Calmodulin stimulates the degradation of brain spectrin by calpain

Brain spectrin has been shown to be a preferential substrate of calcium-dependent proteases (Baudry, Bundman, Smith, and Lynch: Science 212:937-938, 1981) and a major calmodulin-binding protein (Kakiuchi, Sobue, and Fujita: FEBS Lett. 132:144-148, 1981). Since calmodulin, spectrin, and a proteolytically derived spectrin fragment are all components of isolated postsynaptic density preparations (Grab, Berzins, Cohen, and Siekevitz: J. Biol. Chem. 254:8690-8696, 1979; Carlin, Bartelt, and Siekevitz: J. Cell Biol. 96:443-448, 1983), we investigated the functional role of calmodulin binding to brain spectrin with respect to its susceptibility to digestion by proteases. We report that calmodulin's interaction with brain spectrin results in a marked acceleration of the rate of spectrin degradation by calcium-dependent proteases (calpains I and II), but not by chymotrypsin. The cleavage of erythrocyte spectrin (which lacks a high-affinity calmodulin binding site) by calpain I is unaffected by the presence of calmodulin. The stimulatory effect of calmodulin is blocked by trifluoperazine, a calmodulin antagonist, which by itself does not modify brain spectrin proteolysis by calcium-dependent proteases. These results suggest a novel role for calmodulin in neuronal function--namely, a synergistic interaction with calcium-dependent proteases in the regulation of cytoskeletal integrity.     

Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. II. Anatomical studies

Lesions of the various afferents to the hippocampus have been widely used to investigate the mechanisms underlying growth and degeneration in adult mammalian CNS. It has been proposed that disturbances in intracellular calcium and activation of calcium-dependent proteases represent key steps in producing come of the consequences of the lesions. In this study, we show that lesions of the entorhinal cortex or of the commissural pathway result in profound changes in the distribution of brain spectrin. At 2 days after lesions of the entorhinal cortex, immunoreactivity to spectrin is markedly increased in the outer molecular layer (OML) of the dentate gyrus; conversely at 2 days after commissural lesions, immunoreactivity to the same antigen is increased in the inner molecular layer. The increase in immunoreactivity to spectrin varies with survival time after lesions of the entorhinal cortex. By 24 h post lesion, the increase is homogeneous across the OML, and becomes more intense by 48 h. Between 1 and 3 weeks the increase is much less than at 48 h and is concentrated at the inner border of the OML. Pretreatment of the animals with the calpain inhibitor leupeptin reduces the increase in spectrin immunoreactivity normally seen 48 h after the lesion of the entorhinal cortex. Changes in the pattern of immunoreactivity to GFAP are very different to that seen with spectrin antibodies and are consistent with the known modifications in astrocytes that follow lesions of hippocampal afferents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny, compartmentation, and turnover of spectrin isoforms in rat central neurons

A variant of a principal structural protein of erythrocytes, spectrin, is a major neuronal protein. Here we have examined the subcellular and regional distributions, subunit composition, ontogeny, and metabolism of spectrin in rat CNS. While all subcellular fractions, except the mitochondrial, expressed the previously characterized brain form of spectrin (fodrin, or alpha gamma-spectrin), limited brain regions contained, in cytoplasm, a second isoform immunologically related to erythrocyte alpha beta-spectrin. Both alpha gamma- and alpha beta-spectrin are primarily neuronal, as evidenced by immunocytochemistry. The spectrins are distributed between 2 distinct subneuronal compartments: a membrane-associated domain containing alpha gamma-spectrin in relatively constant amounts across brain regions, and a cytoplasmic domain containing both the alpha gamma and alpha beta isoforms in widely varying amounts across brain regions. Although forebrain has considerable alpha beta-spectrin, the diencephalon, mesencephalon, and brain stem are devoid of this isoform. Further evidence for spectrin compartmentation comes from its ontogeny. Membrane-associated alpha gamma-spectrin is present at birth at its adult levels, but cytoplasmic alpha beta-spectrin is expressed only following the second postnatal week. Similarly, the 4-fold difference in cytoplasmic alpha gamma-spectrin content across brain regions develops during the third postnatal week. In this compartment, both spectrin forms may be metabolized in vivo, at least in part, by calcium-activated proteolysis. The presence in mammalian neurons of 2 spectrin isoforms and their compartmentation into distinct domains suggests multiple functions for neuronal spectrin, one of which may be in the stabilization or maturation of forebrain neurons.     

