Cross-talk Between Calpain and Caspase-3 in Penumbra and Core During Focal Cerebral Ischemia-reperfusion

Aims Some data have shown the functional connection between calpain and caspase-3. Here, we investigated the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion. Methods The activities of calpain and the levels of calpastatin, microtubule-associated protein-2 (MAP-2), and spectrin in penumbra and core at 3 or 23 h of reperfusion (R 3 h or R 23 h) after 1-h focal cerebral ischemia in rats were determined in sham- or caspase-3 inhibitor z-DEVD-CHO-treated rats.On the other hand, the determination of the activities of caspase-3 and the levels of MAP-2 and spectrin was done in sham- or calpain-inhibitor I-treated rats. Results z-DEVD-CHO (600 ng/rat, i.c.v.) markedly reduced the μ- and m-calpain activities in penumbra and the m-calpain activities in core at R 3 h and R 23 h, and enhanced the calpastatin levels in penumbra at R 3 h and in core at R 3 h and R 23 h significantly; however, it had no significant effects on the μ-calpain activities in core and the calpastatin levels in penumbra at R 23 h. Calpain inhibitor I (0.8 mg/rat, i.c.v.) markedly reduced the caspase-3 activities in core at R 3 h and R 23 h, but not in penumbra. Both calpain and caspase-3 inhibitors increased the levels of MAP-2 and spectrin in penumbra and core significantly after focal cerebral ischemia-reperfusion. Conclusions Our data provide direct evidence to demonstrate the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion.     

Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase

Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant km. The beta(235 K) subunit of fodrin, but not the alpha(240 K) subunit, was phosphorylated by the kinase. Neither the alpha(240 K) subunit nor the beta(220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.     

In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s).

This report demonstrates that specific proteolysis of brain spectrin by a calcium-dependent protease, calpain I, abolishes association of brain spectrin with the ankyrin-independent binding site(s) in brain membranes. Calpain I cleaves the beta subunit of spectrin at the N-terminal end leaving a 218-kDa fragment and cleaves the alpha subunit in the midregion to produce 150- and 130-kDa fragments. Calpain-proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely destroyed membrane-binding remained associated as a tetramer and retained about 60% of the ability to associate with actin filaments. Cleavage of spectrin occurred at sites distinct from the membrane-binding site which is located on the beta subunit since the isolated 218-kDa fragment of the beta subunit as well as a reconstituted complex of alpha and 218-kDa beta subunit fragment partially regained binding activity. Moreover, cleavage of the alpha subunit alone reduced the affinity of spectrin for membranes by 2-fold. A consequence of distinct sites for calpain I cleavage and membrane-binding is that calpain I can digest spectrin while spectrin is complexed with other proteins and therefore has the potential to mediate disassembly of a spectrin-actin network from membranes.     

Microtubule-Associated Protein 2 as an Early Indicator of Ischemia-Induced Neurodegeneration in the Gerbil Forebrain

Abstract: Microtubule-associated protein 2 (MAP-2) was studied in the gerbil hippocampus and striatum after transient ischemia. Western immunoblot analysis shows that there is a significant decrease of MAP-2 in the dorsolateral sector of the striatum and a slight decrease of MAP-2 in the CA1 region of the hippocampus 6–12 h after ischemia in the gerbil forebrain. The immunohistochemical staining pattern of MAP-2 in these two regions also shows a loss of immunostaining of MAP-2. In particular, a beaded MAP-2 immunostaining pattern at the apical dendritic region of the CA1 neurons of the hippocampus was found within 12 h after ischemia compared with the smooth dendritic immunostaining of MAP-2 in normal CA1 neurons. In vitro assays of MAP-2 degradation suggest that dendritic loss of immunoreactivity after ischemia seen on western blots may be due to calpain I degradation of MAP-2. Loss of MAP-2 in both the striatum and hippocampus was found to occur earlier than spectrin degradation by western blot analysis. These results suggest that loss of MAP-2 may participate in the initial phase of neuronal dysfunction and that dendritic breakdown may be a first sign of neurodegeneration.     

