Pro-NGF secreted by astrocytes promotes motor neuron cell death

Abstract

It is well established that motor neurons depend for their survival on many trophic factors. In this study, we show that the precursor form of NGF (pro-NGF) can induce the death of motor neurons via engagement of the p75 neurotrophin receptor. The pro-apoptotic activity was dependent upon the presence of sortilin, a p75 co-receptor expressed on motor neurons. One potential source of pro-NGF is reactive astrocytes, which up-regulate the levels of pro-NGF in response to peroxynitrite, an oxidant and producer of free radicals. Indeed, motor neuron viability was sensitive to conditioned media from cultured astrocytes treated with peroxynitrite and this effect could be reversed using a specific antibody against the pro-domain of pro-NGF. These results are consistent with a role for activated astrocytes and pro-NGF in the induction of motor neuron death and suggest a possible therapeutic target for the treatment of motor neuron disease.

Figure 1. Expression of sortilin by motor neurons

a) Transversal section of spinal cord from 5 month-old wild-type immunostained with antibody against sortilin (green) and Hoechst 33342 (blue). High power view within the lumbar ventral horn. b–c) E14.5 motor neurons 1 d.i.v. immunostained for sortilin (b) and the motor neuron marker Isl-1 (c). d–e) E14.5 motor neurons 1 d.i.v. immunostained for p75 (d) and the motor neuron marker Isl-1 (e).

Figure 2. Viability of embryonic spinal motor neurons in culture

a) E14.5 motor neurons were visualized with Calcein AM after 48 hours incubation under control conditions. b) Viability of E14.5 SMNs in culture was monitored for 7 days by Calcein AM staining. The graph show the averages of four independent experiments.

Figure 3. ProNGF treatment induces spinal motor neurons apoptosis in culture via a mechanism mediated by p75 and sortilin

a) Induction of motor neuron apoptosis after 24 hours treatment was measured by Hoechst staining and analysis of fragmented nuclei and condensed chromatin. Neither pro-GST nor GST alone induced neuronal apoptosis. b) Motor neuron viability after 48 hours treatment was measured by Calcein AM staining. ProNGF (2 ng/ml), but not mature NGF, reduced motor neuron survival. The simultaneous addition of GST-pro, but not GST alone, eliminated motor neuron death. c) Co-incubation with neurotensin (10 μM) abrogated the proNGF apoptotic activity. d) Co-incubation with a function blocking anti-p75 antibody abrogated the proNGF apoptotic activity. Motor neurons were cultured at all times in complete media supplemented with neurotrophic factors. Experiments were conducted in quadruplicates. Shown are averages of 3 independent experiments. Statistical significance was determined by one-way ANOVA and Tukey’s test. ** = p<0.001.

Figure 4. Stimulated astrocytes production of proNGF

Cultured astrocytes, untreated (a) or treated with 1 mM peroxynitrite (b) were immunostained for the pro-domain of proNGF (red) and counterstained with Hoechst 33342.

Figure 5. Stimulated astrocytes secretion of proNGF

Media from astrocytes cultures treated with peroxynitrite (1 mM, Ln 1), SIN1 (100 μM, Ln 2), degraded peroxynitrite (Ln 3) or vehicle control were subjected to SDS-PAGE. Membranes were probed with an antibody against the pro-domain of proNGF. Recombinant proNGF was loaded as control in lanes 5 and 6, 0.2 and 2 μg respectively. Arrowhead points to the high molecular weight (approx. 30 kD) proNGF species. Lysates from the astrocyte cultures were immunoblotted for tubulin to verify comparable cell numbers.

Figure 6. Astrocyte conditioned media induces motor neuron death in culture

a) Conditioned media from stimulated astrocyte cultures (PN: peroxynitrite; SIN: SIN1; dPN: degraded PN) were used to prepare motor neuron media. The media were supplemented with neurotrophic factors immediately prior to use. Survival of motor neurons was quantified 48 hours after application of the astrocyte-conditioned MN media. b) Prior to survival experiments, astrocyte-conditioned MN media were immuno-depleted using an antibody against the pro-domain of proNGF. c) To confirm that the reduction in MN survival was due to the activation of sortilin, neurotensin was added to the neuronal cultures.