Telomeric DNA damage response mediates neurotoxicity of Aβ42 oligomers in Alzheimer's disease

Abstract

Ageing is the major risk factor for Alzheimer's disease (AD), the most common neurodegenerative disorder. DNA damage is a hallmark of ageing, particularly when occurring at telomeres, genomic regions vulnerable to oxidative damage and often challenging for the cell to repair. Here, we show that brains of 3xTg-AD mice, an established AD model characterized by amyloid-β (Aβ)-induced pathology, exhibit increased activation of DNA damage response (DDR) pathways at telomeres. Exposure of mouse primary hippocampal neurons to 42-residue Aβ (Aβ42) oligomers, a significant pathogenetic contributor to AD, triggers telomeric DDR by increasing the levels of reactive oxygen species caused by calcium imbalance. Antisense oligonucleotides targeting non-coding RNAs generated at damaged telomeres in vivo (in 3xTg-AD mice) and in vitro reduce neurotoxicity in iPSC-derived human cortical neurons and mouse primary neurons while inhibiting Aβ42-induced telomeric DDR, and restore transcriptional pathways altered by Aβ and found dysregulated in AD patients. These results unveil an unexpected role of telomeric DNA damage responses in Alzheimer's disease pathogenesis, and suggest a novel target for the development of RNA-based therapies.

Keywords: ASO; Aging; Alzheimer’s disease (AD); DNA damage response (DDR); Telomeres.

Figure 1. Markers of tDDR activation and pro-apoptotic gene expression are elevated in the brain cortex of 3xTg-AD mice.

(A–H) Brain sections from 12 months old wt and 3xTg-AD mice were analyzed. Each circle in the graphs represents one mouse analyzed. (A) Representative confocal microscopy images of immunofluorescence staining of isocortex from wt and 3xTg-AD mice with antibodies against γH2AX, 53BP1, MAP2 and nuclei were stained with DAPI. Bar graph shows the quantification of the average number of γH2AX foci per nucleus (n = 4 wt and n = 5 3xTg-AD), and 53BP1 foci per nucleus (n = 5 wt and n = 4 3xTg-AD). Arrows point to γH2AX or 53BP1 foci. At least 50 nuclei per mouse section were scored. (B) Representative confocal microscopy images of ImmunoFISH staining combining γH2AX, MAP2 antibodies and FISH for telomeric DNA using a complementary PNA probe (left panel) and co-immunofluorescence with antibodies against γH2AX, MAP2 and the centromeric protein CREST (right panel) of sections from wt and 3xTg-AD mice. Nuclei were stained with DAPI and showed as dash circle in the lower panel. The arrows indicate the colocalizing events. At least 50 nuclei per mouse section were scored. (C) Bar graphs show the percentage of DDR foci (γH2AX foci signal) that localize at telomeres (telomeric PNA probe signal, upper graph)- or centromeres (CREST signal, lower graph)- as detected by immunoFISH (n = 4 for wt and n =  9 3xTg-AD) or immunofluorescence (n = 4 for wt and n = 4 for 3xTg-AD mice), respectively. At least 50 nuclei per mouse section were scored. (D) Bar graphs show the percentage of telomeres—n = 4 for wt and n = 6 for 3xTg-AD—(telomeric PNA probe signal, upper graph) or centromeres—4 wt animals and 3xTg-AD mice—(CREST signal, lower graph)—colocalizing with DDR foci (γH2AX foci signal) as detected by immunoFISH or immunofluorescence, respectively. At least 50 nuclei per mouse section were scored. (E) Bar graphs show the quantification of G-rich and C-rich tdincRNA levels in 3xTg-AD (n = 9/10) mice relative to wt (n = 4) samples as measured by RT-qPCR. (F) Bar graphs show the quantification of p21 mRNA levels in 3XTg-AD (n = 4) mice relative to wt samples (n = 4) in isocortex as measured by RT-qPCR. (G) Bar graph represents the quantification of the ratio between the mRNA levels of Bax over Bcl-2 in 3xTg-AD (n = 5) mice relative to wt (n = 4) samples in isocortex as measured by RT-qPCR. (H) Representative confocal microscopy images of Immunofluorescence staining combining antibodies against MAP2, BAX and BCL-2 of sections from wt and 3xTg-AD mice (left panel). Bar graph represents the quantification of the ratio between the signal of Bax over BCL-2 from immunofluorescence staining in 3xTg-AD (n = 5) mice relative to wt (n = 4) samples in isocortex. For all graphs, unpaired t test was applied. Data are represented as mean ± SEM. Source data are available online for this figure.

