Evidence for different mechanisms of induction of HSP70i: a comparison of cultured rat cortical neurons with astrocytes.

Abstract

This study is a follow-up of previous work which demonstrated that cultured cortical neurons did not synthesize HSP70i immediately after heat stress when compared with cultured cortical astrocytes. We have extended the period of observation for HSP70i induction of cultured cortical neurons and astrocytes up to 24 h after heat stress. Cultured rat cortical neurons derived from 16-day-old fetal rats respond differently to heat stress than cultured rat astrocytes derived from newborn rats. They showed a delayed HSP70i induction in the majority of cultured neurons and the response was heterogeneous and was absent in most smaller neurons. The delayed neuronal induction was accompanied by a prolonged activation of heat-shock transcription factor 1 (HSF-1) and prolonged transcription of HSP70i mRNA. In comparison astrocytes showed a marked early induction of HSP70i mRNA and protein. In addition the induction of HSP70i in astrocytes was followed by translocation of the protein into the nucleus, a finding which we failed to demonstrate in neurons. Immunostaining for HSP70i was more uniform in astrocytes than neurons. Many neurons did not stain for up to 24 h after heat shock in this study. Immunocytochemical staining of HSF-1 and 2 showed major differences between neurons and astrocytes. Astrocytes showed localization of HSF-1 to the nucleus before and after heat stress, while neurons showed HSF-1 localization to the cytoplasm and nucleus before and after heat stress. Finally HSF-2 was undetectable in neurons when compared with astrocytes by Western immunoblot analysis. However, astrocytes and neurons revealed weak immunostaining of HSF-2 in the cytoplasm and nucleus. The staining in the neurons was likely secondary to cross-reactivity to an unidentified protein. We conclude that HSP70i expression after heat shock is delayed in rat cortical neurons when compared with rat cortical astrocytes. In addition most small neurons did not synthesize HSP70i after heat shock. This difference in induction of HSP70i may be secondary to localization and activation of HSF-1 but not HSF-2. Neuronal susceptibility to injury may be related to the delayed induction of HSP70i and also the possible failure of newly synthesized HSP70i to translocate into the nucleus.