Proteolysis of Microtubule-Associated Protein 2 and Tubulin by Cathepsin D

The in vitro degradation of microtubule-associated protein 2 (MAP-2) and tubulin by the lysosomal aspartyl endopeptidase cathepsin D was studied. MAP-2 was very sensitive to cathepsin D-induced hydrolysis in a relatively broad, acidic pH range (3.0-5.0). However, at a pH value of 5.5, cathepsin D-mediated hydrolysis of MAP-2 was significantly reduced and at pH 6.0 only a small amount of MAP-2 was degraded at 60 min. Interestingly, the two electrophoretic forms of MAP-2 showed different sensitivities to cathepsin D-induced degradation, with MAP-2b being significantly more resistant to hydrolysis than MAP-2a. To our knowledge, this is the first clear demonstration that MAP-2 is a substrate in vitro for cathepsin D. In contrast to MAP-2, tubulin was relatively resistant to cathepsin D-induced hydrolysis. At pH 3.5 and an enzyme-to-substrate ratio of 1: 20, only 35% of the tubulin was degraded by cathepsin D at 60 min. The cathepsin D-mediated hydrolysis of tubulin was optimal only at pH 4.5. These results demonstrate that MAP-2 and tubulin are unequally susceptible to degradation by cathepsin D. These data also imply a potential for rapid degradation of MAP-2 in vivo by cathepsin D either in lysosomes or perhaps autophagic vacuoles of the neuron.     

Spectrin subtypes in mammalian brain

Mammalian neural cells contain at least two forms of brain spectrin: brain spectrin (240/235) which is located primarily in the axons and presynaptic terminals of neurons, and brain spectrin (240/235E) which is found in the cell bodies, dendrites and postsynaptic terminals of neurones. Brain spectrin (240/235E) is also found in certain glial cell types. Antibodies against red blood cell spectrin detect only brain spectrin (240/235E), while antibodies against brain spectrin isolated from axonal and synaptic membranes detect brain spectrin (240/235). Previous apparent discrepancies in the literature concerning brain spectrin localization at the light microscope level were undoubtedly due to different laboratories detecting distinct brain spectrin subtypes, based on the particular antibody being utilized for immunohistochemistry. In this review we (1) discuss the data supporting the presence of at least two distinct subtypes of mammalian brain spectrin, (2) explain how these results reconcile previous discrepancies concerning the localization of spectrin within neural cells, and (3) suggest the future implications of these findings.     

Dynamic modulation of cytoskeletal proteins linking integrins to signaling complexes in spreading cells. Role of skelemin in initial integrin-induced spreading

Recently we showed that signaling across beta3-integrin leads to activation of calpain and formation of integrin clusters that are involved in Rac activation. The subsequent activation of Rac and Rho leads to the formation of focal complexes and focal adhesions, respectively. The goal of the present study was to determine whether different proteins link the integrin to the cytoskeleton in the different complexes. We show that talin is present in focal adhesions but not in the calpain-induced clusters. alpha-Actinin colocalized with integrin at various sites, including the calpain-induced clusters. Skelemin, a protein shown recently to interact with beta1- and beta3-integrin in vitro, colocalized with integrin in calpain-induced clusters but was absent from focal adhesions. Cells transiently expressing skelemin C2 motifs, which contain the integrin binding site, failed to form integrin clusters or to spread on a substrate for beta1- and beta3-integrins. These results 1) suggest a dynamic reorganization of integrin complexes during cell spreading, 2) show that different cytoskeletal proteins link integrins in different complexes, and 3) demonstrate that skelemin is responsible for linking integrin to the calpain-induced clusters, and 4) show that the integrin-skelemin interaction is essential for transmission of signals leading to the initial steps of cell spreading.     

Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells

Adult mouse brain contains at least two distinct spectrin subtypes, both consisting of 240-kD and 235-kD subunits. Brain spectrin(240/235) is found in neuronal axons, but not dendrites, when immunohistochemistry is performed with antibody raised against brain spectrin isolated from enriched synaptic/axonal membranes. A second spectrin subtype, brain spectrin(240/235E), is exclusively recognized by red blood cell spectrin antibody. Brain spectrin(240/235E) is confined to neuronal cell bodies and dendrites, and some glial cells, but is not present in axons or presynaptic terminals.     

