Document Type
Article
Publication Date
3-18-2024
Abstract
Background
Biologic TNF-α inhibitors (bTNFIs) can block cerebral TNF-α in Alzheimer’s disease (AD) if these macromolecules can cross the blood–brain barrier (BBB). Thus, a model bTNFI, the extracellular domain of type II TNF-α receptor (TNFR), which can bind to and sequester TNF-α, was fused with a mouse transferrin receptor antibody (TfRMAb) to enable brain delivery via BBB TfR-mediated transcytosis. Previously, we found TfRMAb-TNFR to be protective in a mouse model of amyloidosis (APP/PS1) and tauopathy (PS19), and herein we investigated its effects in mice that combine both amyloidosis and tauopathy (3xTg-AD).
Methods
Eight-month-old female 3xTg-AD mice were injected intraperitoneally with saline (n = 11) or TfRMAb-TNFR (3 mg/kg; n = 11) three days per week for 12 weeks. Age-matched wild-type (WT) mice (n = 9) were treated similarly with saline. Brains were processed for immunostaining and high-resolution multiplex NanoString GeoMx spatial proteomics.
Results
We observed regional differences in proteins relevant to Aβ, tau, and neuroinflammation in the hippocampus of 3xTg-AD mice compared with WT mice. From 64 target proteins studied using spatial proteomics, a comparison of the Aβ-plaque bearing vs. plaque-free regions in the 3xTg-AD mice yielded 39 differentially expressed proteins (DEP) largely related to neuroinflammation (39% of DEP) and Aβ and tau pathology combined (31% of DEP). Hippocampal spatial proteomics revealed that the majority of the proteins modulated by TfRMAb-TNFR in the 3xTg-AD mice were relevant to microglial function (⁓ 33%). TfRMAb-TNFR significantly reduced mature Aβ plaques and increased Aβ-associated microglia around larger Aβ deposits in the 3xTg-AD mice. Further, TfRMAb-TNFR increased mature Aβ plaque-associated microglial TREM2 in 3xTg-AD mice.
Conclusion
Overall, despite the low visual Aβ load in the 11-month-old female 3xTg-AD mice, our results highlight region-specific AD-relevant DEP in the hippocampus of these mice. Chronic TfRMAb-TNFR dosing modulated several DEP involved in AD pathology and showed a largely microglia-centric mechanism of action in the 3xTg-AD mice.
Recommended Citation
Jagadeesan N, Roules GC, Chandrashekar DV, Yang J, Kolluru S, Sumbria RK. Modulation of hippocampal protein expression by a brain penetrant biologic TNF-α inhibitor in the 3xTg Alzheimer’s disease mice. J Transl Med. 2024;22:291. https://doi.org/10.1186/s12967-024-05008-x
Additional file 1: Table S1. Number of outliers removed from Figs. 3 to 5.
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Additional file 2: Fig. S1. Representative images from NanoString GeoMX DSP platform of a sagittal brain section from WT-Saline mice showing the circular hippocampal regions of interest (ROIs) (A). The red-boxed region in the brain section image in the left panel of A, taken from the Allen Institute, represents the hippocampus. The circled ROIs in the right panel of A are shown as high-resolution images for subiculum upper (dorsal subiculum) (B), CA2 (C), subiculum lower (ventral subiculum) (D), and DG (E) subregions labeled with the four morphology markers: Aβ (yellow), Iba1 (red), GFAP (green), and nuclear marker SYTO13 (blue). Scale bar = 500 μm in A and 50 μm in B–E.
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Additional file 3: Fig. S2. Bar plots representing fold change (FC) values expressed in log2 FC showing protein expression in 3xTg-AD mice relative to WT-Saline mice in the upper subiculum region (up-regulation (A) and down-regulation (B)) of the hippocampus (n = 6/group). *p < 0.05.
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Additional file 4: Fig. S3. Bar plots representing fold change (FC) values expressed in log2 FC showing protein expression in 3xTg-AD mice relative to WT-Saline mice in the lower subiculum region (up-regulation (A) and down-regulation (B)) of the hippocampus (n = 6/group). *p < 0.05.
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Additional file 5: Fig. S4. Bar plots representing fold change (FC) values expressed in log2 FC showing protein expression in 3xTg-AD mice relative to WT-Saline mice in the CA2 region (up-regulation (A) and down-regulation (B)) of the hippocampus (n = 6/group). *p < 0.05.
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Additional file 6: Fig. S5. Bar plots representing fold change (FC) values expressed in log2 FC showing protein expression in 3xTg-AD mice relative to WT-Saline mice in the dentate gyrus (DG) region (up-regulation (A) and down-regulation (B)) of the hippocampus (n = 6/group). *p < 0.05.
