Trimethyltin-induced neurotoxicity: gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis.

Damage to the CNS results in a complex series of molecular and cellular changes involving the affected targets and the ensuing glial reaction. The initial gene expression events that underlie these cellular responses may serve as early biomarkers of neurotoxicity. Here, we examined gene expression profiles during the initial phase of hippocampal damage resulting from systemic exposure of rats to the organometallic neurotoxicant, trimethyltin (TMT, 8.0 mg/kg, i.p.). Using TMT as a neurodegeneration tool confers several advantages for evaluating molecular events associated with neural damage: 1) regional and cellular targets and time course of damage are known, 2) the blood-brain barrier is not compromised, which limits the contribution of blood-borne factors, e.g. immune, to neural injury responses and 3) known protein and mRNA signatures of TMT-induced neurotoxicity can be used as positive controls to validate novel expression events associated with exposure to this neurotoxicant. Using Affymetrix Gene Chip® to assess gene expression after TMT, combined with Ingenuity Pathway Analysis®, we observed changes consistent for genes known to be affected in hippocampus, while corresponding changes were not detected in cerebellum, a non-target region. In agreement with previous observations, limited changes in expression of inflammation-related genes were observed. Correlated expression profiles were found after exposure to TMT, including changes in gene ontologies associated with neurological disease, cellular assembly and maintenance, as well as signaling pathways associated with cellular stress, energy metabolism and glial activation. Selected gene changes were confirmed from each category by q-RT-PCR and immunoblot analysis. The canonical relationships identified implicate molecular pathways and processes relevant to detection of early stages of hippocampal damage in the TMT model. These observations provide new insight into early events associated with neuronal degeneration and associated glial activation that may serve as the basis for discovery and development of biomarkers of neurotoxicity.

Chemically induced neuronal damage and gliosis: enhanced expression of the proinflammatory chemokine, monocyte chemoattractant protein (MCP)-1, without a corresponding increase in proinflammatory cytokines(1).

Enhanced expression of proinflammatory cytokines and chemokines has long been linked to neuronal and glial responses to brain injury. Indeed, inflammation in the brain has been associated with damage that stems from conditions as diverse as infection, multiple sclerosis, trauma, and excitotoxicity. In many of these brain injuries, disruption of the blood-brain barrier (BBB) may allow entry of blood-borne factors that contribute to, or serve as the basis of, brain inflammatory responses. Administration of trimethyltin (TMT) to the rat results in loss of hippocampal neurons and an ensuing gliosis without BBB compromise. We used the TMT damage model to discover the proinflammatory cytokines and chemokines that are expressed in response to neuronal injury. TMT caused pyramidal cell damage within 3 days and a substantial loss of these neurons by 21 days post dosing. Marked microglial activation and astrogliosis were evident over the same time period. The BBB remained intact despite the presence of multiple indicators of TMT-induced neuropathology. TMT caused large increases in whole hippocampal-derived monocyte chemoattractant protein (MCP)-1 mRNA (1,000%) by day 3 and in MCP-1 (300%) by day 7. The mRNA levels for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, cytokines normally expressed during the earliest stage of inflammation, were not increased up to 21 days post dosing. Lipopolysaccharide, used as a positive control, caused large inductions of cytokine mRNA in liver, as well as an increase in IL-1beta in hippocampus, but it did not result in the induction of astrogliosis. The data suggest that enhanced expression of the proinflammatory cytokines, TNF-alpha, IL-1beta and IL-6, is not required for neuronal and glial responses to injury and that MCP-1 may serve a signaling function in the damaged CNS that is distinct from its role in proinflammatory events.

Astrogliosis in the adult and developing CNS: is there a role for proinflammatory cytokines?

