Site-specific Tau Phosphorylation Research Articles

Site-specific phosphorylation of tau impacts mitochondrial function and response to stressors.

Phosphorylation of tau at sites associated with Alzheimer's disease (AD) likely plays a role in the disease progression. Mitochondrial impairment, correlating with increased presence of phosphorylated tau, has been identified as a contributing factor to neurodegenerative processes in AD. However, how tau phosphorylated at specific sites impacts mitochondrial function has not been fully defined. We examined how AD-relevant phosphomimetics of tau impact selected aspects of mitochondrial biology. To mimic phosphorylation at AD-associated sites, the serine/threonine (Ser/Thr) sites in wild-type green fluorescent protein (GFP)-tagged tau (T4) were converted to glutamic acid (E) to make pseudo-phosphorylated GFP-tagged Ser-396/404 (2EC) and GFP-tagged Thr-231/Ser-235 (2EM) constructs. These constructs were expressed in immortalized mouse hippocampal neuronal cell lines, and their impact on specific mitochondrial functions and responses to stressors were measured. Phosphomimetic tau altered mitochondrial distribution. Specifically, mitochondria accumulated in the soma of cells expressing either 2EC or 2EM and neurite-like extensions in 2EC cells were shorter. Additionally, adenosine triphosphate levels were reduced in both 2EC- and 2EM-expressing cells, and reactive oxygen species (ROS) production increased in 2EC cells during oxidation of succinate when compared to T4-expressing cells. Thapsigargin reduced mitochondrial membrane potential and increased ROS production in both 2EC and 2EM cells relative to T4 cells, with no significant difference in the effects of rotenone. These results show that tau phosphorylation at specific AD-relevant epitopes negatively affects mitochondria, with the extent of dysfunction and stress response varying according to the sites of phosphorylation. Altogether, these findings show that phosphorylated tau increases mitochondrial susceptibility to stressors and extend our understanding of potential mechanisms whereby phosphorylated tau promotes mitochondria dysfunction in tauopathies, including AD.

Tau phosphorylation and PAD exposure in regulation of axonal growth.

Introduction: Tau is a microtubule associated phosphoprotein found principally in neurons. Prevailing dogma continues to define microtubule stabilization as the major function of tau in vivo, despite several lines of evidence suggesting this is not the case. Most importantly, tau null mice have deficits in axonal outgrowth and neuronal migration while still possessing an extensive microtubule network. Instead, mounting evidence suggests that tau may have a major function in the regulation of fast axonal transport (FAT) through activation of neuronal signaling pathways. Previous studies identified a phosphatase activating domain (PAD) at the tau N-terminal that is normally sequestered, but is constitutively exposed in tauopathies. When exposed, the PAD activates a signaling cascade involving PP1 and GSK3β which affects cellular functions including release of cargo from kinesin. Furthermore, we discovered that PAD exposure can be regulated by a single phosphorylation at T205. Exposure of the PAD is an early event in multiple tauopathies and a major contributing factor to neurodegeneration associated with tau hyperphosphorylation. However, effects of tau PAD exposure on anterograde FAT raised the interesting possibility that this pathway may be a mechanism for physiological regulation of cargo delivery through site-specific phosphorylation of tau and transient activation of PP1 and GSK3β. Significantly, there is already evidence of local control of PP1 and GSK3β at sites which require cargo delivery. Methods: To investigate this hypothesis, first we evaluated cellular localization of tau PAD exposure, pT205 tau phosphorylation, and active GSK3β in primary hippocampal neurons during development. Second, we analyzed the axonal outgrowth of tau knockout neurons following transfection with full length hTau40-WT, hTau40-ΔPAD, or hTau40-T205A. Results and Discussion: The results presented here suggest that transient activation of a PP1-GSK3β signaling pathway through locally regulated PAD exposure is a mechanism for cargo delivery, and thereby important for neurite outgrowth of developing neurons.

Treatment of epilepsy using a targeted p38γ kinase gene therapy.

