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Keywords:

  • apoptosis;
  • calcium;
  • neuroprotection;
  • protease;
  • spectrin

Abstract

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

While caspases have been strongly implicated in delayed neuronal death in a variety of experimental paradigms, other proteases such as calpain can also contribute to neuronal death. To evaluate the relative roles of caspase and calpain, we used a model system wherein UV treatment induced moderate or severe delayed cortical neuronal death, as quantified by propidium iodide and calcein AM. UV treatment led to increases in both caspase and calpain activation. Calpain inhibitor III (MDL-28170) reduced caspase activation, suggesting that caspase activation was mediated by calpain. Calpain contributed to neuronal death, as indicated by strong neuroprotection provided by calpain inhibitor III, calpeptin, or Ca2+-free medium. In contrast, caspase inhibitors were not neuroprotective. These results suggest that UV neurotoxicity is mediated by a loss of Ca2+ homeostasis which leads to a calpain-dependent, caspase-independent cell death. That calpain, but not caspase, may mediate death in instances involving the activation of both proteases may have relevance to other neuronal death models.

Abbreviations used
AP5

2-amino-5-phosphonopentanoic acid

BAF

boc-aspartate-[O-methyl]-fluoromethylketone

Cdk

cyclin-dependent kinase

CHX

cycloheximide

MEM

minimum essential medium

NFH

neurofilament heavy chain

NGF

nerve growth factor

PI

propidium iodide

PMSF

phenylmethylsulfonyl fluoride

SBP

spectrin breakdown products

S-MEM

suspension culture MEM

TUNEL

terminal deoxy-transferase-mediated dUTP-FITC nick-end labeling

UV

ultraviolet light

zVAD-FITC-FMK

carbobenzoxy-valine-alanine-aspartate-fluoroisothiocynate-fluoromethylketone.

While caspase and calpain have been implicated separately in many neuronal death models, our understanding of the interactions between these two proteolytic pathways in cell death is incomplete. Caspases mediate neuronal apoptosis in development (Deshmukh et al. 1996; Troy et al. 1996; Park et al. 1998b) and contribute to apoptosis in certain injury models such as ischemia (Chen et al. 1998; Endres et al. 1998; reviewed in Stennicke and Salvesen 2000). For example, a classic model of developmentally appropriate apoptosis is sympathetic neurons undergoing nerve growth factor (NGF) deprivation-induced death. In this model, pan-caspase inhibitors block death for several days and adding NGF back to these cultures allows the cells to regrow processes, establishing their functional viability (Deshmukh et al. 1996). However, in other neuronal apoptosis models, the same caspase inhibitors block death only marginally. For example, amyloid beta-treated hippocampal neurons are protected from apoptosis only partially (Jordan et al. 1997; Saez-Valero et al. 2000; Selznick et al. 2000). Hence, while caspases are generally attributed to have a requisite role in apoptosis, their actual role depends upon the apoptosis-initiating stimulus.

In contrast, calpain is a Ca2+-dependent protease typically associated with necrosis and its Ca2+ influx concomitant with death (Lipton 1999; Wang 2000). Calpain is activated during cerebellar ischemia and calpain inhibitors decrease ischemia-associated neuronal death (Roberts-Lewis et al. 1994; Markgraf et al. 1998). Moreover, recent work has suggested that calpain is also activated during apoptosis induced by low K+ in cerebellar granule neurons, or by staurosporine or high extracellular Ca2+ concentrations in neural cell lines (McGinnis et al. 1999b; Nath et al. 1996; reviewed in Ray et al. 2000; Wang 2000). Interestingly, calpain has also been implicated in non-neuronal apoptosis models, i.e. dexamethasone-induced thymocyte apoptosis (Squier et al. 1994). Hence, calpain is generally considered to mediate necrotic cell death, but also appears to contribute to apoptosis in some models.

Both calpain and caspases are activated in some cell death models. For example, simultaneous calpain and caspase activation has been reported in staurosporine-treated cerebellar granule neurons, Fas-antibody treated Jurkat cells, and amyloid or high dose glutamate-treated neurons in vitro as well as in traumatic brain injury in vivo (Nath et al. 1996; Jordan et al. 1997; Pike et al. 1998; Buki et al. 2000; Zhao et al. 2000). In each of these models, inhibitor studies suggest that caspases mediate at least a portion of the cell death, whereas the calpain contribution to death appears model-dependent. For example, caspase inhibitors attenuate death in staurosporine-treated neurons and in FAS-mediated cell death, but calpain inhibitors block death only in the former model (Nath et al. 1996; Vanags et al. 1996; Pike et al. 1998). The interactions between caspases and calpains appear complex. In at least one model, calpain activation precedes caspase activation (Waterhouse et al. 1998). Interestingly, calpain appears capable of cleaving pro-caspase-3, yielding a fragment relatively resistant to subsequent activation, and hence may inhibit the caspase pathway (McGinnis et al. 1999a). Conversely, caspases degrade the primary endogenous calpain inhibitor, calpastatin, and thereby may facilitate calpain activation (Wang et al. 1998). These studies are suggestive that the two systems modulate one another, but further studies are necessary to clarify the relationship between these two proteolytic systems and cell death.

