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Keywords:

  • adenovirus;
  • ALS;
  • motor neuron disease;
  • PC12;
  • superoxide dismutase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Aggregates of Cu/Zn superoxide dismutase (SOD) have been demonstrated in familial amyotrophic lateral sclerosis (FALS) and other neurodegenerative diseases; however, their role in disease pathogenesis is unclear. In this study, we investigated the presence of SOD aggregates in nerve growth factor (NGF)-differentiated PC12 cells and cell viability following: (i) transduction with replication-deficient recombinant adenoviruses (AdVs) expressing wild-type SOD (SODWT) or mutant SOD (SODMT, V148G or A4V); (ii) transfection of yellow fluorescent protein-tagged SODWT (SODWT-YFP) or SODMT (SODA4V-YFP, SODV148G-YFP). SOD aggregates were more prominent in cells following transduction of AdSODMT than AdSODWT and following treatment with H2O2, suggesting that mutant SOD leads to oxidation of cellular components. In addition, cells expressing SODMT-YFP yielded SOD aggregates that were significantly larger and more frequent than SOD aggregates in cells expressing SODWT-YFP. Proteasome inhibitors, but not cathepsin B inhibitors, increased aggregate formation but did not increase cell death. In addition, treatments that increased cell viability did not significantly decrease SOD aggregates. Taken together, our data demonstrate that there is no association between SOD aggregates and cell death in FALS.

Abbreviations used
AdV

adenovirus

ALS

amyotrophic lateral sclerosis

FALS

familial amyotrophic lateral sclerosis

GFP

green fluorescent protein

HD

Huntington's disease

MN

motor neuron

MT

mutant

NGF

nerve growth factor

SOD

Cu/Zn superoxide dismutase

WT

wild-type

YFP

yellow fluorescent protein.

About 10% of cases of amyotrophic lateral sclerosis (ALS) are inherited, and about 20% of cases of familial ALS (FALS) cases are associated with mutations in Cu/Zn superoxide dismutase (SOD; reviewed by Cleveland and Rothstein 2001). Mutant SOD is generally believed to lead to motor neuron (MN) death as a result of a toxic gain of function rather than a deficiency of dismutase activity. We have previously reported that mutant SOD induces cell death of cultured neural cells, and that this cell death is increased by inhibiting the rotamase activity of immunophilins (Leeet al. 1999). This observation suggested that the SOD mutation leads to misfolding of the protein, and therefore increases the cell's reliance on rotamase activity. The presentstudy continues investigations of the role of misfolding and aggregation of mutant SOD in the pathogenesis of FALS.

The potential importance of aggregation, which is often a correlate of misfolding, in the pathogenesis of mutant SOD-induced cell death in animals and cultured cells is supported by the following reports. SOD-containing aggregates have been demonstrated in cytoplasmic inclusion bodies in MNs of FALS autopsy tissue (Bruijn et al. 1998), as well as in MNs and astrocytes of mice that carry FALS-linked mutant SOD as a transgene (Bruijn et al. 1998; Watanabe et al. 2001). The aggregates in these transgenic mice are thought to correspond to high-molecular-weight-insoluble SOD protein complexes seen following electrophoresis of CNS lysates (Johnston et al. 2000). Cytoplasmic aggregates of SOD have also been demonstrated in vitro following microinjection of mutant SOD cDNA into primary rat MNs (Bruening et al. 1999) and following transfection of the mutant protein into human embryonic kidney cells (Johnston et al. 2000). Gene transfer of heat shock protein-70 (HSP70) decreased cell death as well as the formation of aggregates in the cultured MNs (Bruening et al. 1999). Of interest, purified SOD is reported to aggregate in vitro following exposure to a radical generating system (Kim et al. 2001), demonstrating that this protein polymerizes as a result of oxidative stress.

In the present study, we investigated whether FALS-associated mutant SOD forms aggregates in neural cells, and explored the relationship between the SOD aggregate formation and cell viability. The A4V mutation we chose to study is the most common one in North America, and one that is associated with a very aggressive form of FALS (Cudkowicz et al. 1997). We found that: invitro cultured neural cells that express mutant SOD (SODMT) contain more frequent evidence of SOD aggregates than cells expressing wild-type SOD (SODWT) or mock cells; an increase in the number of cells with aggregates and the ‘aggregate burden’ per cell had no significant effect on cell viability; and an increase in cell viability was not associated with a decrease in aggregates. These findings have implications not only for FALS, but alsofor other neurodegenerative diseases in which aggregation has been implicated as a key feature in disease pathogenesis.

