Journal: Npj Dementia

Article Title: Progranulin deficiency does not exacerbate TDP-43 pathology in TDP-43 transgenic mouse models

doi: 10.1038/s44400-025-00020-4

Figure Lengend Snippet: A Immunostaining of TDP-43 and NeuN in brain sections from 16-month-old mice WT, TDP-43 Q331K/Q331K (Q331K), Grn −/− , TDP-43 Q331K/Q331K Grn −/− (Q331K Grn −/− ) mice. Representative images from the cortex were shown. Scale bar, 10 µm. B Analysis of TDP-43, phosphorylated TDP-43 (pS409/410), and PGRN levels in cortical lysates from 16-month-old mice of the indicated genotypes. C TDP-43 levels in RIPA- and urea-soluble fractions were quantified. Data are presented as mean ± SEM from 3 mice per group ( n = 3). One-way ANOVA tests with Bonferroni’s multiple comparisons. D PGRN levels in RIPA soluble fractions were quantified and normalized to GAPDH. Data are presented as mean ± SEM from 3 mice per group ( n = 3). E Total RNAs were extracted from the cortex of 10-month-old WT, TDP-43 Q331K/Q331K , Grn −/− , TDP-43 Q331K/Q331K Grn −/− male mice, and the RT-qPCR was performed to analyze the splicing changes in Sort1 exon 17b (left) and Mapt exons 2 and 3 (right). The relative mRNA levels of transcripts including or excluding exons 2 and 3 represent the inclusion of Mapt exons 2 and 3. Data are presented as mean ± SEM ( n = 4 mice per genotype). p -values were determined using one-way ANOVA tests with Bonferroni’s multiple comparisons. F Expression levels of Tardbp in WT and Q331K mice. Total RNAs were extracted from the cortex of 10-month-old WT and Q331K male mice, and the RNA-seq was performed to analyze gene expression changes. Normalized read counts are shown. Data are presented as mean ± SEM ( n = 5-6 mice per genotype). * p < 0.05, unpaired two-tailed Student's t-test.

Article Snippet: The following antibodies were used in this study: rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-CD68 (Bio-Rad, MCA1957), mouse anti-GFAP (Cell signaling, 3670S), rabbit anti-TDP43 (Proteintech Group, 12892-1-AP (C-terminal) and 10782-2-AP (N-terminal)), rabbit anti-phospho-TDP-43 (Ser409/410) (Proteintech group, 80007-1-RR), mouse anti-PLP (Millipore, MAB388), mouse anti-MBP (Millipore, SMI-99), rabbit anti-MAG (Proteintech Group, 14386-1-AP), sheep anti-mouse PGRN (R&D systems, AF2557), and mouse anti-GAPDH (Proteintech Group, 60004-1-Ig).

Techniques: Immunostaining, Quantitative RT-PCR, Expressing, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: Npj Dementia

Article Title: Progranulin deficiency does not exacerbate TDP-43 pathology in TDP-43 transgenic mouse models

doi: 10.1038/s44400-025-00020-4

Figure Lengend Snippet: A Immunostaining of TDP-43 and NeuN using rabbit anti-TDP-43 CTD antibodies and mouse anti-NeuN antibodies, respectively (left panel) or immunostaining of human TDP-43 (right panel) using mouse anti-human TDP-43 in brain sections from 21-day-old WT, hTDP-43 Tg/Tg (Tg/Tg), Grn −/− , and hTDP-43 Tg/Tg Grn −/− mice. Representative images from the cortex were shown. Scale bar, 10 µm. B Analysis of TDP-43 and phosphorylated TDP-43 (pS409/410) and PGRN levels in brain lysates from 21-day-old mice. Antibodies recognizing the C-terminal or N-terminal domain of TDP-43 were used to detect total TDP-43 levels. C TDP-43 and pTDP-43 levels in RIPA- and urea-soluble fractions were quantified. Data are presented as mean ± SEM from 3 mice per group ( n = 3). One-way ANOVA tests with Bonferroni’s multiple comparisons. ** p < 0.01. D PGRN levels in RIPA soluble fractions were quantified and normalized to GAPDH. Data are presented as mean ± SEM from 3 mice per group ( n = 3).

