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

Lesions of the rat entorhinal cortex cause extensive synaptic restructuring and perturbation of calcium regulation in the dentate gyrus of hippocampus. Calpain is a calcium-activated protease which has been implicated in degenerative phenomena in muscles and in peripheral nerves. In addition, calpain degrades several major structural neuronal proteins and has been proposed to play a critical role in the morphological changes observed following deafferentation. In this report we present evidence that lesions of the entorhinal cortex produce a marked increase in the breakdown of brain spectrin, a substrate for calpain, in the dentate gyrus. Two lines of evidence indicate that this effect is due to calpain activation: (i) the spectrin breakdown products observed following the lesion are indistinguishable from calpain-generated spectrin fragments in vitro; and (ii) their appearance can be reduced by prior intraventricular in fusion of leupeptin, a calpain inhibitor. Levels of spectrin breakdown products are increased as early as 4 h post-lesion, reach maximal values at 2 days, and remain above normal to some degree for at least 27 days. In addition, a small but significant increase in spectrin proteolysis is also observed in the hippocampus contralateral to the lesioned side in the first week postlesion. At 2 days postlesion the total spectrin immunoreactivity (native polypeptide plus breakdown products) increases by 40%, suggesting that denervation of the dentate gyrus produces not only an increased rate of spectrin degradation but also an increased rate of spectrin synthesis. These results indicate that calpain activation and spectrin degradation are early biochemical events following deafferentation and might well participate in the remodelling of postsynaptic structures. Finally, the magnitude of the observed effects as well as the stable nature of the breakdown products provide a sensitive assay for neuronal pathology.     

Compartmentation and glycoprotein substrates of calpain in the developing rat brain.

An activated form of calpain I associates with telencephalic membranes in a developmentally regulated fashion during early postnatal ontogeny. During this period, the cytoskeletal component spectrin is available and appears to be differentially susceptible to calpain-mediated cleavage. Lectin blotting techniques demonstrated that the leupeptin-sensitive action of calpain is primarily directed toward large proteins which are glycoconjugate in nature; neuronal cell adhesion molecules are among the glycoproteins whose associations with the telencephalic membranes decrease due to calpain activity. These data suggest that cytoplasmic calpain is translocated to the membrane during early brain development in order to act on the cytoskeletal and adhesive structures responsible in part for neuronal shape and function.     

Effect of calpain on the composition and structure of postsynaptic densities

Endogenous calcium-activated proteases, the calpains, are thought to play a role in the regulation of postsynaptic function. Here we characterize some biochemical and morphological effects of calpain on isolated postsynaptic densities (PSDs). When a PSD preparation from rat forebrain was treated with exogenous calpain, many proteins, including spectrin, tubulin and the α-subunit of calcium calmodulin-dependent protein kinase (α-CaM kinase), were proteolyzed at varying rates, while another major protein, actin, remained intact. The selectivity of calpain action became more apparent in experiments designed to achieve limited proteolysis by using a lower calpain-to-protein ratio; it was possible to obtain extensive breakdown of spectrin with no decrease in the levels of either tubulin, α-CaM kinase, or actin. Electron microscopy of freeze-substituted preparations showed that limited calpain action caused a partial unraveling of the PSD, in which the characteristic central dense lamina became wider and less dense. We interpret these changes as due to calpain-mediated breakdown of cross-bridging elements, leading to a partial unraveling of the central PSD lamina. Opening up of the PSD structure following limited calpain action could facilitate exposure of previously occluded functional sites within the PSD and contribute to the modification of the synaptic function. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

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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.     

Subcellular compartmentalization of calcium-dependent and calcium-independent neutral proteases in brain

