|Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥2,400,000 (Direct Cost: ¥2,400,000)
In the chick embryo spinal cord, two types of programmed cell death (PCD) of motoneurons (MNs) are known to occur, one at a relatively late stage and the other at an earlier stage of differentiation. In the second type, the PCD of MNs occurs only in the non-limb innervating cervical spinal cord between E4 and E5. To elucidate the roles for caspases in this early PCD of MNs in the cervical spinal cord, we examined the activation of caspases and the effects of inhibitors of caspases in vivo.
Caspase-3 like activity was increased in ventral MN containing region of the cervical segments at st 24 (E4.5) when many pyknotic dying neurons are observed. Immunohistochemical observations with an antibody against activated form of caspase-3 suggested that activation of caspase-3 precedes both DNA fragmentation and the morphological manifestation of PCD in cervical MNs. To further investigate roles for casapase-3 like activity, we treated embryos with Ac-DEVD-CHO that inhibits caspase-3 like activit
y. In Ac-DEVD-CHO treated embryos, the nuclei of pyknotic cells showed only moderate condensation. When pyknotic cells were counted, including these moderately pyknotic cells, there was no significant difference between controls and Ac-DEVD-CHO treated embryos. TUNEL staining revealed that DNA fragmentation did not occur in these moderately condensed nuclei, even though electron microscopic observations clearly revealed that degenerative changes were occurring in the cytoplasm. These results suggest that caspase-3 like activity plays a role in the apoptotic nuclear changes.
To investigate involvement of other caspases that are responsible for apoptotic changes of cytoplasm, we treated embryos with Boc-Asp-FMK (BAF) that inhibits broad spectrum of caspases. After treatment with BAF for 12 hours the number of pyknotic cells remarkably fewer than controls. Even moderately pyknotic cell that were observed after treatment with Ac-DEVD-CHO were rarely observed. Instead, there were many cells whose nuclei are smaller and whose cytoplasm was slightly more eosinophilic. These cells lacked DNA fragmentation and retained MN specific marker. Electron microscopic observation revealed that there are many smaller cells that have irregular shaped nuclei, slightly electron dense cytoplasm and apparently normal organelles. Following treatment with BAF for 24 hours, the number of pyknotic cells increased to the level comparable to embryos treated with Ac-DEVD-CHO, although the number of TUNEL positive cells was still fewer than controls. Electron microscopic observation revealed that there are many aberrantly degenerating cells. The total number of surviving MNs was not more than controls and Ac-DEVD-CHO treated embryos. These results suggest that other caspases that can be inhibited by BAF but not by Ac-DEVD-CHO play roles in execution of apoptotic changes in dying MNs. Although inhibition of such caspases considerably delayed the rate of PCD, MNs can not be finally rescued by inhibition of caspases. Less