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. 2021 Jan-Jun:296:100089.
doi: 10.1074/jbc.RA120.016395. Epub 2020 Nov 21.

TNF-α-mediated reduction in inhibitory neurotransmission precedes sporadic Alzheimer's disease pathology in young Trem2R47H rats

Affiliations

TNF-α-mediated reduction in inhibitory neurotransmission precedes sporadic Alzheimer's disease pathology in young Trem2R47H rats

Siqiang Ren et al. J Biol Chem. 2021 Jan-Jun.

Abstract

Alzheimer's disease (AD) is a neurodegenerative dementia associated with deposition of amyloid plaques and neurofibrillary tangles, formed by amyloid β (Aβ) peptides and phosphor-tau, respectively, in the central nervous system. Approximately 2% of AD cases are due to familial AD (FAD); ∼98% of cases are sporadic AD (SAD). Animal models with FAD are commonly used to study SAD pathogenesis. Because mechanisms leading to FAD and SAD may be distinct, to study SAD pathogenesis, we generated Trem2R47H knock-in rats, which carry the SAD risk factor p.R47H variant of the microglia gene triggering receptor expressed on myeloid cells 2 (TREM2). Trem2R47H rats produce human-Aβ from a humanized-App rat allele because human-Aβ is more toxic than rodent-Aβ and the pathogenic role of the p.R47H TREM2 variant has been linked to human-Aβ-clearing deficits. Using periadolescent Trem2R47H rats, we previously demonstrated that supraphysiological tumor necrosis factor-α (TNF-α) boosts glutamatergic transmission, which is excitatory, and suppresses long-term potentiation, a surrogate of learning and memory. Here, we tested the effect of the p.R47H variant on the inhibitory neurotransmitter γ-aminobutyric acid. We report that GABAergic transmission is decreased in Trem2R47H/R47H rats. This decrease is due to acute and reversible action of TNF-α and is not associated with increased human-Aβ levels and AD pathology. Thus, the p.R47H variant changes the excitatory/inhibitory balance, favoring excitation. This imbalance could potentiate glutamate excitotoxicity and contribute to neuronal dysfunction, enhanced neuronal death, and neurodegeneration. Future studies will determine whether this imbalance represents an early, Aβ-independent pathway leading to dementia and may reveal the AD-modifying therapeutic potential of TNF-α inhibition in the central nervous system.