To gain insight into the relative roles of calpain and caspase in neuronal death, we have used a UV-induced apoptosis model. Although UV neurotoxicity is not directly relevant to neuronal death in development or disease, elucidating the intracellular mechanisms whereby neurons are capable of committing apoptosis may suggest potential testable hypotheses into events that underlay cell death in vivo. Indeed, many research groups have used UV irradiation as an experimental tool to induce apoptosis (Griffiths et al. 1998; Godar 1999; Radziszewska et al. 1999, 2000; reviewed in Kulms and Schwarz 2000). Here, we show that UV-treatment activates both caspase and calpain in cortical neurons. Moreover, calpain appears to be upstream of caspase activation because calpain inhibition blocks caspase activation. Lastly, calpain but not caspase activity appears necessary for apoptosis in this model. We interpret these data as suggesting that calpain should be considered as an alternative to caspase in mediating neuronal apoptosis in some cell death models.

Primary rat cortical neuron preparations

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Primary rat cortical neurons were established from embryonic-day-18 Sprague–Dawley rat fetuses as described previously (Estus et al. 1997). Cell suspensions were plated at 3.0 × 105 cells/cm2 in polyethylenimine-coated 35-mm dishes in 2 mL of minimum essential medium (MEM)/B27 (Life Technologies, Rockville, MD, USA) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and B27 (Life Technologies). Cells were maintained in MEM/B27 medium for 1 week with a complete medium change every 2–3 days.

Cell treatments

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

After one week in vitro, cortical neurons were exposed in duplicate to varying amounts of UVC (UVC 500 Crosslinker, Ultraviolet Products, San Francisco, CA, USA) and/or the indicated additional treatments. Dishes were then returned to a water-jacketed incubator for the duration of the experiment (times indicated in results). A mild insult (30 mJ/cm2) and a severe insult (60 mJ/cm2) of UV were determined and used for the majority of the experiments conducted.

Neurons were also treated with additional agents as detailed below. Neurons were treated with cycloheximide (CHX) 1 µg/mL (Sigma, St Louis, MO, USA) to inhibit protein synthesis (Martin et al. 1988) beginning 1 h prior to UV exposure. Active caspases were detected in situ by adding fluoroisothiocynate-carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone [‘CaspACE’ (FITC-VAD-FMK) 5 µm, Promega, Madison, WI, USA] to culture medium 30 min prior to the end of the experiment, as recommended by the manufacturer to detect caspase activity. To inhibit caspase activity, the pan-caspase inhibitor, boc-aspartate-[O-methyl]-fluoromethylketone [BAF, 50 µm (Deshmukh et al. 1996), Enzyme System Products, Livermore, CA, USA] was placed on cells beginning 1 h prior to UV exposure. We used two different exposure paradigms for the calpain inhibitors because they differ in their ability to enter cells. Calpain inhibitor III (MDL 28170, carbonzoxy-valinyl-phenylalaninal, 10 µm (Chen et al. 1997; Calbiochem, La Jolla, CA, USA) was added to cell medium immediately after UV exposure. Because calpeptin (benzyloxycarbonyldipeptidyl aldehyde) is less lipid-soluble, cells were treated with this calpain inhibitor (10 µm, Calbiochem) beginning 24 h before UV. To evaluate the role of Ca2+ in cortical neuron death, medium was changed to Ca2+-free S-MEM (Life Technologies) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and B27 (Life Technologies) beginning 4 h before UV exposure. To evaluate therole of NMDA-type glutamate receptors, cells were treated with 2-amino-5-phosophonopentanoic acid (AP-5, 100 µm, Sigma) beginning 1 h prior to UV exposure.

TUNEL and Hoechst staining

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Chromatin fragmentation was assessed by using a fluorescent TUNEL technique (terminal deoxy-transferase-mediated dUTP-FITC nick-end labeling; In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA). Briefly, neurons were fixed with 4% paraformaldehyde, rinsed with phosphate-buffered saline (PBS), and incubated with terminal deoxy-terminal transferase and FITC-dUTP. Cells were then counterstained with Hoechst 33258 (Molecular Probes, Eugene, OR, USA) and incorporated fluorescein and Hoechst 33258 staining visualized by fluorescence microscopy.

Propidium iodide and calcein AM staining

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

After the indicated treatments, neuronal cultures were maintained until 1 h before neuronal survival was to be assessed. At that time, neurons were stained with propidium iodide (PI, 5 µg/mL, Sigma) and calcein AM (1 µg/mL, Molecular Probes). Fluorescent neuronal images at a final magnification of 400–600× (Nikon Diaphot 300) were then obtained by using an Optronics DEI-470 digital camera and FlashBus FBG version 4.2 (Integral Technologies, Inc., Indianapolis, IN, USA) digital imaging program. These images were then used to conduct counts of cell survival by an observer ‘blinded’ as to the cellular treatments. Cells staining positive for PI or calcein AM were deemed dead or alive, respectively. At least 200 cells were scored per plate. Frequency of cell death was determined by using the ratio of PI-positive cells divided by the total number of cells (PI-positive and calcein AM-positive) counted. Data were analyzed for statistical significance with GB-Stat PPC 6.5.2 (Silver Springs, MD, USA) using a two-way repeated measures anova followed by a Fisher's LSD (protected t)-test.

