FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2290636701> ?p ?o ?g. }

• W2290636701 endingPage "569" @default.
• W2290636701 startingPage "552" @default.
• W2290636701 abstract "Article1 March 2016free access Transparent process The Tau/A152T mutation, a risk factor for frontotemporal-spectrum disorders, leads to NR2B receptor-mediated excitotoxicity Jochen Martin Decker German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Lars Krüger German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Astrid Sydow German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck-Institute for Metabolism Research (Cologne), Hamburg Outstation, Hamburg, Germany Search for more papers by this author Frank JA Dennissen German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Zuzana Siskova German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Eckhard Mandelkow German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck-Institute for Metabolism Research (Cologne), Hamburg Outstation, Hamburg, Germany Caesar Research Center, Bonn, Germany Search for more papers by this author Eva-Maria Mandelkow Corresponding Author German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck-Institute for Metabolism Research (Cologne), Hamburg Outstation, Hamburg, Germany Caesar Research Center, Bonn, Germany Search for more papers by this author Jochen Martin Decker German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Lars Krüger German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Astrid Sydow German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck-Institute for Metabolism Research (Cologne), Hamburg Outstation, Hamburg, Germany Search for more papers by this author Frank JA Dennissen German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Zuzana Siskova German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Eckhard Mandelkow German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck-Institute for Metabolism Research (Cologne), Hamburg Outstation, Hamburg, Germany Caesar Research Center, Bonn, Germany Search for more papers by this author Eva-Maria Mandelkow Corresponding Author German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck-Institute for Metabolism Research (Cologne), Hamburg Outstation, Hamburg, Germany Caesar Research Center, Bonn, Germany Search for more papers by this author Author Information Jochen Martin Decker1,‡, Lars Krüger1,‡, Astrid Sydow1,2, Frank JA Dennissen1, Zuzana Siskova1, Eckhard Mandelkow1,2,3 and Eva-Maria Mandelkow 1,2,3 1German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 2Max-Planck-Institute for Metabolism Research (Cologne), Hamburg Outstation, Hamburg, Germany 3Caesar Research Center, Bonn, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 2284 3302630; E-mail: [email protected] EMBO Rep (2016)17:552-569https://doi.org/10.15252/embr.201541439 See also: S Maeda et al PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract We report on a novel transgenic mouse model expressing human full-length Tau with the Tau mutation A152T (hTauAT), a risk factor for FTD-spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis-sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short- or long-term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage-gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTauAT causes excitotoxicity mediated by NR2B-containing NMDA receptors due to enhanced extracellular glutamate. Synopsis A mouse model of A152T-variant human Tau, a risk factor for frontotemporal dementia spectrum disorders, suggests that neuronal excitotoxicity, leading to neuronal death, contributes to pathogenesis. The expression of Tau/A152T leads to Tau pathology in the form of conformational changes, hyperphosphorylation, mis-sorting, aggregation, neurodegeneration, and neuronal loss in the hippocampus of transgenic mice. The pathophysiology of Tau/A152T includes enhanced presynaptic neurotransmitter release, leading to increased extracellular glutamate, activation of extrasynaptic (NR2B containing) NMDA receptors, elevated postsynaptic calcium, and