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First published online 10 June 2003
doi: 10.1242/jcs.00619
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Research Article |
1 Information et Programmation Cellulaire, UMR6026 CNRS-Université de
Rennes 1, Campus de Beaulieu, Bat. 13, 35042 Rennes Cedex, France
2 CNRS-UPRES-A 8087, Laboratoire de génétique moléculaire
et physiologique de l'EPHE, Université de Versailles/Saint-Quentin,
Bâtiment Fermat, 45 avenue des Etats-Unis, 78035 Versailles Cedex,
France
* Author for correspondence (e-mail: laure.debure{at}univ-rennes1.fr)
Accepted 15 April 2003
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Summary |
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 Top Summary IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain
region strongly accumulated in an ubiquitinated form in juxtanuclear
aggregates meeting the main criterions of aggresomes and leading to profound
alterations of the mitochondrial network. The viability of cells transfected
by intracellular forms of clusterin was improved by overexpression of Bcl-2,
and caspase inhibition was capable of rescuing cells expressing clusterin,
which presented an altered mitochondrial permeability. We propose that,
although it might be an inherently pro-survival and anti-apoptotic protein
expressed by cells under stress in an attempt to protect themselves, clusterin
can become highly cytotoxic when accumulated in the intracellular compartment.
This activity might reconcile the opposite purported influences of clusterin
on cell survival and explain how clusterin can be causally involved in
neurodegeneration.
Key words: Clusterin, Heat-shock protein, Aggresome, Mitochondria, Apoptosis, 
m
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or pathological (Jones
and Jomary, 2002). Clusterin shares chaperone functions with
heat-shock proteins (Hsps) (Humphreys et
al., 1999), which would explain its capacity to interact with a
wide range of partners (Wilson and
Easterbrook-Smith, 2000) and with partially folded stressed
proteins (DeMattos et al.,
2002; Poon et al.,
2002a). Accordingly, the clusterin gene transcription is
responsive to cellular stress (Michel et
al., 1997a). As for certain Hsps, clusterin expression was shown
to confer resistance to apoptosis in some circumstances
(Jomary et al., 1999;
Sensibar et al., 1995).
Conversely, a series of observations supported a link between clusterin and
tissue involution. The clusterin gene expression has been shown induced in
numerous cases of normally occurring apoptosis, such as the involution of
temporary embryonic structures (Buttyan et
al., 1989), the regression of hormone-dependent tissues such as
the mammary gland (Guenette et al.,
1994) or chicken ovarian atretic follicles
(Mahon et al., 1999), and
olfactory neurons after ablation of their synaptic target
(Michel et al., 1994). Based
on its sites of expression, clusterin is considered as a marker for tissue
regression, specially in brain injuries
(May and Finch, 1992).
Clusterin accumulation has been reported in Alzheimer's disease
(May and Finch, 1992),
epilepsy (Dragunow et al.,
1995), excitotoxicity (Michel
et al., 1992), scrapie infection
(Duguid et al., 1989;
Sasaki et al., 2002a),
programmed neuronal degeneration (Norman
et al., 1995) and normal ageing
(Senut et al., 1992). However,
the precise relationships between clusterin and apoptosis are still under
debate. The coincidence between tissue involution and clusterin expression
does not necessarily imply that clusterin is causally involved in apoptosis.
For instance, contradictory results have been reported about the neuron loss
induced by ischemia in clusterin-deficient mice
(Han et al., 2001;
Wehrli et al., 2001). This
situation is further complicated by the complexity of clusterin localization
and biogenesis pathways. Clusterin was formerly identified as a secreted
protein. The clusterin precursor contains a typical secretion signal peptide
and is matured as an heterodisulfide in the endoplasmic reticulum (ER), after
proteolytic cleavage yielding the and ß subunits, and disulfide
bond formation. Alternatively, clusterin can occur as a monomeric
intracellular protein, in mammals (Reddy
et al., 1996; Yang et al.,
2000) and, to a higher extent, in chicken
(Mahon et al., 1999). Besides,
in prostatic cells undergoing apoptosis, clusterin could escape the secretory
pathway and accumulate in the cytoplasmic compartment in a deglycosylated form
(Lakins et al., 1998).
Finally, clusterin could accumulate in cells that do not transcribe the
clusterin gene, possibly through an uptake of extracellular clusterin,
particularly by neurons (Pasinetti et al.,
1994). This last situation has been reported in the injured
nervous system, in which neurons can internalize extracellular clusterin
secreted by glial cells in response to neuronal insults
(Han et al., 2001;
Walton et al., 1996). The
glia-neuron trafficking of clusterin is obvious in the lesioned olfactory
mucosa, in which glial and neuronal cell populations are anatomically well
separated (Michel et al.,
1997b). Consistently, although the clusterin gene is mainly
transcribed in glial cells in the brain
(Danik et al., 1993), the
derived protein is more often found in neurons in Alzheimer's disease
(McGeer et al., 1992), scrapie
infection (Duguid et al.,
1989; Sasaki et al.,
2002a), brain diseases with Lewy bodies
(Sasaki et al., 2002b) and,
particularly, in dying cells (Dragunow et
al., 1995; Han et al.,
2001; McGeer et al.,
1992). Clusterin immunoreactivity also accumulates in brain
neurons whose number increases with age, as cytoplasmic granules with no
visible cell membranes (Senut et al.,
1992). Although the mechanisms responsible for the presence of clusterin in the intracellular compartment remain largely unknown, we tested the biological consequences of intracellular clusterin accumulation by forcing the expression of intracellular versions of clusterin by means of artificial transgenes. This study revealed that intracellular fragments of clusterin can initiate the formation of protein aggregates meeting the major criterions of the recently described aggresomes. When forming aggresomes, clusterin led to a severe disruption of the mitochondrial distribution pattern. All the clusterin fragments tested triggered mitochondria-dependent apoptosis. These observations might help to reconcile the opposite reported influences of clusterin on cell viability and provide a molecular basis for the apoptotic function of clusterin upon exacerbated expression.
