Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism.

Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, ß, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1ß, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.SIGNIFICANCE STATEMENT Tau pathology is closely associated with neurodegeneration in Alzheimer's disease and other tauopathies, but the toxic mechanisms remain a debated topic. We previously proposed that pathologic tau forms induce dysfunction and degeneration through aberrant activation of a PP1-dependent pathway that disrupts axonal transport. Here, we show that tau directly interacts with specific PP1 isoforms, increasing levels of active PP1. Pathogenic tau mutations enhance this interaction, further increasing active PP1 levels and impairing axonal transport in isolated squid axoplasm and primary hippocampal neurons. Mutant-tau-mediated impairment of axonal transport was mediated by PP1γ and a phosphatase-activating domain located at the amino terminus of tau. This work has important implications for understanding and potentially mitigating tau-mediated neurotoxicity in tauopathies.

Nonphosphorylated tau slows down Aß1-42 aggregation, binds to Aß1-42 oligomers, and reduces Aß1-42 toxicity.

The formation of neurofibrillary tangles and amyloid plaques accompanies the progression of Alzheimer's disease. Tangles are made of fibrillar aggregates formed by the microtubule-associated protein tau, whereas plaques comprise fibrillar forms of amyloid-beta (Aß). Both form toxic oligomers during aggregation and are thought to interact synergistically to each promote the accumulation of the other. Recent in vitro studies have suggested that the monomeric nonphosphorylated full-length tau protein hinders the aggregation of Aß1-40 peptide, but whether the same is true for the more aggregation-prone Aß1-42 was not determined. We used in vitro and in vivo techniques to explore this question. We have monitored the aggregation kinetics of Aß1-42 by thioflavine T fluorescence in the presence or the absence of different concentrations of nonphosphorylated tau. We observed that elongation of Aß1-42 fibrils was inhibited by tau in a dose-dependent manner. Interestingly, the fibrils were structurally different in the presence of tau but did not incorporate tau. Surface plasmon resonance indicated that tau monomers bound to Aß1-42 oligomers (but not monomers) and hindered their interaction with the anti-Aß antibody 4G8, suggesting that tau binds to the hydrophobic central core of Aß recognized by 4G8. Tau monomers also antagonized the toxic effects of Aß oligomers in Caenorhabditis elegans. This suggests that nonphosphorylated tau might have a neuroprotective effect by binding Aß1-42 oligomers formed during the aggregation and shielding their hydrophobic patches.

Effects of TYROBP Deficiency on Neuroinflammation of a Alzheimer's Disease Mouse Model Carrying a PSEN1 p.G378E Mutation.

Objective To study the effects of TYRO protein kinase-binding protein (TYROBP) deficiency on learning behavior, glia activation and pro-inflammatory cycokines, and Tau phosphorylation of a new Alzheimer's disease (AD) mouse model carrying a PSEN1 p.G378E mutation.Methods A new AD mouse model carrying PSEN1 p.G378E mutation was built based on our previously found AD family which might be ascribed to the PSEN1 mutation, and then crossed with TYROBP deficient mice to produce the heterozygous hybrid mice (PSEN1G378E/WT; Tyrobp+/-) and the homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/-). Water maze test was used to detect spatial learning and memory ability of mice. After the mice were sacrificed, the hippocampus was excised for further analysis. Immunofluorescence was used to identify the cell that expresses TYROBP and the number of microglia and astrocyte. Western blot was used to detect the expression levels of Tau and phosphorylated Tau (p-Tau), and ELISA to measure the levels of pro-inflammatory cytokines. Results Our results showed that TYROBP specifically expressed in the microglia of mouse hippocampus. Absence of TYROBP in PSEN1G378E mutation mouse model prevented the deterioration of learning behavior, decreased the numbers of microglia and astrocytes, and the levels of interleukin-6, interleukin-1ß and tumor necrosis factor-α in the hippocampus (all P < 0.05). The ratios of AT8/Tau5, PHF1/Tau5, pT181/Tau5, pT231/Tau5 and p-ERK/ERK were all higher in homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/- mice) compared with PSEN1G378E/G378E mice (all P < 0.05). Conclusions TYROBP deficiency might play a protective role in the modulation of neuroinflammation of AD. However, the relationship between neuroinflammation processes involving microglia and astrocyte activation, and release of pro-inflammatory cytokines, and p-Tau pathology needs further study.

