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Keywords:

  • apoptosis;
  • Bcl-2;
  • head injury;
  • Hsc70;
  • Hsp70;
  • trauma

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

The stress response in injured brain is well characterized after experimental ischemic and traumatic brain injury (TBI); however, the induction and regulation of the stress response in humans after TBI remains largely undefined. Accordingly, we examined injured brain tissue from adult patients (n = 8) that underwent emergent surgical decompression after TBI, for alterations in the inducible 72-kDa heat shock protein (Hsp70), the constitutive 73-kDa heat shock protein (Hsc70), and isoforms of the chaperone cofactor BAG-1. Control samples (n = 6) were obtained postmortem from patients dying of causes unrelated to CNS trauma. Western blot analysis showed that Hsp70, but not Hsc70, was increased in patients after TBI versus controls. Both Hsp70 and Hsc70 coimmunoprecipitated with the cofactor BAG-1. The 33 and 46, but not the 50-kDa BAG-1 isoforms were increased in patients after TBI versus controls. The ratio of the 46/33-kDa isoforms was increased in TBI versus controls, suggesting negative modulation of Hsp70/Hsc70 protein refolding activity in injured brain. These data implicate induction of the stress response and its modulation by the chaperone cofactor and Bcl-2 family member BAG-1, after TBI in humans.

Abbreviations used
SDS

sodium dodecyl sulfate

TBI

traumatic brain injury

Heat shock proteins are induced as a part of the stress response in cells exposed to a variety of injurious stimuli (Burdon 1993). During the stress response total protein synthesis is inhibited; however, synthesis of specific proteins, including members of the heat shock protein family, are increased (Lindquist 1986; Hossman 1993). Heat shock proteins were initially described in Drosophila exposed to hyperthermia (Ritossa 1962). Non-thermal stressors, including ischemia and poisoning with heavy metals and sodium arsenite, can also induce heat shock protein synthesis (Lindquist 1986; Massa et al. 1996)). Induction of heat shock proteins has been associated with subsequent protection from otherwise lethal levels of similar and dissimilar stressors. For example, preconditioning with sublethal hyperthermia can protect cells from higher degrees of hyperthermia (Li and Werb 1982), serum withdrawal (Mailhos et al. 1993), exposure to tumor necrosis factor (Jaattela et al. 1992), and ischemia (Papadopoulos et al. 1996).

The heat shock proteins are categorized based on molecular weight, and can be constitutive or inducible. Constitutive members include a 73-kDa heat shock protein (Hsc70), while inducible forms include 10, 72, 90 and 101-kDa proteins (Lindquist 1986; Burdon 1993; Massa et al. 1996; Wells et al. 1998). The 72-kDa inducible heat shock protein (Hsp70) has been well characterized and has been found to stabilize unfolded proteins, assist with proper folding of proteins, facilitate transport of proteins through cell membranes, and regulate protein translation (Beckman et al. 1990; Kang et al. 1990). A role for Hsp70 in the stabilization of certain mRNA has also been reported (Laroia et al. 1999). The chaperone activity of Hsp70 and Hsc70 can be regulated by cofactors, including the Bcl-2 protein family member BAG-1 (Takayama et al. 1997;Stuart et al. 1998). BAG-1 was initially identified because of its capacity to inhibit apoptosis, and was later found to modulate the chaperone activity of both Hsp70 and Hsc70 via binding to their ATPase domains (Takayama et al. 1997; Stuart et al. 1998; Luders et al. 2000b). Distinct isoforms of BAG-1 differentially affect chaperone function. BAG-1S stimulates whereas BAG-1M inhibits protein refolding (Luders et al. 2000b). BAG-1L does not appear to influence protein refolding, its function is primarily related to regulation of transactivation activity of glucocorticoid and androgen receptors (Froesch et al. 1998; Schneikert et al. 1999).

