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T B Shea

Publications and source records attributed to T B Shea.

At least 55 records · Page 3Linked to original sources
PubMed1996

Phosphatase inhibition in human neuroblastoma cells alters tau antigenicity and renders it incompetent to associate with exogenous microtubules.

The abnormal cytoskeletal organization observed in Alzheimer's disease has been suggested to arise from hyperphosphorylation of tau and the resultant elimination of its ability to associate with microtubules. This possibility has been supported by a number of studies under cell-free conditions utilizing various kinases, phosphatases and their corresponding inhibitors each, and by treatment of intact cells with kinase and phosphatase activators and inhibitors. However, in studies utilizing intact cells, it remained difficult to attribute microtubule compromise specifically to tau hyperphosphorylation due to potential influence of inhibitors on tubulin and/or other microtubule-associated proteins which themselves possess assembly-regulatory phosphorylation sites. To address this difficulty, we subjected SH-SY-5Y human neuroblastoma cells to treatment with the phosphatase inhibitor okadaic acid (OA), which has been previously demonstrated to depolymerize microtubules in these cells. OA induced an increase in tau hyperphosphorylation as evidenced by an increase in Alz-50 immunoreactivity and a corresponding decrease in Tau-1 immunoreactivity. When tau-enriched fractions from OA-treated cells were incubated under microtubule assembly-promoting conditions with twice-cycled, tau-free preparations of bovine brain tubulin not exposed to OA, Alz-50-immunoreactive tau isoforms displayed a marked (49%) reduction in ability to co-assemble with bovine microtubules as compared with Tau-1- and 5E2-immunoreactive isoforms. These data indicated that hyperphosphorylated tau has a reduced capacity to associate with microtubules, and support the hypothesis that tau hyperphosphorylation may underlie microtubule breakdown in Alzheimer's disease.

Animals↗
PubMed1996

Role of vimentin in early stages of neuritogenesis in cultured hippocampal neurons.

Vimentin is expressed initially by nearly all neuronal precursors in vivo, and is replaced by neurofilaments shortly after the immature neurons become post-mitotic. Moreover, both vimentin and neurofilaments can be detected transiently within the same neurite, leaving open the possibility that vimentin may play a role in the early stages of neuritogenesis. In the present study, cultured hippocampal neurons, which transiently express vimentin in culture, were treated with sense- and antisense-oriented deoxyoligonucleotides encoding regions of the vimentin sequence that overlap the translation initiation codon. Antisense oligonucleotide treatment reduced vimentin-immunoreactivity to background levels. Moreover, while 90-100% of cultured hippocampal neurons elaborated neurites within the first 24 hr following plating, only 24-30% did so in the presence of vimentin antisense oligonucleotides. Inhibition of neurite outgrowth was reversible following removal of antisense oligonucleotide. These findings substantiate earlier studies in neuroblastoma cells, indicating a possible role for vimentin in the initiation of neurite outgrowth.

Animals↗
PubMed1996

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.

Antigens↗
PubMed1995

Calcium influx recruits an additional class of kinases to hyperphosphorylate tau.

SH-SY-5Y human neuroblastoma cells were treated with combinations of the kinase inhibitors HA-1004, W-7 and H-7 and calcium ionophore A23187. Microdensitometric analyses revealed that, in the absence of ionophore-mediated calcium influx, PHF-1 levels were reduced by approximately half in cultures treated with HA-1004 or W-7, but were not reduced by H-7. By contrast, the doubling in PHF-1 immunoreactivity that resulted following ionophore treatment was prevented by all three inhibitors. These analyses demonstrate the recruitment of an additional kinase or kinases in tau phosphorylation following calcium influx, and underscore the possibility that de novo hyperactivation of calcium-dependent kinases may be involved in the early events that propagate PHF formation.

Antibodies, Monoclonal↗
PubMed1995

Proteolysis of protein kinase C: mM and microM calcium-requiring calpains have different abilities to generate, and degrade the free catalytic subunit, protein kinase M.

Limited proteolysis of protein kinase C (PKC) by calpain under cell free conditions cleaves the regulatory and catalytic PKC subunits, generating a free, co-factor independent catalytic subunit, termed PKM. In the present study, we demonstrate distinct differences in the rate, nature, and lipid-sensitivity of PKC and PKM proteolysis by microM and mM calcium-requiring calpain isozymes (mu calpain or m calpain, respectively). PKC is a preferred substrate for m calpain; not even a 100-fold increase in mu calpain was capable of degrading PKC as fast as in calpain. PKM was generated by both m and mu calpains, but was itself rapidly degraded by m calpain and therefore was only transiently detectable. By contrast, PKM was formed but not degraded by mu calpain, and persisted in the presence of mu calpain long after all PKC had been degraded. Phosphatidyl serine (PS) inhibited PKC hydrolysis by m calpain yet enhanced PKC hydrolysis by mu calpain. The ability of either calpain isoenzyme to degrade [14C]azocasein was unaffected by PS, suggesting that the influence of PS was on PKC conformation. These findings point towards distinct roles for mu and m calpain in PKC regulation.

