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Título: The in vivo neuroprotective role of the normal cellular prion protein /
Autores: Gains, Malcolm J.
Fecha: 2007
Publicador: McGill University - MCGILL
Fuente:
Tipo: Electronic Thesis or Dissertation
Tema: Biology, Neuroscience.
Descripción: The role of prion protein (PrPC) as a crucial factor in the pathogenesis of the Transmissible Spongiform Encephalopathies (TSE's) is well known. However, the physiologic role of this highly conserved protein in vivo is thus far, largely unknown. In vitro evidence shows that PrPC may have a neuroprotective role, inhibiting Bax-mediated apoptosis, by preventing the conformational change of Bax, which is one of the early steps leading to Bax activation and subsequently, apoptosis. In vivo evidence indicates that in some transgenic mouse models overexpressing a mutant prion protein that result in neuronal death, the simultaneous expression of PrP protects against this neuronal death. The pathogenesis of the neuronal death in these in vivo models is unknown. Our objective was to determine if PrPC could protect against Bax-mediated cell death in vivo. We used two experimental paradigms to investigate this neuroprotective role of PrPC in vivo.
The first paradigm involved an external insult (ethanol injection) to 7 day old mice, which induces massive Bax-mediated neuronal death. We then quantified the number of active caspase 3 positive cells, as a downstream marker of Bax activation, induced in mice on various genetic backgrounds, PrP overexpressors (PrpOexp), PrP wild-type (PrP+/+ ) and PrP knockout (PrP0/0). We also examined Bax activation by immunoprecipitation of subcellular fractions, as well as total Bax activation and cytochrome c release directly in ex-vivo mouse brains. In addition, we examined the insertion of active Bax into the mitochondrial membrane, also using subcellular fractionation.
The second paradigm involves an internal insult (physiological Bax-mediated neuronal death during embryonic development). This insult results in massive Bax-mediated cell death in embryonic mice that do not express the natural antagonist of Bax i.e. BClxL. We have therefore created 3 lines of transgenic mice expressing Syrian Hamster PrP (SHaPrP) under the control of the Bclx promoter, to cross with Bclx+/- mice to see if the SHaPrP can assume the role of Bclx in Bclx knockout mice and rescue the neuronal cell death.
In the first paradigm, greater numbers of neurons undergoing apoptosis following ethanol insult were seen in PrP0/0 than in PrP +/+ mice, although the differences were not statistically significant. The ex-vivo examination of mouse brains did not provide definitive results.
In the second paradigm, 3 lines of transgenic mice have been created and are in various stages of breeding to obtain the required genotypes to test our hypothesis. The level of expression of SHaPrP in these mice is below the level of detection via western blot or immunoprecipitation.
The trend shown in our results suggests that in vivo, PrPC may provide some protection for neurons from Bax-mediated cell death as a result of an external insult. Research is ongoing to see if PrPC can protect neurons in vivo against developmental Bax-mediated cell death. The work presented here highlights the variability and technical difficulty inherent with in vivo studies, particularly when the available knowledge of the biochemical pathways involved is incomplete.
Idioma: en