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T B Shea

Publications and source records attributed to T B Shea.

At least 37 records · Page 2Linked to original sources
PubMed1999

Regulation of neurofilament axonal transport by phosphorylation in optic axons in situ.

Axonal transport of neurofilament (NFs) is considered to be regulated by phosphorylation. While existing evidence for this hypothesis is compelling, supportive studies have been largely restricted to correlative evidence and/or experimental systems involving mutants. We tested this hypothesis in retinal ganglion cells of normal mice in situ by comparing subunit transport with regional phosphorylation state coupled with inhibition of phosphatases. NF subunits were radiolabeled by intravitreal injection of 35S-methionine. NF axonal transport was monitored by following the location of the peak of radiolabeled subunits immunoprecipitated from 9x1.1 mm segments of optic axons. An abrupt decline transport rate was observed between days 1 and 6, which corresponded to translocation of the peak of radiolabeled subunits from axonal segment 2 into segment 3. Notably, this is far downstream from the only caliber increase of optic axons at 150 mu from the retina. Immunoblot analysis demonstrated a unique threefold increase between segments 2 and 3 in levels of a "late-appearing" C-terminal NF-H phospho-epitope (RT97). Intravitreal injection of the phosphatase inhibitor okadaic acid increased RT97 immunoreactivity within retinas and proximal axons, and markedly decreased NF transport rate out of retinas and proximal axons. These findings provide in situ experimental evidence for regulation of NF transport by site-specific phosphorylation.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Selective stabilization of microtubules within the proximal region of developing axonal neurites.

This study examined the distribution of labile and stable microtubules (MTs) during axonal neurite elaboration in NB2a/d1 cells using immunocytochemical markers of unmodified (tyrosinated; Tyr), modified (detyrosinated [Glu] and acetylated [Acet]) and total tubulin. Prominent total and Tyr tubulin immunoreactivity was relatively evenly distributed throughout axonal neurites. By contrast, Acet or Glu immunoreactivity was relatively concentrated within the proximal region of the neurite. Ultrastructural analyses demonstrated an array of longitudinal MTs that apparently span the entire neurite length. The observed differential localization of modified tubulin subunits in axonal neurites of these cells may therefore derive from selective stabilization of proximal regions of full-length axonal MTs. This was substantiated by the observation of Acet immunoreactivity on 30-50% of MTs within the most proximal axonal region, along with a proximal-distal decline to < or =5% of Acet immunoreactive MTs, in immunoelectron microscopy (immuno-EM) analyses. Microinjected biotinylated subunits were initially detected in assembled form within soma and proximal neurites, indicative of ongoing tubulin subunit incorporation into MTs within, and/or MT translocation into, proximal neurites. Because acetylation and detyrosination are functions of MT age, their concentration in this region despite deposition and/or transport of biotinylated tubulin suggests that a subset of axonal MTs undergoes subunit turnover and/or translocation at rates vastly slower than that of the majority of axonal MTs. Selective stabilization of the proximal region of a subset of axonal MTs may serve to construct a relatively stationary scaffold against which other axonal elements could translocate to more distal axonal regions for continued axonal outgrowth.

Acetylation↗
PubMed1999

The order of exposure of tau to signal transduction kinases alters the generation of "AD-like" phosphoepitopes.

1. The individual and sequential influence of protein kinase C (PKC), protein kinase A (PKA) and mitogen-activated protein kinase (MAP kinase) on human brain tau was examined. 2. A range of PKC concentrations generated certain phosphoepitopes common with paired helical filaments. These epitopes were masked by higher PKC concentrations, suggesting the presence of multiple tau phosphorylation sites for which PKC exhibited differing affinities and/or conformational alterations in tau induced by sequential PKC-mediated phosphorylation. 3. Prior phosphorylation by PKC enhanced the nature and extent of AD-like tau antigenicity generated by subsequent incubation with MAP kinase yet inhibited that generated by subsequent incubation with PKA. 4. Dephosphorylation of tau prior to incubation with kinases significantly altered the influence of individual and multiple kinase incubation on tau antigenicity in a site-specific manner, indicating that prior in situ phosphorylation events markedly influenced subsequent cell-free phosphorylation. 5. In addition to considerations of the potential impact of tau phosphorylation by individual kinases, these findings extend previous studies which indicate that tau antigenicity, and, presumably, its behavior in situ, is influenced by the sequential and convergent influences of multiple kinases.

