Differential expression of heat shock 70 proteins in primary cultures from rat cerebellum

Abstract

While a number of studies have described the heat shock response in established cell lines and in primary cultures of cells derived from the nervous system, there has been no systematic analysis comparing expression and localization of the inducible heat shock 70 (hsp70) proteins and the constitutively synthesized members of the family (hsc70) in neurons and glia. In the present communication, we utilized specific probes to compare the expression of hsp70 and hsc70 mRNAs and proteins in two types of primary cultures, astroglial and neuro-astroglial, from postnatal rat cerebellum. Conditions were adjusted to maintain physiological numbers of microglia in both types of culture, and cultures were analyzed at a number of different time points following a precisely defined heat shock. The northern, in situ hybridization and immunohistochemical analyses resulted in a number of novel observations concerning the nature of the heat shock response in these neuronal and glial cells. In postnatal day 4–5 cultures, hsp70 mRNA levels were elevated for at least 10 h in both types of culture, but in situ hybridization analysis showed no evidence for hsp70 mRNAs in neurons. Microglia were the only cell type in which hsp70 was detected in non-stressed cultures and this cell type contained the highest concentrations of hsp70 proteins in stressed cultures. Hsc70 mRNA levels were also increased after heat shock, but the increase was more transient. Hsc70 mRNAs and proteins were present in all cell types, again with the highest concentrations being present in microglia. Hsc70 mRNAs and proteins were localized in the cytoplasm at all time points examined, with hsc70 protein also being localized in nucleoli. Hsp70 mRNAs and proteins were diffusely localized over nuclei of astrocytes, as well as of most microglia. Hsp70, but not hsc70, was localized on chromosomes in glia once they had resumed cell division after heat shock, suggesting a role for hsp70 either in targeting damaged chromosomal proteins or in cell division. Some cytoplasmic hsp70 was observed in astrocytes of the mixed neuro-astroglial cultures and a delayed hsp70 immunoreactivity was observed in granule neurons in these cultures, suggesting either that translation of low levels of hsp70 mRNAs was more efficient in neurons, or that glial-neuronal translocation of hsp70 proteins had taken place. These results suggest that metabolism and functions of different heat shock protein family members may not always be identical and that care must be taken in extrapolation of results from one cell type to another.