Article Snippet: The following antibodies were used in this study: rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-CD68 (Bio-Rad, MCA1957), mouse anti-GFAP (Cell signaling, 3670S), rabbit anti-TDP43 (Proteintech Group, 12892-1-AP (C-terminal) and 10782-2-AP (N-terminal)), rabbit anti-phospho-TDP-43 (Ser409/410) (Proteintech group, 80007-1-RR), mouse anti-PLP (Millipore, MAB388), mouse anti-MBP (Millipore, SMI-99), rabbit anti-MAG (Proteintech Group, 14386-1-AP), sheep anti-mouse PGRN (R&D systems, AF2557), and mouse anti-GAPDH (Proteintech Group, 60004-1-Ig).

Techniques: Immunostaining

Journal: eNeuro

Article Title: Effects of Exercise on Progranulin Levels and Gliosis in Progranulin-Insufficient Mice 1 , 2 , 3

doi: 10.1523/ENEURO.0061-14.2015

Figure Lengend Snippet: Exercise (3-4 weeks) produces a small increase in hippocampal progranulin in young adult wild-type mice. Two- to 3-month-old wild-type mice were randomized to sedentary or exercise groups for three to four weeks ( A ). B , Exercise produced a robust increase in BDNF (**** p < 0.0001). C , Representative BDNF and α-tubulin blots. D , Exercise did not significantly increase frontal cortex progranulin protein. E , Exercise produced a small but statistically significant increase in hippocampal progranulin (* p = 0.038). F , Exercise did not affect plasma progranulin. n = 8–16 mice per group. Values in B – F are expressed relative to the sedentary group.

Article Snippet: After electrophoresis, transfer, and blocking as described above, the samples were incubated overnight in a sheep polyclonal anti-progranulin antibody (1:5000, R&D Systems AF2557).

Techniques: Produced, Clinical Proteomics

Journal: eNeuro

Article Title: Effects of Exercise on Progranulin Levels and Gliosis in Progranulin-Insufficient Mice 1 , 2 , 3

doi: 10.1523/ENEURO.0061-14.2015

Figure Lengend Snippet: Exercise (4 weeks) does not increase progranulin protein levels in multiple brain regions of Grn +/− mice. Four- to 8-month-old Grn +/− mice were randomized to sedentary or exercise groups for 4 weeks ( A ). Exercise did not increase progranulin protein levels in frontal cortex ( B ), thalamus ( C ), or hippocampus ( D ), despite producing the expected increase in the number of doublecortin-positive neurons in the dentate gyrus ( E , ** p < 0.01). Values in B – D are expressed relative to sedentary Grn +/+ mice, with sedentary Grn +/− mice set at 0.5 to maintain a consistent scale with Figure 3. n = 11–12 per group.

Article Snippet: After electrophoresis, transfer, and blocking as described above, the samples were incubated overnight in a sheep polyclonal anti-progranulin antibody (1:5000, R&D Systems AF2557).

Techniques:

Journal: eNeuro

Article Title: Effects of Exercise on Progranulin Levels and Gliosis in Progranulin-Insufficient Mice 1 , 2 , 3

doi: 10.1523/ENEURO.0061-14.2015

Figure Lengend Snippet: Measures of progranulin levels

Article Snippet: After electrophoresis, transfer, and blocking as described above, the samples were incubated overnight in a sheep polyclonal anti-progranulin antibody (1:5000, R&D Systems AF2557).

Techniques: Clinical Proteomics

Journal: eNeuro

Article Title: Effects of Exercise on Progranulin Levels and Gliosis in Progranulin-Insufficient Mice 1 , 2 , 3

doi: 10.1523/ENEURO.0061-14.2015

Figure Lengend Snippet: Group housing does not potentiate the effects of exercise on progranulin. Six weeks of wheel running ( A ) failed to increase frontal cortex progranulin mRNA ( B ) or hippocampal progranulin protein ( C ) in either solo- or group-housed wild-type mice aged 3–6 months. D-F , Hippocampal BDNF was increased by exercise ( D ; ANOVA effect of exercise, p = 0.041). F , Representative BDNF and α-tubulin blots for each group. n = 8–14 mice per group. Values in B – E are expressed relative to the solo-housed sedentary group.

Article Snippet: After electrophoresis, transfer, and blocking as described above, the samples were incubated overnight in a sheep polyclonal anti-progranulin antibody (1:5000, R&D Systems AF2557).

