Novel function of Tau in regulating the effects of external stimuli on adult hippocampal neurogenesis
2016; Springer Nature; Volume: 35; Issue: 13 Linguagem: Inglês
10.15252/embj.201593518
ISSN1460-2075
AutoresNoemí Pallas‐Bazarra, Jerónimo Jurado‐Arjona, Marta Navarrete, José A. Esteban, Félix Hernández, Jesús Ávila, María Llorens‐Martín,
Tópico(s)Nerve injury and regeneration
ResumoArticle19 May 2016Open Access Transparent process Novel function of Tau in regulating the effects of external stimuli on adult hippocampal neurogenesis Noemí Pallas-Bazarra Noemí Pallas-Bazarra Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain Search for more papers by this author Jerónimo Jurado-Arjona Jerónimo Jurado-Arjona Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain Search for more papers by this author Marta Navarrete Marta Navarrete Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Jose A Esteban Jose A Esteban Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Félix Hernández Félix Hernández Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Sciences Faculty, Autonoma University, Madrid, Spain Search for more papers by this author Jesús Ávila Corresponding Author Jesús Ávila orcid.org/0000-0002-6288-0571 Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain Search for more papers by this author María Llorens-Martín Corresponding Author María Llorens-Martín Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain Search for more papers by this author Noemí Pallas-Bazarra Noemí Pallas-Bazarra Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain Search for more papers by this author Jerónimo Jurado-Arjona Jerónimo Jurado-Arjona Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain Search for more papers by this author Marta Navarrete Marta Navarrete Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Jose A Esteban Jose A Esteban Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Search for more papers by this author Félix Hernández Félix Hernández Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Sciences Faculty, Autonoma University, Madrid, Spain Search for more papers by this author Jesús Ávila Corresponding Author Jesús Ávila orcid.org/0000-0002-6288-0571 Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain Search for more papers by this author María Llorens-Martín Corresponding Author María Llorens-Martín Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain Search for more papers by this author Author Information Noemí Pallas-Bazarra1,2, Jerónimo Jurado-Arjona1,2, Marta Navarrete1, Jose A Esteban1, Félix Hernández1,3, Jesús Ávila 1,2 and María Llorens-Martín 1,2 1Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain 2Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), Madrid, Spain 3Sciences Faculty, Autonoma University, Madrid, Spain *Corresponding author. Tel: +34 91 196 45 92; Fax: +34 91 196 44 20; E-mail: [email protected] *Corresponding author. Tel: +34 196 45 64; Fax: +34 91 196 44 20; E-mail: [email protected] The EMBO Journal (2016)35:1417-1436https://doi.org/10.15252/embj.201593518 [The copyright line of this article was changed on 17 June 2016 after original online publication.] 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 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. Synopsis Tau regulates the in vivo maturation of newborn granule neurons under physiological conditions. In addition, Tau deficiency prevents both the detrimental and stimulatory actions of acute stress and environmental enrichment, respectively, on the functional maturation of these cells. Altogether, our data shed light on novel functions of Tau protein related to the plastic modulation of adult hippocampal neurogenesis by external stimuli. Tau plays a novel in vivo role in the synaptic maturation of newborn granule neurons under basal conditions. Tau is involved in immature granule neuron cell death caused by acute stress. Tau regulates the stress-induced impairment of newborn granule neuron functional maturation. Tau regulates the increase in newborn neuron survival triggered by environmental enrichment. Tau modulates the stimulatory effects of environmental enrichment on the functional maturation of newborn granule neurons. Introduction Tau is a neuronal microtubule-associated protein (MAP) that promotes microtubule assembly and stabilization (Weingarten et al, 1975). It plays key roles in the establishment of neuronal polarity and migration during embryonic development (Caceres & Kosik, 1990; Dawson et al, 2001; Sapir et al, 2012; Sayas