Phosphorylated Tau Pathology Research Articles

Frontotemporal dementia–amyotrophic lateral sclerosis syndrome locus on chromosome 16p12.1–q12.2: genetic, clinical and neuropathological analysis

Numerous families exhibiting both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) have been described, and although many of these have been shown to harbour a repeat expansion in C9ORF72, several C9ORF72-negative FTD-ALS families remain. We performed neuropathological and genetic analysis of a large European Australian kindred (Aus-12) with autosomal dominant inheritance of dementia and/or ALS. Affected Aus-12 members developed either ALS or dementia; some of those with dementia also had ALS and/or extrapyramidal features. Neuropathology was most consistent with frontotemporal lobar degeneration with type B TDP pathology, but with additional phosphorylated tau pathology consistent with corticobasal degeneration. Aus-12 DNA samples were negative for mutations in all known dementia and ALS genes, including C9ORF72 and FUS. Genome-wide linkage analysis provided highly suggestive evidence (maximum multipoint LOD score of 2.9) of a locus on chromosome 16p12.1–16q12.2. Affected individuals shared a chromosome 16 haplotype flanked by D16S3103 and D16S489, spanning 37.9 Mb, with a smaller suggestive disease haplotype spanning 24.4 Mb defined by recombination in an elderly unaffected individual. Importantly, this smaller region does not overlap with FUS. Whole-exome sequencing identified four variants present in the maximal critical region that segregate with disease. Linkage analysis incorporating these variants generated a maximum multipoint LOD score of 3.0. These results support the identification of a locus on chromosome 16p12.1–16q12.2 responsible for an unusual cluster of neurodegenerative phenotypes. This region overlaps with a separate locus on 16q12.1–q12.2 reported in an independent ALS family, indicating that this region may harbour a second major locus for FTD-ALS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1078-9) contains supplementary material, which is available to authorized users.

Ginsenoside Rd attenuates beta-amyloid-induced tau phosphorylation by altering the functional balance of glycogen synthase kinase 3beta and protein phosphatase 2A

Neurofibrillary tangles are aggregates of hyperphosphorylated tau that are one of the pathological hallmarks of Alzheimer's disease (AD). Tau phosphorylation is regulated by a balance of kinase and phosphatase activities. Our previous study has demonstrated that ginsenoside Rd, one of the principal active ingredients of Pana notoginseng, inhibits okadaic acid-induced tau phosphorylation in vivo and in vitro, but the underlying mechanism(s) is unknown. In this study, we showed that ginsenoside Rd pretreatment inhibited tau phosphorylation at multiple sites in beta-amyloid (Aβ)-treated cultured cortical neurons, and in vivo in both a rat and transgenic mouse model. Ginsenoside Rd not only reduced Aβ-induced increased expression of glycogen synthase kinase 3beta (GSK-3β), the most important kinase involved in tau phosphorylation, but also inhibited its activity by enhancing and attenuating its phosphorylation at Ser9 and Tyr216, respectively. Moreover, ginsenoside Rd enhanced the activity of protein phosphatase 2A (PP-2A), a key phosphatase involved in tau dephosphorylation. Finally, an in vitro biochemical assay revealed that ginsenoside Rd directly affected GSK-3β and PP-2A activities. Thus, our findings provide the first evidence that ginsenoside Rd attenuates Aβ-induced pathological tau phosphorylation by altering the functional balance of GSK-3β and PP-2A.

Tau‐Tubulin Kinase 1 Expression, Phosphorylation and Co‐Localization with Phospho‐Ser422 Tau in the Alzheimer's Disease Brain

Recent reports have implicated tau-tubulin kinase 1 (TTBK1) in the pathological phosphorylation of tau that occurs in Alzheimer's disease (AD). The present study was undertaken to provide an extensive characterization of TTBK1 mRNA and protein expression in human brain from AD cases and non-demented controls so as to better understand the disease relevance of this novel kinase. In situ hybridization and immunohistochemistry revealed abundant expression of TTBK1 in the somatodendritic compartment of cortical and hippocampal neurons of both AD cases and controls. TTBK1 immunoreactivity appeared to vary with the level of phospho-tau staining, and was strong in the somatodendritic compartment of apparently healthy hippocampal neurons as well as in pre-tangle neurons where it co-localized with diffuse phospho-Ser422 tau staining. Ser422 was confirmed as a TTBK1 substrate in vitro, and an antibody towards the site, in addition to labeling AT8-positive neurofibrillary tangles (NFTs), neuritic plaques and neuropil threads, also labeled a small population of neurons that were unlabeled with AT8. These data suggest a role for TTBK1 in pre-tangle formation prior to the formation of fibrillar tau and strengthen the idea that tau is phosphorylated at Ser422 at an early/intermediate stage in NFT formation.

