Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation

Abstract

Microglia maintain homeostasis in the brain, but whether aberrant microglial activation can cause neurodegeneration remains controversial. Here, we use transcriptome profiling to demonstrate that deficiency in frontotemporal dementia (FTD) gene progranulin (Grn) leads to an age-dependent, progressive upregulation of lysosomal and innate immunity genes, increased complement production, and enhanced synaptic pruning in microglia. During aging, Grn(-/-) mice show profound microglia infiltration and preferential elimination of inhibitory synapses in the ventral thalamus, which lead to hyperexcitability in the thalamocortical circuits and obsessive-compulsive disorder (OCD)-like grooming behaviors. Remarkably, deleting C1qa gene significantly reduces synaptic pruning by Grn(-/-) microglia and mitigates neurodegeneration, behavioral phenotypes, and premature mortality in Grn(-/-) mice. Together, our results uncover a previously unrecognized role of progranulin in suppressing aberrant microglia activation during aging. These results represent an important conceptual advance that complement activation and microglia-mediated synaptic pruning are major drivers, rather than consequences, of neurodegeneration caused by progranulin deficiency.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Figure 1. Transcriptome profiling in Grn+/+, Grn+/− and Grn−/− mice reveal age-dependent up-regulation of lysosomal and innate immunity genes in microglia

(A) Diagram showing the procedures to characterize the transcriptomes of specific brain regions in Grn+/+, Grn+/− and Grn−/− mice during aging. (B) Weighted correlation network analysis (WGCNA) identifies highly correlated gene modules that are age-dependently up-regulated in the cerebral cortex of Grn−/− mice. (C) The top 40 genes from the magenta (cerebral cortex) module are highly enriched with microglial genes (inset), and there is extensive topographical overlap among their expression patterns, especially for complements C1qa, C1qb, C1qc and C3, Cd68 and Trem2. (D) Diagram showing the procedures to isolate microglia from 4 and 16 month old Grn+/+ and Grn−/− mice using discontinuous isotonic Percoll gradient, FACS, preparation of amplified antisense RNA (aaRNA), microarray and bioinformatics analyses. (E) Dissociated cells from 16 month old Grn+/+ and Grn−/− mice are incubated with CD11b PE [clone M1/70](Invitrogen), CD45 APC [clone Ly5](eBioscience), and anti-Ly6G PE-Cy7 [clone 1A8](BD Biosciences), and sorted by Beckman-Coulter MoFlo XDPs flow cytometer. (F) Microglia are defined as CD45lo;CD11b+, whereas macrophages are defined as CD45hi;CD11b+ population in FACS. Data are presented as % of total events. Student’s t test, n = 3 for Grn+/+ and Grn−/− mice. ** indicates p < 0.01, ns, not significant. (G) Principle component analysis of the top 500 most variable transcripts from the FACS-sorted microglia from 4 and 16 month old Grn+/+ and Grn−/− mice. (H) Hierarchical clustering analyses of the top 200 transcripts from FACS-sorted microglia from 4 and 16 month old Grn+/+ and Grn−/− mice. See also Figure S1 and Table S1.

Figure 2. Lysosomal defects and increased complement production by Grn−/− microglia

(A–D) Confocal images of Iba-1+ microglia from the ventral thalamus of 4 and 18 month old Grn+/+ and Grn−/− brains show CD68+ lysosomes in the cytoplasm. Images were captured from Grn+/+ and Grn−/− brains (n = 3 per age), and processed for 3D reconstruction of the lysosomes using Imaris software. (E) Quantification of CD68+ lysosome volume in the ventral thalamus of 4 and 18 month old Grn+/+ and Grn−/− mouse brains (left panel) and in 4 month old Grn+/+ and Grn−/− mouse brains after MPTP treatment (right panel). Volumes are expressed as μm3 per microglia. Student’s t test, n = 3 per group. * indicates p < 0.05, ** p < 0.01, and **** p < 0.001. (F–I) Confocal images of primary microglia cultured from neonatal Grn+/+ and Grn−/− mouse brains. Microglia are labeled with antibodies for Lamp1/PGRN/Rab7 or Lamp1/PGRN/Sortilin. Arrows indicate regions in each microglia where fluorescent signal intensity plots are obtained using Nikon NIS-Elements. (F′-I′) Fluorescent signal intensity plots of Lamp1+ (green), PGRN+ (red), Rab7+ (blue) and Sortilin+ (blue) vesicles in Grn+/+ and Grn−/− microglia. Arrows in F′ and H′ indicate partial overlap of Lamp1+;PGRN+ or PGRN+;Sortilin+ signals, respectively. (J–K) Quantification of the size of Lamp1+, Sortilin+ and Rab7+ vesicles in Grn+/+ and Grn−/− microglia. Student’s t test, **** indicates p < 0.001. (L) Diagram showing DQ-BSA assays in cultured microglia. Open circles are quenched DQ-BSA, green circles are dequenched DQ-BSA and red circles are BSA-conjugated with 568nm fluorophore. (M–P) Representative images of Grn+/+ and Grn−/− microglia after 30′ incubation with DQ-BSA (0 hrs) or 4 hrs after washing (4 hrs). Scale bar in P is 10 μm. Insets to the right of each panel represent enlarged images in the boxed area in M-P. Arrows in P indicate dequenched DQ-BSA signals in Lamp1+ lysosomes. (Q) Quantification of the maximal fluorescence intensity (M.F.I.) of dequenched DQ-BSA in Grn+/+ (n = 5) and Grn−/− microglia (n = 7). Bafilomycin inhibits lysosomal acidification and protein degradation in Grn+/+ and Grn−/− microglia. Two-way ANOVA, * indicates p < 0.05. See also Figure S2.