Cell culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Rat pheochromocytoma PC12 cells were plated on 15-mm diameter glass coverslips coated with poly-l-lysine solution (0.01%; Sigma, St Louis, MO, USA) at a density 10 000 cells/coverslip in Dulbecco's modified Eagle's media (DMEM, Gibco, Rockville, MD, USA) supplemented with 10% bovine calf serum (Gibco) and 10 µg/mL penicillin/streptomycin (Sigma). After 24 h in culture, differentiation of the cells was induced with DMEM containing 100 µg/mL of nerve growth factor (NGF; Collaborative Biomedical Products, Inc., Bedford, MA, USA) and 0.3% bovine calf serum.

cDNAs constructs and adenoviruses

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

The generation of wild-type or mutant (A4V or V148G) SOD cDNAs and of replication-deficient recombinant AdVs that express these cDNAs (AdSODWT, AdSODA4V, and AdSOD148G) have been previously detailed (Ghadge et al. 1997). Transduction with AdVs expressing wild-type and mutant SOD produced similar levels of human SOD within cells, comparable to those of endogenous SOD, and with similar enzymatic activities (Ghadge et al. 1997). SODWT-yellow fluorescent protein (YFP) fusion constructs were prepared by polymerase chain reaction of SODWT cDNA followed by cloning into the BamHI–NheI sites of pEYFP-N1 (Clontech, Palo Alto, CA, USA). The accuracy of theresultant clone was confirmed by restriction digests as well as sequencing. Point mutations were introduced into this construct using site-directed mutagenesis (QuikChange Kit, Stratagene, LaJolla, CA, USA) in order to generate SODA4V-YFP and SODV148G-YFP, which were similarly checked for the correct sequence.

Experiments involving cyclophilin A (CyPA) used a wild-type CyPA (CyPAWT) cDNA from rat brain cloned into pcDNA1/AMP vector (Helekar and Patrick 1997; a gift from James Patrick, Baylor College of Medicine), and CD8 cDNA as a control.

Transfection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Aggregate formation was investigated in PC12 cells that had been differentiated with NGF for 2–3 days and then transfected using either polyethyleneimine (PEI, Sigma) or superfect (Qiagen, Valencia, CA, USA) with cDNA of SODWT-YFP, SODMT-YFP or, as a control, an enhanced green fluorescent protein (EGFP; Clontech). For PEI transfection, 2 µL of a 10× stock of PEI was mixed with 98 µL 0.15 m NaCl and the equivalent of 2 µg cDNA, and left to stand for 10 min. This mixture was then added directly to neurons on a glass coverslip in a 12-well dish with conditioned serum-free media. Dishes were spun at 300 g for 10 min in a centrifuge, and then placed in an incubator for 2 h. The neurons were then removed from the PEI-cDNA media and returned to fresh PC12 media. Transfection efficiency was roughly 40–50% for PC12cells. The Superfect transfection protocol followed the manufacturer's instructions.

Drug treatment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Twenty-four hours after viral infection or transfection, cells were treated with lactacystin 1 µm, Cathepsin B inhibitor II (50 µm, Calbiochem-Novabiochem, Corp, San Diego, CA, USA) or ethyl (+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-oxiranecarboxylate (E-64-d) 200 µm (Matreya Inc., Pleasant Gap, PA, USA). The effect of these drugs on SODMT-induced aggregation and cell death was evaluated by comparison with cells that expressed mutant SOD without drug treatment and with cells that expressed wild-type SOD with drug treatment. A group of mock cells that were not transfected or transduced were used to evaluate the toxicity of the drug.

Viability assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Seven to nine days after differentiation, PC12 cells were infected with AdVs in order to express human SODWT or SODMT for viability studies. Four days after AdV infection (and 3 days after drug treatment), cells were stained with fluorescein diacetate and counted in five random fields per coverslip using a 25× objective (Lee et al. 1999).

The presence of apoptosis in cells following infection with AdVs expressing wild-type or mutant SOD was determined using Hoechst 33342 to detect chromatin condensation (Molecular Probes, Eugene, OR, USA; Telford et al. 1992). The stained cells were examined under either a Leitz fluorescence microscope or confocal microscope Zeiss LSM 410.

