| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
|
|---|
| Research Abstract |
In an attempt to elucidate the pathological implications of intracellular accumulation of the amyloid precursor protein (APP) in postmitotic neurons in vivo and in vitro, we transferred APP695cDNA into rat hippocampal neurons, or cultured human postmitotic neurons derived from embryonal carcinoma NTera2 (NT2) cells by using an adenovirus vector. We first improved the efficiency of adenovirus-mediated gene transfer into neurons in vivo by using hypertonic mannitol. when β-galactosidase-expressing recombinant adenovirus suspended in 1M mannitol was injected into a dorsal hippocampal region, a number of neurons in remote areas were positively stained, presumably owing the increased retrograde transport of the virus. When an APP 695-expressing adenovirus was infected into the same site, part of the infected neurons in the hippocampal formation underwent severe degeneration in a few days, whereas astrocytes near the injection site showed no apparent degeneration. These degenerating neurons
… More
showed nuclear DNA fragmentation, and electron microscopic examinations demonstrated that degenerating neurons had shrunken perikarya along with synaptic abnormalities. Microglias were often found in close proximity to degenerating neurons, and in some cases the phagocytosed these neurons. We next transferred the APP-expressing adenoviral vector into human NT2 neurons. Three days after viral infection, NT2 neurons showed the degenerative changes and five days after infection, almost all infected neurons showed severe degeneration, presenting completely retracted neurites, shrunken somata with irregular contours and pyknotic nuclei. These neurons were intensely stained with antibodies against N-and C-termini of APP, suggesting that accumulation of full length APP may be the main cause of apoptosis-like neurodegeneration. Degenerating neurons in vivo or in vitro contained activated caspase-3, and prior to the apoptotic change became apparent, activity of Caspase-3 was increased. These observations suggest that APP is an intrinsic activator of caspase-3 -mediated death machinery in postmitotic neurons. Less
|
