Biomedical subjects

G V Johnson

Publications and source records attributed to G V Johnson.

At least 73 records · Page 4Linked to original sources

PubMed1996

Differential binding of apolipoprotein E isoforms to tau and other cytoskeletal proteins.

The apolipoprotein E4 (apoE4) gene dose is a major risk factor for late-onset and sporadic Alzheimer's disease with 50% of homozygous patients developing the disease by age 70. Previous studies have shown localization of apoE to the cytoplasm of certain neurons within the brain. In addition, apoE3, but not apoE4, forms SDS-stable complexes with the microtubule-associated proteins tau and MAP-2. To extend these studies and quantitate the association of apoE with other proteins, the association of apoE3 and apoE4 with several cytoskeletal proteins was examined using both gel shift and overlay assays. In the gel shift assay, apoE3 formed SDS-stable complexes with the longest isoform of human recombinant tau (T4L), the shortest isoform of human recombinant tau (T3), and the 160-kDa neurofilament protein (NFM). ApoE4 did not bind T3, T4L, or NFM in this assay. The association of apoE3 and apoE4 with T4L, actin, or tubulin was further examined in an overlay assay with known amounts of the cytoskeletal proteins slot-blotted onto nitrocellulose and incubated in 0.15 microM (5 microg/ml) apoE3 or apoE4. In this assay, apoE3 and apoE4 bound T4L and tubulin equally well. In contrast, apoE3 bound actin with a significantly greater affinity than did apoE4. These results indicate that apoE isoforms interact with cytoskeletal proteins with at least two different binding affinities. The more avid interaction results in the formation of complexes which are SDS stable and occurs almost exclusively with apoE3, while the other interactions between apoE and cytoskeletal proteins are specific for apoE3.

Animals↗

PubMed1995

Postnatal changes in serine/threonine protein phosphatases and their association with the microtubules.

The activities and protein levels of three serine/threonine protein phosphatases were determined in homogenates and microtubule preparations from rat brain at various ages from postnatal day 1 (P1) through adulthood. The activities and levels of the calcium/calmodulin-dependent protein phosphatase, phosphatase 2B increased significantly from P1 to P21 in brain homogenates and remained elevated in the adult. The association of phosphatase 2B with microtubules was also found to be increased in the adult compared to the neonate (P3). In contrast, protein phosphatase 2A in brain homogenates decreased significantly from P1 to adult. However, the association of phosphatase 2A with the microtubules was found to increase with age. Finally, the activity and levels of phosphatase 1 in brain homogenates did not change with postnatal age, although the association of phosphatase 1 with microtubules was significantly decreased in the adult brain compared to P3. These studies clearly indicate that the activity, levels and association of these serine/threonine phosphatases with microtubules are independently regulated during postnatal development and suggest unique roles of phosphatase 1, phosphatase 2A and phosphatase 2B in modulating the phosphorylation state and function of microtubule-associated proteins at different postnatal ages.

Animals↗

PubMed1995

Detection of phosphorylated Ser262 in fetal tau, adult tau, and paired helical filament tau.

Paired helical filaments (PHFs) are the major structural elements of Alzheimer's disease neurofibrillary lesions, and these filaments are formed from hyperphosphorylated brain tau known as PHF-tau. Recent studies showed that many previously identified phosphorylated residues in PHF-tau also are phosphate acceptor sites in fetal and rapidly processed adult brain tau. However, Ser262 has been suggested to be uniquely phosphorylated in PHF-tau and a key regulator of the binding of tau to microtubules. For these reasons, we generated a monoclonal antibody (12E8) specific for phosphorylated Ser262 and showed that 12E8 binds to PHF-tau, rat and human fetal brain tau, as well as to rapidly processed adult rat and biopsy-derived human brain tau. Further, phosphorylation Ser262 was developmentally regulated, and endogenous brain phosphatases rapidly dephosphorylated Ser262 in biopsy-derived brain tau isolates. Finally, the phosphorylation of Ser262 did not eliminate the binding of tau to microtubules. Thus, we speculate that the binding of tau to microtubules is regulated by phosphorylation at multiple sites and that the generation of PHF-tau in Alzheimer's disease results from the reduced efficiency of phosphatases leading to the incremental accumulation of hyperphosphorylated tau.