In the present experiments, we studied the subcellular distribution of three types of extralysosomal, neutral proteolytic activities in rat telencephalon: (1) nonthiol proteases (NTP), (2) thiol proteases (TP), and (3) calcium-activated thiol proteases (calpains I and II). Subcellular fractionation was performed by using conventional differential and sucrose-gradient centrifugation techniques. The only significant proteolytic activity detected in crude homogenates could be assigned to calpain II, the high-threshold calcium-activated protease. Within the primary fractions prepared from the homogenates, the highest levels of calpain II were found in S3, or the soluble cytoplasmic fraction. Significant activity of the enzyme was also present in P2, the crude mitochondrial/synaptosomal fraction. In contrast, the specific activity of calpain I was greatest in P2 with somewhat lesser enzymatic activity in P1 and S3. Most of the calpain I in P2 was recovered after differential centrifugation through sucrose gradients and lysis of the resultant subfractions. In marked contrast, only a small percentage of the calpain II activity was recovered in the gradient bands. In all, calpain II appears to be predominantly localized in the soluble cytoplasmic compartment while the greatest concentrations of calpain I are found in the soluble components of small glial and neuronal processes (pinched off during homogenization) that constitute the P2 fraction. The highest specific activity of the calcium-independent proteases was obtained in P3, a fraction essentially devoid of calpain, with a secondary peak in P2. Subfractionation of P2 revealed that calcium-independent TP in P2 was associated with mitochondria while the calcium-independent NTP was more uniformly distributed across myelin, synaptosomes, and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ultrastructural localization of calcium-activated protease "calpain" in rat brain

Calpain I, a calcium-activated neutral protease which degrades a number of cytoskeletal proteins, has been implicated in the rapid turnover of structural proteins that may participate in synaptic plasticity. In the present study, an antibody raised against purified erythrocyte calpain I was biochemically characterized and demonstrated to specifically bind the Mr = 80,000 subunit of both rat erythrocyte and brain calpain I. This antibody was used to examine the cellular distribution of calpain I at the electron microscopic level in rat brain and spinal cord using the avidin-biotin immunocytochemical technique. Reaction product was observed throughout neuronal perikarya, within both axonal and dendritic processes, and within spine heads and necks. Postsynaptic densities in both shaft and spine synapses were also immunoreactive. Glial cell bodies and processes were densely stained. In both neurons and glia, the reaction product was deposited along cytoskeletal elements. The localization of calpain I immunoreactivity to glial processes suggests this degradative enzyme may play a role in the glial hypertrophy and process retraction seen in brain. The presence of the enzyme in spines and postsynaptic densities is consistent with the hypothesis that it is involved in the turnover of synaptic cytoskeleton, thus providing a means through which transient physiological events effect lasting changes in the chemistry and morphology of spines.     

Amelin and synapsin I are 4.1 related spectrin binding proteins in brain

How do synaptic vesicles move towards the presynaptic plasma membrane, fuse with that membrane, and release their contents during synaptic transmission? The answers to these questions at the molecular level are just beginning to be understood. Synapsin I is a neuron specific phosphoprotein that is associated with the cytoplasmic surface of synaptic vesicles. During synaptic transmission, the translocation of the synaptic vesicles to the presynaptic membrane of the neuron is thought to be mediated through changes in the phosphorylation state of synapsin I. It has been suggested that synapsin I is a spectrin binding protein related to the erythrocyte cytoskeletal protein 4.1, which binds to the terminal ends of the erythrocyte spectrin tetramer. The interaction of synapsin I (through brain spectrin) with the neuronal cytoskeleton may be essential for regulating the movement of synaptic vesicles towards the presynaptic plasma membrane. In addition, we have identified another protein in brain that is immunologically and structurally more closely related to erythrocyte 4.1 than is synapsin I. This protein, termed amelin, is localized in the cell body and dendrites of the neuron, whereas synapsin I is found exclusively in the synaptic terminals, suggesting that there is a family of erythrocyte 4.1 related proteins present in brain with distinct subcellular distribution and functions.     

Regional distribution and time-course of calpain activation following kainate-induced seizure activity in adult rat brain

