Tau aggregates improve the Purinergic receptor P2Y12-associated podosome rearrangements in microglial cells

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is associated with protein misfolding, plaque accumulation, neuronal dysfunction, synaptic loss, and cognitive decline. The pathological cascade of AD includes the intracellular Tau hyperphosphorylation and its subsequent aggregation, extracellular Amyloid-β plaque formation and microglia-mediated neuroinflammation. The extracellular release of aggregated Tau is sensed by surveilling microglia through the involvement of various cell surface receptors. Among all, purinergic P2Y12R signaling is involved in microglial chemotaxis towards the damaged neurons. Microglial migration is highly linked with membrane-associated actin remodeling leading to the phagocytosis of extracellular Tau species. Here, we studied the formation of various actin structures such as podosome, lamellipodia and filopodia, in response to extracellular Tau monomers and aggregates. Microglial podosomes are colocalized with actin nucleator protein WASP, Arp2 and TKS5 adaptor protein during Tau-mediated migration. Moreover, the P2Y12 receptors were associated with F-actin-rich podosome structures, which signify the potential of Tau aggregates in microglial chemotaxis through the involvement of actin remodeling.     

Exploration of multi‐target effects of 3‐benzoyl‐5‐hydroxychromen‐2‐one in Alzheimer’s disease cell and mouse models

Microtubule‐associated protein Tau, abundant in the central nervous system (CNS), plays crucial roles in microtubule assembly and stabilization. Abnormal Tau phosphorylation and aggregation are a common pathogenic hallmark in Alzheimer's disease (AD). Hyperphosphorylation of Tau could change its conformation and result in self‐aggregation, increased oxidative stress, and neuronal death. In this study, we examined the potential of licochalcone A (a natural chalcone) and five synthetic derivatives (LM compounds) for inhibiting Tau misfolding, scavenging reactive oxygen species (ROS) and providing neuroprotection in human cells expressing proaggregant ΔK280 Tau_RD‐DsRed. All test compounds were soluble up to 100 μM in cell culture media and predicted to be orally bioavailable and CNS‐active. Among them, licochalcone A and LM‐031 markedly reduced Tau misfolding and associated ROS, promoted neurite outgrowth, and inhibited caspase 3 activity in ΔK280 Tau_RD‐DsRed 293 and SH‐SY5Y cells. Mechanistic studies showed that LM‐031 upregulates HSPB1 chaperone, NRF2/NQO1/GCLC pathway, and CREB‐dependent BDNF/AKT/ERK/BCL2 pathway in ΔK280 Tau_RD‐DsRed SH‐SY5Y cells. Decreased neurite outgrowth upon induction of ΔK280 Tau_RD‐DsRed was rescued by LM‐031, which was counteracted by knockdown of NRF2 or CREB. LM‐031 further rescued the downregulated NRF2 and pCREB, reduced Aβ and Tau levels in hippocampus and cortex, and ameliorated cognitive deficits in streptozocin‐induced hyperglycemic 3 × Tg‐AD mice. Our findings strongly indicate the potential of LM‐031 for modifying AD progression by targeting HSPB1 to reduce Tau misfolding and activating NRF2 and CREB pathways to suppress apoptosis and promote neuron survival, thereby offering a new drug development avenue for AD treatment.     

Low Micromolar Zinc Accelerates the Fibrillization of Human Tau via Bridging of Cys-291 and Cys-322