et al, 2015), in axonal transport (Ballatore et al, 2007; Hernandez & Avila, 2010) and in intracellular trafficking (Hernandez & Avila, 2010). Under physiological conditions, Tau is located mainly in axonal microtubules (Hirokawa et al, 1996; Aronov et al, 2001), although increasing evidence supports its presence also in dendrites (Ittner et al, 2010) and dendritic spines (Ittner et al, 2010; Mondragon-Rodriguez et al, 2012; Kimura et al, 2014). The affinity of Tau to bind microtubules is finely regulated, depending mainly on the selective expression of various isoforms and post-translational modifications. Tau protein is encoded by mapt gene. Human mapt contains 16 exons, from which several Tau isoforms are generated by alternative splicing (Goedert et al, 1989; Andreadis et al, 1992). Exon 10 encodes one of the four repeat sequences that form the microtubule-binding domain. The presence or absence of exon 10 results in Tau isoforms with four (Tau4R) or three (Tau3R) repeat sequences, Tau4R showing a higher affinity to bind microtubules than Tau3R ones (Lu & Kosik, 2001; Avila et al, 2004). At early developmental stages, Tau3R predominates (Lu & Kosik, 2001; Avila et al, 2004), conferring a lower stability of the cytoskeleton and allowing the morphological differentiation and migration of developing neurons. In contrast, in the adult murine brain, Tau4R is the predominant isoform (Lu & Kosik, 2001; Avila et al, 2004), thereby guaranteeing the stability of the cytoskeleton required to maintain neuronal integrity. It is important to note an exception that occurs during adult hippocampal neurogenesis (AHN), when Tau3R isoforms can be found (Bullmann et al, 2007; Llorens-Martin et al, 2012). Adult neurogenesis occurs in discrete brain regions during adulthood. Among these regions, the hippocampal dentate gyrus (DG) has attracted increasing attention due to the functional relevance of AHN, a process related to hippocampal-dependent learning and mood regulation (Sahay & Hen, 2008; Aimone et al, 2011). AHN encompasses the proliferation of adult neural stem cells, their differentiation into mature neurons, and their incorporation into the hippocampal circuitry (Ming & Song, 2005). Furthermore, the addition of new neurons to the hippocampal circuit is regulated by numerous external factors such as physical activity, environmental enrichment (EE), and stress (Gould et al, 1997; Kempermann et al, 1997), thus conferring an outstanding degree of plasticity to the network. By using a Tau knockout mouse model (Dawson et al, 2001), here we demonstrate that Tau is involved in the morphological differentiation and synaptic integration of newborn hippocampal granule neurons in vivo. Moreover, we report a novel function of Tau in the regulation of the negative consequences of acute stress on AHN. We also demonstrate, for the first time, that Tau regulates the stimulatory effects of EE on AHN. Results Tau protein is not involved in the regulation of the basal rate of adult hippocampal neurogenesis (AHN) We first examined whether Tau protein is involved in the regulation of AHN under basal conditions. Figure 1A–D shows that the absence of Tau did not affect the number of proliferative phospho-histone 3 (PH3)+ cells (t = 0.757; P = 0.471) or the number of apoptotic caspase-cleaved actin (fractin)+ cells (t = −0.771; P = 0.452) in the DG. However, as proliferation and death rates are general parameters that may not reflect changes in specific cell subpopulations, we also analyzed the survival of 1-, 2-, 4-, 6-, and 8-week-old cells labeled with the thymidine analogs 5-iodo-2′-deoxyuridine (IdU) or 5-chloro-2′-deoxyuridine (CldU). Figure 1E shows the experimental design. Tau depletion did not alter cell survival at any of the ages studied (Fig 1F). Hence, we conclude that Tau protein is not involved in the regulation of newborn neuron survival rate. Figure 1G summarizes the main cell markers expressed during the sequential differentiation stages that newborn neurons go through before they become fully mature. We addressed whether Tau deficiency affects the differentiation of newborn granule neurons and their commitment to neural lineage. To explore this notion, the percentage of cells that expressed doublecortin (DCX) and neuronal nuclei (NeuN) markers was evaluated in 1-, 4-, and 8-week-old CldU+ or IdU+ newborn neurons. As expected, DCX expression decreased with time, whereas the opposite effect was observed for NeuN (Fig 1H). No differences in the expression of either of these markers