enhanced epileptiform activity and results in axonal sprouting in the hippocampal CA3 region. Pathological effects of Tau/A152T can be ameliorated by stimulating glutamate uptake by astrocytic glutamate transporters. Introduction Familial tauopathies including FTDP-17 are often caused by mutations in the Tau gene (MAPT), which particularly occur in Tau's microtubule-binding repeat region 1. MAPT mutations can lead to a partial loss of function due to a reduction in Tau's microtubule-binding capability or to a gain of toxic function by increasing the tendency of Tau to aggregate into filaments 23. By contrast, the rare Tau p.A152T substitution is located outside the microtubule-binding domain in the N-terminal half of Tau. It has emerged as a risk factor for both frontotemporal dementias (FTDs) and Alzheimer's disease (AD), including progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) 4567. The mutation decreases Tau's binding to microtubules and increases the formation of Tau oligomers in vitro 7. However, the consequences for neuronal function and the exact course of pathogenesis still remain elusive. We therefore generated a transgenic mouse line expressing the Tau-A152T mutation and characterized its pathology. In healthy conditions, Tau is localized mainly in axons, stabilizes microtubules, and supports axonal transport processes 89. Phosphorylation and detachment of Tau from microtubules following subsequent mis-sorting into cell bodies and dendrites may provoke axonal transport alterations 1011, which in turn may disturb physiological conditions at postsynapses 1213. Presynaptic physiological disturbances in Tau models have been reported as well 1415. Phosphorylated Tau within dendritic spines has been proposed to impair synaptic transmission by decreasing the densities of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors 12 and causes calcium dysregulation 16. Furthermore, dendritic Tau is thought to mediate Aβ-induced excitotoxicity and memory deficits in APP transgenic mice 1317. Tau plays a role in neuronal excitability and is required for Aβ and other excitotoxins to execute their degenerative effect, such as aberrant network activity and cognitive decline 1318. In primary neurons prepared from mice expressing mutant human APP tau allows Aβ oligomers to inhibit axonal transport through activation of GSK3β 19. Genetic ablation of Tau decreases spontaneous seizures in APP transgenic mice and even in a non-AD epilepsy model 2021. Tauopathies are characterized by pathological aggregation of Tau and massive cell loss in particular brain regions. In the case of PSP, this occurs in the basal ganglia and brain stem 22 but also in the hippocampus, which may be one factor contributing to cognitive decline. The molecular mechanisms of Tau-mediated neurotoxicity are not well understood and may be diverse. One suggested mechanism in AD is glutamate-mediated excitotoxicity which may lead to the stimulation of extrasynaptic NMDARs (containing NR2B-subunits) and promote Tau-dependent cell death 23. Recently, impairments in glutamate metabolism were also reported in the hippocampus of transgenic mice overexpressing Tau-P301L 2425. When characterizing the new Tau A152T substitution (hTauAT) mouse model by biochemistry and immunohistochemistry, we noticed that the most prominent Tau pathology occurred in the mossy fiber pathway of the CA3 region. We therefore analyzed this region in acute and organotypic slices for structural and functional abnormalities. This revealed a distinct, excitotoxic phenotype which caused an increase in extracellular glutamate, a rise in intracellular calcium, combined with epileptiform activity and mossy fiber sprouting in the hippocampus. This was reminiscent of the situation in PSP patients, where increased seizure susceptibility was reported 26. The effects were consistent with an overactivation of extrasynaptic NMDA receptors and could be prevented by pharmacological treatments stimulating the uptake of glutamate by astrocytic glutamate transporters (EAAT2/GLT1). These results agree