Chaperones, protein aggregation, and brain protection from hypoxic/ischemic injury
1 Department of Anesthesia, Stanford University, Stanford, CA 94305,
USA
2 Department of Neurosurgery, Stanford University, Stanford, CA 94305,
USA
3 Department of Biology, Stanford University, Stanford, CA 94305,
USA
4 Cerebral Vascular Disease Research Center, University of Miami School of
Medicine, Miami, Florida 33136, USA
* Author for correspondence at address 1 (e-mail: rona.giffard{at}stanford.edu)
Accepted 15 April 2004
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Summary |
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Key words: astrocyte, cell culture, global ischemia, HDJ-2, Hsp70, mouse, rat, CA-1, protein aggregation, apoptosis
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Introduction |
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Recent work has highlighted the ability of Hsp70 to suppress multiple types
of cell death including necrotic death, classical apoptosis, and other
programmed cell death pathways that are independent of caspases and not
blocked by Bcl-2 (Beere et al.,
2000; Jaattela et al.,
1998
; Nylandsted et al.,
2000
; Ravagnan et al.,
2001
; Saleh et al.,
2000
). Since both apoptotic and necrotic cell death are involved
in ischemic brain injury, the ability of Hsp70 to reduce both types of cell
death makes it an appealing candidate for brain protection. Understanding the
aspects of Hsp70 function that are required for protection will identify
pathological processes that contribute to cell death and allow future work to
target identified pathological mechanisms for brain protection.
Unfolded or misfolded proteins have exposed hydrophobic segments that
render them prone to aggregation. Protein aggregates are thought to be toxic
to the cell (Taylor et al.,
2002), so to avoid aggregation, abnormal proteins are either kept
soluble by molecular chaperones or quickly degraded by the
ubiquitin/proteasome system (Hershko and
Ciechanover, 1998
). Under pathological conditions, the level of
abnormal proteins may exceed the ability of the cell to maintain them in a
soluble form or degrade them, allowing aggregation to proceed
(Cohen, 1999
;
Zoghbi and Orr, 2000
). Protein
aggregates can inhibit function of the proteosome, thus further limiting the
cell's ability to dispose of the protein aggregates and interfering with the
normal processing of certain short-lived proteins
(Bence et al., 2001
). Protein
aggregates commonly contain ubiquitin immunoreactivity, suggesting that
proteins targeted for degradation that fail to be degraded may end up in
aggregates (Alves-Rodrigues et al.,
1998
). Protein aggregates have been found in most chronic
neurodegenerative diseases (Kakizuka,
1998
; Taylor et al.,
2002
), in global and focal ischemia (Hu et al.,
2001
,
2000
) as well as hypoglycemic
coma (Ouyang and Hu, 2001
).
These earlier studies showed that ubiquitin immunoreactivity labeled protein
aggregates associated with intracellular vesicles early after injury, and
later associated with mitochondria, Golgi and certain regions of the
plasmalemma (Hu et al.,
2000
).
The Hsp40 family constitutes a major group of Hsp70 cochaperone proteins.
Hdj-2, a human DnaJ member of the Hsp40 family, is highly homologous to the
bacterial DnaJ protein from Escherichia coli. Hdj-2 interacts with
Hsp70 through its J domain (Gebauer et
al., 1997; Minami et al.,
1996
; Tang et al.,
1997
), targeting Hsp70 to specific intracellular tasks and
accelerating the Hsp70 ATPase activity. Hdj-2 has been shown to decrease
injury in models of degenerative brain disease involving protein aggregation.
Cotransfection of Hdj-2 with mutant ataxin-1 resulted in a significant
reduction in aggregate formation (Stenoien
et al., 1999
). In a model of the polyglutamine disease
Huntington's disease, overexpression of Hdj-2 suppressed aggregate formation,
and was associated with decreased toxicity
(Jana et al., 2000
). We
recently demonstrated that overexpression of Hdj-2 reduces ischemia-like
injury in vitro (Qiao et al.,
2003
).