Analysis of SOD aggregates

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Coverslips of PC12 cells transfected with EGFP or SOD-YFP were fixed for 15 min with 4% paraformaldehyde and washed with 0.1 m phosphate-buffered saline (PBS; 3×) prior to immunofluorescence studies. For immunostaining, the fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, and then treated with blocking solution (0.1% Tween-20, 4% bovine serum albumin and 0.1 m PBS) for 1 h. To demonstrate the expression of exogenous wild-type or mutant human SOD, cells were incubated at 4°C overnight with a 1 : 300 dilution of murine anti-human SOD monoclonal antibody (Sigma). Immunoreactive primary antibody was detected by 1 h incubation with a 1 : 500 dilution of anti-mouse IgG antibody-alkaline phosphatase (Boehringer Mannheim Biochemical, Indianapolis, IN, USA) and X-phosphate as a chromogen in blocking medium (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). In some experiments, we used a polyclonal anti-SOD antibody that detected both endogenous rat SOD as well as the human isoform to determine whether aggregates of exogenously expressed wild-type or mutant SOD contain endogenous cytoplasmic SOD. In these cases, cells were incubated at 4°C overnight with a 1 : 300 dilution of anti-SOD polyclonal antiserum (The Binding Site, San Diego, CA, USA), and the immunoreactive primary antibody was detected by a 1 : 500 dilution of anti-sheep IgG conjugated to Cy3, Cy5, or Texas Red (Jackson ImmunoResearch Laboratories).

Aggregates of SOD and their characteristics and distribution within the cell were examined by both conventional and confocal fluorescence microscopy. Specimens were scanned on an IX70 Olympus Fluoview 200 laser-scanning confocal microscope (Olympus, Melville, NY, USA) equipped with two ion lasers, three laser lines (488 nm Ar, 568, 647 nm Kr-Ar), 100× UplanApo (NA 1.35; oil) objective and differential interference contrast optics. The 12-bit detectors were set to accommodate the brightest image and were not altered for other scans. Green (typically used for evaluating autofluorescence) and red (used for SOD or other protein staining) images were collected simultaneously using dual laser line excitation (488 + 568 or 488 + 647) after determining the extent of fluorescence ‘bleed’/cross-talk. Where cross-talk (especially ceroid bleed-through) was significant, images were collected using single laser-line excitation, rescanned for the second fluorophore, and the separate image stacks were later superimposed. Little or no ceroid fluorescence was detectable in the Cy5 (647 nm excitation, 700 nm emission) channel. Maximum intensity Z projections of all optical sections were formed using MetaMorph. To score cells showing SOD aggregates following infection, six random microscopic fields were counted for each coverslip, and a total of 12–24 fields were examined for each treatment under a fluorescence microscope. To score cells showing SOD aggregates following transfection, all fluorescent cells were counted on each coverslip.

In some cases, individual aggregates within a cell were quantitated. For this evaluation, confocal images were collected with 0.3-µm steps over the entire cell thickness and maximum intensity Z projections were constructed. Aggregates were defined as bright non-homogeneities within the cytosol and were identified by means of intensity thresholding. Boundaries of individual differentiated PC12 cells were traced to limit morphometric analyses to single cells. The thresholded area, mean intensity, and intensity integral for entire cells were calculated using MetaMorph software for all complete neurons per field of view, and the process was repeated with the threshold set to include only the bright aggregates. Means were compared by Kolmogorov–Smirnov test.

Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Our initial experiments investigating SOD aggregation involved AdV-mediated delivery of wild-type and mutant SOD cDNA into PC12 cells. Immunostaining with human SOD-specific antiserum showed that adenovirus infection led to expression in approximately 50% of PC12 cells. Immunostaining with a polyclonal antibody that detects both endogenous rat SOD as well as the human isoform demonstrated SOD-containing aggregates in mock-infected cells as well as cells infected with AdSODV148G and AdSODWT (Fig. 1). However, cells infected with AdSODV148G tended to have significantly more aggregation that those infected with either mock or AdSODWT (Fig. 1d; mock vs. WT, p = 0.4; V148G vs. mock, p = 0.01). These data indicated that the presence of SOD aggregates is not specifically related to expression of mutant SOD, but can be seen even with endogenous SOD. The inclusions had a mean diameter of 420 ± 10 nm (n = 230). Aggregation was not dependent on use of a specific primary antibody, as aggregates were seen with the polyclonal antibody as well as the human-specific monoclonal antibody, or a particular conjugated secondary antibody, as aggregation was found with antibody conjugated to either Cy3 or Texas Red.

image

Figure 1. Detection of SOD aggregates in cells infected with mock (a), AdSODWT (b) or AdSODV148G (c). Cells were fixed, labeled with polyclonal anti-SOD antibody followed with Cy3-conjugated secondary antibody; and stained with Hoechst 33342. The staining pattern was examined under a confocal microscope. Images are three-dimensional projections of all the optical sections. There was no clear relationship between the aggregated material (arrow) and the presence of dying cells with pyknotic nuclei and shrunken cell bodies (asterisk). (d) The proportion of cells with aggregates increased following infection with AdVSOD148G.