Figure 3. Increased synaptic pruning activity in Grn−/− microglia requires C1qa

(AC) Diagrams showing microglia-neuron co-cultures and Sholl analyses to quantify synapses around microglia. (D–S) Confocal images showing the presence of synapses (SPH+) around Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− microglia (Iba1+)(D, H, L, P). Imaris 3D image reconstruction of the microglia-neuron co-cultures at a lower magnification (E, I, M, Q). Higher magnification shows the presence of C1qa immediately adjacent synapses (F, J, N, R) and C1qa-tagged synapses inside CD68+ lysosomes in microglia (G, K, O, S). Scale bar 20 μm in D, 5 μm in E, and 1 μm in F and G. (T) Synaptic density around Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− microglia. *** p < 0.005, **** p < 0.001, two-way ANOVA, n = 4 for all genotypes. (U–V) Quantification of the percentage of C1qa-tagged synapses outside microglia (U), and the number of SPH+ synapse inside microglia (V). * p < 0.05, ** p < 0.01, *** p < 0.005, Student’s t test, n = 4 per genotype. See also Figure S3.

Figure 4. Age-dependent C1qa accumulation at the synapses of the ventral thalamus in Grn−/− mice

(A–B) QRT-PCR detects the relative abundance of C1qa and C3 mRNA in cerebral cortex (CTX), hippocampus (HIP), caudate-putamen (CP), cerebellum (CRB) and thalamus (THAL) of 12 month old Grn+/+ and Grn−/− mice. (C–D) QRT-PCR shows the progressive increase of C1qa and C3 mRNA in the thalamus of 4, 9, 12 and 18 month-old Grn−/− mice. (E–F) Western blots showing the relative abundance of C1qa and C3 proteins in the thalamus of 4, 9 and 18 month-old Grn+/+ and Grn−/− mice. * p < 0.05, ** p < 0.01, *** p < 0.005, Student’s t test. Error bars indicate s.e.m. n = 3 per age for Grn+/+ and Grn−/− mice. (G–J) Immunostains in 4 and 18-month old Grn+/+ and Grn−/− mouse brain detect C1qa signals in the VPM and VPL thalamic nuclei (dotted areas). Scale bar in G is 500 μm. (K–P) Confocal images using neuronal marker TuJ1 and C1qa antibodies in the ventral thalamus of 12 month-old Grn+/+ and Grn−/− mice. Scale bar in P is 10 μm. (Q–V) Colocalization of synaptophysin and C1qa in the ventral thalamus of 12 month-old Grn+/+ and Grn−/− mice. Scale bar in V is 10 μm. (W–X) Immunogold EM detects C1qa deposits in synapses in the ventral thalamus of 12 month-old Grn+/+ and Grn−/− mice. Scale bars are 0.5 μm. (Y) Western blots using synaptosomes from 4, 9 and 16 month-old Grn+/+ and Grn−/− brains detect C1qa and cleaved C3 proteins.

