scholarly journals Regional Differences in Heat Shock Protein 25 Expression in Brain and Spinal Cord Astrocytes of Wild-Type and SOD1G93A Mice

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1257
Author(s):  
Rebecca San Gil ◽  
Benjamin E. Clarke ◽  
Heath Ecroyd ◽  
Bernadett Kalmar ◽  
Linda Greensmith
Keyword(s):  
Spinal Cord ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Chronic Stress ◽  
Acute Stress ◽  
Physiological Role ◽  
Wild Type ◽  
Acute And Chronic Stress ◽  
Glial Cultures

Heterogeneity of glia in different CNS regions may contribute to the selective vulnerability of neuronal populations in neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS). Here, we explored regional variations in the expression of heat shock protein 25 in glia under conditions of acute and chronic stress. Hsp27 (Hsp27; murine orthologue: Hsp25) fulfils a number of cytoprotective functions and may therefore be a possible therapeutic target in ALS. We identified a subpopulation of astrocytes in primary murine mixed glial cultures that expressed Hsp25. Under basal conditions, the proportion of Hsp25-positive astrocytes was twice as high in spinal cord cultures than in cortical cultures. To explore the physiological role of the elevated Hsp25 expression in spinal cord astrocytes, we exposed cortical and spinal cord glia to acute stress, using heat stress and pro-inflammatory stimuli. Surprisingly, we observed no stress-induced increase in Hsp25 expression in either cortical or spinal cord astrocytes. Similarly, exposure to endogenous stress, as modelled in glial cultures from SOD1 G93A-ALS mice, did not increase Hsp25 expression above that observed in astrocytes from wild-type mice. In vivo, Hsp25 expression was greater under conditions of chronic stress present in the spinal cord of SOD1 G93A mice than in wild-type mice, although this increase in expression is likely to be due to the extensive gliosis that occurs in this model. Together, these results show that there are differences in the expression of Hsp25 in astrocytes in different regions of the central nervous system, but Hsp25 expression is not upregulated under acute or chronic stress conditions.

Combined Effects of Novel Heat Shock Protein 90 Inhibitor NVP-AUY922 and Nilotinib against Mutant Forms of BCR-ABL

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 724-724
Author(s):  
Tetsuzo Tauchi ◽  
Daigo Akahane ◽  
Seiichi Okabe ◽  
Eishi Ashihara ◽  
Shinya Kimura ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 90 ◽  
Parp Cleavage ◽  
Combined Effects ◽  
Wild Type ◽  
Abl Kinase ◽  
Histopathologic Analysis ◽  
Mutant Forms

Abstract NVP-AUY922 (Novartis) is a novel 4,5-diaryloxazole ATP-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and deplete the levels of HSP90 client protein. Combining AUY922 with ABL kinase inhibitors may provide several advantages, such as enhanced efficacy and reducing the potential emergence of new resistant mutations. Treatment with AUY922 has been shown to exert greater potency against BCR-ABL mutants compared with wild type (wt) BCR-ABL. In the present study, we investigated the combined effects of AUY922 and Nilotinib on mutant forms of BCR-ABL-expressing cells. Co-treatment with AUY922 and Nilotinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL and BCR-ABL mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to AUY922 and Nilotinib even in BaF3 cells expressing T315I. In contrast, we did not observe enhanced effects of AUY922 and imatinib in T315I BCR-ABL-expressing cells. Co-treatment with 500nM of Nilotinib significantly increased AUY922-induced apoptosis in BaF3 cells expressing T315I. Combined treatment with AUY922 and Nilotinib in BaF3 T315I also associated with more PARP cleavage, resulting from increased activation of caspase-3 and -9 during apoptosis. Following co-treatment of BaF3 T315I with AUY922 and Nilotinib caused more attenuation of phospho-T315I BCR-ABL levels and the downstream signal transducer, including phosho-CrkL, phospho-Stat5, and phospho-Akt. These results demonstrate that Nilotinib appears to inhibit T315I BCR-ABL kinase activity in BCR-ABL structurally compromised by loss of HSP90 chaperone activity. To assess the in vivo efficacy of AUY922 and Nilotinib, athymic nude mice were injected i.v. with mixture of BaF3 cells expressing wild type BCR-ABL and mutant forms of BCR-ABL(M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 24 hrs after injection, the mice were divided four groups (5 mice per group), with each group receiving either vehicle, AUY922 (50mg/kg; i.p. two times per week), Nilotinib (30mg/kg; p.o. once every day), AUY922 (50mg/kg; i.p. two times per week) + Nilotinib (30mg/kg; p.o. once every day). Animals treated with either vehicle or Nilotinib-alone died of a condition resembling acute leukemia by 28 days; animals treated with AUY922 alone survived more than 40 days, and those treated with the combination of AUY922 + Nilotinib survived more than 60 days. Histopathologic analysis of vehicle or Nilotinib-alone treated mice revealed infiltration of the spleen and bone marrow with leukemic blasts. In contrast, histopathologic analysis of organs from AUY922 plus Nilotinib-treated mice demonstrated normal tissue architecture and no evidence of residual leukemia. Taken together, these preclinical studies show that the combination of AUY922 and Nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells, including T315I, in an efficacious manner.

scholarly journals Neuroinflammatory processes are augmented in mice overexpressing human heat-shock protein B1 following ethanol-induced brain injury

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Brigitta Dukay ◽  
Fruzsina R. Walter ◽  
Judit P. Vigh ◽  
Beáta Barabási ◽  
Petra Hajdu ◽  
...  
Keyword(s):  
Brain Injury ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Glial Cell ◽  
Inflammatory Cytokines ◽  
Cell Activation ◽  
Mrna Level ◽  
Wild Type ◽  
Pro Inflammatory Cytokines

Abstract Background Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. Methods In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. Results Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions. Conclusions Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.

scholarly journals In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation.