Immunofluorescence

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

After the described treatments, cells were stained with calcein AM (1 µg/mL, Molecular Probes) for 30 min and then rinsed with PBS. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS, and blocked with 5% goat serum in PBST (PBS supplemented with 0.2% Triton X-100) for 60 min. The cells were then labeled with polyclonal antibodies against active caspase-3 (Research and Development Systems, Minneapolis, MN, USA; dilution of 1 : 2000). After PBST washes, primary antibodies were detected with a Alexa Fluor 568-conjugated anti-rabbit secondary antibody (1 : 400, Molecular Probes) and counterstained with Hoechst 33258. Fluorescent neuronal images at a final magnification of 400× (Nikon Diaphot 300) were then obtained by using an Optronics DEI-470 digital camera and FlashBus FBG version 4.2 (Integral Technologies, Inc.) digital imaging program.

Western blots

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Cortical neurons were treated as indicated, washed twice with cold PBS containing 100 µg/mL phenylmethylsulfonyl fluoride (PMSF) and placed on ice. Proteins were extracted from cortical cells using a rubber policemen and 100 µL of a cell lysis buffer containing 50 mm Tris (pH 8.3), 150 mm NaCl, 0.02% NaN3, 100 µg/mL PMSF, 1 µg/mL aprotinin, 1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS). The extract was then placed in a 500-µL Eppendorf tube and sonicated for 20 s. An aliquot of 20 µL was removed to assess protein content by using CuSO4 solution and bicinchoninic acid solution (Sigma). β-mercaptoethanol was then added to the remaining extract. Twenty micrograms of protein extract were added to SDS sample buffer, boiled, and proteins separated by electrophoresis on a 6.5% polyacrylamide gel for subsequent spectrin detection, or 12% polyacrylamide gel for caspase detection. Proteins were transferred to nitrocellulose membranes and probed overnight with either a mouse antispectrin antibody (1 : 5000, mAb 1622, Chemicon, Temecula, CA, USA) or a rabbit antiactive caspase3 antibody (1 : 500, Research and Development Systems). Blots were then washed three times for 10 min each and exposed to horseradish peroxidase (HRP)-conjugated goat anti-mouse (1 : 10 000) or anti-rabbit (1 : 5000) antibody (Jackson Immuno Research, West Grove, PA, USA) for 1 h. Blots were visualized using an ECL kit (Amersham\Pharmacia\Biotech, Piscataway, NJ, USA) and a FujiFilm FLA-2000 imager (Fuji, Stamford, CT, USA). The spectrin and caspase blots were then stripped and probed with mouse antineurofilament-H (NFH-200) 1 : 400, Sigma or rabbit antiactin (1 : 200, Sigma), respectively, to ensure equal protein loading among lanes. Results similar to those described here were also obtained with a separate antiactive caspase-3 antibody preparation (Pharmingen, San Diego, CA, USA). Band intensity was detected and quantified by comparing relative signal intensity in treated and control samples. Data obtained were analyzed with GB-Stat PPC 6.5.2 by using a two-way repeated measures anova followed by a Fisher's LSD (protected t)-test.

UV induces protein synthesis-independent apoptosis in cortical neurons

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

To begin to evaluate the hypothesis that caspase and/or calpain contribute to UV-induced cortical neuron apoptosis, we determined the UV dose that stimulated a delayed cell death response. Cortical neurons plated on 35-mm glass bottom dishes were exposed to a range of UV doses (0, 10, 30, 60, and 120 mJ/cm2). Twenty-four hours later, cortical neurons were stained with calcein AM, which labels live cells, and PI, which binds DNA in cells that have lost membrane integrity. Cell death was quantified by ‘blinded’ scoring of calcein AM- versus PI-stained cells. Weobserved a UV-induced, dose-dependent increase in the frequency ofneurons showing a loss in membrane integrity (Figs 1a and b).

image

Figure 1. UV causes dose-dependent neuronal death that is independent of new protein synthesis. Neurons were exposed to UV at the indicated doses and stained with calcein AM and propidium iodide 24 h later (a). Theneuronal death was quantified by a ‘blinded observer’ (b). These data represent the mean ± SEM of four separate neuronal preparations. This neuronal death did not appear to be dependent upon new protein synthesis because death was not altered by including CHX (1 µg/mL) beginning 1 h before UV treatment (a, b). The time course of cell death is quantified in (c) (mean ± SEM, n = 3). Solvent control (Solv. Con.).

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UV treatment is known to induce many genes (Adler et al. 1996; Paunesku et al. 2000). To evaluate whether the UV-induced cell death described here was modulated by new protein synthesis, cells were treated with UV in the absence or the presence of CHX beginning 1 h before UV exposure. Twenty-four hours after exposure, neurons were stained with calcein AM and PI. CHX showed no effect on the frequency of cell death (Figs 1a and b). These results suggest that newly synthesized proteins do not in toto promote or ameliorate UV-induced neuronal death.

At 30 and 60 mJ/cm2, significant cell death was not observed until 6 h after UV treatment and approached a maximum at 24 h after treatment (Fig. 1c). Hence, the UV-induced neuronal death was time-dependent, with the majority of death occurring many hours after the initial insult, consistent with the possibility that this death was largely apoptotic.