Hyperphosphorylated microtubule-associated protein tau has been implicated in dementia, epilepsy, and other neurological disorders. In contrast, site-specific phosphorylation of tau at threonine 205 (T205) by the kinase p38γ was shown to disengage tau from toxic pathways, serving a neuroprotective function in Alzheimer's disease. Using a viral-mediated gene delivery approach in different mouse models of epilepsy, we show that p38γ activity-enhancing treatment reduces seizure susceptibility, restores neuronal firing patterns, reduces behavioral deficits, and ameliorates epilepsy-induced deaths. Furthermore, we show that p38γ-mediated phosphorylation of tau at T205 is essential for this protection in epilepsy, as a lack of this critical interaction reinstates pathological features and accelerates epilepsy in vivo. Hence, our work provides a scope to harness p38γ as a future therapy applicable to acute neurological conditions.

SIRT1 deficiency increases O-GlcNAcylation of tau, mediating synaptic tauopathy.

Hyperphosphorylation of the microtubule associated protein tau is associated with several neurodegenerative diseases including Alzheimer's Disease (AD), collectively referred to as tauopathies. However, the mechanisms by which tau is linked to synaptic dysfunction and memory impairment remain unclear. To address this question, we constructed a mouse model with brain-specific deficiency of SIRT1 (SIRT1 flox/Cre + ). Here, we show that increase of site-specific phosphorylation of tau is coupled with the strengthened O-GlcNAcylation of tau triggered by reduced O-GlcNAcase (OGA) and increased O-GlcNAc transferase (OGT) protein level in the brain of SIRT1 flox/Cre+ mice. SIRT1 deletion in mice brain changes the synaptosomal distribution of site-specific phospho-tau. Learning and memory deficiency induced by dendritic spine deficits and synaptic dysfunction are revealed via SIRT1 flox/Cre+ mice. Our results provide evidence for SIRT1 as a potential therapeutic target in clinical tauopathies.

Association of BDNF Val66Met With Tau Hyperphosphorylation and Cognition in Dominantly Inherited Alzheimer Disease

Allelic variation in the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism moderates increases in cerebrospinal fluid (CSF) levels of tau and phosphorylated tau 181 (p-tau181), measured using immunoassay, and cognitive decline in presymptomatic dominantly inherited Alzheimer disease (DIAD). Advances in mass spectrometry show that CSF tau phosphorylation occupancy at threonine 181 and 217 (p-tau181/tau181, p-tau217/tau217) increases with initial β-amyloid (Aβ) aggregation, while phosphorylation occupancy at threonine 205 (p-tau205/tau205) and level of total tau increase when brain atrophy and clinical symptoms become evident. To determine whether site-specific tau phosphorylation occupancy (ratio of phosphorylated to unphosphorylated tau) is associated with BDNF Val66Met in presymptomatic and symptomatic DIAD. This cross-sectional cohort study included participants from the Dominantly Inherited Alzheimer Network (DIAN) and Aβ-positive cognitively normal older adults in the Alzheimer's Disease Neuroimaging Initiative (ADNI). Data were collected from 2009 through 2018 at multicenter clinical sites in the United States, United Kingdom, and Australia, with no follow-up. DIAN participants provided a CSF sample and completed clinical and cognitive assessments. Data analysis was conducted between March 2020 and March 2021. Mass spectrometry analysis was used to determine site-specific tau phosphorylation level; tau levels were also measured using immunoassay. Episodic memory and global cognitive composites were computed. Of 374 study participants, 144 were mutation noncarriers, 156 were presymptomatic mutation carriers, and 74 were symptomatic carriers. Of the 527 participants in the network, 153 were excluded because their CSF sample, BDNF status, or both were unavailable. Also included were 125 Aβ-positive cognitively normal older adults in the ADNI. The mean (SD) age of DIAD participants was 38.7 (10.9) years; 43% were women. The mean (SD) age of participants with preclinical sporadic AD was 74.8 (5.6) years; 52% were women. In presymptomatic mutation carriers, compared with Val66 homozygotes, Met66 carriers showed significantly poorer episodic memory (d = 0.62; 95% CI, 0.28-0.95), lower hippocampal volume (d = 0.40; 95% CI, 0.09-0.71), and higher p-tau217/tau217 (d = 0.64; 95% CI, 0.30-0.97), p-tau181/tau181 (d = 0.65; 95% CI, 0.32-0.99), and mass spectrometry total tau (d = 0.43; 95% CI, 0.10-0.76). In symptomatic mutation carriers, Met66 carriers showed significantly poorer global cognition (d = 1.17; 95% CI, 0.65-1.66) and higher p-tau217/tau217 (d = 0.53; 95% CI, 0.05-1.01), mass spectrometry total tau (d = 0.78; 95% CI, 0.28-1.25), and p-tau205/tau205 (d = 0.97; 95% CI, 0.46-1.45), when compared with Val66 homozygotes. In preclinical sporadic AD, Met66 carriers showed poorer episodic memory (d = 0.39; 95% CI, 0.00-0.77) and higher total tau (d = 0.45; 95% CI, 0.07-0.84) and p-tau181 (d = 0.46; 95% CI, 0.07-0.85). In DIAD, clinical disease stage and BDNF Met66 were associated with cognitive impairment and levels of site-specific tau phosphorylation. This suggests that pharmacological strategies designed to increase neurotrophic support in the presymptomatic stages of AD may be beneficial.