were found in response to Tau deficiency. Thus, it can be concluded that Tau is not involved in newborn neuron differentiation under basal conditions. Moreover, the absence of Tau did not alter the number of Sex determining region Y-box 2 (Sox2)+ (t = −1.180; P = 0.261) or brain lipid-binding protein (BLBP)+ (t = −1.015; P = 0.330) neural progenitor cells. In addition, no differences in the number of DCX+ neuroblasts (t = −1.201; P = 0.253) or in the number of calretinin+ immature neurons (t = −1.427; P = 0.179) were found in Tau−/− mice compared to WT (Fig 1I–N). All together, these data suggest that neither is Tau involved in the regulation of AHN rate under basal conditions. Figure 1. Tau protein is not involved in regulating the rate of adult hippocampal neurogenesis under basal conditions A, B. Representative image of a proliferative cell in the DG labeled with an anti-PH3 antibody (green) (A) and quantification of the number of PH3+ cells in the DG of WT and Tau−/− mice (B) (mean ± SEM; n = 10 WT mice, n = 8 Tau−/− mice; Student's t-test). C, D. Representative image of an apoptotic cell in the DG labeled with an anti-fractin antibody (red) (C) and quantification of the number of fractin+ cells in the DG of WT and Tau−/− mice (D) (mean ± SEM; n = 10 WT mice, n = 8 Tau−/− mice; Student's t-test). E, F. Schematic diagram of the cell survival experimental design using thymidine analogs (E) and quantification of the percentage of surviving cells in Tau−/− mice as compared to WT mice at each cell age (1-, 2-, 4-, 6-, and 8-week-old cells) (F) (mean ± SEM (normalized data); Student's t-test). G. Schematic diagram of the cell markers representative of each maturational stage during adult hippocampal neurogenesis. H. Quantification of the percentage of 1-, 4-, and 8-week-old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau−/− mice (χ2 test). I, J. Representative images of progenitor cells in the DG labeled with anti-Sox2 (red) and anti-BLBP (green) antibodies (I) and neuroblasts and immature neurons labeled with anti-DCX (red) and anti-calretinin (green) antibodies, respectively (J). K–N. Quantification of the number of Sox2+ (K), BLBP+ (L), DCX+ (M), and calretinin+ (N) cells in the DG of WT and Tau−/− mice (mean ± SEM; n = 10 WT mice, n = 8 Tau−/− mice; Student's t-test). Data information: The number of animals used for the quantification with thymidine analogs (F, H) is indicated in (E). In representative images, cell nuclei were labeled with DAPI (blue). White scale bars, 10 μm. Red scale bars, 20 μm. GL: granular layer; SGL: subgranular layer; H: hilus. Brightness and contrast of representative confocal microscopy images shown in the figure were minimally adjusted in order to improve visualization. Download figure Download PowerPoint Tau protein is necessary for the dendritic maturation of newborn granule neurons To study the role of Tau in the morphological maturation of newborn neurons, we analyzed the morphology of the dendritic tree of 4- and 8-week-old newborn granule neurons labeled with PSD95-GFP-expressing retroviruses. Figure 2A shows representative images of 4-week-old newborn granule neurons of WT and Tau−/− mice. In these neurons, the absence of Tau decreased the total dendritic length (t = 4.963; P < 0.001) (Fig 2B). Moreover, Sholl's analysis revealed alterations in the complexity of the dendritic tree (Fig 2C). In particular, Tau deficiency decreased dendritic branching in 100–150 μm (t = 2.397; P = 0.020), 150–200 μm (U = 143; P < 0.001), and 200–250 μm (U = 354; P = 0.037) from the cell soma. Figure 2D shows representative images of 8-week-old newborn granule neurons of WT and Tau−/− mice. However, at this cell age, the absence of Tau did not lead to differences in the total dendritic length (t = −1.266; P = 0.210) (Fig 2E) or in the complexity of the dendritic tree (Fig 2F). Figure 2. Tau protein is necessary for the dendritic and synaptic maturation of newborn granule neurons A. Representative images of 4-week-old newborn granule neurons of WT and Tau−/− mice infected by a PSD95-GFP-expressing retrovirus. A schematic representation of Sholl's analysis is shown in the WT image. B, C. Quantification of total dendritic length (B) and Sholl's analysis (C) of 4-week-old newborn granule neurons in WT and Tau−/− mice. D. Representative images of 8-week-old newborn granule neurons of WT and Tau−/− mice infected by a PSD95-GFP-expressing retrovirus. E, F. Quantification of total dendritic length (E) and Sholl's analysis (F) of 8-week-old newborn