To evaluate whether this death manifested chromatin fragmentation, a hallmark of apoptosis, neurons were treated with UV, incubated for 6 or 24 h, fixed with paraformaldehyde, and then subjected to TUNEL labeling. Results revealed a dose-dependent increase in the number of TUNEL-positive cells (Fig. 2). The percentage of neurons manifesting chromatin fragmentation increased between 6 and 24 h, indicating that the DNA fragmentation at the later time point was due to a cellular process and not derived from direct UV-induced DNA strand breaks. Overall, the increased frequency of TUNEL-positive cells coincided with the increased frequency of cell death as assessed by the PI and calcein AM assays (Fig. 1). Although chromatin fragmentation can occur inmodels of necrosis (Grasl-Kraupp et al. 1995; van Lookeren Campagne et al. 1995), we interpret fragmentation here as suggesting that UV-induced cell death includes an apoptotic component because this death is delayed and isalso accompanied by chromatin condensation. This interpretation is consistent with other studies showing thatUV causes apoptotic neuronal death (Park et al. 1998a, 1998b).

image

Figure 2. UV causes dose- and time-dependent chromatin fragmentation. Neurons were treated with the indicated UV doses and maintained for 6 or 24 h. The neurons were then analyzed for chromatin fragmentation by the TUNEL method. The presence of neurons was established by the concurrent Hoechst 33258 DNA labeling. Similar results were obtained in a separate neuronal preparation and treatment.

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Ca2+ contributes to UV-induced cell death

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Loss of Ca2+ homeostasis has been implicated in UV-induced cell death (Ihrig et al. 1997). To determine whether UV activates neuronal death via Ca2+, cortical neuron culture medium was switched to Ca2+-free S-MEM/B27 4 h prior to UV treatment, and maintained in this medium for the duration of the experiment. Twenty-four hours after UV-treatment, the frequency of cell death was determined by using calcein AM and PI. Ca2+-free medium provided significant neuroprotection to cells exposed to UV (Fig. 3). This protection averaged 56 ± 4% (mean ± SEM, n = 3) when corrected for background death.

image

Figure 3. Ca2+ contributes to UV-induced cell death. Cortical neurons were treated with the indicated UV doses in normal or Ca2+-free medium, or with AP5 (100 µm). Cells treated in the absence of Ca2+ showed a significant decrease in UV-induced cell death (a, two-way anova with post-hoc Fishers LSD, p < 0.05). In contrast, AP5 provided no protection from the UV insult (b). These data represent the mean ± SEM of three separate experiments.

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UV has been shown to activate or inactivate cellular proteins directly (Chen et al. 1989; Podda et al. 1998) as well as to activate receptors on the cell membrane (Schwarz 1998), e.g. tumor necrosis factor alpha-receptor, epidermal growth factor receptor and platelet-derived growth factor receptor (Sachsenmaier et al. 1994; Knebel et al. 1996; Schwarz 1998). The NMDA type of glutamate-activated Ca2+ channels leads to cell death via Ca2+ influx and calpain activation (Adamec et al. 1998; Faddis et al. 1997; Hewitt et al. 1998; Tremblay et al. 2000). To evaluate whether UV was causing Ca2+ influx via activation of the NMDA-modulated channels, cortical neurons were treated with an NMDA receptor antagonist, AP5, beginning 1 h prior to UV exposure. Twenty-four hours after the UV insult, cell death was quantified using calcein AM and PI. Cortical neurons treated with AP5 showed no protection from UV-induced cell death (Fig. 3b), suggesting that UV-induced Ca2+ influx is not through the NMDA type of glutamate activated Ca2+ channels.

UV effects on calpain and caspase activity

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Because caspases mediate neuronal apoptosis in many models (Chen et al. 1998; Petersen et al. 1999; Giovanni et al. 2000; Mattson 2000; and reviewed in Wang 2000; Martin 2001), we began to evaluate the mechanisms underlying UV-induced cell death by analyzing whether UV-treatment led to caspase activation. Cortical neurons were treated with CaspACE (10 µm), reported to be an irreversible fluorescent inhibitor of active caspases, 23.5 h after UV exposure. Neurons were treated simultaneously withPI to detect dead cells. Thirty minutes later, cells were rinsed, fixed with paraformaldehyde, and stained with Hoechst 33258. Neurons that were not exposed to UVtreatment typically manifested uniformly dispersed chromatin (Fig. 4a), maintained membrane integrity (Fig. 4b), and seldom showed active caspase staining (Fig. 4c). Consistent with the possibility that caspase contributes to background cell death in this non-UV-treated population, the cells that were positive for PI staining and chromatin condensation also showed caspase activation. Most cells treated with UV (60 mJ/cm2) manifested condensed chromatin (Fig. 4d), loss of membrane integrity (Fig. 4e), and caspase activation (Fig. 4f).

image

Figure 4. In situ evidence that UV treatment leads to caspase activation. Neurons were treated with UV (60 mJ/cm2) and analyzed for chromatin condensation by Hoechst 33258 staining (a and d), loss of membrane integrity by PI staining (b and e), and the presence of activated caspase by using a fluorescent, irreversible caspase inhibitor, CaspACE (c and f). Arrows depict cells that stain positive for caspase activity and chromatin condensation, but have not yet lost membrane integrity. Arrowheads identify representative cells manifesting chromatin condensation, loss of membrane integrity, and caspase activation.