Adult↗
PubMed1999

Hyperactivation of mitogen-activated protein kinase increases phospho-tau immunoreactivity within human neuroblastoma: additive and synergistic influence of alteration of additional kinase activities.

Mitogen-activated protein (MAP) kinase phosphorylates tau in cell-free analyses, but whether or not it does so within intact cells remains controversial. In the present study, microinjection of MAP kinase into SH-SY-5Y human neuroblastoma cells increased tau immunoreactivity toward the phosphodependent antibodies PHF-1 and AT-8. In contrast, treatment with a specific inhibitor of MAP kinase (PD98059) did not diminish "basal" levels of these immunoreactivities in otherwise untreated cells. These findings indicate that hyperactivation of MAP kinase increases phospho-tau levels within cells, despite that MAP kinase apparently does not substantially influence intracellular tau phosphorylation under normal conditions. These findings underscore that results obtained following inhibition of kinase activities do not necessarily provide an indication of the consequences accompanying hyperactivation of that same kinase. Several studies conducted in cell-free systems indicate that exposure of tau to multiple kinases can have synergistic effects on the nature and extent of tau phosphorylation. We therefore examined whether or not such effects could be demonstrated within these cells. Site-specific phospho-tau immunoreactivity was increased in additive and synergistic manners by treatment of injected cells with TPA (which activates PKC), calcium ionophore (which activates calcium-dependent kinases), and wortmannin (which inhibits PIP3 kinase). Alteration in total tau levels was insufficient to account for the full extent of the increase in phospho-tau immunoreactivity. These additional results indicate that multiple kinase activities modulate the influence of MAP kinase on tau within intact cells.

Alzheimer Disease↗
PubMed1999

Kinesin-mediated transport of neurofilament protein oligomers in growing axons.

We examined cytoskeleton-associated forms of NF proteins during axonal neuritogenesis in cultured dorsal root ganglion (DRG) neurons and NB2a/d1 neuroblastoma. In addition to filamentous immunoreactivity, we observed punctate NF immunoreactivity throughout perikarya and neurites. Immuno-electron microscopy revealed this punctate immunoreactivity to consist of non-membrane-bound 75 nm round/ovoid structures consisting of amorphous, fibrous material. Endogenous and microinjected NF subunits incorporated into dots prior to their accumulation within filaments. A transfected GFP-conjugated NF-M incorporated into dots and translocated at a rate consistent with slow axonal transport in real-time video analyses. Some dots converted into a filamentous form or exuded filamentous material during transport. Dots contained conventional kinesin immunoreactivity, associated with microtubules, and their transport into axons was blocked by anti-kinesin antibodies and nocodazole. These oligomeric structures apparently represent one form in which NF subunits are transported in growing axons and may utilize kinesin as a transport motor.

Animals↗
PubMed1998

Neurofilament subunits can undergo axonal transport without incorporation into Triton-insoluble structures.

We examined the form(s) in which NF subunits undergo axonal transport. Pulse-chase radiolabeling analyses with 35S-methioinine revealed that newly synthesized Triton-soluble NF subunits accumulated within axonal neurites elaborated by NB2a/d1 neuroblastoma prior to the accumulation of Triton-insoluble subunits. Gel chromatographic, immunological, ultrastructural, and autoradiographic analyses of Triton-soluble axonal fractions demonstrated that radiolabeled, Triton-soluble subunits were associated with NFs. Triton-soluble, radiolabeled axonal NF subunits were also detected within retinal ganglion cell axons following intravitreal injection of 35S-methioinine. Microinjected biotinylated subunits were prominent within axonal neurites of NB2a/d1 cells and cultured dorsal root ganglion neurons substantially before they were retained following Triton-extraction. Prevention of biotinylated subunit, but not dextran tracer, translocation into neurites by nocodazole confirmed that microinjected subunits did not enter axons merely due to diffusion or injection-based pressure. Immuno-EM confirmed the association of biotin label with axonal NFs. These findings point towards multiple populations of NF subunits within axons and leave open the possibility that axonal NFs may be more dynamic than previously considered.