Translocations of fodrin and its binding proteins

Fodrin, a protein related to erythrocyte spectrin, redistributes within the cell in certain situations. We compare such movements of fodrin and several fodrin binding proteins during the processes of axonal transport in neurons, and capping of surface proteins in lymphocytes. In neurons, three different populations of newly synthesized fodrin appear to be transported down the axons at different velocities corresponding to those of groups of transported proteins designated II, IV, and V. Actin, which can interact with fodrin, is transported at the velocity of group IV. Synapsin, a component of synaptic vesicles, is also reported to bind to fodrin. One population of synapsin is transported more rapidly than fodrin, at the velocity of group I: two additional populations of transported synapsin may overlap fodrin in groups II and IV. We consider possible functional associations of these different populations of fodrin and fodrin binding proteins. We note that the transport of group IV proteins resembles in certain respects the process of capping in lymphocytes, suggesting the possibility of a common mechanism. We outline one of several possible mechanisms.     

Brain spectrin(240/235) and brain spectrin(240/235E): conservation of structure and location within mammalian neural tissue

We demonstrate that the brain spectrin isoforms (240/235) and (240/235E) are present in all mammalian species studied (human, bovine, mouse, and rat). Immunohistochemistry with a panel of eleven polyclonal antibodies have indicated an identical localization of the brain spectrin isoforms in all mammalian species. Brain spectrin(240/235) is found primarily in axons, and brain spectrin(240/235E) primarily in cell bodies and dendrites. Immunoprecipitation and Western blotting studies have indicated that the subunit molecular weights of brain spectrin(240/235) and (240/235E) are identical in all mammalian species. We demonstrate that when proteolysis is not completely blocked during immunoprecipitation studies, the 235 kDa subunits are converted to a 230 kDa polypeptide [brain spectrin(240/235)] and a 232 kDa polypeptide [brain spectrin(240/235E)]. Finally, we show that both the alpha and beta subunits of brain spectrin(240/235) and brain spectrin(240/235E) are antigenically distinct in every species examined. These studies indicate that previous findings on the structure, location, and function of mouse brain spectrin isoforms can now be generalized to all mammalian species.     

Cleavage site of calcium-dependent protease in human platelet membrane glycoprotein Ib

Chicken muscle-derived m-type calcium-dependent protease cleaved purified glycoprotein Ib alpha-chain (GPIb alpha, Mr 130,000) from human platelets into two fragments (Mr 100,000 and Mr 38,000) in the presence of 5 mM calcium. With partially purified glycoprotein Ib (alpha beta-dimer), an appearance of a fragment of Mr 100,000 was also demonstrated after treatment with both the m-type and human platelet-derived mu-type protease. These processes in glycoprotein Ib were inhibited by inhibitors of calcium-dependent proteases, 50 muM E-64-C or 0.2 mM leupeptin and by the chelation of calcium. Using two-dimensional gel electrophoresis system, release of glycocalicin in addition to 100 kDa fragment was demonstrated by calcium-dependent proteases. Then surface-labeled platelets were stimulated with A23187 in the presence of 5mM calcium. Under this condition, endogenous calcium-dependent protease is activated. Of the labeled glycoproteins, glycocalicin and glycoprotein V but not 100 kDa fragment were released from the platelet membrane. The released glycocalicin was further digested into a fragment of Mr 100,000 by the addition of m-type calcium-dependent protease. These results showed (i) that GPIb alpha was hydrolyzed by exogenous calcium-dependent proteases in two points and glycocalicin and 100 kDa fragment were produced and (ii) that endogenous protease cleaved GPIb alpha at one point and released glycocalicin.     