A hallmark of a group of neurodegenerative diseases such as Alzheimer disease is the formation of neurofibrillary tangles, which are principally composed of bundles of filaments formed by microtubule-associated protein Tau. Clarifying how natively unstructured Tau protein forms abnormal aggregates is of central importance for elucidating the etiology of these diseases. There is considerable evidence showing that zinc, as an essential element that is highly concentrated in brain, is linked to the development or progression of these diseases. Herein, by using recombinant human Tau fragment Tau_244–372 and its mutants, we have investigated the effect of zinc on the aggregation of Tau. Low micromolar concentrations of Zn²⁺ dramatically accelerate fibril formation of wild-type Tau_244–372 under reducing conditions, compared with no Zn²⁺. Higher concentrations of Zn²⁺, however, induce wild-type Tau_244–372 to form granular aggregates in reducing conditions. Moreover, these non-fibrillar aggregates assemble into mature Tau filaments when Zn²⁺ has been chelated by EDTA. Unlike wild-type Tau_244–372, low micromolar concentrations of Zn²⁺ have no obvious effects on fibrillization kinetics of single mutants C291A and C322A and double mutant C291A/C322A under reducing conditions. The results from isothermal titration calorimetry show that one Zn²⁺ binds to one Tau molecule via tetrahedral coordination to Cys-291 and Cys-322 as well as two histidines, with moderate, micromolar affinity. Our data demonstrate that low micromolar zinc accelerates the fibrillization of human Tau protein via bridging Cys-291 and Cys-322 in physiological reducing conditions, providing clues to understanding the relationship between zinc dyshomeostasis and the etiology of neurodegenerative diseases.     

Clearance and deposition of extracellular alpha-synuclein aggregates in microglia

Abnormal deposition of α-synuclein in neurons and glia is implicated in many neurological diseases, such as Parkinson’s disease and Dementia with Lewy bodies. Recently, evidence has emerged that this protein and its aggregates are secreted from neuronal cells, and this extracellular protein may contribute to the pathogenic process. Here, we show that all the major brain cell types (neurons, astrocytes, and microglia) are capable of clearing the extracellular α-synuclein aggregates by internalization and degradation. Among these cell types, microglia showed the highest rate of degradation. Upon activation by lipopolysaccharide, the degradation of the internalized α-synuclein aggregates was slowed, causing protein accumulation in the microglial cytoplasm. These results suggest that microglia may be the major scavenger cells for extracellular α-synuclein aggregates in brain parenchyma, and that clearance may be regulated by the activation state of these cells.     

Tau deletion impairs intracellular β-amyloid-42 clearance and leads to more extracellular plaque deposition in gene transfer models

Background

Tau is an axonal protein that binds to and regulates microtubule function. Hyper-phosphorylation of Tau reduces its binding to microtubules and it is associated with β-amyloid deposition in Alzheimer’s disease. Paradoxically, Tau reduction may prevent β-amyloid pathology, raising the possibility that Tau mediates intracellular Aβ clearance. The current studies investigated the role of Tau in autophagic and proteasomal intracellular Aβ1-42 clearance and the subsequent effect on plaque deposition.

Results

Tau deletion impaired Aβ clearance via autophagy, but not the proteasome, while introduction of wild type human Tau into Tau^?/? mice partially restored autophagic clearance of Aβ1-42, suggesting that exogenous Tau expression can support autophagic Aβ1-42 clearance. Tau deletion impaired autophagic flux and resulted in Aβ1-42 accumulation in pre-lysosomal autophagic vacuoles, affecting Aβ1-42 deposition into the lysosome. This autophagic defect was associated with decreased intracellular Aβ1-42 and increased plaque load in Tau^?/? mice, which displayed less cell death. Nilotinib, an Abl tyrosine kinase inhibitor that promotes autophagic clearance mechanisms, reduced Aβ1-42 only when exogenous human Tau was expressed in Tau^?/? mice.

Conclusions

These studies demonstrate that Tau deletion affects intracellular Aβ1-42 clearance, leading to extracellular plaque.

     

Novel function of Tau in regulating the effects of external stimuli on adult hippocampal neurogenesis

Tau is a microtubule‐associated neuronal protein found mainly in axons. However, its presence in dendrites and dendritic spines is particularly relevant due to its involvement in synaptic plasticity and neurodegeneration. Here, we show that Tau plays a novel in vivo role in the morphological and synaptic maturation of newborn hippocampal granule neurons under basal conditions. Furthermore, we reveal that Tau is involved in the selective cell death of immature granule neurons caused by acute stress. Also, Tau deficiency protects newborn neurons from the stress‐induced dendritic atrophy and loss of postsynaptic densities (PSDs). Strikingly, we also demonstrate that Tau regulates the increase in newborn neuron survival triggered by environmental enrichment (EE). Moreover, newborn granule neurons from Tau^?/? mice did not show any stimulatory effect of EE on dendritic development or on PSD generation. Thus, our data demonstrate that Tau^?/? mice show impairments in the maturation of newborn granule neurons under basal conditions and that they are insensitive to the modulation of adult hippocampal neurogenesis exerted by both stimulatory and detrimental stimuli.     