Animals↗
PubMed1998

A 26-30 kDa developmentally-regulated tau isoform localized within nuclei of mitotic human neuroblastoma cells.

Tau isoforms migrating at 46-68 and 97-115 kDa were prominent within heat-stable Triton-soluble material, and were present in lesser concentration with Triton-insoluble cytoskeletons, derived from undifferentiated SH-SY-5Y human neuroblastoma cells. Conversely, a 26-30 kDa tau isoform was enriched in the cytoskeleton and detected at relatively minor levels within cytosolic fractions. Pulse labeling with 35S-methionine indicated that this 26-30 kDa "small tau" did not represent a breakdown product of larger isoforms. Since the nucleus is retained within the Triton-insoluble cytoskeleton, additional cultures were fractionated onto sucrose to obtain purified nuclei. The vast majority of small tau was recovered within purified nuclei. Small tau was reactive with tau antibodies directed towards N-terminal, C-terminal and central epitopes, further confirming that this small isoform was not derived from proteolytic cleavage of larger tau isoforms. Small tau demonstrated alkaline phosphatase-sensitive reactivity with multiple phospho-dependent tau antibodies. Small tau was depleted within 3 days of retinoic acid-induced differentiation, suggesting that the putative function of this isoform may be obsolete following terminal differentiation of neurons.

Blotting, Western↗
PubMed1998

Extensive phosphorylation and axonal transport of triton-soluble neurofilament subunits.

The low abundance of soluble neurofilament (NF) subunits in mature axons has suggested that newly synthesized NF proteins rapidly assemble into highly stable polymers and associate with the Triton X-100-insoluble cytoskeleton. Here we present evidence for multiple populations of NFs and NF subunits, distinguished by differential solubility in Triton, within perikarya and axons of neurons in situ and in culture. We further demonstrate, using microinjection of "tagged" NF subunits and by pulse-chase radiolabeling of endogenous NF subunits, that these soluble NF populations represent precursors for incorporation into the axonal cytoskeleton.

Animals↗
PubMed1997

Phospholipids alter tau conformation, phosphorylation, proteolysis, and association with microtubules: implication for tau function under normal and degenerative conditions.

Discerning the in situ functions of the microtubule-associated protein (MAP) tau is of interest both in terms of neuronal differentiation and homeostasis as well as in terms of neurodegenerative conditions such as Alzheimer's disease. In the present study, exposure to excess phosphatidyl serine (PS) for < 1 min induced antigenic alterations in multiple N-terminal, C-terminal and central epitopes of purified human brain tau. Notably, "AD-like" epitopes (PHF-1, ALZ-50, AT-8) were decreased by PS; other epitopes (e.g., 5E2, Tau-1) increased and others remained relatively unchanged. Inclusion of gamma-AT[32P] during incubations did not reveal any contaminating kinase activity. Direct addition of chloroform:methanol (CM; the initial PS solvent) demonstrated that these changes were not derived from CM-mediated tau denaturation. Phosphatidyl choline induced similar antigenic changes, while phosphatidyl inositol did not. PS inhibited MAP-kinase generation of phospho-dependent tau epitopes and incorporation of phosphates by tau. Inclusion of PS during coincubation of tau and tubulin reduced the extent of cosedimentation of tau with MTs. Finally, PS enhanced the ability of calpain-mediated tau proteolysis. These data suggest that tau antigenicity in situ may be derived from phospholipid-dependent alterations in tau conformation in addition to tau phosphorylation state. These data further suggest that disruption of the normal association of tau with phospholipids may foster accumulation of tau and, in doing so, render tau more susceptible to hyperphosphorylation.