Reduction of advanced tau-mediated memory deficits by the MAP kinase p38γ.

Hyperphosphorylation of the neuronal tau protein contributes to Alzheimer's disease (AD) by promoting tau pathology and neuronal and cognitive deficits. In contrast, we have previously shown that site-specific tau phosphorylation can inhibit toxic signals induced by amyloid-β (Aβ) in mouse models. The post-synaptic mitogen-activated protein (MAP) kinase p38γ mediates this site-specific phosphorylation on tau at Threonine-205 (T205). Using a gene therapeutic approach, we draw on this neuroprotective mechanism to improve memory in two Aβ-dependent mouse models of AD at stages when advanced memory deficits are present. Increasing activity of post-synaptic kinase p38γ that targets T205 in tau reduced memory deficits in symptomatic Aβ-induced AD models. Reconstitution experiments with wildtype human tau or phosphorylation-deficient tauT205A showed that T205 modification is critical for downstream effects of p38γ that prevent memory impairment in APP-transgenic mice. Furthermore, genome editing of the T205 codon in the murine Mapt gene showed that this single side chain in endogenous tau critically modulates memory deficits in APP-transgenic Alzheimer's mice. Ablating the protective effect of p38γ activity by genetic p38γ deletion in a tau transgenic mouse model that expresses non-pathogenic tau rendered tau toxic and resulted in impaired memory function in the absence of human Aβ. Thus, we propose that modulating neuronal p38γ activity serves as an intrinsic tau-dependent therapeutic approach to augment compromised cognition in advanced dementia.

Reduction of advanced tau-mediated memory deficits by the MAP kinase p38γ.

Hyperphosphorylation of the neuronal tau protein contributes to Alzheimer's disease (AD) by promoting tau pathology and neuronal and cognitive deficits. In contrast, we have previously shown that site-specific tau phosphorylation can inhibit toxic signals induced by amyloid-β (Aβ) in mouse models. The post-synaptic mitogen-activated protein (MAP) kinase p38γ mediates this site-specific phosphorylation on tau at Threonine-205 (T205). Using a gene therapeutic approach, we draw on this neuroprotective mechanism to improve memory in two Aβ-dependent mouse models of AD at stages when advanced memory deficits are present. Increasing activity of post-synaptic kinase p38γ that targets T205 in tau reduced memory deficits in symptomatic Aβ-induced AD models. Reconstitution experiments with wildtype human tau or phosphorylation-deficient tauT205A showed that T205 modification is critical for downstream effects of p38γ that prevent memory impairment in APP-transgenic mice. Furthermore, genome editing of the T205 codon in the murine Mapt gene showed that this single side chain in endogenous tau critically modulates memory deficits in APP-transgenic Alzheimer's mice. Ablating the protective effect of p38γ activity by genetic p38γ deletion in a tau transgenic mouse model that expresses non-pathogenic tau rendered tau toxic and resulted in impaired memory function in the absence of human Aβ. Thus, we propose that modulating neuronal p38γ activity serves as an intrinsic tau-dependent therapeutic approach to augment compromised cognition in advanced dementia.

