Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin

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(1)) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms rem ain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit pro minent gliosis and significant motor neuron loss in the cervical, but not i n lumbar, spinal cord segments. To address molecular mechanisms, we evaluat ed "death signals" associated with the multifunctional serine protease, thr ombin, which leads to apoptotic motor neuronal cell death in culture by cle avage of a G-protein coupled, protease-activated receptor 1(PAR-1). Materials and Methods: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were perfor med 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 fra gmentation 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. PA R-1 protein expression was increased, as demonstrated by immunocytochemistr y and confirmed with in situ hybridization, in phenotypic wr/wr motor neuro ns, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar, segments, paralleling motor neuron dege neration. We also found, using reverse transcription polymerase chain react ion (qRT-PCR) with RNA from genotyped embryos, that PAR-1 was already incre ased in wr/wr cords at E12, the earliest time examined. Conclusions: Thus, motor neuron degeneration and death follows PAR-1 expres sion both temporally and topographically in wobbler mice. Since our culture studies show that thrombin mobilized [Ca2+](i) by activating PAR-1, eventu ally leading to motor neuron apoptosis, up-regulation of PAR-1 during devel opment may contribute both to "appropriate" as well as "inappropriate" neur onal death in wobbler.