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Although these results suggest that UV treatment leads to caspase activation, recent reports have questioned the specificity of the zVAD-based, CaspACE reagent (Wolf et al. 1999; Foghsgaard et al. 2001). Therefore, we performed an additional set of assays to identify more accurately the protease(s) activated by UV treatment, i.e. western blots to detect spectrin breakdown products (SBPs). Spectrin is a substrate for bothcaspase-3 and calpain; both caspase-3 and calpain produce a 150-kDa SBP, while only caspase-3 produces an additional 120-kDa SBP (reviewed in Wang 2000). Cortical neurons were exposed to 30 mJ/cm2 or 60mJ/cm2 of UV, doses that were selected due to their ability to induce a moderate or high frequency of delayed cell death, respectively. Protein lysates were analyzed by western blotting for intact spectrin (220 kDa) as well as each spectrin fragment. UV treatment caused a dose-dependent increase inboth the 150- and 120-kDa spectrin fragments (Figs 5and 6). To confirm the role of caspases in this proteolysis, neurons were treated with UV in the presence or absence of BAF, a pan-caspase inhibitor. BAF partially decreased production of the 150-kDa protein and, as expected, completely blocked generation of the 120-kDa protein (Fig. 5). We interpret these data as confirming the preliminary CaspACE data suggesting that caspases are activated by UV.

image

Figure 5. Western blot evidence that UV treatment leads to caspase activation. Cortical neurons were treated with UV (30 or 60 mJ/cm2), maintained in vitro for 24 h in the presence or absence of BAF (50 µm), and then analyzed by western blotting to detect intact spectrin (220 kDa) and 120- and 150-kDa spectrin fragments (a). Blots were stripped and probed with an antibody for neurofilament-H (200 kDa) to evaluate equal protein loading. The ratio of 150- and 120-kDa spectrin breakdown product to intact spectrin was quantified (b, c); these data represent the mean ± SEM of three separate neuronal preparations. UV caused an increase in the production of both the 120- and 150-kDa spectrin breakdown products, each of which was significantly reduced by BAF treatment (anova with post-hoc Fishers LSD, p < 0.05).

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image

Figure 6. Western blot evidence that UV treatment leads to calpain activation. Cortical neurons treated with UV (30 or 60 mJ/cm2) in the presence or absence of calpain inhibitor III were analyzed using Western blotting to detect the 120- and 150-kDa spectrin fragments as well as intact spectrin (220 kDa; a). Blots were stripped and probed with an antibody for neurofilament-H (200 kDa) to evaluate equal protein loading. The ratio of spectrin breakdown product to intact spectrin was quantified (b, c); these data represent the mean ± SEM of four separate neuronal preparations. UV caused an increase in the production of the 120- and 150-kDa spectrin fragments, each of which was significantly reduced by calpain inhibitor III (anova with post-hoc Fishers LSD, p < 0.05).

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To evaluate the role of calpain in UV-induced spectrin proteolysis, neurons were treated with UV with or without calpain inhibitor III. This inhibitor blocked the production of the 150-kDa SBP (Fig. 6). Surprisingly, calpain inhibitor III also blocked production of the caspase-3-specific, 120-kDa SBP (Fig. 6). We interpret these data as suggesting that calpain directly contributes to generation of the 150-kDa fragment and indirectly causes the generation of the 120-kDa fragment. This indirect action of calpain may reflect a scenario wherein calpain is upstream of a series of events leading to caspase activation. In summation, the 150-kDa spectrin fragment is known to be produced by caspase and calpain, while the 120-kDa spectrin breakdown product is considered caspase-3-specific (Wang 2000). In our model, BAF blocked production of the 150-kDa fragment partially and the 120-kDa fragment completely, while calpain inhibitor III blocked the production of both. Therefore, we interpret these data as suggesting that calpain and caspase are both activated in UV-treated neurons and that calpain activity directly or indirectly leads to caspase activation.

Calpain inhibition decreases caspase activity

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

To further investigate the hypothesis that calpain is necessary to activate caspase in this model, we determined the effect of calpain inhibition on caspase activation. These effects were evaluated with three separate assays. First, we used the fluorescent selective caspase inhibitor CaspACE, to detect caspase activation in cortical neurons treated with UV in the presence or absence of calpain inhibitor III. UV-treatment led to a robust increase in cells labeling positive for CaspACE, and this increase was strongly blocked by calpain inhibitor III (Fig. 7a). Second, as CaspACE may not be specific for caspases (Wolf et al. 1999), we examined caspase-3 activation directly. Caspase-3 activation consists of pro-caspase-3 cleavage to a 12- and 17-kDa fragment (reviewed in Wang 2000). Therefore, we used immunofluorescence with an antibody specific for the p17 active fragment to detect caspase activation in UV –treated cortical neurons. UV treatment increased the numbers of cells positive for caspase activation (Fig. 7b). BAF, a pan-caspase inhibitor, was expected to block caspases upstream of caspase-3, and, indeed, blocked production of the p17 caspase-3 fragment. Consistent with the SBP and CaspACE data, calpain inhibitor III also blocked p17 formation (Fig. 7b). Third, to evaluate quantitatively the effect of calpain inhibition on caspase activation, we performed a series of western blots using the p17-specific antibody. UV treatment caused a dose-dependent increase in p17, which was blocked by BAF as would be expected (Fig. 7c). Consistent with the immunofluorescent data, this increase in p17 production was also blocked by calpain inhibitor III (Fig. 7c). Overall, we interpret these data as confirming the results observed in the spectrin western blots, and further implicating calpain incaspase-3 activation in this model of UV-treated cortical neurons.