We present here new results on the ability of Hsp70 to reduce protein aggregation in a model of global ischemia and the ability of Hsp70 induction with geldanamycin to block apoptotic astrocyte death induced by glucose deprivation. We discuss additional results on the cochaperone Hdj-2's ability to reduce injury and protein aggregation and the association of Hsp70 overexpression with increased levels of the anti-apoptotic protein Bcl-2.
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Materials and methods |
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Ischemia model
All experimental protocols carried out on animals were approved by the
Stanford University Administrative Panel on Laboratory Animal Care and were in
accordance with NIH guidelines. Surgical anesthesia was induced in male
Sprague Dawley rats (body mass 350450 g; Simonsen Laboratories, Inc.,
Gilroy, CA, USA) with isoflurane (5%) in a mixture of medical air and oxygen
(700:300 ml min1). Anesthesia was maintained with isoflurane
(2-3%) using a facemask. Coordinates for dorsal hippocampal CA1 injection from
bregma were: anteriorposterior, 3.8 mm; medio-lateral,
±1.7 mm; dorso-ventral, 1.8 mm. The dorsal hippocampal CA1
region of each rat was infused with 3 µl of either Hsp72 or control vector.
17 h after vector delivery, rats were anesthetized again with isoflurane and
subjected to 8 minglobal cerebral ischemia followed by 24 h reperfusion as
previously described (Kelly et al.,
2002). Physiological parameters including temperature were
maintained within the normal range before, during, and after ischemia, with
blood pressure intentionally lowered during the ischemic period by blood
withdrawal. Sham-operated (control) rats underwent similar exposure to
anesthesia and surgical manipulation, but the carotid arteries were not
occluded and blood pressure was not altered. After surgery, all animals were
closely monitored throughout the recovery period. At 24 h post ischemia
animals were euthanized by halothane overdose and were transcardially perfused
with 75 ml ofnormal saline followed by 75 ml of 3% paraformaldehyde (PFA).
After removal brains were cryoprotected in 3% PFA/20% sucrose solution.
Double label immunofluorescence and confocal microscopy
After postfixing in 3% PFA/20% sucrose solution for 12 days, 30
µm frozen sections in the coronal plane were taken at 100 µm increments
1 mm anterior and posterior to the needle track. Slices were stained with
X-gal (5'-bromo-4-chloro-3indoly-ß-D-galactopyranoside;
Molecular Probes, Eugene, OR), a chromogenic substrate for
ß-galactosidase (ß-gal), to identify neurons that had taken up the
vector. Adjacent sections were then washed twice with 0.2% Triton X-100
(TX100) in phosphate-buffered saline (PBS), and nonspecific binding was
blocked with 3% bovine serum albumin (BSA) in PBS/0.2% TX100 for 30 min
followed by incubation with anti-ubiquitin monoclonal antibody at 1:400
dilution in PBS/0.1% TX100 overnight at 4°C. This monoclonal
anti-ubiquitin antibody (MAB1510, Chemicon, Temecula, CA, USA) recognizes both
free and bound ubiquitin and was previously characterized by western and
immunocytochemistry (Morimoto et al., 1997). After three washes with
PBS0.1% TX100 the slices were incubated with fluorescein-labeled
anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) at 1:200
dilution. Sections were then labeled with a second primary antibody, a rabbit
polyclonal antibody against ß-galactosidase (ICN, Aurora, OH, USA,
Catalog number 55976) followed by a rhodamine-conjugated, secondary
anti-rabbit antibody, to identify vector targeted cells. Sections were washed
three times then mounted on glass slides and coverslips fixed using Gelvatol.
The slides were observed on a Bio-Rad (Hercules, CA, USA) MRC 1024
laser-scanning confocal microscope.