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The viability of cells showing SOD aggregation after infection with AdV expressing wild-type or mutant SOD was determined using Hoechst 33342 to detect chromatin condensation. Cells undergoing apoptosis (Fig. 1a, asterisk) were not the same cells as those with SOD aggregates, demonstrating that there was no clear relationship between the presence of aggregates and apoptosis. In addition, there were cells with abundant aggregates that had normal nuclei (Fig. 1). We also found that cells with abundant expression of SOD did not necessarily contain aggregates (Fig. 2b, asterisks), perhaps because oxidative stress was also important in aggregate formation (see later).

image

Figure 2. Relationship of mutant SOD expression and ceroid. Confocal images of PC12 cells infected with AdSODV148G andstained for SOD with polyclonal antibody show clumps of autofluorescent ceroid material (asterisks). Autofluorescence (a) was obtained using 488 nm excitation and 510–540 nm bandpass emission. The sample was then rescanned using 547 nm excitation and 605/45 nm emission (b). Cells with the most intense SOD staining (arrows) did not necessarily contain the most ceroid.

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During these initial investigations of SOD aggregate formation, we found that aggregated fluorescent material was present in the cytoplasm of both cultured differentiated PC12 cells (Fig. 2, arrows) and primary MNs (data not shown) prior to any treatment. These fluorescent aggregates, presumably corresponding to ceroid/lipofuscin, increased with the time that the cells spent in culture, as well as oxidative stress, such as that resulting from exposure to 10 µm H2O2 and NGF withdrawal (data not shown). Because of difficulties differentiating the fluorescent antibody staining for SOD from this autofluorescent ceroid material, we chose for most of the experiments to transfect cells with GFP fused to wild-type or mutant SOD (see below) rather than use AdV-mediated gene delivery followed by an indirect fluorescence assay.

We also examined the effect of SODMT expression on cultured rat primary MNs, and observed a similar increased SOD aggregation of SODMT in differentiated PC12 cells and primary MNs (data not shown). For this reason, and because of the greater ease in performing studies with differentiated PC12 cells, we used PC12 cells for future characterization of mutant SOD-induced aggregation.

Aggregation of SOD-YFP

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

For reasons noted above, we used YFP fused to wild-type or mutant SOD to investigate and characterize aggregation of wild-type and mutant SOD. This also allowed us to observe SOD localization in living cells without the influence of fixatives. We found that both SODWT-YFP (Fig. 3b) and SODMT-YFP (Figs 3c and d) aggregated in differentiated PC12 cells (and primary MNs; data not shown), however, the aggregates were more frequent and larger in size in cells expressing mutant than wild-type SOD or mock (Fig. 3e). The aggregates frequently had a juxtanuclear distribution and a complex structure within the cytoplasm of the cell (Fig. 4b).

image

Figure 3. SOD aggregation in PC12 cells. PC12 cells were transfected with an enhanced form of GFP (EGFP, a), SODWT-YFP (b), SODV148G-YFP (c) or SODA4V-YFP (d). At times aggregates showed a non-uniform distribution of staining (asterisk), whereas PC12 cells expressing EGFP or SODWT tended to have rather homogeneous staining of the cell. Cells expressing SODV148G-YFP tended to have large and non-compact inclusions (arrow). SODA4V-YFP-expressing cells tended to have one or two very large bright inclusions (e). Aggregates were significantly more common in cells expressing mutant SOD, especially SODA4V-YFP (p = 0.04). Oxidative stress following application of 40 µm H2O2 increased the proportion of cells with aggregates.

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image

Figure 4. Characteristics of mutant SOD aggregates. (a) Confocal microscopy of PC12 cells expressing SODV148G-YFP (green channel) and stained with polyclonal anti-SOD followed by Cy5- conjugated anti-sheep antibody (red channel – to eliminate cross-talk of green and red signals) showing single optical planes. The overlay shows that the largest YFP-SOD inclusions appear as dark voids in the Cy5 image; the bordering rim of the largest inclusion is Cy5-positive, and therefore appears as yellow in the overlaid image. Orthogonal views (xz and yz reconstructions) along the cross-hair lines show the localization of the aggregates in the cell. (b) Single plane view of two transfected PC12 cells, one of which has several dense aggregates. The cell is magnified with a 3D projection and orthogonal views, demonstrating the complex structure of the inclusion.

