Phosphorylation Of Microtubule-associated Protein Tau Research Articles

Phos-tag SDS-PAGE analysis of <i>in vivo</i> phosphorylation of microtubule-associated protein tau and its protein kinase GSK3β

Phos-tag SDS-PAGE analysis of <i>in vivo</i> phosphorylation of microtubule-associated protein tau and its protein kinase GSK3β

Corticosteroid-binding-globulin (CBG)-deficient mice show high pY216-GSK3β and phosphorylated-Tau levels in the hippocampus.

Corticosteroid-binding globulin (CBG) is the specific carrier of circulating glucocorticoids, but evidence suggests that it also plays an active role in modulating tissue glucocorticoid activity. CBG polymorphisms affecting its expression or affinity for glucocorticoids are associated with chronic pain, chronic fatigue, headaches, depression, hypotension, and obesity with an altered hypothalamic pituitary adrenal axis. CBG has been localized in hippocampus of humans and rodents, a brain area where glucocorticoids have an important regulatory role. However, the specific CBG function in the hippocampus is yet to be established. The aim of this study was to investigate the effect of the absence of CBG on hippocampal glucocorticoid levels and determine whether pathways regulated by glucocorticoids would be altered. We used cbg-/- mice, which display low total-corticosterone and high free-corticosterone blood levels at the nadir of corticosterone secretion (morning) and at rest to evaluate the hippocampus for total- and free-corticosterone levels; 11β-hydroxysteroid dehydrogenase expression and activity; the expression of key proteins involved in glucocorticoid activity and insulin signaling; microtubule-associated protein tau phosphorylation, and neuronal and synaptic function markers. Our results revealed that at the nadir of corticosterone secretion in the resting state the cbg-/- mouse hippocampus exhibited slightly elevated levels of free-corticosterone, diminished FK506 binding protein 5 expression, increased corticosterone downstream effectors and altered MAPK and PI3K pathway with increased pY216-GSK3β and phosphorylated tau. Taken together, these results indicate that CBG deficiency triggers metabolic imbalance which could lead to damage and long-term neurological pathologies.

Subchronic exposure to acrylamide caused behaviour disorders and related pathological and molecular changes in rat cerebellum

Acrylamide (ACR) is a neurotoxin with moderate acute toxicity. Significant level of ACR exists in diet and drinking water. Occupational exposure causes motor function impairment, but the underlying mechanisms remain poorly defined. This study aims to explore whether microtubule-associated protein tau phosphorylation, excessive activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling pathway and BDNF decline are involved in cerebellar neuron lesions and motor dysfunction after subchronic ACR exposure. The present results displayed that ACR caused gait abnormality and hind foot splay in rats. The HE and Nissl staining results revealed that ACR exposure aggravated cerebellar neuron lesions especially in purkinje cell layer. ACR markedly increased tau phosphorylation at Ser262 and Ser396/404 and inhibited the level of phosphorylation of glycogen synthase kinase 3β (P-GSK3β) at Ser9. The PERK-eukaryotic initiation factor-2α (eIF2α)-activating transcription factor 4 (ATF4) pathway was activated to promote CHOP expression and then to accelerate neuron lesions. Furthermore, ACR significantly decreased P-CREB at Ser133 and BDNF expression, which might be related to the inhibition of upstream signals from extracellular signal-related kinase (ERK) and protein kinase B (Akt). This work helps to elucidate the underlying mechanisms of ACR-induced neurotoxicity and present a potential target for prevention against the neurotoxicity.

Glucose Regulates Microtubule Disassembly and the Dose of Insulin Secretion via Tau Phosphorylation.

