Lein Lab Research Focus: Developmental Neurotoxicity

How do neurotoxic environmental contaminants alter developmental processes that underlie neuronal connectivity?

There are two classes of environmental contaminants that we are investigating in the lab: polychlorinated biphenyls (PCBs) and organophosphate pesticides (OPs). There is wide-spread human exposure to both of these environmental contaminants, and both are suspected of causing developmental neurotoxicity in humans. Identifying the mechanism of action of these chemicals is complicated by the fact that very little is currently known about specific neurodevelopmental processes that are disrupted by either PCBs or OPs. Thus, the goals in our lab are to determine if PCBs or OPs alter neurodevelopmental events that determine neuronal connectivity. Specific questions we are currently pursuing include the following:

Do PCBs derail normal neurodevelopment via interactions with the ryanodine receptor?

Mechanistic studies of PCB developmental neurotoxicity have indicated that ortho-substituted non-coplanar PCBs increase intracellular calcium levels in cultured neurons. The relationship of these calcium changes to PCB-induced alterations of neuronal development, plasticity and higher order function is not well established. Since it is well documented that intracellular calcium levels modulate neuronal apoptosis and dendritic growth, we tested the hypothesis that PCBs perturb these developmental processes. We completed a series of experiments indicating that ortho-substituted non-coplanar, but not coplanar, PCBs induce apoptosis in cultured hippocampal neurons. The pro-apoptotic activity of PCBs is mediated by activation of the ryanodine receptor and can be prevented by vitamin E. These data have been published in Toxicology and Applied Pharmacology (2003).

In addition, we have initiated a series of studies to determine if developmental exposure to PCBs alters dendritic growth. The experimental approach is to integrate studies of structural alterations with functional assessments in the whole animal. We have completed a series of studies using the Morris swim task to test spatial learning and memory in juvenile rats. We are currently analyzing dendritic morphology in brains collected from animals immediately following the conclusion of behavioral studies using various morphometric techniques including immunohistochemical localization and quantitative RT-PCR analyses of MAP2, PSD95, and RC3, and Golgi analysis of dendritic growth in individual neurons.

Is acetylcholinesterase (AChE) the critical target in the developmental neurotoxicity of organophosphorus pesticides (OPs)?

It is reported that OPs cause developmental neurotoxicity at doses significantly below those that inhibit the catalytic activity of AChE. These data have been widely interpreted to mean that OPs target molecules other than AChE. However, recent evidence demonstrating that AChE functions to promote axonal growth in developing but not mature neurons suggests an alternative explanation: OPs target AChE, but the mechanism of developmental neurotoxicity involves disruption of the morphogenic function of AChE not inhibition of its catalytic activity. Disruption of axonal morphogenesis is correlated with functional deficits and the critical exposure period for OP developmental neurotoxicity coincides with timing of axonal outgrowth and synaptogenesis in the developing brain. Thus, interference with the axon-promoting activity of AChE represents a biologically plausible mechanism for explaining the functional deficits observed in animals following developmental exposure to OPs. We are using the recently developed AChE null mice generate cultures of sensory neurons from dorsal root ganglia (DRG) of mice that are either AChE +/+ or AChE -/- to test the hypothesis that OPs disrupt axonal growth by interfering with the morphogenic activity of AChE.

In addition, we are conducting experiments to test the hypothesis that OPs may exert developmental neurotoxicity via effects on phosphorylation of the transcription factor CREB. These studies began as a collaboration with David Jett (formerly a faculty member at Johns Hopkins University, currently a program officer in NIH NINDS) to examine the effects of OPs on phophorylation of CREB. This collaboration resulted in a recent publication (Toxicology and Applied Pharmacology, 2002) demonstrating that chlorpyrifos and its metabolites increase levels of pCREB at concentration that have no inhibitory effect on AChE enzymatic activity. We are currently working to understand the mechanism by which OPs phosphorylate CREB, and are working in collaboration with Jack McCarthy (OGI School of Science & Engineering) to determine if this response can be exploited to develop a sensitive biosensor for OPs.

Determine if there is a physiological link between OP exposure and airway hyperreactivity.

In collaboration with Allison Fryer (Physiology & Pharmacology, OHSU), we are testing the hypothesis that OPs increase airway hyperreactivity. This hypothesis is derived from observations that OPs alter cholinergic neurotransmission in the brain via inhibition of AChE or via interaction with cholinergic receptors. In the lung, airway tone and reactivity are mediated by the cholinergic parasympathetic neurons in the vagi: activation of parasympathetic ganglia releases acetylcholine (ACh), which binds to M3 muscarinic receptors on airway smooth muscles to cause bronchoconstriction. A negative feedback loop exists, however, in which released ACh binds to autoinhibitory M2 muscarinic receptors on the parasympathetic nerve endings to decrease further release of ACh. Thus factors that decrease levels of ACh or inhibit function of M2 potentiate bronchoconstriction mediated by vagal activity. It has been demonstrated that virus-infected and antigen-challenged guinea pigs, as well as patients with asthma, have dysfunctional M2 receptors, which contributes to the airway hyperreactivity observed in these pathological conditions.

Our goal is to use the guinea pig model of airway hyperreactivity to 1) determine if OPs alter vagally-induced bronchoconstriction; and 2) identify the mechanisms by which pesticides cause airway hyperreactivity. We recently published a paper (American Journal of Physiology, 2004) demonstrating increased airway hyperreactivity in guinea pigs exposed to chlorpyrifos that is mediated by inhibition of M2 receptors in the absence of effects on AChE catalytic activity. We have just completed a set of studies showing that other organophosphate pesticides, including diazinon and parathion, but not pyrethroids such as permethrin, induce airway hyperreactivity at concentrations that have no effect on AChE catalytic activity in the lung or blood. We have initiated studies designed to test the hypothesis that sensitization alters the airway response to OPs.

Please send comments, questions, and reports of problems with the Lein Lab Web pages to Pam Lein at leinp@ohsu.edu.