Figure 2. Aβ42 oligomers induce DDR markers accumulation at telomeres in mouse hippocampal primary neurons.

(A–I) Mouse hippocampal primary neurons were treated with vehicle or 10 μM Aβ42 oligomers for 48 h. Each circle represents an independent biological replicate. (A) Representative confocal image of immunofluorescence staining of primary neurons with antibodies against MAP2 (magenta) as neuronal marker, γH2AX, 53BP1 and pATM (yellow) as DDR markers, nuclei were stained with DAPI. Bar graph shows the quantification of the average number of γH2AX, 53BP1 and pATM foci per nucleus (for each condition n = 3/4). At least 50 cells for each condition in each replicate were analyzed. (B) Representative confocal microscopy images of immunoFISH combining γH2AX antibody and FISH for telomeric DNA using a complementary PNA probe (left panel) and double immunofluorescence combining antibodies against γH2AX and the centromeric protein CREST (right panel) of primary neurons. Arrows point to co-localization signals between γH2AX antibody and PNA probe. (C) Bar graphs show the percentage of DDR foci (γH2AX foci signal) that localize at telomeres (telomeric PNA probe signal - white arrows) or centromeres (CREST signal) as detected by immunoFISH or immunofluorescence, respectively (n = 3). At least 50 cells for each condition in each replicate were analyzed. (D) Bar graphs show the percentage of telomeres (telomeric PNA probe signal)- or centromeres (CREST signal) colocalizing with DDR foci (γH2AX foci signal) as detected by immunoFISH (Vehicle n = 3 and Aβ42 oligomers n = 4) or immunofluorescence (Vehicle n = 3 and Aβ42 oligomers n = 3), respectively. At least 50 cells for each condition in each replicate were analyzed. (E) Bar graphs show the quantification of G-rich and C-rich tdincRNA levels in Aβ42 oligomers treated primary neurons relative to vehicle treated primary neurons samples as measured by RT-qPCR. n = 3. (F) Bar graph shows the quantification by RT-qPCR of p21 mRNA in Aβ42 oligomers treated primary neurons relative to vehicle treated primary neurons. n = 3. (G) RNA-sequencing analysis was performed on n = 2 replicates for Aβ samples and n = 2 replicates for Vehicle conditions. Volcano plot showing the Differentially expressed genes (DEGs) in Aβ42 oligomer-treated vs vehicle-treated primary neurons. Each point represents a gene. The differential analysis was performed on R with DESeq2 (Love et al, 2014),  DEGs were defined as genes with p value adjusted (Benjamini–Hochberg) < 0.05 (genes with logFC >0 are considerate as upregulated, genes with logFC <0 as downregulated). (H) Heatmap showing the 142 DEGs in Aβ42 oligomers-treated vs vehicle-treated primary mouse hippocampal neurons that are concordant with human AD patients vs control patients in the indicated brain areas (concordant DEGs). ACC anterior cingulate cortex, CBE cerebellum, DLPFC dorsolateral prefrontal cortex, FP frontal pole, IFG inferior frontal gyrus, PCC posterior cingulate cortex, PHG parahippocampal gyrus, STG superior temporal gyrus, TCX temporal cortex. (I) The dotplot shows the 20 most significant Gene Ontology Biological processes that emerged from the enrichment analysis of “concordant DEGs” performed with ClusterProfiler. The name of the pathway is reported on the left of the graph, the x-axis represents the gene ratio, dots size represents the number of  DEGs present in the pathway and the color indicates the adjusted p value of the pathway enrichment. Statistical analysis was performed with over-representation analysis for Gene Ontology Biological processes on DEGs, performed with ClusterProfiler (Wu et al, 2021). Unpaired t test was applied and data are represented as mean ± SEM in (A–F). Source data are available online for this figure.

