Gail V. W. Johnson

Tau phosphorylation in neuronal cell function and dysfunction

Tau is a group of neuronal microtubule-associated proteins that are formed by alternative mRNA splicing and accumulate in neurofibrillary tangles in Alzheimers disease (AD) brain. Tau plays a key role in regulating microtubule dynamics, axonal transport and neurite outgrowth, and all these functions of tau are modulated by site-specific phosphorylation. There is significant evidence that a disruption of normal phosphorylation events results in tau dysfunction in neurodegenerative diseases, such as AD, and is a contributing factor to the pathogenic processes. Indeed, the abnormal tau phosphorylation that occurs in neurodegenerative conditions not only results in a toxic loss of function (e.g. decreased microtubule binding) but probably also a toxic gain of function (e.g. increased tau-tau interactions). Although tau is phosphorylated in vitro by numerous protein kinases, how many of these actually phosphorylate tau in vivo is unclear. Identification of the protein kinases that phosphorylate tau in vivo in both physiological and pathological processes could provide potential therapeutic targets for the treatment of AD and other neurodegenerative diseases in which there is tau pathology.

p38 kinase is activated in the Alzheimer's disease brain.

Abstract: The p38 mitogen‐activated protein kinase is a stress‐activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimers disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age‐matched normal brain. Intense phospho‐p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle‐bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF‐positive tau did not cross‐react with the phospho‐p38 antibody. These findings suggest a neuroinflammatory mechanism in the AD brain, in which aberrant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade, as well as cytoskeletal components.

The Microtubule-associated Protein Tau Is Extensively Modified with O-linked N-acetylglucosamine

Tau is a family of phosphoproteins that are important in modulating microtubule stability in neurons. In Alzheimers disease tau is abnormally hyperphosphorylated, no longer binds microtubules, and self-assembles to form paired helical filaments that likely contribute to neuron death. Here we demonstrate that normal bovine tau is multiply modified by Ser(Thr)-O-linked N-acetylglucosamine, a dynamic and abundant post-translational modification that is often reciprocal to Ser(Thr)-phosphorylation. O-GlcNAcylation of tau was demonstrated by blotting with succinylated wheat germ agglutinin and by probing with bovine milk β(1,4)galactosyltransferase. Structural analyses confirm the linkage and the saccharide structure. Tau splicing variants are multiply O-GlcNAcylated at similar sites, with an average stoichiometry of greater than 4 mol of O-linked N-acetylglucosamine/mol of tau. However, the number of sites occupied appears to be greater than 12, suggesting substoichiometric occupancy at any given site. A similar relationship between average stoichiometry and site-occupancy has also been described for the phosphorylation of tau. Site-specific or stoichiometric changes in O-GlcNAcylation may not only modulate tau function but may also play a role in the formation of paired helical filaments.

Direct, activating interaction between glycogen synthase kinase-3β and p53 after DNA damage

Glycogen synthase kinase-3β (GSK3β) is a central figure in Wnt signaling, in which its activity is controlled by regulatory binding proteins. Here we show that binding proteins outside the Wnt pathway also control the activity of GSK3β. DNA damage induced by camptothecin, which activates the tumor suppressor p53, was found to activate GSK3β. This activation occurred by a phosphorylation-independent mechanism involving direct binding of GSK3β to p53, which was confined to the nucleus where p53 is localized, and mutated p53 (R175H) bound but did not activate GSK3β. Activation of GSK3 promoted responses to p53 including increases in p21 levels and caspase-3 activity. Thus, after DNA damage there is a direct interaction between p53 and GSK3β, and these proteins act in concert to regulate cellular responses to DNA damage.

Insulin transiently increases tau phosphorylation: Involvement of glycogen synthase kinase-3β and Fyn tyrosine kinase

Abstract : The modulation of tau phosphorylation in response to insulin was examined in human neuroblastoma SH‐SY5Y cells. Insulin treatment resulted in a transient increase in tau phosphorylation followed by a decrease in tau phosphorylation that correlated directly with a sequential activation and deactivation of glycogen synthese kinase‐3β (GSK‐3β). The insulin‐induced increase in tau phosphorylation and concurrent activation of GSK‐3β was rapid (<2 min) and transient, and was associated with increased tyrosine phosphorylation of GSK‐3β. The increase in GSK‐3β tyrosine phosphorylation corresponded directly to an increase in the association of Fyn tyrosine kinase with GSK‐3β, and Fyn immunoprecipitated from cells treated with insulin for 1 min phosphorylated GSK‐3β to a significantly greater extent than Fyn immunoprecipitated from control cells. Subsequent to the increase in GSK‐3β activation and tau phosphorylation, treatment of cells with insulin for 60 min resulted in a dephosphorylation of tau and a decrease in GSK‐3β activity. Thus, insulin rapidly and transiently activated GSK‐3β and modulated tau phosphorylation, alterations that may contribute to neuronal plasticity.