Beta spectrin bestows protein 4.1 sensitivity on spectrin-actin interactions

The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.     

Separation of fodrin subunits by affinity chromatography on calmodulin-Sepharose

Spectrin is composed of two nonidentical subunits, with the 240-kDa subunit of nonerythroid spectrin (fodrin) able to bind calmodulin (CaM) Ca2+-dependently. It was found that in the presence of chaotropic salts this binding site was still expressed, although the subunits of fodrin were dissociated. This has been exploited for separating the fodrin subunits rapidly and quantitatively by affinity chromatography on calmodulin-Sepharose. When bovine fodrin was dissolved in 2 M KI + 1 mM Ca2+ and applied to CaM-Sepharose the beta subunit (235-kDa) passed through unretarded whereas the alpha subunit (240-kDa) bound and could be eluted with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. These subunits would reform the intact molecule when mixed and dialyzed.     

Antigen preservation tests for immunocytochemical detection of cytoskeletal proteins: influence of aldehyde fixatives

The effects of aldehyde fixatives on immunochemical detection of cytoskeletal proteins were demonstrated by applying several quantitative assays to evaluate antigen conservation. Immunologically detectable brain spectrin (240/235) was measured by dot-immunobinding and quantitative immunodot assay using a polyclonal antibody. Paraformaldehyde fixation led to a 43-66% reduction in brain spectrin (240/235) immunodetection, and increasing glutaraldehyde concentrations decreased the immunological detection even more. Quantitative cryosection immunoassay and immunocytochemical localization confirmed the aldehyde sensitivity of brain spectrin (240/235). Brain spectrin (240/235) immunoreactivity decreased with increasing protein crosslinking and was dependent on glutaraldehyde concentration and post-fixation period. The assays were also used to test for conservation of antigenicity of neurofilament proteins by two monoclonal antibodies. Neurofilament detection was abolished in brain tissue after aldehyde fixation. The described methods allow screening within 24 hr of many fixation conditions by use of purified proteins as well as brain tissue samples, and allow an estimate of fixative influence on the conservation of protein antigenicity.     

Mechanisms of cytoskeletal regulation: modulation of membrane affinity in avian brush border and erythrocyte spectrins

The spectrins isolated from chicken erythrocytes and chicken intestinal brush border, TW260/240, share a common alpha subunit and a tissue-specific beta subunit. The ability of these related proteins to bind human erythrocyte inside out vesicles (IOVs) and human erythrocyte ankyrin in vitro have been quantitatively compared with human erythrocyte spectrin. Chicken erythrocyte spectrin binds human IOVs and human ankyrin with affinities nearly identical to that for human erythrocyte spectrin. TW260/240 does not significantly bind to either IOVs or ankyrin. These results demonstrate a remarkable tissue preservation of ankyrin-binding capacity, even between diverse species, and confirm the role of the avian beta-spectrins in modulating this functionality. Avian brush border spectrin may represent a unique spectrin which serves primarily as a filament cross-linker and which does not interact strongly with membrane-associated proteins.     

Rapid proteolysis of brain MAP-1 related cytoskeleton-associated 350kd protein by purified calpain

Microtubule associated protein-1 of brain and its intracellular 350kd analogues were highly sensitive to purified Ca2+-dependent cysteine proteinase (calpain). After 15 second digestion, we detected intermediate degradation products of MAP-1 by immunoblotting using anti-MAP-1 antibody as 290, 260, 220, 170, 140, 112, 80, 68, and 32kd polypeptides. These values corresponded to the molecular weights of the immunoreactive polypeptides of microtubule-enriched cytoskeletons isolated from HeLa and SV-3Y1 cells, suggesting the action of endogenous calpain on intracellular MAP-1 analogues in vivo or during the course of preparation.     

Functional expression of N-terminal truncated alpha-subunits of Na,K-ATPase in Xenopus laevis oocytes.

