The degradation of ␣II-and II-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 ␣II-and II-subunits. Major C-terminal ␣II-spectrin BDPs were detected at Ϸ150, 145, and 120 kDa (␣II-BDP150, ␣II-BDP145, and ␣II-BDP120, respectively); major C-terminal II-spectrin BDPs were at Ϸ110 and 85 kDa (II-BDP110 and II-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that ␣II-BDP150 and ␣II-BDP120 were generated by cleavages at DETD 1185 *S 1186 and DSLD 1478 *S 1479 , respectively. For II-spectrin, a major caspase site was detected at DEVD 1457 *S 1458, and both II-BDP110 and II-BDP85 shared a common N-terminal sequence starting with Ser 1458 . An additional cleavage site near the C terminus, at ETVD 2146 *S 2147 , was found to account for II-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 ␣II-and II-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.The importance of proteases in the expression of mammalian apoptosis has been the subject of many recent studies. The mammalian interleukin-1-converting enzyme (ICE) 1 -like protease family (renamed caspase (1) (5), and its deletion by gene knockout blocks neuronal death during brain development with consequential lethality (6). Besides the caspases, a second family of proteases implicated in the initiation and control of apoptosis are the calpains (7, 8), especially in several hematopoietic and neuronal cells (9 -12). The relationship between these two protease families, the consequences of each on their respective substrates and on cellular physiology, or the conditions under which each is activated remain poorly understood. While many proteins are cleaved during apoptosis, a prominent target of both calpain and caspase action is ␣II-spectrin, the major component of the cortical membrane skeleton. In neurons, calcium-activated calpain cleavage of ␣II-spectrin (non-erythroid ␣-spectrin or ␣-fodrin) accompanies N-methyl-D-aspartic acid receptor activation (13), 2 does not directly cause neuronal toxicity (7,15), and is postulated to be necessary for synaptic and neuronal plasticity (16 -18). Indeed, ␣II-spectrin cleavage by calpain appears to be a molecular mechanism by which skeletal plasticity can be enhanced without complete dissolution of the spectrin skeleton since calpain-mediated cleavage of ␣II-spectrin bestows calmodulin regulation on oligomeric spectrin-actin complexes, but does not dissociate them (unless II-spectrin is also c...
An elongated glutamine tract in mutant huntingtin initiates Huntington's disease (HD) pathogenesis via a novel structural property that displays neuronal selectivity, glutamine progressivity and dominance over the normal protein based on genetic criteria. As this mechanism is likely to involve a deleterious protein interaction, we have assessed the major class of huntingtin interactors comprising three WW domain proteins. These are revealed to be related spliceosome proteins (HYPA/FBP-11 and HYPC) and a transcription factor (HYPB) that implicate huntingtin in mRNA biogenesis. In HD post-mortem brain, specific antibody reagents detect each partner in HD target neurons, in association with disease-related N-terminal morphologic deposits but not with filter trapped insoluble-aggregate. Glutathione S:-transferase partner 'pull-down' assays reveal soluble, aberrantly migrating, forms of full-length mutant huntingtin specific to HD target tissue. Importantly, these novel mutant species exhibit exaggerated WW domain binding that abrogates partner association with other huntingtin isoforms. Thus, each WW domain partner's association with huntingtin fulfills HD genetic criteria, supporting a direct role in pathogenesis. Our findings indicate that modification of mutant huntingtin in target neurons may promote an abnormal interaction with one, or all, of huntingtin's WW domain partners, perhaps altering ribonucleoprotein function with toxic consequences.
We have previously demonstrated cleavage of ␣-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 proapoptotic 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 carbobenzoxyAsp-CH 2 OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Aspfluoromethylketone 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 PAPD 176 *A and CG 201 *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.
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.
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 1 20-kDa nonerythroid ea-spectrin breakdown product (SBDP1 20). Second, inhibitors of the caspase family are effective blockers of such neuronal death. In this study, we report the appearance of caspase-mediated SBDP1 20 in excitotoxin-challenged fetal rat cerebrocortical neurons [N-methyl-o-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate] and rat cerebellar granule neurons (NMDA and kainate). A general caspase inhibitor, carbobenzoxy-Asp-CH 2OC (0) -2 ,6-dichlorobenzene (Z-D-DCB), blocked the formation of SBDP1 20 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 NM DA-treated granule neurons. Lastly, oxygen-glucose deprivation (OGD) -challenged cerebrocortical cultures also showed the appearance of SBDP12O. Again, Z-D-DCB blocked the SBDP12O formation as well as attenuated the LDH release from the OGD-challenged neurons. Taken together, the presence of caspase-specific SBDP1 20 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.
delta 9-Tetrahydrocannabinol and two of its metabolites, 11-hydroxy-delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, can be measured in a single 1-ml sample of blood, plasma, or urine by a new assay which combines a relatively rapid extraction procedure with capillary column gas chromatography and negative ion chemical ionization mass spectrometry. Deuterium-labeled analogs of each cannabinoid are added to the physiological specimen as internal standards. Two extracts are obtained from each sample: a neutral fraction containing delta 9-tetrahydrocannabinol and 11-hydroxy-delta 9-tetrahydrocannabinol, and an acid fraction containing 11-nor-9-carboxy-delta 9-tetrahydrocannabinol. The neutral fraction is derivatized by treatment with trifluoroacetic anhydride; the acid fraction is first treated with BF3-methanol followed by reaction with trifluoroacetic anhydride. Under electron-capture chemical ionization conditions the derivatized delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol give abundant molecular anions ideally suited for selected ion monitoring. The negative ion chemical ionization spectrum of the HO-THC-trifluoroacetate shows no molecular anion. Consequently, quantitation of the hydroxy metabolite is achieved by monitoring a fragment ion formed by loss of CF3CO2 from its molecular anion. The limits of reliable measurement are judged to be 0.1 ng ml-1 for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, 0.2 ng ml-1 for delta 9-tetrahydrocannabinol and 0.5 ng ml-1 for 11-hydroxy-delta 9-tetrahydrocannabinol. Four examples are given of the application of the assay to the analysis of specimens of medico-legal importance.
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