Inhibition of 5α Reductase Impairs Cognitive Performance, Alters Dendritic Morphology and Increases Tau Phosphorylation in the Hippocampus of Male 3xTg-AD Mice

Recent work has suggested that 5α-reduced metabolites of testosterone may contribute to the neuroprotection conferred by their parent androgen, as well as to sex differences in the incidence and progression of Alzheimer’s disease (AD). This study investigated the effects of inhibiting 5α-reductase on object recognition memory (ORM), hippocampal dendritic morphology and proteins involved in AD pathology, in male 3xTg-AD mice. Male 6-month old wild-type or 3xTg-AD mice received daily injections of finasteride (50 mg/kg i.p.) or vehicle (18% β-cyclodextrin, 1% v/b.w.) for 20 days. Female wild-type and 3xTg-AD mice received only the vehicle. Finasteride treatment differentially impaired ORM in males after short-term (3xTg-AD only) or long-term (3xTg-AD and wild-type) retention delays. Dendritic spine density and dendritic branching of pyramidal neurons in the CA3 hippocampal subfield were significantly lower in 3xTg-AD females than in males. Finasteride reduced CA3 dendritic branching and spine density in 3xTg-AD males, to within the range observed in vehicle-treated females. In the CA1 hippocampal subfield, dendritic branching and spine density were reduced in both male and female 3xTg-AD mice, compared to wild type controls. Hippocampal amyloid β levels were substantially higher in 3xTg-AD females compared to both vehicle and finasteride-treated 3xTg-AD males. Site-specific Tau phosphorylation was higher in 3xTg-AD mice compared to sex-matched wild-type controls, increasing slightly after finasteride treatment. These results suggest that 5α-reduced neurosteroids may play a role in testosterone-mediated neuroprotection and may contribute to sex differences in the development and severity of AD.

Issue Cover (January 2020)

Journal of NeurochemistryVolume 152, Issue 1 Issue CoverFree Access Issue Cover (January 2020) First published: 22 December 2019 https://doi.org/10.1111/jnc.14727 Read the full preface: ‘High specificity of widely used phospho-tau antibodies validated using a quantitative wholecell based assay’ by D. Li, Y. K. Cho (J. Neurochem. 2020, vol. 152 (1), pp. 122–135) on doi: 10.1111/jnc.14830 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Front cover: Phospho-tau antibodies are the most common tool for detecting site-specific tau phosphorylation. However, there is no quantitative measure of specificity that guides users. We developed a robust flow cytometry assay that determines a specificity parameter termed ϕ(phi), which measures the fraction of non-specific signal in antibody binding. We applied the assay to measure the specificity of widely used phospho-tau antibodies, and show that phospho-tau antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have nearly perfect specificity. Image content: Representative confocal image showing highly specific recognition of tau phosphorylated at Thr231 (red) among total tau (green), imaged using EGFP-tau. Read the full preface ‘High specificity of widely used phospho-tau antibodies validated using a quantitative wholecell based assay’ by D. Li, Y. K. Cho (J. Neurochem. 2020, vol. 152 (1), pp. 122–135) on doi: 10.1111/jnc.14830 Volume152, Issue1January 2020 RelatedInformation

APOE4 Induces Site-Specific Tau Phosphorylation Through Calpain-CDK5 Signaling Pathway in EFAD-Tg Mice.

APOE4 is the greatest genetic risk factor for Alzheimer's disease (AD), particularly associated with increased levels of amyloid-β (Aβ) and amyloid deposition. However, it remains unclear whether APOE4 is associated with greater tau phosphorylation and neurofibrillary tangle formation, a hallmark of AD leading to structural disruption of the neuronal cytoskeleton. The current study used 3 and 7 month old EFAD mice, which express human APOE and over-express specifically human Aβ42 via 5 familial-AD (FAD) mutations, to investigate APOE genotype-specific effects on site-specific tau phosphorylation. The results reveal that AD-like site-specific tau phosphorylation was increased in E4FAD mice, accompanied by disrupted cortical neuronal morphology, compared to E3FAD mice. Further analysis demonstrated that the levels of CDK5, its regulatory subunits (p35 and p25) and calpain (including calpain1 and calpain2), but not GSK3β, were significantly increased in E4FAD mice compared to E3FAD mice. These results suggest that the APOE4 genotype contributes to increased site-specific tau phosphorylation via activation of the calpain-CDK5 signaling pathway.