Systemic injection of kainic acid (KA) in adult rat elicits a pattern of neuronal pathology which exhibits several features of human temporal lobe epilepsy. KA-induced seizure activity is accompanied by the activation of the calcium-dependent protease calpain in limbic structures. In the present study, we evaluated the spatio-temporal activation of calpain after the onset of seizure activity by immunohistochemistry using an antibody for the spectrin breakdown product (sbdp) generated by calpain-mediated spectrin proteolysis. In addition, we compared the changes in sbdp immunoreactivity with those in immunoreactivity to subunits of the Glu/AMPA receptors (GluR₁ andGluR₂₃). One hour after seizure onset, sbdp accumulation was observed in selected interneurons in stratum oriens and in the hilus of the dentate gyrus. By 4 h, sbdp immunoreactivity was prominent in dendritic fields of the hippocampus as well as in neurons in thalamus and piriform cortex. By 8 h, sbdp immunoreactivity had disappeared from interneurons but was localized in pyramidal cell bodies in hippocampus. Intense labeling of cell bodies and dendritic fields persisted until 5 days following KA treatment. Changes in GluR subunit immunoreactivity were mirror images of those seen for sbdp. In general, increased sbdp immunoreactivity in dendritic fields was associated with decreased (GluR₁ immunoreactivity. However, increased sbdp immunoreactivity in neuronal perikarya was also associated with increased GluR immunoreactivity. These results indicate that calpain activation following seizure onset exhibits a specific spatio-temporal pattern, with activation in restricted interneurons preceding widespread activation in pyramidal neurons. Calpain activation also precedes neuronal pathology and could thus represent an initial trigger for neuronal pathology. Finally, the results suggest that calpain activation produces rapid alterations in GluR subunit properties which could be involved in the hyperexcitability observed following seizure activity.     

Cytoskeletal protein degradation and neurodegeneration evolves differently in males and females following experimental head injury

The resulting neuropathological degeneration that occurs following a traumatic brain injury (TBI) is a consequence of both immediate and secondary neurochemical sequelae. Proteolysis of cytoskeletal proteins, triggered by calcium-mediated events, is believed to be a particularly significant contributor to TBI-induced neuronal death. To date, efforts to associate cytoskeletal degradation and neurodegeneration in TBI have been primarily qualitative or semiquantitative. The objectives of this study were (1). to quantitatively describe, over a posttraumatic time course, the relationship and mechanisms of cytoskeletal degradation (Western blot) and neurodegeneration (silver staining) in male and female mice following a moderately severe weight-drop impact-acceleration head injury; (2). to evaluate gender differences in the response to TBI; and (3). to examine the potential therapeutic window for future pharmacological treatment strategies. In male and female mice, we report a close correlation in the time courses of neurofilament M protein degradation and alpha-spectrin breakdown products (SBDP 150 and 145) with the peak magnitude of neurodegeneration, as quantified by silver staining. Evidence from the increased patterns of SBDPs suggests that both calpain and caspase-3 are involved. In general, males incurred peak protein degradation and neurodegeneration within 3 days after injury, while in females this did not occur until 14 days. The neuroprotective effects of estrogen are believed to be key factors in the superior outcome of female vs male mice following TBI. In mice, the therapeutic window of opportunity for pharmacological intervention aimed at limiting cytoskeletal degradation might be as much as 24 h following injury. Evidence of a protracted time course of cytoskeletal degradation, especially in females, suggests a potential for an extended treatment-duration following TBI.     

尼莫地平对局灶性脑缺血鼠脑血影蛋白的影响

目的 观察大脑中动脉（MCA）缺血后血影蛋白的动态变化,寻找反映脑缺血程度的形态学依据。并评价尼莫地平的脑保护作用。方法 制作大鼠MCA局灶性脑缺血模型。免疫组化法检测血影蛋白含量。缺血后10min,30min,3h,6h和48h观察血影蛋白动态变化。结果 缺血后10min缺血区域血影蛋白表达降低。24h达高峰,尼莫地平可减少缺血区神经元血影蛋白的降解,具有脑保护作用。结论 检测血影蛋白可反映神经元缺血损伤后的早期形态学改变,钙离子拮抗剂一尼莫地平有重要的脑保护作用。     

Active site-directed antibodies identify calpain II as an early-appearing and pervasive component of neurofibrillary pathology in Alzheimer's disease

Calpain proteases influence intracellular signaling pathways and regulate cytoskeleton organization, but the neuronal and pathological roles of individual isoenzymes are unknown. In Alzheimer's disease (AD), the activated form of calpain I is significantly increased while the fate of calpain II has been more difficult to address. Here, calpain II antibodies raised to different sequences within a cryptic region around the active site, which becomes exposed during protease activation, were shown immunohistochemically to bind extensively to neurofibrillary tangles (NFT), neuritic plaques, and neuropil threads in brains from individuals with AD. Additional `pre-tangle' granular structures in neurons were also intensely immunostained, indicating calpain II mobilization at very early stages of NFT formation. Total levels of calpain II remained constant in the prefrontal cortex of AD patients but were increased 8-fold in purified NFT relative to levels of calpain I. These results implicate activated calpain II in neurofibrillary degeneration, provide further evidence for the involvement of the calpain system in AD pathogenesis, and imply that neuronal calcium homeostasis is altered in AD.     

