Rand Posmantur

Regional calpain and caspase-3 proteolysis of α-spectrin after traumatic brain injury

ACTIVITY of calpains and caspase-3 inferred from proteolysis of the cytoskeletal protein α-spectrin into signature spectrin breakdown products (SBDPs) was used to provide the first systematic and simultaneous comparison of changes in activity of these two families of cysteine proteases after traumatic brain injury (TBI) in rats. Distinct regional and temporal patterns of calpain/caspase-3 processing of α-spectrin were observed in brain regions ipsilateral to the site of injury after TBI, including large increases of 145 kDa calpain-mediated SBDP in cortex (up to 30-fold), and enduring increases (up to 2 weeks) of 145 kDa SBDP in hippocampus and thalamus. By contrast, 120 kDa caspase-3-mediated SBDP was absent in cortex and showed up to a 2-fold increase in hippocampus and striatum at early (hours) after TBI. Future studies will clarify the pathological significance of large regional differences in activation of calpain and caspase-3 proteases after TBI.

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.

Temporal relationships between de novo protein synthesis, calpain and caspase 3-like protease activation, and DNA fragmentation during apoptosis in septo-hippocampal cultures

Caspase 3‐like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade. However, the influence of upstream events on calpain/caspase activation and the role of calpain/caspase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis‐related events after staurosporine‐induced apoptosis in mixed glial‐neuronal septo‐hippocampal cell cultures. Following 3 hr exposure to staurosporine (0.5 μM), calpain and caspase 3‐like proteases processed α‐spectrin to their signature proteolytic fragments prior to endonuclease‐mediated DNA fragmentation (not evident until 6 hr), indicating that endonuclease activation is downstream from calpain/caspase activation. Cycloheximide, a general protein synthesis inhibitor, completely prevented processing of α‐spectrin by calpains and caspase 3‐like proteases, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains and caspase 3‐like proteases. Calpain inhibitor II and the pan‐caspase inhibitor Z‐D‐DCB each inhibited their respective protease‐specific processing of α‐spectrin and attenuated endonuclease DNA fragmentation and cell death. Thus, activation of calpains and caspase 3‐like proteases is an early event in staurosporine‐induced apoptosis, and synthesis of, as yet, unknown protein(s) is necessary for their activation. J. Neurosci. Res. 52:505–520, 1998.

Subcellular localization and duration of μ-calpain and m-calpain activity after traumatic brain injury in the rat : A casein zymography study

Casein zymographic assays were performed to identify changes in μ-calpain and m-calpain activity in naive, sham-injured, and injured rat cortex at 15 minutes, 3 hours, 6 hours, and 24 hours after unilateral cortical impact brain injury. Cortical samples ipsilateral and contralateral to the site of injury were separated into cytosolic and total membrane fractions. Marked increases in μ-calpain activity in cytosolic fractions in the ipsilateral cortex occurred as early as 15 minutes, became maximal at 6 hours, and decreased at 24 hours to levels observed at 15 minutes after injury. A similar temporal profile of cytosolic μ-calpain activity in the contralateral cortex was observed, although the increases in the contralateral cortex were substantially lower than those in the ipsilateral cortex. Differences were also noted between cytosolic and total membrane fractions. The detection of a shift in μ-calpain activity to the total membrane fraction first occurred at 3 hours after traumatic brain injury and became maximal at 24 hours after traumatic brain injury. This shift in μ-calpain activity between the two fractions could be due to the redistribution of μ-calpain from the cytosol to the membrane. m-Calpain activity was detected only in cytosolic fractions. m-Calpain activity in cytosolic fractions did not differ significantly between ipsilateral and contralateral cortices, and increased in both cortices from 15 minutes to 6 hours after injury. Relative magnitudes of m-calpain versus μ-calpain activity in cytosolic fractions differed at different time points after injury. These studies suggest that traumatic brain injury can activate both calpain isoforms and that calpain activity is not restricted to sites of focal contusion and cell death at the site of impact injury but may represent a more global response to injury.

