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Microglia TREM2R47H Alzheimer-linked variant enhances excitatory transmission and reduces LTP via increased TNF-α levels

  1. Siqiang Ren
  2. Wen Yao
  3. Marc D Tambini
  4. Tao Yin
  5. Kelly A Norris
  6. Luciano D'Adamio  Is a corresponding author
  1. Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, United States
  2. Brain Health Institute, Jacqueline Krieger Klein Center in Alzheimer's Disease and Neurodegeneration Research, Rutgers, The State University of New Jersey, United States
Research Article
Cite this article as: eLife 2020;9:e57513 doi: 10.7554/eLife.57513
7 figures, 1 table and 1 additional file

Figures

Concentrations of human Aβ species are similar in the CSF of peri-adolescent Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rats.

Levels of Aβ38, Aβ40, and Aβ42/Aβ40 ratio in 6–8 weeks old Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rat CSF. We used the following numbers of samples: Trem2w/w, females n = 12, males n = 16; Trem2R47H/w females n = 6, males n = 10; Trem2R47H/R47H females n = 10, males n = 17. Data are represented as mean ± SEM. Data were analyzed by ordinary one‐way ANOVA. No differences were seen in Aβ38 [F(2, 68)=0.5339, p=0.5887], Aβ40 [F(2, 68)=1.010, p=0.3696], Aβ42 [F(2, 68)=0.4376, p=0.6474] levels and the Aβ42/Aβ40 ratio [F(2, 68)=1.564, p=0.2168].

Levels of TNF-α and other proinflammatory cytokines are increased in the CNS of pre-adolescent Trem2R47H/R47H rats.

Levels of IFN-δ, IL-1β, IL-4, IL-5, IL-6, CXCL1, IL-10, IL-13, and TNF-α in the CNS of 4 weeks old rats were measured by ELISA. IL-1β, CXCL1 and TNF-α are significantly increased in Trem2R47H/R47H rats; IFN-γ, IL-4, IL-5, IL-6, IL-10 and IL-13 are not. We used the following numbers of samples: Trem2w/w, females n = 5, males n = 7; Trem2R47H/w females n = 6, males n = 6; Trem2R47H/R47H females n = 6, males n = 6. Data are represented as mean ± SEM and were analyzed by ordinary one‐way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed significant differences [IFN-γ: F(2, 33)=0.1762, p=0.8392 - IL-10: F(2, 33)=0.1781, p=0.8376 - IL-13: F(2, 33)=2.595, p=0.0898 - IL-1β: F(2, 33)=10.52, p=0.0003***; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.5921 (ns); w/w vs. RH/RH, p=0.0003***; RH/w vs. RH/RH, p=0.0051** - IL-4: F(2, 33)=1.763, p=0.1873 - IL-5: F(2, 33)=0.8137, p=0.4519 - IL-6: F(2, 33)=0.4640, p=0.6328 - KC-GRO: F(2, 33)=4.653, p=0.0166*; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.4516 (ns); w/w vs. RH/RH, p=0.0128*; RH/w vs. RH/RH, p=0.1808 (ns) - TNF-α: F(2, 33)=17.32, p<0.0001****; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.3272 (ns); w/w vs. RH/RH, p<0.0001****; RH/w vs. RH/RH, p=0.0005***].

Levels of TNF-α and other pro-inflammatory cytokines are increased in the CSF of peri-adolescent Trem2R47H/R47H rats.

