults and, consequently, actions should be taken to protect the health of not only agricultural workers, but also of residents in agricultural areas.