P3‐025: Chronic inhibition of the MEK/ERK pathway fails to delay disease progression in a transgenic animal model of tauopathy

Background: Neurofibrillary tangles (NFT), one of the hallmarks of Alzheimer’s disease (AD), are composed of paired helical filaments (PHF) of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD.Methods:We have used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with early AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes pS202, pT231, pS235 and pS396/404) were measured in cell lysates using AlphaScreen assays. Results: GSK3a, GSK3b and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all 4 epitopes. Pathway analysis of all the ‘hits’ suggests the Ras family of GTPases (MAPK family) to be a key regulator of tau phosphorylation. Pathway analysis on subgroups of kinases reveals several mechanisms involved in regulating tau expression levels by inhibition or activation of translation.Conclusions: The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.

Sitagliptin increases tau phosphorylation in the hippocampus of rats with type 2 diabetes and in primary neuron cultures

Increasing evidence supports an association between Alzheimer's disease (AD) and diabetes. In this context, anti-diabetic agents such as rosiglitazone and glucagon-like peptide (GLP)-1 have been reported to reduce pathologies associated with AD, including tau hyperphosphorylation, suggesting that such agents might be used to treat AD. One such anti-diabetic agent is sitagliptin, which acts through inhibition of dipeptidyl peptidase (DPP)-IV to increase GLP-1 levels. Given this action, sitagliptin would be predicted to reduce AD pathology. Accordingly, we investigated whether sitagliptin is effective in attenuating AD pathologies, focusing on tau phosphorylation in the OLETF type 2 diabetic rat model. Unexpectedly, we found that sitagliptin was not effective against pathological tau phosphorylation in the hippocampus of OLETF type 2 diabetes rats, and instead aggravated it. This paradoxically increased tau phosphorylation was attributed to activation of the tau kinase, GSK3β (glycogen synthase kinase 3β). Sitagliptin also increased ser-616 phosphorylation of the insulin receptor substrate (IRS)-1, suggesting increased insulin resistance in the brain. These phenomena were recapitulated in primary rat cortical neurons treated with sitagliptin, further confirming sitagliptin's effects on AD-related pathologies in neurons. These results highlight the need for caution in considering the use of sitagliptin in AD therapy.

GSK-3β and memory formation

In Alzheimer’s disease (AD), tau hyperphosphorylation and neurofibrillary tangle (NFT) formation are strongly associated with dementia, a characteristic and early feature of this disease. Glycogen synthase kinase 3β (GSK-3β) is a pivotal kinase in both the normal and pathological phosphorylation of tau. In the diseased state, hyperphosphorylated tau is deposited in NFTs, the formation of which, drive the disease process. GSK-3β which is also involved in long-term depression induction, interacts with tau to inhibit synaptic long-term potentiation. Strong lines of evidence suggest that the activation of GSK-3β is responsible for the memory deficits seen in both advanced age and AD. In this review, we will focus on the role of GSK-3β in brain function, particularly in memory maintenance. We will examine human and mouse studies which suggest a role for GSK-3β in memory maintenance and the eventual development of memory deficits.

Granulovacuolar Degenerations Appear in Relation to Hippocampal Phosphorylated Tau Accumulation in Various Neurodegenerative Disorders

BackgroundGranulovacuolar degeneration (GVD) is one of the pathological hallmarks of Alzheimer's disease (AD), and it is defined as electron-dense granules within double membrane-bound cytoplasmic vacuoles. Several lines of evidence have suggested that GVDs appear within hippocampal pyramidal neurons in AD when phosphorylated tau begins to aggregate into early-stage neurofibrillary tangles. The aim of this study is to investigate the association of GVDs with phosphorylated tau pathology to determine whether GVDs and phosphorylated tau coexist among different non-AD neurodegenerative disorders.MethodsAn autopsied series of 28 patients with a variety of neurodegenerative disorders and 9 control patients were evaluated. Standard histological stains along with immunohistochemistry using protein markers for GVD and confocal microscopy were utilized.ResultsThe number of neurons with GVDs significantly increased with the level of phosphorylated tau accumulation in the hippocampal regions in non-AD neurodegenerative disorders. At the cellular level, diffuse staining for phosphorylated tau was detected in neurons with GVDs.ConclusionsOur data suggest that GVDs appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, while the presence of phosphorylated tau in GVD-harbouring neurons in non-AD neurodegenerative disorders was indistinguishable from age-related accumulation of phosphorylated tau. Although GVDs in non-AD neurodegenerative disorders have not been studied thoroughly, our results suggest that they are not incidental findings, but rather they appear in relation to phosphorylated tau accumulation, further highlighting the role of GVD in the process of phosphorylated tau accumulation.