The microtubule cytoskeleton of pancreatic islet β-cells regulates glucose-stimulated insulin secretion (GSIS). We have reported that the microtubule-mediated movement of insulin vesicles away from the plasma membrane limits insulin secretion. High glucose-induced remodeling of microtubule network facilitates robust GSIS. This remodeling involves disassembly of old microtubules and nucleation of new microtubules. Here, we examine the mechanisms whereby glucose stimulation decreases microtubule lifetimes in β-cells. Using real-time imaging of photoconverted microtubules, we demonstrate that high levels of glucose induce rapid microtubule disassembly preferentially in the periphery of individual β-cells, and this process is mediated by the phosphorylation of microtubule-associated protein tau. Specifically, high glucose induces tau hyper-phosphorylation via glucose-responsive kinases GSK3, PKA, PKC, and CDK5. This causes dissociation of tau from and subsequent destabilization of microtubules. Consequently, tau knockdown in mouse islet β-cells facilitates microtubule turnover, causing increased basal insulin secretion, depleting insulin vesicles from the cytoplasm, and impairing GSIS. More importantly, tau knockdown uncouples microtubule destabilization from glucose stimulation. These findings suggest that tau suppresses peripheral microtubules turning over to restrict insulin oversecretion in basal conditions and preserve the insulin pool that can be released following stimulation; high glucose promotes tau phosphorylation to enhance microtubule disassembly to acutely enhance GSIS.

Multiplexed Phosphoproteomic Study of Brain in Patients with Alzheimer's Disease and Age-Matched Cognitively Healthy Controls.

Alzheimer's disease (AD) is the most common neurodegenerative disorder caused by neuronal loss that results in cognitive and functional impairment. Formation of neurofibrillary tangles composed of abnormal hyperphosphorylation of tau protein is one of the major pathological hallmarks of AD. Importantly, several neurodegenerative disorders, including AD, are associated with abnormal protein phosphorylation events. However, little is known thus far on global protein phosphorylation changes in AD. We report a phosphoproteomics study examining the frontal gyrus of people with AD and age-matched cognitively normal subjects, using tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography to enrich phosphopeptides. We identified 4631 phosphopeptides corresponding to 1821 proteins with liquid chromatography-mass spectrometry (MS)/MS analysis on an Orbitrap Fusion Lumos Tribrid mass spectrometer. Of these, 504 phosphopeptides corresponding to 350 proteins were significantly altered in the AD brain: 389 phosphopeptides increased whereas 115 phosphopeptides decreased phosphorylation. We observed significant changes in phosphorylation of known as well as novel molecules. Using targeted parallel reaction monitoring experiments, we validated the phosphorylation of microtubule-associated protein tau and myristoylated alanine-rich protein kinase C substrate (MARCKS) in control and AD (Control = 6, AD = 11) brain samples. In conclusion, our study provides new evidence on alteration of RNA processing and splicing, neurogenesis and neuronal development, and metabotropic glutamate receptor 5 (GRM5) calcium signaling pathways in the AD brain, and it thus offers new insights to accelerate diagnostics and therapeutics innovation in AD.

Human neuroblastoma SH-SY5Y cells treated with okadaic acid express phosphorylated high molecular weight tau-immunoreactive protein species

BackgroundEarly stages of Alzheimer's disease (AD) are characterized by high phosphorylation of microtubule-associated protein tau, which may result from the downregulation of protein phosphatases. New methodIn order to model phosphatase downregulation and analyze its effect on tau aggregation in vitro, we treated neuroblastoma SH-SY5Y cells with okadaic acid (OA), a protein phosphatase inhibitor, and examined high molecular weight phospho-tau species. Results and comparison with existing methodsOA treatment led to the appearance of heat-stable protein species with apparent molecular weight around 100 kDa, which were immunoreactive to anti-tau antibodies against phosphorylated Ser202 and Ser396. As these high molecular weight tau-immunoreactive proteins (HMW-TIPs) corresponded to the predicted size of two tau monomers, we considered the possibility that they represent phosphorylation-induced tau oligomers. We attempted to dissociate HMW-TIPs by urea and guanidine, as well as by alkaline phosphatase treatment, but HMW-TIPs were stable under all conditions tested. These characteristics resemble properties of certain sodium dodecyl sulfate (SDS)-resistant tau oligomers from AD brains. The absence of HMW-TIPs detection by anti-total tau antibodies Tau46, CP27 and Tau13 may be a consequence of epitope masking and protein truncation. Alternatively, HMW-TIPs may represent previously unreported phosphoproteins cross-reacting with tau. ConclusionsTaken together, our data provide a novel characterization of an OA-based cell culture model in which OA induces the appearance of HMW-TIPs. These findings have implications for further studies of tau under the conditions of protein phosphatase downregulation, aiming to explain mechanisms involved in early events leading to AD.