Hsp70 is induced in the CNS after exposure to thermal injury (Li et al. 1992), epilepsy (Vass et al. 1989), ischemia (Simon et al. 1991; Chen et al. 1993; Planas et al. 1997), subarachnoid hemorrhage (Matz et al. 1996) and traumatic brain injury (TBI; Tanno et al. 1993; Raghupathi et al. 1995; Chen et al. 1998). Relevant to CNS injury, sublethal heat stress protects cultured neurons from glutamate-induced excitotoxic injury (Lowenstein et al. 1991; Rordorf et al. 1991). Excitotoxicity is an important mechanism of neuronal death after TBI in both experimental models (Faden et al. 1989; McIntosh et al. 1990) and in humans (Bullock et al. 1994; Ruppel et al. 2001). The stress response can also protect cells from apoptotic or programmed cell death (Mailhos et al. 1993).

Despite over 200 publications examining heat shock proteins in experimental in vitro and in vivo models of ischemic and TBI (including Brown et al. 1989; Tanno et al. 1993; Lowenstein et al. 1994; Raghupathi et al. 1995; Chen et al. 1998; Dutcher et al. 1998a), what is known about induction of heat shock proteins in brain after acute injury in humans is limited to examination of postmortem tissue after death by asphyxia (Gotohda et al. 2000), immunohistochemical analysis of brain tumors (Kato et al. 1992), and a single report examining Hsp70 after TBI (Dutcher et al. 1998b). To our knowledge there have been no reports showing alterations in BAG-1 after TBI in humans or experimental models. Accordingly, we sought to broaden the previous observations to evaluation of the stress response in injured human brain, by characterizing expression of Hsp70, Hsc70, and isoforms of the chaperone cofactor BAG-1 in brain tissue resected during emergent decompressive craniectomy after severe TBI in humans.

Human brain tissue samples

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

All studies were approved by the University of Pittsburgh Institutional Review Board, informed consent was obtained from the legal next of kin for each patient (approval number 9502101), or approval was granted for existing tissue specimens (approval number 970873). Patients in the study were admitted to the University of Pittsburgh Medical Center between August 1995 and November 1996. Brain tissue samples were obtained from eight adult TBI patients undergoing emergent surgical decompression for the management of life-threatening intracranial hypertension. All patients had clinical or radiographic evidence of cerebral herniation. Surgical decision making was independent of the present study. Samples were immediately frozen to − 70° and stored for batch analysis. Control samples from temporal lobe cortex were obtained from six patients dying of causes unrelated to CNS trauma and were originally collected between April and October 1996. Separate samples from these patients have also been used for analysis of other proteins, results of which have been previously published (Clark et al. 1999). Tissue was first analyzed for Hsp70 and Hsc70. Due to available sample volumes, analysis of BAG-1 was limited to five of eight TBI patients. Analysis of BAG-1 was performed in all six control patients.

Western blot analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

To determine relative levels of Hsp70, Hsc70, and BAG-1 isoforms, western blot analysis was performed as previously described (Clark et al. 1999). Briefly, samples from injured tissue, adjacent to, but not including the contusion if possible, were homogenized in lysis buffer containing 0.1 m NaCl, 0.01 m Tris, 0.1 mm EDTA, and the protease inhibitors chymostatin 2 µg/mL, leupeptin 2 µg/mL, pepstatin 2 µg/mL, and phenylmethylsulfonyl fluoride 100 µg/mL. Lysates were centrifuged at 16 000 gfor 30 min at 4°C and boiled in sodium dodecyl sulfate (SDS) gel loading buffer for 5 min. Total protein concentration in each sample was determined by measuring the ultraviolet absorbance at a wavelength of 280 nm. Relative protein recovery rates were from 11 to 57 µg protein/mg brain tissue. Fifty micrograms of total protein from each sample were loaded on SDS–polyacrylamide gels and separated electrophoretically. Proteins were transferred to nitrocellulose membranes at room temperature overnight. Membranes were blocked with 5% dry milk, then incubated in either a 1 : 1000 dilution of mouse monoclonal antibody against Hsp70 (SPA-810, Stressgen, Victoria, British Columbia, Canada), a 1 : 1000 dilution of rat monoclonal antibody against Hsc70 (SPA-815, Stressgen), or a 1 : 500 dilution of mouse antihuman BAG-1 (clone 3.9F1E11, Upstate Biotechnology, Lake Placid, NY, USA) at room temperature for 1 h. The membranes were washed then incubated in the appropriate alkaline phosphatase-conjugated secondary antibody at a 1 : 3000 dilution for 1 h at room temperature. The membranes were washed then incubated in chemiluminescent detection reagents (New England Nuclear Life Science Products, Boston, MA, USA) and exposed to X-ray film. Expression of Hsp70 and Hsc70 were semiquantified using a gel densitometric scanning program (MCID, St. Catherine's, Ontario). For each protein analyzed two gels were run with equal numbers of control and TBI samples on each gel (in most instances three control and four TBI samples). Electrophoresis, transfer and detection for each pair of gels were performed at the same time. Paired gels were placed on the same X-ray film and multiple exposures were developed to determine response linearity and avoid signal saturation. The relative protein levels were determined from the optical densities of the corresponding protein bands after subtraction of background values obtained in the same lane.