Figure 3. Reactive oxygen species mediate the Aβ42 oligomers-induced DDR at telomeres in mouse hippocampal primary neurons.

(A–G) Mouse primary neurons were treated for 30 min with 1 mM N-acetyl cysteine (NAC) before exposing them to Aβ42 oligomers or vehicle. Each circle represents an independent biological replicate. (A) Representative confocal microscopy images of primary neurons incubated with CM-H₂DCFDA probe, and the green fluorescence arises from the oxidation of the CM-H₂DCFDA probe. White dashed-line circles indicate the nuclei of neurons. Bar correspond to 20 μm. (B) Bar graph represents semiquantitative analysis of intracellular ROS detected by CM-H₂DCFDA. At least 40–50 cells for each condition in each replicate were analyzed (Vehicle, n = 5; Aβ42 oligomers, n = 4; Aβ42 + NAC, n = 4; H₂O₂, n = 6). (C) Bar graph represents cell viability as determined by CellTiter-Glo shown as a percentage of surviving cells compared to the vehicle. (Vehicle, n = 3; Aβ42 oligomers, n = 3; Aβ42 + NAC, n = 3). (D) Representative confocal images of immunofluorescence staining of primary neurons with antibodies against MAP2 (magenta) as neuronal marker, γH2AX (green) as DDR markers, nuclei were stained with DAPI. (E) Bar graph shows the quantification of average number of DDR foci (γH2AX foci signal) per nucleus. At least 50 cells for each condition in each replicate were analyzed. (Vehicle, n = 3; Aβ42 oligomers, n = 3; Aβ42 + NAC, n = 3). (F) Bar graphs show the percentage of DDR foci (γH2AX foci signal) that localize at telomeres (telomeric PNA probe signal) as detected by immunoFISH. (Vehicle, n = 4; Aβ42 oligomers, n = 4; Aβ42 + NAC, n = 4). (G) Bar graphs show the quantification of G-rich and C-rich tdincRNA levels relative to vehicle-treated mouse primary neurons samples as measured by RT-qPCR. (Vehicle, n = 5; Aβ42 oligomers, n = 6; Aβ42 + NAC, n = 6 for G-rich strand and Vehicle, n = 4; Aβ42 oligomers, n = 4; Aβ42 + NAC, n = 4 for C-rich strand. Ordinary one-way ANOVA was applied, and data are represented as mean ± SEM. Source data are available online for this figure.

Figure 4. ROS-induced DDR upon synthetic Aβ42 oligomers administration in mouse hippocampal primary neurons depends on altered Ca²⁺ flux.