Immunoblot Analyses of the Relative Contributions of Cysteine and Aspartic Proteases to Neurofilament Breakdown Products Following Experimental Brain Injury in Rats

Analyses using either one or two-dimensional gel electrophoresis were performed to identify the contribution of several proteases to lower molecular weight (MW) neurofilament 68 (NF68) break down products (BDPs) detected in cortical homogenates following unilateral cortical impact injury in rats. One dimensional immunoblot of BDPs obtained from in vitro cleavage of enriched neurofilaments (NF) by purified μ-calpain, m-calpain, cathepsin, B, cathepsin D, and CPP32 (caspase-3) were compared to in vivo samples from rats following traumatic brain injury (TBI). Comparison of these blots provided information on the relative contribution of different cysteine or aspartic proteases to NF loss following brain injury. As early as 3 hrs post-injury, cortical impact resulted in the presence of several lower MW NF68 immunopositive bands having patterns similar to those previously reported to be produced by calpain mediated proteolysis of neurofilaments. Only μ-calpain and m-calpain in vitro digestion of enriched neurofilaments contributed to the presence of the low MW 57 kD NF68 break down product (BDP) detected in post-TBI samples. Cathepsin B, cathepsin D, and caspase-3 failed to produce either the 53 kD or 57 kD NF BDPs. Further, 1 and 2 dimensional peptide maps containing a 1:1 ratio of in vivo and in vitro tissue samples showed complete comigration of lower MW immunopositive spots produced by TBI or in vitro incubation with m-calpain, thus providing additional evidence for the potential role of calpain activation to the production of NF68 BDPs following TBI. More importantly, 2-dimensional gel electrophoresis detected that immunopositive NF68 spots shifted to the basic pole (+) suggesting that dephosphorylation of the NF68 subunit pool may be associated with NF protein loss following TBI, an observation not previously noted in any model of experimental brain injury.

pH dependency of μ-calpain and m-calpain activity assayed by casein zymography following traumatic brain injury in the rat

Studies employing casein zymographic assays analyzed the effects of varying pH (from pH 6.8 to pH 8.0) on changes in μ-calpain and m-calpain activity in naive, sham-injured and injured rat cortex 3 h following unilateral cortical impact injury. μ-Calpain activity following cortical impact injury was enhanced between pH values of 7.2 and 7.8, with pH 7.5 being optimal. m-Calpain activity was readily detected only between pH values of 7.2 and 7.4, with pH 7.3 producing the most prominent proteolytic activity. These observations suggest that strict control of pH is an important consideration in assessments of brain pH activation by casein zymography. Moreover, activation of different calpain isoforms, especially after traumatic brain injury, may be differentially influenced by smaller changes in physiological pH than previously recognized.

cAMP delays beta-amyloid (25-35) induced cell death in rat cortical neurons.

β-Amyloid (Aβ) accumulation is believed to contribute to neuronal cell death in Alzheimers disease. To understand the role of cAMP in the regulation of Aβ induced cell death, we used 8-chlorophenylthio-cAMP (8-CPT-cAMP, a cAMP analog) to raise intracellular cAMP levels. Exposure of rat cortical neurons to Aβ(25-35) resulted in a gradual increase in lactate dehydrogenase (LDH) over 48 h, which was preceded by a transient elevation in caspase-3-like activity. In the presence of 8CPT-cAMP, both caspase-3 activity and LDH release was significantly reduced. These data suggest that elevation of intracellular cAMP levels attenuate Aβ-induced neurotoxicity and may delay or prevent the onset of Aβ-induced neurode-generation.

Apoptotic death induced in MOLT-4 T-lymphoblasts by purine nucleoside phosphorylase inhibition

Apoptotic death induced in MOLT-4 T-lymphoblasts by purine nucleoside phosphorylase inhibition R. B. Gilbertsen, R. Posmantur, R. Nath and K. K. W. Wang Department of Immunopathology, Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, MI 48105, USA, Fax +1 313 996 4333, e-mail: GILBERR@AA.WL.COM Department of Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, MI 48105, USA