Neuronal intermediate filament protein alpha-internexin facilitates axonal neurite elongation in neuroblastoma cells.

We examined the localization and role of alpha-IN vs. other neuronal intermediate filaments before and during differentiation. Vimentin but not alpha-IN localized within filopodia-like neurites of undifferentiated cells. During differentiation, alpha-IN immunoreactivity accumulated within axonal neurites following vimentin but, as previously describe in neurons in situ, before the appearance of NF-L. We therefore manipulated alpha-IN synthesis, accumulation, and function in attempts to determine whether or not this intermediate filament species played a role in axonal development. Intracellular delivery of anti-alpha-IN antisense oligonucleotides and antibodies was permissive for neuritogenesis, yet compromised neurite elongation; this effect was further reflected in diminished levels of stabilized axonal microtubules. These data suggest that alpha-IN plays a role in the development of neuronal polarity. Relatively more alpha-IN than NF-L accumulated within the plastic axonal neurites induced following serum-deprivation, while stable, dbcAMP-induced neurites treatment contained equivalent levels of each. Protease inhibition increased NF-L and NF-H but not alpha-IN immunoreactivity within serum-deprived neurites, suggesting that proteolysis restricts NF-L accumulation pending neurite stabilization. To test the possibility that NF-H accumulation is dependent upon NF-L and cannot be mediated by alpha-IN, we examined levels of NF-H co-precipitated from cells with alpha-IN and NF-L. Virtually all newly synthesized NF-H co-precipitated with NF-L, while only a small percentage co-precipitated with alpha-IN. Finally, NF-H or NF-M were absent from the axon hillock or perikaryal area at the base of neurites, where alpha-IN immunoreactivity is prominent. These data extend earlier cell-free demonstrations that NF-H preferentially associates with NF-L rather than alpha-IN.

Coding sequence, genomic organization, chromosomal localization, and expression pattern of the signalosome component Cops2: the mouse homologue of Drosophila alien.

The Drosophila alien gene is highly homologous to the human thyroid receptor interacting protein, TRIP15/COPS2, which is a component of the recently identified signalosome protein complex. We identified the mouse homologue of Drosophila alien through homology searches of the EST database. We found that the mouse cDNA encodes a predicted 443-amino-acid protein, which migrates at approximately 50 kDa. The gene for the mouse alien homologue, named Cops2, includes 12 coding exons spanning approximately 30 kb of genomic DNA on the central portion of mouse chromosome 2. Mouse Cops2 is widely expressed in embryonic, fetal, and adult tissues beginning as early as E7.5. Mouse Cops2 cDNA hybridizes to two mRNA bands in all tissues at approximately 2.3 and approximately 4 kb, with an additional approximately 1.9-kb band in liver. Immunostaining of native and epitope tagged proteins localized the mouse Cops2 protein in both the cytoplasm and the nucleus, with larger amounts in the nucleus in some cells.

Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons.

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma, epsilon, zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.

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.

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.

Calcium influx into human neuroblastoma cells induces ALZ-50 immunoreactivity: involvement of calpain-mediated hydrolysis of protein kinase C.

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 mu-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 tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau 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 tau to A68 by hyperactivation of the calpain/PKC system.

Phospholipids inhibit proteolysis of protein kinase C alpha by mM calcium-requiring calpain.

The alpha isoform of protein kinase C (PKC alpha) is rapidly hydrolyzed by mM Ca(2+)-requiring calpain (calcium-activated neutral proteinase) under cell-free conditions (Shea et al, 1994, FEBS Lett. 350:223). In the present study, we demonstrate that this hydrolysis is inhibited by phosphatidyl serine, diacylglycerol, phosphatidyl choline, phosphatidyl inositol, and phosphatidic acid. With the exception of phosphatidic acid, these phospholipids did not directly inhibit calpain activity as evidenced by degradation of [14C]azocasein, suggesting that the nature of inhibition of calpain-mediated PKC alpha degradation is due to an effect of phospholipids on PKC alpha conformation. These findings suggest that m calpain-mediated PKC alpha hydrolysis may be specifically minimized at the plasma membrane, and leave open the possibility that such a mechanism exists in situ. In addition, the unique inhibition of calpain activity by phosphatidic acid suggests the existence of a specific mechanism by which this phospholipid regulates PKC alpha activity.

