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.

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.

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.

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.

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.

Ethanol sustains phosphorylated tau protein in the cultured neonatal rat hippocampus: Implications for fetal alcohol spectrum disorders.

Fetal Alcohol Spectrum Disorders (FASDs) are comprised of developmental, behavioral, and cognitive abnormalities caused by prenatal alcohol exposure, affecting an estimated 2%-5% of children and costing $4 billion annually in the United States. While some behavioral therapies help, the neurobiological mechanisms that underpin FASDs need further elucidation for development of effective pharmacotherapeutics. The role of the tau protein in the hippocampus is likely to be involved. Tau catalyzes microtubule polymerization in developing neurons. However, this function can become disrupted by hyperphosphorylation. Many of the cognitive deficits observed in neurodegenerative tauopathies overlap to some degree with what is observed in juvenile developmental disabilities, such as FASDs (e.g., selective memory, executive dysfunction). Thus, tau protein phosphorylation may be one important mechanism of dysfunction in FASDs. The purpose of this study is to provide an empirical basis for a tauopathic characterization of FASDs. To do so, hippocampal slices were extracted from rats at postnatal day 10 (PND10); hippocampal slices were then exposed to 5 days of 50-mM ethanol between 6 days in vitro (DIV) and 11DIV. Immunoblots were taken for Total and p-Tau (Threonine231) at 12DIV and 24DIV. Immunohistochemical fluorescent images were taken for p-Tau (Threonine231) at 12DIV and 24DIV. Separate p-Tau measures were taken for the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG). Total Tau protein expression remained unchanged between 12DIV and 24DIV regardless of ethanol condition. In the control group, longer DIV was associated with decreased p-Tau. However, in the ethanol-exposed group, p-Tau was sustained across DIV. This is the first study to show that ethanol exposure sustains tau Threonine231 phosphorylation in the perinatal hippocampus regardless of Total Tau expression. These findings could lead to innovative pharmacotherapeutic targets for the treatment of cognitive deficits seen in FASDs.

Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1-amino acid-mTORC1 signaling.

BACKGROUND: Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer's disease (AD). This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy. METHODS: The primary hippocampal neurons, N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy, which was analysed by Student's two-tailed t-test. The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1 (mTORC1) activity and the vacuolar H+-ATPase (v-ATPase) activity, respectively, which were analysed by One-way ANOVA with post hoc tests. The Western blotting, co-immunoprecipitation and immunofluorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations, as analysed by Student's two-tailed t-test or One-way ANOVA with post hoc tests. The autophagosome formation was detected by immunofluorescence staining and transmission electron microscopy. The amino acids (AA) levels were detected by high performance liquid chromatography (HPLC). RESULTS: We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits. Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1 (PRD-TIA1) and this association significantly increased the intercellular level of amino acids (Leucine, P = 0.0038; Glutamic acid, P = 0.0348; Alanine, P = 0.0037; Glycine, P = 0.0104), with concordant upregulation of mTORC1 activity [phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1), P < 0.0001; phosphorylated 70 kDa ribosomal protein S6 kinase 1 (p-p70S6K1), P = 0.0001, phosphorylated unc-51-like autophagy-activating kinase 1 (p-ULK1), P = 0.0015] and inhibition of autophagosome formation [microtubule-associated protein light chain 3 II (LC3 II), P = 0.0073; LC3 puncta, P < 0.0001]. As expected, this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation. Importantly, we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1, downregulating the endogenous TIA1 expression by shRNA, or downregulating tau protein level by a small proteolysis targeting chimera (PROTAC) could remarkably attenuate tau-induced autophagy impairment. CONCLUSIONS: Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway, and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treatment and that of related tauopathies.

Blood pressure, antihypertensive drugs, and incident frailty: The Multidomain Alzheimer Preventive Trial (MAPT).

