Microglial NFκB-TNFα hyperactivation induces obsessive-compulsive behavior in mouse models of progranulin-deficient frontotemporal dementia

Abstract

Frontotemporal dementia (FTD) is the second most common dementia before 65 years of age. Haploinsufficiency in the progranulin (GRN) gene accounts for 10% of all cases of familial FTD. GRN mutation carriers have an increased risk of autoimmune disorders, accompanied by elevated levels of tissue necrosis factor (TNF) α. We examined behavioral alterations related to obsessive-compulsive disorder (OCD) and the role of TNFα and related signaling pathways in FTD patients with GRN mutations and in mice lacking progranulin (PGRN). We found that patients and mice with GRN mutations displayed OCD and self-grooming (an OCD-like behavior in mice), respectively. Furthermore, medium spiny neurons in the nucleus accumbens, an area implicated in development of OCD, display hyperexcitability in PGRN knockout mice. Reducing levels of TNFα in PGRN knockout mice abolished excessive self-grooming and the associated hyperexcitability of medium spiny neurons of the nucleus accumbens. In the brain, PGRN is highly expressed in microglia, which are a major source of TNFα. We therefore deleted PGRN specifically in microglia and found that it was sufficient to induce excessive grooming. Importantly, excessive grooming in these mice was prevented by inactivating nuclear factor κB (NF-κB) in microglia/myeloid cells. Our findings suggest that PGRN deficiency leads to excessive NF-κB activation in microglia and elevated TNFα signaling, which in turn lead to hyperexcitability of medium spiny neurons and OCD-like behavior.

Keywords: FTD; OCD; TNF; microglia; progranulin.

Fig. 1.

GRN mutation carriers exhibit obsessive–compulsive behaviors and subregion gray matter atrophy. (A) GRN mutation increases repetitive and compulsive behaviors in an FTD cohort. n = 35 (GRN carriers), 25 (noncarriers). P = 0.0001, Fisher’s exact test. (B) Gray matter atrophy in 20 of the 23 symptomatic GRN mutation carriers described in A. Group difference maps derived by voxel-based morphometry show extensive, bilateral atrophy of gray matter in frontotemporoparietal cortex and subcortical regions. Maps are thresholded at a t-threshold of P < 0.05 (corrected for familywise error). Color bars represent t-scores, and statistical maps are superimposed on the Montreal Neurological Institute template brain. The left side of the axial and coronal images corresponds to the left side of the brain.

Fig. 2.

Obsessive–compulsive behavior and hyperexcitability of MSNs is rescued by reducing TNFα levels in Grn^−/− mice. (A) Increased self-grooming of Grn^−/− mice is reduced to WT levels in Grn^−/−Tnfα^+/− mice. n = 13, 12, 15, 10 for Grn^+/+, Grn^−/−, Grn^−/−Tnfα^+/−, and Grn^−/−Tnfα^−/− mice, respectively. P = 0.003, F(3,46) = 5.276, one-way ANOVA. **P < 0.01; Tukey–Kramer post hoc test. (B) Morphology of targeted neurons in the nucleus accumbens core (Left) by labeling biocytin-filled cells with fluorescently conjugated streptavidin (Right). Representative image of more than three independent experiments. Ac, accumbens; LV, lateral ventricle; sStr, dorsal striatum. (C) Representative action potential firing of MSNs in Grn^+/+, Grn^−/−, Grn^−/−Tnfα^+/−, and Grn^−/−Tnfα^−/− mice at 250 pA. (D) Grn^−/− neurons had higher instantaneous firing frequencies than Grn^+/+, Grn^−/−Tnfα^+/−, or Grn^−/−Tnfα^−/− neurons at various current intensities. (E) Quantification of instantaneous action potential firing frequency at 250 pA. P = 0.0063, one-way ANOVA. *P < 0.05, post hoc analyses with Holm correction for multiple comparisons. (F) Quantification of the slopes of the FI curves shown in D. *P < 0.05, one-way ANOVA and Holm correction for multiple comparisons. Number of Grn^+/+, Grn^−/−, Grn^−/−Tnfα^+/−, and Grn^−/−Tnfα^−/− neurons (mice): 107 (16), 99 (14), 69 (9), 39 (6).

Fig. S1.

Nociception is unaltered in PGRN-deficient mice. Performance in the hot plate test was similar in Grn^+/+, Grn^−/−, Grn^−/−Tnfα^+/−, and Grn^−/−Tnfα^−/− mice. n = 15, 16, 13, 11 mice, one-way ANOVA, Tukey–Kramer post hoc test.

Fig. S2.

Social Deficits in Grn^−/− are not affected by TNFα reduction. n = 22, 24, 20, 17, P = 0.021, one-way ANOVA, Kruskal–Wallis test, Dunn‘s multiple comparison post hoc test, *P < 0.05 (P = 0.03, Grn^+/+ vs. Grn^−/−; P = 0.04, Grn^+/+ vs. Grn^−/−/Tnfα^−/−).