Phosphorylation of microtubule-associated protein tau by mitogen-activated protein kinase in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease in the elderly. The presence of neurofibrillary tangles (NFTs), which primarily contain self-aggregated hyper phosphorylated tau protein, is one of the major pathological characteristics of AD brains. Tau is a microtubule-associated protein important for regulating microtubule assembly and stability. Abnormal hyper phosphorylation of tau decreases its microtubule-binding capacity and disrupts microtubule stability. A number of protein kinases have been implicated in the abnormal phosphorylation of tau including mitogen-activated protein kinases (MAPKs). This article reviews the hyper phosphorylation of tau in the pathogenesis of AD and discusses the role of MAPKs in the phosphorylation of tau.

Isoform-independent and -dependent phosphorylation of microtubule-associated protein tau in mouse brain during postnatal development

Tau is a microtubule (MT)-associated protein that regulates MT dynamics in the axons of neurons. Tau binds to MTs via its C-terminal MT-binding repeats. There are two types of tau, those with three (3R) or four (4R) MT-binding repeats; 4R tau has a stronger MT-stabilizing activity than 3R tau. The MT-stabilizing activity of tau is regulated by phosphorylation. Interestingly, both the isoform and phosphorylation change at the time of neuronal circuit formation during postnatal development; highly phosphorylated 3R tau is replaced with 4R tau, which is less phosphorylated. However, it is not known how the transition of the isoforms and phosphorylation are regulated. Here, we addressed this question using developing mouse brains. Detailed analysis of developing brains revealed that the switch from 3R to 4R tau occurred during postnatal day 9 (P9) to P18 under the same time course as the conversion of phosphorylation from high to low. However, hypothyroidism, which is known to delay brain development, delayed the timing of tau dephosphorylation but not the exchange of isoforms, indicating that isoform switching and phosphorylation are not necessarily linked. Furthermore, we confirmed this finding by using mouse brains that expressed a single isoform of human tau. Human tau, either 3R or 4R, reduced phosphorylation levels during development even though the isoform did not change. We also found that 3R tau and 4R tau were phosphorylated differently in vivo even at the same developmental days. These results show for the first time that the phosphorylation and isoform alteration of tau are regulated differently during mouse development.

Pathologic Thr175 tau phosphorylation in CTE and CTE with ALS.

ObjectiveTo investigate whether chronic traumatic encephalopathy (CTE) and CTE with amyotrophic lateral sclerosis (CTE-ALS) exhibit features previously observed in other tauopathies of pathologic phosphorylation of microtubule-associated protein tau at Thr175 (pThr175 tau) and Thr231 (pThr231 tau), and glycogen synthase kinase–3β (GSK3β) activation, and whether these pathologic features are a consequence of traumatic brain injury (TBI).MethodsTau isoform expression was assayed by western blot in 6 stage III CTE cases. We also used immunohistochemistry to analyze 5 cases each of CTE, CTE-ALS, and 5 controls for expression of activated GSK3β, pThr175 tau, pThr231 tau, and oligomerized tau within spinal cord tissue and hippocampus. Using a rat model of moderate TBI, we assessed tau pathology and phospho-GSK3β expression at 3 months postinjury.ResultsCTE and CTE-ALS are characterized by the presence of all 6 tau isoforms in both soluble and insoluble tau isolates. Activated GSK3β, pThr175 tau, pThr231 tau, and oligomerized tau protein expression was observed in hippocampal neurons and spinal motor neurons. We observed tau neuronal pathology (fibrillar inclusions and axonal damage) and increased levels of pThr175 tau and activated GSK3β in moderate TBI rats.ConclusionsPathologic phosphorylation of tau at Thr175 and Thr231 and activation of GSK3β are features of the tauopathy of CTE and CTE-ALS. These features can be replicated in an animal model of moderate TBI.