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Materials and Methods |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tubulin
polyclonal (1:100) were generous gifts from, respectively, M. Rauche and Y. A.
Komarova. The following antibodies were also used for immunocytochemistry:
mouse anti-vimentine monoclonal antibody, clone V9 (V6630, Sigma, 1:40);
rabbit polyclonal antibody against subunit MSS1 of the 19S proteasome (PW8165,
Affinity, 1:40); mouse anti-HSP70 monoclonal antibody (clone W27, NeoMarkers,
1:40). Anti-COX6c antibodies were from Molecular Probes. Immunofluorescence
detection was performed with donkey anti-mouse or donkey anti-rabbit
rhodamine-red-X-conjugated secondary antibodies (715-295-150, 711-295-152,
Jackson ImmunoResearch Laboratories, 1:50). For western blotting, donkey
anti-goat horseradish-peroxidase-conjugated secondary antibody (sc-2020, Santa
Cruz Biotechnology, 1:5000) and enhanced chemiluminescence detection reagent
(Amersham Pharmacia) were used. Nocodazole (final concentration 10 µg
ml-1) and DAPI [1,4-diazabicyclo(2,2,)octane, final concentration
0.2 µg ml-1] were purchased from Sigma, MG-132 (final
concentration 10 µM) from TEBU, Mowiol from Calbiochem, MitoTracker Red
CMXRosTM (M-7512, final concentration 50 nM or 100 nM) from Molecular
Probes and ZVAD (Z-Val-Ala-DL-Asp-fluoromethylketone) (final concentration 100
µM) from Bachem.
DNA constructs
The monomeric full-length clusterin was produced from a truncated rat
clusterin cDNA deleted from its secretion signal coding sequence and then
fused to the C-terminus of GFP (GFP-SP-clu construct). We also
constructed expression plasmids yielding the and ß subunits alone
fused to GFP (the GFP--clu and GFP-ß-clu constructs,
respectively). The appropriate clusterin cDNA fragments were obtained by PCR
and then inserted between blunted XhoI and EcoRI restriction
sites of the pEGFP-C3 expression plasmid (Clontech). The upstream (up) and
downstream (down) primers used for PCR contained StuI and
EcoRI restriction sites, respectively. Primers were the following:
5'-GGGGAGGCCTTACCATGGAGCAGGAGTTCTCTGAC-3' (up) and
5'-GGGGAATTCATTCCATGCGGCTTTTC-3' (down) for pEGFP-SP-clu
plasmid; 5'-GGGGAGGCCTTACCATGGAGCAGGAGTTCTCTGAC-3' (up) and
5'-GGGGAATTCAGCGGACCAAGCGGGACTTG-3' (down) for pEGFP--clu
plasmid; and 5'-GGGGAGGCCTTACCATGAGCCTCATGCCTCTCTCC-3' (up) and
5'-GGGGAATTCATTCCATGCGGCTTTTC-3' (down) for pEGFP-ß-clu
plasmid. Equivalent constructs devoid of GFP (SP-clu and -clu)
were obtained with the same cloning strategy by inserting PCR fragments
downstream of the simian virus 40 (SV40) promoter. The Hsp70 expression vector
was a kindly provided by Y. Argon (Dul et
al., 2001). The normal and mutant ubiquitin vectors are described
in Sourisseau et al. (Sourisseau et al.,
2001).
Cell culture and transfection
COS-7 cells were grown (15,000 cells cm-2) at 37°C in 5%
CO2, in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum (Life Technologies) in 35 mm dishes for fluorescence
microscopy and 82 mm dishes for immunoblotting experiences. At 70% of
confluence, the cells were transiently transfected using a calcium phosphate
co-precipitation method with 4 µg of DNA for fluorescence microscopy and 17
µg total DNA for immunoblotting. When necessary, nocodazole or MG-132 were
added 36 hours after transfection for 6 or 12 hours, respectively. HeLa/Bcl-2
cells were plated in 35 mm dishes. At 70% of confluence, the cells were
transfected with 1 µg total DNA per dish of either GFP, GFP-SP-clu,
GFP--clu or GFP-ß-clu encoding vectors mixed with LipofectAMINE
PLUSTM reagent (Gibco-BRL) according to manufacturer's recommendations.
Flow cytometry analyses were performed 24 hours after transfection. When
tested, ZVAD was added at the same time as transfecting DNA.
Immunocytochemistry
COS-7 cells were grown on poly-D-lysine coated glass coverslips and
transiently transfected with 4 µg of pEGFP--clu or
pEGFP-SP-clu. After 36 hours, cells were rinsed in PBS and fixed in
-20°C methanol for 6 minutes. The plated cells were washed in PBS and
incubated in permeabilizing and blocking buffer (0.2% Triton X-100 and 5%
non-fat milk in PBS) at room temperature for 2 hours. Immunostaining was
performed by incubation at 4°C overnight with the appropriate primary
antibody. Coverslips were washed, incubated for 2 hours with secondary
antibody, washed in PBS, counterstained with DAPI and mounted on slides with
Mowiol.