Primary astrocyte cultures
Cultures were prepared as previously described
(Dugan et al., 1995). Briefly,
newborn Swiss-Webster mice were anesthetized and then killed according to a
protocol approved by the Stanford University Administrative Panel on
Laboratory Animal Care, in accordance with the NIH guide. In brief, brains
were removed, freed of meninges, and the cortices minced and treated with
0.09% trypsin for 20 min at 37°C. After centrifugation at 400
g, cells were resuspended in plating medium containing Eagle's
minimal essential medium (Gibco, Grand Island, NY, USA) supplemented with 10%
equine serum (Hyclone, Logan, UT, USA), 10% fetal bovine serum (Hyclone), 21
mmol l1 glucose (Sigma, St Louis, MO, USA), 2 mmol
l1 glutamine (Gibco), 26.8 mmol l1
NaHCO3 and 10 ng ml1 epidermal growth factor
(Sigma) and triturated. The single cell suspension was plated in 24-well
plates (Becton-Dickinson, Franklin Lakes, NJ, USA) at a density of 2
hemispheres/10 ml, or on 25 mm coverslips precoated with
poly-D-lysine (Sigma) (Dugan et
al., 1995
). Astrocyte cultures are
95% glial fibrillary acidic
protein immunoreactive cells, with the majority of remaining cells microglia,
as identified by isolectin or CD11b staining; essentially no oligodendrocytes
are present as determined by O4 antibody staining
(Xu et al., 2001
). Early or
young cultures were subjected to injury after 57 days in culture while
mature or older cultures were used after more than 25 days in culture.
Transfection to express chaperones
-2 or Phoenix packaging cells (a gift from Garry Nolan, Stanford
University) were transfected with a retroviral plasmid containing inducible
Hsp70 (Papadopoulos et al.,
1996
), or FLAG-hdj-2 (a gift from Don DeFranco;
Qiao et al., 2003
). Cells were
infected with viral supernatant from the packaging cells as previously
described (Papadopoulos et al.,
1996
). Uninfected astrocytes and astrocytes expressing the control
gene ß-galactosidase (lac-Z) were used as controls
(Papadopoulos et al., 1996
;
Xu and Giffard, 1997
).
Injury paradigms
Once confluent, transfected cells were subjected to glucose deprivation
(GD) by exchanging growth medium for balanced salt solution lacking glucose
(BSS0); wash control cells had their medium changed to
BSS5.5 (balanced salt solution with 5.5 mmol l1
glucose). The medium was replaced in each well three times
(Papadopoulos et al., 1998).
Cell death was assessed 24 h after beginning GD. For ubiquitin staining, 8 h
GD was followed by 16 h recovery in the presence of glucose prior to staining.
Combined oxygen and glucose deprivation (OGD) was performed by placing
transfected and control cells in an anoxic chamber in an atmosphere of 85%
N2 10% H2 and 5% CO2, with triple exchange of
the medium with deoxygenated BSS0 (bubbled with N2).
Oxygen and 5.5 mmol l1 glucose were present in controls.
After incubation at 37°C in the anoxic chamber, glucose and oxygen were
restored to the medium. Cell survival was determined after 24 h recovery in
the 37°C 5% CO2/room air incubator. Injury was quantified
either by assaying lactate dehydrogenase (LDH) release into the medium, or by
Trypan Blue or propidium iodide (PI) vital staining and cell counting
(Papadopoulos et al., 1998
;
Qiao et al., 2003
). Apoptotic
vs necrotic cell death was quantitated based on nuclear morphology by
counting cells after staining with Hoechst 33258 dye and PI to identify cells
that had lost membrane integrity. Cells showing clear apoptotic nuclear
morphology consisting of apoptotic bodies or bright condensed nuclei were
counted as apoptotic. Cells with diffusely PI-stained normal sized nuclei were
counted as necrotic and normal Hoechst-stained nuclei as viable cells
(Xu et al., 2003
).
