Proteolysis Of Tau Research Articles

Proteolysis of tau by granzyme A in tauopathies generates fragments that are aggregation prone.

Tauopathies, including Alzheimer's disease, corticobasal degeneration and progressive supranuclear palsy, are characterised by the aggregation of tau into insoluble neurofibrillary tangles in the brain. Tau is subject to a range of post-translational modifications, including proteolysis, that can promote its aggregation. Neuroinflammation is a hallmark of tauopathies and evidence is growing for a role of CD8+ T cells in disease pathogenesis. CD8+ T cells release granzyme proteases but what role these proteases play in neuronal dysfunction is currently lacking. Here, we identified that granzyme A (GzmA) is present in brain tissue and proteolytically cleaves tau. Mass spectrometric analysis of tau fragments produced on digestion of tau with GzmA identified three cleavage sites at R194-S195, R209-S210 and K240-S241. Mutation of the critical Arg or Lys residues at the cleavage sites in tau or chemical inhibition of GzmA blocked the proteolysis of tau by GzmA. Development of a semi-targeted mass spectrometry approach identified peptides in tauopathy brain tissue corresponding to proteolysis by GzmA at R209-S210 and K240-S241 in tau. When expressed in cells the GzmA-cleaved C-terminal fragments of tau were highly phosphorylated and aggregated upon incubation of the cells with tauopathy brain seed. The C-terminal fragment tau195-441 was able to transfer between cells and promote aggregation of tau in acceptor cells, indicating the propensity for such tau fragments to propagate between cells. Collectively, these results raise the possibility that GzmA, released from infiltrating cytotoxic CD8+ T cells, proteolytically cleaves tau into fragments that may contribute to its pathological properties in tauopathies.

Human Tau Isoforms and Proteolysis for Production of Toxic Tau Fragments in Neurodegeneration.

The human tau protein is implicated in a wide range of neurodegenerative “tauopathy” diseases, consisting of Alzheimer’s disease (AD) and frontotemporal lobar degeneration which includes progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, and FTLD-tau (frontotemporal dementia with parkinsonism caused by MAPT mutations). Tau gene transcripts in the human brain undergo alternative splicing to yield 6 different tau protein isoforms that are expressed in different ratios in neurodegeneration which result in tau pathology of paired-helical filaments, neurofibrillary tangles, and tau fibrillar aggregates with detrimental microtubule destabilization. Protease-mediated tau truncation is an important post-translational modification (PTM) which drives neurodegeneration in a tau fragment-dependent manner. While numerous tau fragments have been identified, knowledge of the proteolytic steps that convert each parent tau isoform into specific truncated tau fragments has not yet been fully defined. An improved understanding of the relationships between tau isoforms and their proteolytic processing to generate neurotoxic tau fragments is important to the field. This review evaluates tau isoform expression patterns including PTMs and mutations that influence proteolysis of tau to generate toxic fragments that drive cognitive deficits in AD and other tauopathy models. This assessment identifies the gap in the field on understanding the details of proteolytic steps used to convert each tau isoform into fragments. Knowledge of the processing mechanisms of tau isoforms can lead to new protease targeted drug strategies to prevent the formation of toxic tau fragments in tauopathy neurodegenerative diseases.

The interrelationship of proteasome impairment and oligomeric intermediates in neurodegeneration.

Various neurodegenerative diseases are characterized by the accumulation of amyloidogenic proteins such as tau, α-synuclein, and amyloid-β. Prior to the formation of these stable aggregates, intermediate species of the respective proteins—oligomers—appear. Recently acquired data have shown that oligomers may be the most toxic and pathologically significant to neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The covalent modification of these oligomers may be critically important for biological processes in disease. Ubiquitin and small ubiquitin-like modifiers are the commonly used tags for degradation. While the modification of large amyloid aggregates by ubiquitination is well established, very little is known about the role ubiquitin may play in oligomer processing and the importance of the more recently discovered sumoylation. Many proteins involved in neurodegeneration have been found to be sumoylated, notably tau protein in brains afflicted with Alzheimer’s. This evidence suggests that while the cell may not have difficulty recognizing dangerous proteins, in brains afflicted with neurodegenerative disease, the proteasome may be unable to properly digest the tagged proteins. This would allow toxic aggregates to develop, leading to even more proteasome impairment in a snowball effect that could explain the exponential progression in most neurodegenerative diseases. A better understanding of the covalent modifications of oligomers could have a huge impact on the development of therapeutics for neurodegenerative diseases. This review will focus on the proteolysis of tau and other amyloidogenic proteins induced by covalent modification, and recent findings suggesting a relationship between tau oligomers and sumoylation.