N-terminal deletion mutants of Na,K-ATPase alpha 1 isoforms initiating translation at Met34 (alpha 1T1) or at Met43 (alpha 1T2) were expressed in X. laevis oocytes. Compared to beta 3 cRNA injected controls, the co-expression of alpha 1wt, alpha 1T1, alpha 1T2 with beta 3 subunits results in a 2- to 3-fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K-pump current. The apparent K1/2 for potassium activation of the alpha 1T2/beta 3 Na,K-pumps is significantly higher than that of the alpha 1wt/beta 3 or alpha 1T1/beta 3 Na,K-pumps expressed at the cell surface. Total deletion of the lysine-rich N-terminal domain thus allows the expression of active Na,K-pump but with distinct cation transport properties.     

Pharmacological analysis of the cortical neuronal cytoskeletal protective efficacy of the calpain inhibitor SNJ‐1945 in a mouse traumatic brain injury model

The efficacy of the amphipathic ketoamide calpain inhibitor SNJ‐1945 in attenuating calpain‐mediated degradation of the neuronal cytoskeletal protein α‐spectrin was examined in the controlled cortical impact (CCI) traumatic brain injury (TBI) model in male CF‐1 mice. Using a single early (15 min after CCI‐TBI) i.p. bolus administration of SNJ‐1945 (6.25, 12.5, 25, or 50‐mg/kg), we identified the most effective dose on α‐spectrin degradation in the cortical tissue of mice at its 24 h peak after severe CCI‐TBI. We then investigated the effects of a pharmacokinetically optimized regimen by examining multiple treatment paradigms that varied in dose and duration of treatment. Finally, using the most effective treatment regimen, the therapeutic window of α‐spectrin degradation attenuation was assessed by delaying treatment from 15 min to 1 or 3 h post‐injury. The effect of SNJ‐1945 on α‐spectrin degradation exhibited a U‐shaped dose–response curve when treatment was initiated 15 min post‐TBI. The most effective 12.5 mg/kg dose of SNJ‐1945 significantly reduced α‐spectrin degradation by ~60% in cortical tissue. Repeated dosing of SNJ‐1945 beginning with a 12.5 mg/kg dose did not achieve a more robust effect compared with a single bolus treatment, and the required treatment initiation was less than 1 h. Although calpain has been firmly established to play a major role in post‐traumatic secondary neurodegeneration, these data suggest that even brain and cell‐permeable calpain inhibitors, when administered alone, do not show sufficient cytoskeletal protective efficacy or a practical therapeutic window in a mouse model of severe TBI. Such conclusions need to be verified in the human clinical situation.     

Biosynthesis of spectrin and its assembly into the cytoskeletal system of Friend erythroleukemia cells

Friend erythroleukemia cells, grown in the presence of dimethyl sulfoxide for 3 d, synthesize unequal amounts of the two chains (alpha and beta) of spectrin with approximately 15-30% more beta than alpha spectrin. When cells were ruptured by nitrogen cavitation, nascent alpha and beta spectrin were found to be associated with a membranous cell fraction and were not detected in the soluble cytoplasmic cell fraction. Nascent membrane-bound spectrin appeared not to be protected by membranes, since it was susceptible to trypsin degradation in the absence of detergent. On fractionation of cells with 1% Triton X-100, more (1.75-fold) nascent spectrin was found in the Triton-soluble fraction than in the Triton-insoluble fraction (cytoskeleton). In the Triton-soluble fraction, there was 55% more nascent beta spectrin than alpha spectrin, while the cytoskeleton contained nearly equal amounts of alpha and beta spectrin. Cells were pulse-labeled with L-[35S]methionine for 2 min and chase incubated for varying periods of time from 15 to 90 min with nonradioactive L-methionine. Radioactive spectrin accumulated in the Triton-soluble fraction for the first 15 min of chase incubation and then dropped by 25% in the next hour. By contrast, the amount of radioactive spectrin in the Triton-insoluble fraction rose gradually for 1 h of the chase period. This indicates that, in Friend erythroleukemia cells, a pool of membrane-bound spectrin containing an excess of the beta polypeptide is used to form the cytoskeletal system which is composed of equal molar amounts of alpha and beta spectrin. The location of spectrin was determined by immunoelectron microscopy. Small amounts of spectrin were detected in cells not treated with dimethyl sulfoxide and in these cells it was located on the surface membrane and within the cytoplasm. On treatment with dimethyl sulfoxide, complex vacuolar structures containing viruses appeared in the cells. In cells treated with dimethyl sulfoxide for 3 d 30% of the spectrin was near the outer membrane and 25% was associated with vacuolar structures, whereas in cells treated for 5 and 7 d the majority of spectrin (57-61%) was located in the vacuolar areas.     

