Repeated exposure to sublethal doses of the organophosphorus compound VX activates BDNF expression in mouse brain.

The highly toxic organophosphorus compound VX [O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonate] is an irreversible inhibitor of the enzyme acetylcholinesterase (AChE). Prolonged inhibition of AChE increases endogenous levels of acetylcholine and is toxic at nerve synapses and neuromuscular junctions. We hypothesized that repeated exposure to sublethal doses of VX would affect genes associated with cell survival, neuronal plasticity, and neuronal remodeling, including brain-derived neurotrophic factor (BDNF). We examined the time course of BDNF expression in C57BL/6 mouse brain following repeated exposure (1/day × 5 days/week × 2 weeks) to sublethal doses of VX (0.2 LD(50) and 0.4 LD(50)). BDNF messenger RNA expression was significantly (p < 0.05) elevated in multiple brain regions, including the dentate gyrus, CA3, and CA1 regions of the hippocampal formation, as well as the piriform cortex, hypothalamus, amygdala, and thalamus, 72 h after the last 0.4 LD(50) VX exposure. BDNF protein expression, however, was only increased in the CA3 region of the hippocampus. Whether increased BDNF in response to sublethal doses of VX exposure is an adaptive response to prevent cellular damage or a precursor to impending brain damage remains to be determined. If elevated BDNF is an adaptive response, exogenous BDNF may be a potential therapeutic target to reduce the toxic effects of nerve agent exposure.

Susceptibility of Manduca sexta to Cry1Ab toxin of Bacillus thuringiensis correlates directly to developmental expression of the cadherin receptor BT-R(1).

The cadherin receptor BT-R(1), localized in the midgut epithelium of the tobacco hornworm, Manduca sexta, is coupled to programmed oncotic-like cell death, which is triggered by the univalent binding of the Cry1Ab toxin of Bacillus thuringiensis (Bt) to the receptor. Kinetic analysis of BT-R(1) expression during larval development reveals that the density of BT-R(1) on the midgut surface increases dramatically along with an equivalent rise in the concentration of Cry1Ab toxin molecules needed to kill each of the five larval stages of the insect. The increase in the number of BT-R(1) molecules per midgut surface area requires additional toxin molecules to kill older versus younger larvae, as evidenced by the corresponding LC(50) values. Based on these observations, we developed a mathematical model to quantify both the expression of BT-R(1) and the susceptibility of M. sexta larvae to the Cry1Ab toxin. Interestingly, the toxin-receptor ratio remains constant during larval development regardless of larval size and mass. This ratio apparently is critical for insecticidal activity and the decrease in toxin effectiveness during larval development is due primarily to the number of effective toxins and available receptors in the larval midgut. Evidently, susceptibility of M. sexta to the Cry1Ab toxin of Bt correlates directly to the developmental expression of BT-R(1) in this insect.

Global changes in the expression patterns of RNA isolated from the hippocampus and cortex of VX exposed mice.

One of the established activities of the nerve agent VX is inhibition of the enzyme acetylcholinesterase (AChE). This inhibition affects the cholinergic nervous system by decreasing the activity of the neurotransmitter-hydrolyzing enzyme cholinesterase (ChE). In an effort to gain a more comprehensive understanding of the molecular pathways affected by low-level exposure to VX, an expression profiling approach was used to identify genes with altered RNA expression patterns after exposure.Specifically, mice were exposed to 0.1, 0.2, 0.4, and 0.6 LD50 VX for a period of 2 weeks. At 2 h, 72 h, and 2 weeks after the final exposure, RNA was isolated from both the hippocampus and the cortex. Changes in gene expression levels were assessed by DNA microarray technology and grouped according to their expression patterns. Data presented here demonstrate that 2 weeks postexposure all up-regulated gene expression has returned to pre-exposure levels, including genes related to the central nervous system. Additionally, this investigation has revealed non-AChE pathway genes involved in other neuronal functions that display altered expression profiles after VX exposure.

Expression of a midgut-specific cadherin BT-R1 during the development of Manduca sexta larva.

The btr-1 gene of Manduca sexta (GenBank AF319973) encodes a cadherin, BT-R(1) (210-kDa), which contains 12 ectodomain modules in association with a number of motifs potentially involved in interactions with cadherin and integrin. The molecule is a target receptor for Bacillus thuringiensis Cry1A toxins that bind to BT-R(1) with high affinity and specificity. BT-R(1) is localized exclusively in the midgut epithelium. The amount of BT-R(1) protein increases dramatically during larval development, paralleling accumulation of its mRNA. The 5'-UTR of the btr-1 gene contains sequence motifs that most likely recruit specific transcription factors, particularly, those that determine posterior patterning and that control intestinal cell proliferation, differentiation and identity during development. The increase in abundance of BT-R(1) may be required to support not only the differentiation of the epithelial cells but also the establishment of physiological function and structural integrity of the midgut during larval development in M. sexta. We believe that BT-R(1) is essential to larval midgut epithelial organization during rapid cell proliferation and tissue growth in M. sexta because disruption of such organization and functionality occasioned by the binding of the Cry1A toxins of B. thuringiensis to BT-R(1) causes death to the insect.

Proteolytic cleavage of the developmentally important cadherin BT-R1 in the midgut epithelium of Manduca sexta.

BT-R1 (M(r) = 210 kDa) represents a new type of insect cadherin that is expressed specifically in the midgut epithelium during growth and development of Manduca sexta larvae. It also is a target receptor for the Cry1A toxins of the entomopathogenic bacterium Bacillus thuringiensis. Expression of BT-R1, which varies during larval development, correlates with the abundance of the protein and with the differential cleavage of the molecule at each developmental stage. The cleavage of BT-R1 is calcium dependent, and consequently, Ca2+ directly influences the structural integrity of BT-R1. Indeed, removal of calcium ions by chelating agents promotes cleavage of the BT-R1 ectodomain, resulting in formation of fragments that are similar to those observed during larval development. Partial purification of proteins from brush border membrane vesicles (BBMVs) by gel filtration chromatography hinders the cleavage of BT-R1 in the presence of EDTA and EGTA, indicating that there is specific proteolytic activity associated with the BBMV. This specific proteolytic cleavage of BT-R1 not only alters the integrity of BT-R1 but it most likely is implicated in cell adhesion events during differentiation and development of M. sexta midgut epithelium. We propose a model for calcium-dependent protection of BT-R1 as well as a cleavage pattern that may modulate the molecular interactions and adhesive properties of its ectodomain. Molecular characterization of such a protection mechanism should lead to a better understanding of how the function of specific cadherins is modulated during tissue differentiation and insect development.