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Notes: Abstract: We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal (“C1”) and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down-regulation following TPA treatment. Cell-free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.

Notes: Abstract: Calcium-activated neutral proteases (calpains) are regulated by specific endogenous protein inhibitors, the calpastatins, which are widely distributed in mammalian tissues. Calpastatins from different species or in various tissues from the same species exhibit considerable size heterogeneity on sodium dodecyl sulfate (SDS) gels, reflecting both transcriptional and posttranslational regulation. This heterogeneity has complicated previous biochemical characterizations. In this study, we purified bovine brain calpastatin to homogeneity. The inhibitor was purified 2,463-fold from a cytosolic fraction of fresh bovine cerebral cortex by chromatographies on diethylaminoethyl cellulose, Ultrogel AcA44, phenyl-Sepharose, concanavalin A-Sepharose, and Q-Sepharose. The major calpastatin displayed a native molecular mass of 250–300 kDa by gel filtration and was composed of 125-kDa polypeptide chains by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Small amounts of a 68-kDa calpastatin fragment were detected particularly in molecules exhibiting smaller native molecular mass (250 kDa). When electroeluted from SDS gels, the 125- and 68-kDa polypeptides each inhibited calpain. The purified protein was strongly immunoreactive toward antibodies raised against a synthetic peptide, CEKLGEKEETIPPDYR, shown to be a conserved repetitive motif in the calpastatin gene or a recombinant polypeptide corresponding to domains L and 1 of human calpastatin. Calpastatins purified from bovine and human erythrocytes exhibited molecular masses of 78 and 68 kDa, respectively, by SDS-PAGE. Both erythrocyte calpastatins reacted strongly with antibodies against the conserved sequence but not with antibodies raised against domains L and 1 of human calpastatin, indicating that the erythrocyte inhibitors lack these two domains. The heat-stable inhibitor was effective toward calpain I and calpain II and showed no inhibitory activity toward other proteases, including trypsin, chymotrypsin, cathepsin D, bromelain, and papain. Brain calpastatin therefore shares molecular properties with calpastatins from other tissues but is distinct from the erythrocyte form.

Notes: Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-α and -β1 were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-δ and -ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-β-phorbol or staurosporine, and that protein kinase C-ɛ is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-α and -ɛ decreased, and protein kinase C-β1, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.

Notes: Abstract: Exposure of each of the three neurofilament proteins (NFPs) to AlCl3 resulted in their failure to migrate into sodium dodecyl sulfate (SDS)-containing gels. This effect was dependent on length of incubation (minimum, 2 h) and AlCl3 concentrations (minimum, 50 μM) and was not reversed by 20% SDS, 6 M urea, freeze-thawing, boiling, or extensive dialysis. The migration of vimentin and glial fibrillary acidic protein was not affected by AlCl3. The high-molecular-weight neurofilament subunit (NF-H) entered SDS-containing gels after exposure to aluminum lactate but migrated aberrantly as a long high-molecular-weight streak. Migration of the 160-kDa α-chymotryptic cleavage product of NF-H, which contains the higher phosphorylated tail domain, was also prevented from migrating into SDS-containing gels by AlCl3. Dephosphorylation of NF-H and the middle-molecular-weight neurofilament subunit (NF-M) eliminated these effects on gel migration. EDTA, EGTA, MgCl2, CaCl2, or FeCl3 had no effect on NF-H or NF-M migration; furthermore, preincubation with, or simultaneous exposure to, CaCl2 or FeCl3 did not alter the effect of AlCl3. One interpretation of these results is that Al3+ interacts with phosphate groups on extensively phosphorylated C-terminal sidearms of NFPs, resulting in intermolecular cross-linking. These findings demonstrate a direct effect of aluminum on NFPs and provide a possible mechanism for neurofilament accumulation in perikarya during aluminum intoxication.

Notes: Abstract: The millimolar form of calcium-activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000 g centrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion-exchange chromatography on DEAE-cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X-100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.

Notes: Abstract: We studied the effects of aluminum salts on the degradation of human neurofilament subunits (NF-H, NF-M, and NF-L, the high, middle, and low molecular weight subunits. respectively) and other cytoskeletal proteins using calcium-activated neutral proteinase (calpain) purified from human brain. Calpain-mediated proteolysis of NF-L, tubulin, and glial fibrillary acidic protein (GFAP), three substrates that displayed constant digestion rates in vitro, was inhibited by AlCl3 (IC50= 200 μM) and by aluminum lactate (IC50= 400 μM). Aluminum salts inhibited proteolysis principally by affecting the substrates directly. After exposure to 400 μM aluminum lactate and removal of unbound aluminum, human cytoskeletal proteins were degraded two- to threefold more slowly by calpain. When cytoskeleton preparations were exposed to aluminum salt concentrations of 100 μM or higher, proportions of NF-M and NF-H formed urea-insoluble complexes of high apparent molecular mass, which were also resistant to proteolysis by calpain. Complexes of tubulin and of GFAP were not observed under the same conditions. Aluminum salts irreversibly inactivated calpain but only at high aluminum concentrations (IC50= 1.2 and 2.1 μM for aluminum lactate and A1C13, respectively), although longer exposure to the ion reduced by twofold the levels required for protease inhibition. These interactions of aluminum with neurofilament proteins and the effects on proteolysis suggest possible mechanisms for the impaired axoplasmic transport of neurofilaments and their accumulation in neuronal perikarya after aluminum administration in vivo.

Notes: Abstract: Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in μ-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total τ (migrating at 46–52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive τ isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total τ levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive τ isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive τ isoforms. These data therefore indicate that calpain may regulate τ levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered τ phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive τ isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of τ to A68 by hyperactivation of the calpain/PKC system.

Notes: Abstract: Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH-SY5Y differentiates when exposed to 12-tetradecanoyl-13-acetyl-β-phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron-specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF-H). TPA, 12-deoxy-13-tetradecanoyl-β-phorbol, and staurosporine, but not DAG or 4-O-methyl-TPA, caused neurite outgrowth. Neuron-specific enolase was expressed in cells treated with TPA and 12-deoxy-13-tetradecanoyl-β-phorbol but not with DAG, staurosporine, or 4-O-methyl-TPA. NF-H increased in the perikarya of cells treated with DAG and 4-O-methyl-TPA, in processes and to varying degrees in perikarya of TPA- and 12-deoxy-13-tetradecanoyl-β-phorbol-treated cells, but much more in the processes than in the perikarya of staurosporine-differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH-SY5Y cells by different mechanisms, and that the five-membered/seven-membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.

Notes: Abstract: Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hi-rudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucyl-leucyl-norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10-6M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucyl-leucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain.

Notes: Abstract: In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton-insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/dl neuroblastoma cells also contain Triton-soluble, extensively phosphorylated 200-kDa high molecular weight neurofilament subunits (NF-H). High-speed centrifugation (100,000 g) of the Triton-soluble fraction for 1 h sedimented some, but not all, soluble NF-H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF-H subunits in this fraction are assembled into (Triton-soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton-soluble 200-kDa NF-H variants. Because only extensively phosphorylated NF-H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse-chase analyses, radiolabeled 200-kDa NF-H migrated into the 100,000 g particulate fraction of Triton-soluble extracts before its arrival in the Triton-insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton-insoluble, extensively phosphorylated NF-H, contain a sizeable pool of Triton-soluble extensively phosphorylated NF-H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF-H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF-H into neurofilaments and integration into the Triton-insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides. Because NF-H can be extensively phosphorylated in perikarya, additional mechanisms besides differential localization of the responsible kinase systems must account for the segregation of Triton-insoluble NF-H in NB2a/d1 neurites.