Synaptic vulnerability to amyloid-β and tau pathologies differentially disrupts emotional and memory neural circuits

ARTICLE OPEN Synaptic vulnerability to amyloid-βand tau pathologies differentially disrupts emotional and memory neural circuits Maria Dolores Capilla-López 1,2 , Angel Deprada 1,2 , Yuniesky Andrade-Talavera 3 , Irene Martínez-Gallego 3 , Heriberto Coatl-Cuaya 3 , Paula Sotillo 1,2 , José Rodríguez-Alvarez 1,2 , Antonio Rodríguez-Moreno 3 , Arnaldo Parra-Damas 1,2 ✉and Carlos A. Saura 1,2 ✉ © The Author(s) 2025 Alzheimer’s disease (AD) is characterized by memory loss and neuropsychiatric symptoms associated with cerebral amyloid-β(Aβ) and tau pathologies, but whether and how these factors differentially disrupt neural circuits remains unclear. Here, we investigated the vulnerability of memory and emotional circuits to Aβand tau pathologies in mice expressing mutant human amyloid precursor protein (APP), Tau or both APP/Tau in excitatory neurons. APP/Tau mice develop ageand sex-dependent Aβand phosphorylated tau pathologies, the latter exacerbated at early stages, in vulnerable brain regions. Early memory deficits were associated with hippocampal tau pathology in Tau and APP/Tau mice, whereas anxiety and fear appeared linked to intracellular Aβin the basolateral amygdala (BLA) of APP and APP/Tau mice. Transcriptome hippocampal profiling revealed gene changes affecting myelination and RNA processing in Tau mice, and inflammation and synaptic-related pathways in APP/Tau mice at 6 months. At 9 months, we detected common and region-specific changes in astrocytic, microglia and 63 AD-associated genes in the hippocampus and BLA of APP/Tau mice. Spatial learning deficits were associated with synaptic tau accumulation and synapse disruption in the hippocampus of Tau and APP/Tau mice, whereas emotional disturbances were linked to Aβpathology but not synaptic tau in the BLA. Interestingly, Aβand tau exhibited synergistic detrimental effects in long-term potentiation (LTP) in the hippocampus but they counteract with each other to mitigate LTP impairments in the amygdala. These findings indicate that Aβ and tau pathologies cause region-specific effects and synergize to induce synaptic dysfunction and immune responses, contributing to the differing vulnerability of memory and emotional neural circuits in AD. Molecular Psychiatry; https://doi.org/10.1038/s41380-025-02901-9 INTRODUCTION Alzheimer’s disease (AD), the major cause of memory loss in the elderly, is accompanied by early neuropsychiatric symptoms that act as early risk factors for conversion to dementia [1,2]. Cognitive and emotional disturbances are associated with pathological changes in the hippocampus and amygdala at early AD stages [3], but how these brain regions cooperate to cause emotional-related memory changes in AD is unclear. Age and sex affect differentially regional accumulation of amyloid-β(Aβ) and tau pathologies, and cognitive and mood disturbances (depression, anxiety, fear, apathy) [4–7]. In AD mouse models, females with enhanced neuropathology are first affected by anxiety and cognitive deficits [8–14]. Young 3xTg-AD females show higher anxiety and reduced memory retention compared to males [13], whereas sexual dimorphism in behavior, neuropathology and inflammatory molecules are also evident in a tauopathy mouse model [14]. However, how Aβand tau interact to cause cognitive and neuropsychiatric symptoms remain unclear, in part because emotional factors are largely underrepresented in basic, pathological and clinical studies [15]. Recent evidence in human demonstrates dissociation effects but also a crosstalk of pathological tauand amyloid-related neural circuit dysconnectivity in memory impairments of AD patients [16–21]. Spatiotemporal pathological changes of tau and Aβare linked with cognitive decline in aging, and they cause selective regional vulnerability associated with differential gene profiles and cognitive dysfunction in AD [22,23]. Several studies indicate that Aβand/or amyloid plaques increase tau phosphorylation, aggregation and seeding [24–28], and tau inactivation ameliorates Aβ-induced memory deficits independently of amyloid pathology [29,30]. Importantly, Aβand tau induce synapse dysfunction and loss [31], a pathological feature that tightly correlates with cognitive decline [32–34]. Synaptic tau induces synapse dysfunction, instability and loss [35–38], and it dominates over Aβon neural circuit disruption [39,40]. Aβand tau affect both presynaptic and postsynaptic mechanisms impairing excitatory glutamatergic transmission [35,41,42], but whether synaptic tau is responsible and/or synergize with Aβto induce synapse pathology and behavioral changes is unclear. Moreover, the mechanisms of