Immunocytochemistry
Fluorescence immunocytochemistry for ubiquitin was performed on cell
cultures on coverslips as previously described
(Ouyang and Giffard, 2003;
Qiao et al., 2003
) using the
same Chemicon antibody as above. Cells on coverslips were fixed with 4%
paraformaldehyde for 1 h, washed twice in PBS for 5 min at room temperature
and then in PBS containing 0.2% TX100 for 30 min. Non-specific binding sites
were blocked with 3% BSA in PBS/0.2% TX100 for 30 min. The cells were
incubated with primary antibody (1:500 dilution in PBS/0.1% TX100 and 1% BSA)
overnight at 4°C, then washed in PBS/0.1% TX100, 3 times. The primary
antibody was visualized with fluorescein-labeled anti-mouse secondary
antibody. Coverslips were washed several times in PBS/0.1% TX100, mounted on
glass slides using Fluoromount-G (Southern Biotechnology Associates, Inc.,
Birmingham, AL 35226, USA) and observed using an epifluorescence microscope
(Nikon Diaphot, Nikon Corporation, Tokyo, Japan).
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Results |
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To pursue the issue of protein aggregation in astrocyte cell culture injury
where the effects of different genes can be more rapidly assessed, we
developed a ubiquitin staining method that allowed us to visualize protein
aggregates following 8 h glucose deprivation and 16 h recovery. We observed
diffuse labeling of control cells with greater immunoreactivity in the nucleus
(Fig. 2A,B) which changed
dramatically after injury to show reduced nuclear staining, increased
cytoplasmic staining and the occurrence of fine to coarse clumps in the
cytoplasm (Fig. 2CI).
Sometimes a reticular pattern was observed in the cytoplasm
(Fig. 2F). These patterns
observed in astrocytes resembled those mentioned above with respect to CA1
neurons following ischemia. We have previously tested the ability of Hdj-2 to
protect astrocytes and found it reduced both GD and OGD injury
(Qiao et al., 2003).
Protection from GD was associated with reduced redistribution of ubiquitin
staining, suggesting reduced protein aggregation
(Fig. 3).Overexpression of
Hdj-2 was associated with a two thirds reduction in GD-induced cell death and
a similar reduction in the number of cells showing aggregates revealed by
ubiquitin immunohistochemistry (Qiao et
al., 2003
).
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The last aspect of Hsp70 protection that we considered was the ability to
block apoptotic cell death compared to necrotic cell death. Earlier work in a
variety of cells has demonstrated that Hsp70 can block apoptosis. We directly
compared the ability of Hsp70 to protect astrocytes from apoptosis or necrosis
induced by two ischemia-like insults, OGD and GD. We took advantage of the
observation that early cultures, less than 8 days in culture, underwent
largely apoptotic death in response to stress while cells allowed to mature in
culture for more than 20 days showed essentially only necrotic death (Xu et
al., 2003,
2004
). Increased levels of
Hsp70 were induced pharmacologically using geldanamycin (GA) at 0.1 µg
ml1 (Xu et al.,
2003
). GA has previously been shown to induce increased Hsp70
expression and to protect from focal cerebral ischemia in vivo
(Lu et al., 2002
). We observed
that both early and mature cultures were less injured after GD
(Fig. 4) when pretreated with
GA to induce Hsp70 or when transfected with a retroviral vector to overexpress
Hsp70 (Papadapoulos et al., 1996; Xu et
al., 2003
). GA reduced apoptosis of young cultures by about half
(Fig. 4E). We previously showed
that retroviral overexpression of Hsp70 reduced GD-induced necrotic astrocyte
death by about 50% (Xu and Giffard,
1997
), and GA treatment reduced death of older cultures by about
3040% (Fig. 4F).
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Discussion |
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We have shown that both induction of Hsp70 with geldanamycin and by
retroviral overexpression can result in protection of astrocytes from
ischemia-like injury. Protection by retroviral expression is somewhat greater
in some paradigms. This may in part reflect the added stress of reduced Hsp90
levels caused by geldanamycin. Geldanamycin is known to bind the
amino-terminal ATP binding site of Hsp90, disrupting its function and
activating heat shock factor 1, leading to increased levels of Hsp70 and Hsp25
(Zou et al., 1998).
