Sheila J. Rush

Induction of a heat shock gene at the site of tissue injury in the rat brain

Our objective was to investigate whether localized tissue injury induces expression of a gene encoding the major 70 kd heat shock protein (hsp70) in the mammalian nervous system. A small surgical cut was made in the rat cerebral cortex. By 2 hr postsurgery a dramatic and highly localized induction of hsp70 mRNA was detected at the lesion site using in situ hybridization with labeled riboprobe. By 12 hr the intensity of the signal had diminished, and by 24 hr only a few cells along the walls of the cut demonstrated a high level of hsp70 mRNA. Both neurons and glial cells at the site of the surgical cut responded to tissue injury by induction of hsp70 mRNA. Induction was not observed in other brain regions, nor was the pattern of constitutive expression affected by the surgical procedure.

Tissue-specific differences in heat shock protein hsc70 and hsp70 in the control and hyperthermic rabbit.

The ability to resolve protein members of the hsp70 multigene family by two‐dimensional Western blotting permitted the characterization of antibodies which were specific in discriminating constitutively expressed hsc70 isoforms from stress‐inducible hsp70 isoforms. This antibody characterization demonstrated that basal levels of hsp70 isoforms were present in the cerebellum of the control rabbit and that these were elevated following hyperthermia, whereas levels of hsc70 were similar in control and hyperthermic tissue. Multiple isoforms of hsp70 were detected but tissue‐specific differences were not apparent in various organs of the rabbit. However, species differences were observed as fewer hsp70 isoforms were noted in rat and mouse. In the control rabbit, higher levels of hsc70 protein were present in neural tissues compared to non‐neural tissues. Following physiologically relevant hyperthermia, induction of hsp70 was greatest in non‐neural tissues such as liver, heart, muscle, spleen, and kidney compared to regions of the nervous system. These studies suggest that the amount of preexisting constitutive hsc70 protein may influence the level of induction of hsp70 in the stress response. Given this observation, caution is required in the employment of hsp70 induction as an index of cellular stress since endogenous levels of hsc70, and perhaps hsp70, may modulate the level of induction. J. Cell. Physiol. 170:130–137, 1997.

Localization of the Heat-Shock Protein Hsp70 to the Synapse Following Hyperthermic Stress in the Brain

Abstract: Heat‐shock proteins are induced in response to cellular stress. Although heat‐shock proteins are known to function in repair and protective mechanisms, their relationship to critical neural processes, such as synaptic function, has received little attention. Here we investigate whether the major heat‐shock protein Hsp70 localizes to the synapse following a physiologically relevant increase in temperature in the mammalian nervous system. Our results indicate that hyperthermia‐induced Hsp70 is associated with pre‐ and postsynaptic elements, including the postsynaptic density. The positioning of Hsp70 at the synapse could facilitate the repair of stress‐induced damage to synaptic proteins and also contribute to neuroprotective events at the synapse.

Cellular localization of the heat shock transcription factors HSF1 and HSF2 in the rat brain during postnatal development and following hyperthermia.

The heat shock transcription factor HSF1 mediates the induction of heat shock genes in response to temperature elevation and other traumatic events. The induced hsps play roles in cellular repair and protective mechanisms. Immunocytochemistry revealed that in the unstressed rat, HSF1 was already prepositioned in the nucleus at abundant levels in both neuronal and glial cell types. Following a fever-like temperature, glial cells rapidly induced hsp70 whereas populations of large neurons did not. The lack of hsp70 induction in these neurons in vivo did not appear to be due to deficiencies in levels of nuclear HSF1. During postnatal development of the cerebellum, levels of HSF1 increased progressively from day 1 to 30. Members of the hsp gene set are also constitutively expressed in the unstressed animal and play roles as molecular chaperones. HSF2, which has been proposed as a developmental regulator of constitutive heat shock gene expression, demonstrated a developmental alteration in cellular localization, namely a nuclear distribution in neurons at postnatal day 2 and a cytoplasmic localization at day 30. During postnatal development the overall levels of neural HSF2 declined. This profile showed no obvious correlation with previously observed levels of constitutive hsp expression during postnatal neural development.

Heat shock transcription factors and the hsp70 induction response in brain and kidney of the hyperthermic rat during postnatal development.

Abstract : Heat shock transcription factor (HSF) 1 levels increase in brain regions and decline in kidney during postnatal rat development. In both neonatal and adult rats, levels of HSF1 protein in brain and kidney are proportional to the levels of HSF DNA‐binding activity and the magnitude of heat shock protein hsp70 induction after thermal stress. There appears to be more HSF1 protein in adult brain than is needed for induction of hsp70 after thermal stress, suggesting that HSF1 may have other functions in addition to its role as a stress‐inducible activator of heat shock genes. HSF2 protein levels decline during postnatal rat development in brain regions and kidney. Gel mobility shift analysis shows that HSF2 is not in a DNA‐binding form in the neonatal brain and kidney, suggesting that HSF2 may not be involved in the constitutive expression of hsps in early postnatal development. There is no apparent relationship between levels of HSF2 protein and basal levels of hsp90, hsp70, heat shock cognate protein hsc70, and hsp60.