Keywords: Alzheimer's disease; Aβ; GABA; TNF-α; Trem2; animal model; neurodegenerative disease; rats; synaptic plasticity.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Inhibitory GABAergic synaptic transmission is decreased in Trem2R47Hrats.A, representative traces of PPF of GABAergic transmission. B, plot of PPF at 50-ms ISI and the representative traces. [F (2, 50) = 8.968, p = 0.0005∗∗∗∗; post hoc Tukey's multiple comparisons test: w/w vs. RH/w, p = 0.9485 (ns); w/w vs. RH/RH, p = 0.0014∗∗; RH/w vs. RH/RH, p = 0.0022∗∗]. C, the plot of PPF at 200-ms ISI. [F (2, 50) = 5.106, p = 0.0096∗∗; post hoc Tukey's multiple comparisons test: w/w vs. RH/w, p = 0.9082 (ns); w/w vs. RH/RH, p = 0.0461∗; RH/w vs. RH/RH, p = 0.0118∗]. D, representative traces of mIPSCs. E, the plot of the frequency of mIPSCs. [F (2, 41) = 1.734, p = 0.1893(ns)]. Notably, RH mutant rats show mIPSCs with decreased frequency, albeit this decrease did not reach a statistical significance. F, the plot of the amplitude of mIPSCs. [F (2, 41) = 17.04, p < 0.0001∗∗∗∗; post hoc Tukey's multiple comparisons test: w/w vs. RH/w, p = 0.0002 ∗∗∗; w/w vs. RH/RH, p < 0.0001∗∗∗∗; RH/w vs. RH/RH, p = 0.3947(ns)]. G, the plot of the decay time of mIPSCs. [F (2, 41) = 5.254, p = 0.0093∗∗; post hoc Tukey's multiple comparisons test: w/w vs. RH/w, p = 0.1257(ns); w/w vs. RH/RH, p = 0.0070∗∗; RH/w vs. RH/RH, p = 0.3890 (ns)]. Representative averaged mIPSCs traces are shown on the right. Note that the amplitude and decay time of mIPSCs are significantly increased in RH mutant rats. H, the plot of the cumulative probability of mIPSCs interevent intervals. I, the plot of the cumulative probability of mIPSC amplitude. Data are represented as mean ± SD and were analyzed by ordinary one-way ANOVA followed by post hoc Tukey's multiple comparisons test when ANOVA showed significant differences. For each type of recordings, we indicate the number of animals by the genotype and sex, plus the number of recording by genotype and sex as follows: (1) genotypes: w/w = Trem2w/w, RH/w = Trem2R47H/w, RH/RH = Trem2R47H/R47H;(2) sex: F, female, M, males; (3) the number of animals and the number of recordings from animals: n/n’, where n = the number of animals and n’ = the number of recordings from the n animals. For example, the w/w: F = 5/10; M = 3/6 in panel A indicates that data for PPF for the Trem2w/w rats were obtained from 5 female and 3 male rats and that 10 recordings were obtained from the 5 female and 6 recordings from the 3 male rats. GABA, γ-aminobutyric acid; ISI, inter stimulus interval; PPF, paired-pulse facilitation; mIPSCs, miniature inhibitory postsynaptic currents.
Figure 2
Figure 2
The reduced inhibitory GABAergic synaptic transmission in Trem2R47Hrats is restored by reducing TNF-α function.A, representative traces of PPF of GABAergic transmission. Representative traces are averaged from 20 sweeps. B, the plot of PPF at 50-ms ISI. [F (3, 56) = 6.021, p = 0.0013∗∗; post hoc Tukey's multiple comparisons test: w/w + anti–TNF-α vs. RH/RH + anti–TNF-α, 0.2643 (ns); w/w + anti–TNF-α vs. RH/RH + isotype, 0.0452∗; w/w + anti–TNF-α vs. w/w + isotype, 0.9897 (ns); RH/RH + anti–TNF-α vs. RH/RH + isotype, p = 0.0005∗∗∗; RH/RH + anti–TNF-α vs. w/w + isotype p = 0.4452 (ns); RH/RH + isotype vs. w/w + isotype, p = 0.0267∗]. C, the plot of PPF at 200-ms ISI. [F (3, 56) = 5.361, p = 0.0026∗∗; post hoc Tukey's multiple comparisons test: w/w + anti–TNF-α vs. RH/RH + anti–TNF-α, 0.9485 (ns); w/w + anti–TNF-α vs. RH/RH + isotype, 0.0348∗; w/w + anti–TNF-α vs. w/w + isotype, 0.5884 (ns); RH/RH + anti–TNF-α vs. RH/RH + isotype, p = 0.0128∗; RH/RH + anti–TNF-α vs. w/w + isotype p = 0.9030 (ns); RH/RH + isotype vs. w/w + isotype, p = 0.0017∗∗]. Note that the increases in PPF of mIPSCs at 50 and 200 ms are reversed by anti–TNFα antibody application in RH mutant rats. D, representative traces of mIPSCs. E, the plot of the frequency of mIPSCs. [F (3, 53) = 0.9519, p = 0.4223 (ns)]. F, the plot of the amplitude of mIPSCs. [F (3, 53) = 4.562, p = 0.0065∗∗∗; post hoc Tukey's multiple comparisons test: w/w + anti–TNF-α vs. RH/RH + anti–TNF-α, 0.9945 (ns); w/w + anti–TNF-α vs. RH/RH + isotype, 0.0142∗; w/w + anti–TNF-α vs. w/w + isotype, 0.9989 (ns); RH/RH + anti–TNF-α vs. RH/RH + isotype, p = 0.0458∗; RH/RH + anti–TNF-α vs. w/w + isotype p = 0.9996 (ns); RH/RH + Isotype vs. w/w + isotype, p = 0.0350∗]. G, the plot of the decay time of mIPSCs. [F (3, 53) = 6.587, p = 0.0007∗∗∗; post hoc Tukey's multiple comparisons test: w/w + anti–TNF-α vs. RH/RH + anti–TNF-α, 0.6728 (ns); w/w + anti–TNF-α vs. RH/RH + isotype, p = 0.0005∗∗∗; w/w + anti–TNF-α vs. w/w + isotype, 0.6144 (ns); RH/RH + anti–TNF-α vs. RH/RH + isotype, p = 0.0342∗; RH/RH + anti–TNF-α vs. w/w + isotype p = 0.9997 (ns); RH/RH + isotype vs. w/w + isotype, p = 0.0436∗]. The representative averaged traces of the mIPSCs are shown on the right. Note that the decreased amplitude and decay time of mIPSCs in RH/RH rats was restored by anti–TNF-α antibody application. H, the plot of the cumulative probability of mIPSC interevent intervals. I, the plot of the cumulative probability of mIPSC amplitude. All data represent means ± SD. Data were analyzed by ordinary one-way ANOVA followed by post hoc Tukey's multiple comparison test when ANOVA showed significant differences. GABA, γ-aminobutyric acid; PPF, paired-pulse facilitation; mIPSCs, miniature inhibitory postsynaptic currents.