Spectrin expression in neuroblastoma cells

Mouse neuroblastoma cells express a spectrin-related molecule containing 240 kDal (kiloDalton) and 235 kDal subunits in a 1:1 ratio. The 240 kDal and 235 kDal subunits are nearly identical to the alpha and beta subunits respectively of brain spectrin by two dimensional chymotryptic peptide mapping analysis. The neuroblastoma cells do not express a measureable quantity of a red blood cell (rbc)-type spectrin molecule. Neuroblastoma spectrin has been localized throughout the cell body, and neurites of these cells by indirect immunofluorescence studies. As neuroblastoma cells are homogeneous, neuron-like, available in large quantity, and synthesize a single variant of spectrin which is closely related to brain spectrin(240/235), it is the best available model system for the study of the synthesis, assembly and turnover of a neuronal spectrin subtype.     

Study of cytoskeletal proteins in fibroblasts cultured from familial Alzheimer''s disease

Cytoskeletal proteins of the cultured fibroblasts obtained from Alzheimer's disease patients were studied. Western blotting studies of tubulin, actin, and vimentin showed no difference between Alzheimer and the control fibroblasts. Western blotting studies of vimentin revealed five partial degradation products in 50 K-57 K Da. molecular size region, but no difference in the degradation pattern was noticed between Alzheimer and the control fibroblasts. The size of fodrin molecule, however, was quite different between Alzheimer and the control fibroblasts. Comparing the molecular size of fodrin purified from the bovine brain, it is concluded that fodrin in Alzheimer fibroblasts is not degraded, while significant amount of fodrin in the control fibroblasts is partially degraded resulting in the smaller size of the 160 K and 200 K Da. molecular weight products.     

Brain spectrin( ) and brain spectrin( ): Conservation of structure and location within mammalian neural tissue

We demonstrate that the brain spectrin isoforms (240/235) and (240/235E) are present in all mammalian species studied (human, bovine, mouse, and rat). Immunohistochemistry with a panel of eleven polyclonal antibodies have indicated an identical localization of the brain spectrin isoforms in all mammalian species. Brain spectrin( ) is found primarily in axons, and brain spectrin(240/235E) primarily in cell bodies and dendrites. Immunoprecipitation and Western blotting studies have indicated that the subunit molecular weights of brain spectin( ) and ( ) are identical in all mammalian species. We demonstrate that when proteolysis is not completely blocked during immunoprecipitation studies, the 235 kDa subunits are converted to a 230 kDa polypeptide [brain spectrin( )] and a 232 kDa polypeptide [brain spectrin( )]. Finally, we show that both the α and β subunits of brain spectrin(240/235) and brain spectrin(240/235E) are antigenically distinct in every species examined. These studies indicate that previous findings on the structure, location, and function of mouse brain spectrin isoforms can now be generalized to all mammalian species.     

Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes

Protease-activated receptor-1 (PAR1) is a G-protein coupled receptor that is proteolytically activated by blood-derived serine proteases. Although PAR1 is best known for its role in coagulation and hemostasis, recent findings demonstrate that PAR1 activation has actions in the central nervous system (CNS) apart from its role in the vasculature. Rodent studies have demonstrated that PAR1 is expressed throughout the brain on neurons and astrocytes. PAR1 activation in vitro and in vivo appears to influence neurodegeneration and neuroprotection in animal models of stroke and brain injury. Because of increasing evidence that PAR1 has important and diverse roles in the CNS, we explored the protein localization and function of PAR1 in human brain. PAR1 is most intensely expressed in astrocytes of white and gray matter and moderately expressed in neurons. PAR1 and GFAP co-localization demonstrates that PAR1 is expressed on the cell body and on astrocytic endfeet that invest capillaries. PAR1 activation in the U178MG human glioblastoma cell line increased PI hydrolysis and intracellular Ca(2+), indicating that PAR1 is functional in human glial-derived tumor cells. Primary cultures of human astrocytes and human glioblastoma cells respond to PAR1 activation by increasing intracellular Ca(2+). Together, these results demonstrate that PAR1 is expressed in human brain and functional in glial tumors and cultures derived from it. Because serine proteases may enter brain tissue and activate PAR1 when the blood brain barrier (BBB) breaks down, pharmacological manipulation of PAR1 signaling may provide a potential therapeutic target for neuroprotection in human neurological disorders.     