Measurement of cytokines present in the CSF of 6–8 weeks old rats shows that IL-1β, IL-6, IL-10, IL-13 and TNF-α are significantly increased in Trem2R47H/R47H rats; IFN-δ, IL-1β, IL-4, IL-5 and CXCL1 are not. The same samples used for the experiments shown in Figure 1 were used here. Data are represented as mean ± SEM and were analyzed by ordinary one‐way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed significant differences [IFN-γ: F(2, 68)=0.7008, p=0.4997- IL-10: F(2, 68)=5.651, p=0.0054**; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.8335 (ns); w/w vs. RH/RH, p=0.0051**; RH/w vs. RH/RH, p=0.0779 (ns) - ANOVA summary of IL-13: F(2, 68)=26.21, p<0.0001****; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.0066**; w/w vs. RH/RH, p<0.0001****; RH/w vs. RH/RH, p=0.0090** - IL-1β: F(2, 68)=2.473, p=0.0919 - IL-4: F(2, 68)=2.504, p=0.0893 - IL-5: F(2, 68)=3.489, p=0.0361*; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.9545 (ns); w/w vs. RH/RH, p=0.069 (ns); RH/w vs. RH/RH, p=0.0753 (ns) - IL-6: F(2, 68)=9.016, p=0.0003***; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.361 (ns); w/w vs. RH/RH, p=0.0002***; RH/w vs. RH/RH, p=0.0707 (ns) – CXCL1: F(2, 68)=1.847, p=0.1656 - TNF-α: F(2, 68)=9.720, p=0.0002***; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.0565 (ns); w/w vs. RH/RH, p=0.0001***; RH/w vs. RH/RH, p=0.3403 (ns)].

Treml1 mRNA expression is normal in Trem2R47H rats.

(A) Levels of Treml1 mRNA were measured and normalized to Gapdh mRNA expression. We used microglia purified from 2 male and 2 female 5/6 weeks old rats for each genotype (Trem2w/w and Trem2R47H/R47H). Data were analyzed by unpaired student’s t-test (p=0.5198), and presented as average (Treml1/Gapdh)± SEM. (B) Treml1 mRNA expression was measured in total brains. We used 5 female and 5 male (6–8 weeks of age) for each genotype (Trem2w/w, Trem2R47H/w and Trem2R47H/R47H). Data are represented as (Treml1/Gapdh) mean ± SEM and were analyzed by ordinary one‐way ANOVA (F(2, 27)=1.940, p=0.1632).

The Trem2R47H variant alters glutamatergic synaptic transmission in a gene dosage-dependent manner in young rats.

(A) Representative recording traces of mEPSC at SC–CA3 >CA1 pyramidal cell synapses. (B) The Trem2R47H variant causes a significant increase in mEPSC amplitude [F(2, 30)=7.371, p=0.0025**; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.1093 (ns); w/w vs. RH/RH, p=0.0017**; RH/w vs. RH/RH, p=0.1630 (ns)]. (C) In contrast, decay time of mEPSC was not changed [F(2, 30)=1.396, p=0.2632]. (D) Frequency of mEPSC was enhanced by the Trem2R47H variant [F(2, 30)=7.092, p=0.0030**; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.4881 (ns); w/w vs. RH/RH, p=0.0025**; RH/w vs. RH/RH, p=0.0345*]. (E) Average mEPSC of the three groups depicts differences in amplitude. As can also be noted in B, Trem2R47H/w rats show mEPSC with increased amplitude, albeit this increase did not reach statistical significance. Cumulative probability of AMPAR-mediated mEPSC amplitudes (F) and inter event intervals (G). (H) AMPA/NMDA ratio is significantly increased in both Trem2R47H/R47H and Trem2R47H/w rats in a gene dosage dependent manner [F(2, 27)=11.75, p=0.0002***; post-hoc Tukey's multiple comparisons test: w/w vs. RH/w, p=0.0265*; w/w vs. RH/RH, p=0.0001***; RH/w vs. RH/RH, p=0.1160 (ns)]. Representative traces are shown on of the graph (traces are averaged from 20 sweeps). (I) Average PPF at 50 ms (left panel) and 200 ms (right panel) Inter stimulus Interval (ISI) [PPF at 50 ms ISI: F(2, 49)=0.0949, p=0.9096; PPF at 200 ms ISI: F(2, 49)=0.1397, p=0.8700]. Representative traces are shown on top of the panels. Data are represented as mean ± SEM 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 genotype and sex, plus the number of recording by genotype and sex as follow: 1) genotypes: w/w = Trem2 w/w, RH/w = Trem2 R47H/w, RH/RH = Trem2R47H/R47H;2) sex: R47H/R47H = female, M = males; 3) number of animals and number of recordings from animals: n/n’, were n = number of animals, n’=number of recordings from the n animals. For example, the w/w: F = 4/6; M = 4/5 in A indicates that data for mEPSC for the Trem2w/w rats were obtained from 4 females and 4 males, and that 6 recordings were obtained from the 4 females and 5 recordings from the 4 males.