Astrogliosis, characterized by the enhanced expression of GFAP, represents a remarkably homotypic response of astrocytes to all types of injuries of the CNS, including injuries of the developing CNS. As such, astrocytes serve as microsensors of the injured microenvironment regardless of their location in the CNS. The diversity of insults that engender astrogliosis and the brain-wide nature of the astrocytic response suggest that common injury factors serve as the trigger of this cellular reaction. One prominent theme that has emerged in recent years is that proinflammatory cytokines and chemokines serve as a stimulus for induction of astrogliosis. Here we present a brief critique of this hypothesis based on a review of literature and some of our own recentfindings. Studies of astrocytes, in vitro, clearly indicate that these cell types are responsive to a variety of growth factors, including cytokines and chemokines. A somewhat different picture, however, can be seen from data obtained in vivo. It is true that trauma and diseases of the nervous system, as well as some exposures to neurotoxic chemicals, can be associated with the expression in brain of large varieties of cytokines and chemokines. That these same conditions result in astrogliosis has fostered the circumstantial link between cytokine/chemokine expression and the induction of astrogliosis. Several lines of evidence argue against this view, including (a) suppression of cytokine expression does not suppress gliosis, (b) gliosis can occur in the absence of enhanced expression of cytokines, (c) elevations in brain cytokines can occur in the absence of gliosis and (d) the patterns of cytokine expression in the adult and developing CNS are more consistent with a trophic role for these chemical messengers rather than a role in the induction of inflammation. Enhanced expression of cytokines and chemokines after brain injury appear to be signal transduction events unrelated to the induction of astrogliosis.

A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors.

Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.

Decreases in brain glial fibrillary acidic protein (GFAP) are associated with increased serum corticosterone following inhalation exposure to toluene.

Toluene and other neurotoxicants can cause both increases and decreases in the concentration of GFAP in the brain. While increased GFAP concentration is widely regarded as evidence for reactive gliosis, toxicant-induced decreases in GFAP have received less attention. In order to identify conditions under which inhalation exposure to toluene results in decreased GFAP concentration, rats were subjected to repeated inhalation of toluene for up to 7 days. Adult male F344 rats received inhalation exposure to air or to 1000 ppm toluene, 6 hr/day, for 3 or 7 days. This toluene exposure replicated the previously-observed decreases in GFAP in the thalamus. Serum Corticosterone was significantly elevated in the same rats that exhibited decreases in brain GFAP concentration. These results show that decreases in brain GFAP might be a consequence of disruption of the hypothalamic-pituitary-adrenal axis and/or hormonal homeostasis. Changes in GFAP and in Cort were not accompanied by a change in body weight. More research is needed to firmly establish cause and effect between increased serum glucocorticoid levels and GFAP decreases following toluene inhalation and to determine whether these decreases indicate toxicity or adaptive changes.

alpha-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor.

alpha-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin I alpha was previously described as a high affinity alpha-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of alpha-latrotoxin with neurexin I alpha cannot explain how alpha-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of alpha-latrotoxin (CIRL), which is a second high affinity alpha-latrotoxin receptor that may be the major mediator of alpha-latrotoxin's effects. CIRL appears to be a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL has two subunits of the 120 and 85 kDa that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain where it is enriched in the striatum and cortex. Expression of CIRL in chromaffin cells increases the sensitivity of the cells to the effects of alpha-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an alpha-latrotoxin affinity column and forms stable complexes with this receptor in vitro. Interaction of CIRL with a specific presynaptic neurotoxin and with a component of the docking-fusion machinery suggests its role in regulation of neurosecretion.

The calcium-independent receptor of alpha-latrotoxin is not a neurexin.

alpha-Latrotoxin (alpha-LTx), a vertebrate neurotoxin isolated from Black Widow Spider venom, causes massive spontaneous neurotransmitter release. The molecular mechanism(s) by which the toxin exerts its effect is largely unknown. Here we report identification and purification of a novel membrane receptor with high affinity for alpha-LTx. Unlike neurexin Ia, a previously described high affinity alpha-LTx receptor, this novel protein binds alpha-LTx independently of Ca2+ presence and therefore may be a mediator of the calcium-independent stimulation of neurotransmitter release by alpha-latrotoxin. The major protein component of calcium-independent alpha-LTx receptors is a novel M(r) 120,000 protein which does not belong to the neurexin family. Among several tissues tested, the M(r) 120,000 protein was found only in brain.

Exposure to methylmercury results in serum autoantibodies to neurotypic and gliotypic proteins.