Fluorescence microscopy
Immunofluorescence microscopy analysis was performed using an Olympus AX70
and AnalySIS software with the following Olympus filters: MWB for GFP
(excitation at 450-480 nm, emission above 515 nm); MWIY for rhodamine red X
(excitation at 545-580 nm, emission above 610 nm); and MWU for DAPI
(excitation at 330-385 nm, emission above 420 nm). Mitochondrial localization
was assessed with the mitochondrion-selective dye MitoTracker RedTM.
Incubation at 37°C for 30 minutes (100 nM) allowed its accumulation in
mitochondria of living cells. Observation was performed immediately after
washing with fresh medium under an Olympus IX70 microscope. Filters MWB and
MWG (excitation at 510-550 nm, emission above 590 nm) were used, respectively,
for enhanced GFP (EGFP) and MitoTrackerTM fluorescence.
Immunoblotting
COS-7 cells were transiently transfected with 3 µg pCMV-ß-gal, 7
µg of pEGFP-SP-clu and 7 µg of pCMV-FLAG-Ubi or
pCMV-FLAG-K48R-Ubi or empty vector pCMV-FLAG. If necessary, after 36 hours, 10
µM of MG-132 was added for 6 hours before protein extraction. Cell extracts
were prepared by freeze-thaw lysis in 20 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid,
pH 7.9), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol and protease inhibitor cocktail (CompleteTM, Roche). The
soluble and pellet fractions were obtained by ultracentrifugation at 100,000
g for 5 minutes in a Beckman TL100. For immunoblotting, cell
extracts were normalized with ß-galactosidase activities. The reporter
gene (in pCMV-ß-gal) was co-transfected with GFP-chimera and ubiquitin
expression plasmids to be used as a control for transfection efficiency. 10%
of soluble cell extracts were incubated at 37°C in 0.1 M sodium phosphate
buffer, pH 7.5, 1.25 mM MgCl2, 56 mM 2-mercapthoethanol, 1.1 mg
ml-1 2-nitrophenyl-ß-D-galactopyranoside. ß-Galactosidase
activities were measured by optical density at 405 nm. Supernatants and
pellets were solubilized in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS,
8% glycerol and 5% 2-mercapthoethanol) and resolved by 10% SDS-PAGE and
transferred to nitrocellulose. Clusterin was detected using a goat polyclonal
antibody, a horseradish-peroxidase-conjugated secondary antibody and enhanced
chemiluminescence.
Flow cytometry analyses
Mitochondrial membrane potential was assessed by the retention of
Mitotracker Red (CMX-Ros). This cationic lipophilic fluorochrome is a
cell-permeable marker that, at low doses (50 nM), specifically accumulates in
mitochondria in mammalian cells in proportion to m
(Poot et al., 1996). Cell
staining were performed as follows. Cells were harvested 24 hours after
transfection, centrifuged and resuspended in complete medium at a
concentration of 106 cells ml-1. Cells were then loaded
with 50 nM Mitotracker Red for 30 minutes at 37°C in an humidified 5%
CO2/95% air incubator. Flow cytometry measurements were performed
on a XL3C flow cytometer (Beckman-Coulter France). Fluorescence excitation was
obtained using the blue wavelength (488 nm) of an argon ion laser operating at
15 mW. Green fluorescence of GFP was collected with a 525 nm band pass filter
and red fluorescence of Mitotracker Red with a 620 nm band-pass filter.
Analyses were performed on 104 cells and data were stored in list
mode. Light-scattering values were measured on a linear scale of 1024 channels
and fluorescence intensities on a logarithmic scale of fluorescence.
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Results |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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SP-clu
(Fig. 1A), mimicking the
full-length monomeric clusterin produced in situ. Because an important aim is
to determine the subsequent fate of clusterin internalized by cells and
probably leading to neuron death, we also made constructs allowing the
synthesis of GFP fusions with or ß chains, corresponding to the
isolated subunits of secreted heterodisulfide clusterin (GFP--clu and
GFP-ß-clu, Fig. 1A).
Proteins derived from these constructs exhibited different but reproducible
subcellular patterns (Fig. 1B).
GFP-SP-clu fluorescence was mainly diffuse in the cytosol, with a
juxtanuclear aggregate. GFP--clu appeared mostly aggregated in a
cellular background completely devoid of soluble fluorescence. The size and
form of aggregates were dependent on the efficiency of transfection and on the
time after transfection, ranging from a juxtanuclear dot
(Fig. 1B) to massive crescent-
or ring-shaped perinuclear aggregates (Fig.
1D). GFP-ß-clu was almost exclusively diffused except for a
faint perinuclear ring (Fig.