Adult↗

PubMed1995

Modulation of the phosphorylation state of tau in situ: the roles of calcium and cyclic AMP.

Alterations in situ in the phosphorylation state of the microtubule-associated protein tau were examined in response to increasing intracellular levels of Ca2+ through N-methyl-D-aspartate (NMDA)-receptor activation, or activating cyclic AMP (cAMP)-dependent protein kinase (cAMP-PK), in rat cerebral-cortical slices. Increasing intracellular concentrations of Ca2+ by treatment of the brain slices with the glutamate analogue NMDA in depolarizing conditions (55 mM KCl) resulted in dephosphorylation of tau. Addition of KCl+NMDA to the slices resulted in a 40% decrease in 32P incorporation into tau, whereas addition of KCl or NMDA alone had no effect on tau phosphorylation. The KCl+NMDA-induced dephosphorylation of tau was blocked by the non-competitive NMDA-receptor antagonist MK801. Determine the involvement of the Ca2+/calmodulin-dependent phosphatase, calcineurin, in the KCl+NMDA-induced dephosphorylation of tau, slices were pretreated with the calcineurin inhibitor Cyclosporin A. Pretreatment of the rat brain slices with Cyclosporin A completely abolished the dephosphorylation of tau induced by the addition of KCl+NMDA. The dephosphorylation of tau in situ was site-selective, as indicated by the loss of 32P label from only a few select peptides. Activation of cAMP-PK by stimulating adenylate cyclase in rat cerebral-cortical slices with forskolin resulted in a 73% increase over control levels in 32P incorporation into immunoprecipitated tau. Two-dimensional phosphopeptide mapping revealed that most of the sites on tau phosphorylated in brain slices in response to increased cAMP levels were the same as those phosphorylated on isolated tau by purified cAMP-PK. Although the state of tau phosphorylation is certainly regulated by many protein phosphatases and kinases in vivo, to our knowledge this study provides the first direct evidence of a specific protein phosphatase and kinase that modulate the phosphorylation state of tau in situ.

Animals↗

PubMed1995

Localization and in situ phosphorylation state of nuclear tau.

The localization and phosphorylation state of tau in LA-N-5 neuroblastoma cells was examined. Our results demonstrate that there are two populations of tau in LA-N-5 cells: cytosolic tau and nuclear tau. Indirect immunofluorescent microscopy revealed that nuclear tau is specifically localized to the nucleolus while cytosolic tau is diffusely distributed. To localize and quantitate tau in LA-N-5 cells by subcellular fractionation, a method was developed to extract tau from the nucleus while preserving the endogenous state of the protein. These studies revealed that 16% of the total tau protein in LA-N-5 cells is located in the nucleus and more specifically was found predominantly in the chromatin fraction containing DNA, chromatin, and associated proteins. The phosphorylation state of nuclear and cytosolic tau was examined by labeling LA-N-5 cells with 32Pi and immunoprecipitating tau from the different fractions. These data demonstrated that nuclear tau and cytosolic tau are phosphorylated approximately to the same extent. To determine if the phosphorylation of nuclear tau occurs in the nucleus, LA-N-5 nuclei were isolated, incubated with [gamma-32P]ATP, extracted, and tau was immunoprecipitated. Although numerous nuclear proteins were 32P-labeled, tau was not phosphorylated. These results suggest that nuclear tau is not phosphorylated in the nucleus but rather in the cytosol prior to transport into the nucleus. The specific localization of nuclear tau strongly suggests that it has a functional role in the nucleus. However, further studies are necessary to determine the function of nuclear tau and how it may be regulated by phosphorylation.

Autoradiography↗

PubMed1995

Metal-catalyzed oxidation of bovine neurofilaments in vitro.