neuropathological crosstalk between Aβ Received: 19 April 2024 Revised: 22 December 2024 Accepted: 16 January 2025 1 Institut de Neurociències, Department de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain. 2 Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain. 3 Department of Physiology, Anatomy and Cell Biology, Universidad Pablo de Olavide, Sevilla, Spain. ✉email: arnald[email protected]; [email protected] www.nature.com/mp Molecular Psychiatry 1234567890();,: and tau leading to synaptic dysfunction and vulnerability of emotional and memory neural circuits are still unknown. To better elucidate the cellular mechanisms and factors contributing to disruption of cognitiveand emotion-related neural circuits in AD, we generated novel double APP/Tau transgenic mice that recapitulate early synaptic, behavioral, and transcriptional alterations linked to AD pathophysiology. Interestingly, whereas spatial learning and memory deficits were associated with synaptic tau pathology in the hippocampus, emotional disturbances were linked to Aβin the basolateral amygdala (BLA). Transcriptional profiling revealed hippocampal gene signatures enriched in inflammatory and synaptic pathways affected by early concomitant Aβ/tau pathology but not by Aβor tau alone. At late stages, specific and coordinated transcriptional responses, including astrocytic, microglia and AD risk genes identified by genome-wide association studies (GWAS) occur in the hippocampus and amygdala of APP/ Tau mice. Our study reveals that Aβand tau affect differentially emotional and memory neural circuits by exerting distinct effects at the transcriptional, functional and behavioral levels. MATERIALS AND METHODS Behavioral, biochemical, transcriptomic, immunohistiochemical and electrophysiological methods are extensively described in Supplementary Information. Mice Control (WT), APP, Tau and APP/Tau transgenic mice were obtained by crossing heterozygous APP Sw,Ind (line J9; C57BL/6) and Tau P301S (line PS19, JAX #008169; C57BL/6) mice [36,43], and housed under standard conditions (n=4–6/cage; 22 ± 2 °C, 12 h light:dark cycle). Sex, age and genotypes are indicated in the figure legends. Experimental procedures were approved by the Animal and Human Ethical Committee (CEEAH) of the Universitat Autònoma de Barcelona and local government (CEEAH/DMAH: 2895/10571, 4750/10839) following European Union regulations (2010/63/EU). Behavioral tests General and anxiety-like behaviors were studied in the open field and dark/light box (DLB) tests [44,45] (see Supplementary Information). For cued fear conditioning (CFC), 6 month-old mice were exposed in context A to a conditioned sound stimulus (CS, 2800 Hz, 80 dB; 30 s) followed by an electric footshock (unconditioned stimulus, US,0.8mA;2s).Freezingbehaviorwas automatically recorded (Video Freeze Software, Med Associates) immediately after the shock (2 min) and again 24 h later in a novel chamber (context B) before (pre-CS; 2 min) and during (CS; 3 min) tone presentation [45]. Mice at 9–10 months were conditioned with two CS-US pairings (context A; US, 1 mA; 2 s). Freezing behavior was recorded during the 2-min interval between pairings and immediately after the shock. At 24 h, 3-min pre-CS period was followed by 16 CS presentations (30 s each, 5-s inter-CS) in context B [46]. Immunohistochemistry Deparaffinized coronal brain sections (5 μm) were antigen-retrieved with citrate buffer (10 mM, pH 6.0) for tau or with formic acid (60%, 5 min) for Aβor Aβ/tau, and incubated with anti-phosphorylated (p)tau (Ser202/ Thr205, AT8, 1:50; Ser202, CP13, 1:50) or anti-Aβ(6E10, 1:1000) antibodies before biotin-conjugated anti-mouse secondary antibodies (1:200), DAB peroxidase staining (Vector laboratories) and imaging (Nikon Eclipse 80i microscope). For astrocytic and microglial stainings, deparaffined coronal sections (5 μm) were antigen-retrieved (citrate buffer) and incubated with anti-GFAP (Dako Z0334; 1:500) or anti-Iba1 (Wako 019-19741, 1:250) antibodies followed by AlexaFluor-488/594-conjugated goat IgGs (1:400) and Hoechst (1:5000) before imaging (Confocal Zeiss LSM 700 microscope). Immunofluorescence staining with APP/Aβ, anti-Aβ42, pTau and neuronal markers, Iba1 and GFAP quantification [47] and brain atrophy and expansion methods are described in Supplementary Information. Bulk RNA transcriptional profiling Bulk RNA-sequencing (RNA-seq) of hippocampus and BLA from 6 and 9 month-old females was performed on an Illumina NextSeq 6000/2000. RNA-seq data analyses, including alignment to the reference genome and differential expression analysis were performed using QuasR/Rhisat2 and DESeq2 packages, and Gene ontology (GO) and functional enrichment analyses using enrichR in Bioconductor [48–50]. Statistical