Proposed mechanisms by which Pb exposure stimulates astrocyte VEGF expression and extends survival in ALS mice. Pb induces VEGF expression specifically in SOD1^G93A bearing astrocytes abrogating their neurotoxic phenotype to motor neurons. On the contrary, non-Tg astrocytes Pb failed to induce VEGF expression and instead induced a neurotoxic phenotype. Other cytoprotective and/or cytotoxic pathways are likely also stimulated by Pb with yet unknown impact on motor neuron survival.

Lead exposure increases VEGF protein levels in SOD1^G93A transgenic astrocytes. A) Representative western blot of SOD1^G93A and non-transgenic astrocytes treated for 24 h with increasing concentrations of PbAc (Pb). Note the increase in VEGF levels with Pb treatment. No signal was detected in non-transgenic astrocytes. B) Relative densitometric analysis of VEGF expression in SOD1^G93A astrocytes. Values represent the mean ± SEM of four different experiments. * p < 0.05 compared with SOD1^G93A vehicle-treated group.

Exposure to low-Pb levels increases survival of SOD1^G93A transgenic mice. PbAc (Pb) or sodium acetate (vehicle) was administered in drinking water from weaning to death as detailed in materials and methods. A) Disease onset was not modified by Pb administration when compared to vehicle-treated group (vehicle: 101±14 days; Pb: 110±11 days; Kaplan-Meier curve, p= 0.29, Log-rank test). B) Administration of Pb resulted in a significant increase in the survival of SOD1^G93A transgenic mice when compared to vehicle-treated group (vehicle: 125 ± 1 days; Pb: 137 ± 2 days; Kaplan- Meier curve, p< 0.0001, Log-rank test).

Effects of Pb exposure on GFAP immunoreactivity in the lumbar spinal cord. A) GFAP immunofluorescence was almost undetectable in the ventral horns of lumbar spinal cord from non-transgenic animals treated with vehicle (top panels, a), or with Pb treatment (top panels, b). Lower panels show that the increased GFAP immunoreactivity displayed by vehicle-treated SOD1^G93A mice (c) was greatly reduced by Pb treatment (d). Scale bar: 40μm. B) Quantification of GFAP immunofluorescence in the ventral horn showed a reduction of more than 50% in Pb treated SOD1^G93A animals compared to vehicle treated transgenic mice. GFAP immunofluorescence was less than 0.1 % in PbAc or NaAc treated non-transgenic animals. Values represent the mean ± SEM of measured areas of at least 15 spinal cord sections per group. * p < 0.05 compared with non-transgenic vehicle-treated group; ** p < 0.05 compared with vehicle-treated SOD1^G93A group.

VEGF expression is increased in the lumbar spinal cord of SOD1^G93A transgenic mice exposed to Pb. VEGF immunoreactivity expression was analyzed in the lumbar spinal cord of SOD1^G93A and non-transgenic mice exposed to PbAc (Pb) or sodium acetate (Vehicle). A) Representative immunoblots (a) and relative densitometric analysis (b) of VEGF expression in vehicle- and Pb-treated non-transgenic and SOD1^G93A mice. Values represent the mean ± SEM of three different experiments compared by one-way ANOVA. * p < 0.05 compared with SOD1^G93A vehicle-treated group. B) Representative VEGF immunohistochemistry in the ventral horn of spinal cord. Immunoreactivity is typically detected in motor neuron soma, both in non-transgenic (a) and SOD1^G93A (b) mice; note the increase of VEGF immunostaining in the neuropil of SOD1^G93A transgenic mice (b), which was augmented by Pb treatment (c). Arrows show VEGF imunoreactive cells surrounding motor neurons. Scale bar: 30μm.

Effects of Pb-astrocyte exposure on motor neuron survival. A) Lead toxicity in motor neuron cell culture. Wild type motor neurons were exposed during 48 h to different concentrations of PbAc in the presence of GDNF (1 ng/ml). The dose response curve revealed significant motor neuron death beginning at 10μM lead acetate. (*) p<0.05: different from control (GDNF). NONE: trophic factor withdrawal. B) Increased motor neuron survival in transgenic astrocytes pre-exposed to Pb. Astrocyte monolayers were exposed for 24 h to 10 μM of PbAc or vehicle. Wild type motor neurons were plated upon the monolayer after washing out Pb. VEGF-blocking antibody (αVEGF) was added 2 h later to the culture media. Motor neuron survival was analyzed 48 h later. The graph shows the percentage of survival vs. vehicle-treated non-transgenic group (black bar). * p<0.05: statistically different among the indicated groups. C) Lead was toxic for motor neurons co-cultured on Pb-exposed non-transgenic astrocyte monolayers. Non-transgenic astrocyte monolayers were exposed for 24 h to increasing concentrations of Pb. Media were changed and non-transgenic motor neurons were plated on the top and counted after 48h. Significant motor neuron death was evident beginning at 1 μM PbAc. * p<0.05: different from vehicle treated cells.