Neurofilaments (NF) are important determinants of the shape and size of nerve cells. The oxidation of NF, relevant to aging, neurodegenerative disorders, and axonal (Wallerian) degeneration, has not been studied. In this investigation, we have combined biochemical and ultrastructural methods to study the metal-catalyzed oxidation (MCO) of bovine NF using an ascorbate/Fe+3/O2 system. The oxidation of NF proteins was documented by increases in carbonyl content, which were time- and concentration-dependent. Polyacrylamide gel electrophoresis (PAGE) and immunoblot analyses revealed the fragmentation of oxidized NF proteins, predominantly NF-H and NF-M. Electron microscopy (EM) showed that oxidized NF formed dense aggregates and bundles of laterally aggregated filaments. Finally, we also demonstrated that oxidized NF proteins were more susceptible to calpain proteolysis. In view of the growing evidence supporting increased oxidative stress on the nervous system in aging and the report of Cu/Zn superoxide dismutase mutation in familial motor neuron disease, oxidative injury of NF may be relevant to cell atrophy and degeneration of nerve cells and to the formation of abnormal cytoskeletal structures.

Animals↗

PubMed1995

Tau self-association: stabilization with a chemical cross-linker and modulation by phosphorylation and oxidation state.

tau is a major component of paired helical filaments found in the neurofibrillary tangles of Alzheimer's diseased brain. However, the mechanism or mechanisms responsible for the association of tau to form these aggregates remains unknown. In this study, the role of intermolecular disulfide bonds in the formation of higher order oligomers of bovine tau and the human recombinant tau isoform T3 was examined using the chemical cross-linking agent disuccinimidylsuberate (DSS). In addition, the role of phosphorylation and oxidation state on the in vitro self-association of tau was studied using this experimental model. Stabilization of tau-tau interactions with DSS indicated that intermolecular disulfide bonds probably play a predominant role in dimer formation, but the formation of higher order oligomers of tau cannot be attributed to these bonds alone. tau-tau interactions were significantly decreased either by blocking Cys residues or by exposing the tau to a reducing (nitrogen and dithiothreitol), instead of an oxidizing, environment. tau self-association was also significantly decreased by prior phosphorylation with calcium/calmodulin-dependent protein kinase II. Phosphorylation by cyclic AMP-dependent protein kinase or dephosphorylation by alkaline phosphatase did not alter tau self-assembly. These data suggest a role for several factors that may modulate tau self-association in vivo.

Animals↗

PubMed1995

Phosphorylation of tau in situ: inhibition of calcium-dependent proteolysis.

In this study, the in situ phosphorylation and subsequent calcium-activated proteolysis of tau protein were examined in human neuroblastoma (LA-N-5) cells, which were differentiated into a neuronal phenotype. The phosphorylation of tau was increased by treating the cells with forskolin and rolipram, which elevate cyclic AMP levels, by treating with the phosphatase inhibitor okadaic acid, or by treating with a combination of both treatments. Phosphorylated tau migrated slightly slower on sodium dodecyl sulfate-polyacrylamide gels than tau from untreated cells. Immunostaining with the phosphate-sensitive monoclonal antibody Tau-1 was also decreased in cells treated with okadaic acid, indicating an increase in the phosphorylation of specific Ser-Pro motifs within the molecule. Calcium-dependent, in situ proteolysis of tau protein was induced by treating the cells with the calcium ionophore A23187. Tau protein was proteolyzed to a significantly lesser extent in cells treated with forskolin and rolipram, okadaic acid, or both than in cells in which phosphorylation was not increased. Partially purified tau protein from cells treated with a combination of forskolin, rolipram, and okadaic acid was also more resistant to proteolysis by calpain in vitro compared with tau isolated from control cells. These data suggest a possible role for phosphorylation in the regulation of tau metabolism and in pathological conditions in which the balance between protein kinases and phosphatases is disrupted.

Calcimycin↗

PubMed1995

Transglutaminase cross-linking of the tau protein.