1996 ◽  
Vol 135 (2) ◽  
pp. 329-339 ◽  
Author(s):  
N Shulga ◽  
P Roberts ◽  
Z Gu ◽  
L Spitz ◽  
M M Tabb ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Nuclear Pore Complex ◽  
Heat Shock Protein 70 ◽  
Nuclear Transport ◽  
T Antigen ◽  
Nuclear Pore ◽  
Wild Type

The transport of proteins into the nucleus is a receptor-mediated process that is likely to involve between 50-100 gene products, including many that comprise the nuclear pore complex. We have developed an assay in Saccharomyces cerevisiae for the nuclear transport of green fluorescent protein fused to the SV-40 large T antigen nuclear localization signal (NLS-GFP). This assay allows the measurement of relative NLS-GFP nuclear import rates in wild-type and mutant cells under various physiological conditions. Probably the best understood component of the nuclear transport apparatus is Srp1p, the NLS receptor, which binds NLS-cargo in the cytoplasm and accompanies it into the nucleus. When compared to SRP1+ cells, NLS-GFP import rates in temperature-sensitive srp1-31 cells were slower and showed a lower temperature optimum. The in vivo transport defect of the srp1-31 cells was correlated with the purified protein's thermal sensitivity, as assayed by in vitro NLS peptide binding. We show that the kinetics of NLS-directed nuclear transport in wild-type cells is stimulated by the elevated expression of SSA1, which encodes a cytoplasmic heat shock protein 70 (Hsp70). Elevated Hsp70 levels are sufficient to suppress the NLS-GFP import defects in srp1-31 and nup82-3 cells. NUP82 encodes a protein that functions within the nuclear pore complex subsequent to docking. These results provide genetic evidence that Hsp70 acts during both targeting and translocation phases of nuclear transport, possibly as a molecular chaperone to promote the formation and stability of the Srp1p-NLS-cargo complex.

scholarly journals The Heat Shock Protein Ssa2p Is Required for Import of Fructose-1,6-Bisphosphatase into Vid Vesicles

2000 ◽  
Vol 150 (1) ◽  
pp. 65-76 ◽  
Author(s):  
C. Randell Brown ◽  
Jameson A. McCann ◽  
Hui-Ling Chiang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Binding Proteins ◽  
The Other ◽  
Atp Binding ◽  
Wild Type ◽  
Fructose 1,6 Bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Δssa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Δssa1, Δssa3, or Δssa4 strains. Likewise, in vitro import was impaired for the Δssa2 strain, but not for the other Δssa strains. The cytosol was identified as the site of the Δssa2 defect; Δssa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Δssa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Δssa2 Vid vesicles, providing Δssa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.

Blood Pressure and Heat Shock Protein Expression in Response to Acute and Chronic Stress

Hypertension ◽  
1995 ◽  
Vol 25 (4) ◽  
pp. 539-544 ◽  
Author(s):  
Michael J. Blake ◽  
Leslie M. Klevay ◽  
Edward S. Halas ◽  
Ann M. Bode
Keyword(s):  
Blood Pressure ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Expression ◽  
Chronic Stress ◽  
Acute And Chronic Stress ◽  
Heat Shock Protein Expression

scholarly journals The Truncated Form of the Bacterial Heat Shock Protein ClpB/HSP100 Contributes to Development of Thermotolerance in the Cyanobacterium Synechococcus sp. Strain PCC 7942

2000 ◽  
Vol 182 (24) ◽  
pp. 7092-7096 ◽  
Author(s):  
Adrian K. Clarke ◽  
Mats-Jerry Eriksson
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Distinctive Feature ◽  
Functional Role ◽  
Wild Type ◽  
Truncated Form ◽  
Synechococcus Sp ◽  
Pcc 7942 ◽  
Dual Translation

ABSTRACT ClpB is a highly conserved heat shock protein that is essential for thermotolerance in bacteria and eukaryotes. One distinctive feature of all bacterial clpB genes is the dual translation of a truncated 79-kDa form (ClpB-79) in addition to the full-length 93-kDa protein (ClpB-93). To investigate the currently unknown function of ClpB-79, we have examined the ability of the two different-sized ClpB homologues from the cyanobacterium Synechococcus sp. strain PCC 7942 to confer thermotolerance. We show that the ClpB-79 form has the same capacity as ClpB-93 to confer thermotolerance and that the ClpB-79 protein contributes ca. one-third of the total thermotolerance developed in wild-type Synechococcus, the first in vivo demonstration of a functional role for ClpB-79 in bacteria.

scholarly journals Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1228-1239 ◽  
Author(s):  
Miroslav Koulnis ◽  
Ermelinda Porpiglia ◽  
P. Alberto Porpiglia ◽  
Ying Liu ◽  
Kelly Hallstrom ◽  
...  
Keyword(s):  
Mouse Models ◽  
Chronic Stress ◽  
Acute Stress ◽  
Dynamic Responses ◽  
Functional Specialization ◽  
Myeloproliferative Disease ◽  
Response Dynamics ◽  
Acute And Chronic Stress ◽  
Survival Pathways

Abstract Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and β-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-xL is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-xL pathway “resets,” allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using “knock-in” mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-xL response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-xL induction provides a “stop-gap” in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-xL may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms.

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