Casein zymogram assessment of mu-calpain and m-calpain activity after traumatic brain injury in the rat in vivo.
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Animals↗
Biomedical subjects
R L Hayes
Publications and source records attributed to R L Hayes.
Concurrent assessment of calpain and caspase-3 activity by means of western blots of protease-specific spectrin breakdown products.
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Animals↗
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.
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Sphingosine-1-phosphate induces apoptosis of cultured hippocampal neurons that requires protein phosphatases and activator protein-1 complexes.
In this study, we report that mobilization of internal Ca2+ by sphingosine-1-phosphate, a metabolite of ceramide, induces apoptosis in cultured hippocampal neurons. This sphingosine-1-phosphate-induced apoptosis is dependent upon the activation of protein phosphatases, possibly calcineurin and phosphatase 2A (or a related phosphatase). In addition, pretreatment of neurons with double-stranded oligonucleotides containing the metallothionein phorbol-12-myristate-13-acetate response element sequence as transcription factor decoys suppressed apoptosis. In contrast, double-stranded oligonucleotides containing either the c-jun or SV40 phorbol-12-myristate-13-acetate response element sequences were ineffective. Electrophoretic mobility shift assays and supershift assays revealed that c-Fos-containing activator protein- complexes preferentially bound the metallothionein phorbol-12-myristate-13-acetate response element sequence-containing oligonucleotides. Furthermore, antisense oligonucleotides to c-fos and c-jun were also protective. The apoptotic death of hippocampal neurons has been hypothesized to contribute to the cognitive impairments observed following insults to the brain. While increases in intracellular calcium are thought to be key mediators of neuronal apoptosis, the biochemical cascade(s) activated as a result of increased Ca2+ which mediates apoptosis of hippocampal neurons is (are) not well understood. The findings presented in this study suggest that mobilization of internal calcium via prolonged exposure of sphingosine-1-phosphate induces apoptosis of hippocampal neurons in culture. Sustained increases in intracellular calcium activate a phosphatase cascade that includes calcineurin and a phosphatase 2A-like phosphatase, and leads to the expression of genes containing metallothionein phorbol-12-myristate-13-acetate response element (TGAGTCA)-type enhancer sequences. The expression of genes containing TGAGTCA-type enhancer sequences appears to be essential for sphingosine-1-phosphate-induced apoptosis of hippocampal neurons.
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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 alpha-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 alpha-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.
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IGF-I gene transfer in thermally injured rats.
Exogenous insulin-like growth factor-I (IGF-I) is known to improve the pathophysiology of a thermal injury, however, deleterious side-effects have limited its utility. Cholesterol-containing cationic liposomes that encapsulate complementary DNA (cDNA) are nonviral carriers used for in vivo gene transfection. We propose that liposome IGF-I gene transfer will accelerate wound healing in burned rats and attenuate deleterious side-effects associated with high levels of IGF-I. To test this hypothesis IGF-I gene constructs, encapsulated in liposomes, were studied for their efficacy in modulating the thermal injury response. Thirty adult male Sprague-Dawley rats were given a 60% TBSA scald burn and randomly divided into three groups to receive weekly subcutaneous injections of liposomes plus the lacZ gene coding for beta-galactosidase, liposomes plus cDNA for IGF-I and beta-galactosidase or liposomes plus the rhIGF-I protein. Body weights and wound healing were measured. Muscle and liver dry/wet weights and IGF-I concentrations in serum, skin and liver were measured by radioimmunoassay. Transfection was confirmed by histochemical staining for beta-galactosidase. Rats receiving the IGF-I cDNA constructs exhibited the most rapid wound re-epithelialization and greatest increase in body weight and gastrocnemius muscle protein content (P < 0.05). Local IGF-I protein concentrations in the skin were higher when compared to liposomes containing only the lacZ gene (P < 0.05) Transfection was apparent in the cytoplasm of myofibroblasts, endothelial cells and macrophages of the granulation tissue. Liposomes containing the IGF-I gene constructs proved effective in preventing muscle protein wasting and preserving total body weight after a severe thermal injury.
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Liposome-mediated NGF gene transfection following neuronal injury: potential therapeutic applications.
We have systematically investigated the therapeutic potential of cationic liposome-mediated neurotrophic gene transfer for treatment of CNS injury. Following determination of optimal transfection conditions, we examined the effects of dimethylaminoethane-carbamoyl-cholesterol (DC-Chol) liposome-mediated NGF cDNA transfection in injured and uninjured primary septo-hippocampal cell cultures and rat brains. In in vitro studies, we detected an increase of NGF mRNA in cultures 1 day after transfection. Subsequent ELISA and PC12 cell biological assays confirmed that cultured cells secreted soluble active NGF into the media from day 2 after gene transfection. Further experiments showed that such NGF gene transfection reduced the loss of chol- ine acetyltransferase (ChAT) activity in cultures following calcium-dependent depolarization injury. In in vivo studies, following intraventricular injections of NGF cDNA complexed with DC-Chol liposomes, ELISA detected nine- to 12-fold increases of NGF in rat CSF. Further studies showed that liposome/NGF cDNA complexes could attenuate the loss of cholinergic neuronal immunostaining in the rat septum after traumatic brain injury (TBI). Since deficits in cholinergic neurotransmission are a major consequence of TBI, our studies demonstrate for the first time that DC-Chol liposome-mediated NGF gene transfection may have therapeutic potential for treatment of brain injury.