Tau phosphosignatures discriminate Alzheimer Disease from other tauopathies

Neurofibrillary tangles (NFTs) comprised of highly phosphorylated tau are a core pathological feature of Alzheimer's disease (AD) and several other neurodegenerative diseases collectively termed tauopathies, which include progressive supranuclear palsy, corticobasal degeneration and certain frontotemporal dementias. There are six isoforms of human tau in brain and over 40 serine (S), threonine (T), and tyrosine (Y) tau phosphorylation sites have been identified. Phosphorylation of tau at specific S/T/Y sites is hypothesized to cause both functional deficits as well as gain‐of‐function toxicity that ultimately lead to NFT formation, synapse loss and cognitive dysfunction. Despite being clinically distinct diseases it remains unclear whether site‐specific tau phosphorylation differs between AD and other tauopathies. To address this question we employed immobilized metal affinity chromatography followed by liquid chromatography‐tandem mass spectrometry to identify and quantify tau phosphopeptides from individual AD, tauopathy and age‐matched control postmortem human brain tissues (n= 7 per group). In total 77 tau phosphopeptides representing 32 S/T/Y sites were analyzed by weighted correlation network analysis to delineate tau phosphosites co‐enriched in a disease specific manner. Our findings suggest that the mechanisms underlying site‐specific tau phosphorylation differ between AD and other tauopathies.

Enhanced taupathy and AD-like pathology in aged primate brains decades after infantile exposure to lead (Pb)

Late Onset Alzheimer Disease (LOAD) constitutes the majority of AD cases (∼90%). Amyloidosis and tau pathology, which are present in AD brains, appear to be sporadic in nature. We have previously shown that infantile lead (Pb) exposure is associated with a change in the expression and regulation of the amyloid precursor protein (APP) and its beta amyloid (Aβ) products in old age. Here we report that infantile Pb exposure elevated the mRNA and protein levels of tau as well as its transcriptional regulators namely specificity protein 1 and 3 (Sp1 and Sp3) in aged primates. These changes were also accompanied by an enhancement in site-specific tau phosphorylation as well as an increase in the mRNA and protein levels of cyclin dependent kinase 5 (cdk5). There was also a change in the protein ratio of p35/p25 with more Serine/Threonine phosphatase activity present in aged primates exposed to Pb as infants. These molecular alterations favored abundant tau phosphorylation and immunoreactivity in the frontal cortex of aged primates with prior Pb exposure. These findings provide more evidence that neurodegenerative diseases may be products of environmental influences that occur during the development.

Centella asiatica Protects Against the Toxic Effects of Intracellular beta-amyloid Accumulation

Centella asiatica has been used in Ayurvedic medicine as a nerve tonic and to enhance cognition. These effects have been confirmed experimentally in animals as well as human subjects. We have previously reported that the water extract of Centella asiatica (CAW) corrects β-amyloid (Aβ)-induced cognitive deficits in a transgenic mouse model of Alzheimer's disease and prevents Aβ toxicity in vitro. In this study we evaluated CAW's protective effects on Aβ-induced alterations in the MC65 neuroblastoma cell line, and identified potentially active compounds within the extract. In MC65 cells, CAW treatment reduced intracellular Aβ-induced cell death without changing Aβ accumulation. Concurrently, CAW attenuated Aβ-induced increases in total tau expression and site specific phosphorylation of tau. CAW also robustly increased expression of the antioxidant response gene NRF2 (nuclear factor erythroid-derived factor 2) as well as its target genes. While more studies are needed to confirm mechanisms, the effects on NRF2 suggest that the protective effect of CAW could be due, at least in part, to antioxidant actions. High performance liquid chromatography (HPLC) coupled to high-resolution mass spectrometry (HRMS) confirmed the absence in CAW of the triterpene compounds, asiatic acid, madecassic acid, asiaticoside and madecassoside, commonly found in the plant, and reported to have potent biological activities. Instead, using HRMS, several caffeoylquinic acids were determined to be present in CAW. Studies are currently underway to determine the efficacy of these compounds in the MC65 model system in order to evaluate their role in the neuroprotective effects of CAW.

