Expression of a Cu,Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of neuroblastoma cells to infectious injury

Abstract

Background: Infections can aggravate the course of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in the anti-oxidant enzyme Cu,Zn superoxide dismutase (EC 1.15.1.1, SOD1) are associated with familial ALS. Streptococcus pneumoniae, the most frequent respiratory pathogen, causes damage by the action of the cholesterol-binding virulence factor pneumolysin and by stimulation of the innate immune system, particularly via Toll-like-receptor 2.

Methods: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) and SH-SY5Y neuroblastoma cells transfected with wildtype SOD1 were both exposed to pneumolysin and in co-cultures with cultured human macrophages treated with the Toll like receptor 2 agonist N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam3CSK4). Cell viability and apoptotic cell death were compared morphologically and by in-situ tailing. With the help of the WST-1 test, cell viability was quantified, and by measurement of neuron-specific enolase in the culture supernatant neuronal damage in co-cultures was investigated. Intracellular calcium levels were measured by fluorescence analysis using fura-2 AM.

Results: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) were more vulnerable to the neurotoxic action of pneumolysin and to the attack of monocytes stimulated by Pam3CSK4 than SH-SY5Y cells transfected with wild-type human SOD1. The enhanced pneumolysin toxicity in G93A-SOD1 neuronal cells depended on the inability of these cells to cope with an increased calcium influx caused by pores formed by pneumolysin. This inability was caused by an impaired capacity of the mitochondria to remove cytoplasmic calcium. Treatment of G93A-SOD1 SH-SY5Y neuroblastoma cells with the antioxidant N-acetylcysteine reduced the toxicity of pneumolysin.

Conclusion: The particular vulnerability of G93A-SOD1 neuronal cells to hemolysins and inflammation may be partly responsible for the clinical deterioration of ALS patients during infections. These findings link infection and motor neuron disease and suggest early treatment of respiratory infections in ALS patients.

Figure 1

Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1 neuroblastoma cells. Cell viability measured by the WST-1 test after 3 h of incubation with pneumolysin (PLY) at a concentration of 0.5 μg/ml. Values are given in % of mitochondrial metabolic activity of unstimulated control cells ± standard deviation (SD). G93A-SOD1 mutant SH-SY5Y cells were more vulnerable to the action of PLY than Wt-SOD1 SH-SY5Y cells (p < 0.0001).

Figure 2

Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1 neuroblastoma cells. Hemalum staining showed a substantially higher density of living Wt-SOD1 SH-SY5Y cells than G93A-SOD1 SH-SY5Y cells after PLY treatment. Please note the shrinkage and clustering of severely damaged/dead G93A-SOD1 neuroblastoma cells.

Figure 3

Toxicity of pneumolysin for G93A-SOD1 neuroblastoma cells. Apoptotic G93A-SOD1 SH-SY5Y cells after incubation with 0.5 μg/ml PLY for three hours (in-situ tailing, apoptotic cells marked by arrows).

Figure 4

Toxicity of pneumolysin for G93A-SOD1 neuroblastoma cells. Apoptotic G93A-SOD1 SH-SY5Y cells after incubation with 0.5 μg/ml PLY for three hours detected by immunocytochemistry for activated caspase-3.

Figure 5

Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1 neuroblastoma cells. Rate of apoptotic cells in Wt-SOD1 SH-SY5Y and G93A-SOD1 SH-SY5Y neuroblastoma cells after incubation with 0.5 μg/ml PLY for three hours (all cells = 100%). Please note the strong difference of the rate of apoptosis in wild-type and G93A-SOD1 transgenic cells exposed to pneumolysin (p < 0.001).

Figure 6

Strongly elevated calcium influx into G 93A-SOD1 neuroblastoma cells in comparison to wild-type SOD1 cells. Representative recordings of intracellular calcium concentrations in single cells as measured by the fura-2 AM method.

Figure 7

Strongly elevated calcium influx into G 93A-SOD1 neuroblastoma cells in comparison to wild-type SOD1 cells. Comparison of the intracellular calcium concentration-versus-time curves (n = 25 cells each; means ± SD; p < 0.0001).

Figure 8

Strongly elevated calcium influx into G 93A-SOD1 neuroblastoma cells in comparison to wild-type SOD1 cells. Comparison of the pneumolysin-induced peak calcium intracellular concentrations (n = 25 cells each, means ± SD, p < 0.0001).

Figure 9

Radical scavenging by N-acetyl-cysteine. The pneumolysin-induced neuronal injury in G93A-SOD1 neuroblastoma cells, but not in Wt-SOD1 cells, was attenuated by pre-incubation for 72 hours with the anti-oxidant N-acetylcysteine (NAC) in a concentration of 1 mM (p < 0.001).

Figure 10

Radical scavenging by N-acetyl-cysteine. 24 hours of pre-incubation with the anti-oxidant N-acetylcysteine (NAC) in a concentration of 1 mM also resulted in an attenuation of the pneumolysin-induced neuronal injury in G93A-SOD1 (p < 0.0001), but not in Wt-SOD1 neuroblastoma cells.

Figure 11

Vulnerability of G93A-SOD1 and wild-type SOD1 neuroblastoma cells to the attack of monocytes stimulated with Pam₃CSK₄. Release of neuron-specific enolase (values expressed in per cent ± SD of the NSE release induced by cell lysis). After stimulation of monocytes with the Toll-like receptor 2 agonist Pam₃CSK₄ G93A-SOD1 SH-SY5Y cells were more severely injured by activated macrophages than Wt-SOD1 cells.

Figure 12

Vulnerability of G93A-SOD1 and wild-type SOD1 neuroblastoma cells to the attack of monocytes stimulated with Pam₃CSK₄. After staining of Pam₃CSK₄-stimulated co-cultures with light green and macrophage staining with CD 68 less neuronal cell somata of G93A-SOD1 SH-SY5Y (lower panel) cells than of Wt-SOD1 cells in equally treated co-cultures (upper panel) were visible. Please note the clustering of severely damaged/dead G93A-SOD1 cells and the area devoid of neuronal cells in the vicinity of groups of macrophages.

Figure 13

Vulnerability of G93A-SOD1 neuroblastoma cells to the attack of monocytes stimulated with Pam₃CSK₄. In-situ tailing shows a macrophage internalising an apoptotic nucleus of a G93A-SOD1 neuroblastoma cell (arrow).