(A–D) Primary neurons were treated for 1 h with 500 μM EGTA or 10 μM Memantine and 5 μM CNQX before treatment with Aβ42 oligomers or vehicle. Each circle represents an independent biological replicate. (A) Representative confocal microscopy images of mouse primary neurons incubated with Fluo-4 probe: green fluorescence is generated upon Ca²⁺ binding to the Fluo-4 probe. White dashed-line circles indicate the nuclei of neurons. Bar correspond to 20 μm The bar graph represents semiquantitative analysis of intracellular Ca²⁺-derived fluorescence. At least 40-50 cells for each condition in each replicate were analyzed. (Vehicle, n = 5; Aβ42 oligomers, n = 6; Aβ42 + EGTA, n = 4; Aβ42+Mem/CNQX, n = 3). (B) Representative confocal microscopy images of mouse primary neuron with CM-H₂DCFDA probe: the green fluorescence arises from oxidation of CM-H₂DCFDA probe. White dashed-line circles indicate the nuclei of neurons. The bar graph represents semiquantitative analysis of intracellular ROS intracellular ROS detected by CM-H₂DCFDA. White dashed-line circles indicate the nuclei of neurons. Bar correspond to 20 μm. At least 40-50 cells for each condition in each replicate were analyzed. (Vehicle, n = 3; Aβ42 oligomers, n = 3; Aβ42 + EGTA, n = 3; Aβ42+Mem/CNQX, n = 3). (C) Representative confocal images of immunofluorescence staining of mouse primary neurons with antibodies against MAP2 (magenta) as neuronal marker and γH2AX (yellow) as DDR marker, nuclei were stained with DAPI. (D) Bar graph represents the quantification of the average number of DDR foci per nucleus. At least 50 cells for each condition in each replicate were analyzed. (Vehicle, n =6; Aβ42 oligomers, n = 6; Aβ42 + EGTA, n = 3; Aβ42+Mem/CNQX, n = 3). Ordinary one-way ANOVA was applied and data are represented as mean ± SEM. Source data are available online for this figure.

Figure 5. Antisense oligonucleotides against tdincRNAs inhibit Aβ42 oligomers-induced DDR activation in mouse hippocampal primary neurons.

(A–D) Mouse primary neurons were treated with ASO vehicle (PBS) or 10 μM anti-TeloG ASO or anti-Telo C ASO or Control ASO for 24 h before treatment with Aβ42 oligomers or vehicle. Each circle represents an independent biological replicate. (A, B) Bar graph represents the quantification of the average number of 53BP1 or γH2AX foci per nucleus, respectively. At least 50 cells for each condition in each replicate were analyzed. (n = 3 for each condition). (C) Representative confocal images of immunofluorescence staining of mouse primary neurons with antibodies against MAP2 (red) as neuronal marker, γH2AX (cyan) and 53BP1 (yellow) as DDR markers, nuclei were stained with DAPI. (D) Bar graph shows the ratio between the number of 53BP1 foci normalized on the number of γH2AX foci per nucleus per neuron. (n = 3 for each condition). Ordinary one-way ANOVA was applied and data are represented as mean ± SEM. Source data are available online for this figure.

Figure 6. Telomeric DDR inhibition reduces Aβ42 oligomers-induced neurotoxicity in mouse hippocampal primary neurons.

