Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Dec 3;28(49):13038-55.
doi: 10.1523/JNEUROSCI.4407-08.2008.

Hsp27 protects against ischemic brain injury via attenuation of a novel stress-response cascade upstream of mitochondrial cell death signaling

R Anne Stetler  1 , Guodong Cao, Yanqin Gao, Feng Zhang, Suping Wang, Zhongfang Weng, Peter Vosler, Lili Zhang, Armando Signore, Steven H Graham, Jun Chen
Affiliations

Hsp27 protects against ischemic brain injury via attenuation of a novel stress-response cascade upstream of mitochondrial cell death signaling

R Anne Stetler et al. J Neurosci. .

Abstract

Heat shock protein 27 (Hsp27), a recently discovered member of the heat shock protein family, is markedly induced in the brain after cerebral ischemia and other injury states. In non-neuronal systems, Hsp27 has potent cell death-suppressing functions. However, the mechanism of Hsp27-mediated neuroprotection has not yet been elucidated. Using transgenic and viral overexpression of Hsp27, we investigated the molecular mechanism by which Hsp27 exerts its neuroprotective effect. Overexpression of Hsp27 conferred long-lasting tissue preservation and neurobehavioral recovery, as measured by infarct volume, sensorimotor function, and cognitive tasks up to 3 weeks following focal cerebral ischemia. Examination of signaling pathways critical to neuronal death demonstrated that Hsp27 overexpression led to the suppression of the MKK4/JNK kinase cascade. While Hsp27 overexpression did not suppress activation of an upstream regulatory kinase of the MKK/JNK cascade, ASK1, Hsp27 effectively inhibited ASK1 activity via a physical association through its N-terminal domain and the kinase domain of ASK1. The N-terminal region of Hsp27 was required for neuroprotective function against in vitro ischemia. Moreover, knockdown of ASK1 or inhibition of the ASK1/MKK4 cascade effectively inhibited cell death following neuronal ischemia. This underscores the importance of this kinase cascade in the progression of ischemic neuronal death. Inhibition of PI3K had no effect on Hsp27-mediated neuroprotection, suggesting that Hsp27 does not promote cell survival via activation of PI3K/Akt. Based on these findings, we conclude that overexpression of Hsp27 confers long-lasting neuroprotection against ischemic brain injury via a previously unexplored association and inhibition of ASK1 kinase signaling.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Neuroprotective effects of HSP27 transgenic overexpression in mice against focal ischemia. A, Representative photographs of TTC-stained brain coronal sections recovered from 60 min of MCAO and 48 h reperfusion. Data in B represents the total infarct volume determined in wild-type (n = 7) or HSP27 transgenic mice (Tg-HSP27L, low-overexpression mice, n = 8; Tg-HSP27H, high-overexpression mice, n = 8) at 48 h after transient MCAO (tMCAO) or permanent MCAO (pMCAO), whereas data in C illustrates the infarct areas at 48 h after tMCAO taken from consecutive coronal sections beginning +3.0 mm from the bregma and progressing every 1 mm through the entire MCA territory. D, Infarct volume determined at 21 d after 60 min of MCAO in cresyl violet-stained brain sections (n = 9 per group). All data are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 versus wild-type mice. E, F, The neuroprotective effect of HSP27 overexpression is independent of rCBF changes after focal ischemia. Representative [14]-iodoantipyrine autoradiographs obtained at 30 min after the onset of MCAO show similar distribution of low-flow regions between Tg-HSP27H and wild-type brains (E). Data are expressed as low perfusion (<50, 40, 30, or 20% levels of contralateral nonischemic hemisphere) volumes in the ischemic hemisphere (F), n = 7 mice per group.
Figure 2.
Figure 2.
HSP27 transgenic overexpression improves motor function and cognitive performance after focal ischemia and reperfusion. A–C, Assessment of sensorimotor function in sham control mice and ischemic mice. Tg-HSP27H or wild-type mice were subjected to rotarod (A), foot fault (B), and corner tests (C) over 7 d following 60 min of MCAO. D, E, Assessment of cognitive function in mice using Morris water maze 2–3 weeks following MCAO both in the presence of the platform to assess the ability of task acquiring and learning (D) and with the subsequent removal of the platform to assess memory on the position of platform (time spent in goal quadrant, E). All three groups showed similar swim speed during the probe trial (F). At the end of the testing, mice were killed for infarct size measurement (results presented in Fig. 1). All data are presented as mean ± SEM, where *p < 0.05, **p < 0.01, ***p < 0.001, n = 9 mice per group.