1B), suggesting the existence of some nuclear targeting signal,
and rare cells presenting a small juxtanuclear aggregate (not shown). We then
wanted to check whether the propensity to aggregate of GFP-SP-clu and
GFP--clu could be an artefact caused by the GFP fusion. Indeed, GFP was
shown to profoundly modify the solubility of fusion proteins in an
unpredictable manner when fused to either the N- or C-terminus of a protein
(Garcia-Mata et al., 1999). To
test such a possibility for clusterin, we examined by immunochemistry the
distribution of -subunit and full-length clusterin devoid of any fusion
protein. As shown in Fig. 1C,
their distributions were similar to those of GFP-fused chimeras, either in
small or large juxtanuclear deposits. Interestingly, advanced stages of
GFP--clu aggregation yielded images with massive perinuclear aggregates
that were very reminiscent of those obtained after clusterin immunostaining in
neurons from senescent rat brain and described as punctate cytoplasmic
labeling and intensely stained granules with no visible cell membrane
(Senut et al., 1992)
(Fig. 1D).
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Structural characterization of juxtanuclear -clu deposits as
aggresomes
We checked clusterin aggregates for the cytological criterions of the
recently described structures known as aggresomes. Aggresomes are perinuclear
inclusions forming specifically at the centrosome and ensheathed in
membrane-free vimentin cages, and were initially described as misfolded,
ubiquitinated protein inclusions (Johnston
et al., 1998). Aggresomes were also shown to recruit proteasome
subunits as well as Hsps, and are believed to be involved in the balance
between protein folding, aggregation and degradation
(Fabunmi et al., 2000). COS-7
cells were transfected with GFP--clu and fixed to proceed to
immunochemistry against the markers of aggresomes. Immunostaining against
-tubulin, a component of the centrosome and of the pericentriolar
material, showed two discrete dots corresponding to centrioles, recognizable
in mitotic cells (Fig. 2, arrowhead shows a centriole during metaphase). GFP--clu deposits were
detected at the centrosome location and a strong -tubulin
immunostaining was observed within the aggregates. Juxtanuclear inclusions of
GFP-SP-clu showed the same location at the centrosome (data not shown).
In cells overexpressing GFP--clu, a network of vimentin collapsed and
concentrated at the level of juxtanuclear deposits, in contrast to
untransfected control cells from the same dishes, in which vimentin
immunostained filaments formed a network extending from a juxtanuclear focus
(the intermediate filament organization center) to the periphery
(Fig. 2). Immunostaining of the
MSS1 subunit of the 19S proteasomal complex revealed a dotted distribution in
control cells but a strong enrichment at the level of GFP--clu
aggregates in transfected cells (Fig.
2). Finally, we tested the localization of the Hsp70 protein
chaperone. Hsp70 immunostaining was mainly homogeneous and cytoplasmic in
untransfected cells but, in transfected cells, it was concentrated at the
level of GFP--clu inclusions (Fig.
2). These colocalizations were systematically observed in all
cells expressing constructs, in at least two distinct experiments. Together,
these co-localization experiments allowed us to classify GFP--clu
inclusions as aggresomes.
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Intracellular GFP--clu aggregates share functional features
with aggresomes
In addition to the presence of the molecular markers listed above,
aggresomes also have functional characteristics. Aggresome formation strictly
requires functional microtubules (Kopito,
2000) and can be antagonized by the protein chaperone Hsp70
(Dul et al., 2001). We tested
the effect of the microtubule-disrupting agent nocodazole on GFP--clu
aggregation. Incubation with nocodazole dramatically prevented the formation
of juxtanuclear clusterin aggregates and stabilized peripheral cytoplasmic
fluorescent dots (Fig. 3C,D).
Treatment with nocodazole reduced from 75% to 25% (n=200) the
proportion of cells presenting one large juxtanuclear aggregate. This result
is a strong indication that clusterin deposits form at the microtubule
organizing center (MTOC) by pericentriolar accretion of peripheral small
protein aggregates. Aggresome formation is also believed to be initiated by
misfolded proteins, abnormally exposing buried domains that are hydrophobic
and pro-aggregative. The protein chaperone Hsp70, binding with high affinity
to such domains, has frequently been shown to be capable of preventing or
reversing protein precipitation processes. We tested this possibility in the
case of the GFP--clu aggresomes. As shown in
Fig. 3E,F, co-transfection of
COS-7 cells with a fourfold excess of Hsp70 expression vector relative to
GFP--clu led to a striking reduction in the size of aggresomes, in
parallel with an increase in the diffuse fluorescence background. Hsp70
co-transfection increased the number of GFP--clu-postive cells
presenting soluble GFP fluorescence from 30% to 80% (n=200). Similar
effects of Hsp70 and nocodazole have been observed on the smaller
GFP-SP-clu juxtanuclear aggregates (data not shown).
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Ubiquitination of intracellular clusterin
Because aggresomes frequently immunostain for ubiquitin, we tested for the
presence of ubiquitin in clusterin inclusions. Transfection of COS-7 cells
with the two clusterin constructs forming aggregates led to a redistribution
of the cellular ubiquitin content (Fig.
4A), with a strong and reproducible concentration at the level of
clusterin aggregates. Such a co-localization of ubiquitin into aggresomes does
not necessarily imply that aggregated proteins are ubiquitinated. Considering
the striking superposition of ubiquitin immunoreactivity and GFP-clusterin
fluorescence, we wanted to check whether this co-localization was the
consequence of a mere accumulation of ubiquitin into aggresomes or instead of
a covalent ligation of ubiquitin to clusterin. For this purpose, we
co-transfected COS-7 cells with GFP-SP-clu and plasmids expressing
ubiquitin, either normal or carrying the K48R amino acid substitution. This
mutation acts as a K48-G76 polyubiquitin chain terminator, leading to an
enrichment in monoubiquitinated forms (Chau
et al., 1989). Transfected cells were treated or not with the
proteasome inhibitor MG-132 before western blot clusterin immunodetection. In
conditions of wild-type ubiquitin overexpression and MG-132 treatment,
clusterin immunostaining revealed a series of high molecular weight bands in
both supernatant and pellet fractions (Fig.