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In order to determine whether the expression of SOD-YFP co-localized with endogenous SOD, PC12 cells that had been transfected with SODWT-YFP or SODMT-YFP were immunohistochemically stained with a polyclonal antibody against SOD which recognized both human as well as rodent SOD. These studies showed that the transfected SODV148G and endogenous SOD co-localized (Fig. 4). The co-localization was primarily seen on the surface of the large aggregates with no immunostaining with the polyclonal SOD antibody within the interior of the SOD-YFP aggregate, presumably because the antibody was unable to efficiently penetrate the aggregate.

Oxidative stress increases SOD aggregation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Our previous studies suggested that mutant SOD expression increased the sensitivity of cells to oxidative stress (Ghadge et al. 1997): microfluorimetry studies demonstrated a slightly increased rate of superoxide accumulation in PC12 cells expressing mutant SOD, especially in cells undergoing oxidative stress; drugs that counteracted the effect of oxidative stress, such as glutathione and SOD mimics, decreased mutant SOD-induced cell death. To determine whether an increase in free radicals plays a role in SOD aggregation, we treated PC12 cells expressing SODWT or SODMT with H2O2. Figure 3(e) shows that H2O2 treatment significantly enhanced SOD aggregation induced by wild-type and mutant SOD. This result indicates that free radical accumulation and oxidative stress contribute to SOD aggregation in neural cells.

The effect of various agents on SOD aggregation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Because aggregates can be degraded by the cellular proteasome or lysosome, we questioned whether inhibition of the proteasome by means of lactacystin or inhibition of the lysosome by means of cathepsin B inhibitors might enhance SOD aggregate formation. We found that following transfection of SOD-YFP, lactacystin treatment (1 µm) increased aggregate formation, both the size of aggregates and the number of cells that contain aggregates; the increase in aggregation was most prominent in the case of SODMT-YFP (Figs 5 and 6). The aggregated SODMT-YFP in PC12 cells tended to have a complex structure and became large in size(Figs 5 and 6), although usually maintaining a juxtanuclear localization. Interestingly, the inclusions seen with SODA4V-YFP generally had a slightly different morphology than with SODV148G-YFP. SODA4V-YFP aggregates tended to be more concentrated, compact and bead-like (Fig. 5c), with little homogeneous staining seen in the cytosol. Also of interest was the occasional presence of SOD-YFP staining in the nucleus (Fig. 5a, see ‘n’ label), both in the case of wild-type and mutant. In contrast to lactacystin, the cathepsin B inhibitor had little effect on the size or frequency of aggregate formation (Fig. 6).

image

Figure 5. Confocal Z projection reconstruction images of YFP-SOD in PC12 cells following treatment with lactacystin (1 µm) showing varying morphologies of accumulated SOD. Cells expressing SODWT-YFP (a) generally had a rather homogeneous YFP fluorescence, although rare cells showed a relatively poorly defined juxtanuclear accumulation of YFP fluorescence (arrows). In contrast, cells expressing SODV148G-YFP (b) and SODA4V-YFP (c) tended to have much more frequent well-formed juxtanuclear inclusions (arrows). The inclusions in cells expressing SODA4V-YFP tended to be compact and bead-like and varied in number from many small ones to a single large inclusion; there tended to be little detectable fluorescence in the rest of the cytosol in cells with these compact inclusions. At times, cells expressing SODWT-YFP or SODMT-YFP had evidence of YFP-SOD staining in the nucleus (n).

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image

Figure 6. Graph showing the proportion of cells with aggregates after treatment with DMSO (control), lactacystin (lacta; 1 µm), and cathepsin B inhibitor (catBi; 50 µm). Lactacystin significantly increased the proportion of aggregates in cells expressing the mutant SODs (WT, p = 0.10; V148G, p = 0.00005; A4V, p = 0.001). The effect of cathepsin B inhibitor was not significant (p = 0.8, p = 0.3, and p = 0.17, respectively).