Fodrin degradation by calcium-activated neutral proteinase (CANP) in retinal ganglion cell neurons and optic glia: preferential localization of CANP activities in neurons

The activity of calcium-activated neutral proteinases (CANPs) toward endogenous substrates was measured in axons of retinal ganglion cell (RGC) neurons and separately in adjacent optic glia under in vitro conditions that preserved the ultrastructure and anatomic relationships between these cellular elements. RGC neurons and optic glia both expressed CANP activity. In contrast to RGC axons, which contained at least two CANP activities with calcium requirements in the millimolar (CANP A) and micromolar (CANP B) range (Nixon et al., 1985), CANP activity in optic glia was detectable only at millimolar calcium concentrations. When maximally activated, CANP(s) in optic glia exhibited a broad specificity for endogenous proteins but degraded larger proteins at a faster rate. The cytoskeletal protein fodrin (brain spectrin) was among the most susceptible endogenous substrates in RGC axons or glia. The similar properties of fodrin in neurons and glia, including its susceptibility to a purified millimolar calcium-sensitive brain CANP (mCANP), provided the basis for using this protein as a substrate to compare the relative activity of neuronal and glial CANPs in situ. Fodrin degradation mediated by CANPs proceeded at least 6 X more rapidly in intact RGC axons than in optic glia. Comparable differences in the relative degradation rates of the total neuronal and glial protein pools were also observed. These results indicate that the potential activity of CANPs is substantially greater in RGC neurons than in glia. The enormous potential activity and preferential localization of multiple CANP activities in RGC neurons support previously hypothesized roles for CANPs in the processing of axonally transported proteins and in the regulation of neuronal cytoskeletal dynamics and geometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cue reactivity in nicotine and tobacco dependence: a “multiple-action” model of nicotine as a primary reinforcement and as an enhancer of the effects of smoking-associated stimuli

The present paper proposes a model for the identification and the validation of brain processes and mechanisms underlying smokers' cue reactivity. Smoking behaviour is maintained by the reinforcing properties of nicotine, but it was also proposed that nicotine enhances the conditioned value of smoking and nicotine-associated stimuli. In fact, it is widely reported that the exposure of smokers to smoking/nicotine-associated stimuli induces cue reactivity, which is a vast array of physiological, psychological and behavioural responses. Imaging studies are revealing neuroanatomical correlates of cue reactivity in brain areas involved in motivational, emotional, cognitive processes and in their integration. Behavioural studies in laboratory animal models have shown analogies between the effects of nicotine-associated stimuli and cue reactivity effects in smokers. Lesion and mapping studies with nicotine reported brain activation patterns in cortico-limbic areas similarly to those obtained with imaging studies in humans. Although only limited studies have been done with nicotine-associated stimuli in animals, the identification of molecular mechanisms underlying other drugs of abuse-associated cue effect may help to propose potential common molecular mechanisms for nicotine cues. These findings suggest that smoking/nicotine-associated stimuli are processed at two levels: (i), bottom-up, automatic processing in a parallel fashion through ascendant pathways, to activate attentional functions; (ii), top-down, in a serial fashion from cortical areas, to modulate sensory inputs and motor control. It appears that nicotine increase information processing at both levels so as to establish and to amplify the conditioned value of smoking cues.     

Neural (N-) cadherin,a synaptic adhesion molecule,is induced in hippocampal mossy fiber axonal sprouts by seizure

Aberrant mossy fiber sprouting and synaptic reorganization are plastic responses in human temporal lobe epilepsy, and in pilocarpine-induced epilepsy in rodents. Although the morphological features of the hippocampal epileptic reaction have been well documented, the molecular mechanisms underlying these structural changes are not understood. The classic cadherins, calcium-dependent cell adhesion molecules, are known to function in development in neurite outgrowth, synapse formation, and stabilization. In pilocarpine-induced status epilepticus, the expression of N-cadherin mRNA was sharply upregulated and reached a maximum level (1- to 2.5-fold) at 1- to 4 weeks postseizure in the granule cell layer and the pyramidal cell layer of CA3. N-cadherin protein was correspondingly increased and became concentrated in the inner molecular layer of the dentate gyrus, consistent with the position of mossy fiber axonal sprouts. Moreover, N-cadherin labeling was punctate; colocalized with definitive synaptic markers, and partially localized on polysialated forms of neural cell adhesion molecule (PSA-NCAM)-positive dendrites of granule cells in the inner molecular layer. Our findings show that N-cadherin is likely to be a key factor in responsive synaptogenesis following status epilepticus, where it functions as a mediator of de novo synapse formation.     