Functional Implications of Glycogen Synthase Kinase‐3‐Mediated Tau Phosphorylation

Tau is primarily a neuronal microtubule-associated protein that has functions related to the stabilisation of microtubules. Phosphorylation of tau is an important dynamic and regulatory element involved in the binding of tau to tubulin. Thus, highly phosphorylated tau is more likely to be present in the cytosolic compartment of neurons, whereas reduced phosphate burden allows tau to bind to and stabilise the microtubule cytoskeleton. Highly phosphorylated forms of tau are deposited in the brain in a range of neurodegenerative disorders including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal lobar degeneration associated with Pick bodies. A key candidate kinase for both physiological and pathological tau phosphorylation is glycogen synthase kinase-3 (GSK-3). Multiple phosphorylation sites have been identified on tau exposed to GSK-3 in vitro and in cells. In this review, we highlight recent data suggesting a role for GSK-3 activity on physiological tau function and on tau dysfunction in neurodegenerative disease.

Tau and neurodegenerative disorders.

The mechanisms that render tau a toxic agent are still unclear, although increasing evidence supports the assertion that alterations of tau can directly cause neuronal degeneration. In addition, it is unclear whether neurodegeneration in various tauopathies occurs via a common mechanism or that specific differences exist. The aim of this review is to provide an overview of tauopathies from bench to bedside. The review begins with clinicopathological findings of familial and sporadic tauopathies. It includes a discussion of the similarities and differences between these two conditions. The second part concentrates on biochemical alterations of tau such as phosphorylation, truncation and acetylation. Although pathological phosphorylation of tau has been studied for many years, recently researchers have focused on the physiological role of tau during development. Finally, the review contains a summary of the significance of tauopathy model mice for research on neurofibrillary tangles, axonopathies, and synaptic alteration.

Animal models reveal role for tau phosphorylation in human disease

Many proteins that are implicated in human disease are posttranslationally modified. This includes the microtubule-associated protein tau that is deposited in a hyperphosphorylated form in brains of Alzheimer's disease patients. The focus of this review article is on the physiological and pathological phosphorylation of tau; the relevance of aberrant phosphorylation for disease; the role of kinases and phosphatases in this process; its modeling in transgenic mice, flies, and worms; and implications of phosphorylation for therapeutic intervention.

New Approaches to the Discovery of cdk5 Inhibitors

Cyclin-dependent kinase 5 (cdk5) is a member of the serine-threonine kinase family of cyclin-dependent kinases. This family is known for its role in the cell cycle, but cdk5 differs due to its interaction with activators p35 or p39, both abundant in post-mitotic neurons. Cdk5 is not known to have a role in cell cycle regulation at all, but is known to be an important modulator of neuronal activity. Cdk5 has been an attractive target for CNS diseases for a number of years. Among its attractions is the possibility that inhibitors will prevent the pathological phosphorylation of tau and neurofibrillary pathology in both Alzheimer's disease and tauopathies. More recently, there has been evidence that cdk5 is involved in the processing of pain and therefore inhibitors would also have potential therapeutic value for acute pain. Several classes of potent chemical inhibitors for cdk5 have been identified but most are competitive with the ATP binding site, resulting in a lack of specificity among the other cyclin-dependent kinases as well as other ATP-dependent kinases. We are working to discover specific inhibitors that might disrupt the interaction of tau and cdk5 at sites other than the ATP binding site. We are screening our compound library of 110,000 compounds using the full length tau as a substrate and will separate ATP competitive from non-competitive binders. In addition, we are taking a computational approach with virtual screening to identify non-ATP-competitive binders. These two approaches may lead to the discovery of site-specific inhibitors for tau and cdk5 interactions rather than competitive inhibitors for ATP binding. The hope is that non-ATP competitive compounds will more likely be selective and will be better therapeutics.