Rapid reduction of excess TNF-α activity normalizes amplitude of glutamatergic synaptic responses in young Trem2R47H/R47H rats.

(A) Representative mEPSC traces. (B) The increase in mEPSC amplitude caused by the Trem2R47H variant is occluded by anti-TNF-α application but not by the IgG isotype control [F(3, 54)=18.79, p<0.0001****; post-hoc Tukey's multiple comparisons test: w/w + anti-TNF-α vs. RH/RH + anti-TNF-α, p=0.7884 (ns); w/w + anti-TNF-α vs. RH/RH + Isotype, p<0.0001****; w/w + anti-TNF-α vs. w/w + Isotype, p=0.9252 (ns); RH/RH + anti-TNF-α vs. RH/RH + Isotype, p<0.0001****; RH/RH + anti-TNF-α vs. w/w + Isotype p=0.4299 (ns); RH/RH + Isotype vs. w/w + Isotype, p<0.0001****]. (C) Decay time of mEPSC was not changed by either genotype or treatments [F(3, 54)=2.716, p=0.0536]. (D) The increased frequency of mEPSC observed in Trem2R47H/R47H rats was not significantly affected by either anti-TNF-α or isotype control IgG [F(3, 54)=12.05, p<0.0001****; post-hoc Tukey's multiple comparisons test: w/w + anti-TNF-α vs. RH/RH + anti-TNF-α, p=0.0046**; w/w + anti-TNF-α vs. RH/RH + Isotype, p<0.0001****; w/w + anti-TNF-α vs. w/w + Isotype, p=0.9947 (ns); RH/RH + anti-TNF-α vs. RH/RH + Isotype, p=0.5147 (ns); RH/RH + anti-TNF-α vs. w/w + Isotype p=0.0109*; RH/RH + Isotype vs. w/w + Isotype, p<0.0001****]. (E) Average mEPSC of the four groups. Cumulative probability of AMPAR-mediated mEPSC amplitudes (F) and inter event intervals (G). (H) The increase of AMPA/NMDA ratio observed in Trem2R47H/R47H rats was reversed by anti-TNF-α but not IgG isotype control [F(3, 36)=13.50, p<0.0001****; post-hoc Tukey's multiple comparisons test: w/w + anti-TNF-α vs. RH/RH + anti-TNF-α, p=0.6507 (ns); w/w + anti-TNF-α vs. RH/RH + Isotype, p<0.0001****; w/w + anti-TNF-α vs. w/w + Isotype, p=0.2880 (ns); RH/RH + anti-TNF-α vs. RH/RH + Isotype, p=0.0002***; RH/RH + anti-TNF-α vs. w/w + Isotype p=0.9171 (ns); RH/RH + Isotype vs. w/w + Isotype, p<0.0015**]. Representative traces are shown above the graph (traces are averaged from 20 sweeps). (I) Neither genotype nor treatment changed average PPF at 50 ms (left panel) and 200 ms (right panel) ISI [PPF at 50 ms ISI: F(3, 58)=0.7420, p=0.5313; PPF at 200 ms ISI: F(3, 58)=2.356, p=0.0812]. Representative traces are shown above the panels. Data are represented as mean ± SEM and were analyzed by ordinary one‐way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed significant differences. Number of animals and of recordings are shown as explained in Figure 5.

LTP is impaired in RH mutant rats, and the impairment could be rescued by application of anti-TNF-α antibody.