Alzheimer Disease↗
PubMed1997

Beta-amyloid and ionophore A23187 evoke tau hyperphosphorylation by distinct intracellular pathways: differential involvement of the calpain/protein kinase C system.

SH-SY-5Y human neuroblastoma cells were treated with 22 microM of a synthetic peptide corresponding to amino acid residues 25-35 of beta-amyloid (betaA) or 3 microM calcium ionophore A23187 in culture medium containing 1.8 mM extracellular calcium. Both agents increased tau immunoreactivity towards antibodies (PHF-1, ALZ-50) that recognize epitopes common with paired helical filaments (PHFs) and towards an antibody (5E2) that recognized a phosphate-independent tau epitope. However, only ionophore increased immunoreactivity with an additional phosphate-dependent antibody (AT-8) that recognized an epitope of tau when phosphorylated, and induced a corresponding decrease in immunoreactivity towards an additional antibody (Tau-1) that recognizes the same site when that site is not phosphorylated. Moreover, the ionophore-mediated increase in PHF-1 was blocked by EGTA, by the calpain inhibitor calpeptin and by the PKC inhibitor H7, while that evoked by betaA treatment was not inhibited by any of these treatments. Since ionophore-mediated calpain activation induces proteolytic PKC activation, we further examined the influence of PKC inhibition on betaA and ionophore-mediated PHF-1 induction. Antisense oligonucleotide-mediated downregulation of PKCepsilon in a stable transfectant SH-SY-5Y subclone diminished the ionophore-mediated, but not the betaA-mediated, increase in PHF-1 immunoreactivity. These data indicate specific differences in the intracellular cascade of events invoked by betaA and ionophore A23187. Moreover, although betaA invoked calcium influx in these cells, our findings further suggest that the induction of tau hyperphosphorylation by betaA may not be due to calcium influx.

Amyloid beta-Peptides↗
PubMed1997

Triton-soluble phosphovariants of the heavy neurofilament subunit in developing and mature mouse central nervous system.

The low abundance of soluble neurofilament (NF) subunits in mature axons has suggested that newly synthesized NF proteins rapidly assemble into highly stable polymers and associate with the Triton X-100-insoluble cytoskeleton. The dynamic nature of these subunit associations in vivo remains unresolved, and the applicability of this assembly model to NFs in other neuronal compartments or to developing neurons is unknown. Here, we report that a unique pool of Triton X-100-soluble, extensively phosphorylated, high molecular weight NF subunits (NF-H, or H-200) are abundantly expressed in the mouse CNS during early postnatal development and persist in the perikaryal compartment of some mature neurons. Triton-soluble H-200 subunits appeared at postnatal day 14 (P14) and remained high through P60, beyond which the percentage declined to marginal levels by P120. Medium and low molecular weight NF (NF-M and NF-L, respectively) were at all times only detectable within the cytoskeleton. Comparison of soluble and cytoskeleton-associated H-200 immunoreactivity indicated that certain phosphorylation-dependent epitopes were confined to the cytoskeleton. Pulse-chase radiolabeling analyses in optic pathway demonstrated that some Triton-soluble NF-H subunits are extensively phosphorylated within retinal perikarya before they are incorporated into Triton-insoluble structures. These findings indicate that the assembly behaviors of NF-H differ substantially from those of NF-M and NF-L, and that the interaction of NF-H with NFs may be more dynamic than is generally recognized, especially during brain development and within specific compartments of mature neurons.

Animals↗
PubMed1997

Restriction of microM-calcium-requiring calpain activation to the plasma membrane in human neuroblastoma cells: evidence for regionalized influence of a calpain activator protein.