Aluminum treatment of intact neuroblastoma cells alters neurofilament subunit phosphorylation, solubility, and proteolysis.

Addition of 400 microM AlCl3 to the culture medium for 72 h has been previously shown to induce perikaryal whorls of intermediate-sized filaments in intact mouse NB2a/d1 neuroblastoma cells. Immunoblot analyses demonstrated that in vivo treatment of cells with aluminum induced the de novo appearance of extensively phosphorylated NF-H isoforms in cytoskeletons of undifferentiated cells and increased levels of these isoforms in differentiated cells. Neurofilament subunits isolated from intact cells treated with aluminum were resistant to dephosphorylation in vitro by alkaline phosphatase and to in vitro degradation by endogenous calcium-dependent protease(s). These alterations were accompanied by a greater tendency of neurofilaments to form insoluble aggregates after isolation. These findings demonstrate direct effects of aluminum on neurofilament subunits within intact neuronal cells similar to those previously demonstrated following in vitro exposure of isolated neurofilaments to aluminum.

Enhancement of neurite outgrowth following calpain inhibition is mediated by protein kinase C.

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.

Prehospital resuscitation--outcome in an urban area.

All prehospital resuscitations performed by emergency physicians in the city of Bochum, Germany, were recorded and evaluated prospectively from 1 August 1989 to 30 September 1990. Initially successful cardiopulmonary resuscitation (CPR) was achieved in 33.8% (alive at admission), and definitive success in 10.4% (discharged alive). Of the patients who presented with cardiac arrest before the arrival of the emergency physician, 28.4% could be resuscitated initially and 7.6% survived definitively. In patients who suffered circulatory arrest on or after the arrival of the emergency physician, the initial success rate of CPR was 51.7%, and the definitive success rate 11.1%. Although the initial success rate was significantly more favourable, the definitive outcome did not differ statistically between these two groups. Two time periods were compared. During the first period of 5 months only one base with two ambulances staffed with emergency physicians was available. During the second period of 9 months an additional base with a physician-staffed ambulance was established. This reorganization resulted in the reduced call to arrival time falling from 8.5 +/- 2.4 min to 7.6 +/- 2.4 min (univariate variance analysis: f = 8.89, d.f. = 1.31, p < 0.01). This decrease, however did not improve either the initial or the definitive success of CPR. From these results we conclude that further improvement of prehospital resuscitation can only be achieved to a small extent by reducing the call to arrival time of ambulances staffed with emergency physicians. Improvement is more likely to be seen when immediate resuscitation is performed by bystanders present at the scene.

Relative susceptibility of cytoskeleton-associated and soluble neurofilament subunits to aluminum exposure in intact cells. A possible mechanism for reduction of neurofilament axonal transport during aluminum neurotoxicity.