Adenosine A3 receptor is involved in ADP-induced microglial process extension and migration

The extension of microglial processes toward injured sites in the brain is triggered by the stimulation of the purinergic receptor P2Y(12) by extracellular ATP. We recently showed that P2Y(12) stimulation by ATP induces microglial process extension in collagen gels. In the present study, we found that a P2Y(12) agonist, 2-methylthio-ADP (2MeSADP), failed to induce the process extension of microglia in collagen gels and that co-stimulation with adenosine, a phosphohydrolytic derivative of ATP, and 2MeSADP restored the chemotactic process extension. An adenosine A3 receptor (A3R)-selective agonist restored the chemotactic process extension, but other receptor subtype agonists did not. The removal of adenosine by adenosine deaminase and the blocking of A3R by an A3R-selective antagonist inhibited ADP-induced process extension. The A3R antagonist inhibited ADP-induced microglial migration, and an A3R agonist promoted 2MeSADP-stimulated migration. ADP and the A3R agonist activated Jun N-terminal kinase in microglia, and a Jun N-terminal kinase inhibitor inhibited the ADP-induced process extension. An RT-PCR analysis showed that A1R and A3R were expressed by microglia sorted from adult rat brains and that the A2AR expression level was very low. These results suggested that A3R signaling may be involved in the ADP-induced process extension and migration of microglia.     

UDP Facilitates Microglial Phagocytosis Through P2Y6 Receptors

Microglia engage in the clearance of dead cells or dangerous debris. When neighboring cells are injured, the cells release or leak ATP into extracellular space and microglia rapidly move toward or extend a process to the nucleotides as chemotaxis through P2Y12 receptors. In the meanwhile, microglia express the metabotropic P2Y6 receptors, the activation of which by uridine 5′-diphosphate (UDP) triggers microglial phagocytosis in a concentration-dependent fashion. UDP/UTP was leaked when hippocampal neurons were damaged by kainic acid in vivo and in vitro. Systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in mRNA for P2Y6 receptors in activated microglia. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and would function as a sensor for phagocytosis by sensing diffusible UDP signals.Key Words: microglia, phagocytosis, P2Y6 receptors, UDPAccumulating findings indicate that nucleotides play an important role in neuron to glia communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y) (Fig. 1). P2X receptors (seven types; P2X₁-P2X₇) contain intrinsic pores that open by binding with ATP. P2Y (eight types; P2Y_{1,2,4,6 and 11–14}) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins.¹ Microglia express P2X₄, P2X₇, P2Y₂, P2Y₆ and P2Y₁₂¹ and are known as resident macrophages in CNS, accounting for 5–10% of the total population of glia.^2,3 When neurons are injured or dead, microglia are activated, resulting in their interaction with immune cells, active migration to the site of injury, release of pro-inflammatory substances and the phagocytosis of damaged cells or debris. For such activation of microglial motilities, extracellular nucleotides have a central role. Extracellular ATP functions as a chemoattractant. Microglial chemotaxis by ATP via P2Y₁₂ receptors was originally found by Honda et al.,⁴ and has recently been confirmed in vivo in P2Y₁₂ receptor knockout animals.⁵ Neuronal injury results in the release or leakage of ATP that appears to be a “find-me” signal from damaged neurons to microglia to cause chemotaxis. In addition to microglial migration by ATP, another nucleotide, UDP, an endogenous agonist of the P2Y₆ receptor, greatly activates the motility of microglia and orders microglia to engulf damaged neurons.⁶Open in a separate windowFigure 1P2 purinergic receptors (ATP receptors).Phagocytosis is a specialized form of endocytosis taking relatively large particles (> 1.0 µm) into vacuoles and has a central role in tissue remodeling, inflammation and the defense against infectious agents.⁷ Phagocytosis is initiated by the activation of cell-surface phagocytosis receptors, including Fc receptors, complement receptors, integrins, endotoxin receptors (CD18, CD14), mannose receptors and scavenger receptors⁸ which are activated by corresponding extracellular ligands called as “eat-me” signals. Since recognition is the most important step for phagocytosis, extensive studies on phagocytosis receptors have been reported. With regard to apoptotic cells, it is well known that dying cells express so called “eat-me” signals such as phosphatidylserine (PS) on their surface membrane,⁸ by which microglia recognize the apoptotic cells in order to catch and remove them.⁸ As for amyloid β protein (Aβ), a key molecule that mediates Alzheimer''s disease, microglia remove Aβ presumably via Fc receptor-dependent phagocytosis.^9,10 It, however, is unclear how phagocytotic cells come to the target cells or debris. Our findings suggest that nucleotides might be the molecules to guide phagocytotic cells to the targets.We found that exogenously applied UDP caused microglial phagocytosis through P2Y₆ in a concentration-dependent manner, and that neuronal injury caused by kainic acid (KA) upregulated P2Y₆ receptors in microglia, the KA evoked neuronal injury resulted in an increase in extracellular UTP, which was immediately metabolized into UDP in vivo and in vitro. We also found that UDP leaked from injured neurons caused P2Y₆ receptor-dependent phagocytosis in vivo and in vitro. Thus, UDP could be a diffusible molecule that signals the crisis of damaged neurons to microglia, triggering phagocytosis. Nucleotides seem to have the ability to act as “eat-us” signals for necrotic cells suffering traumatic or ischemic injury because such necrotic cells cause swelling, followed by shrinkage, leading to the leakage of cytoplasmic molecules including a large amount of ATP and UTP and extracellular nucleotides are immediately degraded by ecto-nucleotideases, suggesting that leaked nucleotides could be transient and localized signals that alert to the crisis created by the presence of the necrotic cells. These findings suggest that microglia might be attracted by ATP/ADP^4,5,11,12 and subsequently recognize UDP, starting to recognize “eat-me” signals attached to the targets and engulf them (Fig. 2). It is interesting that ATP/ADP is not able to efficiently activate P2Y₆ receptors, nor can UDP act on P2Y₁₂ receptors. Thus, adenine and uridine nucleotides would regulate microglial motilities, i.e. chemotaxis and phagocytosis, in a coordinated fashion.Open in a separate windowFigure 2Illustration of nucleotide-activated microglial chemotaxix and phagocytosis. Activated microglia might be attracted by ATP/ADP is not able to efficiently activate P2Y6 receptors, nor ca UDP act on P2Y12 receptors.     

