There is an increasing body of evidence that hsp27 protects cells against various insults. In addition, hsp27 has been found to interfere with the apoptotic cascade. We have recently shown that hsp27 expression is increased in motoneurons following neonatal nerve injury (Kalmar et al. 2002). Moreover, those motoneurons that survive injury and express hsp27 do not co-express markers of apoptosis such as activated caspase-3 (Kalmar et al. 2002). These results suggest that increased hsp27 expression may protect injured motoneurons from cell death. In this study we examined the effect of exogenous application of hsp27 protein on cell survival in an in vitro model of motoneuron degeneration.

Mixed cultures of ventral horn cells were prepared from E14 rat embryos (Camu & Henderson, 1994). All animals were humanely killed. At 7 days in vitro cells were exposed to stimuli that are known to induce apoptosis, such as staurosporin, H₂O₂ or the glutamate agonist AMPA, in the presence or absence of hsp27 (2 µg ml^-1; Stressgen). Control cultures were treated with vehicle (20 mM Tris pH 7.5, 10 mM NaCl, 1 mM EDTA, 1 mM DTT). Twenty-four hours later the cultures were stained with trypan blue in order to assess the extent of neuronal death. The cultures were then fixed and processed for peripherin immunocytochemistry. Peripherin is a specific marker of motoneurons in cultures of ventral horn cells. The effect of these treatments on motoneuron survival was assessed by counting the number of trypan blue-positive, peripherin-immunoreactive cells. Results are means ± S.E.M., and n = 5 in all cases. Significance was assessed by a Mann-Whitney U test and significance level was set at 0.05.

In untreated cultures only 5.1 ± 1 % of peripherin-positive cells were also positive for trypan blue. Treatment with apoptotic stimuli, however, resulted in an increase in motoneuron death, which is significantly reduced by co-treatment with hsp27. The results show that in staurosporin-treated cultures, 20.2 ± 4 % of motoneurons take up trypan blue compared with only 7.2 ± 2 % of those cultures co-treated with hsp27. Similarly, application of H₂O₂ onto motoneurons induces the uptake of trypan blue in 25.5 ± 5 % of motoneurons, which is significantly reduced in the presence of hsp27 to only 10.8 ± 3 %. Treatment with AMPA results in even more extensive motoneuron death, so that 31.7 ± 3 % of motoneurons are positive for trypan blue. In cultures co-treated with hsp27 and AMPA, motoneuron death was reduced to 14.8 ± 1.2 %.

These results show that manipulation of cellular hsp27 levels by exogenous application of hsp27 can rescue motoneurons from cell death induced by a variety of apoptotic stimuli.

This work was supported by the BRT.