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Calpains are ubiquitously expressed cysteine proteases that are thought to affect a variety of cellular functions and to regulate intracellular signaling, proliferation, and differentiation (Patel and Lane 1999). They have been implicated in the pathophysiology of various disease states including Alzheimer's disease (Chen and Fernandez 1999). We found that the cell-permeable calpain inhibitor E-64-d had little effect on the size or frequency of SOD aggregation (data not shown).

An increase in SOD aggregation has no significant effect on cell death

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

Our previous studies (Ghadge et al. 1997) showed that AdV-mediated mutant SOD delivery causes significant neural cell death compared to wild-type SOD and mock; infection with AdSODA4V and AdSODV148G led to death in 5 days in approximately 50% of the PC12 cells. In contrast, there was no significant decline in cell viability 5 days after infection with AdSODWT when compared with mock-infected cells. In order to determine the effect of aggregation on cell death we tested whether increasing SOD aggregation with lactacystin affected cell viability. We found that lactacystin had little effect on cell viability (Fig. 7). In fact, there was a non-significant trend for increased survival following lactacystin treatment of cells expressing mutant SOD.

image

Figure 7. Graph of cell viability of mock PC12 cells and following infection with adenoviruses expressing SODWT, SODV148G, and SODA4V without (unshaded) and following lactacystin (shaded) treatment. There was no increase in cell death following lactacystin.

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These data show that an increase in the number of cells with aggregates does not increase cell death. We then questioned whether an increase in the amount of aggregated material within individual cells had an effect on cell death. In order to assess individual aggregates we used automated morphometry to measure the area and the average and integrated intensities of each aggregated particle. We specifically tested whether lactacystin increased the aggregate content per cell following expression of SODA4V-YFP (presumably because lactacystin would block its degradation via the proteasome). The mean ‘aggregate burden’ (corresponding to the number of aggregates per cell) of a sample of 10 cells transfected with SODA4V-YFP was 5.3, while the mean of a sample of 17 cells transfected with SODA4V-YFP followed by treatment with lactacystin was 10.5. Thus, lactacystin treatment doubled the aggregate burden. The mean integrated intensity (which roughly corresponds to theYFP-A4V content) was not significantly greater (1.447 × 107 ± 3.35 × 106 vs. 1.415 × 107 ± 3.35 × 106 intensity units; p = 0.67, n.s), indicating that the cells had similar expression levels. The mean intensity within individual aggregates increased from 1939 ± 118 to 3006 ± 45 intensity units (p < 0.001), suggesting that much of the YFP-A4V was being sequestered into the aggregates. Because we found no significant change in cell viability following lactacystin treatment, these data showed that celldeath was not affected by a doubling of the aggregate burden per cell and by a significant increase in the amount ofmutant SOD sequestered within individual aggregates.

An increase in cell viability has no significant effect on SOD aggregation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

In order to determine whether drugs that increase cell viability have an effect on SOD aggregation, we tested the effect of CyPA. Transfection of CyPAWT cDNA decreased AdSODV148G-induced cell death by 50% compared to that seen with transfection of a control cDNA, CD8 (Lee et al. 1999). PC12 cells were exposed to this same amount of AdSODV148G, and were then transfected with either CyPAWT cDNA or CD8 cDNA or mock, fixed after 3 days, and labeled with anti-SOD polyclonal antibody and Hoechst 33342. Aggregates were seen in 52.85 ± 6.7% of cells (in 12 fields of view) that had been transduced with AdSODV148G and had not been transfected with cDNA. Transfection of CyPAWT cDNA decreased the percentage of cells with aggregates to 44.19 ± 2.7%, which was not significant (p = 0.24; t-test). Transfection of CD8 cDNA had no effect on the level of cells with aggregates (52.86 ± 2.2%). This result demonstrated that cell viability increased significantly with CyPA expression with only a small, non-significant effect on SOD aggregation.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References

The identification of SOD mutation as a cause of 20% of cases of FALS was a breakthrough in our understanding of this disease (reviewed by Cleveland and Rothstein 2001) and a potential clue to a better understanding of neurodegenerative disease in general. However, despite the passage of 10 years since this discovery, we remain uncertain about the pathogenesis of FALS and the mechanism by which mutant SOD causes MN degeneration.