Figure 3.
Figure 3.
Transgenic overexpression of HSP27 attenuates cell death and mitochondrial prodeath signaling after ischemia in vivo. A–C, Tg-HSP27H or wild-type mice were subjected to 60 min of MCAO and brain sections were processed for in situ detection of DNA fragmentation at 24 h of reperfusion and immunohistochemistry for cleaved caspase-3. Representative fluorescence images (Aa–c) demonstrate the reduced number of caspase-3-positive cell in Hsp27 transgenic brain than in the wild-type brain after ischemia (a, wild-type nonischemic control; b, wild-type ischemic brain; c, transgenic ischemic brain). Images in Ad–e show the reduced density of DNA damage in cortex (CTX) and striatum (Str) in Hsp27 transgenic brain than in the wild-type brain after ischemia; high power images are presented in panels Af–i, showing that DNA damage occurred mainly in neurons (insert in g, costained with NeuN) and astrocytes (GFAP-positive, arrows in h). The numbers of caspase-3 immunoreactive cells (B) and DNA-damaged (C) were quantified using stereology principles at the coronal levels between −1.5 and 1.5 mm from the bregma. Data are mean ± SEM, **p < 0.01, ***p < 0.001 versus wild-type mice, n = 7 per group. D, E, Activation of caspases 3–24 h following 60 min of MCAO. Cortical cell lysates were immunoblotted against active caspase-9 and active caspase-3 (D) or assessed for DEVDase activity using the DEVD.afc substrate-based enzymatic assay (E). F, Mitochondrial release of cytochrome c (Cyto c) and Smac was detected by Western blot following subcellular fractionation (cyto, cytosol; mito, mitochondria) in ischemic cortex 3–24 h following MCAO. The graph in the lower panel illustrates the semiquantitative results from four independent sets of data. Data are presented as mean ± SEM, where *p < 0.05, **p < 0.01, ***p < 0.001 versus wild-type mice.
Figure 4.
Figure 4.
Overexpression of HSP27 prevents mitochondrial prodeath signaling following in vitro ischemia. A, B, Cortical neuronal cultures derived from either wild-type or Tg-HSP27H mice were challenged at 12 DIV with OGD for 45 or 60 min, then returned to normal culture conditions for 24 h. Cell viability was assessed using Alamar blue (A), while cell death was quantified by measuring LDH release from neurons (B, 100% is based on total cell lysis by Triton X-100). C, D, Cortical neurons from rat embryos were infected with AAV-GFP or AAV-HSP27 for 3 d and then challenged at 12 DIV with 60 min of OGD. Cell viability (C) and cell death (D) were determined at 24 h after OGD using Alamar blue and Hoescht staining (percentages of condensed/fragmented nuclei), respectively. E, Representative triple-label fluorescence images from AAV-infected cortical cultures 3 d after infection either without OGD (top) or subjected to 60 min of OGD followed by 24 h of reperfusion (middle and bottom). Neurons were transfected with GFP alone (top, middle) or coinfected with GFP and HSP27 (bottom), and labeled (GFP, green; Hoechst, blue; active caspase-3, red; arrows indicate dying neurons). F–I, AAV-infected cortical cultures were subjected to 60 min of OGD. At 6 and 16 h after OGD, neurons were assessed for active caspase-9 and -3 (F) or fluorogenic substrate-based DEVDase activity assay (G), release of cytochrome c by Western blot following subcellular fractionation (H) or immunocytochemistry with Hoescht counterstaining (Ia–d: empty AAV, e–h: AAV-HSP27; arrows in c, d indicate neurons that lost cytochrome c immunoreactivity and contained apoptotic nuclei at 16 h after OGD). All data are presented as mean ± SEM; for cell viability or cell death assessment, each data point was from at least 12 culture wells out of 3 independent experiments, where **p < 0.01, ***p < 0.001 versus wild-type neurons; for DEVDase activity and cytosolic cytochrome c analysis, data were derived from 4 experiments, *p < 0.05 versus empty AAV- or AAV-GFP infected cultures.
