Ian R. Brown

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.

Heat shock proteins and protection of the nervous system.

Abstract:  Manipulation of the cellular stress response offers strategies to protect brain cells from damage induced by ischemia and neurodegenerative diseases. Overexpression of Hsp70 reduced ischemic injury in the mammalian brain. Investigation of the domains within Hsp70 that confers ischemic neuroprotection revealed the importance of the carboxyl‐terminal domain. Arimoclomol, a coinducer of heat shock proteins, delayed progression of amyotrophic lateral sclerosis (ALS) in a mouse model in which motor neurons in the spinal cord and motor cortex degenerate. Celastrol, a promising candidate as an agent to counter neurodegenerative diseases, induced expression of a set of Hsps in differentiated neurons grown in tissue culture. Heat shock “preconditioning” protected the nervous system at the functional level of the synapse and selective overexpression of Hsp70 enhanced the level of synaptic protection. Following hyperthermia, constitutively expressed Hsc70 increased in synapse‐rich areas of the brain where it associates with Hsp40 to form a complex that can refold denatured proteins. Stress tolerance in neurons is not solely dependent on their own Hsps but can be supplemented by Hsps from adjacent glial cells. Hence, application of exogenous Hsps at neural injury sites is an effective strategy to maintain neuronal viability.

Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/SPARC.

We describe the cloning of SC1, a novel cDNA that was selected from a rat brain expression library using a mixed polyclonal antibody directed against synaptic junction glycoproteins. SC1 detects a 3.2 kb mRNA expressed throughout postnatal development of the brain and present at high levels in the adult. In situ hybridization reveals that the SC1 mRNA is expressed widely in the brain and is present in many types of neurons. DNA sequence data suggest that the SC1 product is a secreted, calcium binding glycoprotein. Strikingly, the carboxy-terminal region of the SC1 protein shows substantial similarity to the extracellular matrix glycoprotein osteonectin/BM40/SPARC. These data are consistent with the hypothesis that SC1 is an extracellular matrix glycoprotein in the brain.

Selective induction of a heat shock gene in fibre tracts and cerebellar neurons of the rabbit brain detected by in situ hybridization

In situ hybridization reveals striking regional differences in the expression of constitutive and inducible heat shock genes in the rabbit brain. Fibre tracts throughout the brain show a dramatic induction of heat shock mRNA after hyperthermia, a pattern consistent with a strong glial response to heat shock. While neurons in the cerebellum exhibit a marked induction of heat shock mRNA, neurons of the hippocampus do not. Constitutive expression of a heat shock gene is observed in neuronal laminae of the hippocampus and cerebellum.

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.

The effect of hyperthermia on the induction of cell death in brain, testis, and thymus of the adult and developing rat

Abstract This paper is dedicated to the memory of Professor David A. Walsh (1945–2000), of the University of New South Wales, Australia, who pioneered research on the heat shock response during early mammalian development. Stressful stimuli can elicit 2 distinct reactive cellular responses, the heat shock (stress) response and the activation of cell death pathways. Most studies on the effects of hyperthermia on the mammalian nervous system have focused on the heat shock response, characterized by the transient induction of Hsps, which play roles in repair and protective mechanisms. This study examines the effect of hyperthermia on the induction of cell death via apoptosis, assayed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling and active caspase 3 cytochemistry, in the adult rat brain, testis, and thymus. Results show that a fever-like increase in temperature triggered apoptosis in dividing cell populations of testis and thymus, but not in mature, postmitotic cells of the adult cerebellum. These differential apoptotic responses did not correlate with whole-tissue levels of Hsp70 induction. We further investigated whether dividing neural cells were more sensitive to heat-induced apoptosis by examining the external granule cell layer of the cerebellum at postnatal day 7 and the neuroepithelial layers of the neocortex and tectum at embryonic day 17. These proliferative neural regions were highly susceptible to hyperthermia-induced apoptosis, suggesting that actively dividing cell populations are more prone to cell death induced by hyperthermia than fully differentiated postmitotic neural cells.

Neuronal expression of constitutive heat shock proteins: implications for neurodegenerative diseases

Abstract Neurodegenerative disorders such as Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis have been termed “protein misfolding disorders.” These diseases differ widely in frequency and impact different classes of neurons. Heat shock proteins provide a line of defense against misfolded, aggregation-prone proteins and are among the most potent suppressors of neurodegeneration in animal models. Analysis of constitutively expressed heat shock proteins revealed variable levels of Hsc70 and Hsp27 in different classes of neurons in the adult rat brain. The differing levels of these constitutively expressed heat shock proteins in neuronal cell populations correlated with the relative frequencies of the previously mentioned neurodegenerative diseases.

Association of heat shock proteins and neuronal membrane components with lipid rafts from the rat brain

Lipid rafts are specialized plasma membrane microdomains enriched in cholesterol and sphingolipids that serve as major assembly and sorting platforms for signal transduction complexes. Constitutively expressed heat shock proteins Hsp90, Hsc70, Hsp60, and Hsp40 and a range of neurotransmitter receptors are present in lipid rafts isolated from rat forebrain and cerebellum. Depletion of cholesterol dissociates these proteins from lipid rafts. After hyperthermic stress, flotillin‐1, a lipid raft marker protein, does not show major change in levels. Stress‐inducible Hsp70 is detected in lipid rafts at 1 hr posthyperthermia, with the peak levels attained at 24 hr, suggesting that Hsp70 may play roles in maintaining the stability of lipid raft‐associated signal transduction complexes following neural stress.

Constitutive expression of heat shock proteins Hsp90, Hsc70, Hsp70 and Hsp60 in neural and non-neural tissues of the rat during postnatal development

Heat shock proteins (Hsps) are a group of highly conserved proteins, that are constitutively expressed in most cells under normal physiological conditions. Previous work from our laboratory has shown that neurons in the adult brain exhibit high levels of Hsp90 and Hsc70 mRNA and protein, as well as basal levels of Hsp70 mRNA. We have now investigated the expression of Hsp90, Hsc70, Hsp60 and Hsp70 in neural and non-neural tissues of the rat during postnatal development, a time of extensive cell differentiation. Western blot analysis revealed constitutive expression of these Hsps early in postnatal development. Developmental profiles of these Hsps suggest that they are differentially regulated during postnatal development of the rat. For example, while levels of Hsp90 decrease somewhat in certain developing brain regions, levels of Hsp60 show a developmental increase, and Hsc70 protein is abundant throughout postnatal neural development. Low basal levels of Hsp70 are also observed in the developing and adult brain. A pronounced decrease in Hsp90 and Hsc70 was observed during postnatal development of the kidney while levels of Hsp60 increased. In addition, tissue-specific differences in the relative levels of these Hsps between brain and non-brain regions were found. Immunocytochemical studies demonstrated a neuronal localization of Hsp90, Hsc70 and Hsp60 at all stages of postnatal development examined as well as in the adult, suggesting a role for Hsps in both the developing and fully differentiated neuron. The developmental expression of subunit IV of cytochrome oxidase was similar to that of Hsp60, a protein localized predominantly to mitochondria.

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.