Immunoprecipitation of Hsp70 and Hsc70 with BAG-1

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

To determine whether BAG-1 forms heteromeric complexes with Hsp70 and/or Hsc70, brain tissue lysates were immunoprecipitated with anti-BAG-1, followed by western blotting with anti-Hsp70 or anti-Hsc70. Fifty micrograms of protein lysate from injured (n = 2) and control (n = 2) samples were diluted in cold lysis buffer, then incubated in normal mouse IgG agarose conjugate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 30 min with gentle agitation. Samples were centrifuged at 1000 g at 4°C for 5 min and the supernatants were incubated with 2 µg anti-BAG-1 at 4°C for 1 h with agitation. Protein A/G PLUS-agarose (Santa Cruz) was then added, mixed well, and incubated at 4°C overnight with agitation. Immunoprecipitates were collected by centrifugation at 1000 g at 4°C for 5 min. The pellets were washed, resuspended in electrophoresis sample buffer, boiled for 5 min, then chilled on ice. Peptides were separated on 12% SDS–PAGE and gels stained with Sypro Ruby (Molecular Probes, Eugene, OR, USA) followed by visualization under UV light. Peptides were then transferred onto a polyvinylidene difluoride membrane and western blotting was completed as described above, with the exception that an anti-Hsp70 antibody (sc-1060; Santa Cruz) with an available blocking peptide was used, to confirm the specificity of the immunoprecipitation experiment. For this a 5 : 1 ratio of blocking peptide (Santa Cruz) to anti-Hsp70 was applied as primary antibody and western blotting was completed as described above. For Hsc70, the same primary and secondary (anti-rat IgG) antibodies were used as described above.

Immunohistochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

To determine cellular localization of Hsp70, immunocytochemical analysis was performed as previously described (Clark et al. 1999). Briefly, previously frozen specimens from four TBI and three control patients were postfixed for 1 h in ice-cold 2% paraformaldehyde, then snap frozen in 2-methylbutane in liquid nitrogen. Specimens were cut in 10-µm sections using a cryostat and mounted on glass slides. Slides were washed and non-specific activity was blocked with 5% normal goat serum. Brain sections were then incubated overnight in a 1 : 100 dilution of the antibody against Hsp70 used above. Sections were then washed and incubated for 1 h in a 1 : 3000 dilution of anti-mouse Cy3.18 immunoconjugate (Jackson Immunochemicals, West Grove, PA, USA). Sections were again washed, mounted in gelvatol, and cover slipped for light microscopy. To label nuclei, bis-benzamide (Sigma, St. Louis, MO, USA) diluted in sterile water was applied for 30 s prior to coverslipping. Sections were examined with an Olympus light microscope equipped for epifluorescent illumination. Images were collected using an integrated three-chip Sony color video camera (700 × 600 pixels) equipped with a color frame grabber board. All images were collected while integrating at 4 frames/s using excitation/emission wavelengths of 550/565 nm (red) and 346/460 nm (blue). In sections from each specimen, the primary antibody was omitted to assess for non-specific binding of the secondary antibody.