image

Figure 7. Calpain inhibitor III decreases caspase-3 activation. Cortical neurons were treated with UV at the indicated doses in the presence or absence of calpain inhibitor III. (a) Cells were labeled with CaspACE from 23.5 to 24 h after UV treatment; Hoechst 33258 staining was used to establish the presence of cells. (b) Cells were treated as indicated, and active caspase detected 24 h later by staining with an antibody specific for active caspase-3. Concurrent calcein AM and Hoechst 33258 staining were used to provide an indication of cellular viability. Cells that were healthy, as defined by calcein AM staining anduniformly distributed chromatin, stained negatively for active caspase-3 (arrows). However, cells susceptible to UV toxicity, as discerned bycondensed chromatin, and negative calcein AM staining, stained positively for active caspase-3 (arrow heads). (c) Protein lysates fromcells treated as indicated were analyzed by western blotting to detect the p17 active caspase fragment. Blots were stripped and probed with an antibody for β-actin (43 kDa) to evaluate equal protein loading. The accompanying graphs depict the relative quantities of p17present in each of the treatments; these data represent the mean ± SEM of three separate neuronal preparations. UV treatment increased caspase-3 activity, as demonstrated by enhanced CaspACE labeling (a), increased cellular p17 detection in situ (b), and increased total p17 present in the cultures (c). This increased active caspase was inhibited by both BAF and calpain inhibitor III. The inhibitory actions of BAF and calpain inhibitor III on p17 generation described in (c) were statistically significant (anova with post-hoc Fishers LSD, p < 0.05).

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Calpain but not caspase contributes to UV-induced neuronal apoptosis

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Both calpains and caspases have been implicated in neuronal apoptosis (reviewed in Wang 2000; Martin 2001). Calpains are cysteine proteases that are present in an inactive form and become active in a process stimulated by Ca2+ (Donkor 2000). Because we observed that Ca2+ was important for UV-induced cell death, we evaluated whether calpain was necessary for cell death. Cortical neurons were treated with calpain inhibitor III, immediately after exposure to UV. Twenty-four hours later, cells were stained with calcein AM and PI and the frequency of cell death determined. Calpain inhibitor III strongly decreased the frequency of neuronal death (Figs 8a and b). When corrected for background cell death, calpain inhibitor III decreased the frequency of UV-induced neuronal death by 54 ± 2% (mean ± SEM, n = 4).

image

Figure 8. Calpain inhibitors decrease the frequency of UV-induced neuronal death. Neurons were exposed to UV at the indicated doses in the presence or absence of a calpain inhibitor. Both calpain inhibitor III (10 µm; a and b) and calpeptin (10 µm; c) significantly protected from UV-induced cell death (anova with post-hoc Fishers LSD, p< 0.01). Data for calpain inhibitor III and calpeptin represent the mean ± SEM for four and three separate experiments, respectively.

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To confirm the role of calpain in this neuronal death, we evaluated the effects of another calpain inhibitor, calpeptin. Because this peptide inhibitor is not highly membrane permeant, cells were treated with calpeptin beginning 24 h before exposure to UV. Similar to the results of calpain inhibitor III, calpeptin decreased neuronal death significantly (Fig. 8c). Overall, these results strongly implicate calpain as contributing to UV-induced apoptosis.

To evaluate whether Ca2+ has actions in addition to its effects on calpain, we compared the neuroprotection derived from Ca2+-free medium in the presence of calpain inhibitor III versus Ca2+-free medium alone; the neuroprotection observed for neurons treated with both calpain inhibitor III and Ca2+-free medium (50 ± 4%, mean ± SEM, n = 3) was unchanged from that observed for neurons treated in parallel with Ca2+-free medium alone (56 ± 4%, mean ± SEM, n=3). Both of these values are similar to that observed for calpain inhibitor III alone (above). We interpret these results as suggesting that the toxic effects of Ca2+ are mediated solely by calpain.

Because we observed that caspases are activated by UV insult, we hypothesized that caspase activity may also contribute to death. To evaluate this hypothesis, cells were treated with BAF (50 µm) and death quantified 24 h after UV treatment. Consistent with our prior results, these doses of UV induced moderate to high levels of neuronal death (Fig. 9). However, BAF did not alter cell death significantly (Fig. 9). To evaluate further whether caspase and calpain may act together to modulate cell death, neurons were exposed to UV and treated with either calpain inhibitor III alone, or with both calpain inhibitor III and BAF. However, the addition of BAF was no more protective than calpain inhibitor III alone (Fig. 9b). We interpret these results as suggesting that UV causes caspase activation but caspases are not required for cell death. Rather, calpain activation upstream of caspase activation is sufficient for regulating death in this model.

image

Figure 9. BAF does not alter UV-induced neuronal death. Neurons were treated with UV at the indicated doses in the presence or absence of BAF (50 µm), calpain inhibitor III (10 µm), or both, and the frequency of cell death determined 24 h later. These data (b) represent the mean ± SEM (n = 4). BAF did not significantly alter UV-induced neuronal death (p > 0.05).