Modulation of the in Situ Activity of Tissue Transglutaminase by Calcium and GTP

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situtransglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r 2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly lessin situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTGin situ.

Degradation of Microtubule-Associated Protein 2 and Brain Spectrin by Calpain: A Comparative Study

Abstract: The in vitro degradation of microtubule‐associated protein 2 (MAP‐2) and spectrin by the calcium‐dependent neutral protease calpain was studied. Five major results are reported. First, MAP‐2 isolated from twice‐cycled microtubules (2XMT MAP‐2) was extremely sensitive to calpain‐induced hydrolysis. Even at an enzyme‐to‐substrate ratio (wt/wt) of 1:200, 2XMT MAP‐2 was significantly degraded by calpain. Second, MAP‐2 purified from the total brain heat‐stable fraction (total MAP‐2) was significantly more resistant to calpain‐induced hydrolysis compared with 2XMT MAP‐2. Third, MAP‐2a and MAP‐2b were proteolyzed similarly by calpain, although some relative resistance of MAP‐2b was observed. Fourth, the presence of calmodulin significantly increased the extent of calpain‐induced hydrolysis of the α‐subunit of spectrin. Fifth, the two neuronal isoforms of brain spectrin (240/235 and 240/235E, referred to as α/βN and α/βE, respectively) showed different sensitivities to calpain. αN‐spectrin was significantly more sensitive to calpain‐induced degradation compared to αE‐spectrin. Among other things, these results suggest a role for the calpain‐induced degradation of MAP‐2, as well as spectrin, in such physiological processes as alterations in synaptic efficacy, dendritic remodeling, and in pathological processes associated with neurodegeneration.

Histone deacetylase 6 interacts with the microtubule‐associated protein tau

Histone deacetylase 6 (HDAC6), a unique cytoplasmic deacetylase, likely plays a role in neurodegeneration by coordinating cell responses to abnormal protein aggregation. Here, we provide in vitro and in vivo evidence that HDAC6 interacts with tau, a microtubule‐associated protein that forms neurofibrillary tangles in Alzheimer’s disease. This interaction is mediated by the microtubule‐binding domain on tau and the Ser/Glu tetradecapeptide domain on HDAC6. Treatment with tubacin, a selective inhibitor of tubulin deacetylation activity of HDAC6, did not disrupt HDAC6–tau interaction. Nonetheless tubacin treatment attenuated site‐specific tau phosphorylation, as did shRNA‐mediated knockdown of HDAC6. Proteasome inhibition potentiated HDAC6–tau interactions and facilitated the concentration and co‐localization of HDAC6 and tau in a perinuclear aggresome‐like compartment, independent of HDAC6 tubulin deacetylase activity. Furthermore, we observed that in Alzheimer’s disease brains the protein level of HDAC6 was significantly increased. These findings establish HDAC6 as a tau‐interacting protein and as a potential modulator of tau phosphorylation and accumulation.

Transglutaminase activity is increased in Alzheimer's disease brain

Transglutaminase is a calcium-activated enzyme that crosslinks substrate proteins into insoluble, often filamentous aggregates resistant to proteases. Because the neurofibrillary tangles in Alzheimers disease have similar characteristics, and because tau protein, the major component of these tangles is an excellent substrate of transglutaminase in vitro, transglutaminase activity and levels were measured in control and Alzheimers disease brain. Frozen prefrontal cortex and cerebellum samples from Alzheimers disease and control cases matched for age and postmortem interval were used in the analyses. Total transglutaminase activity was significantly higher in the Alzheimers disease prefrontal cortex compared to control. In addition the levels of tissue transglutaminase, as determined by quantitative immunoblotting, were elevated approximately 3-fold in Alzheimers disease prefrontal cortex compared to control. To our knowledge, this is the first demonstration that transglutaminase is increased in Alzheimers disease brain. There were no significant differences in transglutaminase activity or levels in the cerebellum between control and Alzheimers disease cases. Because the elevation of transglutaminase in the Alzheimers disease samples occurred in the prefrontal cortex, where neurofibrillary pathology is usually abundant, and not in the cerebellum, which is usually spared in Alzheimers disease, it can be suggested that transglutaminase could be a contributing factor in neurofibrillary tangle formation.

Proteolysis of tau by calpain

The calpain-induced proteolysis of tau associated with twice-cycled microtubules or from a total brain heat-stable fraction was studied. Twice-cycled microtubule tau was rapidly hydrolyzed by calpain. In contrast, tau purified from the total brain heat-stable fraction was very resistant to degradation by calpain. These results clearly demonstrate that there are at least 2 populations of tau in the brain based on calpain-sensitivity, a calpain-sensitive form that is associated with microtubules and a calpain-resistant form that may represent another population of tau in the brain.