Mary Lou Beermann

Calcium-Activated Neutral Proteinase (Calpain) System in Aging and Alzheimer's Diseasea

Calpains (CANPs) are a family of calcium-dependent cysteine proteases under complex cellular regulation. By making selective limited proteolytic cleavages, they activate or alter the regulation of certain enzymes, including key protein kinases and phosphatases, and induce specific cytoskeletal rearrangements, accounting for their suspected involvement in intracellular signaling, vesicular trafficking, and structural stabilization. Calpain activity has been implicated in various aging phenomena, including cataract formation and erythrocyte senescence. Abnormal activation of the large stores of latent calpain in neurons induces cell injury and is believed to underlie neurodegeneration in excitotoxicity, Wallerian degeneration, and certain other neuropathologic states involving abnormal calcium influx. In Alzheimers disease, we found the ratio of activated calpain I to its latent precursor isoform in neocortex to be threefold higher than that in normal individuals and those with Huntingtons or Parkinsons disease. Immunoreactivity toward calpastatin, the endogenous inhibitor of calpain, was also markedly reduced in layers II-V of the neocortex in Alzheimers disease. The excessive calpain system activation suggested by these findings represents a potential molecular basis for synaptic loss and neuronal cell death in the brain in Alzheimers disease given the known destructive actions of calpain I and its preferential neuronal and synaptic localization. In surviving cells, persistent calpain activation may also contribute to neurofibrillary pathology and abnormal amyloid precursor protein trafficking/processing through its known actions on protein kinases and the membrane skeleton. The degree of abnormal calpain activation in the brain in Alzheimers disease strongly correlated with the extent of decline in levels of secreted amyloid precursor protein in brain. Cytoskeletal proteins that are normally good calpain substrates become relatively calpain resistant when they are hyperphosphorylated, which may contribute to their accumulation in neurofibrillary tangles. As a major effector of calcium signals, calpain activity may mirror disturbances in calcium homeostasis and mediate important pathologic consequences of such disturbances.

Calpain I activation in rat hippocampal neurons in culture is NMDA receptor selective and not essential for excitotoxic cell death

Administration of glutamate (100 microM) to primary cultures of rat hippocampal neurons for 1 h led to calpain I activation as determined by monitoring the extent of spectrin breakdown with the antibodies designed to specifically recognize the calpain I-mediated spectrin breakdown products. Based on the studies with subtype selective antagonists of glutamate receptors, glutamate caused calpain I activation specifically through the activation of the NMDA receptor. In parallel experiments, the magnitude and the temporal profiles of Ca2+ rise were determined by Fura-2 microfluorimetry. Ca2+ influx through voltage-sensitive Ca2+ channels, even though leading to substantial Ca2+ rise, did not by itself activate calpain I. These results indicate that for calpain I activation, the source of Ca1+ influx is more important than the magnitude of Ca2+ rise. Glutamate-mediated calpain I activation was fully blocked by preincubation (30 min) of the cultures with calpain inhibitor I, calpain inhibitor II, or calpeptin (all 10 microM). The presence of calpain inhibitors did not, however, in any way ameliorate the massive excitotoxicity resulting from 16 h exposure to glutamate, indicating that calpain I activation and excitotoxicity are not causally related events. Similarly, preincubation with any of the tested calpain inhibitors was detrimental to the clearance of neuritic from a 10-min exposure to glutamate. Additionally, the presence of calpain inhibitors was detrimental to the clearance of neuritic varicosities resulting from a short-term sublethal exposure to glutamate, suggesting that a physiological level of calpain I activation might actually play an important homeostatic role in the restoration of normal cytoskeletal organization.

Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH-SY5Y Neuroblastoma Cells: Modifications During Differentiation

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.

Calcium Influx into Human Neuroblastoma Cells Induces ALZ-50 Immunoreactivity: Involvement of Calpain-Mediated Hydrolysis of Protein Kinase C

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.

Degradation of protein kinase Cα and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions: Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC

Proteolytic cleavage of protein kinase C (PKC) under cell‐free conditions generates a co‐factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH‐SY‐5Y cells with 2‐O‐tetradecanoylphorbol 13‐acetate resulted in complete down‐regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore‐mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell‐free degradation of partially purified SH‐SY‐5Y PKCα by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain‐mediated degradation of purified rat brain PKCα. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an ‘unregulated’ kinase, since its persistence is apparently strictly regulated by proteolysis.

Enhancement of Neurite Outgrowth Following Calpain Inhibition Is Mediated by Protein Kinase C

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.

Multiple Proteases Regulate Neurite Outgrowth in NB2a/dl Neuroblastoma Cells

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.

Aluminum Alters the Electrophoretic Properties of Neurofilament Proteins: Role of Phosphorylation State

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.

Acute rise in the concentration of free cytoplasmic calcium leads to dephosphorylation of the microtubule-associated protein tau.

The objective of this study was to asses the response of the microtubule-associated protein tau to acute rise in the concentration of free cytoplasmic calcium ([Ca2+]i) in rat cortical neurons and mouse cerebellar granule cells in culture. One-hour exposure to glutamate (100 microM), N-methyl-D-aspartate (100 microM), KCl (50 mM), and ionomycin (5 microM) led to tau protein dephosphorylation as indicated by an appearance of additional faster moving bands on Western immunoblots with a phosphorylation-independent antibody and an increase in the tau-1 immunoreactivity associated with the appearance of an additional faster moving band. Lowering the extracellular concentration of Ca2+ to less than 1 microM fully prevented the drug-induced tau protein dephosphorylation indicating a dependence on Ca2+ influx from the extracellular environment. Administration of okadaic acid (inhibitor of phosphatase 1/2A) simultaneously with the above mentioned drugs decreased the drug-mediated dephosphorylation. Pre-incubation with okadaic acid fully prevented the dephosphorylation. Treatment with cypermethrin (inhibitor of phosphatase 2B) was without effect when administered either alone, simultaneously with the drugs, or pre-incubated. These findings indicate that, independently of the influx pathway, [Ca2+]i elevation leads to dephosphorylation of the microtubule-associated protein tau and implicate phosphatase 1 and/or 2A in the process.

Staurosporine-induced morphological differentiation of human neuroblastoma cells

SH-SY-5Y human neuroblastoma cells rapidly elaborated an extensive network of neuritic processes following treatment with staurosporine, an inhibitor of protein kinase C. These neurites were retracted within 24hr following removal of inhibitor. Another inhibitor of protein kinase C, H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], also induced rapid, reversible neurite outgrowth. However, neurites induced by these two inhibitors were morphologically distinct: staurosporine-treated cells elaborated a branching neuritic network adjacent to cell bodies, with some longer, unbranching neurites extending out of this network, while H7-treated cells elaborated only long, unbranching neurites. HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], which inhibits of cAMP- and cGMP-dependent protein kinases but not protein kinase C, did not induce neuritogenesis. Staurosporine-induced neurite outgrowth did not require protein synthesis but did require microtubule assembly, suggesting that cells contained the necessary components for neuritogenesis, and that alterations in protein phosphorylation alone was sufficient to initiate neurite outgrowth by rearrangement of existing structures or cytoskeletal precursors. These results implicate phosphorylation in the regulation of neuronal differentiation and neuritogenesis.