Data analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

Data are presented as median (range) as data failed tests for normality and equal variance. Comparisons of relative protein levels between TBI patients and control patients were made using the Mann–Whitney rank sum test. A p < 0.05 was considered significant. Statistics were performed using sigmastat software (Jandel Scientific, San Rafael, CA, USA). Analysis of immunohistochemical and immunoprecipitation data was descriptive.

Patient population

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

Patient characteristics are summarized in Tables 1 and 2, and patient numbers correspond to the patient identifiers in the figures. Aspects of the molecular biology of programmed cell death have been previously reported in a separate study using the same patient population (Clark et al. 1999).

Table 1.  Patient population
PatientAge (years)SexInjuryInitial GCSGOS (6 months)Brain regionInjury day
  1. GCS, Glasgow coma score; GOS, Glasgow outcome score (1, dead; 2, persistent vegetative state; 3, severely disabled; 4, moderately disabled; 5, normal); M, male; F, female; MVA, motor vehicle accident.

T140MFall 73Temporal lobe cortex1
T221MAuto-Pedestrian 43Frontal lobe cortex4
T329MMVA 33Temporal lobe cortex1
T432FAssault 55Temporal lobe cortex9
T557MMVA 31Temporal lobe cortex1
T622FFall155Temporal lobe cortex1
T748MMVA 64Frontal lobe cortex1
T838MIndustrial accident 53Temporal lobe cortex1
Table 2.  Control population
PatientAge (years)SexDiagnosisCause of deathPost-mortem time (h)
  1. M, male; F, female.

C116FMalignant melanomaMalignant melanoma11
C261MAtherosclerosisRuptured aortic aneurysm20
C377MMyocardial infarctionCardiac arrest11
C447FChronic neurological disease: unspecifiedPulmonary embolus 2.5
C542FDisseminated viral infectionCardiac arrest 8
C670FHepatic cirrhosisGastrointestinal hemorrhage10.5

Relative levels of Hsp70, Hsc70, and BAG-1 isoforms

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

Figure 1 shows the results of western blot analysis for Hsp70, Hsc70, and BAG-1. For Hsp70, a robust, single, approximately 70-kDa protein band was detected in all eight TBI patients. Relative protein levels for Hsp70 were increased in TBI patients compared with control patients [Fig. 1b; 1359 (373–2986) versus 131 (71–308), respectively; p = 0.002]. In two of the six control patients (C4 and C5), detectable bands corresponding to Hsp70 were seen that were similar to the two TBI patients with the lowest relative optical densities (T2 and T6). Patient C4 had a chronic unspecified neurological disease and Patient C5 died from a disseminated viral infection (Table 2), conditions which could elicit the stress response in brain. These findings are consistent with reports showing that Hsp70 mRNA is increased in postmortem tissue from patients dying with chronic neurodegenerative disease (Pardue and Morrison-Bogorad 1994) and with agonal fever (Morrison-Bogorad et al. 1995).

image

Figure 1. (a) Western blots demonstrating expression of Hsp70, Hsc70 and BAG-1 isoforms in brain tissue samples. (b) Relative Hsp70 (○) and Hsc70 (▿) protein abundance in samples from TBI (filled symbols) and control (open symbols) patients. (c) Relative BAG-1S (33 kDa; ◊), M (46 kDa; ▵) and L (50 kDa; hexagons) protein abundance, and BAG-1 M/S ratio (□) in samples from TBI (filled symbols) and control (open symbols) patients. Relative optical densities for corresponding protein bands were determined using an image analysis system. Lines represent median values, *p < 0.01, Mann–Whitney rank sum test.