(A–G) Mouse primary neurons were treated with ASO vehicle (PBS) or 10 μM ASO anti-Telo G or Control ASO for 24 h before treatment with Aβ42 oligomers or vehicle. Each circle represents an independent biological replicate (A) Bar graph shows the quantification of p21 mRNA levels relative to vehicle as measured by RT-qPCR. Controls showed as PBS (full dots) and control ASO (empty dots). (Vehicle, n = 3; Controls = 9, anti-TeloG, n = 4). (B) Bar graph represents cell viability as determined by CellTiter-Glo shown as a percentage of surviving cells compared to the vehicle. Controls are shown as PBS (full dots) and control ASO (empty dots). (Vehicle, n = 7; Controls = 13, anti-TeloG, n = 6). (C) Bar graph represents the quantification of the ratio between the mRNA levels of Bax and Bcl-2 relative to vehicle as measured by RT-qPCR. Controls showed as PBS (full dots) and control ASO (empty dots). (Vehicle, n = 3; Controls = 6, anti-TeloG, n = 3). (D) RNA-sequencing analysis was performed on n = 3 biological replicates for Aβ oligomers ASO Control and n = 3 replicates for Aβ 42 oligomers anti-teloG. Volcano plot showing significant differences in gene expression in primary neurons treated with Aβ42 oligomers Anti-telo G vs Aβ42 oligomers control ASO (differentially expressed genes (DEGs)). Each point represents a gene. The differential analysis was performed on R with DESeq2 (Love et al, 2014), differentially expressed genes were defined as genes with p value adjusted (Benjamini–Hochberg) < 0.05 (genes with logFC >0 are considerate as upregulated, genes with logFC <0 as downregulated). (E) Venn diagram indicating DEGs common between Aβ42 oligomers vs vehicle (549) and Aβ42 oligomers anti-Telo G vs Aβ42 oligomers control ASO (431). 41 of these genes are reverted in the two conditions (Reverted DEGs). (F) The dotplot shows the significant Gene Ontology Biological processes from the enrichment analysis of “Reverted DEGs” performed with ClusterProfiler. The name of the pathway is reported on the left of the graph, the x-axis represents the gene ratio, dots size represents the number of differentially expressed genes present in the pathway and the color indicates the adjusted p value of the pathway enrichment. Statistical analysis was performed with over-representation analysis for Gene Ontology Biological processes on DEGs, performed with ClusterProfiler (Wu et al, 2021). (G) Heatmap showing the nine reverted genes that are concordant with human AD patients vs control patients in the indicated brain areas (concordant reverted DEGs). ACC anterior cingulate cortex, CBE cerebellum, DLPFC dorsolateral prefrontal cortex, FP frontal pole, IFG inferior frontal gyrus, PCC posterior cingulate cortex, PHG parahippocamapal gyrus, STG superior temporal gyrus, TCX temporal cortex. In (A–C), ordinary one-way ANOVA was applied and data are represented as mean ± SEM. Source data are available online for this figure.

Figure 7. Antisense oligonucleotides against tdincRNAs inhibit DDR activation induced by Aβ42 oligomers and reduce Aβ42 oligomer-induced neurotoxicity in human cortical neurons derived from iPSCs.

(A–G) Human cortical neurons derived from iPSCs were treated with ASO vehicle (PBS) or 10 μM ASO anti-Telo G or ASO anti-Telo C or ASO control for 24 h before treatment with Aβ42 oligomers or vehicle. Each circle represents an independent biological replicate. (A–D) DDR analyses. (A, B) Bar graph represents the quantification of the average number of 53BP1 or γH2AX foci per nucleus, respectively. At least 50 cells for each condition in each replicate were analyzed. (n = 3 for each condition). (C) Representative confocal images of immunofluorescence staining of mouse primary neurons with antibodies against MAP2 (red) as neuronal marker, γH2AX (cyan) and 53BP1 (yellow) as DDR markers, nuclei were stained with DAPI. (D) Bar graph represents the quantification of ratio between 53BP1 foci normalized on the numbers of γH2AX foci per nucleus in each neuron. At least 50 cells for each condition in each replicate were analyzed. (n = 3 for each condition). (E) Bar graph shows the quantification of p21 mRNA levels relative to vehicle as measured by RT-qPCR. Controls showed as PBS (full dots) and control ASO (empty dots). (Vehicle, n = 4; Controls = 8, anti-TeloG, n = 4). (F) Bar graph represents cell viability as determined by CellTiter-Glo shown as percentage of surviving cells compared to vehicle. Controls are shown as PBS (full dots) and control ASO (empty dots). (Vehicle, n = 10; Controls = 17, anti-TeloG, n = 10). (G) Bar graph represents the quantification of the ratio between the mRNA levels of Bax and Bcl-2 relative to vehicle as measured by RT-qPCR. Controls are shown as PBS (full dots) and control ASO (empty dots). Vehicle, n = 3; Controls = 6, anti-TeloG, n = 3) Ordinary one-way ANOVA was applied and data are represented as mean ± SEM. Source data are available online for this figure.

Figure 8. Proposed model for tDDR generation and impact of its inhibition by tASO in neurons.

