Kim M. McGinnis

Simultaneous degradation of alphaII-and betaII-spectrin by caspase 3 (CPP32) in apoptotic cells

The degradation of αII- and βII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both αII- and βII-subunits. Major C-terminal αII-spectrin BDPs were detected at ≈150, 145, and 120 kDa (αII-BDP150, αII-BDP145, and αII-BDP120, respectively); major C-terminal βII-spectrin BDPs were at ≈110 and 85 kDa (βII-BDP110 and βII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that αII-BDP150 and αII-BDP120 were generated by cleavages at DETD1185*S1186 and DSLD1478*S1479, respectively. For βII-spectrin, a major caspase site was detected at DEVD1457*S1458 , and both βII-BDP110 and βII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146*S2147, was found to account for βII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both αII- and βII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.

Effects of ICE-like protease and calpain inhibitors on neuronal apoptosis

Both ice-like protease and calpain have been shown to be involved in apoptosis in non-neuronal cells. Cultured rat cerebellar granule neurons undergo apoptosis when exposed to low potassium-containing medium. Calpain inhibitors 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and N-acetyl-Leu-Leu-Met-CHO (calpain inhibitor II) as well as interleukin-beta 1 converting enzyme (ICE)-like protease inhibitor Z-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) protect against such apoptotic death. They also reduce DNA laddering and the number of apoptotic nuclei. Staurosporine treatment also evokes apoptosis in human neuroblastoma SH-SY5Y. While Z-D-DCB is again anti-apoptotic, calpain inhibitors only provide modest effects in this model. Our results suggest that ICE-like protease plays a critical role in neuronal apoptosis whereas the contributions of calpain are more cell-type dependent.

Calcium/calmodulin-dependent protein kinase IV is cleaved by caspase-3 and calpain in SH-SY5Y human neuroblastoma cells undergoing apoptosis.

We have previously demonstrated cleavage of α-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996)Biochem. J. 319, 683–690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the pro-apoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Asp-fluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD176*A and CG201*A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.

Evidence for activation of caspase-3-like protease in excitotoxin- and hypoxia/hypoglycemia-injured neurons.

Abstract: Caspase activation has been shown to be a critical step in several models of neuronal apoptosis such as staurosporine treatment of human neuroblastoma SH‐SY5Y cells and potassium deprivation of rat cerebellar granule neurons. One common event is the appearance of caspase‐mediated 120‐kDa nonerythroid α‐spectrin breakdown product (SBDP120). Second, inhibitors of the caspase family are effective blockers of such neuronal death. In this study, we report the appearance of caspase‐mediated SBDP120 in excitotoxin‐challenged fetal rat cerebrocortical neurons [N‐methyl‐d‐aspartate (NMDA), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, and kainate] and rat cerebellar granule neurons (NMDA and kainate). A general caspase inhibitor, carbobenzoxy‐Asp‐CH2OC(O)‐2,6‐dichlorobenzene (Z‐D‐DCB), blocked the formation of SBDP120 under these conditions and attenuated the observed NMDA‐induced lactate dehydrogenase (LDH) release in both cell types. Furthermore, hydrolytic activity toward a caspase‐3‐preferred synthetic peptide substrate, acetyl‐DEVD‐7‐amido‐4‐methylcoumarin, was significantly elevated in NMDA‐treated granule neurons. Lastly, oxygen‐glucose deprivation (OGD)‐challenged cerebrocortical cultures also showed the appearance of SBDP120. Again, Z‐D‐DCB blocked the SBDP120 formation as well as attenuated the LDH release from the OGD‐challenged neurons. Taken together, the presence of caspase‐specific SBDP120 and the neuroprotective effects of Z‐D‐DCB strongly suggest that caspase activation contributes at least in part to excitotoxin‐ and OGD‐induced neuronal death.