Neuronal recovery after moderate hypoxia is improved by the calpain inhibitor MDL28170

The role of calcium-activated proteolysis in hypoxic neuronal injury was investigated using an in vitro slice model of moderate hypoxia that mimics many features of an ischemic penumbra. The calpain inhibitor, MDL28170, significantly improved the recovery of synaptic responses in hippocampal slices following prolonged, moderate hypoxia without hypoxic depolarization. This finding further implicates calpain-mediated proteolysis in the development of neuronal injury following moderate metabolic challenge such as occurs in regions of partial ischemia.     

Neuropathological changes in the rat brain cortex in vitro: effects of kainic acid and of ion substitutions

The ionic mechanisms that may contribute to the neurotoxicity of kainic acid, were studied in a system of rat thin neocortical slices superfused in vitro. Slices superfused for 3 h under control conditions showed an essentially normal aspect when studied by light microscopy. Presence of 30 microM kainate in the superfusion fluid induced neuronal swelling, nuclear condensation and signs of necrosis in some cells, while other neurons, especially in deeper layers, appeared dark and condensed, with microvacuolation. The neuropil presented numerous profiles of swollen dendrites. When the slices were superfused with chloride-free medium, a large number of pyknotic neurons was seen. This was further enhanced by 30 microM kainate, which produced no swelling in this medium. These effects of Cl-free medium were almost entirely prevented in Cl-free medium without calcium and with 0.1 mM of EGTA. Sodium-free medium induced a marked neuronal swelling that was not much changed by kainate. When calcium in an otherwise normal superfusion fluid was reduced to 0.1 mM, a large number of pyknotic neurons, some with incrustations, were seen. Kainate (30 microM) in this low calcium medium led to a very large swelling and destruction of neurons, and to a spongy neuropil. These effects of kainate were greatly intensified in calcium-free-EGTA (0.1 mM) medium. Ca-free-EGTA medium by itself induced considerable neuronal and neuropil swelling. It is concluded that kainate induces neuronal swelling by a sodium- and chloride-dependent mechanism, and the enhancement of swelling in low calcium is due to an increased sodium uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylated MAP-1B isoforms in the developing mouse barrel cortex

Developmental expression of two phosphorylation modes of microtubule-associated protein 1B (MAP-1B) has been studied in the barrel cortex of mice at postnatal days (P)5, P12, P21 and P90 using immunocytochemistry with antibodies 125 and 150 that recognize phosphorylation modes II and I, respectively. The antibody 125 immunoreactive processes, identified as dendrites, are not yet detectable at P5; they are already present at P12 and become more evident at P21. In the barrel cortex of P90 animals the antibody 125 immunopositive dendrites are still present, although they are much less pronounced. The antibody 150 punctate immunostaining seen at P5 is not detectable at P12. At P21, however, thin immunopositive fibres appear, implicating a re-expression of the microtubule-associated protein 1B phosphorylation mode I in a portion of axons. The antibody 150 immunopositive axons are no longer present in the P90 barrel cortex. The re-expression of the MAP-1B phosphorylation mode I, which is a juvenile isoform characteristic for growing axons, may imply induction of mechanisms providing mouse barrel cortex neurons with the potency for plastic changes at a terminal stage of synaptogenesis.     

Anti-brain spectrin immunoreactivity in Alzheimer's disease: degradation of spectrin in an animal model of cholinergic degeneration

In a previous work, we described the existence of anti-brain spectrin auto antibodies in Alzheimer's disease (AD) patients (J. Neuroimmunol. 68 (1996) 39–44). In this report, we further support our previous observations, showing that sera from 9 out of 18 AD patients, but none of 14 control subjects, immunoreacted with spectrin synthesized by PC12 cells. In addition, degradation of brain spectrin was found to be greatly enhanced in the frontal cortex of rats subjected to an animal model of cholinergic degeneration. Our data suggest that spectrin degradation and generation of anti-spectrin auto antibodies may be related to the cholinergic degeneration encountered in AD.     