Tissue transglutaminase (EC 2.3.2.13) is a calcium-activated enzyme that cross-links specific substrate proteins into insoluble, protease-resistant, high molecular weight complexes. Because the neurofibrillary tangles in Alzheimer disease have similar biochemical characteristics, and because the microtubule-associated protein tau is the predominant component of these structures, the substrate properties of tau with respect to transglutaminase were investigated. Bovine tau and recombinant human tau isoforms rapidly form high molecular weight, cross-linked polymers on incubation with transglutaminase. Polyamine incorporation assays indicate that bovine tau is an excellent substrate of transglutaminase, with a Km of 10.4 +/- 2.2 microM and a Vmax of 40.9 +/- 4.5 nmol/mg of enzyme/min. Individual recombinant human tau isoforms are not equivalent with respect to transglutaminase, as the smallest isoform T3 (352 amino acids) is not as good a substrate as the larger isoforms T4 (383 amino acids) and T4L (441 amino acids). To determine which segments of the tau protein are susceptible to modification by transglutaminase, tau was labeled with [3H]putrescine by transglutaminase and proteolyzed with alpha-chymotrypsin, and the breakdown products were analyzed. These experiments demonstrate that the enzyme modifies tau at only one or a few discrete sites, primarily in the carboxyl half of the molecule. Thus, the reaction is specific for only a small number of the many glutamine residues in tau. Furthermore, a tau deletion construct (T264) containing a portion of the microtubule-binding domains, which is a substrate of transglutaminase, cannot be cross-linked by the enzyme. This provides evidence that the cross-linking reaction is specific, and requires that the substrates be appropriately associated for cross-linking to occur.

Alzheimer Disease↗

PubMed1994

Monoclonal antibody Alz-50 reacts with bovine and human serum albumin.

Alz-50, a monoclonal antibody originally prepared using Alzheimer brain homogenates, reacts with PHF-tau and normal tau on immunoblots, and stains specific neuronal populations in sections from Alzheimer's disease brain. Although the Alz-50 epitope has been mapped to amino acids 2-10 present in all human tau isoforms, minimal Alz-50 immunoreactivity is present in tissue from control brain, suggesting Alz-50 binding may be dependent on tau conformational differences. The absence of conclusive results concerning Alz-50 binding presents the possibility of Alz-50 immunoreactivity with proteins other than tau. The present study demonstrates Alz-50 cross-reactivity with denatured bovine serum albumin (BSA) and human serum albumin (HSA). Using LA-N-5 neuroblastoma cells, BSA from serum-containing media was present in cell homogenates and was found to be Alz-50-reactive on immunoblots. In fact, Alz-50 (0.1 microgram/ml) recognized as little as 78 ng of BSA and 312 ng of HSA. Since Alz-50 does not recognize native BSA, blocking of immunoblots with 3% BSA did not alter Alz-50 reactivity with tau from LA-N-5 cells. On SDS-polyacrylamide gels, HSA (approximately 69 kDa) migrates very closely to the pattern of A68 (PHF-tau) from Alzheimer brain homogenates. Hence, the presence of BSA or other albumins in cell or brain homogenates may be an important concern when using the Alz-50 antibody.

Alzheimer Disease↗

PubMed1994

Transglutaminase facilitates the formation of polymers of the beta-amyloid peptide.

One of the major pathological characteristics of Alzheimer's disease is the increased number of amyloid-containing senile plaques within the brain. The dense cores of these plaques are composed primarily of highly insoluble aggregates of a 39-43-residue peptide referred to as the beta-amyloid peptide (beta A). The mechanisms by which these insoluble extracellular deposits of beta A are formed remain unknown. In this study, the cross-linking of beta A by the calcium-dependent enzyme, transglutaminase was examined. Transglutaminases are a family of enzymes which are found in brain, and catalyse the cross-linking of specific proteins into insoluble polymers. Synthetic beta A (1-40) was readily cross-linked by transglutaminase, forming multimers in a time-dependent fashion. Furthermore, a second peptide with a substitution similar to that in the Dutch-type hereditary amyloidosis mutation (Glu22 to Gln) was also found to be a substrate for transglutaminase. Since transglutaminase covalently cross-links proteins through glutamine residues, it is suggested that transglutaminase contributes to amyloid deposition in Dutch-type hereditary amyloidosis, and possibly Alzheimer's disease.