The C-terminus of tau protein plays an important role in its stability and toxicity.

The identification of tau fragments generated by proteolysis in the neurons of AD patients and in neurofibrillary tangles encourages research on the toxicity of truncated tau. However, the detailed mechanism underlying the proteolysis-induced toxicity of tau is not clear and even controversial in some cases. In the present study, we used Drosophila as a model to evaluate the toxicity of a set of truncated tau fragments in vivo and found that the flies harboring C-terminal-truncated tau exhibited less toxicity due to the unstable characteristic of C-terminal-truncated tau fragments. Further study carried out in cultured Drosophila Kc cells revealed that C-terminal-truncated tau fragments degrade faster than full-length tau or N-terminal-truncated fragments. Collectively, our data implicate proteolysis of tau as an important pathway mediating tau degradation and neurotoxicity in vivo.

P33: Nitrite prevents hypoxic injury of neurons through suppression of caspase-6-mediated cleavage of Tau

It has been shown clearly that hypoxic stress associated with various pathological conditions, such as ischemic stroke or obstructive sleep apnea, impairs signal transduction of neurons, thus causing severe destruction of brain functions. To date the underlying mechanism of hypoxia-induced neuronal injuries remains elusive. It is also not known what strategy might be used to prevent neurons from hypoxic damages. Here we show that exposure of differentiated Neuro-2a (N2a) cells and primary rat cortical neurons with hypoxia resulted in significant neurite/axon retraction, a hallmark of neuronal injury. Interestingly, this detrimental effect of hypoxia on neurites was completely prevented by nitrite treatment, suggesting a protective role of nitrite in neuronal functions. We further illustrated that hypoxic stress induces collapse and disassembly of microtubule structure, the main framework composing neurites, leading to retraction of neurites. The disruption of microtubule in neurites is caused at least in part by the loss of integrity of Tau, a microtubule-associated protein that stabilizes microtubule structure, through a caspase-dependent process. Our data showed that cleavage of Tau and retraction of neurites is induced by caspase-6, which is robustly activated in hypoxic neurons. Importantly, in the presence of nitrite, caspase-6 was inactivated through S -nitrosylation of its active-site Cys residue, thus being unable to catalyze the proteolysis of Tau. Together these results are the first to demonstrate that nitrite plays a critical role in the protection of neuronal function against hypoxic insult. Our findings show that nitrite holds great potential for the treatment of diseases associated with acute or chronic hypoxia-induced disorder of neuronal homeostasis. Disclosure Nothing to disclose.

Implicating Calpain in Tau-Mediated Toxicity In Vivo

Alzheimer's disease and other related neurodegenerative disorders known as tauopathies are characterized by the accumulation of abnormally phosphorylated and aggregated forms of the microtubule-associated protein tau. Several laboratories have identified a 17 kD proteolytic fragment of tau in degenerating neurons and in numerous cell culture models that is generated by calpain cleavage and speculated to contribute to tau toxicity. In the current study, we employed a Drosophila tauopathy model to investigate the importance of calpain-mediated tau proteolysis in contributing to tau neurotoxicity in an animal model of human neurodegenerative disease. We found that mutations that disrupted endogenous calpainA or calpainB activity in transgenic flies suppressed tau toxicity. Expression of a calpain-resistant form of tau in Drosophila revealed that mutating the putative calpain cleavage sites that produce the 17 kD fragment was sufficient to abrogate tau toxicity in vivo. Furthermore, we found significant toxicity in the fly retina associated with expression of only the 17 kD tau fragment. Collectively, our data implicate calpain-mediated proteolysis of tau as an important pathway mediating tau neurotoxicity in vivo.