Figure 3
Figure 3
Levels of human Aβ species are similar in the brain of periadolescent Trem2w/w, Trem2R47H/w, and Trem2R47H/R47Hrats.A, levels of Aβ38, Aβ40, and Aβ42/Aβ40 ratio in 7- to 8-week-old Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rat brains. We used 5 male and 5 female rats for each genotype. Data are represented as mean ± SD. Data were analyzed by ordinary one-way ANOVA. No differences were seen in Aβ38 [F (2, 27) = 1.931, p = 0.1645], Aβ40 [F (2, 27) = 1.132, p = 0.3374], and Aβ42 [F (2, 27) = 1.668, p = 0.2074] levels and the Aβ42/Aβ40 ratio [F (2, 27) = 0.2683, p = 0.7667]. B, the same samples analyzed in panel A were used to determine levels of Aβ oligomers by dot blots using the oligomer-specific antibody A11. Before immunoblot analysis, membranes were stained with Ponceau red. Quantitative analysis of A11 blot was normalized to the Ponceau red quantitative analysis. The “mix” lane represents an equal mixture of all 5 samples of the same sex-genotype. Quantitation of the data is shown below the blots. Data are represented as mean ± SD. Data were analyzed by ordinary one-way ANOVA. No significant differences were seen among genotypes [F (2, 27) = 0.5667, p = 0.5740].
Figure 4
Figure 4
Immunohistochemistry staining in 3-month-old Trem2w/w, Trem2R47H/w, and Trem2R47H/R47Hrats. Representative images of the anterior hippocampus and overlaying somatosensory cortex of 3-month-old male Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rat brains. Illustrates of, from the top to bottom, H&E, NeuN, GFAP, IBA-1, Aβ, pTau, and Bielschowski Silver staining, respectively. No observable differences in morphology (H&E) or neuronal (NeuN) cellularity are observed. The staining intensity of the microglial (IBA-1) and astrocytic (GFAP) markers are similar across all three genotypes. However, no Aβ or pTau expression can be observed, and no Bielschowski silver–stained plaques or tangles are present. Immunohistochemistry staining was performed on Trem2w/w (4 male and 5 female rats), Trem2R47H/w (4 male and 4 female rats), and Trem2R47H/R47H (4 male and 4 female rats). The scale bar is equivalent to 500 microns. GFAP, glial fibrillary acidic protein.
Figure 5
Figure 5
Qualitative assessment of the NeuN, IBA-1, and GFAP staining in 3-month-old Trem2w/w, Trem2R47H/w, and Trem2R47H/R47Hrats. Immunohistochemistry staining was scored in Trem2w/w (4 male and 5 female rats), Trem2R47H/w (4 male and 4 female rats), and Trem2R47H/R47H (4 male and 4 female rats). Data are represented as mean ± SD of the qualitative score within the cortex and hippocampus-CA1 regions. Data were analyzed by ordinary one-way ANOVA within each brain region. No statistically significant differences were seen in NeuNCx [F (2, 21) = 1.736, p = 0.200], NeuNHC-CA1 [F (2, 21) = 0.533, p = 0.594], IBA-1Cx [F (2, 22) = 0.375, p = 0.692], IBA-1HC-CA1 [F (2, 22) = 0.507, p = 0.609], GFAPCx [F (2, 22) = 0.159, p = 0.854], or GFAPHC-CA1 [F (2, 22) = 0.0237, p = 0.977]. GFAP, glial fibrillary acidic protein.
Figure 6
Figure 6
Astrocyte and microglia immunohistochemistry staining in 3-month-old Trem2w/w, Trem2R47H/w, and Trem2R47H/R47Hrats. Representative images illustrate microglia (A, IBA-1) and astrocyte (B, GFAP) staining with a red-brown chromagen in the dorsal hippocampus-CA1, hippocampus-CA3, and hippocampus-dentate gyrus (DG), of 3-month-old male Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rat brains (left to right). The scale bar is equivalent to 500 microns. GFAP, glial fibrillary acidic protein.
Figure 7
Figure 7
Model depicting how the p.R47H TREM2 variant may enhance the excitatory/inhibitory balance and cause neurodegeneration.A, astrocytes and microglia set physiological excitatory/inhibitory balance and LTP via TNF-α. B, TREM2 expression is restricted to microglia: thus, it is likely that microglia expressing the p.R47H variant are the source of supraphysiological TNF-α 1. Trem2R47H microglia may also promote TNF-α production by other cell types, such as astrocytes 2. Finally, TNF-α produced by microglia may modulate the synaptic regulatory functions of astrocytes 3. These possibilities are not mutually exclusive. Supraphysiological TNF-α impairs LTP and increases the excitatory/inhibitory balance by enhancing glutamate transmission and reducing GABA transmission. A swift surface exocytosis of AMPA receptors and endocytosis of GABA receptors at postsynaptic termini could be one mechanism by which supraphysiological TNF-α increases the excitatory/inhibitory balance and reduces LTP. These early dysfunctions may enhance glutamate excitotoxicity and neuronal cell death, culminating into obvert cognitive deficits, brain pathology, and neurodegeneration decades later. C, resetting TNF-α activity at physiological levels normalizes excitatory/inhibitory balance and LTP and could prevent neurodegeneration. D, supraphysiological TNF-α may promote neurodegeneration via early and late pathogenic mechanisms. Early pathogenic mechanisms would include interfering with synaptic transmission; late pathogenic mechanisms would include progressing amyloid pathology by favoring Aβ production over clearance. GABA, γ-aminobutyric acid; LTP, long-term potentiation.

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