Calpain induces proteolysis of neuronal cytoskeleton in ischemic gerbil forebrain

We investigated the relationship between the activity of calcium-dependent protease (calpain) and the ischemic neuronal damage. We also investigated the mechanism of ischemic resistance in astrocytes. In gerbil, a 10-min forebrain ischemia was induced by occlusion of both common carotid arteries. The calpain-induced proteolysis of cytoskeleton (fodrin) was examined by immunohistochemistry. Immunolocalization of micro and m-calpain was also examined. Intact fodrin was observed both in neurons and astrocytes, but proteolyzed fodrin was not observed in normal brain. Fifteen minutes after ischemia, proteolysis of fodrin took place in putamen, parietal cortex and hippocampal CA1. The proteolysis extended to thalamus 4 h after ischemia after which the immunoreactivity faded down in all areas except hippocampus. On day 7, the proteolysis was still observed only in hippocampus. Neurons with the proteolysis of soma resulted in neuronal death. Throughout the experiment, the proteolysis was not observed in astrocytes. micro -Calpain was observed only in neurons but m-calpain was observed both in neurons and astrocytes. The ischemia induced only micro -calpain activation, which resulted in fodrin proteolysis of neurons with differential spatial distribution and temporal course. The proteolysis was developed rapidly and was completed within 24 h in all vulnerable regions except hippocampal CA1. The proteolysis preceded the neuronal death. The mechanism of the proteolysis seemed to be involved by Ca(2+) influx via glutamate receptor and rapid neuronal death seemed reasonable. The reason why neuronal death in CA1 evolved slowly was not clarified. In astrocytes, fodrin was not proteolyzed by m-calpain. The low Ca(2+)-sensitivity of m-calpain may be the reason of ischemic resistance in astrocytes.     

Sodium butyrate induces major morphological changes in C6 glioma cells that are correlated with increased synthesis of a spectrin-like protein

Butyrate induced flattening and development of cell processes in rat glioma (C6) cells and this change was correlated with an increase in the synthesis of a polypeptide doublet with an apparent molecular weight of about 200 kDa. Blot analysis revealed that at least one of these polypeptides was a spectrin-like protein. Indirect immunofluorescence studies with the spectrin antiserum indicated that the antigen was present in the cell bodies, and also in the cell processes. Thus fodrin may be one the major targets for the action of butyrate on C6 cells.     

Effect of clonidine on the secretion of anterior pituitary hormones in heroin addicts and normal volunteers

Neuroendocrine effects of intravenous injections of clonidine, 0.15 mg, were investigated in 13 heroin addicts and 14 normal control subjects. The study was designed to determine whether continuous opiate administration leads to the development of hypersensitive alpha 2-adrenergic receptors. The peak increments in levels of plasma growth hormone (GH) and beta-endorphin induced by clonidine did not differ between heroin addicts and normal control subjects. At no time interval could the clonidine-induced rise in GH levels in addicts be differentiated from that induced by placebo. Clonidine failed to alter plasma prolactin, gonadotropin, or thyrotropin levels in either heroin addicts or controls. Since clonidine's neuroendocrine effects are reportedly due to the activation of postsynaptic alpha 2-adrenoceptors, it appears that (1) continuous opiate use does not lead to the development of hypersensitive alpha 2-adrenergic receptors involved in neuroendocrine mechanisms and (2) brain norepinephrine does not play a role in the regulation of tonic prolactin, gonadotropin, and thyrotropin secretion in man.     