Tau cleavage by calpain: The intermediate at Mr 17kD / MW 11kD is metastable but not toxic

The amyloid cascade hypothesis of AD posits that Aß triggers the neurofibrillary pathology of Tau. It was proposed that a toxic “17 kD” fragment of Tau, generated by the protease calpain, was induced by exposure of neurons to Aß. Its size (residues 45-230, MW=18.7kD) was estimated on the basis of potential cleavage sites in Tau by calpain (Park & Ferreira, J. Neurosci. 2005). In the current study, we sought to identify the exact nature of the “17 kD” fragment of Tau generated by calpain and its mode of toxicity. The limited proteolysis of Tau by calpain in vitro or in cultured neurons caused by treatment with Aß oligomers was analysed by western blotting. Calpain-induced fragments of Tau were identified by protein sequencing, mass spectrometry, and antibody labelling. Cytotoxicity of Tau fragments was monitored by LDH release assay or apoptotic assay. The presence of fragments in Alzheimer or control brains was assessed by immunoblotting. The “Mr=17 kD” fragment is actually much smaller than assumed so far, containing only residues 125-230 (MW=10.7kD). On SDS gels it runs at Mr∼17kD because of the anomalous behavior of Tau. Inducing Tau hyperphosphorylation by okadaic acid or mimicking phosphorylation by multiple Glu mutations did not prevent the generation of this fragment. The fragment was induced not only by Aß oligomers, but also by other cell stressors, e.g. thapsigargin or glutamate in cortical neurons. However, overexpression of neither Tau45-230 nor Tau125-230 fragment was toxic to neurons. Finally, the calpain-induced fragment was observed both in AD brains and in control normal human brains. The Mr=17 kD Tau fragment is a metastable cleavage product of calpain and comprises residues 125-230. The fragment is induced by cell stress, including exposure to Aß, but it is not a mediator of Aß-induced toxicity. This suggests that events upstream of calpain activation might cause both Tau fragmentation and toxicity. - Supported by: EU/Memosad, BMBF/KNDD, Breuer Fund, Metlife Fund.

Proteolytic processing of tau

Tau aggregation is a hallmark of several neurodegenerative diseases, including AD (Alzheimer's disease), although the mechanism underlying tau aggregation remains unclear. Recent studies show that the proteolysis of tau plays an important role in both tau aggregation and neurodegeneration. On one hand, truncation of tau may generate amyloidogenic tau fragments that initiate the aggregation of tau, which in turn can cause toxicity. On the other hand, truncation of tau may result in tau fragments which induce neurodegeneration through unknown mechanisms, independently of tau aggregation. Blocking the truncation of tau thus may represent a promising therapeutic approach for AD or other tauopathies. In the present paper, we summarize our data on tau cleavage in a cell model of tauopathy and major results on tau cleavage reported in the literature.

Involvement of puromycin‐sensitive aminopeptidase in proteolysis of tau protein in cultured cells, and attenuated proteolysis of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) mutant tau

In tauopathies, tau protein is hyperphosphorylated, ubiquitinated, and accumulated in the brain; however, the mechanisms underlying this accumulation remain unclear. To gain an understanding of the role of proteases in the metabolism of tau protein, in the present study we evaluated the effects of protease inhibitors in SH-SY5Y human neuroblastoma cells and COS-7 cells transfected with the tau gene. When cells were treated with 0.1-10 micromol/L of lactacystin and 1.0-20 micromol/L of MG-132 (inhibitors of proteasome), 0.1-10 micromol/L of CA-074Me (a cathepsin inhibitor), and 0.1-2 micromol/L of puromycin (a puromycin-sensitive aminopeptidase (PSA) inhibitor) for up to 24 h, there were no significant changes in tau protein levels. However, pulse-chase experiments demonstrated that the proteolysis of tau protein in SH-SY5Y cells was attenuated following treatment of cells with 200 nmol/L puromycin. Increased tau protein levels were also observed in SH-SY5Y cells treated with short interference (si) RNA to PSA to inhibit the expression of PSA. These data suggest that PSA is a protease that catalyses tau protein predominantly in SH-SY5Y cells. The protein metabolism of tau-containing mutations of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) was also investigated using pulse-chase experiments. The results indicate attenuated proteolysis of tau in cells transfected with mutant tau genes after 48 h. Further immunocytochemical analysis and subcellular fractionation experiments revealed that the mutations did not alter the intracellular distribution of tau and suggested that impaired accessibility of tau to PSA is unlikely to account for the attenuated proteolysis of tau protein. Western blotting with phosphorylation-dependent antibodies revealed that phosphorylation levels of tau at Thr(231), Ser(396), and Ser(409) were increased in cells transfected with V337M, R406W, and R406W mutant tau genes, respectively. Together, the data suggest that attenuated proteolysis of FTDP-17 mutant tau may be explained by increased phosphorylation levels, resulting in resistance to proteolysis.