Previous studies have demonstrated the appearance of phosphorylated neurofilament (NF) subunits within perikaryal cytoskeletons following aluminum exposure. In order to examine the mechanisms leading to this altered distribution of NF subunits, we carried out biochemical analyses of NF subunits in Triton-insoluble and -soluble fractions derived from aluminum-treated NB2a/d1 cells. In addition to increases in the Triton-insoluble cytoskeleton, increases in all three NF subunits were also detected within the Triton-soluble fraction of aluminum-treated cells. To address the nature of this increase in Triton-soluble subunits, aluminum-treated and untreated cultures were harvested in the absence of Triton and fractionated by established procedures to yield fractions greatly enriched for perikarya and neurites, respectively. Each of these subcellular fractions was then subjected to further homogenization in the presence of 1% Triton and centrifugation to yield Triton-insoluble cytoskeletons and Triton-soluble material derived from perikarya and axonal neurites, respectively. Resulting Triton-soluble fractions were "clarified" by high-speed centrifugation to eliminate oligomeric assemblies or soluble neurofilaments. Immunoblot analysis demonstrated quantitative recovery of the aluminum-induced increase in Triton-soluble NF subunits in the perikaryal fraction. Additional aluminum-treated and untreated cultures were pulse-chase radiolabeled with [35S]methionine and fractionated into Triton-insoluble and soluble fractions from isolated perikarya and axonal neurites. Autoradiographic analysis of immunoprecipitated NF subunits revealed that aluminum treatment delayed the translocation of newly synthesized subunits into neurites and resulted in the accumulation of radiolabeled subunits within the Triton-soluble fraction of perikarya. These findings suggest that aluminum may exert a relatively greater effect on NF subunits that have not yet undergone axonal transport and/or incorporation into Triton-insoluble structures vs those that have already deposited into axons. This possibility was supported by the observation that a higher concentration of aluminum was required to alter the electrophoretic migration of in vitro reassembled neurofilaments vs that required for unassembled NF subunits. These findings provide possible mechanisms for the accumulation of NF subunits in perikarya during aluminum intoxication.

Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer's disease.

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 Alzheimer's 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 Huntington's or Parkinson's disease. Immunoreactivity toward calpastatin, the endogenous inhibitor of calpain, was also markedly reduced in layers II-V of the neocortex in Alzheimer's 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 Alzheimer's 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 Alzheimer's 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.

Degradation of protein kinase C alpha 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 PKC alpha 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 PKC alpha. 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.

Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization.

The respective roles of neurofilaments (NFs), microtubules (MTs), and the microtubule-associated proteins (MAPs) MAP 1B and tau on neurite outgrowth and stabilization were probed by the intracellular delivery of specific antisera into transiently permeabilized NB2a/d1 cells during treatment with dbcAMP. Intracellular delivery of antisera specific for the low (NF-L), middle (NF-M), or extensively phosphorylated high (NF-H) molecular weight subunits did not prevent initial neurite elaboration, nor did it induce retraction of existing neurites elaborated by cells that had been previously treated for 1 d with dbcAMP. By contrast, intracellular delivery of antisera directed against tubulin reduced the percentage of cells with neurites at both these time points. Intracellular delivery of anti-NF-L and anti-NF-M antisera did not induce retraction in cells treated with dbcAMP for 3 d. However, intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, tau, or tubulin induced similar levels of neurite retraction at this time. Intracellular delivery of monoclonal antibodies (RT97 or SMI-31) directed against phosphorylated NF-H induced neurite retraction in cell treated with dbcAMP for 3 d; a monoclonal antibody (SMI-32) directed against nonphosphorylated NF-H did not induce neurite retraction at this time. By contrast, none of the above antisera induced retraction of neurites in cells treated with dbcAMP for 7 d. Neurites develop resistance to retraction by colchicine, first detectable in some neurites after 3 d and in the majority of neurites after 7 d of dbcAMP treatment. We therefore examined whether or not colchicine resistance was compromised by intracellular delivery of the above antisera. Colchicine treatment resulted in rapid neurite retraction after intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, or tau into cells that had previously been treated with dbcAMP for 7 d. By contrast, colchicine resistance was not compromised by the intracellular delivery of antisera directed against NF-L, NF-M, or tubulin. These findings support previous studies indicating that MT polymerization mediates certain aspects of axonal neurite outgrowth and suggest that NFs do not directly participate in these events. These findings further suggest that NFs function in stabilization of the axonal cytoskeleton, apparently by interactions among NFs and MTs that are mediated by NF-H and MAPs.

Multiple interactions of aluminum with neurofilament subunits: regulation by phosphate-dependent interactions between C-terminal extensions of the high and middle molecular weight subunits.