Alzheimer Disease↗

PubMed1994

Pavlovian conditioning alters cortical microtubule-associated protein-2.

Three experiments indicate that Pavlovian conditioning to tone alters microtubule-associated protein-2 (MAP-2) in the temporal cortex. First, increased MAP-2 immunohistochemistry was evident in temporal cortex following tone-shock pairings but not light-shock pairings. In the second experiment, animals given tone paired with shock (compared with animals trained with tone unpaired with shock or given tone only) showed MAP-2 immunohistochemical changes in the temporal cortex, as well as in the frontal and cingulate cortex, the hippocampus and amygdala. In experiment 3, quantitative immunoblots showed decreased intact MAP-2 and increased breakdown products selectively in temporal cortex following fear conditioning to tone. Conditioning to tone also increased sizes of MAP-2 rich pyramidal somata and apical dendrites in temporal and frontal cortex.

Acoustic Stimulation↗

PubMed1994

Casein kinase II preferentially phosphorylates human tau isoforms containing an amino-terminal insert. Identification of threonine 39 as the primary phosphate acceptor.

The in vitro phosphorylation of the microtubule-associated protein tau by casein kinase II was studied. Purified human brain tau was phosphorylated by casein kinase II to a stoichiometry of 0.7 mol of 32P/mol of tau. Individual recombinant human tau isoforms were phosphorylated to stoichiometries ranging from 0.2 to 0.8 mol of 32P/mol of tau. Casein kinase II catalyzed a 4-fold greater incorporation of phosphate into the tau isoform containing a 58-amino acid insert near its amino terminus (T4L) than the isoforms without the 58-amino acid insert (T3 and T4). Phosphopeptide mapping of casein kinase II phosphorylated human tau and recombinant tau isoforms suggested that the isoforms containing an amino-terminal insert constitute the major substrates for casein kinase II within the tau family. The sites of phosphorylation on T4L were identified by digesting phosphorylated T4L with the protease Asp-N, separating the peptides by reversed phase high performance liquid chromatography, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase amino-terminal sequencing. Thr39 was identified as the predominant phosphorylation site, which is located 5 residues from the amino-terminal insert in T4L. Phosphopeptide mapping of tau isolated from LA-N-5 neuroblastoma cells indicates that Thr39 is phosphorylated in situ. To our knowledge, this is the first demonstration of a differential phosphorylation of the human tau isoforms, with the isoforms containing the acidic amino-terminal insert being the preferred substrates of casein kinase II.

Amino Acid Sequence↗

PubMed1994

Compromised mitochondrial function results in dephosphorylation of tau through a calcium-dependent process in rat brain cerebral cortical slices.

Mitochondria play an important role in modulating intracellular levels of calcium, and therefore compromised mitochondrial function often leads to disruptions in calcium homeostasis. In this study, the effects of two uncouplers of oxidative phosphorylation, carbonyl cyanide-3-chlorophenylhydrazone (CCCP) and p-trifluoromethoxyphenylhydrazone (FCCP), on calcium-mediated modifications of the microtubule-associated protein, tau, in rat brain slices were examined. Incubation of slices with CCCP or FCCP resulted in an increase in electrophoretic mobility of several of the tau isoforms, with no apparent loss of intact tau or the appearance of degradation products. These data indicated that disrupting mitochondrial function by dissipating the transmembrane potential resulted in the dephosphorylation of tau. This finding was confirmed by using a front phosphorylation assay to demonstrate a CCCP-induced decrease in the phosphorylation state of tau. The dephosphorylation of tau induced by the proton-ionophores appeared to be calcium-dependent since the effect was blocked by EGTA. In addition, the CCCP-induced dephosphorylation of tau was blocked by cyclosporin A, a selective inhibitor of the calcium-dependent phosphatase, calcineurin. These data strongly indicate that tau is a substrate for calcineurin in vivo. Finally, the levels of ATP were depleted to a similar extent in brain slices incubated in the presence of CCCP or CCCP and EGTA. These results demonstrated depletion of ATP alone was not sufficient to stimulate the dephosphorylation of tau in this experimental paradigm.