Nucleotides released from Aβ₁₋₄₂ -treated microglial cells increase cell migration and Aβ₁₋₄₂ uptake through P2Y₂ receptor activation

Amyloid β-protein (Aβ) deposits in brains of Alzheimer's disease patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aβ. Nucleotides released from apoptotic cells activate P2Y(2) receptors (P2Y(2) Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y(2) Rs in the phagocytosis and clearance of Aβ. Treatment of mouse primary microglial cells with fibrillar (fAβ(1-42) ) and oligomeric (oAβ(1-42) ) Aβ(1-42) aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAβ(1-42) and oAβ(1-42) treatment for 24 h caused an increase in P2Y(2) R gene expression. Treatment with fAβ(1-42) and oAβ(1-42) aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y(2) R agonists ATP and UTP caused significant uptake of Aβ(1-42) by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aβ(1-42) uptake by primary microglial cells isolated from P2Y(2) R(-/-) mice. Inhibitors of α(v) integrins, Src and Rac decreased UTP-induced Aβ(1-42) uptake, suggesting that these previously identified components of the P2Y(2) R signaling pathway play a role in Aβ phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aβ(1-42) degradation by microglial cells, but not in cells isolated from P2Y(2) R(-/-) mice. Taken together, our findings suggest that P2Y(2) Rs can activate microglial cells to enhance Aβ clearance and highlight the P2Y(2) R as a therapeutic target in Alzheimer's disease.     

Protein Disulfide Isomerase Interacts with Tau Protein and Inhibits Its Fibrillization

Background

Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI) is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization.

Methodology/Principal Findings

As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau_244–372) monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau_244–372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau_244–372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau_244–372 fibrillization more strongly than full-length human PDI.

Conclusions/Significance

We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau fibrillization, and have applications for developing novel strategies for treatment and early diagnosis of Alzheimer disease.     