Effect of intracerebellar microinjection of cdc2-siRNA on the coordination of mice with Niemann-Pick disease type C

Objective To observe the effect of inhibiting the abnormal activation of cdc2 gene on the coordination of mice with Niemann-Pick disease type C(NPC). Methods Recombinant adeno-associated virus( rAAV) encoding cdc2-siRNA was packaged, and then was injected into the cerebellum of 2 weeks old npc-/- mice. Footprint test and vertical screen test were performed to assess the coordination of mice at the age of 8 weeks. Purkinje cells visualized by HE staining in cerebellum were counted, and the phosphorylation of microtubule-associated protein Tau recognized by PHF-1 antibody was detected by immunoblotting technology. Results (1) Footprint test showed that the stride length in cdc2-siRNA npc-/- group((4.92±0.31) cm) was markedly longer than that in empty vector npc-/- group((4.05±0.19) cm)(P<0.05). (2)Vertical screen test showed that the latency to turn head upwards or reach the upper edge of the screen in cdc2-siRNA npc-/- group((26.01±1.82) s, (50.93±1.98) s) was significantly shorter than that in the empty vector npc-/- group((31.96±3.47)s, (56.89±2.97)s), respectively(P<0.05 for all comparisons). (3)The number of Purkinje cells in cerebellum was dramatically increased in cdc2-siRNA npc-/- group(11.0±2.5) compared with the empty vector npc-/- group(5.1±2.2)(P<0.05). (4)The relative optical densities of cdc2 and phosphorylated Tau immunoreactive bands in cdc2-siRNA npc-/- group(1.42±0.22, 0.95±0.31)were significantly lower than those in the empty vector npc-/- group(2.11±0.29, 2.61±0.62), respectively(P<0.05 for all comparisons). Conclusion Inhibiting the abnormal activation of cdc2 gene can improve the coordination of npc-/- mice by ameliorating Purkinje cell’s loss and reducing the hyperphosphorylation of Tau in cerebellum. Key words: Niemann-Pick disease type C; Coordination; Cell division cycle 2; Purkinje cell

Traumatic Brain Injury in hTau Model Mice: Enhanced Acute Macrophage Response and Altered Long-Term Recovery.

Traumatic brain injury (TBI) induces widespread neuroinflammation and accumulation of microtubule associated protein tau (MAPT): two key pathological features of tauopathies. This study sought to characterize the microglial/macrophage response to TBI in genomic-based MAPT transgenic mice in a Mapt knockout background (called hTau). Two-month-old hTau and age-matched control male and female mice received a single lateral fluid percussion TBI or sham injury. Separate groups of mice were aged to an acute (3 days post-injury [DPI]) or chronic (135 DPI) post-injury time point. As judged by tissue immunostaining for macrophage markers, microglial/macrophage response to TBI was enhanced at 3 DPI in hTau mice compared with control TBI and sham mice. However, MAPT phosphorylation increased in hTau mice regardless of injury group. Flow cytometric analysis revealed distinct populations of microglia and macrophages within all groups at 135 DPI. Unexpectedly, microglial reactivity was significantly reduced in hTau TBI mice compared with all other groups. Instead, hTau TBI mice showed a persistent macrophage response. In addition, TBI enhanced MAPT pathology in the temporal cortex and hippocampus of hTau TBI mice compared with controls 135 DPI. A battery of behavioral tests revealed that TBI in hTau mice resulted in compromised use of spatial search strategies to complete a water maze task, despite lack of motor or visual deficits. Collectively, these data indicate that the presence of wild-type human tau alters the microglial/macrophage response to a single TBI, induces delayed, region-specific MAPT pathology, and alters cognitive recovery; however, the causal relationship between these events remains unclear. These results highlight the potential significance of communication between MAPT and microglia/macrophages following TBI, and emphasize the role of neuroinflammation in post-injury recovery.

Role of microtubule-associated protein tau phosphorylation in Alzheimer's disease.

As a major microtubule-associated protein, tau plays an important role in promoting microtubule assembly and stabilizing microtubules. In Alzheimer's disease (AD) and other tauopathies, the abnormally hyperphosphorylated tau proteins are aggregated into paired helical filaments and accumulated in the neurons with the form of neurofibrillary tangles. An imbalanced regulation in protein kinases and protein phosphatases is the direct cause of tau hyperphosphorylation. Among various kinases and phosphatases, glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) are the most implicated. Accumulation of the hyperphosphorylated tau induces synaptic toxicity and cognitive impairments. Here, we review the upstream factors or pathways that can regulate GSK-3β or PP2A activity mainly based on our recent findings. We will also discuss the mechanisms that may underlie tau-induced synaptic toxicity.

Involvement of aberrant cyclin-dependent kinase 5/p25 activity in experimental traumatic brain injury.

