Generation of PGRN-deficient mice. (A) Mouse pgrn locus, targeting vector, and predicted floxed and deleted alleles. Gray boxes denote exons. Sequences are indicated for the neomycin/kanamycin resistance gene (neo/kan; light blue), diphtheria toxin/ampicillin gene (DTX/Amp; pink), LoxP sites (blue triangles), and flippase recombination target sites (red triangles). The red bar marks the 3′ probe. (B) Genotyping of PGRN-deficient mice after crossing PGRN floxed mice with CAG-Cre mice. Genomic DNA was restricted with ScaI for Southern blotting with the 3′ probe. Het, heterozygous; KO, homozygous knockout. (C) PGRN expression monitored by RT-PCR compared with GAPDH as a control (left). Quantification of PGRN transcripts in brain by real-time RT-PCR is shown (right). Results are means ± SEM. B, brain; In, intestine; K, kidney; Li, liver; Sk, skin; Sp, spleen. (D) Hippocampal (top) and cortical (bottom) sections from brains of WT and PGRN-deficient (KO) mice were stained with antibody against PGRN. Bar, 200 µm. (E) Mature BMDMs from 2-mo-old WT or PGRN-deficient mice (KO) were cultured in serum-free media for 48 h. Protein contents of conditioned media were blotted with anti-PGRN (right). Ponceau S staining of the same membrane is shown (left).

PGRN restrains macrophage inflammatory responses by synergizing with LPS for induction of IL-10 transcription. (A) Effect of anti–IL-10 antibody on TNF and MCP-1 production from BMDMs. 1 µg/ml anti–IL-10 or control IgG was added together with LPS and cytokines quantitated by ELISA as in Fig. 2 B. Results are expressed as means ± SEM of triplicate samples from one out of four experiments. (B) PGRN regulates IL-10 expression at the transcriptional level. Quantitative RT-PCR for nascent IL-10 transcripts (left) or spliced IL-10 transcripts (right) was measured 1 h after exposure of BMDMs to 100 ng/ml LPS. Results are expressed as means ± SEM of triplicate samples from one out of two independent experiments. (C) Recombinant PGRN synergized with LPS in IL-10 induction. RAW264.7 cells were incubated with the indicated concentrations of LPS and PGRN for 20 h. IL-10 in the conditioned media was measured by ELISA. Results are expressed as means ± SEM of three independent experiments. *, P < 0.05 using the Student’s t test.

Augmented activation of microglia and astrocytes in the brain of aged PGRN-deficient mice. (A) Activation of microglia and astrocytes in PGRN-deficient mice. Hippocampal, cortical, and thalamic sections from 18-mo-old WT and PGRN-deficient (KO) mice (n = 6) were immunostained using antibody against CD68 or GFAP. (B) Activated CD68⁺ and GFAP⁺ cells were expressed as cells per square millimeter within indicated regions of both cerebral hemispheres of each animal. Results are means ± SEM from three independent experiments. *, P < 0.01 using the Student’s t test. Bar, 80 µm.

Increased ubiquitination and phosphorylation of TDP-43 in the PGRN-deficient brain. (A) Enhanced ubiquitin immunostaining in the hippocampus of old PGRN-deficient mice. Sections from 18-mo-old WT and PGRN-deficient (KO) mice (n = 5) were stained using antibody against ubiquitin. Representative sections from two different mice in each group are shown. Bar, 80 µm. (B) Phosphorylation of TDP-43 and its cytosolic translocation in aged PGRN-deficient (KO) mice. Hippocampal and thalamic sections from 18-mo-old WT and PGRN-deficient mice (n = 5) were stained with antibodies against TDP-43 (top) or phosphorylated TDP-43 (bottom). Bars: (left and middle) 80 µm; (right) 12 µm.

