Brian R. Pike

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.

Temporal Profile of Apoptotic-like Changes in Neurons and Astrocytes Following Controlled Cortical Impact Injury in the Rat

Apoptotic cell death has been observed in both neurodegenerative diseases and acute neurological traumas such as ischemia, spinal cord injury, and traumatic brain injury (TBI). Recent studies employing different models of TBI have described morphological and biochemical changes characteristic of apoptosis following injury. However, no study has examined the temporal profile of apoptosis following controlled cortical impact (CCI) injury in the rat. In addition, the relative frequency of apoptotic profiles in different cell types (neurons versus glia) following CCI has yet to be investigated. In the present experiments, injured cortex was subjected to DNA electrophoresis, and serial sections from the contusion area were stained with hematoxylin and eosin or Hoechst 33258 or double-labeled with TUNEL and neuronal or glial markers. The results of the present study indicate that CCI produces a substantial amount of DNA damage associated with both apoptotic-like and necrotic-like cell death phenotypes primarily at the site of cortical impact and focal contusion. DNA damage, as measured by TUNEL and DNA electrophoresis, was most apparent 1 day following injury and absent by 14 days post-TBI. However, quantitative analysis showed that the majority of TUNEL-positive cells failed to exhibit apoptotic-like morphology and were probably undergoing necrosis. In addition, apoptotic-like morphology was predominantly observed in neurons compared to astrocytes. The present study provides further evidence that apoptosis is involved in the pathology of TBI and could contribute to some of the ensuing cell death following injury.

Accumulation of non-erythroid αII-spectrin and calpain-cleaved αII-spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats

Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of specificity for defining neuropathological cascades. Identification of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Non‐erythroid αII‐spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase‐3 cysteine proteases. As we have previously demonstrated, cleavage of αII‐spectrin by calpain and caspase‐3 results in accumulation of protease‐specific spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of αII‐spectrin and αII‐SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native αII‐spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain‐specific SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain‐specific 145 kDa SBDP in CSF were 244%, 530% and 665% of sham‐injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase‐3‐specific SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of αII‐spectrin and αII‐SBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase‐3.

Temporal Profile and Cell Subtype Distribution of Activated Caspase‐3 Following Experimental Traumatic Brain Injury

Abstract: This study investigated the temporal expression and cell subtype distribution of activated caspase‐3 following cortical impact‐induced traumatic brain injury in rats. The animals were killed and examined for protein expression of the proteolytically active subunit of caspase‐3, p18, at intervals from 6 h to 14 days after injury. In addition, we also investigated the effect of caspase‐3 activation on proteolysis of the cytoskeletal protein α‐spectrin. Increased protein levels of p18 and the caspase‐3‐specific 120‐kDa breakdown product to α‐spectrin were seen in the cortex ipsilateral to the injury site from 6 to 72 h after the trauma. Immunohistological examinations revealed increased expression of p18 in neurons, astrocytes, and oligodendrocytes from 6 to 72 h following impact injury. In contrast, no evidence of caspase‐3 activation was seen in microglia at all time points investigated. Quantitative analysis of caspase‐3‐positive cells revealed that the number of caspase‐3‐positive neurons exceeded the number of caspase‐3‐positive glia cells from 6 to 72 h after injury. Moreover, concurrent assessment of nuclear histopathology using hematoxylin identified p18‐immunopositive cells exhibiting apoptotic‐like morphological profiles in the cortex ipsilateral to the injury site. In contrast, no evidence of increased p18 expression or α‐spectrin proteolysis was seen in the ipsilateral hippocampus, contralateral cortex, or hippocampus up to 14 days after the impact. Our results are the first to demonstrate the concurrent expression of activated caspase‐3 in different CNS cells after traumatic brain injury in the rat. Our findings also suggest a contributory role of activated caspase‐3 in neuronal and glial apoptotic degeneration after experimental TBI in vivo.

Accumulation of Calpain and Caspase-3 Proteolytic Fragments of Brain-Derived αII-Spectrin in Cerebral Spinal Fluid after Middle Cerebral Artery Occlusion in Rats

Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid αII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature αII-spectrin breakdown products (αII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved αII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length αII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas αII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific αII-SBDPs were significantly increased in CSF after injury. However, caspase-3 αII-SBDPS were observed in CSF of some injured animals. These results indicate that αII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.

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.