analysis Statistical analysis was performed using parametric oneor two-way Analysis of Variance (ANOVA) or non-parametric Kruskal-Wallis tests according to D’Agostino-Pearson omnibus normality test (Prism software, GraphPad 8.0.2). For multiple comparisons, we used Sidak’s or Tukey’s post hocs for parametric tests and Dunn’s post hoc for non-parametric test. Parametric unpaired two-tailed Student’s t-test or non-parametric MannWhitney test, according to D’Agostino-Pearson omnibus normality test, were used when two groups were compared. Pvalues less than 0.05 were considered significant. Adjusted Pvalues (padj) less than 0.1 were considered significant in the transcriptomic analysis. The sample size was calculated using the “Power and Precision”software to ensure adequate power while adhering to the 3Rs principle. Randomization was performed by an independent investigator who assigned animal codes and sample order, ensuring unbiased allocation and blindness to the genotypes during data acquisition. Grubbs’test was used to identify outliers. RESULTS Aβand tau accumulation in excitatory neurons results in agedependent cerebral AD pathology To investigate the specific contribution of Aβand tau on memory and emotional disturbances, we crossed APP Sw,Ind and Tau P301S transgenic mice to generate double APP/Tau transgenic mice that express human APP and Tau translated transcripts in hippocampal excitatory CaMKIIαbut not inhibitory parvalbumin neurons (Supplementary Fig. 1A). Immunofluorescence analysis revealed high localization of hAPP/Aβ(6E10) and total/phosphorylated (p) Tau in excitatory neurons (CaMKIIα, vGlut1, L-glutamate) but not inhibitory interneurons (GAD-67, parvalbumin, somatostatin) in APP/Tau hippocampus, and some colocalization of Aβwith GAD-67 in the BLA (Supplementary Fig. 1B). APP and APP/Tau mice at 6 months show elevated human APP (∼2 fold, APP CTF antibody;∼17 fold, 6E10), APP α-CTFs and intraneuronal Aβ42 (MOAB-2) but unchanged APP β-CTFs (Supplementary Fig. 2A, B). Tau was similar in Tau and APP/Tau mice (∼25 fold) but phosphorylated (p)Tau (Ser202/Thr205) levels were significantly increased in APP/Tau mice (Supplementary Fig. 2A; P<0.01). APP and APP/Tau mice show similar intracellular Aβstaining in CA1/ CA3 hippocampus with no staining in the entorhinal cortex (EC) and BLA at 6 months (Kruskal-Wallis test, CA1: P< 0.0001; CA3: P< 0.0001; EC: P> 0.05; BLA: P> 0.05; Fig. 1A). At 9 months, male and female APP and APP/Tau mice show similar number of Aβ-positive neurons and amyloid plaques in hippocampal and cortical regions, except for an increase of Aβ-positive neurons in the EC of females (Two-way ANOVA, EC, genotype effect: F (3, 37) =12.78, P< 0.0001; sex effect: F (1, 37) =10.10, P< 0.01; interaction: F (3, 37) =3.43, P< 0.05; Fig. 1B; Supplementary Fig. 3). The number of pTau (Ser202/Thr205)-positive neurons was significantly increased in APP/Tau mice at 6 months (Kruskal-Wallis test, CA1: P<0.001;CA3:P< 0.001; one-way ANOVA, EC: P< 0.001; BLA: P< 0.001; Fig. 1A).At9months,pTau(Ser202,CP13)was prominently increased in neuronal cell bodies and fibers of hippocampus and cortex of male and female Tau and APP/Tau mice (∼20–40 fold; Fig. 1B, Supplementary Fig. 3), whereas, compared to males, APP/Tau females show elevated pTau in CA3 (Two-way ANOVA, CA3, genotype effect: F (3, 35) =43.57, P< 0.0001; sex effect: F (1, 35) =7.87, P< 0.01) (Fig. 1B). Interestingly, APP/Tau mice develop prominent hippocampal atrophy at 9 months (P<0.01–0.0001; Supplementary Fig. 2C). These results demonstrate that APP/Tau mice develop age-dependent Aβand tau pathologies with no major sex differences. Hippocampal-dependent learning/memory deficits in young Tau and APP/Tau mice Analysis of general behavior in the open field revealed no significant differences among genotypes at 6 months in total M.D. Capilla-López et al. 2 Molecular Psychiatry travelled distance, time inactive and percentage of time in the center (C) and periphery (P) at days 1 and 2 (Supplementary Fig. 4A). DLB and cued fear conditioning (CFC) tests revealed no significant changes in anxiety, neophobia, and fear memory among the transgenic lines at this age (Kruskal-Wallis test and two-way ANOVA, P> 0.05; Fig. 2A, B). In the Morris water maze (MWM), all groups showed similar swimming speeds (P> 0.05) and decreased latencies during spatial training (Two-way ANOVA, training effect: F (4, 390) =37.40, P< 0.0001), although Tau and APP/Tau mice exhibited significantly longer latencies starting at day 2 (Genotype effect: F (3, 390) =25.95, P< 0.0001; Fig. 2C, D). In the probe trial, control mice displayed a preference for the target quadrant (P< 0.05), whereas APP, Tau and APP/Tau mice showed reduced target quadrant occupancies (Two-way