Inhibition of calpain-mediated cell death by a novel peptide inhibitor

Calpains are calcium- and thiol-dependent proteases whose overactivation and degradation of various substrates have been implicated in a number of diseases and conditions such as cardiovascular dysfunction and ischemic stroke. With increasing evidence for calpain's role in cellular damage, the development of calpain inhibitors continues to be an important objective. Previously, we identified a highly specific calcium-dependent, calpain interacting peptide L-S-E-A-L, that showed homology to domains A and C of the only known endogenous inhibitor of calpains, calpastatin. This suggested that LSEAL had a calpain inhibitory function and synthetic LSEAL inhibited calpain I and II proteolysis of two calpain substrates, tau and alpha-synuclein. In the present study, we demonstrate that synthetic LSEAL is membrane permeable and is a potent inhibitor in two established models of calpain-mediated cell death using primary rat cortical neurons and SH-SY5Y neuroblastoma cells. In addition, we show that LSEAL inhibits calpain activity towards protein substrates as detected by an antibody to a calpain-specific breakdown product of spectrin. Taken together, these results suggest that LSEAL may represent a novel calpastatin mimetic with the potential for benefit in conditions of increased calpain activity such as stroke, traumatic brain injury or heart attack.     

Brain spectrin: A review

Red blood cell spectrin, along with actin and several other proteins, forms a skeletal meshwork on the cytoplasmic surface of the erythrocyte plasma membrane. This structure is thought to maintain red blood cell shape, membrane structural stability, and cellular elasticity, as well as controlling the lateral mobility of integral membrane proteins and the transbilayer movement of phospholipids. It is now clearly established that spectrin-related molecules are ubiquitous structural elements subjacent to the plasma membrane of mammalian and avian nonerythroid cells. In this review, we present the current knowledge concerning brain spectrin. Brain spectrin is an approximately 11S, approximately 1,000,000 molecular weight (alpha beta)2 tetramer containing subunits of 240,000 (alpha) and 235,000 (beta) molecular weight. It is present in the cortical cytoplasm of all neuronal cell bodies and processes, and to a lesser extent in glial cells. Its involvement in the actin-membrane interaction, as well as other proposed functions in the nervous system is discussed.     

Evidence for caspase-mediated cleavage of AMPA receptor subunits in neuronal apoptosis and Alzheimer's disease.

In Alzheimer's disease (AD) synapses degenerate and neurons die in brain regions involved in learning and memory processes. Although the cellular and molecular mechanisms underlying the neurodegenerative process in AD are unclear, increasing evidence suggests roles for amyloid beta-peptide (Abeta) and biochemical cascades associated with a form of programmed cell death called apoptosis. Cysteine proteases of the caspase family are activated in neurons undergoing apoptosis and apparently play a major role in the cell death process by cleaving yet-to-be-identified substrates. We now report that caspase activity is increased in brain tissue and neurons from AD patients, and in cultured hippocampal neurons undergoing apoptosis after exposure to amyloid beta-peptide (Abeta). Western blot analyses using antibodies against different subunits of 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types of ionotropic glutamate receptors indicate that AMPA receptor subunits (GluR1, GluR2/3, and GluR4), but not NMDA receptor subunits (NR1 and NR2A), are proteolytically cleaved after exposure of hippocampal neurons to apoptotic insults, including Abeta, and that the caspase inhibitor zVAD-fmk suppresses such cleavage. Western blot analysis of brain tissue from AD patients and age-matched controls revealed evidence for increased proteolysis of AMPA receptor subunits in AD. Our data suggest roles for caspase-mediated cleavage of AMPA receptor subunits in modifying neuronal responsivity to glutamate and in the neurodegenerative process in AD.     

Proteolysis of NR2B by calpain in the hippocampus of epileptic rats

Overactivation of N-methyl-D-aspartate receptors is known to mediate excitotoxicity due to excessive entry of calcium, leading to the activation of several calcium-dependent enzymes. Calpains are calcium-activated proteases that appear to play a role in excitotoxic neuronal death. Several cellular proteins are substrates for these proteases, particularly the N-methyl-D-aspartate receptor. Recently, cleavage of NR2B subunits has been implicated in excitotoxic neurodegeneration in ischemia. In this work, we investigated the proteolysis by calpains of NR2B subunits of the N-methyl-D-aspartate receptor in the hippocampus of epileptic rats. Our results show that cleaved forms of NR2B subunits are formed after status epilepticus, in the same areas of the hippocampus where calpain activation was detected by immunohistochemical staining of calpain-specific spectrin breakdown products.