Translational suppression of calpain I reduces NMDA-induced spectrin proteolysis and pathophysiology in cultured hippocampal slices

Transfection of cultured hippocampal slices for five days with antisense oligonucleotides directed against mRNA encoding calpain I resulted in an approximately 60% decrease in the amount of caseinolytic activity stimulated by 10 μM calcium. Increases in a single proteolytic fragment of spectrin produced by 10–20 min of NMDA receptor stimulation were substantially ( 50%) reduced in antisense treated slices; this effect was not obtained in slices exposed to NMDA for 45 min. Attenuation of NMDA receptor-induced spectrin proteolysis by the antisense oligonucleotides was confirmed in immunoassays using antibodies that recognize multiple spectrin breakdown products and in immunocytochemical experiments with an antibody that defects an individual calpain I-mediated fragment. Translational suppression of calpain I did not detectably affect evoked synaptic responses but markedly improved their recovery from a 15 min infusion of NMDA. These results indicate that spectrin breakdown products provide a useful index of in situ calpain I activity and support the hypothesis that the protease plays a significant role in excitoxicity.     

Distribution of calcium-activated protease calpain in the rat brain

Calpain is a calcium-activated neutral protease that degrades a number of cytoskeletal proteins. It may participate in the maintenance of the cytoskeleton and in the rapid turnover of structural proteins associated with synaptic plasticity. Calpain may also be involved in the neurodegeneration that accompanies aging and age-related diseases. To aid in the interpretation of disease-related alterations in staining patterns, the present study examined calpain's normal distribution in the mammalian brain and spinal cord. A monoclonal antibody was employed with the avidin-biotin-peroxidase immunocytochemical technique on samples of rat tissue. Glia (astrocytes, microglia) and virtually all neurons were immunopositive, although neuronal processes exhibited varying staining patterns. The axonal staining pattern depended upon either the origin or destination of the process: those axons remaining within the brain (e.g., corpus callosum) were only lightly immunoreactive, whereas spinal cord and peripheral axons (trigeminal nerve) were more darkly labeled. The architecture of the dendritic tree determined the dendritic staining pattern: neurons with prominent apical and basal dendritic trees (e.g., pyramidal cells) were immunolabeled along their entire extent; labeling of multipolar cells (e.g., hilar cells of dentate gyrus) was limited to the proximal dendrites. The ubiquitous distribution of calpain argues against a primary role for the enzyme in the regional pattern of neuronal death seen in Alzheimer's disease. An alteration in the concentration, localization, or inhibition of the enzyme could, however, lead to the abnormal accumulations of cytoskeletal elements seen with the disease.     

Multimodal signaling by the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) promotes neurite extension

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains associate with hyaluronan and various secreted proteins to form a complex of extracellular matrix (ECM) that inhibits neural plasticity in the central nervous system (CNS). Chondroitinase treatment depletes PGs of their CS side chains and enhances neurite extension. Increasing evidence from in vivo models indicates that proteolytic cleavage of the PG core protein by members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of glutamyl-endopeptidases also promotes neural plasticity. The purpose of this study was to determine whether proteolytic action of the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of primary rat neurons with ADAMTS4 cDNA induced longer neurites, whether the neurons were grown on a monolayer of astrocytes that secrete inhibitory PGs or on laminin/poly-L-lysine substrate alone. Similar results were found when neurons were transfected with a construct encoding a proteolytically inactive, point mutant of ADAMTS4. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures also enhanced neurite extension in a dose-dependent manner, an effect demonstrated to be dependent on the activation of MAP ERK1/2 kinase. These results suggest that ADAMTS4 enhances neurite outgrowth via a mechanism that does not require proteolysis but is dependent on activation of the MAP kinase cascade. Thus a model to illustrate multimodal ADAMTS activity would entail proteolysis of CS-bearing PGs to create a loosened matrix environment more favorable for neurite outgrowth, and enhanced neurite outgrowth directly stimulated by ADAMTS signaling at the cell surface.