4B). These bands are likely to correspond to polyubiquitinated
clusterin, because they accumulated upon proteasome inhibition and were no
longer visible when using mutant ubiquitin, except for the smallest one, which
probably corresponds to monoubiquitinated clusterin. In control conditions, in
which only empty vector was used, high molecular weight bands were also
observed in presence of proteasome inhibitor but only in the insoluble
fraction corresponding to aggresomes. Thus, under these conditions,
ubiquitination of clusterin seems to favor its precipitation. Together, these
observations strongly suggest that intracellular clusterin is a major
substrate for polyubiquitination and that aggresomes are enriched in
multiubiquitinated clusterin.
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Alteration of the mitochondrial distribution upon clusterin aggresome
formation
Permanent aggresomes have been shown to compromise cell viability but the
cellular and molecular basis of their toxicity remains puzzling. We observed
that clusterin aggresome formation led to dramatic mitochondrial changes. We
first compared the distribution of the mitochondrial dye MitoTracker Red
between untransfected and GFP--clu-overexpressing cells. The top panels
of Fig. 5A show that
MitoTracker Red was strongly concentrated at the level of clusterin aggresomes
and that the cytoplasmic mitochondrial density was lowered (compare
transfected and non-transfected cells). Because the localization of artificial
dyes such as MitoTracker Red is not fully specific and might be influenced by
the presence of protein aggregates, we wanted to verify the relocalization of
mitochondria through an immunological detection of a mitochondrial protein.
Immunostaining of COX6C, the cytochrome c oxidase subunit VIc,
yielded images perfectly similar to those obtained with MitoTracker Red
(Fig. 5A). However, the close
overlap between mitochondrial markers and clusterin aggresomes did not reflect
a precise localization of clusterin at the level of mitochondria.
Fig. 5B clearly shows that
clusterin and mitochondria co-localization depended on the degree of
aggregation of clusterin. In cells still presenting scattered aggregates, the
most peripheral clusterin and mitochondrial signals were clearly dissociated
except in densely stained areas. Hence, GFP--clu aggresome formation
led to a strong concentration of mitochondria and to a decrease of the
peripheral mitochondrial density. These changes were also observed for
GFP-SP-clu aggregates but not for GFP-ß-clu, which did not form
aggresomes (data not shown).
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Intracellular clusterin fragments trigger a mitochondria-mediated
apoptotic pathway
Transfection of cells with clusterin constructs often led to the appearance
of apoptotic nuclei and of floating fluorescent cells with highly condensed
cytoplasm, reminiscent of apoptotic cells. Although these figures were
transient and rapidly lost their GFP fluorescence, they could be detected by
fluorimetric analysis. As shown in Fig.
6, more small fluorescent bodies were detected after transfection
with GFP-clusterin fusions compared with GFP alone
(Fig. 6A, compare left panels
for small sizes and right panels for large sizes between all constructs; see
Fig. 6B for mean values). We
wanted to evaluate the possible induction of mitochondrion-mediated apoptosis
in cells containing clusterin aggregates by examining the effects of Bcl-2
overexpression and caspase inhibition. For this purpose, we have developed a
stable HeLa cell line overexpressing Bcl-2 in a tetracycline-regulated manner.
In the presence of tetracycline, expression of Bcl-2 is inhibited and removal
of tetracycline allows Bcl-2 overexpression. Cells overexpressing or not Bcl-2
were transfected with the different GFP-clusterin constructs. We first
observed that the proportion of small fluorescent cells was strikingly lowered
upon Bcl-2 induction in cell populations transfected with GFP-clusterin
constructs compared with those transfected with GFP alone
(Fig. 6A, compare left and
right panels; see Fig. 6B for
mean values).
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The protective effect of Bcl-2 suggested that mitochondrial defect could be
involved in the triggering of the apoptotic program in cells containing
clusterin. It has been shown that the mitochondrial membrane potential
(m) drop can be an early apoptosis event in promoting
mitochondrial outer membrane permeabilization and activation of the
mitochondrial apoptotic pathway (Desagher
and Martinou, 2000;
Vayssière et al.,
1994). Therefore, we asked whether GFP-clusterin indeed provokes
m collapse by the mean of flow cytometry analyses. We
assessed mitochondrial membrane potential using MitoTracker Red, a lipophilic
cation taken up by mitochondria in proportion to m
(Macho et al., 1996). This
study was performed on GFP-clusterin-transfected cells overexpressing or not
Bcl-2 and, in the latter case, in the presence or not of ZVAD, a
broad-spectrum caspase inhibitor. For each sample, mitochondrial membrane
potential and cell size, as respectively measured by MitoTracker Red
fluorescence and forward scatter value (FSC) (reported in cytogram MitoTracker
Red versus size) were recorded. Only GFP-positive cells with large cell size
are considered in multiparametric cytograms. Both late apoptotic cells with
condensed cytoplasm and apoptotic bodies were excluded from analyses. In all
cases, it appeared that cells can be separated into two populations based on
their MitoTracker Red fluorescence: one with high fluorescence (top) and the
other with lower fluorescence (bottom). Cells in the upper panels are living
cells with normal m, whereas cells in the lower panels
are early apoptotic cells presenting a reduced m
(Kroemer et al., 1995). We
observed that GFP-SP-clu, GFP--clu and GFP-ß-clu expression
increased the portion of apoptotic cells (28%, 23% and 27%, respectively)
(Fig. 7Aa,d,g for cytograms and
Fig. 7B for mean values)
compared with GFP-only expression (9%). Simultaneous overexpression of Bcl-2
greatly reduced the percentage of cells presenting a m
drop, to 15%, 8% and 9% for GFP-SP-clu, GFP--clu, and
GFP-ß-clu, respectively (Fig.