OBJECTIVES: To examine the association of (1) high and low blood pressure (BP) and (2) antihypertensive (AH) drug use with incident frailty. STUDY DESIGN: We conducted a secondary analysis of data from the Multidomain Alzheimer Preventive Trial (MAPT), in which 1394 non-frail community-dwelling participants aged ≥70 years were followed up for 5 years. BP was measured once at baseline in a lying position using a validated electronic device. High BP was defined as systolic BP ≥ 140 mm Hg and/or diastolic BP ≥ 90 mm Hg, and low BP as systolic BP ≤ 110 mm Hg and/or diastolic BP ≤ 70 mm Hg. AH drugs were assessed at baseline and classified according to the Anatomical Therapeutic Chemical (ATC) code. MAIN OUTCOME MEASURES: Incident frailty over the 5 years was assessed using the Fried phenotype. Cox proportional hazards models were used for the analyses. RESULTS: Low BP was associated with a greater risk of frailty (HR = 1.43, 95% CI [1.07-1.92], p = 0.02) after adjustment for age, sex, education, AH drug use, BMI, diabetes, ischemic heart disease, congestive heart failure, AF, stroke, MAPT randomization group, sit-to-stand chair test and pre-frailty. Participants with low BP and those on two or more AH drugs were at the greatest risk of frailty. Neither high BP (HR = 0.84, 95% CI [0.63-1.22], p = 0.24) nor AH drug use (HR = 1.21, 95% CI [0.89-1.64], p = 0.22) was independently associated with incident frailty. CONCLUSIONS: Low BP could be used as a new marker for identifying older adults at higher risk of frailty. CLINICALTRIALS: gov registration number: NCT00672685.

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.

Aggregated Tau-PHF6 (VQIVYK) Potentiates NLRP3 Inflammasome Expression and Autophagy in Human Microglial Cells.

Intra-neuronal misfolding of monomeric tau protein to toxic ß-sheet rich neurofibrillary tangles is a hallmark of Alzheimer's disease (AD). Tau pathology correlates not only with progressive dementia but also with microglia-mediated inflammation in AD. Amyloid-beta (Aß), another pathogenic peptide involved in AD, has been shown to activate NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3), triggering the secretion of proinflammatory interleukin-1ß (IL1ß) and interleukin-18 (IL18). However, the effect of tau protein on microglia concerning inflammasome activation, microglial polarization, and autophagy is poorly understood. In this study, human microglial cells (HMC3) were stimulated with the unaggregated and aggregated forms of the tau-derived PHF6 peptide (VQIVYK). Modulation of NLRP3 inflammasome was examined by qRT-PCR, immunocytochemistry, and Western blot. We demonstrate that fibrillar aggregates of VQIVYK upregulated the NLRP3 expression at both mRNA and protein levels in a dose- and time-dependent manner, leading to increased expression of IL1ß and IL18 in HMC3 cells. Aggregated PHF6-peptide also activated other related inflammation and microglial polarization markers. Furthermore, we also report a time-dependent effect of the aggregated PHF6 on BECN1 (Beclin-1) expression and autophagy. Overall, the PHF6 model system-based study may help to better understand the complex interconnections between Alzheimer's PHF6 peptide aggregation and microglial inflammation, polarization, and autophagy.

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.

Learning Deficits Accompanied by Microglial Proliferation After the Long-Term Post-Injection of Alzheimer's Disease Brain Extract in Mouse Brains.

BACKGROUND: Human tauopathy brain injections into the mouse brain induce the development of tau aggregates, which spread to functionally connected brain regions; however, the features of this neurotoxicity remain unclear. One reason may be short observational periods because previous studies mostly used mutated-tau transgenic mice and needed to complete the study before these mice developed neurofibrillary tangles. OBJECTIVE: To examine whether long-term incubation of Alzheimer's disease (AD) brain in the mouse brain cause functional decline. METHODS: We herein used Tg601 mice, which overexpress wild-type human tau, and non-transgenic littermates (NTg) and injected an insoluble fraction of the AD brain into the unilateral hippocampus. RESULTS: After a long-term (17-19 months) post-injection, mice exhibited learning deficits detected by the Barnes maze test. Aggregated tau pathology in the bilateral hippocampus was more prominent in Tg601 mice than in NTg mice. No significant changes were observed in the number of Neu-N positive cells or astrocytes in the hippocampus, whereas that of Iba-I-positive microglia increased after the AD brain injection. CONCLUSION: These results potentially implicate tau propagation in functional decline and indicate that long-term changes in non-mutated tau mice may reflect human pathological conditions.