Adenosine Triphosphate↗

PubMed1993

In vitro polymerization of oxidized tau into filaments.

Paired helical filaments (PHF) are abnormal neuronal polymers characteristic of Alzheimer's disease (AD). Although tau appears to be a major constituent of PHF, the mechanism for the polymerization of tau or its integration into PHF remains unknown. Here, we show that the oxidation of bovine tau in vitro induces an apparent dimerization of this protein and polymerization into filaments. These observations suggest that the oxidation of tau in vivo may contribute to the development of PHF in individuals with AD.

Actin Cytoskeleton↗

PubMed1993

Calpain-mediated proteolysis of microtubule-associated protein 2 (MAP-2) is inhibited by phosphorylation by cAMP-dependent protein kinase, but not by Ca2+/calmodulin-dependent protein kinase II.

The effects of cAMP-dependent protein kinase (cAMP-PK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation on the calpain-mediated degradation of microtubule-associated protein 2 (MAP-2) were studied. Both cAMP-PK and CaMKII readily phosphorylated MAP-2. However, cAMP-PK phosphorylated MAP-2 to a significantly greater extent than did CaMKII (4.5 mol 32P/mol MAP-2 and 1.4 mol 32P/mol MAP-2, respectively). Phosphorylation of MAP-2 by cAMP-PK, but not by CaMKII, significantly inhibited the calpain-induced hydrolysis of MAP-2. These results demonstrate that the phosphorylation of sites on the MAP-2 molecule accessible to cAMP-PK, but not to CaMKII, result in increased resistance to calpain proteolysis.

Animals↗

PubMed1993

Phosphorylation, calpain proteolysis and tubulin binding of recombinant human tau isoforms.

In this study, the phosphorylation, calpain hydrolysis and tubulin binding of three recombinant human tau isoforms were examined. The three isoforms used in these studies were tau with three (T3) or four (T4) tandemly repeated tubulin binding domains located in the carboxy-terminal half of the molecule; and tau with four-tandem repeats and a 58-amino acid insert in the amino terminus (T4L). Both cAMP-dependent protein kinase (cAMP-PK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) readily phosphorylated the three human tau isoforms, although cAMP-PK phosphorylated them to a significantly greater extent than CaMKII. Phosphorylation of T3, T4 and T4L by cAMP-PK or CaMKII resulted in the slowed migration of the protein bands on sodium dodecyl sulfate-polyacrylamide gels and a shift of the isoelectric variants to more acidic positions on two-dimensional non-equilibrium pH gradient electrophoresis gels compared with controls. However, the phosphorylation-induced changes in the electrophoretic migration of the tau isoforms were unique for each kinase. Two-dimensional phosphopeptide maps and sequential phosphorylation experiments indicate that cAMP-PK phosphorylates sites in the human tau isoforms that are phosphorylated by CaMKII, as well as unique sites that are not phosphorylated by CaMKII. T3, T4 and T4L were hydrolyzed similarly by calpain; however, the calpain proteolysis of the recombinant tau isoforms was significantly faster than the proteolysis of human or bovine tau. Phosphorylation of the isoforms by either cAMP-PK or CaMKII did not alter the rate or extent of calpain proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1993

Metal (Fe3+) affinity chromatography: differential adsorption of tau phosphoproteins.

Tau is a neuronal cytoskeletal protein consisting of a group of isoforms with apparent molecular masses ranging from 45 to 62 kDa. Tau purified from brain exists in multiple phosphorylated forms and abnormally phosphorylated tau appears to play an important role in the neuropathology of Alzheimer's disease. To separate the differentially phosphorylated populations of tau, a chromatographic technique using ferric ions adsorbed onto iminodiacetic acid substituted Sepharose was developed. Several distinct populations of tau were isolated based on the phosphorylation state. These preparations can be used for further investigation of how each specific phosphorylation state modulates the metabolism and function of tau.

Adsorption↗