7Ab,e,h and Fig
7B). ZVAD addition, by contrast, induced a significant increase of
dying cells, from 28% to 64% for GFP-SP-clu, from 23% to 62% for
GFP--clu and from 27% to 58% for GFP-ß-clu, but it had only minor
effects on the proportion of dying GFP-expressing cells
(Fig. 7Ac,f,i,l and
Fig. 7B). Taking into account
the effects of Bcl-2 and ZVAD on the viability of cells, it can be proposed
that Bcl-2 exerts its protective role by preventing commitment of
GFP-SP-clu, GFP--clu or GFP-ß-clu expressing cells to
apoptosis, whereas ZVAD-mediated caspase inhibition rescues early apoptotic
cells with lower m in delaying completion of the
apoptotic program (McCarthy et al.,
1997). These data suggest that intracellular clusterin can trigger
the mitochondrial signaling pathway of apoptosis.
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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-B-crystallins
clearly tend to form aggregates in certain neurological pathologies
(Iwaki et al., 1989;
Tamaoka et al., 1995). Hsp104
can enhance the formation of proteins inclusions in yeast
(Newnam et al., 1999) and
Hsp40 can increase protein aggregate formation in mammalian cells
(Wyttenbach et al., 2000). Our
work suggests that clusterin, which has also been proposed to be a protein
chaperone largely composed of sticky amphipathic -helices and
membrane-interacting and aggregation-prone domains
(Bailey et al., 2001), can also
enhance aggresome formation when accumulating inside cells.
Aggresome-forming activity of clusterin
We showed that -chain of clusterin has a strong aggresome-forming
capacity compared with previously described aggresome-forming proteins, and
that this activity was obtained in the absence of proteasome inhibition. Under
the same conditions, full-length clusterin (which exhibited a cytosolic,
soluble distribution) also formed smaller juxtanuclear inclusions. Aggresomes
are recently defined structures, primarily identified as sites of accumulation
of misfolded proteins such as the mutated cystic fibrosis conductance
regulator (CFTR) (Johnston et al.,
1998). The established functional and structural features of
aggresomes were reproduced for GFP--clu aggregates, such as the
formation at centrosomes by accretion of peripheral aggregates via
microtubular tracts, and the co-localization of components of protein quality
control systems: proteasome subunits, ubiquitin and protein chaperones. The
relationships between nuclear or cytoplasmic protein aggregation structures
and pathologies remain obscure. Although it was first associated with protein
diseases, aggresome formation has since been interpreted as a mechanism of
protection against the deleterious spreading of misfolded proteins in the cell
(Chung et al., 2001). However,
certain proteins appear to resist to these degradation factories, such as
polyglutamine-rich or misfolded proteins with exposed hydrophobic
aggregation-prone domains. These resistant aggresomes can cause secondary
cellular troubles, leading to cell death. The present work suggests that this
can be the case for clusterin accumulated in diseased tissues and whose
expression is exacerbated in situations of tissue stresses. The physiological
existence of isolated clusterin -chain has not been demonstrated. It
could correspond to fragment dissociated after uptake of secreted clusterin in
the reducing intracellular compartment in presence of glutathione, but data
from Humphreys et al. (Humphreys et al.,
1999) showed that the subunits remain associated when reduced in
presence of 20 mM DTT and held in a physiological buffer. The existence of
isolated -chain clusterin in the cytosol remains to be tested in vivo,
especially under stress conditions, when uptake of clusterin has been strongly
suggested. However, our comparatives studies of full-length and isolated
clusterin chains indicated that the aggregation activity of clusterin clearly
map at the level of the -chain but is absent from the ß-chain
region. This striking behavior might be surprising, because hydrophobic
domains have been described all along the clusterin precursor
(Bailey et al., 2001). These
results suggest that the various intracellular clusterin fragments proposed in
the literature, either full-length or truncated and carrying the
region, should have comparable effects when accumulating inside cells. Indeed,
monomeric full-length or truncated clusterin fragments seem capable of
accumulating intracellularly after in situ synthesis, sometimes without
passing through the ER-to-Golgi traffic. Such situations have been reported in
chicken (Mahon et al., 1999)
and mammals (Lakins et al.,
1998; Mahon et al.,
1999; Reddy et al.,
1996; Yang et al.,
2000).
Clusterin aggregation and the ubiquitin-proteasome system
The way by which intracellular protein aggregation is deleterious for cells
remains elusive. Some hypotheses have been proposed, such as a dysregulation
of the ubiquitin-proteasome pathway. If unfolded proteins escape degradation,
19S particles of proteasomes are supposed to be sequestered by the growing
protein aggregates, resulting in a depletion of proteasomal activity and
cellular dysfunction. This possibility cannot be ruled out for clusterin
aggresomes because we showed that they are also enriched in proteasome
subunits. Another classical feature of aggresomes is the accumulation of
ubiquitin, suggesting that they contain misfolded or abnormally folded
proteins potentially targeted for but resistant to proteasomal degradation.