In vivo activation of neural heat shock transcription factor HSF1 by a physiologically relevant increase in body temperature.

Molecular mechanisms which underlie the heat shock response have commonly been analyzed using tissue culture systems, with less investigation of the intact mammal. In tissue culture, a temperature elevation of 5°C is required to activate mammalian heat shock transcription factor 1 (HSF1) to the DNA‐binding form. We demonstrate that a physiologically relevant increase in body temperature of 2.5 ± 0.2°C, similar to that attained during fever reactions, is sufficient to activate HSF1 in the rabbit nervous system. Maximal HSF activation, as measured by gel mobility shift assay, was attained at 1 hr with the cerebellum showing the strongest signal. Supershift experiments with antibodies specific to HSF1 and HSF2 demonstrated that the signal reflected activation of HSF1. Western blot analysis showed that cerebellum exhibited high levels of HSF1 protein.

Expression of mRNA species encoding heat shock protein 90 (hsp90) in control and hyperthermic rabbit brain

Northern blot and in situ hybridization were employed to investigate regional and cell type differences in the expression of hsp90 mRNA species in control and hyperthermic rabbit brain. Riboprobes specific to hsp90 α and β mRNA species were utilized in time‐course Northern blot studies on cerebral hemispheres and the cerebellum. Following hyperthermia, levels of hsp90 α and β mRNA were elevated in both brain regions; however, the magnitude of induction was more robust in the cerebellum than in cerebral hemispheres. The pattern of expression of hsp90 genes in rabbit brain was analyzed by in situ hybridization. These studies revealed that hsp90 genes are preferentially expressed in neuronal cell populations in the unstressed mammalian brain. The distribution of hsp90 α and β mRNA was similar, though the signal for the latter was stronger. Following hyperthermia, changes were not detected in the pattern of hsp90 β mRNA expression in the hippocampus. In the cerebellum, a rapid induction of hsp90 β mRNA was apparent in the neuron‐enriched granule cell layer, followed by a delayed accumulation in Purkinje neurons. Unlike hsp70, induction of hsp90 was not detected in glial cells of hyperthermic rabbit brain. The localization of hsp90 to neurons suggests that this heat shock protein plays an important role in neuronal function.

Effect of hyperthermia on the transcription rate of heat-shock genes in the rabbit cerebellum and retina assayed by nuclear run-ons.

The induction of heat‐shock protein 70 (hsp70) mRNA in the hyperthermic rabbit brain has been examined previously by using Northern blotting and in situ hybridization procedures that measure steady‐state levels of mRNA, which may be influenced by transcript stability and transcription rate. In the present investigation, the in vivo transcription rate of hsp70 has been examined by using run‐on transcription assays on isolated brain nuclei. A major up‐regulation in the transcription rate of hsp70 was observed between 0.75 and 1.50 hours after hyperthermia in the cerebellum and the retina. Gel‐mobility shift assays revealed that the time course of conversion of heat‐shock transcription factor (HSF1) to a DNA‐binding form paralleled the transcriptional induction profile of hsp70. The transcription rates of several nonheat‐shock genes were also studied in the hyperthermic brain, and little change was noted relative to the induction of hsp70. Thus, a physiologically relevant increase in temperature of 2.5°C induces a major up‐regulation in the in vivo transcription rate of hsp70 in the nervous system with little affect on the transcription rates of other genes. J. Neurosci. Res. 52:538–548, 1998.

Expression of SC1 is associated with the migration of myotomes along the dermomyotome during somitogenesis in early mouse embryos.

Abstract SC1 is a secreted glycoprotein with a high amino acid sequence similarity to SPARC (Secreted Protein, Acidic, Rich in Cysteine). SC1 transcripts were first detected in mouse embryos after day 8.5 post coitus (p.c.) in somites at the medial lip of the dermomyotome. Expression of SC1 transcripts by the progenitor cells continued as they began involuting under the dermomyotome and during their migration along the lateral wall of the dermomyotome. After myotome migration was completed, SC1 mRNA expression was downregulated in the trunk region. The data indicate that SC1 expression is restricted to the initial stages of epaxial myotome differentiation and migration, undergoing rapid downregulation prior to myotome emigration from the somitic environment.

Induction of a heat shock gene (hsp 70) in rabbit retinal ganglion cells detected by in situ hybridization with plastic-embedded tissue

Elevation of body temperature by 2–3°C induces a 2.7 kilobase hsp70 mRNA species in the rabbit retina within 1 hr. In situ hybridization with thin sections derived from plastic-embedded tissue permitted a higher level of resolution of retinal cell types compared to procedures which involved the use of frozen tissue sections. A prominent induction of hsp70 mRNA in retinal ganglion cells was observed when an hsp70 riboprobe was utilized for in situ hybridization. These results indicate that this neuronal cell type responds rapidly to fever-like body temperatures by inducing one of the major heat shock genes.