Pathological Tau Tangles Localize to Focal Cortical Dysplasia in Older Patients

Reactivation of neurodevelopmental processes may contribute to neurodegeneration. For example, the proteins cyclin dependent kinase 5 (cdk5) and glycogen synthase kinase 3 beta (GSK3beta), which are essential to normal cortical development, can hyperphosphorylate tau and might contribute to the pathogenesis of Alzheimer's disease. Focal cortical dysplasia (FCD) is an important neurodevelopmental cause of refractory human epilepsy within which dysplastic neurons exhibit increased immunoreactivity for cdk5 and GSK3beta as well as neurofilamentous accumulations. We therefore hypothesized that the developmentally abnormal cortex of FCD might be more susceptible to tau-mediated neurodegeneration than adjacent histologically normal cortex. We examined a series of 15 cases of FCD, spanning a wide age range, for beta-amyloid, pathologically phosphorylated tau and neurofibrillary tangles using silver staining, immunohistochemistry for tau, AT8, RD3, RD4 and two-dimensional cell counting. Beta-amyloid plaques, aberrantly phosphorylated tau and neurofibrillary tangles are only found in older patients. The hyperphosphorylated tau tangles are confined to dysplastic neurons. Immunoreactivity for 3- and 4-repeat tau was again only detected within regions of FCD in older patients. With increasing age, the dysplastic cortex became hypocellular and a higher proportion of dysplastic neurons exhibited pathological tau phosphorylation. In older patients, FCD appears more susceptible to formation of pathologically phosphorylated tau neurofibrillary tangles than adjacent histologically normal cortex. Our results suggest a novel convergence of pathological neurodevelopment with pathological age-related neurodegeneration.

2.017 Pattern of α-synuclein and phosphorylated tau pathology in the olfactory bulb, brainstem and limbic regions in aged individuals

2.017 Pattern of α-synuclein and phosphorylated tau pathology in the olfactory bulb, brainstem and limbic regions in aged individuals

2.302 Pattern of alpha-synuclein and phosphorylated tau pathology in the olfactory bulb, brainstem and limbic regions in aged individuals

2.302 Pattern of alpha-synuclein and phosphorylated tau pathology in the olfactory bulb, brainstem and limbic regions in aged individuals

O2-05-07: Minocycline reduces tau phosphorylation and protects against Aβ-induced neurotoxicity

Minocycline is a semi–synthetic, second–generation tetracycline derivative. Recent research has indicated that in addition to its antimicrobial properties, minocycline is also a potent anti–inflammatory, anti–apoptotic and neuroprotective agent when tested in models of neurodegenerative disease. To determine if minocycline may have therapeutic value for the treatment of Alzheimer's disease by preventing tau hyperphosphorylation and amyloid–induced neurotoxicity. We have investigated the effect of minocycline, both in primary neuronal cultures, and in NFT–forming tau transgenic mice (htau line). Treatment of cultures with fibrillar and oligomeric Aβ42 over a period of 48 hours resulted in progressive neuronal death, which was prevented by pre–treatment of cultures with minocycline. Application of minocycline to control cultures also resulted in increased cell survival, demonstrating the neuroprotective properties of this agent. There is increasing evidence to suggest that kinase activation resulting in tau phosphorylation is an important factor in the demise of neurons that results from exposure to Aβ. To investigate this mechanism we have examined the effects of minocycline treatment on pathological tau phosphorylation in response to Aβ in culture, and also during tangle development in htau mice. We find that minocycline treatment results in significantly reduced tau phosphorylation at several epitopes of relevance to AD, including the CP13 and PHF1 epitopes at S202 and S396/404 respectively. The reduced tau phosphorylation we have observed correlates well with decreased cell death following treatment. Furthermore, after minocycline treatment we find reduced activity of specific tau protein kinases, which may account for the decreased tau phosphorylation we have observed. In addition to modulating kinase activities, we find reduced levels of GFAP after minocycline treatment indicating that minocycline can induce suppression of glial responses. These results suggest a dual neuroprotective effect of minocycline with inhibition of kinase activities preventing pathological tau phosphorylation and suppression of glial responses preventing neuroinflammation. At the doses used minocycline is known to be both safe and well tolerated. These results suggest that minocycline may be an effective therapeutic agent for the treatment of Alzheimer's disease and related neurodegenerative disorders. This work was supported by a fellowship grant from the Alzheimer's Society.