well with observations from another mouse model based on the same mutation 27, which shows age-dependent neuronal loss, cognitive impairments, and spontaneous nonconvulsive epileptiform activity. Results Generation and phenotypic characterization of hTauAT mice We generated a novel transgenic mouse model (hTauAT) expressing human full-length Tau (hTau40, 2N4R) with the PSP-related point mutation p.A152T (Fig 1A). The transgene is located in the ROSA26 locus 28 and its expression is controlled by the neuron-specific Thy1.2 promoter. This results in moderate overexpression levels in the hippocampus of heterozygous (+/−) and homozygous (+/+) hTauAT mice at an age of 14 months (Fig 1B). The molecular ratios of mutant hTauAT to endogenous mouse Tau protein in the hippocampus are ~1:1 (heterozygous) and 1.8:1 (homozygous mice) (Fig 1C). The hTauAT mRNA levels in transgenic heterozygous (1.00 ± 0.35 A.U.) and homozygous (2.19 ± 0.27 A.U.) mice (16 month) were in good agreement with the hTau/mTau protein expression ratios in hippocampi from transgenic animals (compare Fig 1C to D). Immunohistological analysis with the human Tau-specific antibody HT7 confirmed the higher hTauAT expression in the homozygous hTauAT mice (Fig 1E). Both types of mice show intense staining of the CA3-mossy fibers (Fig 1E, asterisks) and mis-sorting of the human TauAT into the somato-dendritic compartment, a typical early feature of AD (Fig 1E, arrowheads). The hTauAT-expressing mice show hyperphosphorylated Tau (by antibodies pT217, AT180 (pSer235, pSer202), PHF1 (pSer396/pSer404), and AT8 (pSer202/pThr205); Fig 1F–I, respectively) and a pathological Tau conformation (antibody Alz-50, Fig 2A). Transgenic Tau is prominent in the axonal mossy fibers of the CA3 region (Fig 1E and H, asterisks) and is mis-sorted into the cell bodies of hippocampal pyramidal neurons (Fig 1E–I, arrowheads). Using two complementary techniques, Gallyas silver staining (Fig 2B) and sarcosyl extraction (Fig 2C). we could identify aggregated Tau species in 14-month-old mice. Interestingly, both hTauAT and mouse Tau was found in the sarcosyl-insoluble fraction indicating strong co-aggregation (Fig 2C). The ratio of mutant hTauAT to endogenous Tau within the sarcosyl-insoluble fraction is 0.7:1 (+/−) and 1.4:1 (+/+), demonstrating that endogenous mouse Tau is incorporated into Tau aggregates (Fig 2D). Figure 1. Expression of hTauAT causes Tau phosphorylation and mis-sorting in aged mouse hippocampi The diagram shows the domain structure of wild-type human Tau and the location of the substitution A152T (hTauAT). Tau domains are broadly divided into the N-terminal “projection domain” (amino acids M1-Y197) and the C-terminal “assembly domain” (amino acids S198-L441). The C-terminal assembly domain includes three or four pseudo-repeats (˜31 residues each, R1–R4), which together with their proline-rich flanking regions (P1 and P2) constitute the microtubule-binding region. Repeat R2 (exon10) and the two near-N-terminal inserts (N1 and N2, exons 2 and 3) may be absent due to alternative splicing. Blots showing expression of hTauAT (hTau, Mr ˜67 kDa) and/or endogenous mouse Tau (mTau, Mr ˜45–55 kDa) in the hippocampus of 10-month-old heterozygous (+/−) and homozygous (+/+) hTauAT mice in comparison to age-matched control (Ctrl) detected by pan-Tau antibody K9JA. Actin at 42 kDa serves as a loading control. Quantification of (B). Ratio of hTau/mTau indicating a higher hTauAT expression in homozygous mice (˜1.8) compared to heterozygous mice (˜1.0). Each bar represents an average of n = 4 animals, error bars represent SEM. Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16-month-old mice (n = 4 animals per group). Error bars indicate mean ± SEM. Distribution of hTauAT visualized by the human Tau-specific antibody HT7 in the hippocampal CA3-region of hetero- and homozygous hTauAT mice at the age of 14 months. Notice the mis-sorted hTauAT in cell bodies and dendrites (arrowheads) of pyramidal neurons of the CA3 region and immunreactivity of the axons (mossy fibers, asterisks). By contrast, the control shows no immunoreactivity. Elevated hTauAT expression causes Tau pathology inside area CA3 of the hippocampus. Note the increase in Tau phosphorylation probed with the antibody against pT217, a site upstream of the repeat domain. Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3. Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231 + pSer235 (arrowheads denote somato-dendritic mislocalized tau). Antibody PHF-1 (phosphorylation sites pSer396 + pSer404) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum (asterisks) of area CA3 and pyramidal cell bodies (arrows). Tau phosphorylation detected by antibody AT8 (pS202 + pT205) in stratum pyramidale of area CA3 (arrowhead). Note that immunoreactivity increases with expression level. Download figure Download PowerPoint Figure 2. Expression of hTauAT leads to pathological conformation and aggregation of Tau in the hippocampus of aged mice Conformational change of Tau detected by ALZ-50 antibody immunoreactivity in stratum lucidum (asterisks) of area CA3 and somata in stratum pyramidale (arrowheads). Tau aggregation confirmed by Gallyas silver staining of NFTs bearing neurons in hetero- and homozygous hTauAT mice at 14 months of age compared to non-reactive control littermate mice. In homozygous hTauAT mice, the extent of neurofibrillary tangles (NFTs) visualized by Gallyas silver staining is enhanced compared to heterozygous hTauAT mice (white arrowheads). The control shows no silver-reactive Tau aggregates. Western blot analysis using the pan-Tau antibody K9JA indicates sarcosyl-insoluble Tau species of human hTauAT (upper band) and mouse Tau (mTau, lower band). Note the enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice and control mice. Quantification of (C). The ratio of hTauAT/mTau indicates a stronger aggregation in the homozygous hTauAT mice (˜1.4) compared to heterozygous hTauAT mice (˜0.7) (n = 4 animals, error bars represent SEM). Electron micrograph of CA3 region of the hippocampus illustrating typical synapse with a perforated postsynaptic density (asterisks) in a control animal at 12 months. ds, dendritic spine; pt, presynaptic terminal; m, mitochondrium. Example of a degenerating synaptic bouton (arrow) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density (asterisks) in a transgenic animal at 13 months. Electron-dense neuronal cytoplasm with dark nucleoplasm and various vacuoles (arrow) detected in CA3 pyramidal neurons (arrow) at 13 months in transgenic animals in proximity to normally appearing neurons (asterisks), degenerating apical dendrite (d). Neuropil of a transgenic animal (13 months) with a degenerating neuritic profile (arrow). Degenerating dendrite (arrow) in a transgenic animal with darkened cytoplasm and abnormally distributed mitochondria (arrow). Degenerating dendrite containing electron-dense whorling membrane fragments (arrow) in proximity to normally appearing synapses (synapse with a perforated postsynaptic density, asterisks). Download figure Download PowerPoint Electron microscopy revealed typical ultrastructural hallmarks of hippocampal CA3 neuropil, including distinct mossy fiber boutons in the stratum lucidum that appeared intact, densely packed with clear synaptic vesicles in control animals (Fig 2E). By contrast, in transgenic age-matched animals, some presynaptic terminals were electron dense and filled with swollen synaptic vesicles (Fig 2F, arrow). Interestingly, relatively high numbers of synapses, specifically with perforated postsynaptic densities, appeared intact in the stratum lucidum of transgenic animals with no obvious signs of degeneration (Fig 2F and J, asterisks). This finding is in apparent contrast to other animal models including a tau-aggregation model associated with the ΔK280 mutation and the classical murine models overexpressing APP/PS1 mutations 2930. Neuropathology in these models was generally associated with a profound and early reduction in the numbers of excitatory hippocampal synapses that was not apparent in our study even at late disease stage (12–13 months). Signs of