Left side of the scheme: Aβ42 oligomers induce increased intracellular Ca²⁺ levels mediated by NMDA/AMPA receptors. This leads to a rise in ROS, causing telomeric DDR (tDDR), which is associated with reduced neuronal viability. Right side of the scheme: inhibition of Ca²⁺ availability in the culture medium by EGTA, Ca²⁺ influx by NMDA/AMPA blockers, or ROS generation by NAC, all cause decreased tDDR activation. Inhibition of tDDR by tASO decreases tDDR activation, normalizes gene expression, and increases neuronal viability. Dashed arrows represent the functional consequences of the events described in the graph. Created with BioRender.com.

Figure EV1. Markers of tDDR activation and pro-apoptotic gene expression are elevated in the hippocampus of 3xTg-AD mice.

(A–D) Hippocampal regions from 12-month-old wt and 3xTg-AD mice were analyzed. Each circle in the graphs represents one mouse analyzed. (A) Representative confocal images of immunofluorescence staining of hippocampal regions from wt and 3xTg-AD mice with antibodies against γH2AX, 53BP1, MAP2, nuclei were stained with DAPI. Bar graph shows quantification of the average number of γH2AX (n = 4 wt and n = 4 3xTg-AD), and 53BP1 foci per nucleus (n = 5 wt and n =  53xTg-AD). At least 50 nuclei per mouse section were scored. (B) Bar graphs show the quantification of G-rich (n = 4 wt and n = 8 3xTg-AD) and C-rich (n = 5 wt and n = 10 3xTg-AD) tdincRNA levels in 3xTg-AD mice relative to wt samples in brain hippocampus as measured by RT-qPCR. (C) Bar graph shows the quantification of p21 mRNA levels in 3xTg-AD mice relative to wt samples in the hippocampus as measured by RT-qPCR. (n = 4 wt and n = 4 3xTg-AD). (D) Bar graph represents the quantification of the ratio between the mRNA levels of Bax over Bcl-2 in 3xTg-AD mice relative to wt samples in the hippocampus as measured by RT-qPCR (n = 4 wt and n = 4 3xTg-AD). (E) Representative confocal microscopy images of Immunofluorescence stainings combining antibodies against MAP2, BAX and BCL-2 of sections from wt and 3xTg-AD mice (left panel). Bar graph represents the quantification of the ratio between the signal of BAX over BCL-2 from immunofluorescence in 3xTg-AD mice relative to wt samples in hippocampal regions (n = 4 wt and n = 4 3xTg-AD). Unpaired t test was applied and data are represented as mean ± SEM.

Figure EV2. Experimental descriptions of Aβ42 oligomers preparation and their specificity.

In all experiments in this manuscript, a fresh batch of Aβ42 oligomers was prepared as described in the “Methods and protocols” section. (A) Scheme of Aβ42 oligomer preparation and analysis of the oligomer’s solution. Each Aβ42 oligomers batch was tested by immunoblotting for oligomerization status before use. (B) Representative images of western blots using the Oligomers A11 polyclonal antibody recognizing selectively the oligomeric forms only, or the 6E10 monoclonal antibody recognizing the C terminal fragment (CTF) of the peptide. Control peptide prepared in parallel but unable to undergo oligomerization, was used as negative control. Polyacrylamide gel stained with blue Coomassie-based solution was used to check the Aβ42 oligomers and control peptide. (C) Representative images of immunofluorescence staining of mouse primary neurons treated with Vehicle, Aβ42 oligomers and solutions containing control peptide and stained with antibodies against MAP2 (magenta) as neuronal marker, γH2AX (cyan) and 53BP1 (yellow) as DDR markers, nuclei were stained with DAPI. Source data are available online for this figure.

Figure EV3. Aβ42 oligomers induce DDR and accumulation at telomeres in HT-22 cells.