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For Hsc70, a predominant ∼70-kDa protein band was detected in samples from both control and TBI patients. There was no difference in relative levels of Hsc70 between TBI and control patients [Fig. 1b; 2191 (1143–4409) versus 3657 (range 538–4879), respectively; p = 0.85]. Given that Hsc70 was not detectable in the sample from control patient 3, it is possible that this sample was degraded. To ensure that this result did not influence interpretation of the data, statistical analysis was performed with this sample excluded; however, a difference between relative levels of Hsc70 in TBI versus control patients was still not found (p = 0.102).

Three BAG-1 isoforms, ∼33 kDa (BAG-1S), ∼46 (BAG-1M), and ∼50 (BAG-1L) were detected in samples from TBI and control patients (Fig. 1a). The 33 and 46, but not the 50-kDa BAG-1 isoforms were increased in patients after TBI patients versus control [Fig. 1c; 72.5 (56.3–91.0) versus 46.3 (21.0–57.5), p = 0.007; 12.6 (7.1–20.0) versus 0.0 (0–5.7), p = 0.004; 39.1 (11.8–48.9) versus 16.5 (0–63.4), p = 0.53; respectively]. Because BAG-1S stimulates whereas BAG-1 M inhibits chaperone activity, the ratio of the 46/33-kDa isoforms was examined. BAG-1M/S was increased in TBI versus control [18 (8–28) versus 0 (0–11)%, p = 0.002].

Results of the immunoprecipitation experiments are shown in Fig. 2. Both Hsp70 and Hsc70 coimmunoprecipitated with BAG-1. An ∼70-kDa band corresponding to Hsp70 was detected with relatively higher optical density in TBI patients versus controls. A band corresponding to mouse IgG used for immunoprecipitation was also detected using the anti-mouse secondary antibody. Peptide blocking experiments verified the specificity of the Hsp70 immunoprecipitation (data not shown). A single ∼70-kDa band corresponding to Hsc70 was detected with similar optical density in TBI patients versus controls. Note that mouse IgG was not detected with the anti-rat secondary antibody used for the Hsc70 primary antibody. A blocking peptide was not available for Hsc70. Sypro Ruby staining for total protein demonstrated that the predominant proteins collected after immunoprecipitation were ∼70 kDa corresponding to Hsp/Hsc70, and ∼25–60 kDa corresponding to BAG-1 isoforms and IgG, although other proteins were detected in the sample from TBI patient 3.

image

Figure 2. Immunoprecipitation of Hsp70 and Hsc70 in brain tissue. Representative control and TBI samples were immunoprecipitated with mouse monoclonal antihuman BAG-1 antibody, then western blot was performed using mouse monoclonal antihuman Hsp70 or rat monoclonal antihuman Hsc70 antibodies. Both Hsp70 and Hsc70 coprecipitated with BAG-1. A protein band corresponding to mouse IgG was detected using the anti-mouse IgG (for the Hsp70 western blot) but not anti-rat IgG (for the Hsc70 western blot) secondary antibody. Bottom panel: Sypro Ruby staining showing all immunoprecipitated proteins.

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Cellular localization of Hsp70

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

Immunocytochemical results (Fig. 3) for the most part confirmed the western blot findings showing an increase in Hsp70 immunoreactivity in sections from three of the four patients with TBI assayed (T3, T5, and T6, but not T2), compared with sections from the three control patients assayed (C1, C4, and C5). Sections from C1 are shown in Fig. 3(a) and sections from T5 are shown in Fig. 3(b–f). Labeling throughout the sections examined was heterogeneous and particulate autofluorescence was observed in all sections (including the primary delete); therefore, only cells with distinctly labeled cell bodies and identifiable nuclei stained with Hoechst were considered to have increased immunoreactivity. In TBI patients, increased Hsp70 immunoreactivity was seen in approximately 30% of cells (identified by Hoechst) with the morphologic appearance of neurons and glia. Increased Hsp70 immunoreactivity was also observed in occasional vascular smooth muscle and endothelium. Cellular labeling was not seen in sections incubated without primary antibody (Fig. 3f). The parenchyma was for the most part intact, but there were areas of microscopic hemorrhage in sections from some TBI patients. Areas of frank necrosis were not seen in the sections examined. It is unclear why increased Hsp70 immunoreactivity was not observed in sections from patient T2, given that Hsp70 was detectable using western blot; although the heterogeneous pattern of cell injury seen after TBI in both experimental models and in humans (Chen et al. 1998; Dutcher et al. 1998a) is one possible explanation.