Traumatic brain injury (TBI) is associated with adverse effects on brain functions, including sensation, language, emotions and/or cognition. Therapies for improving outcomes following TBI are limited. A better understanding of the pathophysiological mechanisms of TBI may suggest novel treatment strategies to facilitate recovery and improve treatment outcome. Aberrant activation of cyclin-dependent kinase 5 (Cdk5) has been implicated in neuronal injury and neurodegeneration. Cdk5 is a neuronal protein kinase activated via interaction with its cofactor p35 that regulates numerous neuronal functions, including synaptic remodeling and cognition. However, conversion of p35 to p25 via Ca(2+) -dependent activation of calpain results in an aberrantly active Cdk5/p25 complex that is associated with neuronal damage and cell death. Here, we show that mice subjected to controlled cortical impact (CCI), a well-established experimental TBI model, exhibit increased p25 levels and consistently elevated Cdk5-dependent phosphorylation of microtubule-associated protein tau and retinoblastoma (Rb) protein in hippocampal lysates. Moreover, CCI-induced neuroinflammation as indicated by increased astrocytic activation and number of reactive microglia. Brain-wide conditional Cdk5 knockout mice (Cdk5 cKO) subjected to CCI exhibited significantly reduced edema, ventricular dilation, and injury area. Finally, neurophysiological recordings revealed that CCI attenuated excitatory post-synaptic potential field responses in the hippocampal CA3-CA1 pathway 24h after injury. This neurophysiological deficit was attenuated in Cdk5 cKO mice. Thus, TBI induces increased levels of p25 generation and aberrant Cdk5 activity, which contributes to pathophysiological processes underlying TBI progression. Hence, selectively preventing aberrant Cdk5 activity may be an effective acute strategy to improve recovery from TBI. Traumatic brain injury (TBI) increases astrogliosis and microglial activation. Moreover, TBI deregulates Ca(2+) -homeostasis triggering p25 production. The protein kinase Cdk5 is aberrantly activated by p25 leading to phosphorylation of substrates including tau and Rb protein. Loss of Cdk5 attenuates TBI lesion size, indicating that Cdk5 is a critical player in TBI pathogenesis and thus may be a suitable therapeutic target for TBI.

Introducing a Developed Model of Reversible Cardiac Arrest to Produce Global Brain Ischemia and Its Impact on Microtubule-Associated Protein Tau Phosphorylation at Ser396

Introducing a Developed Model of Reversible Cardiac Arrest to Produce Global Brain Ischemia and Its Impact on Microtubule-Associated Protein Tau Phosphorylation at Ser396

Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells

Event Abstract Back to Event Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells Taeko Kimura1, Koichi Ishiguro2, Masato Hasegawa3 and Shin-ichi Hisanaga1* 1 Tokyo Metropolitan University, Molecular Neurosicence/Biological Sciences/Science and Engineering, Japan 2 Juntendo University, Neurology, Japan 3 Tokyo Metropolitan Institute of Medical Science, Dementia and Higher Brain Function, Japan Tau, a microtubule-associated protein, is a major component of aggregates seen in brains of tauopathies such as Alzheimer’s disease (AD) and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Tau is hyperphosphorylated in the aggregates and therefore the mechanism of hyperphosphorylation has been an intense research subject. Here, we applied the Phos-tag SDS-PAGE technique to characterize tau phosphorylation by cyclin-dependent kinase 5 (Cdk5) in vitro and in cultured cells. Cdk5-p25 phosphorylated tau at Ser404, Ser235, Thr205 and probably Se202 in that order. Among those four major in vitro Cdk5 phosphorylation sites, Ser404 was preferentially phosphorylated by Cdk5 in cultured cells, whereas Thr205 was not. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau existed in 12 different phosphorylation states in COS-7 cells with Ser202, Thr231 and Ser235 as major phosphorylation sites. p35-Cdk5 and p25-Cdk5 phosphorylated tau to a similar extent, when their expression levels were adjusted. Phosphorylation of FTDP-17 tau mutants was also analyzed by this method. While the mutants in the N-terminal or microtubule-binding region were phosphorylated identically as was wild type tau, the C-terminal mutants showed slightly different banding patterns from wild type tau. In particular, it was clear that R406W mutation resulted in loss of Ser404 phosphorylation. These results not only include novel and interesting information on tau phosphorylation but also indicate usefulness of Phos-tag SDS-PAGE in the analysis of site-specific and cellular phosphorylation pattern of tau. Keywords: Phosphorylation, Alzheimer’s disease, tau, in vitro, protein Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Kimura T, Ishiguro K, Hasegawa M and Hisanaga S (2016). Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00195 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Shin-ichi Hisanaga, Tokyo Metropolitan University, Molecular Neurosicence/Biological Sciences/Science and Engineering, Hachioji, Tokyo, Japan, hisanaga-shinichi@tmu.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Google Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Google Scholar Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga PubMed Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The Microtubule-Associated Protein Tau and Its Relevance for Pancreatic Beta Cells.