(A–C) HT-22 cells were treated with 5 μM Aβ42 oligomers for 24 h or vehicle. Each circle represents an independent biological replicate. (A) Representative confocal image of immunofluorescence staining of HT-22 with antibodies against pS/TQ (cyan) and pATM (yellow) as DDR markers, nuclei were stained with DAPI. Bar graphs show the quantification of average number of DDR foci per nucleus. At least 50 cells for each condition in each replicate were analyzed. (n = 3 for each condition) (B) Representative confocal images of immunofluorescence staining of HT-22 with antibodies against γH2AX (cyan) and 53BP1 (yellow) as DDR markers, nuclei were stained with DAPI. Bar graphs show the average number of DDR foci per nucleus. At least 50 cells for each condition in each replicate were analyzed. (n = 3 for each condition). (C) Bar graphs show the percentage of DDR foci that localize at telomeres or centromeres, as detected by immunoFISH combining γH2AX antibody and FISH for telomeric DNA using a complementary PNA probe and by double immunofluorescence combining antibodies against γH2AX and the centromeric protein CREST of primary neurons. At least 50 cells for each condition in each replicate were analyzed. (n = 3 for each condition). Unpaired t test was applied and data are represented as mean ± SEM.

Figure EV4. Aβ42 oligomers induce an RNA-signature present in AD-affected areas.

(A) The dotplot shows the significant Gene Ontology Biological processes that emerged from the enrichment analysis of DEGs in Aβ42 oligomers versus Vehicle, performed with ClusterProfiler. The name of the pathway is reported on the left of the graph, the x-axis represents the gene ratio, dots size represents the number of DEGs present in the pathway and the color indicates the adjusted p value of the pathway enrichment. Statistical analysis was performed with over-representation analysis for Gene Ontology Biological processes on DEGs, performed with ClusterProfiler (Wu et al, 2021). (B) The histogram shows the extent of correlation between DEG in Aβ42oligomers vs vehicle and known AD-associated genes when compared to random genes or known AD-associated genes. (C) Plot as generated by http://alzcode.xyz showing the number of the interactions of DEGs in Aβ42oligomers vs vehicle and known AD-associated genes (red line) compared to the distribution of interactions between random genes and known AD-associated genes. The significance is calculated by permutation test (Lin et al, 2022). (D) Heatmap showing the 477 DEGs in Aβ42 oligomers-treated vs vehicle-treated primary neurons and in human AD patients vs control patients in the indicated brain areas. ACC anterior cingulate cortex, CBE cerebellum, DLPFC dorsolateral prefrontal cortex, FP frontal pole, IFG inferior frontal gyrus, PCC posterior cingulate cortex, PHG parahippocaml gyrus, STG superior temporal gyrus, TCX temporal cortex.

Figure EV5. Positive control for Ca²⁺ measurements in mouse primary neurons.

Treatment of mouse primary neurons with 2 μM ionomycin for 2 h was used as positive control for Ca²⁺-derived fluorescence signal. Each circle represents an independent biological replicate. (A) Representative confocal microscopy images of mouse primary neurons incubated with Fluo-4 probe, green fluorescence is generated upon Ca²⁺ binding to the Fluo-4 probe. White dashed-line circles indicate nuclei of neurons. White dashed-line circles indicate the nuclei of neurons. Bar correspond to 20 μm. (B) Bar graph shows semiquantitative measures of intracellular Ca²⁺-derived fluorescence. (control, n = 5; Ionomycin, n = 4) Unpaired t test was applied and data are represented as mean ± SEM.

Figure EV6. tASO improves pathways dysregulated by Aβ42 oligomers.

(A) The dotplot shows the significant Gene Ontology Biological processes that emerged from the enrichment analysis of DEGs in Aβ42 oligomers Anti-Telo G-treated vs Aβ42 oligomers Control ASO-treated primary neuron, performed with ClusterProfiler. The name of the pathway is reported on the left of the graph, the x-axis represents the gene ratio, the dot size represents the number of differentially expressed genes present in the pathway and the color indicates the adjusted p value of the pathway enrichment. Statistical analysis was performed with over-representation analysis for Gene Ontology Biological processes on DEGs, performed with ClusterProfiler (Wu et al, 2021).