image

Figure 3. Immunofluorescent labeling of Hsp70 (red) in brain tissue samples. Sections were also treated with bis-benzimide to label cell nuclei (blue). Minimal Hsp70 immunoreactivity was seen in a representative section from a control patient (a). In sections from patients with TBI, increased immuoreactivity was seen in cells with the size and morphologic appearance of neurons (b), glia (c), vascular smooth muscle (d), and endothelium (e). (f) Representative section from a patient with TBI incubated without primary antibody. Size bars ∼100 µm (size bar for panels c–f are the same as for panel b).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

These descriptive findings are consistent with the hypothesis that the stress response, as evidenced by an increase in Hsp70 and alterations in the Hsp70/Hsc70 chaperone cofactor BAG-1, is initiated in human brain following severe head injury. This was demonstrated by western blot analysis in which a significant increase in relative protein abundance of Hsp70 and the BAG-1S and BAG-1M isoforms were seen in injured human brain samples surgically resected for the treatment of life-threatening intracranial hypertension compared with postmortem control brain tissue. Increased expression of Hsp70 was also seen using immunohistochemistry in cells with the morphologic appearance of neurons, glia, vascular smooth muscle, and endothelium in TBI, but not control patients. The constitutive Hsc70, as predicted, was seen both in samples from control and TBI patients. Both Hsp70 and Hsc70 coimmunoprecipitated with BAG-1, consistent with the functional role of BAG-1 as a cofactor that forms heterodimers with Hsp70 and Hsc70.

The results showing increased Hsp70 corroborate the findings reported by Dutcher et al. (1998a), who examined Hsp70 mRNA and protein after human TBI. An approximately threefold increase in Hsp70 protein (versus control) was seen by these investigators, whereas an approximately ninefold increase in Hsp70 protein was seen in our study (versus control). This difference may be related to severity of injury since patients in our study had evidence of cerebral herniation, whereas in the study by Dutcher et al. (1998a), specimens were collected during neurosurgically indicated craniotomy which would include evacuation of hematomas and debridement for penetrating injuries independent of herniation; otherwise, patient populations in regard to age, mechanism of injury, mortality, and time after injury that the specimens were collected were similar. Also, the study by Dutcher et al. (1998a) included control patients with brain tumors, which can induce the stress response (Kato et al. 1992). In our study, two control patients had detectable Hsp70 in brain. These patients succumbed from a chronic neurological disease and a disseminated viral infection (Table 2), conditions which could elicit the stress response in brain (Pardue et al. 1994; Morrison-Bogorad et al. 1995).

Studies such as these have limitations inherent to the use of clinical materials. For instance, we could not control for the region, time after injury, or severity of injury within the TBI patients. Also, there were primary differences in handling of tissues from patient biopsies compared to postmortem controls such as the time of sampling and region of brain examined. While postmortem time does influence levels of Hsp70 and Hsc70 mRNA recoverable in brain (Pardue et al. 1994), it has been reported that a postmortem time of up to 24 h does not influence levels of Hsp70 and Hsc70 protein (Tytell et al. 1998). Furthermore, barring selective degradation of Hsp70, relative levels of Hsc70 and BAG-1S in controls suggests that comparisons can be made between the two groups. All control samples were from the temporal lobe cortex, whereas the location of samples from TBI patients included both temporal and frontal lobe cortex (see Table 1). In addition, the control patients tended to be older than the injured patients, and this may have influenced the results of the study. It is also possible that relative Hsp70 levels may be underrepresented, as supernatants from whole cell homogenates were used for western blot analysis in our study, and it has been shown that Hsp70 redistributes to the insoluble fraction of synaptic densities after stress (Bechtold et al. 2000). Recognizing these limitations, taken together with experimental models of brain injury there appears to be sufficient evidence to support induction of Hsp70 and the stress response in humans after TBI.