Animals↗
PubMed1995

Inhibition of neuronal surface proteases decreases the requirement for GAP-43 in neurite outgrowth.

Intracellular delivery of anti-GAP IgG inhibits the elaboration of neurites by NB2a/d1 cells. However, recent studies indicate that the extent of inhibition is minimized when cells were cultured on poly-L-lysine-coated or laminin-coated versus uncoated plates, suggesting that the role of GAP-43 in neuritogenesis may be specifically related to membrane adhesiveness. We therefore examined the influence of inhibition of thrombin, the neuronal surface protease that restricts neurite outgrowth, on GAP-43-dependent neurite outgrowth. Treatment of cells with the specific thrombin inhibitor hirudin in the presence of serum induced a similar percentage of neurite outgrowth as was observed following serum withdrawal. However, while neurite outgrowth induced by serum deprivation of cells was reduced following intracellular delivery of anti-GAP IgG, neurite outgrowth induced by hirudin treatment of cells was not. That inhibition of neuronal surface protease activity overcomes the inhibition of neurite outgrowth following intracellular delivery of anti-GAP IgG provides further evidence that the role that GAP-43 plays in neuritogenesis is related to membrane adhesiveness.

GAP-43 Protein↗
PubMed1995

Inhibition of neurite outgrowth following intracellular delivery of anti-GAP-43 antibodies depends upon culture conditions and method of neurite induction.

NB2/dl neuroblastoma cells acquire a neuronal phenotype in response to several differentiating agents, including dibutyryl cAMP (dbcAMP) and the withdrawal of serum. As shown previously, antibodies to the growth-associated protein, GAP-43, introduced intracellularly using a lipid carrier, blocked the differentiation induced by dbcAMP. Antibodies to GAP-43, at a low concentration, also blocked neurite outgrowth induced by serum withdrawal when cells were grown on a relatively unadhesive substrate. On more adhesive substrates such as poly-L-lysine and laminin, however, anti-GAP-43 antibodies had less of an effect on neurite outgrowth. Previous studies have shown that the increased adhesivity of laminin allows a small but significant population of neurites to grow from serum-deprived cells, even in the presence of the microtubule-depolymerizing drug, colchicine. The outgrowth of this population of neurites was blocked by antibodies to GAP-43. These results are in conformity with recent studies showing that the requirement for GAP-43 in neuritogenesis may be related to membrane adhesiveness, and may contribute to an understanding of some of the apparent discrepancies in the literature concerning the involvement of GAP-43 in neuronal differentiation.

Antibody Specificity↗
PubMed1995

Differential synthesis and cytoskeletal deposition of neurofilament subunits before and during axonal outgrowth in NB2a/d1 cells: evidence that segregation of phosphorylated subunits within the axonal cytoskeleton involves selective deposition.

NB2a/d1 cells constitutively express and extensively phosphorylate neurofilament (NF) triplet proteins. However, only hypophosphorylated NFs are observed within the Triton-insoluble perikaryal cytoskeletons of undifferentiated and differentiated cells, while phosphorylated NF isoforms accumulate exclusively within the axonal neurites elaborated following treatment with dbcAMP. We examined NF synthesis and distribution of newly synthesized subunits by immunoprecipitation from 35S-methionine-radiolabeled undifferentiated and dbcAMP-treated differentiated cells. Following a 15 min pulse radiolabeling, NF-H isoforms migrating from approximately 160-200 kDa, NF-M isoforms migrating from approximately 97 k-145 Da, and a single 70 kDa NF-L isoform were readily detectable within Triton-soluble fractions from both undifferentiated and differentiated cells. During chase analyses in the absence of radiolabel, the entire spectrum of isoforms was present in Triton-soluble and -insoluble fractions from both undifferentiated and differentiated cells. However, differentiated cells displayed a significant increase in radiolabel associated with each subunit and isoform. Normalization of their NF synthesis levels to those of undifferentiated cells revealed that differentiated cells deposited 10-fold more radiolabeled subunits into the Triton-insoluble cytoskeleton as compared to undifferentiated cells. Similar levels of radiolabeled subunits were observed throughout the 2 hr period in dbcAMP-treated cells. By contrast, radiolabeled subunits and isoforms increased in undifferentiated cytoskeletons during the chase period, although final levels remained substantially lower than those observed in cytoskeletons of dbcAMP-treated cells. These data were considered with respect to potential mechanisms by which the phosphorylated NFs are normally excluded from perikaryal cytoskeletons. The presence of extensively phosphorylated subunits within perikarya indicates the presence of necessary NF kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Hyperphosphorylation of Tau and filopodial retraction following microinjection of protein kinase C catalytic subunits.