Fig. 3.

PGRN-deficient microglia are functionally impaired. (A) Baseline motility of Grn^+/+/Cx3Cr1+/GFP and Grn^−/−/Cx3Cr1+/GFP microglia was measured by intravital two-photon microscopy; the number of process extensions (Green) and retractions (red) were quantified over 10 min. n = 6, six regions of interest (ROIs) from 4, 5 mice. *P < 0.05, unpaired t test. (Scale bar: 10 μm.) (B) Representative projections at different times show the response to a laser ablation (red circle) in Grn^+/+Cx3Cr1+/GFP (Top) or Grn^−/−Cx3Cr1+/GFP mice (Bottom). (Scale bar: 10 μm.) (C) The response of microglia to a laser ablation was measured over 60 min. n = 6, 7 ROIs from five mice per genotype. Two-way-ANOVA, P < 0.0001, genotype effects. *P < 0.05, Bonferroni post hoc test. (D) The chemotactic response of primary microglia from Grn^+/+ or Grn^−/− mice toward 100 μm ATP or ADP was measured by a transwell assay. n = 17, 35 ROIs from four independent experiments. *P < 0.05; ***P < 0.001; n.s., not significant. Kruskal–Wallis test and Dunn’s multiple comparison post hoc test.

Fig. 4.

Microglial PGRN deficiency induces excessive self-grooming. (A) Representative pictures of RFP-expressing microglia in tamoxifen (Tamox)-injected Cx3Cr1-CreERT2/RFPF/F mice. Image shows colocalization (Right, merge) of Iba1 immunoreactivity (Left, microglia, green) and Cre-driven RFP expression (Center, red). Representative of two independent experiments. (Scale bar: 40 μm.) (B–D) Tamoxifen injection in GrnF/F/Cx3Cr1-CreERT2 mice induces selective deletion of PGRN in microglia (B), but not in spleen macrophages (C) or blood cells (D). PGRN mRNA in microglia was measured with quantitative reverse transcription (qRT-PCR). PGRN proteins were measured with ELISA. n = 3 mice per genotype (B), n = 4 mice/genotype (C and D). **P < 0.01, unpaired t test. (E and F) Tamoxifen injection reduces PGRN in the brain, but not in plasma of GrnF/FCx3Cr1-CreERT2 mice. PGRN levels were measured by ELISA. n = 12 mice per genotype (E), n = 4 mice per genotype (F). *P < 0.05, unpaired t test. (G) Tamoxifen-injected GrnF/F/Cx3Cr1-CreERT2 mice show increased self-grooming time at 8 to 10 mo of age. n = 17, 9. ***P < 0.001, unpaired t test.

Fig. 5.

Inhibition of NF-κB signaling abolished FTD-like behavioral abnormalities induced by microglial PGRN deficiency. (A) PGRN mRNA levels in CD11b⁺ microglia from GrnF/F or LysM-Cre/GrnF/F with or without Ikbkb, determined by qRT-PCR. n = 4 mice per group. ***P < 0.001, one-way ANOVA and Tukey’s post hoc test. (B) Attenuation of NF-κB signaling in PGRN-deficient microglia/myeloid cells in LysM-Cre/GrnF/F/IkbkbF/F mice restored the excessive self-grooming in LysM-Cre/GrnF/F mice to GrnF/F (WT) levels. n = 7 to 9 mice per genotype. *P < 0.05, one-way ANOVA and Bonferroni post hoc test. (C) Attenuation of NF-κB signaling in microglia/myeloid cells restored the nesting to WT (GrnF/F) levels. n = 26, 10, 12. *P < 0.05, one–way ANOVA with Newman–Keuls post hoc test. (D) Attenuation of NF-κB signaling in microglia/myeloid cells restored the marble burying to WT (GrnF/F) levels. n = 31, 11, 12. *P < 0.05, one-way ANOVA, one-way ANOVA with Newman–Keuls post hoc test. (E) Attenuation of NFκB signaling in microglia/myeloid cells restored social interaction to WT (GrnF/F) levels. n = 31, 11, 12. *P < 0.05, one-way ANOVA, one-way ANOVA with Newman–Keuls post hoc test.

Fig. S3.

PGRN deficiency exacerbates TNFα-induced NFκB. (A) Grn^+/+ or Grn^−/− microglia were infected with 5xκB-GFP lentivirus for 3 d and stimulated with TNFα. GFP fluorescence was measured after 8 h. Representative of two independent experiments. (Scale bar: 40 μm.) (B) Grn^+/+ or Grn^−/− microglia were infected with pGreenFire-5xκB lentivirus for 3 d and stimulated with TNFα. Cells were lysed, and luciferase activity was measured after 8 h (n = 4 wells per condition, two-way ANOVA with Bonferroni post hoc test).

Fig. S4.

Breeding scheme to selectively inactivate IKKβ in microglia/myeloid cells with reduced PGRN.