R. William Currie

Benign focal ischemic preconditioning induces neuronal Hsp70 and prolonged astrogliosis with expression of Hsp27

We have established a focal preconditioning (PC) paradigm that produces significant and prolonged ischemic tolerance (IT) of the brain to subsequent permanent middle cerebral artery occlusion (MCAO). PC using 10 min of MCAO induces brain tolerance at 1-7 days of reperfusion that requires active protein synthesis. The protective protein(s) involved are unknown. In these studies the increased transcription and translation of the inducible 70-kDa heat shock protein (Hsp70) and the 27-kDa heat shock protein (Hsp27), and astrogliosis/glial fibrillary acidic protein (GFAP) were determined by Northern analysis and immunohistochemistry following PC. Cellular localization of proteins was determined by double labeling. PC produced no brain injury but did increase Hsp70 mRNA transiently at 6 h and increased Hsp27 mRNA later at 24 h for at least 5 days. Protein expression induced by PC exhibited a similar profile. Hsp70 protein was primarily expressed in neurons from 1 to 5 days post-PC throughout the PC cortex. Hsp27 protein expression was initiated later for a much longer period of time. A remarkable astroglyosis was verified with increased astrocytic Hsp27 from 1 to 7 days after PC. Gliosis with increased Hsp27 in the PC cortex was still present but reduced 4 weeks after PC. Therefore, PC that results in brain tolerance/neuroprotection increases neuronal Hsp70 in the PC cortex and activated astrocytic Hsp27 in the PC cortex in a temporal fashion associated with developing IT. The short duration of benign ischemia (PC) that produces IT produces a robust, long-lived cellular and protein synthetic response that extends throughout the entire cortex (i.e. well beyond the MCA perfusion territory). The resulting IT is associated with changes in astrocyte-activation that might provide increased support and protection from injury. Although both Hsp70 and Hsp27 may participate in the neuroprotection/brain tolerance induced by PC, the temporal expression patterns of these proteins indicate that they are not solely responsible for the tolerance to brain injury.

Constitutive expression of the 25-kDa heat shock protein Hsp25 reveals novel parasagittal bands of Purkinje cells in the adult mouse cerebellar cortex

Despite the reported absence of the 25‐kDa heat shock protein Hsp25 in the rodent cerebellum, we have determined that Hsp25 is constitutively expressed in a subset of Purkinje cells in the adult cerebellum of the mouse. No other cerebellar neurons are Hsp25 immunoreactive, but there is weak staining associated with blood vessels. In the vermis, Hsp25‐immunoreactive Purkinje cells are confined to two regions: one in lobules VI/VII, the other in lobules IX/X. In each region, only a subset of the Purkinje cells is immunoreactive. These cells are grouped in five parasagittal bands arranged symmetrically about the midline. The boundaries of these expression domains correspond to transverse zones previously inferred from other expression patterns. A third Hsp25‐immunopositive domain is seen in the paraflocculus and flocculus. Again, only a subset of Purkinje cells within the paraflocculus and flocculus express Hsp25, revealing three distinct bands. Previous descriptions of compartmentation antigens have not differentiated between adult populations of Purkinje cells in these regions, suggesting that Hsp25 is a novel compartmentation antigen in the adult cerebellum. J. Comp. Neurol. 416:383–397, 2000.

Effects of ischemia and perfusion temperature on the synthesis of stress-induced (heart shock) proteins in isolated and perfused rat hearts*

Isolated and perfused rat hearts were examined by 2-dimensional gel electrophoresis and liquid scintillation counting to determine the effect of ischemia or perfusion temperature on protein synthesis. Isolated hearts were subjected to ischemia at either 4 degrees, 20 degrees, or 30 degrees C and then perfused at 37 degrees C and radio-labeled with [3H]-leucine from 0.5 to 2.5 h of perfusion. Following 0.5 h of 4 degrees C or 0.2 h of 20 degrees C ischemia, minimal or no effect was seen by fluorography on the patterns of protein synthesis and there was no apparent synthesis of the stress-induced (heat shock) protein with Mr = 71,000 (SP71). After 4.0 h of 4 degrees C or 0.5 h of 20 degrees C ischemia, SP71 was detectable on fluorograms. After 17 h of 4 degrees C or 1 h of 20 degrees C or 30 degrees C ischemia, SP71 was a prominent spot on fluorograms; the percentage incorporation of precursor into SP71 was significantly increased and overall incorporation of precursor into protein appeared depressed. Following 17 h at 4 degrees C, during which time the buffer was continuously oxygenated, percentage incorporation of precursor into SP71 was only moderately increased, suggesting that hypoxia was responsible for the high level of SP71 synthesis. The effect of perfusion temperature was initially examined using a perfusion method which results in a moderate synthesis of SP71 at 37 degrees C between 2.5 and 4.5 h of perfusion. Fluorograms revealed synthesis of some normal proteins at all temperatures, with little or no synthesis of SP71 at 31 degrees and 34 degrees C, moderate synthesis at 37 degrees C, and intense synthesis at 40 degrees C. The percentage incorporation of precursor into SP71 and total incorporation of precursor appeared depressed at 31 degrees C and 34 degrees C, from that at 37 degrees C, while at 40 degrees C, percentage incorporation of precursor into SP71 was significantly increased but overall incorporation of precursor into protein appeared depressed. When SP71 synthesis was induced by 17 h of 4 degrees C ischemia, hearts were sensitive to a lower perfusion temperature (34 degrees C); the percent incorporation of precursor into SP71 was significantly reduced from that seen at 37 degrees C. Interestingly, the effect of prolonged ischemia on protein synthesis (increased synthesis of SP71; suppression of overall synthesis) is the same as the heat shock response seen after perfusion at 40 degrees C.