It is clear that FALS is not caused by a deficiency of SOD activity as FALS SOD transgenic mice that develop disease have an increased amount of dismutase activity and animals completely deficient in SOD activity fail to develop MN disease (reviewed by Cleveland and Rothstein 2001). These findings suggest that mutant SOD causes FALS because of toxicity from a new function or an enhancement of a known non-dismutase function of the mutated protein. Several hypotheses have been proposed concerning possible mechanisms of toxicity (Cleveland and Rothstein 2001): peroxynitrite, the product of superoxide with nitric oxide, may react in an enhanced way with mutant SOD, leading to nitration of proteins at tyrosine residues and the subsequent death of MNs; mutant SOD may have an enhanced peroxidase activity which leads to the formation of damaging hydroxyl radicals; there may be decreased binding (or ‘shielding’) of metals by the mutant SOD; a loss of the EAAT2 transporter on astrocytes, possibly because of oxidative damage from mutant SOD, may lead to excitotoxic degeneration of MNs; mutant SOD leads to misfolding and aggregation of proteins which interferes with the function/viability of the MN (e.g. by sequestering factors critical to cell survival). It is this last proposed mechanism that we investigated in the present study. However, it may be that more than one mechanism is involved in the development of ALS.

Our previously published studies supported the importance of mutant SOD misfolding in the pathogenesis of FALS (Ghadge et al. 1997; Lee et al. 1999). Our studies demonstrated that mutant SOD caused apoptosis in neural cells and that cyclosporine A enhanced this cell death. These results suggested that the inhibition of the peptidyl-prolyl cis-trans isomerase activity of cyclophilin A by cyclosporine A was critical for this drug's enhancement of cell death. We presumed that: misfolding of the mutant SOD might make the mutant SOD protein more sensitive than wild-type to further changes in its conformation; misfolding of mutant SOD may perturb protein–protein or protein–nucleic acid interactions leading tocellular dysfunction and cell death. A similar interference with protein–protein interactions has been proposed in triplet repeat diseases such as Huntington's disease (HD) and bulbospinal muscular atrophy (Patel and Lane 1999).

SOD aggregates have been seen in neurons of the CNS of FALS patients and in both neurons and glia of FALS transgenic mice. In addition, aggregates have been demonstrated in vitro in two other reports. In the first, aggregates appeared following intracellular inoculation of mutant SOD cDNA into neurons. The inoculated primary neurons underwent apoptosis, but were rescued by expression of HSP-70 (Bruening et al. 1999). A potential criticism of this study related to the small number of cells that were used. The second study described aggregate formation following transient transfection of human embryonic kidney cells with mutant SOD cDNA (Johnston et al. 2000). A limitation of this study related to the use of human embryonic kidney cells rather than neural cells; in addition, the relationship between the mutant protein's aggregation and cell death was not clarified.

In order to carry out our studies, we made use of AdV-mediated gene delivery (which leads to robust expression in these cells) and transfection of SOD-YFP cDNA. The use of YFP avoided difficulties related to distinguishing aggregates of mutant SOD from autofluorescent ceroid material in neural cells. Our studies demonstrated that cytoplasmic aggregates were significantly more frequent following expression of mutant SOD than wild-type SOD. These aggregates were frequently juxtanuclear in location with a complex structure. Interestingly, aggregates of the two different mutant SODs tended to have distinct and different morphologies; this heterogeneity may be the in vitro reflection of the varying inclusion bodies and aggregates that have been seen in different transgenic mice (Watanabe et al. 2001). We found that the mutant SOD co-localized with endogenous SOD. Although we identified endogenous SOD on the surface of aggregates, we were unable to demonstrate its presence within the aggregate probably because of a failure in penetration of the relevant antibody. This failure in penetration in antibody of SOD aggregates may underlie the variation that has been seen in the case of co-localization of other proteins in aggregates on the basis of immunohistochemical staining (Watanabe et al. 2001). We found that the SOD aggregates increased in frequency with lactacystin, a proteasome inhibitor. The characteristics of these depositions were typical of those seen with aggresomes, as first proposed by Johnston et al. (2000). A similar increase in aggregate formation following treatment with proteasome inhibitors has been described in a number of the triplet repeat diseases (Chai et al. 1999; Jana et al. 2001; Wyttenbach et al. 2001). The aggregates in these diseases have generally been found in both the nucleus and the cytoplasm. Interestingly, we found that both wild-type and mutant SOD were present at times in the nucleus as well as in the cytoplasm following overexpression of the respective proteins,however, we did not identify aggregates in the nucleus.