Techniques:

Journal: The American journal of pathology

Article Title: Accelerated lipofuscinosis and ubiquitination in granulin knockout mice suggest a role for progranulin in successful aging.

doi: 10.2353/ajpath.2010.090915

Figure Lengend Snippet: Figure 1. PGRN immunoreactivity is reflective of genotypes and increases in older GRN/ and GRN/ mice. As shown in 12-month-old mice, GRN/ mice (A) have higher PGRN immunoreactivity than GRN/ mice (B), whereas GRN/ mice (C) have no detectable PGRN expression. Higher magnification insets correspond to the areas identified by black squares. D: In a subset of GRN/ (green) and GRN/ (orange) mice, PGRN immuno- reactivity was quantified by image analysis. Consistent with genotypes, GRN/ mice had significantly reduced PGRN levels compared with GRN/

Article Snippet: Membranes were blocked in 5% milk in Tris-buffered saline-0.1% Triton X-100 and incubated overnight in anti-mouse PGRN (1:500; R&D Systems, Minneapolis, MN) or anti-glyceraldehyde-3-phosphate dehydrogenase (1:10,000; BioSource International, Camarillo, CA) at 4°C.

Techniques: Expressing

Journal: The American journal of pathology

Article Title: Accelerated lipofuscinosis and ubiquitination in granulin knockout mice suggest a role for progranulin in successful aging.

doi: 10.2353/ajpath.2010.090915

Figure Lengend Snippet: Figure 7. Hypothetical model of neurodegeneration based on the chrono- logical appearance of pathology in GRN/ mice. 1 month: young neurons are free of any pathology and microglia are in a ramified or resting state; seven months: through an unknown mechanism, the lack of PGRN results in intraneuronal accumulation of lipofuscin pigment in aging neurons resulting in cellular stress which is detected by nearby microglia; 12 months: neuronal cytoplasm is full of lipofuscin pigment which adversely affects cellular func- tion and long-term neuronal survival, signaling the activation of microglia; 23 months: slow and progressive loss of neurons, resulting in expulsion of neuronal lipofuscin into extracellular space, most of which is phagocytosed by activated microglia with some lipofuscin accumulating in the extracellular space. The lack of PGRN may also affect the normal function of activated microglia resulting in accumulation of endogenous microglial lipofuscin. A severe, sustained, and/or an inappropriate microglia (inflammatory) re- sponse (attributable to absence of microglial PGRN) may further enhance neuronal loss.

Article Snippet: Membranes were blocked in 5% milk in Tris-buffered saline-0.1% Triton X-100 and incubated overnight in anti-mouse PGRN (1:500; R&D Systems, Minneapolis, MN) or anti-glyceraldehyde-3-phosphate dehydrogenase (1:10,000; BioSource International, Camarillo, CA) at 4°C.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

doi: 10.1371/journal.pone.0065542

Figure Lengend Snippet: (A) Schematic diagram of DNA injection. 6- to 8-week old female BALB/c mice were injected subcutaneously with ALD-DNA (50 µg/mice) plus CFA at week 0, followed by two booster injections of ALD-DNA (50 µg/mice) emulsified with IFA at week 2 and week 4 after initial injection. (B) Serum anti-dsDNA IgG levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (C) 8 weeks after initial injection, glomerular immune deposition were detected by direct immunofluorescence for IgG in frozen kidney section from ALD-DNA-injected SLE mice or control mice. Representative images (magnification×200) of 8 mice are shown for each group. (D) Nephritic pathology was evaluated by H&E staining of renal tissues. Images (magnification×200) are representative of 8 mice in each group. (E) The kidney score was assessed using paraffin sections stained with H&E in (D). n = 8. (F) Urine protein levels of the mice were assessed by BCA Protein Assay Kit every 2 weeks. Data are means ± SD from 8 mice in each group. (G) Serum GRN levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (H) The correlation between serum GRN level and kidney score in lupus model. Correlation analysis was performed by Pearson correlation analysis. Each symbol indicates an individual mouse (n = 21). (I) The correlation between serum GRN level and urine protein level in lupus model. Pearson correlation analysis was used to carry out the correlation study. Each symbol indicates an individual mouse (n = 21). *, P <0.05.