Upregulation of the immediate early gene arc in the brains of rats exposed to environmental enrichment: implications for molecular plasticity.

Exposure to an enriched environment, a procedure that induces plasticity in the cerebral cortex, is associated with pronounced morphological changes, including higher density of dendritic spines, enlargement of synaptic boutons, and other putative correlates of altered neurotransmission. Recently, it has been demonstrated that animals reared in an enriched environment setting for 3 weeks have less neuronal damage as a result of seizures and have decreased rates of spontaneous apoptosis. Even though clear morphological modifications are observed in the cerebral cortex of animals exposed to heightened environmental complexity, the molecular mechanisms that underlie such modifications are yet to be described. In the present work, we investigated the expression of the immediate early gene arc in the cortex of animals exposed to an enriched environment. Animals were exposed daily, for 1 h, to an enriched environment, for a total period of 3 weeks. Brains were processed for in-situ hybridization against arc mRNA. We found a marked upregulation of arc mRNA in the cerebral cortex of animals exposed to the enriched environment, when compared to undisturbed controls, an effect that was most pronounced in cortical layers III and V. Animals in an additional control group that were handled for 5 min daily, displayed intermediate levels of arc mRNA. Furthermore, arc expression was upregulated in the CA1, CA2 and CA3 hippocampal subfields and in the striatum, but to a lesser extent in the dentate gyrus of animals exposed to an enriched environment, as compared to the two control groups. Our results support the association between the upregulation of the immediate early gene arc and plasticity-associated anatomical changes in the cerebral cortex of the adult mammal.

Expression of heat-shock protein Hsp25 in mouse Purkinje cells during development reveals novel features of cerebellar compartmentation.

The small heat shock protein Hsp25 is constitutively expressed in the adult mouse cerebellum by parasagittal stripes of Purkinje cells confined to the caudal central zone (∼lobules VI and VII), the nodular zone (∼ventral lobule IX and lobule X), and the paraflocculi/flocculi. During development several distinct phases in Hsp25 expression can be distinguished. Hsp25‐immunopositive Purkinje cells are first seen at birth, when four clusters are visible in the vermis of lobules IV/V, and scattered Hsp25‐immunoreactive Purkinje cells are seen in lobule VIII. By postnatal day 2/3, six narrow parasagittal stripes of Hsp25‐immunopositive Purkinje cells are seen in the vermis of the anterior lobe. In the posterior lobules, most Purkinje cells in the vermis of lobules VIII and IX express Hsp25. This initial limited expression is followed by a phase of widespread expression (postnatal days 6–9) in which Hsp25 immunoreactivity is detected in virtually all Purkinje cells. This global cerebellar expression of Hsp25 then gradually disappears, first in the anterior zone and the hemispheres and subsequently in the posterior zone, to leave the restricted adult expression pattern. Western blotting analysis and immunoprecipitation with anti‐Hsp25 suggest that all immunocytochemistry can be attributed the expression of Hsp25. Furthermore, visual deprivation had no effect on the development of Hsp25 expression in Purkinje cells, suggesting that visuomotor input is not responsible for the establishment of constitutive Hsp25 expression in the cerebellar cortex. J. Comp. Neurol. 429:7–21, 2001.

Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1? mRNA in ischemic brain tolerance

A short duration of ischemia (i.e., ischemic preconditioning) was shown to result in significant tolerance to subsequent ischemic injury. Since previous reports suggest that interleukin‐1β (IL‐1β) may be involved in both ischemic damage and neuroprotection, the present work examined the expression of IL‐1β mRNA in cortical brain tissue after an established preconditioning (PC) stimulus known to produce significant brain tolerance to focal stroke after 1–7 days. Significant induction of IL‐1β mRNA was observed in the ipsilateral cortex at 6 hr (87 ± 9 copies of the mRNA per microgram of brain tissue compared to 16 ± 5 copies in sham‐operated samples, P < 0.001, n = 4) and 8 hr (46 ± 4 copies, P < 0.01, n = 4) after PC by means of real‐time Taqman polymerase chain reaction (PCR). The peak expression of IL‐1β mRNA after PC was significantly (P < 0.01) lower than that after permanent occlusion of the middle cerebral artery (MCAO), i.e., 87 ± 9 and 546 ± 92 copies of RNA per microgram tissue at peak levels for PC and focal stroke, respectively. Immunohistochemistry studies revealed a parallel induction of IL‐1β in the ipsilateral cortex after PC. The maximal expression of IL‐1β was observed during the first week post‐PC, showing marked parallelism with the duration of ischemic tolerance. These data suggest that the significant but low levels of IL‐1β induction after PC may contribute to ischemic brain tolerance. J. Neurosci. Res. 59:238–246, 2000

Huntingtin inclusion bodies are iron-dependent centers of oxidative events.

Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1‐polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion‐dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1‐polyQ polypeptides. Analysis of live COS‐7 cells as well as neuronal SK‐N‐SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1‐polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion‐dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation‐dependent morphologic alterations in the vicinity of httEx1‐polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1‐polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron‐dependent oxidative events that can be deleterious to the cell.

Confocal Microscopic Localization of Constitutive and Heat Shock–Induced Proteins HSP70 and HSP27 in the Rat Heart

BackgroundHeat-shock treatment of rats elevates expression of heat-shock proteins, which play a role in improving the contractile recovery and reducing infarct size in hearts after ischemic injury. However, the location of these proteins in the heart is unknown. Methods and ResultsAnesthetized rats were heat-shocked by elevation of body temperature to 42°C to 42.5°C for 15 minutes, followed by 24 hours of recovery. Control and heat-shocked hearts were extirpated and perfused briefly with saline followed by 2% paraformaldehyde in PBS. Confocal immunofluorescence microscopy of control hearts revealed that HSP27 was localized in cardiomyocytes in a pattern reminiscent of Z bands and was colocalized with neuronal markers in somata and axons. No obvious change in HSP27 content or distribution occurred after heat shock. Confocal microscopy revealed little or no HSP70 in control hearts. After heat shock, HSP70 was detected neither in cardiomyocytes nor in neuronal elements within the heart, but HSP70 was abundant in small blood vessels found between the ventricular cardiomyocytes. ConclusionsHeat shock induces a cell type–specific expression of HSP70 in blood vessels but not myocytes or intrinsic cardiac neurons, suggesting that blood vessels play a primary role in myocardial protection.

Constitutive expression of heat shock protein HSP25 in the central nervous system of the developing and adult mouse

Immunohistochemistry and in situ hybridization have been used to survey constitutive heat shock protein (HSP)25 expression in the brain and spinal cord of the developing and adult mouse. The data reveal both transient and sustained patterns of expression and demonstrate robust differences between mice and rats. During development, HSP25 is transiently expressed in neurons of the inferior colliculus, various thalamic subnuclei, and the majority of Purkinje cells in the cerebellum. Sustained expression into adulthood is seen in neurons of the cranial nerve nuclei, spinal cord motoneurons, median preoptic nucleus, and a subset of Purkinje cells. Differences in HSP25 expression between adult rats and mice include the somatic motor nuclei innervating the extraocular muscles, which are HSP25 immunoreactive only in the rat. Similar differences in HSP25 expression are seen during the development of the inferior colliculus, thalamus, and cerebellum, where expression is restricted to mice. J. Comp. Neurol. 434:262–274, 2001.

Hyperthermic induction of the 27-kDa heat shock protein (Hsp27) in neuroglia and neurons of the rat central nervous system

The 27‐kDa heat shock protein (Hsp27) is constitutively expressed in many neurons of the brainstem and spinal cord, is strongly induced in glial cells in response to ischemia, seizures, or spreading depression, and is selectively induced in neurons after axotomy. Here, the expression of Hsp27 was examined in brains of adult rats from 1.5 hours to 6 days after brief hyperthermic stress (core body temperature of 42°C for 15 minutes). Twenty‐four hours following hyperthermia, Western blot analysis showed that Hsp27 was elevated in the cerebral cortex, hippocampus, cerebellum, and brainstem. Immunohistochemistry for Hsp27 revealed a time‐dependent, but transient, increase in the level of Hsp27 immunoreactivity (Hsp27 IR) in neuroglia and neurons. Hsp27 IR was detected in astrocytes throughout the brain and in Bergmann glia of the cerebellum from 3 hours to 6 days following heat shock. Peak levels were apparent at 24 hours, gradually declining thereafter. In addition, increases in Hsp27 IR were detected in the ependyma and choroid plexus . Hyperthermia induced Hsp27 IR in neurons of the subfornical organ and the area postrema within 3 hours and reached a maximum by 24 hours with a return to control levels 4–6 days after hyperthermia. Specific populations of hypothalamic neurons also showed Hsp27 IR after hyperthermia. These results demonstrate that hyperthermia induces transient expression of Hsp27 in several types of neuroglia and specific populations of neurons. The pattern of induced Hsp27 IR suggests that some of the activated cells are involved in physiological responses related to body fluid homeostasis and temperature regulation. J. Comp. Neurol. 428:495–510, 2000.