Increased expression and processing of caspase‐12 after traumatic brain injury in rats

Traumatic brain injury (TBI) disrupts tissue homeostasis resulting in pathological apoptotic activation. Recently, caspase‐12 was reported to be induced and activated by the unfolded protein response following excess endoplasmic reticulum (ER) stress. This study examined rat caspase‐12 expression using the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase‐12 proform (∼60 kDa) and processed form (∼12 kDa), with peak induction observed within 24 h post‐injury in the cortex (418% and 503%, respectively). Hippocampus caspase‐12 proform induction peaked at 24 h post‐injury (641%), while processed form induction peaked at 6 h (620%). Semi‐quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis confirmed elevated caspase‐12 mRNA levels after TBI. Injury severity (1.0, 1.2 or 1.6 mm compression) was associated with increased caspase‐12 mRNA expression, peaking at 5 days in the cortex (657%, 651% and 1259%, respectively) and 6 h in the hippocampus (435%, 451% and 460%, respectively). Immunohistochemical analysis revealed caspase‐12 induction in neurons in both the cortex and hippocampus as well as in astrocytes at the contusion site. This is the first report of increased expression of caspase‐12 following TBI. Our results suggest that the caspase‐12‐mediated ER apoptotic pathway may play a role in rat TBI pathology independent of the receptor‐ or mitochondria‐mediated apoptotic pathways.

TNF-α stimulates caspase-3 activation and apoptotic cell death in primary septo-hippocampal cultures

Primary septo‐hippocampal cell cultures were incubated in varying concentrations of tumor necrosis factor (TNF‐α; 0.3–500 ng/ml) to examine proteolysis of the cytoskeletal protein α‐spectrin (240 kDa) to a signature 145 kDa fragment by calpain and to the apoptotic‐linked 120‐kDa fragment by caspase‐3. The effects of TNF‐α incubation on morphology and cell viability were assayed by fluorescein diacetate‐propidium iodide (FDA‐PI) staining, assays of lactate dehydrogenase (LDH) release, nuclear chromatin alterations (Hoechst 33258), and internucleosomal DNA fragmentation. Incubation with varying concentrations of TNF‐α produced rapid increases in LDH release and nuclear PI uptake that were sustained over 48 hr. Incubation with 30 ng/ml TNF‐α yielded maximal, 3‐fold, increase in LDH release and was associated with caspase‐specific 120‐kDa fragment but not calpain‐specific 145‐kDa fragment as early as 3.5 hr after injury. Incubation with the pan‐caspase inhibitor, carbobenzosy‐ Asp‐CH2‐OC (O)‐2‐6‐dichlorobenzene (Z‐D‐DCB, 50‐140 μM) significantly reduced LDH release produced by TNF‐α. Apoptotic‐associated oligonucleosomal‐sized DNA fragmentation on agarose gels was detected from 6 to 72 hr after exposure to TNF‐α. Histochemical changes included chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Results of this study suggest TNF‐α may induce caspase‐3 activation but not calpain activation in septo‐hippocampal cultures and that this activation of caspase‐3 at least partially contributes to TNF‐α‐induced apoptosis. J. Neurosci. Res. 64:121–131, 2001.

Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures.

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen–glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of α-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3–mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen–glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.

Maitotoxin induces calpain but not caspase-3 activation and necrotic cell death in primary septo-hippocampal cultures

Maitotoxin is a potent toxin that activates voltage and receptor-mediated Ca2+ channels, resulting in Ca2+ overload and rapid cell death. We report that maitotoxin-induced cell death is associated with activation of calpain but not caspase-3 proteases in septo-hippocampal cell cultures. Calpain and caspase-3 activation were examined by accumulation of protease-specific breakdown products to α-spectrin. Cell death manifested exclusively necrotic-like characteristics including round, shrunken nuclei, even distribution of chromatin, absence of DNA fragmentation and failure of protein synthesis inhibition to reduce cell death. Necrotic cell death was observed in neurons and astroglia. Calpain inhibitor II inhibited calpain-specific processing of α-spectrin and significantly reduced cell death. The pan-caspase inhibitor, Z-D-DCB, nominally attenuated cell death. Results suggest that: (1) calpain, but not caspase-3, is activated as a result of maitotoxin-induced Ca2+ influx; (2) necrotic cell death caused by maitotoxin exposure is partially mediated by calpain activation; (3) maitotoxin is a useful tool to investigate pathological mechanisms of necrosis.