(A) The input-output slope is significantly increased in Trem2R47H/R47H rats [two-way ANOVA, stimulation intensity x genotype interaction F(6, 144)=6.745, p<0.0001****; post-hoc Sidak's multiple comparisons test: 1.2 mV p=0.0175*; 1.4 mV: p=0.0021**; 1.6 mV: p=0.0001***] Representative traces of fEPSPs in response to increasing stimulus from 0.4 to 1.6 mA are shown on the top. (B) LTP is impaired in Trem2R47H/R47H rats. Average traces of the baseline and the last 5mins of LTP are shown on top. (C) Plot of fEPSP slope change of the last 10 min of LTP in B (unpaired t test, p<0.0001****). (D) The increase of input-out slope in Trem2R47H/R47H rats is reversed by application of anti-TNF-α [ANOVA for repeated measures F(18, 300)=6.579, p<0.0001****; post-hoc Tukey's multiple comparisons test: 0.8 mV RH/RH + Isotype vs. w/w + anti-TNF-α p=0.0479*; 1.0 mV RH/RH + Isotype vs. RH/RH + anti-TNF-α p=0.0061**, RH/RH + Isotype vs. w/w + anti-TNFα p=0.0072**, RH/RH + Isotype vs. w/w + Isotype p=0.0113*; 1.2 mV RH/RH + Isotype vs. RH/RH + anti-TNFα p=0.0009***, RH/RH + Isotype vs. w/w + anti-TNF-α p=0.0009***, RH/RH + Isotype vs. w/w + Isotype p=0.0029**; 1.4 mV RH/RH + Isotype vs. RH/RH + anti-TNF-α p<0.0001****, RH/RH + Isotype vs. w/w + anti-TNF-α p<0.0001****, RH/RH + Isotype vs. w/w + Isotype p=P < 0.0001****; 1.6 mV RH/RH + Isotype vs. RH/RH + anti-TNF-α p<0.0001****, RH/RH + Isotype vs. w/w + anti-TNF-α P p<0.0001****, RH/RH + Isotype vs. w/w + Isotype p<0.0001****]. Representative fEPSP traces are shown on top. (E) The impaired LTP is restored by application of anti-TNF-α antibody. The average traces of the baseline and the last 5mins of LTP are shown on top. (F) Plot of fEPSP slope change of the last 10 min of LTP in E [one-way ANOVA, F(3, 36)=1490, p<0.0001; post-hoc Tukey's multiple comparisons test: RH/RH + Isotype vs. RH/RH + anti-TNF-α p<0.0001****, RH/RH + Isotype vs. w/w + anti-TNF-α P p<0.0001****, RH/RH + Isotype vs. w/w + Isotype p<0.0001****].

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (Rattus Norvegicus)ApphTambini et al., 2019. Aging Cell 18: e13033Rat App allele with humanize Aβ region
Genetic reagent (Rattus Norvegicus)Trem2R47HTambini and D'Adamio, 2020 Sci Rep 10: 4122Rat Trem2 allele with R47H mutation
Commercial assay or kitV-PLEX Plus Aβ Peptide
Panel 1
Meso Scale DiscoveryCat# K15200GUsed following manufacturer’s recommendations
Commercial assay or kitV-PLEX Proinflammatory Panel 2Meso Scale DiscoveryCat# K15059DUsed following manufacturer’s recommendations
Commercial assay or kitCD11b/c (Microglia) Micro-Beads, rat antibody Cat# 130-105-634Miltenyi BiotecRRID:AB_2783886Used following manufacturer’s recommendations
Commercial assay or kitAdult Brain Dissociation KitMiltenyi BiotecCat# 130-107-677Used following manufacturer’s recommendations
Commercial assay or kitRNeasy RNA
Isolation kit
QiagenCat# 74106Used following manufacturer’s recommendations
Commercial
assay or kit
High-Capacity cDNA RT kitThermoCat# 4368814)Used following manufacturer’s recommendations
Commercial assay or kitTaqMan
Fast Advanced Mix
ThermoCat# 4444556Used following manufacturer’s recommendations
Commercial assay or kitGapdh Real-Time PCRThermoRn01775763_g1Used following manufacturer’s recommendations
Commercial assay or kitTreml1 Real-Time PCRThermoRn01511908_g1Used following manufacturer’s recommendations
AntibodyPolyclonal Goat IgG anti-Rat TNFα Cat# AF-510-NAR and D SystemsRRID:AB_35451110 ng/ml in ACSF
AntibodyPolyclonal Goat IgG. antibody Cat# AB-108-CR and D SystemsRRID:AB_35426710 ng/ml in ACSF
Software, algorithmLinRegPCR softwarehartfaalcentrum.nl
Software, algorithmpCLAMP10 softwareMolecular Devices,
Software, algorithmImage Lab softwareBioradRRID:SCR_014210
Software, algorithmGraphPad PrismRRID:SCR_002798

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