U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy

Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.     

Pseudophosphorylation of tau at serine 422 inhibits caspase cleavage: in vitro evidence and implications for tangle formation in vivo

The tangles of Alzheimer's disease (AD) are comprised of the tau protein displaying numerous alterations, including phosphorylation at serine 422 (S422) and truncation at aspartic acid 421 (D421). Truncation at the latter site appears to result from activation of caspases, a class of proteases that cleave specifically at aspartic acid residues. It has been proposed that phosphorylation at or near caspase cleavage sites could regulate the ability of the protease to cleave at those sites. Here, we use tau pseudophosphorylated at S422 (S422E) to examine the effects of tau phosphorylation on its cleavage by caspase 3. We find that S422E tau is more resistant to proteolysis by caspase 3 than non-pseudophosphorylated tau. Additionally, we use antibodies directed against the phosphorylation site and against the truncation epitope to assess the presence of these epitopes in neurofibrillary tangles in the aged human brain. We show that phosphorylation precedes truncation during tangle maturation. Moreover, the distribution of the two epitopes suggests that a significant length of time (perhaps as much as two decades) elapses between S422 phosphorylation and cleavage at D421. We further conclude that tau phosphorylation at S422 may be a protective mechanism that inhibits cleavage in vivo.     

Ubiquitin ligase CHIP suppresses cancer stem cell properties in a population of breast cancer cells

Cancer stem cells (CSCs) have several distinctive characteristics, including high metastatic potential, tumor-initiating potential, and properties that resemble normal stem cells such as self-renewal, differentiation, and drug efflux. Because of these characteristics, CSC is regarded to be responsible for cancer progression and patient prognosis. In our previous study, we showed that a ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) suppressed breast cancer malignancy. Moreover, a recent clinical study reported that CHIP expression levels were associated with favorable prognostic parameters of patients with breast cancer. Here we show that CHIP suppresses CSC properties in a population of breast cancer cells. CHIP depletion resulted in an increased proportion of CSCs among breast cancers when using several assays to assess CSC properties. From our results, we propose that inhibition of CSC properties may be one of the functions of CHIP as a suppressor of cancer progression.     

Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation

Neurofibrillary tangles, which are major pathological hallmarks of Alzheimer's disease (AD), are composed of paired helical filaments (PHFs) containing hyperphosphorylated tau. Specific kinases regulate tau phosphorylation and are closely linked to the pathogenesis of AD. We have characterized a human tau-tubulin kinase 1 (TTBK1) gene located on chromosome 6p21.1. TTBK1 is a serine/threonine/tyrosine kinase that is conserved among species and belongs to the casein kinase 1 superfamily. It is specifically expressed in the brain, especially in the cytoplasm of cortical and hippocampal neurons. TTBK1 phosphorylates tau proteins in both a Mg2+- and a Mn2+-dependent manner. Phosphopeptide mapping and immunoblotting analysis confirmed a direct tau phosphorylation by TTBK1 at Ser198, Ser199, Ser202 and Ser422, which are also phosphorylated in PHFs. TTBK1 also induces tau aggregation in human neuronal cells in a dose-dependent manner. We conclude that TTBK1 is a neuron-specific dual kinase involved in tau phosphorylation at AD-related sites and is also associated with tau aggregation.     

CHIP interacts with heat shock factor 1 during heat stress

Heat shock factor 1 (HSF1) is a major transactivator of heat shock genes in response to stress and mediates cell protection against various harmful conditions. In this study, we identified the interaction of CHIP (carboxyl terminus of the heat shock cognate protein 70-interacting protein) with the N-terminus of HSF1. Using GST full-down assay, we found that CHIP directly interacts with C-terminal deleted HSF1 (a.a. 1-290) but not with full-length HSF1 under non-stressed conditions. Interestingly, interaction of CHIP with full-length HSF1 was induced by heat shock treatment. The structural change of HSF1 was observed under heat stressed conditions by CD spectra. These observations demonstrate the direct interaction between HSF1 and CHIP and this interaction requires conformational change of HSF1 by heat stress.     