Figure 5.
Figure 5.
Hsp27 is neuroprotective upstream of mitochondrial cell death signaling. A–D, Apoptosis induced by microinjection of cytochrome c or BH3 peptide is not protected by Hsp27 overexpression. Cortical neurons derived from Hsp27 transgenic mice or their wild-type littermates were microinjected with either cytochrome c or the BH3 peptide at the indicated concentrations combined with the tracer rhodamine dextran. BH3 microinjection was also performed using neurons from Bcl-xL transgenic mice. Neurons were counterstained with Hoescht at 6 h after cytochrome c injection or 2 h after BH3 peptide injection to visualize apoptosis. Representative images (A, C) show cytochrome c- or BH3-induced apoptosis (arrows) in neurons; quantitative analysis of apoptosis under various conditions are presented as graphs (B and D; in B, the caspase inhibitor z-DEVD.fmk was added at 100 μm to wild-type cultures 30 min before cytochrome c injection). All data are mean ± SEM, where *p < 0.05, **p < 0.01, ***p < 0.001 versus wild-type cultures; from five independent experiments, each data point was derived from at least 150 neurons. E, Hsp27 or Bcl-x-L transgenic cultures were infected with adenovirus carrying Bax, Puma, or BimEL and assessed for cell survival at 16 h after infection. Data were analyzed based on three experiments, ***p < 0.001 versus wild-type cultures. F, G, Hsp27 overexpression inhibits bleomycin- or OGD-induced translocation of proapoptotic proteins from cytosol to mitochondria and release of cytochrome c from mitochondria. Cortical cultures were infected with AAV-Hsp27 or AAV-GFP for 3 d, exposed to bleomycin (10 μm) or 60 min of OGD followed by reperfusion, then subjected to cellular fractionation (cyto, cytosol; mito, mitochondria) and Western blotting. H, Bleomycin-induced cytochrome c release is attenuated in neurons derived from Bax or Puma knock-out mice, compared with the wild-type cultures. Blots are representatives of 3 sets of independent experiments. I, Hsp27 overexpression inhibits bleomycin-induced apoptosis. Cortical cultures were infected with AAV-Hsp27 or AAV-GFP for 3 d, exposed to bleomycin (10 μm) for 16 h, and then assessed for apoptosis using Hoescht nuclear staining. Data are presented as mean ± SEM, where ***p < 0.001 versus noninfected or AAV-GFP-infected cultures.
Figure 6.
Figure 6.
Alteration in JNK, but not Akt/PKB, signaling contributes to Hsp27 neuroprotection. A, Overexpression of HSP27 suppresses OGD-induced JNK and c-Jun phosphorylation. Cortical neurons were infected with AAV-HSP27 or AAV-GFP for 3 d and then exposed to 60 min of OGD. Cell lysates obtained at 1, 2, and 6 h following OGD were subjected to Western blot using phospho-specific antibodies. Optical density was quantified by averaging four independent experiments. B–D, JNK activity plays a critical role in OGD- or DNA damage-induced cytochrome c release and cell death. Cortical neurons were treated with the JNK inhibitor SP600125 (3 μm) for 30 min and then exposed to either 60 min of OGD (B) or bleomycin (10 μm) (C). Cytochrome c was assessed by Western blot in cytosolic (cyto) and mitochondrial (mito) fractions, respectively, at 6 and 16 h after OGD (B) and at 2 and 6 h after bleomycin exposure (C); cell viability was determined 24 h after OGD or bleomycin by Alamar blue (D). E, HSP27 overexpression leads to sustained expression of phospho-Akt following OGD or bleomycin exposure. Neurons were infected with AAV vectors for 3 d before OGD or bleomycin exposure. The blots are representatives of four sets. F, Inhibition of Akt activation does not affect HSP27-mediated neuroprotection against OGD or bleomycin toxicity. The PI3K/Akt inhibitor LY294002 (10 μm) was added to cortical cultures 30 min before bleomycin or OGD challenge, and assessed for cell survival 24 h later using Alamar blue. All data are presented as mean ± SEM, where *p < 0.05, **p < 0.01 versus vehicle controls.
