Journal
MOLECULAR MEDICINE
Volume 6, Issue 5, Pages 410-429Publisher
SPRINGER
DOI: 10.1007/BF03401784
Keywords
-
Ask authors/readers for more resources
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 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). Materials and 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
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available