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Altered calpastatin protein levels following traumatic brain injury in rat.
Pathological activation of the intracellular Ca2+-dependent proteases calpains may be responsible for the neuronal pathology associated with neurodegenerative diseases and acute traumas to the central nervous system. Though calpain activation has been shown definitively in traumatic brain injury (TBI), no studies have investigated calpastatin (CAST), the calpains' endogenous and specific inhibitor, after TBI. The present study examined temporal changes in CAST protein following controlled cortical impact injury in the rat. Western blot analyses of CAST in cortex and hippocampus detected two bands corresponding to molecular weights of 130 kDa [high-molecular-weight (HMW)] and 80 kDa [low-molecular-weight (LMW)]. A modest decrease in the HMW band in conjunction with a significant increase in the LMW band was observed in cortex ipsilateral to the site of impact following TBI. Examination of ipsilateral hippocampus revealed an increasing trend in the LMW band after injury, while no changes were observed in the HMW band. Thus, observable changes in CAST levels appear to occur several hours after reported calpain activation and cleavage of other substrates. In addition, a new analysis was performed on previously published data examining calpain activity in the same tissue samples used in the present study. These data suggest an association between decreases in calpain activity and accumulation of LMW CAST in the ipsilateral cortex following TBI. The present study cannot exclude proteolytic processing of CAST to LMW forms. However, the absence of reciprocity between changes in LMW and HMW bands in consistent with other data suggesting that rat brain could contain different CAST isoforms.
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Regional calpain and caspase-3 proteolysis of alpha-spectrin after traumatic brain injury.
Activity of calpains and caspase-3 inferred from proteolysis of the cytoskeletal protein alpha-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 alpha-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.
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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 microM), calpain and caspase 3-like proteases processed alpha-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 alpha-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 alpha-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.
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pH dependency of mu-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 mu-calpain and m-calpain activity in naive, sham-injured and injured rat cortex 3 h following unilateral cortical impact injury. Mu-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.
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Activation of phosphatidylinositol 3-kinase by brain-derived neurotrophic factor gene transfection in septo-hippocampal cultures.
Brain-derived neurotrophic factor (BDNF) has therapeutic potential for treatment of the injured central nervous system. BDNF induces both differentiation and survival of neurons by binding to trkB receptors. This interaction stimulates the intrinsic tyrosine kinase activity of trkB, initiating a signal cascade involving the phosphorylation of intracellular protein on tyrosine, serine, and threonine residues. The purpose of this investigation was to examine the effects of cationic lipid-mediated gene transfection of BDNF on phosphatidylinositol 3 (PI3)-kinase activity in primary septo-hippocampal cell cultures. Thirty-six hours after BDNF gene transfection in the primary CNS cell culture, PI3-kinase activity was significantly increased. The increased PI3-kinase activity was inhibited by wortmannin, a selective and irreversible inhibitor of PI3-kinase. In addition, wortmannin blocked neurofilament increases induced by BDNF gene transfection. This result suggests a possible role of PI3-kinase activation in neuroprotective effects produced by BDNF gene transfection.
Androstadienes↗
Social skill as determinant of social networks and perceived social support in schizophrenia.
Factors influencing supportive social networks of people with schizophrenia are little understood. Data from 46 outpatients with schizophrenia were analysed using structural equation modelling to test plausible sets of inter-relationships between social skill, social networks, and social support. The data supported a tentative model about the causal relationships between variables. Paths showed that people with greater social skill had larger social networks, but did not necessarily perceive greater support from these networks. Negative symptoms accounted for some of the effect of social skill on social networks. Whereas groups of single-admission and multiple-admission participants did not differ in terms of social skill, social networks, or support, the age of the participants influenced their social skill and the size of their social networks. Younger participants had greater social skill and larger social networks. The results appear to suggest the importance of early intervention for young people with first-episode psychosis.
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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 micro-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 micro-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.
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Protective effects of moderate hypothermia on behavioral deficits but not necrotic cavitation following cortical impact injury in the rat.