We found that increasing aggregates of mutant SOD had little effect on cell death caused by this mutant protein. In addition, treatment of mutant SOD-expressing cells with CyPA increased cell viability, but had little effect on aggregate formation. The relationship between aggregation and cell death has been investigated in the case of other neurodegenerative diseases, especially the triplet repeat diseases; however, an understanding of this relationship remains incomplete. In some cases, aggregates may act as an extra ‘burden’ for the proteasome, and lead to altered proteasomal function with increased pathology (Jana et al. 2001). For example, the function of the ubiquitin–proteasome system may become impaired following aggregate formation from in vitro expression of the mutant cystic fibrosis membrane conductance regulator or mutant huntingtin gene with an expanded repeat (Bence et al. 2001). On the other hand, it has been questioned whether aggregation in some cases may actually represent a protective mechanism rather than a pathogenic one. For example, cellular aggregates in autopsy HD tissue tend to be present in the spared interneurons, with few or no aggregates within more vulnerable neuron types (Kuemmerle et al. 1999). In addition, mice that carry mutant ataxin as a transgene (which causes spinocerebellar degeneration type 1) and also lack E6-associated protein (AP) ubiquitin ligase have a decrease in nuclear inclusions but an enhanced pathology, again suggesting that intranuclear inclusions might be part of a strategy to rid the cell of toxic material (Cummings et al. 1999). Lastly, some reports have noted that there is no correlation between aggregates and cell death. For example, transfection of varied mutant huntingtin constructs into cultured striatal neurons may have a similar proapoptotic effect but a varying propensity to generate intranuclear inclusion bodies (Saudou et al. 1998). Neural cells are known to survive for prolonged periods with some aggregates such as lipofuscin.

Some studies have shown that the expression of varied chaperone proteins can decrease aggregates as well as cell death. For example, expression of fragments of the bacterial chaperone GroEL and the full-length yeast heat shock protein HSP104 reduced both aggregate formation and cell death in mammalian cell models of HD (Carmichael et al. 2000). In addition, a recent study found that expression of HSP70 along with mutant SOD led to a decrease in aggregates and cell death (Bruening et al. 1999). However, the protective effect of HSP70 was not found in mice containing both mutant SOD and overexpressed HSP70 (Ward et al. 2001). Treatment with chaperone proteins may decrease cell death, not because of a specific decrease in aggregation, but because of a decrease in misfolding or an inhibition of caspase activity (Zhou et al. 2001).

How can we align these varied reports? In the case of FALS (as well as the triplet repeat diseases), the critical pathogenic feature may be misfolding of the mutant protein, perhaps because the abnormal conformation disruptsprotein–protein or protein–nucleic acid interactions that are important for cell survival. Aggregation may result from misfolding of the mutant protein and also from oxidative modification of cellular components (Kim et al. 2001) caused by mutant SOD-induced increased levels of free radicals (Ghadge et al. 1997). Our studies, however, suggest that aggregation by itself is not a key pathogenic feature of the disease and does not lead to cell death. Mutant SOD may cause cell death even though few aggregates are present, presumably because the disaggregated mutant protein still remains misfolded (with resultant pathogenic disrupted protein–protein interactions). An increase in oxidative stress may enhance the misfolding of SOD (Kim et al. 2001), leading to further disruption of the protein–protein interactions.

Although a number of the reports noted above have shown that aggregation impairs the ubiquitin–proteasome system (because of an increase of the proteasomal burden) and leads to cell death (Bence et al. 2001), we found no evidence for enhanced mutant SOD-induced cell death after treatment with a proteasome inhibitor despite the increase in aggregates. It is relevant to note that PC12 neural cells are reported to be resistant to lactacystin-induced apoptosis (Lee et al. 2001). It may be that a pathogenic overload of proteasomes and increased aggregate formation requires a more robust or more prolonged duration of expression of the mutant protein or may be more likely to occur in a particular cell type (Shinder et al. 2001). It also may be that not all aggregates have similar pathogenicity, as suggested by studies of the properties of different lengths of amyloid beta peptide and polyglutamine repeats (Chen et al. 2001). Our data show that increased aggregate burden or proteasome inhibition with respect to mutant SOD does not necessarily lead to neuronal death.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. cDNAs constructs and adenoviruses
  6. Transfection
  7. Drug treatment
  8. Viability assay
  9. Analysis of SOD aggregates
  10. Results
  11. Formation of SOD aggregates in PC12 cells expressing wild-type or mutant SOD
  12. Aggregation of SOD-YFP
  13. Oxidative stress increases SOD aggregation
  14. The effect of various agents on SOD aggregation
  15. An increase in SOD aggregation has no significant effect on cell death
  16. An increase in cell viability has no significant effect on SOD aggregation
  17. Discussion
  18. Acknowledgement
  19. References
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