Regulation of the microM-calcium-requiring form of calpain (mu calpain) was studied in SH-SY-5Y human neuroblastoma cells. Immunoblot analysis demonstrated that the vast majority of mu calpain is localized within cytosolic pools. Calpain activation was monitored as a function of autolysis within intact cells following calcium influx from the culture medium by calcium ionophores A23187 or ionomycin, or following release of calcium from intracellular stores by thapsigargin. Within intact neuronal cells, following an influx of calcium into the cytosolic from either extracellular or intracellular sources, mu calpain is preferentially activated at the plasma membrane as evidenced by autolytic generation of faster-migrating isoforms. By contrast, similar autolytic profiles for mu calpain in membrane or cytosolic fractions following addition of calcium were observed under cell-free conditions and within cells following death due to extended ionophore-mediated calcium influx. These differential activation profiles for cytosolic mu calpain within living cells and following cellular fractionation/cell death indicate the presence of a regulatory system within neuronal cells. As in previous studies in other systems, we demonstrate the presence of a calpain activator protein. Cycloheximide treatment depleted the autolytic capacity of membrane-associated mu calpain within 4-6 hr without a corresponding decline in total mu calpain protein levels, indicating that the activator protein undergoes rapid turnover in comparison to calpain; pulse-chase radiolabeling confirmed the half-life of mu calpain to exceed 24 hr. Our data suggest that this labile protein represents a major rate-limiting step for in situ calpain activation within neuronal cells, and that, given the tremendous latent mu calpain activity within the cytosol, the interplay of the activator protein and the endogenous inhibitor calpastatin are crucial for maintaining neuronal homeostasis.

Calcimycin↗
PubMed1997

Calpains and calpastatin in SH-SY5Y neuroblastoma cells during retinoic acid-induced differentiation and neurite outgrowth: comparison with the human brain calpain system.

Calpains have importance in human neurodegenerative disease pathogenesis, but these mechanisms are difficult to study in postmortem tissues. To establish a cellular model of the human calpain and calpastatin system, we characterized calpain I, calpain II, and calpastatin in SH-SY5Y human neuroblastoma cells in relation to their counterparts in human brain and investigated their expression and activity after inducing cellular differentiation with retinoic acid (RA), a physiological effector of normal brain development. Calpain I in both SH-SY5Y cells and human brain existed in the cytosolic and particulate fractions as three isoforms (80, 78, and 76 kDa) and exhibited atypical isoelectric focusing behavior. Calpain II in SH-SY5Y cells, as in human brain, migrated as a single predominantly cytosolic 76-kDa protein with an isoelectric point ranging from 5.9 to 6.3. Calpastatin from both sources was also 90% cytosolic. In the cells it was composed of four discrete bands, ranging in molecular weight from 110 to 127 kDa. Levels of activated (76 and 78 kDa) and precursor (80 kDa) calpain I isoforms rose 54% (P < 0.0001) in the particulate fraction and 26% (P < 0.0001) in the soluble fraction after 3 days of RA exposure. Because levels and activity of calpastatin remain unchanged during the first 7 days of RA exposure, the increased abundance of calpain I implies a net activation of the calpain system during differentiation. Calpain I activation may contribute to the remodeling of cell shape and neurite extension/retraction associated with neuronal differentiation.

Brain↗
PubMed1997

Phosphorylation events mediated by protein kinase C alpha and epsilon participate in regulation of tau steady-state levels and generation of certain "Alzheimer-like" phospho-epitopes.