Characterization of CPP32-Like Protease Activity Following Apoptotic Challenge in SH-SY5Y Neuroblastoma Cells

Abstract: We characterized the activation of interleukin‐1β‐converting enzyme (ICE)‐like proteases (caspases) in human neuroblastoma cells (SH‐SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti‐poly(ADP‐ribose) polymerase (anti‐PARP) and anti‐CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl‐DEVD‐7‐amido‐4‐methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32‐like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH‐SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH‐SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy‐Asp‐CH2OC(O)‐2,6‐dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.

Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells

Abstract: The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH‐SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase‐3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase‐3‐like, but not calpain, activity. Pan‐caspase inhibitors protected against EGTA‐, but not CaCl2−, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase‐3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death‐mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase‐3‐like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.

Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis.

Abstract: Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH‐SY5Y human neuroblastoma cells undergoing staurosporine‐ or EGTA‐mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA‐treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase‐3‐like activity. An increase in caspase‐3‐like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase‐3‐like activity in staurosporine‐treated SH‐SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K+‐mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.

Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis.

Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478)*SVEAL). Here, by using a synthetic peptide (NH(2)-SVEALC) mimicking the neo-N-terminal of SBDP120 as antigen, we report the development of chicken antibodies that specifically recognize the SBDP120 generated by in vitro caspase-3 digestion of bovine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to human neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-spectrin nor its other fragments on Western blots. These anti-SBDP120 work equally well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical studies, these antibodies also specifically stained apoptotic SH-SY5Y or CGNs undergoing apoptosis in a caspase- inhibitor-sensitive manner. These anti-SBDP120s might become powerful markers for apoptotic neurons in various neurological or neurodegenerative conditions in vivo.

Cytochrome c translocation does not lead to caspase activation in maitotoxin-treated SH-SY5Y neuroblastoma cells

Cytosolic cytochrome c elevation has been associated with activation of caspase-3-like proteases. In this study, we demonstrate that treatment with the neurotoxin and potent calcium channel opener maitotoxin (MTX) induces cytochrome c release from the mitochondria that is not accompanied by caspase activation. Cytochrome c translocation in MTX-treated SH-SY5Y cells was readily apparent after 30 min and peaked at 2.5h. We assayed caspase activity by acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) hydrolysis and by immunoblotting for caspase-3 processing and proteolysis of alphaII-spectrin and PARP. In contrast, treatment with pro-apoptosis agent staurosporine (STS) induced both cytochrome c release and caspase-3 activation after 2h. In addition, with MTX treatment, we found no evidence of caspase activation at any time point or MTX concentration used. Instead, we observed that caspase-9, Apaf-1 and caspase-3 were all partially truncated by calpain under these conditions. These combined effects likely contribute to the lack of caspase activation cascade in MTX-treated cells, despite the presence of cytochrome c in the cytosol.

Cytosolic Calmodulin Is Increased in SK‐N‐SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK‐N‐SH. Increasing the intracellular Ca2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca2+ pools in the muscarinic receptor‐mediated increases in cytosolic CaM in SK‐N‐SH cells. Stimulus‐mediated changes in intracellular Ca2+ were monitored in fura‐2‐loaded cells, and CaM was measured by radioimmunoassay in the 100,000‐g cytosol and membrane fractions. The influx of extracellular Ca2+ normally seen with carbachol treatment in SK‐N‐SH cells was eliminated by pretreatment with the nonspecific Ca2+ channel blocker Ni2+. Blocking the influx of extracellular Ca2+ had no effect on carbachol‐mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca2+ from the medium did not affect carbachol‐mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol‐mediated increase of intracellular free Ca2+ by pretreatment with the cell‐permeant Ca2+ chelator BAPTA/AM did attenuate the carbachol‐mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca2+ into the cell by K+ depolarization was assessed. Incubation of SK‐N‐SH cells with 60 mM K+ elicited an immediate and persistent increase in intracellular free Ca2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca2+, which releases Ca2+ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K+ depolarization. These data demonstrate that release of Ca2+ from the intracellular store is important for the carbachol‐mediated redistribution of CaM in human neuroblastoma SK‐N‐SH cells.