granule neurons in WT and Tau−/− mice. G. Representative images of 4-week-old newborn granule neurons of WT and Tau−/− mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green). H, I. Quantification of the number of PSDs/μm (H) and PSD area (I) in each dendritic branching order of 4-week-old newborn granule neurons in WT and Tau−/− mice. J. Representative images of 8-week-old newborn granule neurons of WT and Tau−/− mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green). K, L. Quantification of the number of PSDs/μm (K) and PSD area (L) in each dendritic branching order of 8-week-old newborn granule neurons in WT and Tau−/− mice. Data information: In (B, C, E, F, H, I, K, L), data are presented as mean ± SEM; n = 3 mice per genotype; *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test or Mann–Whitney U-test). In representative images, cell nuclei were labeled with DAPI (blue). White scale bars, 50 μm. Red scale bars, 5 μm. Brightness and contrast of representative confocal microscopy images shown in the figure were minimally adjusted in order to improve visualization. Download figure Download PowerPoint Taken together, these results show that Tau is involved in the morphological maturation of newborn granule neurons, since its absence causes a transient alteration of the dendritic arborization of these cells. Tau protein is necessary for the formation of postsynaptic densities (PSDs), dendritic spines, and mossy fiber terminals (MFTs) in granule neurons In addition to the morphology of newborn neurons, we examined the participation of Tau in the functional maturation of these cells. Figure 2G shows representative images of 4-week-old WT and Tau−/− newborn granule neurons infected with PSD95-GFP-expressing retroviruses and their respective high-power magnifications, in which PSDs (afferent synapses) can be observed. No differences were found in PSD density along the dendritic tree (Fig 2H). However, the average PSD area was higher in the 2nd (U = 56,105; P < 0.001) and 4th (U = 992,492; P < 0.001) branching orders of Tau−/− dendrites compared to WT ones (Fig 2I). The absence of Tau in 8-week-old WT and Tau−/− newborn neurons (Fig 2J), fully integrated into the circuitry and infected with PSD95-GFP-expressing retroviruses, decreased the density (t = 2.034; P = 0.047) (Fig 2K) and area (U = 1,282,519; P < 0.001) (Fig 2L) of PSDs in the 5th dendritic branching order. Conversely, the PSD area was higher in the remaining branching orders (2nd (U = 81,074; P = 0.069); 3rd (U = 769,008; P < 0.001), and 4th (U = 1,304,378; P = 0.001)). In light of these results, we examined whether the Tau deficiency has additional effects on the morphology of the dendritic spines of 8-week-old newborn neurons, which had shown the most remarkably alterations in PSDs. It should be noted that each dendritic spine can contain one, none, or more than one PSD (Fig EV1A). The absence of Tau increased the density of spines in the 4th branching order dendrites (U = 232; P = 0.022), whereas a trend to decrease this parameter was observed in 5th branching order dendrites (t = 1.815; P = 0.077) (Fig EV1B). Moreover, the diameter of the head of the spines located in 5th branching order dendrites was reduced due to Tau deficiency (U = 177,552; P < 0.001) (Fig EV1C). In addition, we classified the dendritic spines into three categories (stubby, thin, and mushroom) and quantified the percentages of each type of spine (Fig EV1D). Tau deficiency reduced the percentage of mushroom spines in 3rd (χ2 = 6.109; P = 0.014) and 5th (χ2 = 12.92; P < 0.001) branching order dendrites. Furthermore, it increased the percentage of stubby spines in the 3rd (χ2 = 7.076; P = 0.008) and that of thin spines in the 5th (χ2 = 8.173; P = 0.004), respectively. Click here to expand this figure. Figure EV1. Tau protein deficiency causes morphological alterations in the dendritic spines of newborn granule neurons A. Representative image of a dendrite of an 8-week-old newborn granule neuron infected by a PSD95-GFP-expressing retrovirus. Arrows indicate examples of the different types of dendritic spines (stubby, thin and mushroom) (shown in red). Note that each dendritic spine can contain several PSDs (shown in green), as indicated by the white asterisk. Scale bar, 3 μm. B, C. Quantification of the number of spines/μm (B) and spine head diameter (C) in each dendritic branching order (mean ± SEM; n = 3 mice per genotype; *P < 0.05, ***P < 0.001; Student's t-test or Mann–Whitney U-test). D. Quantification of the