Amyloid-ß and p-Tau Anti-Threat Response to Herpes Simplex Virus 1 Infection in Primary Adult Murine Hippocampal Neurons.

Alzheimer's Disease (AD) is the sixth leading cause of death in the United States. Recent studies have established a potential link between herpes simplex virus 1 (HSV-1) infection and the development of AD. HSV-1 DNA has been detected in AD amyloid plaques in human brains, and treatment with the antiviral acyclovir (ACV) was reported to block the accumulation of the AD-associated proteins beta-amyloid (Aß) and hyper-phosphorylated tau (p-tau) in Vero and glioblastoma cells. Our goal was to determine whether the accumulation of AD-related proteins is attributable to acute and/or latent HSV-1 infection in mature hippocampal neurons, a region of the brain severely impacted by AD. Primary adult murine hippocampal neuronal cultures infected with HSV-1, with or without antivirals, were assessed for Aß and p-tau expression over 7 days postinfection. P-tau expression was transiently elevated in HSV-1-infected neurons, as well as in the presence of antivirals alone. Infected neurons, as well as uninfected neurons treated with antivirals, had a greater accumulation of Aß42 than uninfected untreated neurons. Furthermore, Aß42 colocalized with HSV-1 latency-associated transcript (LAT) expression. These studies suggest that p-tau potentially acts as an acute response to any perceived danger-associated molecular pattern (DAMP) in primary adult hippocampal neurons, while Aß aggregation is a long-term response to persistent threats, including HSV-1 infection.IMPORTANCE Growing evidence supports a link between HSV-1 infection and Alzheimer's disease (AD). Although AD is clearly a complex multifactorial disorder, an infectious disease etiology provides alternative therapy opportunities for this devastating disease. Understanding the impact that HSV-1 has on mature neurons and the proteins most strongly associated with AD pathology may identify specific mechanisms that could be manipulated to prevent progression of neurodegeneration and dementia.

Activated microglia desialylate their surface, stimulating complement receptor 3-mediated phagocytosis of neurons.

The glycoproteins and glycolipids of the cell surface have sugar chains that normally terminate in a sialic acid residue, but inflammatory activation of myeloid cells can cause sialidase enzymes to remove these residues, resulting in desialylation and altered activity of surface receptors, such as the phagocytic complement receptor 3 (CR3). We found that activation of microglia with lipopolysaccharide (LPS), fibrillar amyloid beta (Aß), Tau or phorbol myristate acetate resulted in increased surface sialidase activity and desialylation of the microglial surface. Desialylation of microglia by adding sialidase, stimulated microglial phagocytosis of beads, but this was prevented by siRNA knockdown of CD11b or a blocking antibody to CD11b (a component of CR3). Desialylation of microglia by a sialyl-transferase inhibitor (3FAx-peracetyl-Neu5Ac) also stimulated microglial phagocytosis of beads. Desialylation of primary glial-neuronal co-cultures by adding sialidase or the sialyl-transferase inhibitor resulted in neuronal loss that was prevented by inhibiting phagocytosis with cytochalasin D or the blocking antibody to CD11b. Adding desialylated microglia to glial-neuronal cultures, in the absence of neuronal desialylation, also caused neuronal loss prevented by CD11b blocking antibody. Adding LPS or Aß to primary glial-neuronal co-cultures caused neuronal loss, and this was prevented by inhibiting endogenous sialidase activity with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid or blockage of CD11b. Thus, activated microglia release a sialidase activity that desialylates the cell surface, stimulating CR3-mediated phagocytosis of neurons, making extracellular sialidase and CR3 potential treatment targets to prevent inflammatory loss of neurons.

Extracellular tau induces microglial phagocytosis of living neurons in cell cultures.

Tau is a microtubule-associated protein, found at high levels in neurons, and its aggregation is associated with neurodegeneration. Recently, it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are unclear. In this study, we investigated whether extracellular tau2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were examined for neuronal loss, death, and phosphatidylserine exposure, as well as for microglial phagocytosis by fluorescence microscopy. Aggregation of tau2N4R was assessed by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2 days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the 'eat-me' signal phosphatidylserine on the surface of living neurons, and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.

