title: Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin.
abstract: BACKGROUND
  Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated "death signals" associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1 (PAR-1).
  
  
  METHODS
  Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridizations. Cell death was monitored with in situ DNA fragmentation assays.
  
  
  RESULTS
  In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) littermates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wr motor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar, segments, paralleling motor neuron degeneration. We also found, using reverse transcription polymerase chain reaction (qRT-PCR) with RNA from genotyped embryos, that PAR-1 was already increased in wr/wr cords at E12, the earliest time examined.
  
  
  CONCLUSIONS
  Thus, motor neuron degeneration and death follows PAR-1 expression both temporally and topographically in wobbler mice. Since our culture studies show that thrombin mobilized [Ca2+]i by activating PAR-1, eventually leading to motor neuron apoptosis, up-regulation of PAR-1 during development may contribute both to "appropriate" as well as "inappropriate" neuronal death in wobbler.
authors.affiliation: Department of Veterans Affairs Medical Center, Kansas City, Missouri 64128, USA. serpin@eagle.cc.ukans.edu
authors.forename: B W
authors.initials: BW
authors.lastname: Festoff
authors.forename: M R
authors.initials: MR
authors.lastname: D'Andrea
authors.forename: B A
authors.initials: BA
authors.lastname: Citron
authors.forename: R M
authors.initials: RM
authors.lastname: Salcedo
authors.forename: I V
authors.initials: IV
authors.lastname: Smirnova
authors.forename: P
authors.initials: P
authors.lastname: Andrade-Gordon
mesh_terms: D000818:Animals; D016923:Cell Death; D003433:Crosses, Genetic; D015342:DNA Probes; D005260:Female; D005838:Genotype; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D005911:Gliosis; D006720:Homozygote; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D016472:Motor Neuron Disease / Q000235:genetics / Q000503:physiopathology; D009046:Motor Neurons / Q000166:cytology / Q000473:pathology / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000235:genetics*; D013116:Spinal Cord / Q000473:pathology* / Q000503:physiopathology; D013482:Superoxide Dismutase / Q000235:genetics; D013917:Thrombin / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D015342:DNA Probes; D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013482:Superoxide Dismutase; D013917:Thrombin