percentage of each type of spine (stubby, thin, and mushroom) in each dendritic branching order (n = 3 mice per genotype; *P < 0.05, **P < 0.01, ***P < 0.001; χ2 test). Data information: Brightness and contrast of representative confocal microscopy image shown in the figure was minimally adjusted in order to improve visualization. Download figure Download PowerPoint In order to further characterize the morphological alterations caused by Tau deficiency on the postsynaptic elements of granule neurons, we next analyzed the ultrastructure of the afferent synapses in their dendritic spines by electron microscopy (Fig EV2A). This analysis was performed in three separate regions of the molecular layer (ML) of the DG: external (EML), medial (MML), and inner (IML). We found that Tau deficiency reduced the density of synapses (number of synapses per mm2) in the whole ML ((EML: t = 2.363; P = 0.0019); (MML: t = 9.282; P < 0.001); (IML: t = 2.371; P = 0.019)) (Fig EV2B). Moreover, it increased the size of the synaptic cleft ((EML: t = −11.216; P < 0.001); (MML: t = −12.385; P < 0.001); (IML: t = −8.343; P < 0.001)) (Fig EV2C) and caused alterations in PSDs, which showed a reduced area ((EML: t = 2.64; P = 0.0087); (MML: t = 2.322; P = 0.021); (IML: t = 2.641; P = 0.0087)) (Fig EV2D) and length ((EML: t = 2.668; P = 0.0079); (MML: t = 0.818; P = 0.413); (IML: t = 4.036; P < 0.001)) (Fig EV2E), and an increased depth ((EML: t = −2.669; P = 0.0079); (MML: t = −0.488; P = 0.625); (IML: t = −3.051; P = 0.0024)) (Fig EV2F) than WT ones. Click here to expand this figure. Figure EV2. Tau protein deficiency alters the ultrastructure of the afferent synapses of granule neurons in the ML of the DG A. Representative electron microscopy images of the ML of WT and Tau−/− mice showing the density (a, b) and the ultrastructural details (c, d) of afferent synapses in granule neurons. Arrows indicate synapses. Brackets indicate PSDs. Asterisks indicate synaptic clefts. White scale bar, 1 μm. Red scale bar, 250 nm. B–F. Quantification of the number of synapses/mm2 (B), the synaptic cleft size (C) and the area (D), length (E) and depth (F) of the PSDs in the EML, MML, and IML of WT and Tau−/− mice (mean ± SEM; n = 3 mice per genotype; *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test). Data information: IML, inner molecular layer; MML, medial molecular layer; EML, external molecular layer. Brightness and contrast of representative images shown in the figure were minimally adjusted in order to improve visualization. Download figure Download PowerPoint In order to evaluate whether Tau deficiency also has an impact on the morphology of newborn neuron MFTs (efferent synapses), we quantified the area of each individual MFT in 8-week-old retrovirally labeled newborn granule neurons. Figure EV3A and B shows representative images of the whole hippocampus (A), as well as high-power magnifications of MFTs in the CA3 field (B) of WT and Tau−/− mice. No differences were found between WT and Tau−/− animals (U = 17,886; P = 0.166) (Fig EV3C). In addition, and given the high variability in size of the MFT population, we plotted the percentages of MFT grouped by size in WT and Tau−/− mice (Fig EV3D). As shown, no differences in the distribution of MFT sizes were observed to be caused by the absence of Tau (K–S Z = 1.089; P = 0.187). Click here to expand this figure. Figure EV3. Tau protein deficiency alters the ultrastructure of the efferent synapses of granule neurons in CA3 A. Representative image showing 8-week-old newborn granule neurons in the DG (infected by a PSD95-GFP-expressing retrovirus) projecting axons into the CA3 field (indicated by a white square). Red scale bar, 300 μm. B. Representative images of MFTs of 8-week-old newborn granule neurons in the CA3 region of WT and Tau−/− mice. Green scale bar, 20 μm. C. Quantification of the area of MFTs of 8-week-old newborn granule neurons in WT and Tau−/− mice (mean ± SEM; n = 3 mice per genotype; Mann–Whitney U-test). D. Representation of the distribution of the size of MFTs in 8-week-old newborn granule neurons of WT and Tau−/− mice. MFTs were grouped on the basis of size, and the percentage of MFTs in each size group was represented (n = 3 mice per genotype; Kolmogorov–Smirnov Z-test). E. Representative electron microscopy images of the ultrastructure of efferent synapses of granule neurons in the CA3 region of WT and Tau−/− mice. Brackets indicate the active zone of the presynaptic terminal. Asterisks indicate synaptic clefts. White scale bar, 