Dysregulated inflammatory responses of PGRN-deficient macrophages to bacterial endotoxin in vitro. (A) Reduced cytokine expression by PGRN-deficient macrophages in response to LPS. Quantitative RT-PCR at indicated times after exposure to 10 ng/ml LPS is shown. (B) Reduced cytokine release by PGRN-deficient macrophages in response to LPS. ELISAs were done with conditioned media collected 12 or 24 h after adding the indicated concentrations of LPS. (C) Enhanced IL-10 production by PGRN-deficient macrophages in response to LPS. IL-10 induction was determined as in A and B. Results are means ± SEM for triplicates from three to five similar experiments. *, P < 0.05 using the Student’s t test. ND, nondetectable.

Defective host defense of PGRN-deficient mice. (A) Enhanced MCP-1 levels in PGRN-deficient mice in response to infection. WT and PGRN-deficient mice (n = 10 per group) were infected intravenously with 3 × 10³ L. monocytogenes. MCP-1 levels in serum and spleen homogenates were determined 24 h after infection by ELISA. Hippocampal expression of MCP-1 was determined by real-time RT-PCR 5 d after infection and expressed as relative levels after normalization with GAPDH mRNA. Results are means ± SEM. *, P < 0.05 using the Student’s t test. ND, nondetectable. (B) Decreased monocyte recruitment to infected spleens of PGRN-deficient mice. Cells from naive or infected spleen (24 h after infection) were tested for the expression of CD11b and Ly6C (for monocyte population). Numbers in dot plots indicate the percentage of Ly6C^hiCD11b⁺ cells. Dot plots are of individual mice; bar graphs represent the mean of five mice per group. Experiments were repeated three times with similar results. *, P < 0.05 using the Student’s t test. (C) Inability to rapidly resolve bacterial infection by PGRN-deficient mice. WT and PGRN-deficient (KO) mice were infected intravenously with 5 × 10³ L. monocytogenes. Bacterial burdens in the spleen, liver, and brain were measured as CFUs at the times indicated (n = 5 per genotype for days 3 and 5 after infection; n = 10 per genotype for day 7 after infection). Results are means ± SEM from one out of three similar experiments. (D) Exaggerated tissue inflammation in PGRN-deficient mice, as assessed by hematoxylin and eosin staining at day 3 (liver) or 5 (brain) after intravenous infection with 5 × 10³ L. monocytogenes. Results are means ± SEM from one out of three similar experiments. Bars, 200 µm.

Enhanced neurotoxic potential of PGRN-deficient macrophages and microglia, and increased vulnerability of PGRN-deficient neurons to stress. (A) BMDM-induced cell death in cultured hippocampal slices. WT coronal hippocampal slices were incubated with WT (n = 29) or PGRN-deficient (KO; n = 36) BMDMs with (LPS/IFN-γ) or without (Non) LPS and IFN-γ for 5 d. Cell death was assessed with PI staining. (top) Representative images. (bottom) Quantitative evaluation is expressed as the percentage of PI staining relative to maximal PI staining (Max) induced with 0.1% Triton X-100. The results are means ± SEM from five independent experiments. *, P < 0.001 using the Student’s t test. Bar, 1 mm. (B) Activated microglia-induced cell death in hippocampal slices. Slices (n = 12 per group) from WT or PGRN-deficient mice were incubated with 10 ng/ml GM-CSF (3 d), followed by stimulation with LPS and IFN-γ in the presence of 10 µg/ml anti–IL-10 or control IgG for 3 d. Cell death was measured as in A. The results are means ± SEM from three independent experiments. *, P < 0.002 using the Student’s t test. (C) Effect of OGD on hippocampal slices from PGRN-deficient (KO) and WT mice (n = 6). Slices were cultured for 2 wk, followed by OGD treatment. Neuronal viability was assessed by PI staining before and after OGD. Maximal neuronal death was induced by exposure to 1 mM NMDA after OGD treatment. The results are means ± SEM from three independent experiments. *, P < 0.001 using the Student’s t test.