Antibody Uptake into Neurons Occurs Primarily via Clathrin-dependent Fcγ Receptor Endocytosis and Is a Prerequisite for Acute Tau Protein Clearance

Tau immunotherapy is effective in transgenic mice, but the mechanisms of Tau clearance are not well known. To this end, Tau antibody uptake was analyzed in brain slice cultures and primary neurons. Internalization was rapid (<1 h), saturable, and substantial compared with control mouse IgG. Furthermore, temperature reduction to 4 °C, an excess of unlabeled mouse IgG, or an excess of Tau antibodies reduced uptake in slices by 63, 41, and 62%, respectively (p = 0.002, 0.04, and 0.005). Uptake strongly correlated with total and insoluble Tau levels (r² = 0.77 and 0.87 and p = 0.002 and 0.0002), suggesting that Tau aggregates influence antibody internalization and/or retention within neurons. Inhibiting phagocytosis did not reduce uptake in slices or neuronal cultures, indicating limited microglial involvement. In contrast, clathrin-specific inhibitors reduced uptake in neurons (≤78%, p < 0.0001) and slices (≤35%, p = 0.03), demonstrating receptor-mediated endocytosis as the primary uptake pathway. Fluid phase endocytosis accounted for the remainder of antibody uptake in primary neurons, based on co-staining with internalized dextran. The receptor-mediated uptake is to a large extent via low affinity FcγII/III receptors and can be blocked in slices (43%, p = 0.04) and neurons (53%, p = 0.008) with an antibody against these receptors. Importantly, antibody internalization appears to be necessary for Tau reduction in primary neurons. Overall, these findings clarify that Tau antibody uptake is primarily receptor-mediated, that these antibodies are mainly found in neurons with Tau aggregates, and that their intracellular interaction leads to clearance of Tau pathology, all of which have major implications for therapeutic development of this approach.     

Phosphorylation of Tau at Thr212, Thr231, and Ser262 Combined Causes Neurodegeneration

Abnormal hyperphosphorylation of the microtubule-associated protein Tau is a hallmark of Alzheimer disease and related diseases called tauopathies. As yet, the exact mechanism by which this pathology causes neurodegeneration is not understood. The present study provides direct evidence that Tau abnormal hyperphosphorylation causes its aggregation, breakdown of the microtubule network, and cell death and identifies phosphorylation sites involved in neurotoxicity. We generated pseudophosphorylated Tau proteins by mutating Ser/Thr to Glu and, as controls, to Ala. These mutations involved one, two, or three pathological phosphorylation sites by site-directed mutagenesis using as backbones the wild type or FTDP-17 mutant R406W Tau. Pseudophosphorylated and corresponding control Tau proteins were expressed transiently in PC12 and CHO cells. We found that a single phosphorylation site alone had little influence on the biological activity of Tau, except Thr²¹², which, upon mutation to Glu in the R406W background, induced Tau aggregation in cells, suggesting phosphorylation at this site along with a modification on the C-terminal of the protein facilitates self-assembly of Tau. The expression of R406W Tau pseudophosphorylated at Thr²¹², Thr²³¹, and Ser²⁶² triggered caspase-3 activation in as much as 85% of the transfected cells, whereas the corresponding value for wild type pseudophosphorylated Tau was 30%. Cells transfected with pseudophosphorylated Tau became TUNEL-positive.     