neuronal degeneration were obvious in pyramidal neurons; degenerating profiles were identified by a shrunken cell membrane plus a darkened cytoplasm, as well as darkened nucleoplasm and crenated nucleolemma within cell soma (Fig 2G, arrow). Furthermore, various degrees of structural abnormalities were detected in dendrites and axons (Fig 2G–J). Electron opaque, degenerating neuritic profiles were identified by an increase in mitochondrial density, presence of whorling membrane fragments, and numerous lysosome-like vacuoles (Fig 2J, arrow). Tau is hyperphosphorylated and mis-sorted due to hTauAT expression in hippocampal slice cultures To further clarify whether hTauAT is mis-sorted into dendrites and dendritic spines, we used immunofluorescence in organotypic hippocampal slices. Organotypic hippocampal slices were derived from young heterozygous transgenic hTauAT mice and maintained in culture for several weeks. Western blotting with pan-Tau antibody K9JA revealed a ~onefold overexpression in heterozygous slice cultures when compared to endogenous Tau levels (Fig 3A and B), well comparable with hippocampal extracts from adult hTauAT mice (Fig 1C). To detect the subcellular location of hTauAT, we used immunohistochemistry with human Tau-specific antibody TauY9 or pan-Tau antibody K9JA. In slices from non-transgenic littermates, we could not detect any signal with antibody TauY9 (Fig 3C) but found an axonal distribution of Tau with the pan-Tau antibody (Fig EV1A). In contrast, in hTauAT slices the human Tau appeared both in axons (Fig 3C asterisk) and dendrites (Fig 3C, right panel). Whereas the dendritic marker MAP2 was restricted to the dendritic shaft, Tau was also apparent in dendritic spines when hTauAT was expressed (Fig 3E, see also Fig EV1B, inset). The somato-dendritically mis-sorted Tau showed enhanced phosphorylation at the PHF1 epitope (pSer394/pSer404) already at DIV 10, but not in control littermate slices (Fig 3D). Figure 3. hTauAT expression causes Tau phosphorylation and mis-sorting in organotypic hippocampal slice cultures Western blot analysis with pan-Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau (mTau). Slice homogenates from non-transgenic littermate (lane 1, Ctrl), heterozygous hTauAT (lane 2 (+/−)), or homozygous hTauAT slices (lane 3 (+/+)) were analyzed after three weeks in culture. Actin served as a loading control. Quantification of hTauAT expression levels (n = average of 3–6 slice homogenates per group, error bars indicate mean ± SEM). The ratio between hTauAT and endogenous mouse Tau is shown. Immunohistochemistry with antibodies against human Tau (TauY9 antibodies; middle panel) and dendritic marker MAP2 (left panel) in slice cultures at DIV 10. Stratum radiatum of area CA3 is shown. Although MAP2 staining is apparent, no signal is detected by the TauY9 antibody in control slices. In contrast, in hTauAT slices human Tau is detected in both dendrites and axons (see asterisk in merged image). Immunohistochemistry against phosphorylated Tau at the PHF1 epitope in area CA3 of a control (upper lane) or hTauAT slice (bottom lane). PHF1 staining is slightly visible in some axons in control slices. In contrast, PHF1-phosphorylated Tau is apparent in somata, dendrites and axons of neurons in hTauAT slice cultures. Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 (red) and Tau (K9JA antibody, green). Tau staining is seen in dendritic spines (asterisks) in contrast to MAP2, which is restricted to the dendritic shaft. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Expression of hTauAT causes missorting in organotypic hippocampal slice cultures Immunohistochemistry highlighting the dendritic marker MAP2 and the axonal marker Tau protein (by pan-Tau antibody K9JA recognizing both human and mouse Tau) in slice cultures from non-transgenic littermate at DIV 10. Overview images (upper panel) and higher magnification of area CA3 (lower panel). Tau is restricted to axons and not seen in dendrites. Immunohistochemistry highlighting the dendritic marker MAP2 and axonal Tau protein (by pan-Tau antibody K9JA recognizing both human and mouse Tau) in slice cultures from hTauAT at DIV 10. Overview images (upper panel) and higher magnification of area CA3 (lower panel). In hTauAT slices, Tau is seen both in axons and dendrites. Higher magnification of apical dendrite (inset) displays Tau in dendritic spines in contrast to MAP2, which is restricted to the dendritic shaft. Download figure Download PowerPoint Expression of hTauAT elevates intracellular Ca2+ concentrations in CA3 neurons at resting state and after membrane depolarization through NR2B-containing NMDA receptors It was reported earlier that mis-sorted, phosphorylated Tau within dendritic spines of primary neurons impairs synaptic transmission by altering the densities of postsynaptic receptors such as AMPARs and NMDARs 12 and causes abnormal changes in intracellular Ca2+ ([Ca2+]i) 17. We therefore investigated [Ca2+]i by Fura-2AM imaging in organotypic hippocampal slices. Experiments were performed in area CA3 of slice cultures at DIV 15 (Fig 4A and B), when the NR2B to NR2A ratio in synapses is comparable to acute slices 313233. We observed a pronounced increase in resting [Ca2+]i in hTauAT slices (~129 nM) compared to control littermate slices (~82 nM, Fig 4A–D). Next, slices were incubated and imaged for [Ca2+]i in the presence of drugs to identify the source of the [Ca2+]i increase. We applied the L-type voltage-gated calcium channel (L-VGCC) blocker nifedipine (NIF), the AMPAR antagonist CNQX, the NMDAR antagonists APV and ifenprodil (IFEN, inhibitor of NMDARs composed of NR1 and NR2B subunits 34), memantine (MEM: preferentially blocking extrasynaptic NMDA receptor currents; 35), the sodium channel blocker tetrodotoxin (TTX), and the presynaptic vesicle release inhibitor tetanus neurotoxin (TeNT), or imaged in the absence of extracellular Ca2+ (see Table EV1 for a summary of all drugs and concentrations used). In control littermate slices, the resting [Ca2+]i was not significantly affected by these drugs (Fig 4D). However, as expected, the removal of extracellular Ca2+ leads to a pronounced decrease in [Ca2+]i (−50%, ~40 nM, P < 0.05, Fig 4D). In agreement, Ca2+-free saline caused the strongest [Ca2+]i reduction in hTauAT slices (−55%, ~55 nM, P < 0.0001, Fig 4D) but additionally the blockade of NMDAR with APV leads to a reduction in resting [Ca2+]i (~83.5 nM, P < 0.05, Fig 4D). Figure 4. hTauAT causes elevation of Ca2+ levels in CA3 hippocampal neurons at resting state and after membrane depolarization through extrasynaptic NMDA receptors Slices (DIV 15) from control mice were loaded with the Ca2+-sensitive dye Fura-2AM. Ratiometric images are presented at baseline and after application of high potassium. Note the increase in intracellular Ca2+ ([Ca2+]i) after KCl application. Slices (DIV 15) from hTauAT mice were loaded with the Ca2+-sensitive dye Fura-2AM. Ratiometric images at baseline and after application of high potassium chloride in area CA3 are depicted. Note the increase in [Ca2+]i (false color legend) under both conditions in hTauAT slices compared to controls. Electrophysiological example traces (control, black; hTauAT, red) of excitatory postsynaptic field potentials (fEPSP) depict examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale (s.p.) of area CA3 in slices in which calcium imaging experiments were conducted. The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph. The graph shows the mean of [Ca2+]i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura-2AM-loaded hippocampal slices. After depolarization, [Ca2+]i rises up to ˜600 nM in hTauAT slices (red trace, n = 6 slices, prepared from at least three animals), compared to ˜300 nM in control slices (black trace, n = 6 slices; prepared from at least three animals). Note that even under resting conditions, [Ca2+]i is elevated to ˜120 nM due to hTauAT expression. Quantification of the [Ca2+]i from ratiometric images as depicted in (A) and (B) after background subtraction and selection of regions of interest (10 circles of fixed diameter) in stratum radiatum border to stratum