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Discussion

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

The primary findings of the work reported are that: (i) UV activates both caspase and calpain in cortical neurons; (ii) calpain inhibition blocks caspase-3 activation; (iii) two separate calpain inhibitors each protected from UV toxicity, suggesting calpain contributes to this neuronal death; and (iv) caspase inhibition did not inhibit cell death, suggesting that caspases are not necessary for apoptosis in this model. These results suggest that while the caspase pathway is activated by calpain during UV-induced neuronal death, caspases do not contribute to neuronal apoptosis. Rather, death here is attributable to the actions of calpain. Hence, in at least some neuronal apoptosis models involving caspase activation, cell death may actually depend on calpain. These results renew interest in the role of calpain in neuronal death, especially in instances wherein death is not modulated by caspase inhibitors.

The mechanisms underlying UV-induced cell death

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Multiple mechanisms have been implicated in UV-induced cell death that have relevance here. First, death can result from direct DNA damage mediated by the formation of pyrimidine dimers (Schreiber et al. 1995; Struthers et al. 1995; McGregor 1999). This pathway of death involves activation of p53 and the resultant increased transcription of cell cycle and DNA repair genes (Sanchez and Elledge 1995; Hiyama and Reeves 1999; reviewed in Shackelford et al. 1999). The hypothesis would be that neurons attempting to re-enter the cell cycle are forced into apoptosis (Freeman et al. 1994). The possibility that p53-mediated gene induction leads to death in UV-treated neurons appears unlikely here because death in this model was independent of new protein synthesis.

Second, in some models, oxidative stress contributes to UV toxicity (Podda et al. 1998). UV-induced production of superoxides can lead to the formation of other pro-oxidants including hydrogen peroxide and peroxynitrite. Some UV effects on cell membrane and cytoplasmic proteins may be mediated by oxidative stress, e.g. lactate dehydrogenase is inhibited by UV radiation (Chen et al. 1989; A. T. McCollum and S. Estus, personal observation). This mechanism does not appear to be responsible for cell death here as two different antioxidants, a vitamin E derivative (trolox) and N-acetyl-l-cysteine, did not alter UV-induced cell death (A.T.McCollum and S. Estus, personal observation).

A third mechanism implicated by others in UV toxicity involves increased cytoplasmic Ca2+ (Kataoka and Ohmori 1994; Ihrig et al. 1997) and calcium signaling (Schieven and Ledbetter 1993; Schieven et al. 1993). Recently, Ca2+ chelators have been shown to protect HeLa cells from UV-induced death, suggesting a role for the loss of Ca2+ homeostasis in UV toxicity (Pu and Chang 2001). Three lines of evidence support the possibility that loss of Ca2+ homeostasis leading to calpain activation contributes to the UV-induced death in cortical neurons reported here. First, neuronal death was inhibited by Ca2+-free medium, implicating Ca2+ in the death process. Second, death was inhibited by two separate calpain inhibitors, implicating calpain directly and a loss of Ca2+ homeostasis indirectly in the apoptotic process. Third, Ca2+-free medium and calpain inhibitor III provided similar levels of partial protection that were not additive. We interpret these data as suggesting that UV kills a subpopulation of neurons via a loss of Ca2+ homeostasis leading to calpain activation. In a second neuronal population susceptible to UV toxicity, death is Ca2+- and calpain-independent, and hence proceeds by a presently unknown death pathway. This differential susceptibility to loss of Ca2+ homeostasis may reflect differential expression of calbindin, a protein known to be differentially expressed in neuronal populations and to mediate protection from inappropriate Ca2+ levels (Cheng et al. 1994; Masliah et al. 1995; D'Orlando et al. 2001).

How does calpain induce cell death?

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Calpain has a large number of substrates that may potentiallycontribute to neuronal death. Interestingly, several calpain substrates are also caspase substrates, including caspases themselves, β-actin, amyloid-beta precursor protein, poly(ADP)ribose polymerase, focal adhesion kinase, protein kinase C, and spectrin (Wang 2000). Although calpain apparently activated the caspase pathway here, caspase activity was not necessary for neuronal death. Overall, we interpret these results as suggesting the possibility that calpain assumes the role of caspases in some aspects of neuronal apoptosis. Recent studies have implicatedadditional calpain substrates in cell death, including Bax, c-Fos/c-Jun, cyclin D1, cdk5, and tau (Wang 2000). These studies have suggested that cell cycle signaling plays a significant role in neuronal apoptosis induced by UV treatment (Park et al. 1998a; Hiyama and Reeves 1999; Wisdom 1999; Wisdom et al. 1999). In particular, the cdk inhibitors flavopiridol and olomoucine decrease UV-induced neuronal death (Park et al. 1998a). A potential role for inappropriate cdk activation in calpain-mediated cell death has also been suggested by studies involving cells exposed to 4-hydroxynonenal, hypoxic stress, or Ca2+ influx; each of these insults leads to the activation of calpain, which in turn proteolyses the cdk5 activator protein, p35, into a truncated form, p25 (Kusakawa et al. 2000; Lee et al. 2000; Nath et al. 2000; Kerokoski et al. 2001). p25 causes prolonged activation and inappropriate localization of cdk5. This in turn can lead to tau hyperphosphorylation, cytoskeletal disruption, and apoptosis. In summary, whether calpain toxicity in this model is the result of calpain actions on cytoskeletal proteins, cell cycle proteins, and/or kinase regulators such as p35 will require further study.