The J domain of Hdj-2 is the region of the protein responsible for
mediating the binding of Hdj-2 to Hsp70
(Hartl, 1996). Hdj-2 binds to
and stimulates the ATPase activity of Hsp70, thereby enhancing the chaperone
function of Hsp70 (Hartl,
1996
). The fact that Hdj-2 is still able to suppress aggregation
when the J domain is deleted suggests that this suppression is independent of
interactions with Hsp70 (Chai et al.,
1999
). It has been suggested that Hdj-2 alone can bind misfolded
proteins, suppress aggregation, and facilitate delivery of misfolded
polypeptides to the cellular machinery for proteolytic degradation
(Stenoien et al., 1999
). It
has been speculated that the chaperone function of Hdj-2 in the brain may be
one of the factors responsible for the relative resistance of brain cells to
damage (Jana et al., 2000
;
Miller et al., 1990
). Since
Hsp70 levels are not increased in parallel with Hdj-2 in our astrocytes
(Qiao et al., 2003
), the
protection observed in this study is likely to reflect the direct effects of
Hdj-2. However, we cannot rule out a contribution from modulation of the
function of Hsp70 that is present in the cells. It will be interesting to test
deletion mutants of Hdj-2 in future experiments to identify the domains
required for protection from ischemia-like injury.
Using retroviral vectors, we previously showed that Hsp70 overexpression
protects astrocytes from glucose deprivation, combined oxygen-glucose
deprivation, and H2O2 exposure
(Papadopoulos et al., 1996;
Xu and Giffard, 1997
).
Overexpression of Hsp70 in cultured neurons is also associated with protection
(Amin et al., 1996
;
Beaucamp et al., 1998
;
Fink et al., 1997
;
Uney et al., 1993
), and
overexpression of Hsp70 in astrocytes was found to protect cocultured wild
type neurons (Xu et al.,
1999
). These in vitro injury models mimic some of the
aspects of injury involved in ischemic damage during stroke and suggest
several ways in which Hsp70 could provide protection. Astrocytes protected
from injury by Hsp70 had higher levels of glutathione than did control cells
subjected to the same stress (Xu and
Giffard, 1997
). The ability to refold proteins or prevent
aggregation may allow the cell to conserve glutathione and possibly ATP.
Neurons overexpressing Hsp70 were also found to overexpress the anti-apoptotic
protein bcl-2, both in vitro and in vivo
(Kelly et al., 2002
). The
ability of astrocytes to better protect neurons when only the astrocytes
overexpress Hsp70 raises several intriguing issues about the ways in which
astrocytes interact with neurons during and following ischemia. Protection may
be due to better antioxidant support of neurons and/or even direct provision
of Hsp70 from astrocytes to neurons
(Guzhova et al., 2001
).
Recently studies from several laboratories have begun to define the ways in
which Hsp70 can inhibit the apoptosis signal transduction pathway. Studies
performed in cell lines and immune cells have shown that Hsp70 can block
apoptosis at both early (Gabai et al.,
1998) and late (Jaattela et
al., 1998
) steps in the cascade. Apoptosis is one of the ways
neurons and astrocytes die after ischemia
(Graham and Chen, 2001
). A
functional analysis of the role of the different Hsp70 subdomains in brain
cells subjected to ischemia-like injury will begin to elucidate how Hsp70
protein modulates apoptosis in this setting, and could lead to new therapeutic
approaches. Because Hsp70 can block both apoptotic and necrotic cell deaths,
it is an especially interesting target for anti-ischemic therapy. Future work
with mutants of Hsp70 will help define those protein domains and activities
necessary to inhibit apoptosis in brain cells stressed by ischemia and
reperfusion.
In conclusion, chaperones and cochaperones are interesting candidates for brain protection from ischemiareperfusion injury since they can block multiple modes of cell death. Identifying those actions of the chaperones that are most important for blocking injury will likely lead to the development of novel approaches to reduce damage from both chronic and acute neurodegeneration. Reduction of protein aggregation is one likely direction. Structurefunction studies of chaperones will identify which activities and which proteinprotein interactions are most relevant to different injuries and are most broadly protective.
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Acknowledgments |
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