Tau Contributes to Sevoflurane-induced Neurocognitive Impairment in Neonatal Mice.

BACKGROUND: Sevoflurane anesthesia induces Tau phosphorylation and cognitive impairment in neonatal but not in adult mice. This study tested the hypothesis that differences in brain Tau amounts and in the activity of mitochondria-adenosine triphosphate (ATP)-Nuak1-Tau cascade between the neonatal and adult mice contribute to the age-dependent effects of sevoflurane on cognitive function. METHODS: 6- and 60-day-old mice of both sexes received anesthesia with 3% sevoflurane for 2 h daily for 3 days. Biochemical methods were used to measure amounts of Tau, phosphorylated Tau, Nuak1, ATP concentrations, and mitochondrial metabolism in the cerebral cortex and hippocampus. The Morris water maze test was used to evaluate cognitive function in the neonatal and adult mice. RESULTS: Under baseline conditions and compared with 60-day-old mice, 6-day-old mice had higher amounts of Tau (2.6 ± 0.4 [arbitrary units, mean ± SD] vs. 1.3 ± 0.2; P < 0.001), Tau oligomer (0.3 ± 0.1 vs. 0.1 ± 0.1; P = 0.008), and Nuak1 (0.9 ± 0.3 vs. 0.3 ± 0.1; P = 0.025) but lesser amounts of ATP (0.8 ± 0.1 vs. 1.5 ± 0.1; P < 0.001) and mitochondrial metabolism (74.8 ± 14.1 [pmol/min] vs. 169.6 ± 15.3; P < 0.001) in the cerebral cortex. Compared with baseline conditions, sevoflurane anesthesia induced Tau phosphorylation at its serine 202/threonine 205 residues (1.1 ± 0.4 vs. 0.2 ± 0.1; P < 0.001) in the 6-day-old mice but not in the 60-day-old mice (0.05 ± 0.04 vs. 0.03 ± 0.01; P = 0.186). The sevoflurane-induced Tau phosphorylation and cognitive impairment in the neonatal mice were both attenuated by the inhibition of Nuak1 and the treatment of vitamin K2. CONCLUSIONS: Higher brain Tau concentrations and lower brain mitochondrial metabolism in neonatal compared with adult mice contribute to developmental stage-dependent cognitive dysfunction after sevoflurane anesthesia.

Adiponectin Attenuates Streptozotocin-Induced Tau Hyperphosphorylation and Cognitive Deficits by Rescuing PI3K/Akt/GSK-3ß Pathway.

Clinical studies have demonstrated that decreased adiponectin is associated with the development of Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD). We focused on determining the neuroprotective effect offered by adiponectin against streptozotocin-induced brain damage in ICV-STZ rat model. We found that adiponectin supplements significantly restored the cognitive deficits in ICV-STZ rat model including shorter escape latency, more crossing times and increased time spent in the target quadrant. Adiponectin supplements also increased number of dendritic branches and mushroom percentage. In addition, adiponectin supplements attenuated tau hyperphosphorylation at multiple AD-related sites through activation of protein Ser9-phosphorylated glycogen synthase kinase-3ß (Ser9-GSK-3ß) with increased the Akt and PI3K activity. Our data suggest that adiponectin supplements have neuroprotective effects on the ICV-STZ rat model, which may be mediated by the activation of the PI3K/Akt/GSK-3ß signaling pathway.

Fully human MAP-fusion protein selectively targets and eliminates proliferating CD64(+) M1 macrophages.

Classical immunotoxins compromise a binding component (for example, a ligand, antibody or fragment thereof) and a cytotoxic component, usually derived from bacteria or plants (for example, Pseudomonas exotoxin A or ricin). Despite successful testing in vitro, the clinical development of immunotoxins has been hampered by immunogenicity and unsatisfactory safety profiles. Therefore, research has focused on fully human pro-apoptotic components suitable for the development of cytolytic fusion proteins (CFP). We recently reported that human microtubule-associated protein tau (MAP) can induce apoptosis when delivered to rapidly proliferating cancer cells. Here, we describe a new fully human CFP called H22(scFv)-MAP, which specifically targets CD64(+) cells. We show that H22(scFv)-MAP can efficiently kill proliferating HL-60 pro-monocytic cells in vitro. In addition, the human CFP specifically eliminates polarized M1 macrophages in a transgenic mouse model of cutaneous chronic inflammation. Because M1 macrophages promote the pathogenesis of many chronic inflammatory diseases, targeting this cell population with H22(scFv)-MAP could help to treat diseases such as atopic dermatitis, rheumatoid arthritis and inflammatory bowel disease.