Insoluble tau aggregates induce neuronal death through modification of membrane ion conductance, activation of voltage-gated calcium channels and NADPH oxidase.

Most neurodegenerative disorders are associated with aggregation and accumulation of misfolded proteins. One of these proteins, tau, is involved in a number of pathologies including Alzheimer's disease and frontotemporal dementia. Aggregation and phosphorylation of tau have been shown to be a trigger for abnormal signal transduction and disruption of cellular homeostasis. Here, we have studied the effect of extracellular tau at different stages of aggregation in cortical co-cultures of neurons and astrocytes, to understand how this process affects tau pathogenicity. We found that the species formed after prolonged in vitro aggregation of tau (longer than 1 day) are able to stimulate reactive oxygen species (ROS) production through the activation of NADPH oxidase without decreasing the level of the endogenous antioxidant glutathione. The same late insoluble aggregates of tau induced calcium signals in neurons and a gradual increase in the ionic current of artificial membranes. Both tau-induced calcium signals and ROS production in NADPH oxidase were reduced in the presence of the inhibitor of voltage-gated calcium channels (VGCC) nifedipine. This suggests that insoluble aggregates of tau incorporate into the membrane and modify ionic currents, changing plasma membrane potential and activating VGCCs, which induces a calcium influx that triggers ROS production in NADPH oxidase. The combination of all these effects likely leads to toxicity, as only the same insoluble tau aggregates which demonstrated membrane-active properties produced neuronal cell death.

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.

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.

Intracerebral seeding of amyloid-ß and tau pathology in mice: Factors underlying prion-like spreading and comparisons with α-synuclein.

Alzheimer's disease (AD) is characterized neuropathologically by progressive neurodegeneration and by the presence of amyloid plaques and neurofibrillary tangles. These plaques and tangles are composed, respectively, of amyloid-beta (Aß) and tau proteins. While long recognized as hallmarks of AD, it remains unclear what causes the formation of these insoluble deposits. One theory holds that prion-like templated misfolding of Aß and tau induces these proteins to form pathological aggregates, and propagation of this misfolding causes the stereotyped progression of pathology commonly seen in AD. Supporting this theory, numerous studies have been conducted in which aggregated Aß, tau, or α-synuclein is injected intracerebrally into pathology-free host animals, resulting in robust formation of pathology. Here, we review this literature, focusing on in vivo intracerebral seeding of Aß and tau in mice. We compare the results of these experiments to what is known about the seeding and spread of α-synuclein pathology, and we discuss how this research informs our understanding of the factors underlying the onset, progression, and outcomes of proteinaceous pathologies.

Tau-induced mitochondrial membrane perturbation is dependent upon cardiolipin.

Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer's disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity.

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.

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.

Dephosphorylated rather than hyperphosphorylated Tau triggers a pro-inflammatory profile in microglia through the p38 MAPK pathway.

Tauopathies are a broad set of neurodegenerative dementias characterized by the aggregation of Tau protein. Activated microglia and elevated levels of pro-inflammatory molecules are also pathological hallmarks of tauopathies. In these diseases, intracellular Tau is secreted to the extracellular space, where it interacts with other cells, such as neurons and glia, promoting inflammation. However, the mechanism through which extracellular Tau triggers pro-inflammatory responses in microglia remains unknown. Primary microglia cultures were treated with extracellular Tau in its hyperphosphorylated, dephosphorylated or non-phosphorylated form. Protein cytokine arrays, real-time PCR, inhibition of the p38 MAPK pathway, phosphatase assays, and quantification of proteins through immunoblotting were used to analyze the effect of extracellular Tau on the pro-inflammatory response of microglia. The main finding of this work is that extracellular non-phosphorylated and dephosphorylated forms of Tau, rather than hyperphosphorylated Tau, activate the p38 MAPK pathway in microglia, thus triggering a pro-inflammatory response in these cells.