Figure 7.
Figure 7.
Hsp27 inhibits and forms a physical association with ASK1. A, Hsp27 overexpression decreases the activation of MKK4 but does not affect the phosphorylation of ASK1 following OGD. Cortical cultures were infected with AAV-Hsp27 or AAV-GFP for 3 d and then challenged with 60 min of OGD and immunoblotted using phospho-specific antibodies at 1, 2, and 6 h after OGD. The graph in the bottom panel illustrates the semiquantitative results for p-MKK4 and p-MKK7, based on 3 independent experiments. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01 versus AAV-GFP-infected cultures. B, Hsp27 directly inhibits in vitro ASK1, but not MKK4/7 or JNK1/3, activity. Recombinant active kinases were incubated with recombinant Hsp27 protein at the indicated concentrations and then assessed for the incorporation of radiolabeled phosphate onto the substrate protein (MBP) as evidence of kinase activity. Data are presented as mean ± SEM, ***p < 0.001 versus reactions without adding Hsp27 protein. C, Hsp27 and ASK1 form a physical association. Cortical neurons were infected with HSP27 or GFP (both HA-tagged) for 3 d and exposed to 60 min of OGD, followed by 2 h of reperfusion. Lysates were collected for coimmunoprecipitation using anti-HA (left) or anti-ASK1 (right) and immunoblotted for HA, ASK1, or MLK3. Blots are representatives of 3 sets.
Figure 8.
Figure 8.
Characterization of functional domains required for Hsp27 and ASK1 interaction and suppression of ASK1 activity. A, Schematic representation of various ASK1 constructs. All ASK1 constructs were Flag-tagged. B, Interaction with Hsp27 requires the kinase domain of ASK1. Human HEK293 cells were cotransfected with Flag-tagged ASK1 constructs and HA-tagged full-length Hsp27 for 16 h. Cell lysates were immunoprecipitated with either anti-HA or anti-Flag antibody and immunoblotted against HA or Flag. C, Interaction with ASK1 requires the N-terminal domain of Hsp27. HSP27 deletion mutants lacking either the C-terminal (ΔC) or the N-terminal (ΔN) domain were HA-tagged and cotransfected with full-length Flag-tagged ASK1 into HEK293 cells for 16 h. Cell lysates were immunoprecipitated with either anti-HA or anti-Flag antibody and immunoblotted against HA or Flag. D, E, The N-terminal domain of Hsp27 efficiently suppresses ASK1 activity. HEK293 cells were cotransfected with Flag-tagged ASK1 and either HA-tagged full-length Hsp27 (D) or HA-tagged ΔC truncated Hsp27 (E) at the indicated plasmid quantity (μg) for 16 h. Cell lysates were immunoprecipitated with anti-Flag and assessed for ASK1 activity present in cell lysates. All blots presented in B–E are representatives of at least 3 independent experiments. F, The N-terminal domain is required for Hsp27-mediated neuroprotection against OGD. Cortical cultures were infected with AAV carrying full-length Hsp27 or truncated Hsp27 lacking either the C-terminal (ΔC) or the N-terminal (ΔN) domain. Three days after infection, cultures were exposed to 60 min of OGD and assessed for viability using Alamar blue. All data are presented as mean ± SEM, *p < 0.05, ***p < 0.001, and each data point is derived from at least 12 culture wells based on 3 independent experiments.
Figure 9.
Figure 9.