Proteasome inhibition increases tau accumulation independent of phosphorylation

An intrinsic link between proteasome and tau degradation in Alzheimer's disease (AD) has been suggested, however, the role of proteasome in the proteolysis of tau is still uncertain. Here, we investigated the influence of proteasome inhibition on the accumulation, phosphorylation, ubiquitination, solubility of tau and the memory retention in rats. We observed that lactacystin inhibited the proteasome activities and increased the level and insolubility of different tau species, including phosphorylated tau. The elevation of the phosphorylated tau was no longer present and the level of pS214 and pT231 tau was even lower than normal level after normalized to total tau. Inhibition of proteasome resulted in activation of cAMP-dependent protein kinase, glycogen synthase kinases-3β and cyclin-dependent kinase-5, and inhibition of protein phosphatase-2A and c-Jun N-terminal kinase (JNK). Proteasome inhibition did not affect the memory retention of the rats. We conclude that proteasome inhibition increases accumulation and insolubility of tau proteins independent of tau phosphorylation, and JNK inhibition may be partially responsible for the relatively decreased phosphorylation of tau in the rat brains.

Neurodegeneration and Alzheimer’s disease: the lesson from tauopathies

The Amyloid Cascade Hypothesis suggests that the decisive event in Alzheimer's disease (AD) is the deposition of fibrils of beta-amyloid protein (Abeta). The main objection to this hypothesis is the weak correlation between plaque load and severity of dementia. The good correlation between synaptic loss and dementia suggests that AD may be regarded as a synaptic failure. The toxicity of Abeta depends on its state of aggregation. The most important implication derived from the studies of tau gene mutations in a familial form of frontotemporal dementia (FTDP-17) is that the mutation itself is sufficient to cause neuronal loss. Several recent data suggest that apoptotic mechanisms may represent the missing link between Abeta deposition and proteolysis of tau, an early event in the pathogenic sequence of AD. Collectively, these observations suggest a model of AD whereby overproduction or reduced clearance of Abeta initiates a cascade of events that lead to neuronal loss directly or through post-translational modification of tau.

Tau phosphorylation and proteolysis: Insights and perspectives

In 1992 little was known about the specific protein kinases that phosphorylate tau and the proteases that regulate tau turnover. Although we had already demonstrated that tau was a substrate of the calcium-activated protease calpain (Johnson et al. (1989), Biochem Biophys Res Commun 163, 1505-1511), our publication entitled, "Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain" (Litersky and Johnson (1992), J Biol Chem 267, 1563-1568) was the first demonstration that phosphorylation by a specific kinase could inhibit the proteolysis of tau by calpain. At the time these findings suggested that the abnormal phosphorylation of tau in Alzheimer's disease brain could result in impaired tau turnover and thus result in an abnormal accumulation of the protein that could contribute to the formation of pathological lesions. Since this initial finding, much has been learned about the proteolysis of tau, not only by calpain, but by other proteases as well. However, much remains unknown about how phosphorylation regulates tau turnover in vivo and the specific proteases involved. In this article we give a brief history of our initial findings and then discuss subsequent studies from our laboratory, as well as others, on tau proteolysis and modulation by phosphorylation and how these findings contribute to our understanding of the posttranslational processing of tau in Alzheimer's disease.