Irreversible aggregation of protein synthesis machinery after focal brain ischemia

Focal brain ischemia leads to a slow type of neuronal death in the penumbra that starts several hours after ischemia and continues to mature for days. During this maturation period, blood flow, cellular ATP and ionic homeostasis are gradually recovered in the penumbral region. In striking contrast, protein synthesis is irreversibly inhibited. This study used a rat focal brain ischemia model to investigate whether or not irreversible translational inhibition is due to abnormal aggregation of translational complex components, i.e. the ribosomes and their associated nascent polypeptides, protein synthesis initiation factors and co-translational chaperones. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons, but clumped into large abnormal aggregates in penumbral neurons subjected to 2 h of focal ischemia followed by 4 h of reperfusion. The abnormal ribosomal protein aggregation lasted until the onset of delayed neuronal death at 24-48 h of reperfusion after ischemia. Biochemical study further suggested that translational complex components, including small ribosomal subunit protein 6 (S6), large subunit protein 28 (L28), eukaryotic initiation factors 2alpha, 4E and 3eta, and co-translational chaperone heat-shock cognate protein 70 (HSC70) and co-chaperone Hdj1, were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational complex components were also highly ubiquitinated. This study clearly demonstrates that focal ischemia leads to irreversible aggregation of protein synthesis machinery that contributes to neuronal death after focal brain ischemia.     

Kinase activities increase during the development of tauopathy in htau mice

Hyperphosphorylated tau aggregates are the core constituent of neurofibrillary tangles. Recent research has shown a division between the presence of tangles, neurodegeneration and subsequent memory impairment, raising the possibility that an earlier pre-aggregated form of tau may be toxic. To gain further insight into the relationship between abnormal forms of tau, we have analyzed pathological changes in tau during tauopathy development in tangle-forming transgenic mice. In addition, we have quantified changes in the endogenous levels of a panel of protein kinases. We show progressive increases in aggregated tau and disease-specific conformational change, with hyperphosphorylation occurring in an age-dependent manner at specific sites. There were significant correlations between specific phosphorylation changes and amounts of aggregated tau and and abnormal tau conformations. Of the protein kinases tested, we found increases in phosphorylated (activated) p38 and the cyclin-dependent kinase-5 neuronal activators, p35 and p25, with aging, in the htau line, but not in non-tangle-forming control mice. Changes in tau kinases correlated with the amount of tau present in abnormal conformations and with insoluble tau in htau mice. These data suggest that cdk5 and p38 may be associated with pathological changes in wild-type human tau during the progressive development of tauopathy.     

Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms

Neurofibrillary tangles are composed of insoluble aggregates of the microtubule-associated protein tau. In Alzheimer's disease the accumulation of neurofibrillary tangles occurs in the absence of tau mutations. Here we present mice that develop pathology from non-mutant human tau, in the absence of other exogenous factors, including beta-amyloid. The pathology in these mice is Alzheimer-like, with hyperphosphorylated tau accumulating as aggregated paired helical filaments. This pathologic tau accumulates in the cell bodies and dendrites of neurons in a spatiotemporally relevant distribution.     

Cholesterol-dependent modulation of tau phosphorylation in cultured neurons

One of the hallmarks of Alzheimer's disease (AD) is the abnormal state of tau. It is both highly phosphorylated and aggregated into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). However, the mechanism underlying the hyperphosphorylation of tau in NFTs and neuronal degeneration in AD remains to be elucidated. The fact that hyperphosphorylation of tau in NFTs are also found in the patients with Niemann-Pick disease, type C (NPC), which is a cholesterol storage disease associated with defective intracellular trafficking of exogenous cholesterol, implies that perturbation of cholesterol metabolism may be involved in tau phosphorylation and neurodegeneration. Here, we report that cholesterol deficiency induced by inhibition of cholesterol biosynthesis in cultured neurons results in hyperphosphorylation of tau, accompanied by axonal degeneration associated with microtubule depolymerization. These changes were prevented by concurrent treatment with beta-migrating very low-density lipoprotein (beta-VLDL) or cholesterol. We propose that intracellular cholesterol plays an essential role in the modulation of tau phosphorylation and the maintenance of microtubule stability.     