250 nm. F–H. Quantification of the number of synaptic vesicles/μm2 (F), the length of the active zone (G) and the synaptic cleft size (H) of efferent synapses of granule neurons in the CA3 region of WT and Tau−/− mice (mean ± SEM; n = 3 mice per genotype; **P < 0.01, ***P < 0.001; Student's t-test). Data information: DG, dentate gyrus; H, hilus; SS, synaptic spine; MFT, mossy fiber terminal. Brightness and contrast of representative images shown in the figure were minimally adjusted in order to improve visualization. Download figure Download PowerPoint Finally, we analyzed the effects of Tau deficiency on the ultrastructure of the MFTs under an electron microscope (Fig EV3E). In this regard, we studied the stratum lucidum and stratum pyramidale of the CA3 region. The density of presynaptic vesicles (number of vesicles per μm2) (t = 3.548; P < 0.001) (Fig EV3F) and the length of the presynaptic active zone (t = 5.589; P < 0.001) (Fig EV3G) were reduced, whereas the size of the synaptic cleft (t = −7.409; P < 0.001) (Fig EV3H) was increased in Tau−/− animals compared to WT ones. In summary, these results suggest that Tau participates in the functional maturation of granule neurons, since its absence alters PSD, dendritic spine, and MFT morphology. The absence of Tau modifies the electrophysiological properties of granule neurons In order to determine whether the morphological alterations caused by Tau deficiency affect the basal electrophysiological properties of granule neurons, we recorded the membrane properties and synaptic activity of these cells under whole-cell configuration. Figure 3A and B shows representative traces of miniature excitatory postsynaptic currents (mEPSCs) recorded at a holding potential of −65 mV from WT (black traces) and Tau−/− (red traces) mice. The resting membrane potential of Tau−/− cells was hyperpolarized compared to that of WT cells (t = 2.217; P = 0.038) (Fig 3C). Figure 3. Tau protein deficiency affects basal synaptic activity in granule neurons A. Representative traces of mEPSCs recorded at a holding potential of −65 mV from WT (black traces) and Tau−/− (red traces) mice. B. Exemplary traces of mEPSCs from one cell from WT (black trace) and Tau−/− (red trace) mice. C. Resting membrane potential of WT and Tau−/− mice (mean ± SEM; n = 13 WT cells, n = 10 Tau−/− cells; *P < 0.05, Student's t-test). D, E. mEPSC amplitude (D) and mEPSC frequency (E) of WT and Tau−/− animals (mean ± SEM; n = 13 WT cells, n = 10 Tau−/− cells; Student's t-test or Mann–Whitney U-test). F. Cumulative probability plot of the mEPSC amplitudes, as well as a detailed representation of small (i) and big (ii) mEPSCs from both WT and Tau−/− mice (black and red traces, respectively) (***P < 0.001; Kolmogorov–Smirnov Z-test). Download figure Download PowerPoint We focused our analyses on the mEPSCs to assess whether synaptic inputs were altered by the absence of Tau. Granule cells of the DG of Tau−/− mice did not exhibit alterations in the amplitude (t = −0.256; P = 0.800) (Fig 3D) neither in the frequency (U = 53,000; P = 0.483) (Fig 3E) of mEPSCs compared to WT ones. However, examination of the cumulative probability distribution of amplitudes of mEPSCs revealed two distinct populations of mEPSCs altered in opposite directions in Tau−/− mice (Fig 3F). Thus, the amplitudes of small mEPSCs of Tau−/− mice were statistically larger than those of WT animals (K–S Z = 6.306; P < 0.001) (Fig 3F (i)). In contrast, when comparing the mEPSCs of larger amplitudes, those of Tau−/− mice were smaller than those of WT animals (K–S Z = 9.820; P < 0.001) (Fig 3F (ii)). This result is indicative of a redistribution of excitatory synaptic weights in Tau−/− mice. Tau protein is required for stress-induced death of newborn granule neurons Given that AHN is regulated by external stimuli, we next examined whether Tau protein is involved in the negative regulation of AHN exerted by acute stress. We administered CldU thymidine analog to WT and Tau−/− animals in order to label dividing newborn neurons, and then split these animals into two experimental conditions: mice subjected to the Porsolt test (P), which is considered an acute stress model; and control mice not subjected to this test (CNP). Animals were sacrificed 1 week after CldU administration. Mice were subjected to the Porsolt test on the 2 days prior to sacrifice (Fig 4A and B). Figure 4C shows representative images of 1-week-old CldU-labeled hippocampal newborn neurons of WT CN
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