Structural and biochemical alterations of the microtubule-associated protein tau (MAPT) are associated with degenerative disorders referred to as tauopathies. We have previously shown that MAPT is present in human islets of Langerhans, human insulinomas, and pancreatic beta-cell line models, with biophysical similarities to the pathological MAPT in the brain. Here, we further studied MAPT in pancreatic endocrine tissue to better understand the mechanisms that lead to functional dysregulation of pancreatic beta cells. We found upregulation of MAPT protein expression in human insulinomas when compared to human pancreatic islets of Langerhans and an imbalance between MAPT isoforms in insulinomas tissue. We cloned one 3-repeat domain MAPT and transduced this into a beta-cell derived rodent cell line Rin-5F. Proliferation experiments showed higher growth rates and metabolic activities of cells overexpressing MAPT protein. We observed that a MAPT overexpressing cell line demonstrates altered insulin transcription, translation, and insulin secretion rates. We found the relative insulin secretion rates were significantly decreased in a MAPT overexpressing cell line and these findings could be confirmed using partial MAPT knock-down cell lines. Our findings support that MAPT may play an important role in insulin granule trafficking and indicate the importance of balanced MAPT phosphorylation and dephosphorylation for adequate insulin release.

Human tau expression reduces adult neurogenesis in a mouse model of tauopathy

Accumulation of hyperphosphorylated and aggregated microtubule-associated protein tau (MAPT) is a central feature of a class of neurodegenerative diseases termed tauopathies. Notably, there is increasing evidence that tauopathies, including Alzheimer’s disease, are also characterized by a reduction in neurogenesis, the birth of adult neurons. However, the exact relationship between hyperphosphorylation and aggregation of MAPT and neurogenic deficits remains unclear, including whether this is an early- or late-stage disease marker. In the present study, we used the genomic-based hTau mouse model of tauopathy to examine the temporal and spatial regulation of adult neurogenesis during the course of the disease. Surprisingly, hTau mice exhibited reductions in adult neurogenesis in 2 different brain regions by as early as 2 months of age, before the development of robust MAPT pathology in this model. This reduction was found to be due to reduced proliferation and not because of enhanced apoptosis in the hippocampus. At these same time points, hTau mice also exhibited altered MAPT phosphorylation with neurogenic precursors. To examine whether the effects of MAPT on neurogenesis were cell autonomous, neurospheres prepared from hTau animals were examined in vitro, revealing a growth deficit when compared with non-transgenic neurosphere cultures. Taken together, these studies provide evidence that altered adult neurogenesis is a robust and early marker of altered, cell-autonomous function of MAPT in the hTau mouse mode of tauopathy and that altered adult neurogenesis should be examined as a potential marker and therapeutic target for human tauopathies.

Thiamet-G-mediated inhibition of O-GlcNAcase sensitizes human leukemia cells to microtubule-stabilizing agent paclitaxel

Although the microtubule-stabilizing agent paclitaxel has been widely used for treatment of several cancer types, particularly for the malignancies of epithelia origin, it only shows limited efficacy on hematological malignancies. Emerging roles of O-GlcNAcylation modification of proteins in various cancer types have implicated the key enzymes catalyzing this reversible modification as targets for cancer therapy. Here, we show that the highly selective O-GlcNAcase (OGA) inhibitor thiamet-G significantly sensitized human leukemia cell lines to paclitaxel, with an approximate 10-fold leftward shift of IC50. Knockdown of OGA by siRNAs or inhibition of OGA by thiamet-G did not influence the cell viability. Furthermore, we demonstrated that thiamet-G binds to OGA in competition with 4-methylumbelliferyl N-acetyl-β-d-glucosaminide dehydrate, an analogue of O-GlcNAc UDP, thereby suppressing the activity of OGA. Importantly, inhibition of OGA by thiamet-G decreased the phosphorylation of microtubule-associated protein Tau and caused alterations of microtubule network in cells. It is noteworthy that paclitaxel combined with thiamet-G resulted in more profound perturbations on microtubule stability than did either one alone, which may implicate the underlying mechanism of thiamet-G-mediated sensitization of leukemia cells to paclitaxel. These findings thus suggest that a regimen of paclitaxel combined with OGA inhibitor might be more effective for the treatment of human leukemia.