ANOVA, interaction effect: F (3, 156) =5.86, P< 0.001) (Fig. 2C, D). This result suggests that spatial learning deficits are associated with hippocampal tau pathology in Tau and APP/Tau mice. Fig. 1 Aβpotentiates early tau pathology in AD vulnerable brain regions in APP/Tau mice. Coronal brain sections of control (WT), APP, Tau and APP/Tau mice at 6 months (A) and 9 months (B) were stained with anti-human Aβ/APP (6E10; top) and pTau (Ser202, AT-8 (A) or CP13 (B); bottom) antibodies. Left images: Representative low and high (insets) magnified images of Aβand pTau-stained neurons in CA1 and CA3 hippocampus, entorhinal cortex (EC) and basolateral amygdala (BLA). Amyloid plaques in a 9 month-old APP/Tau mouse are visualized in the top right inset of CA3 region. Objective: 20×. Scale bar: 50 μm. Right diagrams: Quantification of Aβ-positive cells in WT (white symbols), APP (red symbols), Tau (blue symbols), and APP/Tau (grey symbols) mice. Data represent mean number ± SEM of APP/Aβ-and pTau-positive cells/μm 2 (×10 −5 )(n=3–4 slices/mouse). Number of mice (male/female), 6 months: WT (2/6), APP (1/5), Tau (0/5) and APP/Tau (3/5); 9 months: WT (6/6), APP (5/6), Tau (5/5) and APP/Tau (4-5/6-7). Statistical analysis was performed using one-way ANOVA or non-parametric Kruskal-Wallis tests according to the D’Agostino-Pearson omnibus normality test, followed by Tukey’s or Dunn’s post hoc test, respectively (A), and two-way ANOVA followed by Sidak’s (sex comparison) and Tukey’s (genotypes comparison within each sex) multiple comparison tests (B). * P< 0.05, ** P< 0.01, *** P< 0.001, **** P< 0.0001 vs WT or the indicated group. M.D. Capilla-López et al. 3 Molecular Psychiatry Age-dependent emotional disturbances in APP and APP/ Tau mice General behavior evaluated in the open field revealed no significant differences in travelled distances in all male and female groups at 9–10 months, except for increased activity on day 1 in APP/Tau females, indicating similar ambulatory locomotor activities (Supplementary Fig. 4B). In the DLB test, 9 month-old APP and APP/Tau mice of both sexes, but not Tau mice, showed reduced entries (One-way ANOVA, male: F (3, 36) =6.88, P< 0.001; females: F (3, 29) =5.16, P< 0.01), and higher latencies (except male APP/Tau mice; male: F (3, 36) =2.98, P< 0.05; females: F (329) =4.47, P< 0.05) into the light compartment (Fig. 2E, G), suggesting increased anxiety in APP and APP/Tau mice. In CFC, innate freezing responses before shock (neophobia) and immediately after shock are similar in female groups (Two-way ANOVA, genotype effect: P> 0.05), whereas males show a main significant effect of genotype (F (3, 108) =5.23, P< 0.01) and treatment (F (2, 108) =15.70, P< 0.0001) (Fig. 2F, H). At 24 h, all groups showed similar and significant CS-induced freezing responses indicating fear memory consolidation. However, there was a significant effect of genotype and tone Fig. 2 Differential Aβand tau effects on spatial memory and emotional disturbances in AD transgenic mice. A–DBehavior of AD transgenic mice at 6 months. ANumber of entries and latencies to light zone in the DLB test. BAssociative memory in the fear conditioning test. Freezing responses in the CFC during conditioning (day 1, left) and training (day 2, right) of WT, APP, Tau and APP/Tau mice. CSpatial memory training in the MWM for 5 days. D% Time in target quadrant vs others on day 5 probe trial in the MWM. Heat maps show decreased occupancies and trajectories of transgenic mice in the target quadrant (marked in white). Data represent mean ± SEM. For A,Bnumber of mice (male/female): WT (4/6), APP (5/5), Tau (2/8) and APP/Tau (4-5/4). For C,Dnumber of mice (male/female): WT (12/15), APP (11/6), Tau (7/8) and APP/Tau (9/14). E–HBehavior of AD transgenic mice at 9 months. E,GAnxiety behavior of male (E) and female (G) mice in the dark/light test. F,H Associative memories of male (F) and female (H) mice in the CFC test. Freezing responses during conditioning (day 1) and training (CS 1: conditioned stimulus 1) or extinction (CS 2–8) on day 2 in the CFC. The mean ± SEM of time freezing (%) during CS (period from 2 to 8) is represented at the right. In all cases, data represent mean ± SEM. Number of mice (male/female) for E–H: WT (14/8, white bars/symbols) APP (9/8, red bars/symbols), Tau (10/9, blue bars/symbols) and APP/Tau (7/8, grey bars/symbols). Statistical analysis was determined by one-way or nonparametric Kruskal-Wallis tests followed by Tukey’sorDunn’s post hoc test (A,E,G) or two-way ANOVA (B–D,F,H) followed by Tukey’s post hoc test for genotype comparisons. * P< 0.05, ** P< 0.01, *** P< 0.001, **** P< 0.0001 vs the indicated group or control mice (C,F,H). M.D. Capilla-López et al. 4 Molecular Psychiatry during extinction training (males: genotype effect: F (3, 324) =9.57, P< 0.0001; tone effect: F (8, 324) =9.51, P< 0.0001; females: genotype effect: F (3, 306) =12.56, P< 0.0001; tone effect: F (8, 306) =11.48, P< 0.0001) (Fig. 2F, H). Particularly, APP and APP/Tau mice of both sexes show enhanced freezing responses compared to control and Tau mice (One-way ANOVA, male: F (3, 24) =7.00, P< 0.01; females: F (3, 24) =12.51, P< 0.0001) (Fig. 2F, H). These results indicate that fear-related emotional symptoms are associated with Aβaccumulation in the amygdala of APP and APP/Tau mice. M.D. Capilla-López et al. 5 Molecular Psychiatry Early Aβ/tau-induced transcriptional responses in the hippocampus are related to synapse disruption and inflammation To identify transcriptional responses related to biological pathways affected by early Aβand tau pathologies, we performed genome-wide bulk RNAseq analysis followed by pathway enrichment analysis in the hippocampus of littermate female WT, APP, Tau and APP/Tau mice at 6 months. Gene expression analysis revealed differentially expressed genes (DEGs) in the hippocampus of APP (94: 39 up, 55 down), Tau (1551: 743 up, 808 down) and APP/Tau (1129: 808 up, 321 down) mice (Fig. 3A). In APP mice, we found significant upregulation of genes related to metabolic processes (Idh1,Ugt2b34) and oxidative stress (Prdx1,Gstm1), the latter shared with APP/Tau mice but not Tau mice. GO analysis revealed that the top significantly enriched pathways of the upregulated genes were related to gliogenesis and axon ensheathment/myelination in Tau mice (Bmp2,Dlx2,Cst7,Itgax, Opalin, Tenm4), and immune responses in APP/Tau mice (Aif1,C3, Cd9,Clu, Cnp, Cxcl5, Sox10). By contrast, downregulated genes were related to RNA processing/splicing in Tau mice (Cdk9,Celf3, Celf5, Mettl3, Srsf7, Ybx2), and synapse organization, transporters and receptors in APP/Tau mice (Bdnf,Cacnb3,Fgf13,Dlg4,Negr1, Nptxr). The common upregulated (178) and downregulated (54) genes shared between Tau and APP/Tau mice were related to myelination/glial differentiation and synapse organization/transmission pathways, respectively (not shown). Consistently, cellular pathways related to immune/leukocyte responses (upregulation) and synapse function/ion transporters/neurotransmitter receptors (downregulation) were altered in APP/Tau hippocampus at 6 and 9 months of age (Supplementary Fig. 5A). These results indicate that Aβand tau cooperate to induce synaptic disruption and immune responses at the transcriptional level. We next examined preand post-synaptic proteins and tau in purified hippocampal synaptosomes and/or brain sections of 6 month-old mice. Biochemical analysis revealed no major changes in synaptic proteins in hippocampal lysates of transgenic mice despite ∼45% of APP/Tau mice showed decrease of these proteins (Fig. 3B). In purified hippocampal synaptosomes, we found decrease of Homer-1 in APP mice, and synaptophysin, Homer-1 and PSD95 in Tau mice, whereas all analyzed synaptic proteins and β-actin were decreased in APP/Tau mice (Fig. 3B). Tissue expansion revealed high colocalization of pTau (Ser 202) with the glutamatergic postsynaptic protein Homer1 in the hippocampus (53%) but not in the BLA (15%) of APP/Tau mice (Fig. 3C). Changes in preand post-synaptic proteins occur in parallel with synaptic tau accumulation (Fig. 3B), which suggests that synaptic pathological tau contributes to hippocampal synapse pathology in APP/Tau mice. Tau and Aβcooperate to induce differential brain regional effects in synaptic plasticity We next investigated the functional effects of neuronal amyloid and tau pathologies on hippocampal and amygdalar synaptic transmission and plasticity. Input-output curves in Schaffer collateral CA3/CA1 synapses were similarly decreased in all mutant transgenic female mice at 6 months (Two-way ANOVA, stimulus effect: F (8, 180) =19.90, P< 0.0001; genotype effect: F (3, 180) =34.34, P< 0.0001; interaction effect: F (24,180) =2.24, P< 0.01; Fig. 4A). Short-term synaptic plasticity (STP), and longterm potentiation (LTP) induction, measured as early-LTP (60 min) and late-LTP (120 min) were significantly impaired in APP/Tau hippocampus compared with the rest of groups (One-way ANOVA, STP: F (3, 24) =4.06, P< 0.05; E-LTP: F (3, 24) =3.41, P< 0.05; L-LTP: F (3, 24) =7.00, P< 0.01; Fig. 4A–C). Paired-pulse facilitation (PPF) ratio was reduced in APP and APP/Tau mice (F (3, 21) =5.37, P< 0.01; Fig. 4D). Whole-cell patch-clamp recordings revealed impaired NMDA responses and NMDA/AMPA ratio in APP/Tau mice (F (3, 36) =3.44, P< 0.01; Fig. 4E). These results demonstrate that tau and Aβcooperate to disrupt hippocampal synaptic plasticity through postsynaptic mechanisms. In thalamic-LA synapses, basal synaptic transmission was