Hyperactivation of protein kinase C (PKC) in intact neuroblastoma cells by several methods increases site-specific tau phosphorylation as shown by increases in paired helical filament-I (PHF-I) and ALZ-50 but not AT-8 immunoreactivity. In the present study, the influence of PKC on tau metabolism was further examined by isoform-specific antisense oligonucleotide-mediated PKC downregulation in human SH-SY-5Y neuroblastoma cells and by generation of stably-transfected subclones expressing isoform-specific anti-PKC mRNA sequences. Downregulation of PKC epsilon by both of these methods reduced PHF-I and ALZ-50 immunoreactivity, suggesting that this PKC isoform, perhaps via downstream kinase cascades, regulated tau phosphorylation events that normally generate these epitopes. By contrast, downregulation of either PKC epsilon or PKC alpha reduced immunoreactivity towards the phosphate-independent anti-tau antibodies 5E2 and JM, suggesting that both of these isoforms participated in regulation of tau steady-state levels. Downregulation of PKC beta did not affect any of the above changes. The above roles were apparently unique for PKC epsilon and PKC alpha, since activation of multiple PKC isoforms by phorbol ester treatment and/or other calcium-dependent kinase(s) by ionophore-mediated calcium influx could not compensate for downregulation of PKC alpha or PKC epsilon in maintaining tau steady-state levels or PHF-I/ALZ-50 immunoreactivity, respectively. These findings suggest that hyperactivation of signal transduction pathways, including those regulated by PKC, could evoke changes in neuronal cells reminiscent of those seen in affected neurons in Alzheimer's disease.

Alzheimer Disease↗
PubMed1997

Selective activation by bryostatin-1 demonstrates unique roles for PKC epsilon in neurite extension and tau phosphorylation.

Phorbol esters such as 12-O-tetradeonyl phorbol-13 acetate (TPA) induce a time-dependent biphasic effect on protein kinase C (PKC)-mediated events by fostering translocation of cytosolic (latent) PKC to the plasma membrane (where it is activated). Continued treatment, however, depletes the cell's entire PKC complement and induces a functional stake of PKC inhibition. Previous studies from several laboratories have demonstrated that long-term TPA treatment, like treatment with PKC inhibitors, induces neuronal differentiation. Bryostatin-1 also induces translocation and overall downregulation of PKC following long-term treatment, yet, unlike TPA or PKC inhibitors, does not induce neuronal differentiation, promoting controversy regarding the role of PKC inhibition in neuronal differentiation. We demonstrate herein that, despite overall downregulation in human neuroblastoma cells, membrane-associated levels of one PKC isoform (PKC epsilon) are actually increased following long-term bryostatin-1 treatment. Since previous studies have implicated this PKC isoform in phosphorylation of the microtubule-associated protein tau and in neuritogenesis, we examined the consequences of long-term bryostatin treatment on these phenomena. Treatment with 25 n-100 M bryostatin-1 for 72 h increased tau phosphorylation and inhibited neuritogenesis. By contrast, treatment with either TPA or the PKC inhibitor staurosporine did not induce tau phosphorylation and induced neurite elaboration. Bryostatin-1 antagonized neurite induction by staurosporine. These findings provide additional evidence for a unique role of PKC epsilon in the regulation of tau phosphorylation and neuronal differentiation, and demonstrate that bryostatin-1 can function under certain conditions as a selective PKC epsilon activator even following long-term treatment.

Bryostatins↗
PubMed1996

Spectrin-actin interaction is required for neurite extension in NB 2a/dl neuroblastoma cells.

Spectrin is an actin-binding membrane skeleton protein involved in the maintenance of cell shape and generation of distinct membrane protein domains. Actin binds to the N-terminal domain of beta-spectrin. To examine the function of spectrin-actin interaction in neurons, we sought to disrupt this interaction in differentiating NB 2a neuroblastoma cells by microinjecting an N-terminal domain-specific anti-beta-spectrin antibody. We found that microinjection of the affinity-purified N-terminal domain-specific anti-beta-spectrin inhibited the extension of the neurites in NB 2a/dl cells. The microinjected cells remained flat, and put out many filopodia-like processes; but these processes failed to extend when the cells were induced to differentiate in the presence of dbc AMP or in serum-free medium. The N-terminal domain-specific anti-beta-spectrin also inhibited the binding of spectrin to actin. By contrast, the microinjection of monospecific anti-alpha-spectrin(G) did not inhibit neurite extension. These results suggest that beta-spectrin-actin interaction may be required for neurite extension, which is critical for development of polarity in nerve cells.

Actins↗