Characterization of the in vitro phosphorylation of human tau by tau protein kinase II (cdk5/p20) using mass spectrometry

Hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies, and tau protein kinase II is reported to play a role in the pathogenesis of Alzheimer's disease. Recently, we reported that tau protein kinase II (cdk5/p20)-phosphorylated human tau inhibits microtubule assembly, and tau protein kinase II (cdk5/p20) phosphorylation of microtubule-associated tau results in dissociation of phosphorylated tau from the microtubules and tubulin depolymerization. In the studies reported here, a combination of mass spectrometric techniques was used to study the phosphorylation of human recombinant tau by recombinant tau protein kinase II (cdk5/p20) in vitro. The extent of phosphorylation was determined by measuring the molecular mass of phosphorylated tau using mass spectrometry. Reaction of human recombinant tau with tau protein kinase II (cdk5/p20) resulted in the formation of two major species containing either five or six phosphate groups. The specific amino acid residues phosphorylated were determined by analyzing tryptic peptides by tandem mass spectrometry via either MALDI/TOF post-source decay or by electrospray tandem mass spectrometry. Based on these experiments, we conclude that tau protein kinase II (cdk5/p20) can phosphorylate human tau at Thr(181), Thr(205), Thr(212), Thr(217), Ser(396) and Ser(404).     

N-glycosylation induced changes in tau protein dynamics reveal its role in tau misfolding and aggregation: A microsecond long molecular dynamics study

Various posttranslational modifications like hyperphosphorylation, O-GlcNAcylation, and acetylation have been attributed to induce the abnormal folding in tau protein. Recent in vitro studies revealed the possible involvement of N-glycosylation of tau protein in the abnormal folding and tau aggregation. Hence, in this study, we performed a microsecond long all atom molecular dynamics simulation to gain insights into the effects of N-glycosylation on Asn-359 residue which forms part of the microtubule binding region. Trajectory analysis of the stimulations coupled with essential dynamics and free energy landscape analysis suggested that tau, in its N-glycosylated form tends to exist in a largely folded conformation having high beta sheet propensity as compared to unmodified tau which exists in a large extended form with very less beta sheet propensity. Residue interaction network analysis of the lowest energy conformations further revealed that Phe378 and Lys353 are the functionally important residues in the peptide which helped in initiating the folding process and Phe378, Lys347, and Lys370 helped to maintain the stability of the protein in the folded state.     

The role of tau phosphorylation in transfected COS-1 cells

Tau cDNAs from each of the six human isoforms were transfected into COS- 1 cells and, in every case, more than one peptide was observed. The diversity of expressed isoforms was due to different levels of tau phosphorylation. Tau phosphorylation results in a decrease of the protein electrophoretic mobility. The major contribution to this mobility shift is due to the phosphorylation at the at the C-terminus of the molecule, as inferred from the expression of tau fragments. Phosphorylation takes place in some of the sites modified in neural cells and in the basis of AD patients. Copolymerization studies indicate that the level of phosphorylation, as well as the localization of the modified residues, may affect the binding of the protein to microtubules. These results indicate that phosphorylation regulates tau function inside the cell.     

Structure-activity relationship of cyclic thiacarbocyanine tau aggregation inhibitors

Macrocyclic bis-thiacarbocyanines are efficacious inhibitors of tau protein aggregation. To extend the structure-activity relationship of this inhibitor class, N,N’-alkylene bis-thiacarbocyanines linked by chains of three to eight methylene carbons were synthesized and examined for inhibitory activity against recombinant human tau aggregation in vitro. At 10 micromolar concentration, inhibitory activity varied with linker length, with four methylene units being most efficacious. On the basis of absorbance spectroscopy measurements, linker length also affected compound folding and aggregation propensity, with a linker length of four methylene units being optimal for preserving open monomer conformation. These data suggest that inhibitory potency can be optimized through control of linker length, and that a contributory mechanism involves modulation of compound folding and aggregation.     

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.     