JNK2 Translocates to the Mitochondria and Mediates Cytochrome c Release in PC12 Cells in Response to 6-Hydroxydopamine

6-Hydroxydopamine (6-OHDA) causes death of dopaminergic neurons by mitochondrial dysfunction with JNKs as central mediators. Here we provide novel insights into specific actions of JNK isoforms in 6-OHDA-induced death of PC12 cells. Twenty five mum 6-OHDA enhanced total JNK activity in the cytoplasm, nucleus, and at the mitochondria. Inhibition of JNKs by 2 mum SP600125 or transfection with dominant-negative JNK2 (dnJNK2) rescued more than 60% of the otherwise dying PC12 cells after 24 h, whereas transfection with dnJNK1 had no protective effects. In contrast to constitutively present JNK1, JNK2 amounts increased in the nucleus and at the mitochondria after 6-OHDA stimulation. JNK inhibition by SP600125 or transfection of dnJNK2 reduced the pool of active JNKs in the nucleus, the release of cytochrome c, as well as the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase-1. Transfection with dnJNK1, however, had no effects on the translocation of JNKs to the mitochondria or the release of cytochrome c. Our data provide novel functional insights into the pathological role of individual JNK isoforms, the signalosome at the mitochondria, and the mode of JNK-induced release of cytochrome c.

Increased MAP kinase activity in Alzheimer's and Down syndrome but not in schizophrenia human brain

Abnormal phosphorylation of tau is a feature of Alzheimer's disease (AD), which develops prematurely in Down syndrome (DS) patients. Cognitive impairment is also recognized as a clinical characteristic of schizophrenia, which does not appear to be associated with tau-aggregate formation. Several kinases can phosphorylate tau in cell-free assays. Here we show increased activity of mitogen-activated protein kinases (MAPKs) (including ERK1/2, SAPKs and p38) in post mortem AD and DS brains, which could not be accounted for by expression changes. In contrast, glycogen synthase kinase-3 activity (GSK-3 alpha beta) was reduced significantly. Examination of tau in AD and DS using antibodies selective for MAPK phosphorylation sites showed increased immunoreactivity. In addition, phosphorylation of S(199), reportedly a selective substrate for cyclin-dependent kinase-5 (cdk5) or GSK-3 alpha beta was only observed in AD samples, which showed a concomitant increase in the expression of p25, the enhancing cofactor for cdk5 activity. However, in schizophrenia brain, MAPK-phosphorylated tau was unchanged compared to matched controls, despite similar expression levels to those in AD. The activities of the MAPKs and GSK-3 alpha beta were also unchanged. These data demonstrate that in AD and DS, enhanced MAPK activity, which has an established role in regulating neuronal plasticity and survival, can account for irregular tau phosphorylation, and that the molecular processes involved in these neurodegenerative disorders are distinct from those in schizophrenia. These data also question the significance of GSK-3 alpha beta, as much previous work carried out in vitro has placed this kinase as a favoured candidate for involvement in the pathological phosphorylation of tau.

MARK4 Is a Novel Microtubule-associated Proteins/Microtubule Affinity-regulating Kinase That Binds to the Cellular Microtubule Network and to Centrosomes

The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease.

Parahippocampal tau pathology in healthy aging, mild cognitive impairment, and early Alzheimer's disease.

Abnormally phosphorylated tau accumulates as neurofibrillary tangles and neuropil threads in older persons with and without Alzheimer's disease. The relationship between neurofibrillary tangles and neuropil threads and how they relate to cognitive function is unknown. This study investigated the relationship between phosphorylated tau lesions and cognitive function in 31 persons participating in the Religious Orders Study, a prospective, longitudinal clinicopathological study of aging and Alzheimer's disease. All subjects underwent detailed neuropsychological performance testing within a year of death and evidenced a spectrum of cognitive performance ranging from normal abilities to mild dementia. Measures of neurofibrillary tangle density and phosphorylated tau immunoreactive structures (predominantly neuropil threads) in the entorhinal and perirhinal cortices by quantitative image analysis were significantly correlated (r = 0.5). In multiple linear regression analyses controlling for age, sex, and education, parahippocampal neurofibrillary tangles and neuropil threads were significantly lower in persons without cognitive impairment compared to those with mild cognitive impairment and/or Alzheimer's disease. Further, neurofibrillary tangles were significantly correlated to measures of episodic memory but not other cognitive abilities; neuropil tangles were not significantly related to memory or other cognitive functions. These data indicate that phosphorylated tau pathology in the ventromedial temporal lobe develop prior to the onset of clinical dementia and their presence is associated with cognitive impairment, particularly impairment of episodic memory.

The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and Thr-Pro motifs

Tau protein can be transformed into an Alzheimer-like state by phosphorylation with a kinase activity from brain [Biernat et al. (1992) EMBO J. 11, 1593–1597]. Here we show that the phosphorylation at Ser-Pro motifs strongly decreases tau's affinity for Microtubules. The major reduction occurs during the first of the three main stages of phosphorylation. The data explain the lower Stability of microtubules resulting from the pathological tau phosphorylation.