Environmental exposure to methylmercury (MeHg) continues to pose a threat to humans, making early detection of neurotoxic effects a pressing concern. An enzyme-linked immunosorbent assay (ELISA) to measure serum autoantibodies (lg) to neurotypic and gliotypic proteins [neurofilament triplet (NF68; NF160; NF200), myelin basic protein (MBP) and glial fibrillary acid protein (GFAP)] as markers of subclinical neurotoxicity was developed and tested in Fisher 344 rats exposed orally to 16 or 32 ppm MeHg. Both levels of MeHg resulted in serum lg to all 5 proteins, not normally seen in controls. For anti-NFs and anti-GFAP, lgM isotype predominated significantly (p < 0.05) over lgG. lg for MBP were of the lgG isotype, lgM were not detected. Significant differences (p < 0.05) between 16 and 32 ppm MeHg in levels of anti-NF 68 and GFAP, lgM, were evident at 7 days of exposure, but not at 14 days. Anti-NF 160, lgM, was significantly (p < 0.01) elevated in rats exposed to 32 ppm vs 16 ppm at 14 days. However, at both dose levels anti-NF 68 titers were the most elevated of the three NF proteins (p < 0.0001). For anti-NF 200 and anti-MBP it was the lgG isotype that was significantly (p < 0.01) elevated in the 32 ppm group at 7 days. GFAP levels as a marker of neurotoxicity were determined in the cortex, hippocampus and cerebellum. Exposure to 32ppm MeHg resulted in decreased (p < 0.05) levels in the cortex at 14 days. Both levels of MeHg resulted in increased GFAP in the cerebellum at 14 days. This study suggests that assay of autoantibodies against nervous system proteins may provide a means of assessing the early neurotoxic effects of environmental MeHg exposure.

Exposure to methyl mercury results in serum autoantibodies to neurotypic and gliotypic proteins.

Environmental exposure to methyl mercury (MeHg) continues to pose a threat to humans, making early detection of neurotoxic effects a pressing concern. An enzyme-linked immunosorbent assay (ELISA) to measure serum autoantibodies (Ig) to neurotypic and gliotypic proteins [neurofilament triplet (NF68; NF160; NF200), myelin basic protein (MBP) and glial fibrillary acid protein (GFAP)] as markers of subclinical neurotoxicity was developed and tested in Fisher 344 rats exposed orally to 16 or 32 ppm MeHg. Both levels of MeHg resulted in serum Ig to all 5 proteins, not normally seen in controls. For anti-NFs and anti-GFAP, IgM isotype predominated significantly (p < 0.05) over IgG.Ig for MBP were of the IgG isotype, IgM were not detected. Significant differences (p < 0.05) between 16 and 32 ppm MeHg in levels of anti-NF 68 and GFAP, IgM, were evident at 7 days of exposure, but not at 14 days. Anti-NF 160, IgM, was significantly (p < 0.01) elevated in rats exposed to 32 ppm vs 16 ppm at 14 days. However, at both dose levels anti-NF 68 titers were the most elevated of the three NF proteins (p < 0.0001). For anti-NF 200 and anti-MBP it was the IgG isotype that was significantly (p < 0.01) elevated in the 32 ppm group at 7 days. GFAP levels as a marker of neurotoxicity were determined in the cortex, hippocampus and cerebellum. Exposure to 32ppm MeHg resulted in decreased (p < 0.05) levels in the cortex at 14 days. Both levels of MeHg resulted in increased GFAP in the cerebellum at 14 days. This study suggests that assay of autoantibodies against nervous system proteins may provide a means of assessing the early neurotoxic effects of environmental MeHg exposure.

Trimethyl lead neurotoxicity in the rat: changes in glial fibrillary acidic protein (GFAP).

The literature on the toxicology of lead provides little evidence of the neurotoxicity of organic lead compounds. Toxicant-induced changes in the concentration of glial fibrillary acidic protein (GFAP) in the brain may help clarify at which stage of neurotoxicity astrocytes are affected and whether GFAP may provide an index of toxicity. Male F344 rats (> 42 days old) were exposed to 0 (control), 8 or 16 ppm lead as trimethyl lead (TMPb) in drinking water for up to 14 days. Weight Gain was significantly reduced in both exposed groups. Control rats had the expected brain regional pattern of GFAP concentration with the highest in the hippocampus and cerebellum and lowest in the cerebral cortex. The hippocampus was the region very sensitive to TMPb, with increased GFAP in rats exposed to 8 and 16 ppm TMPb with decreases in GFAP in rats exposed to 8 and 16 ppm TMPb for 14 days. There was a significant time-response in rats exposed to 8 ppm TMPb with decreases in GFAP on day 7 and increases on day 14. A hypothesis concerning this biphasic change in GFAP concentrations is discussed. The results indicate that GFAP may be used to indicate the role of the astrocyte in the neurotoxicity of TMPb. GFAP concentration, as biomarker of TMPb effect, was as sensitive to TMPb as body weight and thus may provide a marker of neurotoxicity.