pyramidale of area CA3. Under resting conditions, [Ca2+]i was elevated in transgenic slice cultures (˜120 nM, n = 11) compared to controls (˜80 nM, n = 8). Slices were incubated with one of the following channel blockers: nifedipine (NIF, VGCC; Ctrl n = 8; A152T: n = 7), APV (NMDAR; Ctrl n = 7; A152T n = 8), ifenprodil (IFEN, NMDAR; Ctrl n = 8; A152T n = 7), memantine (MEM, NMDAR; Ctrl/A152T n = 6), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPAR; Ctrl n = 8; A152T n = 7)), tetrodotoxin (TTX, VGNaC; Ctrl n = 4; A152T n = 5), tetanus neurotoxin (TeNT, neurotransmitter release; A152T n = 6) or in Ca2+-free buffer (Ctrl n = 10; A152T n = 7). For each experiment, slice preparations from at least three different mice were used. One-way ANOVA followed by Tukey's post hoc test; Ctrl: F(7/51) = 3,533; P = 0.0036; A152T: F(8/55) = 5,230; P < 0.0001. For an overview of drug concentrations see Table EV1. Quantification of the maximum peak increase in intracellular calcium after high KCl stimulation. One-way ANOVA followed by Tukey's post hoc test Ctrl: F(7/45) = 4,080; P = 0.0015; A152T: F(8/53) = 7,580; P < 0.0001). Data information: Error bars represent SEM; one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint The inhibition of extrasynaptic NMDARs (either by IFEN or by low-dose MEM) or the blockage of L-VGCC (by NIF) was sufficient to reduce resting [Ca2+]i in hTauAT-expressing slices to control levels (IFEN: ~70 nM, P < 0.05; MEM: ~83 nM, P < 0.01; NIF: ~83 nM, P < 0.05, Fig 4D). [Ca2+]i levels were not significantly altered in presence of either TTX, TeNT, or CNQX in both control and hTauAT slices under resting conditions (Fig 4D). Next, changes in calcium influx after membrane depolarization were monitored by applying high potassium chloride (KCl). After activity induction, we observed a steep rise in [Ca2+]i levels in hTauAT slices (~575 nM, Fig 4E), whereas control slices achieved only maximum values of ~300 nM (Fig 4E). KCl-evoked Ca2+ influx was blocked by nifedipine (NIF: −47%, P < 0.05, Fig 4E) and less prominently by APV (−36%, Fig 4E) in control littermate slices. In contrast, treatment of hTauAT slices with APV caused a stronger inhibition of Ca2+ influx than application of NIF (−56%, P < 0.001; APV: −69%, P < 0.001, Fig 4E). Comparison of the effects of MEM and IFEN on KCl-evoked Ca2+ influx in control (MEM: −1%; IFEN: −13%, Fig 4E) and in hTauAT slices (MEM: −44%, P < 0.05; IFEN: −48%, P < 0.01, Fig 4E) demonstrated a prominent contribution of NR2B-mediated Ca2+ influx to the overall [Ca2+]i increase in hTauAT slices. hTauAT expression is neurotoxic by increasing extracellular glutamate, thereby activating the CREB shutoff pathway Adult excitatory neurons of the hippocampus express NMDARs with mainly two subunit compositions, NR1/NR2A and NR1/NR2B 3637. Synaptic NMDARs are predominantly of the types NR1/NR2A or NR1/NR2A/NR2B (ifenprodil insensitive), whereas somatic or extrasynaptic NMDARs are predominantly of the type NR1/NR2B (ifenprodil sensitive) 36. The pathway downstream of NMDAR activation is dependent both on the subunit composition and on the location of the receptors 3839. For example, Ca2+ flux through synaptic NMDARs initiates changes in synaptic efficacy and promotes pr" @default.
• W2290636701 created "2016-06-24" @default.
• W2290636701 creator A5003261774 @default.
• W2290636701 creator A5032440702 @default.
• W2290636701 creator A5034349864 @default.
• W2290636701 creator A5047141760 @default.
• W2290636701 creator A5059641759 @default.
• W2290636701 creator A5060906393 @default.
• W2290636701 creator A5066503488 @default.
• W2290636701 date "2016-03-01" @default.
• W2290636701 modified "2023-10-10" @default.
• W2290636701 title "The Tau/A152T mutation, a risk factor for frontotemporal‐spectrum disorders, leads to <scp>NR</scp> 2B receptor‐mediated excitotoxicity" @default.
• W2290636701 cites W1530155692 @default.
• W2290636701 cites W1577577364 @default.
• W2290636701 cites W1597597862 @default.
• W2290636701 cites W192077740 @default.
• W2290636701 cites W1965859168 @default.