Presence of calpain‐induced proteolysis in retinal degeneration and dysfunction in a rat model of acute ocular hypertension

The purpose of this study was to determine if calpain-induced proteolysis was associated with retinal degeneration or dysfunction in the rat acute ocular hypertensive model. Acute glaucoma was produced by elevation of IOP to 120 mm Hg for 1 hr. Retinal degeneration was evaluated by H&E staining and apoptosis was determined by TUNEL staining in histologic sections of retina. Electroretinogram (ERG) was carried out to evaluate changes in functionality. Activation of calpains was determined by casein zymography and immunoblotting. Total calcium in retina was measured by atomic absorption spectrophotometry. Proteolysis of alpha-spectrin, tau, cdk5, and p35 (a regulator of cdk5) were evaluated by immunoblotting. The thickness of inner plexiform layer (IPL) and inner nuclear layer (INL), and the number of cells in the ganglion cell layer (GCL) decreased after ocular hypertension. Numerous cells in the INL stained positive for TUNEL and some cells in the outer nuclear layer (ONL) showed TUNEL staining. The a-wave in ERG was temporarily decreased after ocular hypertension and then recovered to normal. In contrast, the b-wave was completely lost. Calpains were activated after ocular hypertension. Activation of calpains was associated with increased calcium in retina. Calpain-dependent proteolysis of alpha-spectrin, tau, and p35 were observed in retina after ocular hypertension. The results suggested that increased calcium and subsequent proteolysis by activated calpains was associated with the death of inner retinal cells due to acute ocular hypertension in the rat model. Calpain inhibitors may be candidate drugs for treatment of retinal degeneration and dysfunction resulting from glaucoma.

Concurrent calpain and caspase-3 mediated proteolysis of αII-spectrin and tau in rat brain after methamphetamine exposure: A similar profile to traumatic brain injury

Neurotoxicity in rat cortex and hippocampus following acute methamphetamine administration was characterized and compared to changes following traumatic brain injury. Doses of 10, 20, and 40 mg/kg of methamphetamine produced significant increases in calpain- and caspase-cleaved αII-spectrin and tau protein fragments, suggesting cell injury or death. Changes in proteolytic products were significantly increased over vehicle controls. Use of fragment specific biomarkers detected prominent calpain-mediated protein fragments in the cortex and hippocampus while caspase-mediated protein fragments were also detected in the hippocampus. Remarkably, proteolytic product increases at the 40 mg/kg dose after 24 h were as high as those observed in experimental traumatic brain injury. Use of calpain and caspase proteolytic inhibitors may be useful in preventing methamphetamine-induced neurotoxicity.

Proteolysis of Non-phosphorylated and Phosphorylated Tau by Thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.

Modifications of tau protein during neuronal cell death.

Accumulation of hyperphosphorylated tau protein and progressive neuronal loss are among the major hallmarks of certain neurodegenerative disorders including Alzheimer's disease. However, the relationship between tau phosphorylation and neuronal cell death remains unclear. Here we have analyzed tau modifications during two forms of neuronal death, apoptosis and necrosis, using primary cultures of cerebellar granule neurons as a simple model system. Induction of neuronal apoptosis by inhibition of phosphatidylinositol 3-kinase results in a rapid increase in the phosphorylation of tau, which is followed by the dephosphorylation and cleavage of the protein. In contrast, necrosis triggered by high salt shock or glutamate treatment leads to a rapid dephosphorylation and an almost complete proteolysis of tau. These data suggests that a transient tau hyperphosphorylation occurs at an early stage of apoptosis, whereas tau is dephosphorylated and cleaved during the late phase of apoptosis as well as in necrotic neurons.

Relationship of calbindin D28K-immunoreactive cells and neuropathological changes in the hippocampal formation of Alzheimer's disease.

Previous studies have reported that calcium binding proteins, which have important functions in regulating the intracellular ion concentration, may influence the vulnerability of neurons in neurodegenerative disease. It has been observed that the neurons containing calbindin D28K (CB) may in certain circumstances be more resistant to excitotoxic and ischemic injury. In the present study the susceptibility of hippocampal neurons containing CB to develop NFT was studied, and the distribution of CB cells was compared with hippocampal plaque density in the Alzheimer's disease (AD) brain. Interestingly CB-positive hippocampal neurons did not contain tangles and could be seen next to degenerating tau-positive pyramidal cells. Comparison of the hippocampal plaque distribution with that of CB neurons showed that in general CB-positive neurons were found in areas with a low plaque burden. Further comparison of cases with differing degrees of severity indicated that CB-positive neurons were relatively preserved in cases with moderate plaque and tangle content but that in severe cases the CB-positive pyramidal cells were lost. These findings indicate that CB cells may be protected in the earlier stages of the disease but that this resistance ability is lost in the late stages of AD. The observation that CB-positive pyramidal cells do not accumulate NFT suggests that proteolysis of tau differs in CB-negative and CB-positive cells.