Polymeric alkylpyridinium salts permit intracellular delivery of human Tau in rat hippocampal neurons: requirement of Tau phosphorylation for functional deficits.

Patients suffering from tauopathies including frontotemporal dementia (FTD) and Alzheimer's disease (AD) present with intra-neuronal aggregation of microtubule-associated protein Tau. During the disease process, Tau undergoes excessive phosphorylation, dissociates from microtubules and aggregates into insoluble neurofibrillary tangles (NFTs), accumulating in the soma. While many aspects of the disease pathology have been replicated in transgenic mouse models, a region-specific non-transgenic expression model is missing. Complementing existing models, we here report a novel region-specific approach to modelling Tau pathology. Local co-administration of the pore-former polymeric 1,3-alkylpyridinium salts (Poly-APS) extracted from marine sponges, and synthetic full-length 4R recombinant human Tau (hTau) was performed in vitro and in vivo. At low doses, Poly-APS was non-toxic and cultured cells exposed to Poly-APS (0.5 µg/ml) and hTau (1 µg/ml; ~22 µM) had normal input resistance, resting-state membrane potentials and Ca(2+) transients induced either by glutamate or KCl, as did cells exposed to a low concentration of the phosphatase inhibitor Okadaic acid (OA; 1 nM, 24 h). Combined hTau loading and phosphatase inhibition resulted in a collapse of the membrane potential, suppressed excitation and diminished glutamate and KCl-stimulated Ca(2+) transients. Stereotaxic infusions of Poly-APS (0.005 µg/ml) and hTau (1 µg/ml) bilaterally into the dorsal hippocampus at multiple sites resulted in hTau loading of neurons in rats. A separate cohort received an additional 7-day minipump infusion of OA (1.2 nM) intrahippocampally. When tested 2 weeks after surgery, rats treated with Poly-APS+hTau+OA presented with subtle learning deficits, but were also impaired in cognitive flexibility and recall. Hippocampal plasticity recorded from slices ex vivo was diminished in Poly-APS+hTau+OA subjects, but not in other treatment groups. Histological sections confirmed the intracellular accumulation of hTau in CA1 pyramidal cells and along their processes; phosphorylated Tau was present only within somata. This study demonstrates that cognitive, physiological and pathological symptoms reminiscent of tauopathies can be induced following non-mutant hTau delivery into CA1 in rats, but functional consequences hinge on increased Tau phosphorylation. Collectively, these data validate a novel model of locally infused recombinant hTau protein as an inducer of Tau pathology in the hippocampus of normal rats; future studies will provide insights into the pathological spread and maturation of Tau pathology.

Oligomer formation of tau protein hyperphosphorylated in cells.

Abnormal phosphorylation ("hyperphosphorylation") and aggregation of Tau protein are hallmarks of Alzheimer disease and other tauopathies, but their causative connection is still a matter of debate. Tau with Alzheimer-like phosphorylation is also present in hibernating animals, mitosis, or during embryonic development, without leading to pathophysiology or neurodegeneration. Thus, the role of phosphorylation and the distinction between physiological and pathological phosphorylation needs to be further refined. So far, the systematic investigation of highly phosphorylated Tau was difficult because a reliable method of preparing reproducible quantities was not available. Here, we generated full-length Tau (2N4R) in Sf9 cells in a well defined phosphorylation state containing up to ∼20 phosphates as judged by mass spectrometry and Western blotting with phospho-specific antibodies. Despite the high concentration in living Sf9 cells (estimated ∼230 µm) and high phosphorylation, the protein was not aggregated. However, after purification, the highly phosphorylated protein readily formed oligomers, whereas fibrils were observed only rarely. Exposure of mature primary neuronal cultures to oligomeric phospho-Tau caused reduction of spine density on dendrites but did not change the overall cell viability.