饥饿对小鼠脑中tau蛋白磷酸化和O-GlcNAc糖基化的影响

为了探讨大脑中葡萄糖摄取和代谢障碍在阿尔茨海默病(Alzheimer$sdisease,AD)神经退行性病变中的作用,将昆明种小鼠进行饥饿和再喂食处理,并使用多种磷酸化tau蛋白特异性的抗体和蛋白O-GlcNAc糖基化特异性抗体进行检测,观察饥饿及恢复喂养后不同时间点大脑皮质中tau蛋白糖基化及多个位点磷酸化的变化.结果显示:饥饿处理引起小鼠大脑皮质中总蛋白和tau蛋白的O-GlcNAc糖基化水平降低,同时tau蛋白磷酸化水平升高,饥饿引起的tauO-GlcNAc糖基化和磷酸化改变均在恢复进食后逆转成正常水平.该研究结果提示:大脑中tau蛋白的磷酸化和O-GlcNAc糖基化之间存在相互调节,脑中葡萄糖代谢障碍可能通过下调tau蛋白O-GlcNAc糖基化水平使tau蛋白产生异常过度磷酸化,进而促发AD的病理进程.这一结果为在早期阶段通过逆转tau蛋白异常过度磷酸化治疗AD成为可能提供了实验基础.     

Neurotoxic dopamine quinone facilitates the assembly of tau into fibrillar polymers

Aberrant aggregation of microtubule associated protein tau is the main characteristic of different disorders known as tauopathies. Different compounds have been described to facilitate tau aberrant aggregation. In this work, we demonstrate that oxidized products of dopamine (neurotoxic dopamine quinone), a neurotransmitter involved in Parkinson's disease, promote tau polymerization. Curiously, neurons expressing dopamine (substantia nigra) show a low content of tau protein and seldom have tau aggregation in tauopathies. In non-dopaminergic neurons, quinone oxidation products may be involved in tau polymerization. These results support a link between oxidative damage and the onset of tauopathies. (Mol Cell Biochem 278: 203–212, 2005)     

Mutant γPKC that causes spinocerebellar ataxia type 14 upregulates Hsp70, which protects cells from the mutant’s cytotoxicity

Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is misfolded, susceptible to aggregation and cytotoxic. Molecular chaperones assist the refolding and degradation of misfolded proteins and prevention of the proteins’ aggregation. In the present study, we found that the expression of mutant γPKC-GFP increased the levels of heat-shock protein 70 (Hsp70) in SH-SY5Y cells. To elucidate the role of this elevation, we investigated the effect of siRNA-mediated knockdown of Hsp70 on the aggregation and cytotoxicity of mutant γPKC. Knockdown of Hsp70 exacerbated the aggregation and cytotoxicity of mutant γPKC-GFP by inhibiting this mutant’s degradation. These findings suggest that mutant γPKC increases the level of Hsp70, which protects cells from the mutant’s cytotoxicity by enhancing its degradation.     

Phosphorylation of tau at serine 416 by Ca2+/calmodulin-dependent protein kinase II in neuronal soma in brain

It is well known that tau is a good in vitro substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). However, it is not clear at present whether CaM kinase II phosphorylates tau in vivo or not. Serine 416, numbered according to the longest human tau isoform, has been reported to be one of the major phosphorylation sites by CaM kinase II in vitro. In this study, we produced a specific antibody against tau phosphorylated at serine 416 (PS416-tau). Immunoblot analysis revealed that the antibody reacted with tau in the rat brain extract which was prepared in the presence of protein phosphatase inhibitors. Developmental study indicated that serine 416 was strongly phosphorylated at early developmental stages in rat brain. We examined the localization of PS416-tau in primary cultured hippocampal neurons and the immortalized GnRH neurons (GT1-7 cells), which were stably transfected with CaM kinase IIalpha cDNA. Immunostaining of these cells indicated that tau was phosphorylated mainly in neuronal soma. Interestingly, tau in neuronal soma in Alzheimer's disease (AD) brain was strongly immunostained by the antibody. These results suggest that CaM kinase II is involved in the accumulation of tau in neuronal soma in AD brain.