In vitro digestion of spectrin, protein 4.1 and ankyrin by erythrocyte calcium dependent neutral protease (calpain I)

1. In whole ghosts, ankyrin, protein 4.1, protein band 3 and spectrin are lysed by purified calpain I in the presence of calcium. 2. Limited calpain lysis of purified ankyrin results in several peptides, including a 85 kD peptide bearing the ankyrin interaction site for the protein band 3 internal fragment (43 kD), and a 55 kD peptide carrying the ankyrin-spectrin interaction site. 3. These peptides are differently phosphorylated: the 85 kD by cytosol casein kinase, and the 55 kD by membrane casein kinase. 4. Protein 4.1 lysis mainly produces a 30 kD peptide resistant to proteolysis. 5. The spectrin beta-chain is more sensitive to calpain cleavage than the alpha chain; both chains seem to be cleaved in a similar sequential manner. 6. Limited proteolysis of spectrin dimer does not impede tetramerization in vitro.     

Rapid Calpain I Activation and Cytoskeletal Protein Degradation Following Traumatic Spinal Cord Injury: Attenuation with Riluzole Pretreatment

Abstract: Immunocytochemical and immunoblotting techniques were used to investigate calpain I activation and the stability of the calpain-sensitive cytoskeletal proteins microtubule-associated protein 2 (MAP2) and spectrin at 1, 4, and 24 h after contusion injury to the spinal cord. Spinal cord injury resulted in the activation of calpain I at all time points examined, with the highest level of activation occurring at 1 h. At the same early time point, there was a loss of dendritic MAP2 staining in spinal cord sections, accompanied by pronounced perikaryal accumulation. The loss in MAP2 staining in the injured spinal cord progressed over the 24-h survival period to affect regions 3 mm distant to the site of injury. The presence of calpain I-specific spectrin degradation was apparent in neuronal cell bodies and fibers as early as 1 h after injury, with the most intense staining occurring within and juxtaposed to the injury site. Spectrin breakdown products in neuronal cell bodies declined rapidly at 4 h and were nearly undetectable at 24 h after injury. Immunoblot studies confirmed the immunocytochemical results by demonstrating a significant increase in calpain I activation, a significant decrease in MAP2 levels, and a significant increase in spectrin breakdown. Finally, treatment of animals with riluzole, an inhibitor of glutamate release, before surgery reduced significantly the loss of MAP2 levels observed at 24 h after injury. These results demonstrate that Ca²⁺-dependent protease activation and degradation of critical cytoskeletal proteins are early events after spinal cord injury and that treatments that minimize the actions of glutamate may limit their breakdown.     

Effects of positive AMPA receptor modulators on calpain-mediated spectrin degradation in cultured hippocampal slices

Positive modulators of AMPA receptors (AMPAr), also known as ampakines, are allosteric effectors of the receptors and have been extensively studied in past years due to their potential use as treatment for various diseases and ailments of the central nervous system such as mild cognitive impairment, schizophrenia, and Alzheimer's disease. Ampakines have been shown to improve performance on memory tasks in animals and in human subjects, an effect linked to their ability to increase agonist-mediated ion influx through AMPAr, thus leading to enhanced synaptic responses and facilitation of long-term potentiation (LTP) induction at glutamatergic synapses. As LTP is associated with calpain activation and spectrin degradation, we determined the effects of ampakine treatment of cultured hippocampal slices on spectrin degradation. Calpain activation was evaluated by determining the levels of the 145-150kDa degradation products of spectrin. Our data indicated that incubation of hippocampal slices with some, but not all positive modulators of AMPA receptors resulted in enhanced spectrin degradation, an effect that was blocked by a calpain inhibitor. In addition, an antagonist of AMPAr but not of NMDAr blocked ampakine-induced spectrin degradation. These results indicate that prolonged treatment with selected ampakines leads to spectrin degradation mediated by activation of the calcium-dependent protease calpain.