Extracellular association of APP and tau fibrils induces intracellular aggregate formation of tau.

Alzheimer's disease (AD) is characterized by extracellular amyloid ß (Aß) deposition and intracellular tau aggregation. Many studies have indicated some association between these processes, but it remains unknown how the two pathologies are linked. In this study, we investigated whether expression of amyloid precursor protein (APP) influences extracellular seed-dependent intracellular tau accumulation in cultured cells. Treatment of tau-expressing SH-SY5Y cells with Aß fibrils did not induce intracellular tau aggregation. On the other hand, in cells expressing both tau and APP, treatment with tau fibrils or Sarkosyl-insoluble tau from AD brains induced intracellular tau aggregation. The seed-dependent intracellular tau aggregation was not induced by expression of APP lacking the extracellular domain. The amount of phosphorylated tau aggregates in cultured cells was dose dependently elevated in response to increased levels of APP on the cell membrane. Our results indicate that the extracellular region of APP is involved in uptake of tau fibrils into cells, raising the possibility that APP, but not Aß, influences cell-to-cell spreading of tau pathologies in AD by serving as a receptor of abnormal tau aggregates.

Beta-amyloid oligomers activate apoptotic BAK pore for cytochrome c release.

In Alzheimer's disease, cytochrome c-dependent apoptosis is a crucial pathway in neuronal cell death. Although beta-amyloid (Aß) oligomers are known to be the neurotoxins responsible for neuronal cell death, the underlying mechanisms remain largely elusive. Here, we report that the oligomeric form of synthetic Aß of 42 amino acids elicits death of HT-22 cells. But, when expression of a bcl-2 family protein BAK is suppressed by siRNA, Aß oligomer-induced cell death was reduced. Furthermore, significant reduction of cytochrome c release was observed with mitochondria isolated from BAK siRNA-treated HT-22 cells. Our in vitro experiments demonstrate that Aß oligomers bind to BAK on the membrane and induce apoptotic BAK pores and cytochrome c release. Thus, the results suggest that Aß oligomers function as apoptotic ligands and hijack the intrinsic apoptotic pathway to cause unintended neuronal cell death.

Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo.

Hyperphosphorylated forms of the microtubule-associated protein (MAP) tau accumulate in Alzheimer's disease and related tauopathies and are thought to have an important role in neurodegeneration. However, the mechanisms through which phosphorylated tau induces neurodegeneration have remained elusive. Here, we show that tau-induced neurodegeneration is associated with accumulation of filamentous actin (F-actin) and the formation of actin-rich rods in Drosophila and mouse models of tauopathy. Importantly, modulating F-actin levels genetically leads to dramatic modification of tau-induced neurodegeneration. The ability of tau to interact with F-actin in vivo and in vitro provides a molecular mechanism for the observed phenotypes. Finally, we show that the Alzheimer's disease-linked human beta-amyloid protein (Abeta) synergistically enhances the ability of wild-type tau to promote alterations in the actin cytoskeleton and neurodegeneration. These findings raise the possibility that a direct interaction between tau and actin may be a critical mediator of tau-induced neurotoxicity in Alzheimer's disease and related disorders.

Senescence Targeting Methods Impact Alzheimer's Disease Features in 3xTg Mice.

Background: Cellular senescence has been associated with neurodegenerative disease and clearance of senescent cells using genetic or pharmaceutical strategies (senolytics) has demonstrated beneficial effects in mouse models investigating individual disease etiologies of Alzheimer's disease (AD). However, it has remained unclear if senescent cell clearance in a mouse model exhibiting both plaque and tau pathologies modifies the disease state (3xTg). Objective: To investigate the effects of senescent cell clearance in the 3xTg mouse model. Methods: 3xTg mice were treated with senolytics (ABT263 (navitoclax; NAVI), a combination of dasatinib and quercetin (D+Q)), or subjected to transgene-mediated removal of p16-expressing cells (via INK-ATTAC). Results: Senolytic treatments consistently reduced microgliosis and ameliorated both amyloid and tau pathology in 3xTg mice. Using RNA sequencing, we found evidence that synaptic dysfunction and neuroinflammation were attenuated with treatment. These beneficial effects were not observed with short-term senolytic treatment in mice with more advanced disease. Conclusions: Overall, our results further corroborate the beneficial effects senescent cell clearance could have on AD and highlight the importance of early intervention for the treatment of this debilitating disease.