Limited proteolysis of protein kinase C (PKC) by calcium-activated proteolysis cleaves the regulatory and catalytic subunits of PKC, generating a free, constitutively activated kinase ("PKM") that, unlike the intact parent enzyme, is not calcium-dependent, and is not restricted to the plasma membrane. These latter properties leave open the possibility that PKM may have access to, and may therefore phosphorylate, substrates normally unavailable to intact PKC. We examined the potential involvement of such aberrant phosphorylation in certain aspects of the neurodegeneration accompanying Alzheimer's disease by microinjecting PKC and PKM, along with a rhodamine-conjugated dextran tracer, into undifferentiated NB2a/d1 mouse neuroblastoma cells. After 4 hr, cultures were fixed and processed for immunofluorescence with monoclonal antibodies (PHF-1, ALZ-50, Tau-1, AT8) directed against tau in various phosphorylation states followed by fluorescein-conjugated secondary antibodies. Microinjected cells were localized via co-injected rhodamine-conjugated dextran tracer under rhodamine illumination, after which antibody immunoreactivity was examined under fluorescein illumination. Microdensitometric analyses indicated that microinjection of PKC did not increase basal immunofluorescent intensities of the antibodies; by contrast, microinjection of PKM induced three- and twofold increases in PHF-1 and ALZ-50 levels, respectively. By contrast, no significant alteration was observed in AT8 and Tau-1 immunofluorescence following either PKC or PKM microinjection. Whereas undifferentiated NB2a/d1 cells typically elaborate short, filopodia-like neurites, phase-contrast microscopy revealed the absence of filopodia or neurites on PKM-injected cells, while a similar percentage of PKC-injected cells. Cell-free analyses confirmed the ability of PKC, in the presence of necessary co-factors, and PKM to increase PHF-1 and ALZ-50 immunoreactivity; no change was observed in AT8 or Tau-1 immunoreactivity. These findings underscore the possibility that an abnormal amplification in limited PKC proteolysis to generate PKM could, under certain pathological conditions, contribute to neuronal degeneration.

Animals↗
PubMed1995

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.

Calpain↗
PubMed1995

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.

Animals↗
PubMed1995

Inhibition of proteolysis enhances aluminum-induced perikaryal neurofilament accumulation but does not enhance tau accumulation.

As observed for neurons in situ, phosphorylated neurofilament (NF) epitopes are normally segregated within the axonal cytoskeleton of NB2a/d1 cells. However, accumulations of phosphorylated NFs develop in NB2a/d1 perikarya following exposure to aluminum salts and following inhibition of proteolysis. In the present study, we observed that perikarya of cells exposed to both aluminum and the protease inhibitor C1 (also known as "AllNal") were more intensely labeled by monoclonal antibodies directed against both nonphosphorylated and phosphorylated epitopes than were cells treated with either aluminum or protease inhibitor alone. Since these monoclonal antibodies crossreact with tau, we also immunostained cells treated under these conditions with monoclonal antibodies directed against phosphate-insensitive (5E2) and phosphorylated (PHF-1) epitopes of tau. Aluminum treatment, but not C1 treatment, induced accumulation of total tau isoforms as judged by an increase in 5E2 immunoreactivity. Neither treatment, either separately or in combination, induced an increase in PHF-1 immunoreactivity. These findings suggest that alterations in immunoreactivity with SMI antibodies reflected increases in NF epitopes. This was confirmed by immunoblot analyses. Since proteolysis is apparently instrumental in maintaining the normal distribution patterns of phosphorylated NF epitopes, these findings implicate deficiencies in proteolytic mechanisms in the development of neurofibrillary pathology, and underscore the possibility of a multiple etiology in human neuropathological conditions.

Aluminum↗
PubMed1995

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.

Blood Physiological Phenomena↗
PubMed1994

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.

Aging↗
PubMed1994

Delivery of anti-GAP-43 antibodies into neuroblastoma cells reduces growth cone size.

We have previously demonstrated that antibodies to the growth-associated protein, GAP-43, introduced intracellularly using a lipid carrier inhibited neurite outgrowth in NB2a/d1 neuroblastoma cells, and that culturing of these cells on adhesive substrates such as laminin or poly-L-lysine overcame this restriction. These findings suggest that GAP-43 may facilitate neuritogenesis by increasing membrane adhesiveness. To address this issue, in the present study we examined the effect of intracellular delivery of this antibody on growth cone size. A statistically significant percentage of those neurites that did elaborate following intracellular delivery of GAP-43 exhibited either no observable growth cones or smaller growth cones versus cells receiving pre-immune IgG. These results support the hypothesis that the requirement for GAP-43 in neuritogenesis may be related to growth cone formation and membrane adhesiveness.

Animals↗
PubMed1994

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.

Calpain↗
PubMed1994

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.

Aluminum↗