The temporal and cellular expression of Hsp70 in human brain after head injury is congruent with rodent models of TBI (Brown et al. 1989; Tanno et al. 1993; Raghupathi et al. 1995; Chen et al. 1998; Dutcher et al. 1998b). Induction of Hsp70 protein occurs within 24 h after injury in experimental models (Raghupathi et al. 1995; Chen et al. 1998; Dutcher et al. 1998b), and an increase in Hsp70 was seen in brain tissue samples from patients resected within 24 h after injury in the present study. In both humans (present study) and rats (Chen et al. 1998), possible cell types demonstrating an increase in Hsp70 protein after TBI include neurons, glia, vascular smooth muscle, and neurons. Whether the increase in Hsp70 was sufficient to afford cellular protection after TBI cannot be determined from this human or the aforementioned rodent studies; although Yenari et al. (1998) have shown that overexpression of Hsp70 using viral vectors can protect neurons in rat models of stroke and epilepsy.

The stress response, often identified via induction of Hsp70, confers cellular protection from injurious stimuli including thermal stress, ischemia, and serum withdrawal (Li et al. 1982; Mailhos et al. 1993; Papadopoulos et al. 1996). The protective effects of Hsp70 may include its role as a molecular chaperone, assisting in protein stabilization, folding, and transport while preventing protein aggregation (Beckmann et al. 1990). Recent reports have shown that these chaperone functions are regulated by the Bcl-2 family member BAG-1. The present study showing increases in both BAG-1S and BAG-1M isoforms, but an increase in the ratio of BAG-1 M/S, would support negative modulation of BAG-1 on the protein refolding activity of Hsp70/Hsc70 in injured brain after trauma. Alternatively, BAG-1 has also been shown to provide a physical link between Hsp70/Hsc70 and the proteasome (Luders et al. 2000a). Although details of the proteasome–Hsp70/Hsc70 interaction related to specific BAG-1 isoforms remain unclear, it is possible that a ratio of BAG-1 isoforms that inhibit Hsp70/Hsc70 refolding activity similarly facilitate degradation of damaged proteins. Taken together, we speculate that after severe TBI BAG-1 may preferentially modulate degradation rather than refolding of proteins; a function that could be either detrimental or homeostatic after injury. Hsp70 and other stress proteins may also confer cellular protection by an inhibitory effect on pro-inflammatory gene expression (Simon et al. 1995; de Vera et al. 1996; Feinstein et al. 1996), cell signaling mechanisms (Someren et al. 1999), or attenuation of apoptotic (Schett et al. 1999) or necrotic cell death (Champagne et al. 1999).

In conclusion, we provide evidence for the stress response and its regulation in human brain following severe head injury. Further study to determine if augmenting the stress response in the injured brain by increasing stress proteins or enhancing their chaperone function via modulation of cofactors such as BAG-1 is warranted.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References

Supported by grants KO8 NS01946, RO1 NS38620, and P50 NS30318 from the National Institute of Neurologic Diseases and Stroke, and the Children's Hospital of Pittsburgh. We would like to thank Dr Joseph Carcillo for critical reading of this manuscript, Patricia Carlier, RN for data collection, J. Eric Loeffert for technical support and Chari Clark for assistance with manuscript preparation.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human brain tissue samples
  5. Western blot analysis
  6. Immunoprecipitation of Hsp70 and Hsc70 with BAG-1
  7. Immunohistochemistry
  8. Data analysis
  9. Results
  10. Patient population
  11. Relative levels of Hsp70, Hsc70, and BAG-1 isoforms
  12. Cellular localization of Hsp70
  13. Discussion
  14. Acknowledgements
  15. References
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