Caloric Restriction Improves Spatial Learning Deficits in Tau Mice.

Background: Caloric restriction (CR) has been recognized for its benefits in delaying age-related diseases and extending lifespan. While its effects on amyloid pathology in Alzheimer's disease (AD) mouse models are well-documented, its effects on tauopathy, another hallmark of AD, are less explored. Objective: To assess the impact of a short-term 30% CR regimen on age-dependent spatial learning deficits and pathological features in a tauopathy mouse model. Methods: We subjected male PS19 tau P301S (hereafter PS19) and age-matched wildtype mice from two age cohorts (4.5 and 7.5 months old) to a 6-week 30% CR regimen. Spatial learning performance was assessed using the Barnes Maze test. Tau pathology, neuroinflammation, hippocampal cell proliferation, and neurogenesis were evaluated in the older cohort by immunohistochemical staining and RT-qPCR. Results: CR mitigated age-dependent spatial learning deficits in PS19 mice but exhibited limited effects on tau pathology and the associated neuroinflammation. Additionally, we found a decrease in hippocampal cell proliferation, predominantly of Iba1+ cells. Conclusions: Our findings reinforce the cognitive benefits conferred by CR despite its limited modulation of disease pathology. Given the pivotal role of microglia in tau-driven pathology, the observed reduction in Iba1+ cells under CR suggests potential therapeutic implications, particularly if CR would be introduced early in disease progression.

High-fat diet induces cognitive impairment through repression of SIRT1/AMPK-mediated autophagy.

AIMS: Recent evidence suggests an association between a high-fat diet (HFD) and cognitive decline. HFD may reduce synaptic plasticity and cause tau hyperphosphorylation, but the mechanisms involved remain unclear. The purpose of this study was to explore whether Sirtuin1 (SIRT1)/AMP-activated protein kinase (AMPK) pathway was involved in this pathogenic effect in the HFD exposed mice. METHODS: C57BL/6 mice at 12 months of age were fed a standard (9% kcal fat) or high-fat (60% kcal fat) diet for 22 weeks, and Neuro-2a (N2a) cells were treated with normal culture medium or a palmitic acid (PA) medium (100uM) for 40 h. After that, cognitive function was tested by Morris water maze (MWM). The levels of proteins involved in SIRT1/AMPK pathway and autophagy were measured using western blotting and immunofluorescence. We also assessed the phosphorylation of tau protein and synapse. RESULTS: The mice presented impaired learning and memory abilities. We further found decreased levels of synaptophysin (Syn) and brain-derived neurotrophic factor (BDNF), increased tau46 and phosphorylated tau protein, and damaged neurons in mice after HFD or in N2a cells treated with PA medium. Moreover, HFD can also reduce the expression of SIRT1, inhibit AMPK phosphorylation, and block autophagic flow in both mice and cells. After treating the cells with the SIRT1 agonist SRT1720, SIRT1/AMPK pathway and autophagy-related proteins were partially reversed and the number of PA-induced positive cells was alleviated in senescence-associated ß-galactosidase (SA-ß-gal) staining. CONCLUSIONS: HFD may inhibit the expression of SIRT1/AMPK pathway and disrupt autophagy flux, and result in tau hyperphosphorylation and synaptic dysfunction during aging, which ultimately lead to cognitive decline.

Rapidly reversible persistent long-term potentiation inhibition by patient-derived brain tau and amyloid ß proteins.