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Additional file 7: Fig. S6. Effect of TfRMAb-TNFR on total 6E10 count/µm2 of the entire hippocampus (A) and intraneuronal 6E10-positive area as a % of tissue area in the plaque-bearing subiculum (B) of 3xTg-AD mice. The data are shown as Mean ± SEM for Tg-Saline (n = 11) and Tg-TfRMAb-TNFR (n = 11) mice. Data were analyzed using the unpaired t-test in A and Mann–Whitney U test in B. ns = not significant for the indicated comparisons.
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Additional file 8: Fig. S7. Effect of TfRMAb-TNFR on AT8-positive area in the hippocampus of 3xTg-AD mice. Representative confocal images of AT8-positive immunofluorescence staining in the subiculum of 3xTg-AD mice with or without TfRMAb-TNFR treatment (A). Scale bar = 25 μm in A. AT8-positive area (B). The data are shown as Mean ± SEM for WT-Saline (n = 5), Tg-Saline (n = 10), and Tg-TfRMAb-TNFR (n = 9) mice. Data were analyzed using the one-way ANOVA with Holm Sidak’s post-hoc test compared to Tg-Saline mice. **p < 0.01 and ns = not significant for the indicated comparisons.
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Additional file 9: Fig. S8. Scatter plots show the correlation between Iba1 and TREM2% positive area in the plaque-bearing (subiculum) (A) and plaque-free (CA2) hippocampus (B), and mature Aβ plaques and TREM2% positive area in the plaque-bearing hippocampus (C) of 3xTg-AD mice by the Pearson correlation coefficient. Bar graph showing no difference in the TREM2 area when normalized to Iba1 area in the plaque-bearing and plaque-free hippocampus (D) consistent with TREM2-positive area % data shown in Figure 5C. Significantly higher 6E10-associated microglia area normalized to 6E10 area with TfRMAb-TNFR in the plaque-bearing subiculum (E) consistent with Figure 4D which shows 6E10-associated microglial MFI. The data are shown as Mean ± SEM for WT-Saline (n = 9), Tg-Saline (n = 10–11), and Tg-TfRMAb-TNFR (n = 8–11) mice. Data were analyzed using the two-way repeated measures ANOVA with Holm Sidak’s post-hoc test in D, and unpaired t-test in E. *p < 0.05 and **p < 0.01 for the indicated comparisons.
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Additional file 10: Fig. S9. Plasma metabolic panel of 3xTg-AD mice with or without TfRMAb-TNFR treatment. Albumin (A), alkaline phosphatase (ALP) (B), alanine transaminase (ALT) (C), amylase (D), total bilirubin (E), BUN (F), calcium (G), phosphorus (H), creatinine (I), glucose (J), sodium (K), potassium (L), total protein (M), globulin (N). The data are shown as Mean ± SEM for WT-Saline (n = 8–9), Tg-Saline (n = 10–11), and Tg-TfRMAb-TNFR (n = 10–11) mice. Data are reported as % of Tg-Saline values and were analyzed using the one-way ANOVA with Holm Sidak’s post-hoc test. The heat map of p values (O). *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons.
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Additional file 11. Materials and methods.
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Additional file 12: Table S2. List of differentially and non-differentially expressed proteins in Tg-TfRMAb-TNFR compared with Tg-Saline 3xTg-AD mice.
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Additional file 13: Fig. S10. Weights, open field, Y-maze, and nest building test in 3xTg-AD mice with or without TfRMAb-TNFR treatment. Body weight of animals during the study (A). Representative trajectory maps of mouse movement in the Y-maze test (B), and discrimination index, latency to novel arm, and % entries in the novel arm (C). Representative trajectory maps showing the mouse activity in the open-field test (D), and total distance traveled and mean speed (E) in the open-field arena. Representative images of nests built by 3xTg-AD mice compared with age-matched WT mice (F), and nesting scores and amount of untorn nestlet (G). The data are shown as Mean ± SEM for WT (n = 9), Tg-Saline (n = 11), and Tg-TfRMAb-TNFR (n = 11) per group. The data were analyzed using two-way repeated measures ANOVA in A, one-way ANOVA with Holm Sidak’s post-hoc test in C and E, and Kruskal–Wallis test with Dunn’s post-hoc test in G. *p < 0.05.
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Comments
This article was originally published in Journal of Translational Medicine, volume 22, in 2024. https://doi.org/10.1186/s12967-024-05008-x