A number of experimental studies have reported that moderate hypothermia can produce significant protection against behavioral deficits and/or morphopathological alterations following traumatic brain injury; a Phase 3 clinical trial is currently examining the therapeutic potential for moderate hypothermia (32 degrees C) to improve outcome following severe traumatic brain injury in humans. The current study examined whether hypothermia (32 degrees C) provided behavioral protection following experimental cortical impact injury. The extent of focal cortical contusion was also examined in the same rats. A total of 30 male Sprague-Dawley rats were trained on beam balance and beam walking tasks prior to injury. Under isoflurane anesthesia, cortical impact was produced on the right parietal cortex of 20 rats. Ten rats underwent all surgical procedures but were not impacted (sham-injured rats). Ten of the injured rats were cooled to 32 degrees C (measured in temporalis muscle) beginning 5 min postinjury, maintained for 2 h and rewarmed slowly for 1 h. In the other 10 injured rats, normothermic temperatures (37.5 degrees C) were maintained for the same duration. Beam balance and beam walking performance was assessed daily for 5 days following injury. At 11 days postinjury, rats were assessed for 5 days on acquisition of the Morris water maze task. Following behavioral assessments, rats were perfused and the brain removed. Coronal sections were cut through the site of cortical impact injury and stained with hematoxylin and eosin. Hypothermic treatment resulted in significantly less beam balance and beam walking deficits than observed in normothermic rats. Hypothermia also significantly attenuated spatial memory performance deficits. Quantitative morphometric analyses failed to detect any significant differences in volumes of necrotic tissue cavitation in cortices of hypothermic and normothermic rats. Hypothermic treatment also had no effect on volumes of dorsal hippocampal tissue or numbers of cells in CA1 or CA3 regions of the hippocampus. These data suggest that hypothermia, consistent with the reports of others, can produce significant behavioral protection following cortical impact injury that is not necessarily correlated with changes in focal cortical necrosis within the first 15 days following injury.
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Subcellular localization and duration of mu-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 mu-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 mu-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 mu-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 mu-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 mu-calpain activity between the two fractions could be due to the redistribution of mu-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 mu-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.
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Survival of patients with glioblastoma multiforme is not influenced by altered expression of p16, p53, EGFR, MDM2 or Bcl-2 genes.
Deregulated expression of one or more growth control genes including p16, p53, EGF receptor (EGFR), MDM2 or Bcl-2 may contribute to the treatment resistance phenotype of GBM and generally poor patient survival. Clinically, GBM have been divided into two major groups defined by (1) histologic progression from a low grade tumor ("progressive" or "secondary" GBM) contrasted with (2) those which show initial clinical presentation without a prior history ("de novo" or "primary" GBM). Using molecular genetic analysis for p53 gene mutations together with immunophenotyping for overexpression of EGFR, up to four GBM variants can be distinguished, including the p53+/EGFR- progressive or the p53-/EGFR+ de novo variant. We examined the survival of 80 adult patients diagnosed with astrocytic GBM stratified by age category (>40, 41-60 or 61-80) to determine whether alterations in any one given growth control gene or whether different genetic variants of GBM (progressive versus de novo) were associated with different survival outcomes. Survival testing using Kaplan-Meier plots for GBM patients with or without altered expression of p16, p53, EGFR, MDM2 or Bcl-2 showed no significant differences by age group or by gene expression indicating a lack of prognostic value for GBM. Also the clinical outcome among patients with GBM showed no significant differences within each age category for any GBM variant including the progressive and de novo GBM variants indicating similar biologic behavior despite different genotypes. Using a pairwise comparison, one-third of the GBM with normal p16 expression showed accumulation of MDM2 protein and this association approached statistical significance (0.01 < P < 0.05) using the Bonferroni procedure. These GBM may represent a variant in which the p19ARF/MDM2/p53 pathway may be deregulated rather than the p16/cyclin D-CDK4/Rb pathway.
Adult↗
Potential contribution of proteases to neuronal damage.
Calpain was first discovered 30 years ago. Two major isoforms were subsequently isolated and purified. The presence of an endogenous protein inhibitor, calpastatin, was later discovered. Calpain activity is tightly regulated by Ca(2+). At physiological levels of Ca(2+), the role of calpain remains poorly understood, but is believed to be involved in mitosis and muscle cell differentiation. Calpain has also been implicated in various membrane fusion events through remodeling of the cytoskeletal network. Calpain activation has been shown to be increased during normal aging and in muscular dystrophy, cataract, arthritis and Alzheimer's disease, and in acute traumas such as traumatic brain injury (TBI), spinal cord injury and cerebral and cardiac ischemia. Early work on calpain inhibitors was limited to protein inhibitors and other nonselective enzyme inhibitors. Peptidyl aldehydes such as leupeptin and antipain are also among the earliest reported calpain inactivators. Irreversible inhibitors such as the E64 family have also been studied, and peptidyl halomethanes and diazomethanes have long been used as protease inhibitors. A variety of calpain inhibitors are under development. From a therapeutic perspective, calpain inhibitors may have several advantages over other more conventional targets such as ion channel blockers and glumate antagonists, since calpain proteolysis represents a later component of a pathway mediating cell death initiated by excitotoxicity and elevated Ca(2+) levels. Although the potential clinical utility of calpain inhibitors seems well established, a number of important considerations remain to be addressed. The role of other proteolytic cascades contributing to neuronal cell damage following TBI must also be considered.
Journal Article↗