Exposure of individual purified neurofilament (NF) proteins to AlCl3 alters their electrophoretic properties in a time- and concentration-dependent manner, as visualized by their failure to migrate into SDS gels. Co-incubation of purified high (NF-H) and middle (NF-M) but not low (NF-L) molecular weight NF subunits prevents this AlCl3-induced alteration in electrophoretic migration. This latter finding suggested that specific interactions between NF-H and NF-M other than filament formation influenced their interaction with AlCl3. Co-incubation of the 160 kDa alpha-chymotryptic cleavage product of NF-H (corresponding to the highly phosphorylated C-terminal sidearm domain) with native NF-M prevented alteration in subunit electrophoretic migration by AlCl3. By contrast, intact, dephosphorylated NF-H subunits were unable to prevent AlCl3-induced alteration of native NF-M electrophoretic migration. Taken together, these findings suggest that phosphate-dependent interactions between the sidearm extensions of NF-H and NF-M diminish the ability of AlCl3 to associate with either subunit in a manner that alters their electrophoretic migration. This interaction of NF-H and NF-M sidearms is SDS-sensitive, while AlCl3-induced alteration in electrophoretic migration of individual subunits is SDS-resistant. Addition of SDS to mixtures of NF-H and NF-M subunits disrupted the protective effect, and promoted AlCl3-induced alterations in subunit electrophoretic migration. These findings support and extend the current hypothesis that the ability of aluminum to interact with NF subunits is a function of subunit phosphorylation, assembly, and extent of neurofilament-neurofilament cross-linking.

Secretion of amyloid precursor protein and laminin by cultured astrocytes is influenced by culture conditions.

Although normally quiescent, astrocytes in the adult brain respond to various types of brain injury by rapidly dividing, swelling, extending cellular processes, and expressing increased amounts of glial fibrillary acidic protein (GFAP). These phenomena are collectively referred to as "astrogliosis." Similarly, astroglia in primary culture stop dividing when they attain confluency, yet, as seen in situ, they retain their proliferative capacity for extended periods and resume rapid division when subcultured. To examine the impact of glial division on secretion of neurite-promoting factors, conditioned medium (CM) was removed from subconfluent, newly confluent, and long-term confluent ("aged") neonatal rat astrocyte cultures, and from aged confluent cultures that had been repassaged, "lesioned" (scraping with a rubber policeman), or triturated 3 days before harvest. Secretion of neurite-promoting factor(s) by glial cells into these CM was then assayed by treating neuroblastoma cultures with these various CM and quantitating neurite elaboration. Extensive neurite sprouting was elicited by CM from cultures just reaching confluency and from repassaged, lesioned, or triturated cultures. CM from aged confluent cultures did not induce sprouting. These results indicate that secretion of neurite-promoting factor(s) is regulated by glial division, and suggest that gliosis in situ may contribute to neurite sprouting by similar mechanisms. Immunoblot analysis demonstrated the presence in CM of varying amounts of laminin and amyloid precursor protein (APP), including isoforms containing the Kunitz-type protease inhibitor domain. CM from subconfluent cultures contained trace amounts of these proteins, but CM from cultures just reaching confluency contained significant amounts. Although CM from aged cultures contained barely detectable levels of either protein, trituration or repassage of aged cultures dramatically increased secretion of these proteins. APP- and laminin-enriched CM fractions promoted neuritogenesis to a similar level as respective unfractionated CM; anti-APP and anti-laminin antisera blocked this effect. Purified human brain APP promoted neuritogenesis when added to non-conditioned medium and aged CM. Increased secretion of APP and laminin therefore mediates at least a portion of CM-induced neuronal sprouting; these proteins may perform analogous functions during astrogliosis in situ.

Transient requirement for vimentin in neuritogenesis: intracellular delivery of anti-vimentin antibodies and antisense oligonucleotides inhibit neurite initiation but not elongation of existing neurites in neuroblastoma.