ASK1 plays a critical role in OGD-induced neuronal death. A–E, Knockdown of ASK1 expression is neuroprotective against OGD. Murine cortical cultures were infected for 3 d with lentivirus-shRNA targeting the ASK1 mouse sequence (Lnt-ASK1t) or a scrambled sequence (Lnt-ASK1 s), or the empty lentivirus (Lnt). Lnt-ASK1t effectively suppressed ASK1 protein expression for >90% (A) and inhibited activation of the downstream kinase MKK4 and JNK at 2 and 6 h after 60 min of OGD (B). Knockdown of ASK1 promoted neuron survival in wild-type mice-derived cultures at 24 h after 60 min of OGD, but failed to confer additional neuroprotection against OGD in Tg-Hsp27H mice-derived cultures (C). Coinfection of HA-tagged human ASK1, which is resistant to Len-ASK1t knockdown (D), restored sensitivity of wild-type cortical cultures to 60 min of OGD (E), confirming that the effects observed from ASK1 knockdown were specific. F, G, Infection of lentivirus encoding an HA-tagged dominant-negative form of ASK1 (F) promoted cell survival assessed by Alamar blue at 24 h after 60 min of OGD (G). H–J, Knockdown of endogenous Hsp27 promotes ASK1 activity and cell death after OGD. Cortical cultures were infected for 3 d with lentivirus-shRNA targeting Hsp27 (Lnt-Hsp27t) or a scrambled sequence (Lnt-Hsp27 s) and then challenged with OGD (60 min) and reperfusion. Cell extracts were subjected to Western blot for Hsp27 (H) or ASK1-pull down for ASK1 activity assay (I). Cell viability was measured at 24 h after OGD (J). All data are presented as mean ± SEM, where *p < 0.05, ***p < 0.001, and each data point was derived from at least 12 culture wells out of 3 independent experiments.
Figure 10.
Figure 10.
Hsp27 transgenic overexpression inhibits ischemia-induced ASK1/JNK signaling pathways in vivo. A–C, Hsp27 transgenic overexpression suppresses ASK1 and JNK1 and JNK3 activities following transient ischemia. Tg-Hsp27H or wild-type mice were subjected to 60 min of MCAO followed by reperfusion. Whole-cell protein extracts prepared from the cortical MCA territory at 4 or 8 h after ischemia or sham operation (S) were assessed for kinase activities (A, ASK1 activity; B, JNK1, JNK2, and JNK3 activities). The graphs in the right panel of A and B illustrate the quantitative results from four independent sets of samples. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01 versus wild-type mice. Brain sections obtained at 4 h of postischemic reperfusion were processed for immunohistochemistry for phospho-c-Jun (C), and wild-type sections were counterstained with the neuronal markers NeuN (C, bottom). The majority of p-c-Jun positive cells are also NeuN positive. Scale bars, 50 μm. D, Hsp27 overexpression inhibits JNK activation but has no effect on the status of p38 or ERK1/2 activation at 4 or 8 h after 60 min of MCAO. Blots are representatives of four sets. E, HSP27 overexpression inhibits mitochondrial translocation of the prodeath proteins Bax and BimEL. Lysates were collected 4 and 8 h after 60 min of MCAO, fractionated to separate mitochondria from cytosol, and immunoblotted. Blots are representatives of four sets. F, Pharmacological inhibition of JNK decreases infarct volume in wild-type mice, but has no additional effect in Tg-Hsp27H mice. Mice were injected (i.p.) with the JNK inhibitor SP6000125 (10 mg/kg) or with vehicle at the onset of postischemic reperfusion. Infarct volume was determined using TTC staining at 48 h after 60 min of MCAO. Data are mean ± SEM, **p < 0.01, ***p < 0.001, n = 8 per group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. An JJ, Lee YP, Kim SY, Lee SH, Lee MJ, Jeong MS, Kim DW, Jang SH, Yoo KY, Won MH, Kang TC, Kwon OS, Cho SW, Lee KS, Park J, Eum WS, Choi SY. Transduced human PEP-1-heat shock protein 27 efficiently protects against brain ischemic insult. FEBS J. 2008;275:1296–1308. - PubMed
    1. Arya R, Mallik M, Lakhotia SC. Heat shock genes—integrating cell survival and death. J Biosci. 2007;32:595–610. - PubMed
    1. Badin RA, Lythgoe MF, van der Weerd L, Thomas DL, Gadian DG, Latchman DS. Neuroprotective effects of virally delivered HSPs in experimental stroke. J Cereb Blood Flow Metab. 2006;26:371–381. - PubMed
    1. Bao XQ, Liu GT. Bicyclol: a novel antihepatitis drug with hepatic heat shock protein 27/70-inducing activity and cytoprotective effects in mice. Cell Stress Chaperones. 2008;13:347–355. - PMC - PubMed
    1. Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, Tailor P, Morimoto RI, Cohen GM, Green DR. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol. 2000;2:469–475. - PubMed

Publication types

MeSH terms