similarly decreased in APP, Tau and APP/Tau mice at 6 months (Two-way ANOVA, stimulus effect: F (12,481) =3.45, P< 0.0001; genotype effect: F (3, 481) =10.11, P< 0.0001; Fig. 4F). Field recordings revealed decreased STP induction in APP, Tau and APP/Tau mice (F (3, 34) =9.01, P< 0.001), and E-LTP and L-LTP deficits in APP and Tau mice but not in APP/Tau mice (E-LTP: F (3, 34) =7.61, P<0.001; L-LTP: F (3, 34) =11.29, P< 0.0001; Fig. 4G, H). Basal PPF was reduced in APP mice and recovered in APP/Tau mice (F (3, 36) =5.13, P< 0.01; Fig. 4I). NMDA/AMPA ratios were not significantly affected in the transgenic groups (F (3, 40) =0.41, P> 0.05; Fig. 4J). Together, these findings indicate that, in contrast to the hippocampus, Aβand tau counteract each other to maintain synaptic plasticity in the amygdala of young mice. Coordinated and divergent transcriptional changes related to inflammation and AD-associated genes occur in the hippocampus and BLA of APP/Tau mice To identify gene signatures and networks differentially affected by late Aβ/tau pathology in the hippocampus and BLA, we next assessed DEGs of female WT and APP/Tau mice at 9 months. We identified 5985 hippocampal genes (3260 up, 2725 down) and 2168 BLA genes (1263 up, 905 down) deregulated in APP/Tau mice, with a high number of DEGs (1423: 24% hippocampus, 65% BLA) coordinately deregulated in both regions (Fig. 5A, B). The most significant enriched pathways affected in both regions were related to neutrophil immune response (upregulation: Arhgap9, Cd33, Cd68, Itgam, Lamp1, Nfam1, Stk10, Ticam2) and neurotransmission/glutamate receptor (downregulation: Camk2a,Dlgap1/4, Gria2/3,Grin1, Homer1, Lrrk2, Mapk8ip2, Mink1, Shank1, Syn1) (Fig. 5C). By contrast, the most significantly divergent enriched pathways of upregulated genes in APP/Tau mice were fatty acid metabolism (Echs1,Eci1,Echdc3,Hadha,Hadhb,Hsd17b4/10) in the hippocampus, and Aβbinding, GTPase regulation and oxidoreductase/NAD (Adrb2,Bdh2,Hspg2, Itgb2,Ncf1/2/4, Rnls,Sirt2,Sirt5, Tlr2) in the BLA (Fig. 5C; Table 1). In addition, genes highly expressed in reactive astrocytes (Gfap, ApoE) and diseaseassociated microglia (DAM) (Ccl6,Cd33,Csf1, Cst7, Cx3cr1,Itgax, Trem2,Tyrobp) were upregulated in hippocampus and/or BLA of APP/Tau mice (Supplementary Table 1). Accordingly, GFAP and Fig. 3 Differential transcriptional profiles and synapse pathology in the hippocampus of AD transgenic mice. A Venn and gene ontology diagrams illustrating the differentially expressed genes and enriched cellular pathways altered in the hippocampus of 6 month-old female APP (n=8), Tau (n=8) and APP/Tau mice (n=10) compared with controls (WT, n=11). BBiochemical analysis of preand post-synaptic proteins and tau (D1M9X antibody) in hippocampal lysates and purified synaptosomes of WT, APP, Tau and/or APP/Tau mice at 6 months (n=8–11 mice/group). Data represent mean levels (fold change) normalized to β-tubulin ± SEM. Number of mice (male/female): 11 WT (6/5, white symbols), 8 APP (5/3, red symbols), 8 Tau (5/3, blue symbols) and 9 APP/Tau (5/4, grey symbols). CConfocal microscope images of cleared expanded hippocampal and BLA sections (expansion factor 3.63x) of 6 month-old WT and APP/Tau mice showing colocalization (yellow; quantification at the right graph) of postsynaptic Homer1 (green) and pTau (Ser 202; red) in the CA1 hippocampus but not in the BLA of APP/Tau mice. Right top insets are magnified images of the indicated squared regions. Scale bars: 10 μm and 36.30 μm (expanded). Statistics were determined by one-way ANOVA or Kruskal-Wallis tests followed by Dunnet’s or Dunn’s multiple comparisons tests according to the normality test, respectively (B) or by two-tailed Student’s t-test (C). * P< 0.05, ** P< 0.01, *** P< 0.001 vs non-transgenic control group. M.D. Capilla-López et al. 6 Molecular Psychiatry Fig. 4 Brain regional specific effects of tau on Aβ-induced synaptic plasticity impairments. A, F Input-output curves showing EPSC amplitudes (A) and fEPSP slope (F)vs the applied stimulus intensity in CA3/CA1 hippocampal (A) and thalamus-lateral amygdala (LA) (F) synapses of control (WT, white), APP (red), Tau (blue), and APP/Tau (grey) mice. B, G Traces (top) and time course (bottom) of fEPSPs before and after LTP induction in CA3-CA1 hippocampal (B) and thalamus-LA synapses (G). C, H Histograms showing short-term synaptic plasticity (STP), early long-term potentiation (E-LTP), late LTP (L-LTP) in CA3-CA1 hippocampal (C) and thalamus-LA synapses (H). D, I Paired-pulse facilitation (PPF) does not change after LTP induction in the hippocampus (D) or amygdala (I), and baseline PPFs of APP are decreased in the hippocampus and amygdala compared to controls. However, PPF of APP/Tau mice is significantly decreased