Vimentin is initially expressed by nearly all neuronal precursors in vivo, and is gradually replaced by neurofilaments shortly after the immature neurons become postmitotic (Cochard and Paulin, 1984, J Neurosci 4:2080; Tapscott et al., 1981, Dev Biol 86:40). A transient increase in neuritic vimentin filaments occurs within the first day of dbcAMP-mediated neurite induction in NB2a/d1 neuroblastoma, after which vimentin levels rapidly decline and neurofilaments increase (Shea, 1990, Brain Res 521:343). In the present study, we tested the possibility that vimentin filaments may function in neurite elaboration by inducing neuritogenesis under conditions where vimentin expression and assembly was inhibited. Intracellular delivery of anti-vimentin antiserum into transiently permeabilized NB2a/d1 cells prevented the initial elaboration of neurites, but did not retract existing neurites. By contrast, intracellular delivery of antiserum directed against the low molecular weight neurofilament subunit or normal rabbit antiserum did not affect neurite outgrowth. Treatment with vimentin antisense oligonucleotides reversibly depleted vimentin synthesis and steady-state levels, and prevented neurite initiation, but did not induce retraction of existing neurites. These findings point toward an hitherto undetected role for vimentin in the initiation of neurite outgrowth.

The protein phosphatase inhibitor okadaic acid increases axonal neurofilaments and neurite caliber, and decreases axonal microtubules in NB2a/d1 cells.

When cells were treated with dbcAMP for 3 days to induce the outgrowth of axonal neurites, the addition of the phosphatase inhibitor okadaic acid (OA; 5 nM) for the last 24 hr markedly increased neurofilament subunit immunoreactivity including phosphate-dependent NF-H epitopes in axonal neurites, increased axonal neurite caliber by approximately 30%, but did not increase neurite contour length. Ultrastructural analysis demonstrated a > 2-fold increase in neurofilaments and indicated that neurofilaments were phosphorylated to a similar extent in the presence and absence of OA. Vimentin immunoreactivity, which undergoes down-regulation during dbcAMP-mediated differentiation, was not increased by OA. OA did not induce the precocious appearance of delayed phosphate-dependent neurofilament epitopes suggesting that it did not induce the activation of additional neurofilament kinases. NF-H subunits from cytoskeletons of OA-treated cells were less susceptible to degradation by an endogenous calcium-dependent protease, providing a possible mechanism for neurofilament accumulation during OA treatment. By contrast, OA decreased axonal neurite microtubules, and eliminated stabilized (acetylated) axonal microtubules. OA treatment at earlier times prevented and reversed neurite outgrowth. Despite increased deposition of phosphorylated neurofilaments, OA did not hasten the development of colchicine resistance to neurites, suggesting that stabilization of the axonal cytoskeletal lattice requires neurofilament-microtubule interaction.

Evidence that the monoclonal antibodies SMI-31 and SMI-34 recognize different phosphorylation-dependent epitopes of the murine high molecular mass neurofilament subunit.

The monoclonal antibodies SMI-31 and SMI-34 react with phosphate-dependent epitopes of the high molecular mass (200 kDa) neurofilament protein (Hphos). Determination of whether or not these monoclonals react with different epitopes would assist in interpretation of post mortem immunocytochemical analyses in neurodegenerative disorders and in normal aging. We therefore examined the relative immunoreactivity of these antibodies against Triton-insoluble (cytoskeleton-associated) and Triton-soluble Hphos variants in NB2a/d1 neuroblastoma and post-natal mouse brain in immunoblot analysis. Densitometric analysis yielded a 'reactivity ratio' (soluble Hphos/insoluble Hphos) for each antibody. This ratio was approximately 44% and 87% less for SMI-34 than for SMI-31 in neuroblastoma and brain, respectively. These findings confirm that the SMI-34 epitope is distinct from that recognized by SMI-31, and, in these systems, is preferentially associated with the cytoskeleton.

Differential expression and subcellular localization of protein kinase C alpha, beta, gamma, delta, and epsilon isoforms in SH-SY5Y neuroblastoma cells: modifications during differentiation.

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 alpha, beta 1, delta, and epsilon isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-alpha and -beta 1 were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-delta and -epsilon 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 alpha isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-beta-phorbol or staurosporine, and that protein kinase C-epsilon 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-alpha and -epsilon decreased, and protein kinase C-beta 1 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 alpha and epsilon in neuritogenesis.