Certain aggresome-forming proteins, such as the mutant CFTR, have been shown
to be polyubiquitinated in aggresomes
(Johnston et al., 1998) but
this has not yet been demonstrated in vivo for polyglutamine-containing
proteins (Sakahira et al.,
2002). We showed that ubiquitin strongly concentrated only in
transfected cells containing clusterin aggregates. In addition, our
biochemical studies strongly suggest that clusterin is a major ubiquitination
substrate. The biological significance of the intense polyubiquitination of
certain aggregated proteins is currently unclear. It can reflect the inability
of proteasome to degrade these ubiquitinated substrates or the aggregation
propensity of polyubiquitinated proteins
(Cyr et al., 2002). Our results
are in line with this last hypothesis because overexpression of a mutant
ubiquitin acting as a K48-G76 polyubiquitin chain terminator inhibited
apparent clusterin polyubiquitination. Alternatively, there is the attractive
possibility that protein modification with monoubiquitin or short
polyubiquitin chains can be a proteasome-independent post-translational
modification of the protein surface avoiding extended aggregation
(Gray, 2001). However, this
hypothesis is not supported for clusterin by our experiment using the K48R
mutant ubiquitin. This polyubiquitin-chain-terminating molecule abolished the
formation of high molecular weight conjugated clusterin, which would not be
the case if these bands corresponded to clusterin monoubiquitinated at the
level of different lysines. We showed that overexpression of Hsp70 can prevent
or decrease aggresome formation by GFP--clu. This feature, reported for
many slowly aggregating disease proteins, suggests that aggregation of
clusterin proceeds through an intermediate unfolded state with exposed
hydrophobic interfaces. This view is supported by the existence of amphipathic
-helices and molten globule-like regions in clusterin
(Bailey et al., 2001).
Alteration of mitochondrial integrity by intracellular clusterin
Intracellular clusterin has already shown long-term toxicity in stable
expression experiments (Yang et al.,
2000). We also failed to obtain cell lines stably expressing
intracellular clusterin, unlike secreted clusterin (data not shown). We show
here that clusterin can initiate a mitochondrion-dependent apoptosis program.
Apoptosis is a mode of cell death used by multicellular organism to eradicate
cells in diverse physiological and pathological settings
(Kaufmann and Hengartner,
2001). Apoptotic cell demolition is orchestrated by a family of
cysteine proteases, the caspases, which act in cascade. One major caspase
activation cascade is triggered by cytochrome c release from the
intermembrane space of mitochondria. In the cytosol, cytochrome c
binds Apaf-1 forming an oligomeric complex (apoptosome), which recruits
procaspase-9 and induces its autoactivation. Caspase-9 in turn activates
downstream caspases including caspase-3. Proteins of the Bcl-2 family are
major regulators of this mitochondrial pathway. Notably, the antiapoptotic
Bcl-2 protein inhibits apoptosis by preserving mitochondrial integrity. The
intracellular accumulation of clusterin fragments clearly triggered an
alteration of mitochondrial parameters, because Bcl-2 overexpression strongly
improved the viability of cells expressing clusterin. Moreover, clusterin
clearly led to mitochondrial changes associated with a decrease in
m. In the mitochondrion-associated apoptosis cascade,
this parameter is considered as an event upstream of caspase activation. The
fact that caspase inhibition can rescue clusterin-positive cells with low
m from cell death is consistent with a causal
relationship between intracellular clusterin accumulation and caspase
activation, although this remains to be established.
We also observed that cells containing GFP--clu aggresomes present
dramatic perinuclear relocalizations of mitochondria. Mitochondrial markers,
such as the dye MitoTracker Red and the enzyme COX6C, shifted from a typical
scattered pattern to an intense staining at the level of condensed aggresomes.
Such mitochondrial clustering had already been observed in cells
overexpressing the proapoptotic proteins Bax
(Desagher and Martinou, 2000)
and t-Bid (Li et al., 1998),
and in cells containing aggresomes driven by mutant huntingtin fragments
(Waelter et al., 2001).
Several hypotheses about the molecular bases of aggresome toxicity have been
proposed, such as sequestration of essential regulatory proteins involved in
key cellular events, such as transcription factors or proteasomes. These
observations also suggest the possibility of a mitochondrial network
breakdown. However, the results obtained with GFP-ß-clu, which does not
form aggresomes, indicate that the mitochondrion-associated apoptosis caused
by clusterin is not necessarily associated with the redistribution of
mitochondria. Two hypotheses can be raised to explain this observation. On the
one hand, the disruption of mitochondrial network caused by clusterin
aggresomes could be irrelevant to the effect on m. The
effect of the relocalization of these organites at the centrosome on cellular
physiology should be elucidated. On the other hand, mitochondrial aggregation
could be causally related to the decrease of mitochondrial potential and
another mechanism could be postulated to explain the ß-chain toxicity.
Considering that the C-terminal portion of clusterin has been shown to be
capable of inducing cell death through an unknown mechanism involving the KU70
nuclear protein (Yang et al.,
2000), it is possible that this toxicity could also be mediated by
mitochondrion-dependent apoptosis.