• W2290636701 cites W1966556202 @default.
• W2290636701 cites W1966599992 @default.
• W2290636701 cites W1967993742 @default.
• W2290636701 cites W1968553823 @default.
• W2290636701 cites W1968937229 @default.
• W2290636701 cites W1970686195 @default.
• W2290636701 cites W1970910654 @default.
• W2290636701 cites W1978457897 @default.
• W2290636701 cites W1980311878 @default.
• W2290636701 cites W1981890392 @default.
• W2290636701 cites W1982864908 @default.
• W2290636701 cites W1984854704 @default.
• W2290636701 cites W1988036480 @default.
• W2290636701 cites W1994030229 @default.
• W2290636701 cites W2001261203 @default.
• W2290636701 cites W2003102216 @default.
• W2290636701 cites W2003171800 @default.
• W2290636701 cites W2005609503 @default.
• W2290636701 cites W2012405577 @default.
• W2290636701 cites W2022532556 @default.
• W2290636701 cites W2022959246 @default.
• W2290636701 cites W2024380057 @default.
• W2290636701 cites W2030377743 @default.
• W2290636701 cites W2030575711 @default.
• W2290636701 cites W2030674642 @default.
• W2290636701 cites W2032653084 @default.
• W2290636701 cites W2033198098 @default.
• W2290636701 cites W2033283296 @default.
• W2290636701 cites W2041142924 @default.
• W2290636701 cites W2041956586 @default.
• W2290636701 cites W2052742260 @default.
• W2290636701 cites W2058829280 @default.
• W2290636701 cites W2059127030 @default.
• W2290636701 cites W2061538653 @default.
• W2290636701 cites W2062605351 @default.
• W2290636701 cites W2068046501 @default.
• W2290636701 cites W2076333797 @default.
• W2290636701 cites W2078167209 @default.
• W2290636701 cites W2079054696 @default.
• W2290636701 cites W2080612159 @default.
• W2290636701 cites W2083204774 @default.
• W2290636701 cites W2085877811 @default.
• W2290636701 cites W2087140031 @default.
• W2290636701 cites W2095151099 @default.
• W2290636701 cites W2096557110 @default.
• W2290636701 cites W2096610613 @default.
• W2290636701 cites W2097717784 @default.
• W2290636701 cites W2103740237 @default.
• W2290636701 cites W2108044151 @default.
• W2290636701 cites W2114444638 @default.
• W2290636701 cites W2116635659 @default.
• W2290636701 cites W2118516203 @default.
• W2290636701 cites W2120675188 @default.
• W2290636701 cites W2125985978 @default.
• W2290636701 cites W2132265466 @default.
• W2290636701 cites W2132844263 @default.
• W2290636701 cites W2133603940 @default.
• W2290636701 cites W2134906491 @default.
• W2290636701 cites W2141260882 @default.
• W2290636701 cites W2146521940 @default.
• W2290636701 cites W2148908036 @default.
• W2290636701 cites W2149497936 @default.
• W2290636701 cites W2150110395 @default.
• W2290636701 cites W2152468922 @default.
• W2290636701 cites W2159643400 @default.
• W2290636701 cites W2167937350 @default.
• W2290636701 cites W2170906442 @default.
• W2290636701 cites W2292902805 @default.
• W2290636701 cites W4210979710 @default.
• W2290636701 doi "https://doi.org/10.15252/embr.201541439" @default.
• W2290636701 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4818782" @default.
• W2290636701 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26931569" @default.
• W2290636701 hasPublicationYear "2016" @default.
• W2290636701 type Work @default.
• W2290636701 sameAs 2290636701 @default.
• W2290636701 citedByCount "87" @default.
• W2290636701 countsByYear W22906367012016 @default.
• W2290636701 countsByYear W22906367012017 @default.
• W2290636701 countsByYear W22906367012018 @default.
• W2290636701 countsByYear W22906367012019 @default.
• W2290636701 countsByYear W22906367012020 @default.

• next