How the two pathognomonic proteins of Alzheimer's disease (AD); amyloid ß (Aß) and tau, cause synaptic failure remains enigmatic. Certain synthetic and recombinant forms of these proteins are known to act concurrently to acutely inhibit long-term potentiation (LTP). Here, we examined the effect of early amyloidosis on the acute disruptive action of synaptotoxic tau prepared from recombinant protein and tau in patient-derived aqueous brain extracts. We also explored the persistence of the inhibition of LTP by different synaptotoxic tau preparations. A single intracerebral injection of aggregates of recombinant human tau that had been prepared by either sonication of fibrils (SτAs) or disulfide bond formation (oTau) rapidly and persistently inhibited LTP in rat hippocampus. The threshold for the acute inhibitory effect of oTau was lowered in amyloid precursor protein (APP)-transgenic rats. A single injection of synaptotoxic tau-containing AD or Pick's disease brain extracts also inhibited LTP, for over two weeks. Remarkably, the persistent disruption of synaptic plasticity by patient-derived brain tau was rapidly reversed by a single intracerebral injection of different anti-tau monoclonal antibodies, including one directed to a specific human tau amino acid sequence. We conclude that patient-derived LTP-disrupting tau species persist in the brain for weeks, maintaining their neuroactivity often in concert with Aß. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Microtubule-associated protein tau facilitates the targeted killing of proliferating cancer cells in vitro and in a xenograft mouse tumour model in vivo.

BACKGROUND: Antibody drug conjugates (ADCs) and immunotoxins (ITs) are promising anticancer immunotherapeutics. Despite their encouraging performance in clinical trials, both ADCs and ITs often suffer from disadvantages such as stoichiometrically undefined chemical linkage of the cytotoxic payload (ADCs) and the potential immunogenicity of toxins derived from bacteria and plants (ITs). METHODS: Human microtubule-associated protein tau (MAP) was cloned in-frame with human EGF, expressed in E. coli and purified by standard chromatographic methods. The in vitro activity was confirmed by flow cytometry, cell viability assays and tubulin polymerisation assay. The in vivo efficacy was demonstrated using noninvasive far-red in vivo imaging. RESULTS: The EGF-MAP selectively induced apoptosis in EGFR-overexpressing proliferating cancer cells through stabilisation of microtubules. Nonproliferating cells were not affected, demonstrating superior selectivity of EGF-MAP for cancer cells. The EGF-MAP was well tolerated at high doses in mice compared with the ETA'-based control. The in vivo efficacy of EGF-MAP was demonstrated in a tumour xenograft mouse model. CONCLUSION: Our data indicate the general mechanism of action for a new class of human immunotherapeutic reagents suitable for the treatment of cancer. This approach combines the binding specificity of targeting ligands with the selective cytotoxicity of MAP towards proliferating cells.

Naringin Protects against Tau Hyperphosphorylation in Aß 25-35-Injured PC12 Cells through Modulation of ER, PI3K/AKT, and GSK-3ß Signaling Pathways.

Alzheimer's disease (AD) is the most common form of dementia and a significant social and economic burden. Estrogens can exert neuroprotective effects and may contribute to the prevention, attenuation, or even delay in the onset of AD; however, long-term estrogen therapy is associated with harmful side effects. Thus, estrogen alternatives are of interest for countering AD. Naringin, a phytoestrogen, is a key active ingredient in the traditional Chinese medicine Drynaria. Naringin is known to protect against nerve injury induced by amyloid beta-protein (Aß) 25-35, but the underlying mechanisms of this protection are unclear. To investigate the mechanisms of naringin neuroprotection, we observed the protective effect on Aß 25-35-injured C57BL/6J mice's learning and memory ability and hippocampal neurons. Then, an Aß 25-35 injury model was established with adrenal phaeochromocytoma (PC12) cells. We examined the effect of naringin treatment on Aß 25-35-injured PC12 cells and its relationship with estrogen receptor (ER), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and glycogen synthase kinase (GSK)-3ß signaling pathways. Estradiol (E2) was used as a positive control for neuroprotection. Naringin treatment resulted in improved learning and memory ability, the morphology of hippocampal neurons, increased cell viability, and reduced apoptosis. We next examined the expression of ERß, p-AKT (Ser473, Thr308), AKT, p-GSK-3ß (Ser9), GSK-3ß, p-Tau (Thr231, Ser396), and Tau in PC12 cells treated with Aß 25-35 and either naringin or E2, with and without inhibitors of the ER, PI3K/AKT, and GSK-3ß pathways. Our results demonstrated that naringin inhibits Aß 25-35-induced Tau hyperphosphorylation by modulating the ER, PI3K/AKT, and GSK-3ß signaling pathways. Furthermore, the neuroprotective effects of naringin were comparable to those of E2 in all treatment groups. Thus, our results have furthered our understanding of naringin's neuroprotective mechanisms and indicate that naringin may comprise a viable alternative to estrogen therapy.