Propagation of Tau Misfolding from the Outside to the Inside of a Cell

Tauopathies are neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau in neurons and glia. Although Tau is normally considered an intracellular protein, Tau aggregates are observed in the extracellular space, and Tau peptide is readily detected in the cerebrospinal fluid of patients. Tau aggregation occurs in many diseases, including Alzheimer disease and frontotemporal dementia. Tau pathology begins in discrete, disease-specific regions but eventually involves much larger areas of the brain. It is unknown how this propagation of Tau misfolding occurs. We hypothesize that extracellular Tau aggregates can transmit a misfolded state from the outside to the inside of a cell, similar to prions. Here we show that extracellular Tau aggregates, but not monomer, are taken up by cultured cells. Internalized Tau aggregates displace tubulin, co-localize with dextran, a marker of fluid-phase endocytosis, and induce fibrillization of intracellular full-length Tau. These intracellular fibrils are competent to seed fibril formation of recombinant Tau monomer in vitro. Finally, we observed that newly aggregated intracellular Tau transfers between co-cultured cells. Our data indicate that Tau aggregates can propagate a fibrillar, misfolded state from the outside to the inside of a cell. This may have important implications for understanding how protein misfolding spreads through the brains of tauopathy patients, and it is potentially relevant to myriad neurodegenerative diseases associated with protein misfolding.Tau filament deposition in Alzheimer disease (AD),² frontotemporal dementia (FTD), and other tauopathies correlates closely with cognitive dysfunction and cell death (1). Mutations in the tau gene cause autosomal dominant tauopathy, implicating Tau as the proximal cause (2–4). Specific disease phenotypes are defined by the early sites of pathology. For example, AD is characterized by memory loss that derives from involvement of hippocampal neurons, whereas FTD is characterized by personality changes that result from frontal lobe involvement (5). Pathology ultimately spreads to involve much larger regions of brain. Studies on patients with AD show a progressive, stereotyped spread of Tau deposits from the transentorhinal cortex to the hippocampus, and eventually to most cortical areas (6–8). Others have correlated the distribution of neurofibrillary tangles of Tau in AD brains with trans-synaptic distance from the affected areas (9). A similar spread affecting different subsets of neurons has been observed in other sporadic tauopathies, such as progressive supranuclear palsy (10). It is unknown why Tau misfolding progresses through the brain, whether it is a sequence of cell autonomous processes or whether a toxic factor is involved. Loss of synaptic connections and cell death may expose healthy cells to toxic factors and decrease available neurotrophins (11, 12). Another possibility is that the Tau protein itself serves as the agent of trans-cellular propagation. For example, it has been shown that extracellular Tau is toxic to cultured neuronal cells (13, 14). This is consistent with the observation that immunotherapy against Tau reduces pathology in a mouse model (15).Tau is well known as an intracellular protein that stabilizes microtubule filaments (16); however, it is readily detected in cerebrospinal fluid (17) and as extracellular aggregates, termed “ghost tangles,” in diseased brain. These are comprised predominantly of the microtubule-binding region (MTBR), the functional and pathogenic core of the Tau protein (18). We hypothesize that Tau aggregates present in the extracellular space enter naive cells and induce misfolding of intracellular Tau. We have tested this idea using cellular studies, biochemistry, and atomic force microscopy (AFM).     

P2Y6 receptor-dependent microglial phagocytosis of synapses mediates synaptic and memory loss in aging

Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y₆ receptor (P2Y₆R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y₆R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y₆R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal–glial co-cultures. In vivo, wild-type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging-induced synaptic loss and internalization were absent in P2Y₆R knockout mice, and these mice also lacked any aging-induced memory loss. Thus, P2Y₆R appears to mediate aging-induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y₆R has the potential to prevent age-associated memory impairment.     

Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP‐evoked production of interleukin‐6 (IL‐6) and its intracellular signals were examined using a mouse microglial cell line, MG‐5. ATP, but not its metabolites, produced IL‐6 in a concentration‐dependent manner. Although ATP activated two mitogen‐activated protein kinases, i.e. p38 and extracellular signal‐regulated protein kinase, only p38 was involved in the IL‐6 induction. However, the activation of p38 was not sufficient for the IL‐6 induction because 2′‐ and 3′‐O‐(4‐benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL‐6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL‐6 production by the cells. The ATP‐evoked IL‐6 production was attenuated by Gö6976, an inhibitor of Ca²⁺‐dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX‐insensitive P2Y receptors activates p38 and Ca²⁺‐dependent PKC, thereby resulting in the mRNA expression and release of IL‐6 in MG‐5. This is a novel pathway for the induction of cytokines in microglia.     

Anti-aggregant tau mutant promotes neurogenesis

Background

The microtubule-associated protein Tau plays a role in neurodegeneration as well as neurogenesis. Previous work has shown that the expression of the pro-aggregant mutant Tau repeat domain causes strong aggregation and pronounced neuronal loss in the hippocampus whereas the anti-aggregant form has no deleterious effects. These two proteins differ mainly in their propensity to form ß structure and hence to aggregate.

