Caspase‐3 is activated following axotomy of neonatal facial motoneurons and caspase‐3 gene deletion delays axotomy‐induced cell death in rodents

Abstract

In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.

Long-Term Adeno-Associated Viral Vector-Mediated Expression of Truncated TrkB in the Adult Rat Facial Nucleus Results in Motor Neuron Degeneration

“…This may be because of the difficulty in detecting a small number of apoptotic cells over a long time span, because apoptosis is a fast process (Rossiter et al, 1996). A lack of apoptotic cells after adult motor neuron injury has been reported by others (Li et al, 1998;Vanderluit et al, 2000). Adult axotomized rat facial motor neurons may undergo a slow, nonapoptotic form of degeneration (Moran et al, 2001).…”

c-Jun N-terminal Kinase 3 Deficiency Protects Neurons from Axotomy-induced Death in Vivo through Mechanisms Independent of c-Jun Phosphorylation

“…Mice were sacrificed 24 h or 4 days following axotomy. For quantitation of survival, every fifth section through the facial nucleus was evaluated by cresyl violet staining and the total number of live motor neurons was evaluated both ispialteral and contralateral to the axotomy as previously described (33).…”

“…Previous work has shown that this results in apoptotic death, which is associated with caspase induction (33,38). Utilizing this model, we first asked whether JNKs might be required for death.…”

Caspase-7 Expanded Function and Intrinsic Expression Level Underlies Strain-Specific Brain Phenotype ofCaspase-3-Null Mice

“…Experimental animals were backcrossed 4 -10 generations onto the B6 strain and 4 -14 generations onto the 129 strain. Genotyping for the Casp3 wild-type (WT) and knock-out (KO) alleles was performed on DNA from adult tail or ear biopsy or embryo tail, paw, or yolk sac by PCR using the previously described oligonucleotide pairs (Vanderluit et al, 2000). Specifically, the WT Casp3 allele 5Ј primer 5Ј-AAGCTG-TCTTCGTCCAGTGAG-3Ј (oEMS1051) corresponding to base pairs 3122-3142, and the 3Ј primer 5Ј-CTAAGTTAACCAAACTGAGC-ACCGC-3Ј (oEMS905) corresponding to base pairs 3475-3451, generated a band of 353 bp.…”