title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Neurology, Georg-August-University of Göttingen, Göttingen, Germany. mlotz@gwdg.de
authors.forename: Miriam
authors.initials: M
authors.lastname: Goos
authors.forename: Wolf-Dieter
authors.initials: WD
authors.lastname: Zech
authors.forename: Manoj Kumar
authors.initials: MK
authors.lastname: Jaiswal
authors.forename: Saju
authors.initials: S
authors.lastname: Balakrishnan
authors.forename: Sandra
authors.initials: S
authors.lastname: Ebert
authors.forename: Timothy
authors.initials: T
authors.lastname: Mitchell
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Carrì
authors.forename: Bernhard U
authors.initials: BU
authors.lastname: Keller
authors.forename: Roland
authors.initials: R
authors.lastname: Nau
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001426:Bacterial Proteins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D018920:Coculture Techniques; D006801:Humans; D007150:Immunohistochemistry; D055666:Lipopeptides; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D009447:Neuroblastoma / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D010455:Peptides / Q000494:pharmacology; D013301:Streptolysins / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D051195:Toll-Like Receptor 2 / Q000037:antagonists & inhibitors; D014162:Transfection; D000097382:CD68 Molecule
chemical_list: D000111:Acetylcysteine; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000975:Antioxidants; D001426:Bacterial Proteins; D002118:Calcium; D053148:Caspase 3; D055666:Lipopeptides; D010455:Peptides; D013301:Streptolysins; D013482:Superoxide Dismutase; D051195:Toll-Like Receptor 2; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C508075:Pam(3)CSK(4) peptide; C495342:TLR2 protein, human; C032290:plY protein, Streptococcus pneumoniae