Figure 5. Reduced microglia number and preservation of synaptic density in the ventral thalamus of Grn−/−;C1qa−/− mutant mice

(A–H) Immunostains for Iba-1 in coronal sections of 12 month-old Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− mouse brains at the level of anterior hippocampus. The square dotted boxes in panels A, C, E and G highlight the ventral thalamus, where the higher magnification images are obtained. Scale bar 500 μm in A and 50 μm in B. (I) Iba-1+ microglial density in the ventral thalamus of Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− mouse brains at 2, 4, 7, 12 and 19 month-old. *** p < 0.005, **** p < 0.001, two-way ANOVA, n = 4 per genotype per age. (J–M) Confocal images of synaptophysin in 12 month-old Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− brains. Scale bar 10 μm in J. (N) Synaptophysin density in Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− brains at 2, 4, 7, 12 and 19 month-old. * p < 0.05, ** p < 0.01, *** p < 0.005, ns, not significant. Student’s t test, n = 4 per genotype per age.

Figure 6. Removing C1qa in Grn−/−;C1qa−/− mice protects synaptic pruning, restores thalamic microcircuit function, mitigate OCD-like behaviors and improves survival

(A–D) Confocal images of parvalbumin (Parv) show the projection of Parv+ neurons in the reticular nucleus (TRN) to the ventroposterior medial (VPM) and ventroposterior lateral (VPL) nuclei in the ventral thalamus of 12 month old Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− mice. Dashed lines highlight VPM and VPL nuclei, and squares regions higher magnification in A′-D′. Scale bar is 500 μm in D, and 20 μm in D′. (E–H) Confocal images of VGAT+ synapses in the ventral thalamus in 19 month old Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− mice. Scale bar is 25 μm in H. (I) Quantification of VGAT+ synaptic density in the ventral thalamus of 8, 12 and 19 month old Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− mice. Student’s t test, * indicates p < 0.05, ** p < 0.01 and ns, not significant. (J) Image of a thalamic slice showing the stimulating electrode in the internal capsule and the 16-channel linear array silicon probe that records multiunit firing in VPM and VPL. (K) Representative multiunit recordings (orange box in J) from the ventral thalamus of Grn+/+, Grn−/− and Grn−/−;C1qa−/− mice. Black circle indicates stimulation artifact. Bottom, rate meters showing consistent evoked spike rate across sweeps of stimulations (X-axis) relative to time (Y-axis). (L) Peri-stimulus time histogram of the population data from 6 Grn+/+, 7 Grn−/− and 4 Grn−/−;C1qa−/− mice. Inset bottom: enlargement of the black dashed box in (L) showing the slope of the response is significantly different among genotypes (***, p < 0.0001, F = 10.5554). Inset top: plot of the relative probability of eliciting AP firing frequencies among Grn+/+, Grn−/−, and Grn−/−;C1qa−/− mice analyzed by the Kolmogorav-Smirnov Test (Grn+/+ vs. Grn−/−, *** p < 0.0001, D = 0.5783; Grn+/+ vs. Grn−/−;C1qa−/−, *** p < 0.0001, D = 0.4980; Grn−/− vs. Grn−/−;C1qa−/−, *** p < 0.0001, D = 0.7751). Error bars, s.e.m., (M) Grooming activities in Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− mice is expressed as percentage of total time. * p < 0.05, ** p < 0.01, ns, not significant, Student’s t test. (N–O) Kaplan-Meier curve for skin lesion onset and survival in Grn+/+, Grn−/−, C1qa−/− and Grn−/−;C1qa−/− mice. * p < 0.05, **** p < 0.001, ns, not significant, Long-rank (Mantel-Cox) test. See also Figures S4, S5 and S6, and Supplemental Movie S1.

Figure 7. Microglial pathology and CSF complement levels in FTLD Grn mutation carriers

(A–H) Immunostains of frontal cortex from control, FTLD Grn carriers and AD patients detect the presence of microglia and C1qa (A–B, D–E, G–H). In addition, immunogold EM detects the presence of C1qa deposits at the synapses in FTLD Grn mutation carriers (C, F). Arrows in A, B, D, E, G and H indicate microglia. Arrowhead in G indicates an amyloid plaque, and in H indicates a blood vessel (BV). Arrowheads in C and F indicate presynaptic terminals, and arrows in F indicate C1qa-positive immunogold particles in synapses. (I) Quantification of Iba-1+ microglia in the frontal cortex of controls (n = 7), FTLD Grn carriers (n = 16), and AD patients (n = 8). *** p < 0.005, **** p < 0.001, ns, not significant, Student’s t test. (J) Quantification of CSF PGRN levels in controls and FTLD GRN carriers. Student’s t test, **** indicates p < 0.0001. (K–L) ELISA assays for C1qa and C3b protein levels in the CSF of controls (n = 23) and FTLD Grn carriers (n = 19). Chi Square Goodness of Fit test to calculate R2 and p values. See also Figure S7, Tables S2 and Table S3.