Opposing effects of membrane-anchored CX3CL1 on amyloid and tau pathologies via the p38 MAPK pathway.

Several Alzheimer's disease (AD) risk genes are specifically expressed by microglia within the CNS. However, the mechanisms by which microglia regulate the pathological hallmarks of AD--extracellular deposition of β-amyloid (Aβ) and intraneuronal hyperphosphorylation of microtubule-associated protein tau (MAPT)--remain to be established. Notably, deficiency for the microglial CX3CR1 receptor has opposing effects on Aβ and MAPT pathologies. CX3CL1, the neuronally derived cognate ligand for CX3CR1, signals both in membrane-anchored and soluble forms. In this study, we sought to determine the relative contribution on membrane-anchored versus soluble CX3CL1 in regulating the microglia-mediated amelioration of Aβ pathology, as well as provide insight into the potential downstream microglial-based mechanisms. As expected, CX3CL1 deficiency reduced Aβ deposition in APPPS1 animals in a similar manner to CX3CR1 deficiency. Surprisingly, however, CX3CL1-deficient APPPS1 animals exhibited enhanced neuronal MAPT phosphorylation despite reduced amyloid burden. Importantly, neither of these phenotypes was altered by transgenic expression of the soluble CX3CL1 isoform, suggesting that it is the membrane-anchored version of CX3CL1 that regulates microglial phagocytosis of Aβ and neuronal MAPT phosphorylation. Analysis of transcript levels in purified microglia isolated from APPPS1 mice with the various CX3CL1/CX3CR1 genotypes revealed increased expression of inflammatory cytokines and phagocytic markers, which was associated with activation of p38 mitogen-activated protein kinase and Aβ internalization within microglia. Together, these studies challenge the "frustrated phagocytosis" concept and suggest that neuronal-microglial communication link the two central AD pathologies.

Proteasome inhibition-induced downregulation of Akt/GSK-3β pathway contributes to abnormality of tau in hippocampal slice.

Proteasome inhibition can induce abnormal accumulation and phosphorylation of microtubule-associated protein tau. The major function of tau protein is to promote microtubules assembly and stabilization, and abnormal tau protein would disturb its microtubule-binding function. In this study, proteasome inhibitor MG132 was used to treat hippocampal slices to explore the role and mechanism of Akt/glycogen synthase kinase-3β (GSK-3β) in proteasome inhibition-induced tau abnormality. During the culture period, we measure the lactate dehydrogenase (LDH) content to assay the viability of hippocampal slices. Following 2.5 and 5 μM MG132 treatment for 6 h, we detected the expression, phosphorylation modification, and microtubule-binding function of tau protein of slices. We also analyzed the changed activities of glycogen synthase kinase-3β (GSK-3β) and protein kinase B (PKB/Akt) and the level of heat shock protein 90 (Hsp90) in the process. In addition, co-immunoprecipitation was used to investigate the interaction between Akt and Hsp90, Akt and protein phosphatase-2A (PP2A) in the MG132-treated organotypic hippocampal slices. Our results indicated that proteasome inhibition led to degradation obstacles and abnormal phosphorylation of tau protein. The downregulated Akt/GSK-3β signaling pathway might be responsible for the abnormal phosphorylation of tau protein at multiple sites which further reduced the microtubule-binding function of tau protein. Furthermore, proteasome inhibition decreased the binding capacity of Akt-Hsp90 while increased the Akt-PP2A binding ability which mediated Akt inactivity. This current study establishes a hippocampal slice model targeting Akt/GSK-3β signaling pathway to explore the pivotal role of proteasome inhibition in tau pathology.