only in hippocampus. E, J NMDA/ AMPA ratio was decreased in hippocampal neurons of APP/Tau mice compared to control, APP, and Tau mice (E). In amygdala (J), NMDA/AMPA ratio was not affected in the transgenic mice. Data represent mean ± SEM from electrophysiological recordings in CA3-CA1 and thalamic-LA synapses of 6 month-old female control (n=7–8), APP (n=5–6), Tau (n=7–8), and APP/Tau (n=6) mice. Statistical analysis was determined by one-way ANOVA or Kruskal-Wallis tests according to the D’Agostino-Pearson omnibus normality test, followed by Tukey’s or Dunn’s post hoc test, respectively (D, E, I, J) or two-way ANOVA (A, F) followed by Tukey’s post-hoc test. * P< 0.05, ** P< 0.01, *** P< 0.001, **** P< 0.0001 vs the indicated group. M.D. Capilla-López et al. 7 Molecular Psychiatry Iba1 stainings were significantly elevated in the CA1/CA3 hippocampus and BLA of APP/Tau mice (One-way ANOVA, F(3, 22) =3.51, P< 0.05; BLA: F(3, 22) =5.44, P< 0.01; F(3, 20) =3.82, P< 0.05; BLA: F(3, 22) =4.10, P< 0.05) (Fig. 5E). Importantly, 63 mouse orthologs of human AD-associated genes previously identified in GWAS (e.g, APOE,BIN1,CD33,CLU,MS4A4A, PICALM,PLCG2, PTK2B, SLC24A4, SORL1,TREM2,USP6NL) were deregulated in the hippocampus and/or BLA of female APP/Tau mice (Fig. 5C; Table 1)[51,52]. Intriguingly, AD-associated genes related to synapse function and ion transport (NCS1, NKAIN2, M.D. Capilla-López et al. 8 Molecular Psychiatry PDE7B, SLC24A4, SLC4A8, TSPAN13) were specifically downregulated in APP/Tau hippocampus at 9 months. In addition, 23 of these genes were distinctly altered in the hippocampus of APP, Tau and APP/Tau mice at 6 months (Supplementary Fig. 5B). Remarkably, ten mouse orthologs of these human AD-related genes (AHNAK,ARHGAP20,BCL3,CELF1,CLU,INPP5D,NKAIN2, PDE7B,STK32B, TREM2) were shared and changed in the same direction in the hippocampus of APP/Tau mice at 6 and 9 months but not in Tau mice (Fig. 5D; Supplementary Table 2). These results suggest that coordinated specific transcriptional responses related to inflammation and synapse function occur in response to Aβ/tau pathology in the hippocampus and BLA. DISCUSSION Recent compelling evidence indicates that Aβand tau pathologies exert synergistic effects on synaptic dysfunction and memory loss, suggesting that therapeutic approaches targeting only one of these factors may not be sufficient for achieving clinical benefits [31,53]. Accordingly, immunotherapy clinical trials targeting Aβor tau showed promising biomarker reductions but limited cognitive benefits [54,55], likely because multiple pathological factors contribute to the disease process [56]. Discerning the pathological mechanisms of Aβand tau crosstalk is critical to develop effective AD therapies. Importantly, understanding how Aβand tau pathologies are mechanistically linked in specific brain circuits may help to elucidate their role in cognitive and neuropsychiatric symptoms. Here, we show that APP/Tau mice replicate the key pathological features of AD, including early intracellular Aβand potentiation of tau pathology, synapse dysfunction, inflammation, neuron loss and deregulation of disease-related gene signatures. Comprehensive analysis of single and double transgenic mice, indicate that Aβenhances neuronal pathological tau at early disease stages, coinciding with tau-induced disruption of synaptic function, memory, and altered transcriptional profiles related to inflammation and synaptic pathways. Conversely, altered emotional responses were linked to the presence of Aβin the amygdala at late pathological stages. Our study reveals distinct pathological vulnerability of memory and emotional circuits, with the presence of pathological tau in hippocampal glutamatergic neurons linked to spatial memory deficits, and Aβaccumulation in the BLA associated with fear emotional disturbances. Hippocampal tau pathology is a strong predictor of memory decline by disconnecting neural networks [18,57], and synergizes with Aβto disrupt hippocampal function and memory performance in older adults [16,17]. The regionspecific susceptibility to pathology may be exacerbated by the effect of Aβon the progression of neuronal tau pathology, as previously shown in double APP-V717I/Tau-P301L mice [58], which can be mediated by cellor non-cell-autonomous (e.g. microglia) effects [59]. As reported in AD [32,33,41,60], synaptic tau was associated with reduced glutamatergic synaptic proteins and impaired hippocampal-dependent learning in both Tau and APP/ Tau mice. This similar phenotype occurs despite significant differences in early hippocampal synaptic plasticity, inflammation, and dysregulation of transcriptional programs, suggesting that alternative cellular and molecular mechanisms converge to