Biological importance of intracellular clusterin accumulation
Considering the present results, the elucidation of the molecular
mechanisms underlying the intracellular accumulation of clusterin is of great
importance. One possibility suggested by the literature is the direct
synthesis in situ of cytosolic forms of clusterin
(Lakins et al., 1998;
Mahon et al., 1999;
Reddy et al., 1996;
Yang et al., 2000). Another
possibility would be an escape from the ER and Golgi apparatus of clusterin
forms destined to secretion. Disruption of the ER and Golgi has been shown for
several misfolded mutant protein (Graves
et al., 2001; Stieber et al.,
2000), which can escape the ER, exceed the proteasomal system and
accumulate in the cytoplasm (Kopito,
2000). Such a phenomenon could occur for clusterin, which is
synthesized in large amounts in insulted tissues and is an intrinsically
disordered protein (Bailey et al.,
2001). This possibility is also in line with the potent
aggregation propensity of clusterin revealed in this study, and could explain
the intracellular accumulation and conformational changes of clusterin
observed in clusterin-synthesizing cells dying in the involuting prostate
(Lakins et al., 1998).
Alternatively, the cellular uptake of extracellular clusterin has
repeatedly been suggested, particularly for neurons
(Han et al., 2001;
Michel et al., 1997b;
Pasinetti et al., 1994;
Walton et al., 1996), but the
precise mode of clusterin internalization is not known. Several endocytosis
receptors for clusterin have been proposed
(Mahon et al., 1999),
including Megalin/gp330, which is also a receptor for ApoE
(Kounnas et al., 1995) [a
protein upregulated in neurons destined to die in the Alzheimer's disease
(LaFerla et al., 1997)]. After
endocytic internalization, clusterin could escape the endosomal compartment to
enter the cytosol and produce its biological effect. The intracellular
trafficking from endosomes to the cytosol has been clearly reported for
certain bacterial toxins, such as diphtheria toxin
(Falnes and Sandvig, 2000).
This toxin is composed of two subunits linked by disulfide bonds that are
derived from a single protein precursor after proteolytic cleavage. After
endocytosis, the acidic pH of early endosomes leads to the exposure of the
lipid-interacting domains of diphtheria toxin and then to its escape in the
cytoplasm. Interestingly, low pH has also been shown to increase the exposure
of hydrophobic regions on clusterin (Poon
et al., 2002b). This behavior is strikingly similar to that of
diphtheria toxin, suggesting the possibility of an escape of clusterin from
endosomes to cytoplasm.
After endocytosis, other toxins undergo retrograde transport to reach the
ER lumen, from where they translocate into the cytosol
(Falnes and Sandvig, 2000).
These toxins probably use the ER-associated protein degradation pathway for
this intracellular transport (Simpson et
al., 1999), like unfolded secretory or membrane proteins also
known to be moved from the ER to the cytoplasm for degradation by the
ubiquitin-proteasome system (Kopito,
2000).
Finally, though clusterin appears to be mainly intracellular in chicken, in
other species, it has been described as an atypical Hsp in that it is secreted
outside synthesizing cells (Poon et al.,
2000). In fact, its capacity for secretion seems to be also a
general feature of classical Hsps, which has also been shown to be released
from cells (Multhoff and Hightower,
1996). Moreover, some molecular chaperones were found capable of
cell-cell trafficking from glia to neurons
(Tytell et al., 1986) through
an unconventional mechanism that allows their entry into the cytosol
(Fujihara and Nadler, 1999).
It is possible, although largely speculative, that the glia-neuron trafficking
of clusterin could be involved in a phenomenon like this whose mechanisms are
unknown.
The present work adds a novel link between neurodegeneration and protein
aggregation (Kakizuka, 1998)
because clusterin, which is regularly found accumulated in neurodegenerative
disorders, can also be strongly pro-aggregative depending on the clusterin
fragments present. Contradictory activities of extracellular brain clusterin
have been reported. Based on its solubilizing action observed in vitro
(Matsubara et al., 1996;
McHattie and Edington, 1999),
clusterin is believed to inhibit neurotoxic agents but, conversely, the
presence of clusterin in amyloid deposits suggests its inability to reverse
protein precipitation. Moreover, studies of transgenic mice have suggested
that clusterin expression promotes amyloid plaque formation in vivo
(DeMattos et al., 2002). The
biological action of intracellular clusterin, which is particularly abundant
in certain neurons (Han et al.,
2001; McGeer et al.,
1992; Sasaki et al.,
2002b; Senut et al.,
1992) remains unknown, but our observations rather suggest a cell
deleterious activity. One must notice that clusterin accumulation is often
associated with intracellular protein aggregation. For example, cerebral
ischemia, which leads to a strong intraneuronal accumulation of clusterin
(Han et al., 2001), is
associated with intense protein aggregation
(Hu et al., 2000). Clusterin
gene expression is also induced in retinitis pigmentosa
(Jones and Jomary, 2002) and
sporadic amyotrophic lateral sclerosis
(Grewal et al., 1999), which
are associated with the abnormal folding and aggregation of rhodopsin and
oxidized superoxide dismutase, respectively
(Rakhit et al., 2002;
Saliba et al., 2002). The
functional relationships between clusterin and intracellular protein
aggregation will be of major importance to elucidate its precise participation
to neurodegeneration.
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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