Synergetic activation of p38 mitogen-activated protein kinase and caspase-3-like proteases for execution of calyculin A-induced apoptosis but not N-methyl-d-aspartate-induced necrosis in mouse cortical neurons.

We examined the possibility that p38 mitogen-activated protein kinase and caspase-3 would be activated for execution of apoptosis and excitotoxicity, the two major types of neuronal death underlying hypoxicischemic and neurodegenerative diseases. Mouse cortical cell cultures underwent widespread neuronal apoptosis 24 h following exposure to 10-30 nM calyculin A, a selective inhibitor of Ser/Thr phosphatase I and IIA. Activity of p38 was increased 2-4 h following exposure to 30 nM calyculin A. Addition of 3-10 microM PD169316, a selective p38 inhibitor, partially attenuated calyculin A neurotoxicity. Activity of caspase-3-like proteases was increased in cortical cell cultures exposed to 30 nM calyculin A for 8-16 h as shown by cleavage of DEVD-p-nitroanilide and phosphorylated tau. Proteolysis of tau was completely blocked by addition of 100 microM N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), a broad-spectrum inhibitor of caspases, but incompletely by 10 microM PD169316. Calyculin A neurotoxicity was partially sensitive to 100 microM z-VAD-fmk. Cotreatment with 10 microM PD169316 and 100 microM z-VAD-fmk showed additive neuroprotection against calyculin A. Neither PD169316 nor z-VAD-fmk showed a beneficial effect against excitotoxic neuronal necrosis induced by exposure to 20 microM NMDA. Thus, caspase-3-like proteases and p38 likely contribute to calyculin A-induced neuronal apoptosis but not NMDA-induced neuronal necrosis.

Calcineurin inhibition prevents calpain‐mediated proteolysis of tau in differentiated PC12 cells

The effects of calcium influx on tau levels and phosphorylation were examined in differentiated PC12 cells. Maitotoxin-induced calcium influx resulted in time- and concentration-dependent tau dephosphorylation and degradation. Incubation of PC12 cells with a membrane-permeable calpain inhibitor blocked maitotoxin-induced tau degradation, suggesting the involvement of calpain in calcium-stimulated tau turnover. Okadaic acid or the calcineurin inhibitor FK520 partially inhibited maitotoxin-induced tau dephosphorylation at the Tau-1 epitope, indicating both phosphatase 2A/1 and calcineurin were involved. In addition, FK520, but not okadaic acid, blocked the maitotoxin-induced tau degradation, demonstrating that dephosphorylation of specific tau epitopes by was essential for calpain-mediated tau degradation. Moreover, maitotoxin effects were likely independent of tau association with microtubules because maitotoxin induced tau degradation and dephosphorylation in the presence of either nocodazole or taxol. These data provide evidence that calpain is involved in tau turnoverin situand calcineurin plays an important role in modulating tau susceptibility to calpain. J. Neurosci. Res. 53:153–164, 1998. © 1998 Wiley-Liss, Inc.

Calcineurin inhibition prevents calpain-mediated proteolysis of tau in differentiated PC12 cells.

The effects of calcium influx on tau levels and phosphorylation were examined in differentiated PC12 cells. Maitotoxin-induced calcium influx resulted in time- and concentration-dependent tau dephosphorylation and degradation. Incubation of PC12 cells with a membrane-permeable calpain inhibitor blocked maitotoxin-induced tau degradation, suggesting the involvement of calpain in calcium-stimulated tau turnover. Okadaic acid or the calcineurin inhibitor FK520 partially inhibited maitotoxin-induced tau dephosphorylation at the Tau-1 epitope, indicating both phosphatase 2A/1 and calcineurin were involved. In addition, FK520, but not okadaic acid, blocked the maitotoxin-induced tau degradation, demonstrating that dephosphorylation of specific tau epitopes by was essential for calpain-mediated tau degradation. Moreover, maitotoxin effects were likely independent of tau association with microtubules because maitotoxin induced tau degradation and dephosphorylation in the presence of either nocodazole or taxol. These data provide evidence that calpain is involved in tau turnover in situ and calcineurin plays an important role in modulating tau susceptibility to calpain.