Methods

To elucidate the basis of these contrasting effects, we analyzed organotypic hippocampal slice cultures (OHSCs) from transgenic mice expressing the repeat domain (RD) of Tau with the anti-aggregant mutation (Tau^RDΔKPP) and compared them with slices containing pro-aggregant Tau^RDΔK. Transgene expression in the hippocampus was monitored via a sensitive bioluminescence reporter gene assay (luciferase).

Results

The expression of the anti-aggregant Tau^RDΔKPP leads to a larger volume of the hippocampus at a young age due to enhanced neurogenesis, resulting in an increase in neuronal number. There were no signs of activation of microglia and astrocytes, indicating the absence of an inflammatory reaction. Investigation of signaling pathways showed that Wnt-5a was strongly decreased whereas Wnt3 was increased. A pronounced increase in hippocampal stem cell proliferation (seen by BrdU) was observed as early as P8, in the CA regions where neurogenesis is normally not observed. The increase in neurons persisted up to 16 months of age.

Conclusion

The data suggest that the expression of anti-aggregant Tau^RDΔKPP enhances hippocampal neurogenesis mediated by the canonical Wnt signaling pathway, without an inflammatory reaction. This study points to a role of tau in brain development and neurogenesis, in contrast to its detrimental role in neurodegeneration at later age.

     

Extracellular Tau Levels Are Influenced by Variability in Tau That Is Associated with Tauopathies

Tauopathies are a class of neurodegenerative diseases marked by intracellular aggregates of hyperphosphorylated Tau. These diseases may occur by sporadic mechanisms in which genetic variants represent risk factors for disease, as is the case in Alzheimer disease (AD). In AD, cerebrospinal fluid (CSF) levels of soluble Tau/pTau-181 are higher in cases compared with controls. A subset of frontotemporal dementia (FTD) cases occur by a familial mechanism in which MAPT, the gene that encodes Tau, mutations are dominantly inherited. In symptomatic FTD patients expressing a MAPT mutation, CSF Tau levels are slightly elevated but are significantly lower than in AD patients. We sought to model CSF Tau changes by measuring extracellular Tau in cultured cells. Full-length, monomeric extracellular total Tau and pTau-181 were detectable in human neuroblastoma cells expressing endogenous Tau, in human non-neuronal cells overexpressing wild-type Tau, and in mouse cortical neurons. Tau isoforms influence the rate of Tau release, whereby the N terminus (exons 2/3) and microtubule binding repeat length contribute to Tau release from the cell. Compared with cells overexpressing wild-type Tau, cells overexpressing FTD-associated MAPT mutations produce significantly less extracellular total Tau without altering intracellular total Tau levels. This study demonstrates that cells actively release Tau in the absence of disease or toxicity, and Tau release is modified by changes in the Tau protein that are associated with tauopathies.     

Surface masking shapes the traffic of the neuropeptide Y Y2 receptor

The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.     

The Rate of Tau Synthesis Is Differentially Regulated During Postnatal Development in Mouse Cerebellum

1. Tau, which is a microtubule-associated protein, with mRNA targeted to the axon and growth cone, is involved in axonal elongation. During postnatal development in mouse, Tau expression in cerebellar granule cells is reduced after the second postnatal week. The aim of this work was to study the regulation of the rate of the synthesis of Tau protein during the period of granule cell axonal growth in mouse cerebellum.2. We found four [³⁵S]methionine-labeled isoforms of Tau synthesized postnataly. Their levels remain constant from postnatal day 9 to 12 (P9–P12), and decreased by P20.3. The rate of Tau synthesis showed differences with the rate of synthesis of total proteins. They also differ from proteins phosphatases 2A and 2B, both associated with the regulation of Tau function. In addition, the turnover of newly synthesized Tau increased at P20, compared with P9 and P12.4. These results imply a specific developmental regulation of mRNA translation of Tau, and indicate that, after the period of synapse formation is complete, and therefore axonal growth has finished (P20), only a limited number of new Tau molecules are synthesized. This might reflect that, after synapse formation is complete, newly synthesized Tau molecules are not longer needed.     

Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.