Tau Hyperphosphorylation is Associated with Spatial Learning and Memory After Exposure to Benzo[a]pyrene in SD Rats

As a representative substance of the polycyclic aromatic hydrocarbons, benzo[a]pyrene (B[a]P) is a widely distributed environmental contaminant. Several studies have indicated that B[a]P exposure could impair learning and memory function in human population and animal models. Abnormal hyperphosphorylation and aggregation of microtubule-associated protein tau play a crucial role in neurodegenerative diseases manifesting deficits of learning and memory. In the present study, we investigated the involvement of tau hyperphosphorylation in memory impairment after espousing to B[a]P in SD rats. Male SD rats were randomly divided into five groups: the blank control group received no treatment and the others received intraperitoneal injection of B[a]P (0, 1.0, 2.5, 6.25mg/kgbw) for 1, 2, and 3months, respectively. Morris water maze was employed to observe the learning and memory impairment. To find the relationship between cognitive functions and tau protein, we measured the site-specific phosphorylation of tau at Ser199, Ser396, Thr181, and Thr231, which are related with spatial and memory deficits in the brain of rats. The spatial and learning of rats were impaired after exposing to B[a]P for 1month, so as 2 and 3months compared to blank control groups, respectively. Rats exposed to 2.5 and 6.25mg/kgbw B[a]P for either 2 or 3months had modified behavior compared to control as indicated by the increased latencies, increased the first time arriving at the target space and decreased number crossing the platform and time in target quadrant. B[a]P led to tau hyperphosphorylation at Ser199, Thr181, and Thr231 epitopes. And with time and dose expanded, tau protein, Ser199, Thr181, and Thr231 expression increased. However, the differences in expression of Ser396 at different doses or points were not statistically significant. Moreover, we observed a correlation between memory impairments and tau phosphorylation levels. The present results indicate that Tau hyperphosphorylation is associated with the observed deficits in spatial learning and memory following exposure to B[a]P in SD rats.

Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation.

Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD). The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β), possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence being site-specific, bi-directional regulation of tau phosphorylation in the mammalian brain.

Characterization of the Brain β-Amyloid Isoform Pattern at Different Ages of Tg2576 Mice

Background: Although genetic and biochemical studies have suggested a cardinal role for β-amyloid (Aβ) in Alzheimer’s disease, the underlying mechanism(s) of how Aβ induces neurodegeneration is still unclear. Our objective was to investigate the consequences of Aβ, especially on tau phosphorylation at specific epitopes important for Alzheimer’s disease. Methods: We used cortices from Tg2576 mice at 7 days to 15 months of age. Results: MALDI-TOF MS revealed an age-dependent shift in the Aβ isoform pattern. Young animals displayed high cortical levels of the shorter Aβ isoforms (Aβ1–16 and Aβ1–17) compared to 15-month-old Tg2576 mice which mainly expressed Aβ1–40 and Aβ1–42. The Aβ1–42 showed an age-dependent increase, whereas total Aβ1–40 levels remained constant. The highest levels of TBS-soluble Aβ oligomers were found at 90 days of age. Brain Aβ build-up did not affect the phosphorylation of tau at the epitopes investigated. Conclusions: This study provides new information about age-dependent Aβ isoforms and oligomers as well as their effect on site-specific tau phosphorylation in this transgenic mouse model. Our observations suggest that the different human Aβ isoforms do not directly cause increased tau phosphorylation and that the cognitive deficits seen in this mouse model are only related to the Aβ overexpression.

