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

Goos M, Zech WD, Jaiswal MK, Balakrishnan S, Ebert S, Mitchell T, Carrì MT, Keller BU, Nau R - BMC Infect. Dis. (2007)

Bottom Line: Mutations in the anti-oxidant enzyme Cu,Zn superoxide dismutase (EC 1.15.1.1, SOD1) are associated with familial ALS.Cell viability and apoptotic cell death were compared morphologically and by in-situ tailing.These findings link infection and motor neuron disease and suggest early treatment of respiratory infections in ALS patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Georg-August-University of Göttingen, Göttingen, Germany. mlotz@gwdg.de

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.

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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.
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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.

Mentions: Incubation with the antioxidant N-acetyl-cysteine (NAC) in a concentration of 1 mM starting 72 and 24 hours in the absence of pneumolysin suggested a slightly higher mitochondrial metabolic activity of G93A-SOD1 cells compared to G93A-SOD1 cells kept in medium without NAC (difference not significant). After 72 hours of NAC pre-treatment, pneumolysin exposure to G93A-SOD1 cells resulted in a metabolic activity of 27.2 ± 3.5% of G93A-SOD1 control cells not exposed to pneumolysin compared to a metabolic activity of 20.8 ± 3.1% of respective pneumolysin-challenged G93A-SOD1 cells in the absence of NAC (p < 0.001) (Fig. 9). After pre-incubation of G93A-SOD1 cells in NAC-containing medium for a period of 24 hours similar results were observed: NAC exerted a neuroprotective effect on G93A-SOD1 cells treated with pneumolysin as determined by the WST-1 test after 3 hours of pneumolysin exposure (22.5 ± 4.1% mitochondrial metabolic activity of control cells in G93A cells with NAC treatment versus 15.1 ± 5.4% without NAC treatment, p < 0.0001) (Fig. 10).


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

Goos M, Zech WD, Jaiswal MK, Balakrishnan S, Ebert S, Mitchell T, Carrì MT, Keller BU, Nau R - BMC Infect. Dis. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211486&req=5

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.
Mentions: Incubation with the antioxidant N-acetyl-cysteine (NAC) in a concentration of 1 mM starting 72 and 24 hours in the absence of pneumolysin suggested a slightly higher mitochondrial metabolic activity of G93A-SOD1 cells compared to G93A-SOD1 cells kept in medium without NAC (difference not significant). After 72 hours of NAC pre-treatment, pneumolysin exposure to G93A-SOD1 cells resulted in a metabolic activity of 27.2 ± 3.5% of G93A-SOD1 control cells not exposed to pneumolysin compared to a metabolic activity of 20.8 ± 3.1% of respective pneumolysin-challenged G93A-SOD1 cells in the absence of NAC (p < 0.001) (Fig. 9). After pre-incubation of G93A-SOD1 cells in NAC-containing medium for a period of 24 hours similar results were observed: NAC exerted a neuroprotective effect on G93A-SOD1 cells treated with pneumolysin as determined by the WST-1 test after 3 hours of pneumolysin exposure (22.5 ± 4.1% mitochondrial metabolic activity of control cells in G93A cells with NAC treatment versus 15.1 ± 5.4% without NAC treatment, p < 0.0001) (Fig. 10).

Bottom Line: Mutations in the anti-oxidant enzyme Cu,Zn superoxide dismutase (EC 1.15.1.1, SOD1) are associated with familial ALS.Cell viability and apoptotic cell death were compared morphologically and by in-situ tailing.These findings link infection and motor neuron disease and suggest early treatment of respiratory infections in ALS patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Georg-August-University of Göttingen, Göttingen, Germany. mlotz@gwdg.de

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.

Show MeSH
Related in: MedlinePlus