Zinc induces a Src family kinase-mediated up-regulation of NMDA receptor activity and excitotoxicity.

Zinc is coreleased with glutamate from excitatory nerve terminals throughout the central nervous system and acutely inhibits N-methyl-d-aspartate (NMDA) receptor activation. Here we report that cultured murine cortical neurons briefly exposed to sublethal concentrations of zinc developed increased intracellular free Na(+), phosphorylation of Src kinase at tyrosine 220, and tyrosine phosphorylation of NMDA receptor 2A/2B subunits, in a fashion sensitive to the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, PP2. Functionally, this zinc exposure produced a delayed increase in NMDA receptor current in perforated patch but not conventional whole-cell recordings, as well as an increase in NMDA receptor-mediated cell death. These observations suggest that the effect of synaptically released zinc on neuronal NMDA receptors may be biphasic: acute block, followed by Src family kinase-mediated up-regulation of NMDA receptor activity and cytotoxicity.

Zinc-induced neuronal death in cortical neurons.

Although Zn2+ is normally stored and released in the brain, excessive exposure to extracellular Zn2+ can be neurotoxic. The purpose of the present study was to determine the type of neuronal cell death, necrosis versus apoptosis, induced by Zn2+ exposure. Addition of 10-50 microM ZnCl2 to the bathing medium of murine neuronal and glial cell cultures induced, over the next 24 hrs., Zn2+-concentration-dependent neuronal death; some glial death also occurred with Zn2+ concentrations above 30 microM. The neuronal death induced by 20 microM Zn2+ was characterized by coarse chromatin condensation, the formation of apoptotic bodies, and internucleosomal DNA fragmentation. It was attenuated in cortical cell cultures prepared from mice null for the bax gene, and by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-CH2F (ZVAD, 100 microM), but not by the NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV, 200 microM ). In contrast, the neuronal death induced by 50 microM Zn2+ was characterized by plasma membrane disruption and random DNA fragmentation; this death was attenuated by D-APV, but exhibited little sensitivity to ZVAD or deletion of bax. These results suggest that Zn2+ can induce cell death with characteristics of either apoptosis or necrosis, depending on the intensity of the Zn2+ exposure.

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.

The neuronal stress response: nuclear translocation of heat shock proteins as an indicator of hyperthermic stress.

Two characteristic features of the heat shock response, (i) induction of hsp70 protein and (ii) nuclear translocation of constitutive hsc70 and stress-inducible hsp70 protein, were utilized as markers of cellular stress in the rabbit brain. Following a physiologically relevant increase in body temperature of 2.7 +/- .3 degrees C, nonneuronal cell types, such as ependymal cells and oligodendrocytes, undergo a stress response as assayed by the above criteria. In contrast, several neuronal cell populations required an increased degree of hyperthermic stress (3.4 +/- .2 degrees C) before exhibiting nuclear translocation of constitutive hsc70 protein. Induction of hsp70 protein was not observed in these neuronal cells at either temperature. The present results suggest that certain neurons in the rabbit brain are buffered against induction of the heat shock response, perhaps due to their high constitutive levels of hsc70 protein.

Temporal and spatial distribution of heat shock mRNA and protein (hsp70) in the rabbit cerebellum in response to hyperthermia.

We have previously investigated the expression of hsp70 genes in the hyperthermic rabbit brain at the mRNA level by Northern blot and in situ hybridization procedures. Our studies have now been extended to the protein level utilizing Western blot and immunocytochemistry. Using an antibody which is specific to inducible hsp70, a prominent induction of hsp70 protein in glial cells of hyperthermic animals was noted. In particular, Bergmann glial cells in the cerebellum are strongly immunoreactive while adjacent Purkinje neurons are immunonegative. Extension of our in situ hybridization studies to a time course analysis revealed that the initial glial induction events were followed by a delayed accumulation of inducible hsp70 mRNA in Purkinje neurons at 10 hr post-heat shock. In control animals, high levels of constitutively expressed hsc70 mRNA and protein were observed in Purkinje neurons. Similar hsc70 and hsp70 mRNA observations were also made in neurons of the deep cerebellar nuclei and in motor neurons of the spinal cord. Our results suggest that these neuronal cell types accumulate hsp70 mRNA in response to hyperthermic treatment; however, the response is delayed when compared to the rapid response seen in glial cells. The high constitutive levels of hsc70 in certain neuronal cell types may play a role in the initial dampening of the hsp70 induction response in these cells.

Distribution of constitutive- and hyperthermia-inducible heat shock mRNA species (hsp70) in the Purkinje layer of the rabbit cerebellum.

In previous studies we have analyzed the effect of hyperthermia on the expression of hsp70 genes in the rabbit cerebellum using an hsp70 riboprobe which hydridized to both constitutively expressed and stress-inducible transcripts. These studies have now been extended utilizing riboprobes which are able to discriminate hyperthermia-inducible hsp70 mRNA of size 2.7 kb and constitutively expressed mRNA of size 2.5 kb. In situ hybridization with the inducible specific riboprobe revealed a prominent induction of the 2.7 kb species 1 hr after a 2-3 degrees C increase in body temperature in the following cerebellar cell types: i) Bergmann glial cells in the Purkinje layer, ii) glial cells in deep white matter fiber tracts and iii) granule neurons. The inducible transcript was not detected in the cerebellum of control animals. The constitutive specific riboprobe detected the 2.5 kb transcript in several neuronal cell types of the cerebellum such as Purkinje and granule neurons with little increase in signal in hyperthermic animals compared to controls.

Expression of heat shock genes (hsp70) in the rabbit spinal cord: localization of constitutive and hyperthermia-inducible mRNA species.

We have previously reported that hyperthermia induces the expression of a heat shock gene in the rabbit brain (Sprang and Brown, Mol Brain Res 3:89-93, 1987). Striking regional and cell type differences in the pattern of induction of the hsp70 mRNA were noted. Tissue injury also induces the rapid induction of hsp70 mRNA in the mammalian brain (Brown et al., Neuron 2:1559-1564, 1989). In the present study, in situ hybridization with 35S-labelled riboprobes specific for constitutive and inducible hsp70 mRNA species was employed to investigate the effect of fever-like temperatures on hsp70 gene expression in the rabbit spinal cord. Expression of constitutive hsp70 mRNA was detected in large motor neurons of both control and hyperthermic animals. Within 1 hr after hyperthermia, a massive induction of inducible hsp70 mRNA was noted in fibre tracts of the spinal cord, a pattern consistent with a strong glial response to heat shock. Induction was not observed in the large motor neurons.

Time course of induction of a heat shock gene (hsp70) in the rabbit cerebellum after LSD in vivo: involvement of drug-induced hyperthermia.

In situ hybridization studies were carried out to determine whether induction of hsp70 mRNA in various cellular layers of the rabbit cerebellum was due to hyperthermic effects of the psychotropic drug LSD. Results indicated that induction was not present when LSD-induced hyperthermia was blocked. The pattern of induction of hsp70 mRNA in various cell types of the cerebellum was similar when hyperthermia was induced by either drug (LSD) or nondrug means (placement of animals in a warm incubator). A time course analysis of the induction of hsp70 mRNA following LSD-induced hyperthermia revealed maximal levels of mRNA at 1 hr in all cerebellar cell layers except the Purkinje layer where highest levels were attained at 5 hr. By 10 hr hsp70 mRNA had returned to constitutive levels in all cellular layers of the cerebellum.