Tau hyperphosphorylation is associated with memory impairment after exposure to 1.5% isoflurane without temperature maintenance in rats

There is increasing interest in studying the role of tau hyperphosphorylation associated with memory impairment. We examined the involvement of tau hyperphosphorylation in memory impairment after hypothermia following isoflurane anaesthesia in rats. Adult rats were randomly divided into three groups: the control group received no treatment and others were subjected to 1.5% isoflurane anaesthesia with or without temperature control for 2 h. On the day before anaesthesia and on postanaesthetic days 1, 3 and 7, cognitive functions were assessed in a Y-maze test paradigm. To find the relationship between memory results and tau, we measured the site-specific phosphorylation of tau at Thr-205 and Ser-396 and the activity of protein phosphatase 2A within the hippocampus. The spatial learning and memory of animals with hypothermia were impaired at day 1 after anaesthesia, compared with nonanaesthetized rats. Anaesthesia and hypothermia led to tau hyperphosphorylation at the Thr-205 and Ser-396 epitopes in the hippocampus. There was no significant difference in the protein phosphatase 2A activity between the control and the postanaesthetic rat hippocampal samples, whereas nearly 45% protein phosphatase 2A inhibition was detected in the anaesthetized without temperature maintenance rat samples. Our results indicate that tau phosphorylation is not a direct result of anaesthesia per se, but it is due to anaesthesia-induced hypothermia and this leads to the inhibition of phosphatase activity as well as tau hyperphosphorylation. Tau hyperphosphorylation is associated with the observed deficits in spatial learning and memory following anaesthesia in hypothermic rats.

Developmental regulation of tau phosphorylation, tau kinases, and tau phosphatases

Tau is a neuronal microtubule-associated protein. Its hyperphosphorylation plays a critical role in Alzheimer disease (AD). Expression and phosphorylation of tau are regulated developmentally, but its dynamic regulation and the responsible kinases or phosphatases remain elusive. Here, we studied the developmental regulation of tau in rats during development from embryonic day 15 through the age of 24 months. We found that tau expression increased sharply during the embryonic stage and then became relatively stable, whereas tau phosphorylation was much higher in developing brain than in mature brain. However, the extent of tau phosphorylation at seven of the 14 sites studied was much less in developing brain than in AD brain. Tau phosphorylation during development matched the period of active neurite outgrowth in general. Tau phosphorylation at various sites had different topographic distributions. Several tau kinases appeared to regulate tau phosphorylation collectively at overlapping sites, and the decrease of overall tau phosphorylation in adult brain might be due to the higher levels of tau phosphatases in mature brain. These studies provide new insight into the developmental regulation of site-specific tau phosphorylation and identify the likely sites required for the abnormal hyperphosphorylation of tau in AD.

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.

P2-142: Decreased protein phosphatase 2A in Down syndrome brain: A cause of neurofibrillary degeneration

Individuals with Down syndrome (DS) develop neurofibrillary tangles, a characteristic brain lesion of Alzheimer's disease (AD), when they reach the fourth decade of life. Neurofibrillary tangles are composed of highly aggregated and phosphorylated form of microtubule associated protein tau. In AD, hyperphosphorylation of tau partially results from down-regulation of protein phosphatase (PP) 2A, a major brain tau phosphatase. The abnormal hyperphosphorylation of tau in DS had not yet been characterized, and its causes were not understood. The levels and site-specific phosphorylation of tau as well as the levels of several brain PPs in the temporal cortices of 6 DS (58.8 ± 3.8 years old) and 6 controls (68.0 ± 9.5 years old) were determined by using quantitative Western blot analysis. Linear correlation between the levels of PP2A catalytic subunit and the level of tau or tau phosphorylation at individual phosphorylation sites was analyzed by using Microsoft Excel 2003. We found that the level of the catalytic subunit of PP2A, but not of PP1, PP2B or PP5, was dramatically decreased. The decrease of PP2A level correlated negatively to tau level and tau phosphorylation at several abnormal hyperphosphorylation sites, including Ser199, Thr205, Thr212, Ser262, Ser396 and Ser422. These findings indicate that the down-regulation of PP2A might be involved in the abnormal hyperphosphorylation and accumulation of tau and thus neurofibrillary degeneration in DS.