Calpain activates caspase-3 indirectly

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

The mechanism whereby calpain leads to caspase-3 activation in this model is unclear. Our results are consistent with those of Waterhouse et al. (1998) who showed in an irradiation model that calpain activation can proceed caspase activation. Currently, only caspase-8 and caspase-9 are widely accepted as capable of activating caspase-3 (Wang 2000). In theory, calpain could act upon caspase-3 directly to generate the p17 fragment. Alternatively, calpain could act upon other macromolecules, which then proceed to causecaspase-8/caspase-9 activation, which then activate caspase-3. The former possibility appears unlikely because calpain has been reported to cleave the 32-kDa pro-caspase-3 only to a 30-kDa fragment that is less susceptible to subsequent caspase-8- or caspase-9-mediated cleavage and activation (McGinnis et al. 1999a; Lankiewicz et al. 2000). Indeed, others have reported that calpain is capable of directly degrading both caspase-8 and -9 as well as APAF-1, at least in cell-free systems (Chua et al. 2000; Lankiewicz et al. 2000; Reimertz et al. 2001). However, we found that the pan-caspase inhibitor BAF blocked p17 production, suggesting that caspase-8 or caspase-9 are required for caspase-3 activation. Hence, we interpret our data overall as consistent with the hypothesis that, in UV-induced neuronal death, calpain activation indirectly leads to caspase-3 activation in a process involving caspase-8 or caspase-9 activity.

If caspases are activated, why aren't they killing the cells?

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References

Initially, we were puzzled by the observation that caspase-3 was activated by UV-treatment but that a pan-caspaseinhibitor did not block death. However, a careful consideration of the available data leads us to suggest the model depicted in Fig. 10. In this model, we propose that UV treatment leads to a loss of Ca2+ homeostasis, which in turn leads to calpain activation. Data supporting this would be that Ca2+ -free medium is neuroprotective, and the studies showing calpain activation. We next propose that the levels of active calpain are sufficient to induce apoptosis in a neuronal subpopulation. Data supporting this contention would include the neuroprotection afforded by calpain inhibitor III and calpeptin. We next propose that within this same neuronal subpopulation, the actions of calpain lead to activation of the caspase pathway through unclear means. That calpain leads to caspase activation in this model is supported by the ability of calpain inhibitor III to block CaspACE labeling, generation of the 120-kDa SBP discerned by western blotting, and p17 generation as discerned by western blotting and immunofluorescence. Lastly, we hypothesize that caspase inhibitors do not block death because the activity of calpain alone precedes caspase activation and is sufficient by itself to induce neuronal death. Whether the levels of active caspase that are produced are sufficient to kill neurons is amoot point because caspase is not activated independent ofcalpain.

image

Figure 10. A model for UV-induced neuronal death. This model summarizes the findings of this manuscript that UV treatment leads to neuronal death in a process involving loss of calcium homeostasis and calpain activation. While caspase-3 is activated, the caspase proteolytic pathway does not appear to contribute to cell death.

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To what extent this model for the interactions between the calpain and caspase proteolytic pathways is applicable to other cell death models is unclear. The caspase pathway can certainly be activated in the absence of the calpain pathway, and hence in some models, caspase alone clearly mediates apoptosis (Deshmukh et al. 1996). However, both calpain and caspase activation has been associated with certain models of cellular insults (Nath et al. 1996; Jordan et al. 1997; Pike et al. 1998; Buki et al. 2000; Zhao et al. 2000). The model proposed here may be applicable in these instances.

In conclusion, the primary findings of the work reported are that UV activates both caspase and calpain in cortical neurons, that calpain is necessary for caspase-3 activation, and that calpain but not caspase contributes to UV-induced neuronal death. The parsimonious model proposed here accounts for our observations with minimal speculation, and suggests a novel interplay between the calpain and caspase pathways that promotes cell death. Calpain may mediate apoptosis in certain neuronal insults wherein calpain leads to caspase activation.

References

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Primary rat cortical neuron preparations
  5. Cell treatments
  6. TUNEL and Hoechst staining
  7. Propidium iodide and calcein AM staining
  8. Immunofluorescence
  9. Western blots
  10. Results
  11. UV induces protein synthesis-independent apoptosis in cortical neurons
  12. Ca2+ contributes to UV-induced cell death
  13. UV effects on calpain and caspase activity
  14. Calpain inhibition decreases caspase activity
  15. Calpain but not caspase contributes to UV-induced neuronal apoptosis
  16. Discussion
  17. The mechanisms underlying UV-induced cell death
  18. How does calpain induce cell death?
  19. Calpain activates caspase-3 indirectly
  20. If caspases are activated, why aren't they killing the cells?
  21. Acknowledgements
  22. References
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