induce hippocampal dysfunction in Tau and APP/Tau mice. Based on our transcriptomic analysis, these alternative mechanisms may implicate molecular changes in RNA processing/splicing and synaptic genes/pathways linked to dementia and intellectual disability (e.g. Bdnf, Cacnb3,Dlg4,Fgf13…) in Tau and APP/Tau mice, respectively [61–63]. The presence of Aβpathology in the EC and BLA coincided with anxiety and emotional disturbances in 9–10 month-old APP and APP/Tau mice, further emphasizing the detrimental effects of Aβon emotional and mood behaviors in AD [45,64,65]. Amygdala pathology affects key cellular pathways related to inflammation, neurotransmission, Aβbinding, GTPase activity, and oxidoreductase/NAD+activity. Given the link among emotional symptoms, inflammation, and neuronal excitability, it is plausible that pharmacological interventions aimed to enhance GABAergic neurotransmission or to mitigate inflammation and oxidative stress could represent promising therapeutic strategies for neuropsychiatric symptoms in dementia [45,64]. Notably, tau counteracted the Aβ-induced synaptic transmission and plasticity deficits in the amygdala at early stages. The mechanisms underlying taumediated protection of synaptic function remain unknown; however, it is intriguing that this coincides with the absence of synaptic tau in this region. As the pathology advances, deficits in this neural circuitry may arise alongside dysregulation of glutamatergic synaptic genes, as observed in APP knock-in mice [66]. Notably, female APP/Tau mice at 9 months exhibited a regionspecificincreaseofAβin EC and pTau in CA3 compared to males. This finding is particularly significant considering that women are more susceptible to emotional disturbances and experience faster cognitive decline. The molecular factors driving sex-specific differences in emotional and memory changes remain poorly understood and warrant further investigation. A potential explanation may be the distinct cellular vulnerability of women to pathological changes, including Aβand/or neurofibrillary tangles [67–70]. The temporal and regional associations of Aβand tau pathologies with specific behavioral changes do not exclude the possibility that both factors may contribute to memory and emotional decline [17]. Besides the strong correlation between cerebral Aβdeposition and anxiety in elderly non-demented individuals [71,72], tau and Aβ/tau pathologies are closely associated with neuropsychiatric symptoms in dementia [73,74]. Considering that anxiety is reversed by anti-tau therapeutic treatments [75–77], the apparent lack of tau effects on emotional symptoms could be explained by dominant effects of Aβover tau, differences in cell-specific transgene expression and/or agedependent pathological differences in APP/Tau mice. In this context, gene profiling analysis identified critical genes related to inflammation and synaptic function in two vulnerable brain regions in AD, which is particularly relevant considering the strong correlation between inflammatory and synaptic genes and the progression of AD [78–80]. Nonetheless, our transcriptomic Fig. 5 Bulk RNAseq reveals differential and common gene expression signatures associated with AD in hippocampus and BLA of APP/ Tau mice. A Number and Venn diagram illustrating the total, up and down differentially expressed transcripts in the hippocampus (HPC) and BLA of 9 month-old female APP/Tau mice (n=9) compared to controls (WT, n=11 for HPC; n=10 for BLA). BVolcano plots showing the fold change of genes differentially expressed in the hippocampus and BLA of APP/Tau mice ( cut-off: Pvalue < 0.01, log2FC > 0.5). Some significant deregulated AD risk genes are indicated (see also Table 1). CFunctional set enrichment of differentially expressed genes in APP/Tau hippocampus and BLA. The plots show the top significantly enriched pathways from the GWAS catalog (2019) (left) and GO Biological Process/ Molecular Function (2021) (right) databases. Human orthologs were used in the analysis with GWAS catalog database. DVenn diagram illustrating the AD-associated genes identified previously by GWAS shared in the hippocampus of 6 and 9 month-old female APP/Tau and/or Tau mice. EConfocal microscope images (left) and quantification (right graphs) of percentage of GFAP (magenta; top images) and Iba1 (magenta; bottom images) areas, and Hoechst (blue) staining in CA1 hippocampus and BLA sections of female 9 month-old WT (n=7), APP (n=7), Tau (n=4−5) and APP/Tau (n=6−7) mice. Insets are magnified images of the indicated regions. Scale bars: 30 μm. Statistical analysis was determined by one-way ANOVA followed by Tukey’s post-hoc test. * P< 0.05, # P=0.06 vs the indicated control group. M.D. Capilla-López et al. 9 Molecular Psychiatry