Article Snippet: To assess protein levels of GRN in murine serum, ELISA assays were performed with commercial mouse GRN ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Control, Staining, Bicinchoninic Acid Protein Assay

Journal: PLoS ONE

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

doi: 10.1371/journal.pone.0065542

Figure Lengend Snippet: BALB/c mice were administrated intramuscularly with 100 µg/mouse pGRN or pcDNA3.1 to overexpress GRN, intravenously injected with LV-shGRN or LV-shNC (2×10 8 molecules/mouse) to down-regulate GRN expression. And 72h later, mice were then injected subcutaneously with ALD-DNA (50 µg/mouse) for total 3 times in 4 weeks. (A) The dynamics of serum GRN levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (B) Nephritic pathology was evaluated by H&E staining of renal tissues. Images (magnification×200) are representative of at least 8 mice in each group. (C) The kidney score was assessed using paraffin sections stained with H&E in (B). n = 8. (D) Urine protein levels of the mice were assessed by BCA Protein Assay Kit every 2 weeks. Data are means ± SD from 8 mice in each group. *, P <0.05.

Article Snippet: To assess protein levels of GRN in murine serum, ELISA assays were performed with commercial mouse GRN ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Bicinchoninic Acid Protein Assay

Journal: PLoS ONE

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

doi: 10.1371/journal.pone.0065542

Figure Lengend Snippet: (A–B) Primary macrophages were stimulated with increasing amounts of ALD-DNA for 24 h. mRNA levels of GRN in macrophages were analyzed by real time PCR analysis (A), and protein levels of GRN in the supernatants of macrophages were analyzed by western blot (B). Above, quantitative results of western blots, the band intensity was measured by Image J; Below, representative western blots. Similar results were obtained in three independent experiments. Data are representative of results obtained in three independent experiments. Primary peritoneal macrophages were stimulated by ALD-DNA (50 µg/mL) with GRN (5 µg/mL) for 24 h (C and F), or were pretreated with elastase inhibitor (100 µM) or DMSO (0.1%) for 12 h (D and G), and then were exposed to ALD-DNA, UnALD-DNA, or PBS for another 24 h. Macrophages were transfected with control siRNA (200 nM) or GRN siRNA (siGRN, 200 nM). 36 h posttransfection, macrophages were stimulated with PBS, UnALD-DNA or ALD-DNA (50 µg/mL) (E and H). (C–E) ELISA assay was used to analyze the levels of TNF-α, IL-1β, IL-6, IL-10, IL-12, and MCP-1 in the culture supernatants of macrophages. Data are means ± SD of three independent experiments. (F–H) Western blot analysis was used to analyze the protein levels of iNOS in macrophages. Data are representative of three separate experiments. Similar results were obtained in three independent experiments. Band intensity was measured by Image J and the ratios of iNOS to β-actin were calculated. *, P <0.05.

Article Snippet: To assess protein levels of GRN in murine serum, ELISA assays were performed with commercial mouse GRN ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Granulin Exacerbates Lupus Nephritis via Enhancing Macrophage M2b Polarization

doi: 10.1371/journal.pone.0065542

Figure Lengend Snippet: (A) Primary macrophages were stimulated with 50 µg/mL ALD-DNA together with 5 µg/mL purified GRN for the indicated time. Phospho-Akt, ERK, JNK, and p38 were detected by immunoblotting. Data are representative of three separate experiments. Similar results were obtained in three independent experiments. (B) Primary macrophages were pretreated with elastase inhibitor (100 µM) for 12 h, and then were stimulated with 50 µg/mL ALD-DNA for the indicated time. Phospho-Akt, ERK, JNK, and p38 were detected by Western blot. Data are representative of three separate experiments. Similar results were obtained in three independent experiments. Left, representative western blots; Right, quantitative results, the band intensity was measured by Image J and the ratios of phospho-Akt, phospho-ERK, phospho-JNK and phospho-p38 to β-actin were calculated. Primary peritoneal macrophages were pretreated with U0126 (10 µM) (C), SP600125 (10 µM) (D), SB203580 (10 µM) (E) for 30 min, and then were stimulated with 50 µg/mL ALD-DNA together with 5 µg/mL purified GRN for 24 h. Cytokine expression levels of TNF-α, IL-1β and IL-10 in the supernatants of macrophages were determined by ELISA assay. Data are means ± SD of three independent experiments. *, P <0.05.

Article Snippet: To assess protein levels of GRN in murine serum, ELISA assays were performed with commercial mouse GRN ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Purification, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Progranulin deficiency causes impairment of autophagy and TDP-43 accumulation

doi: 10.1084/jem.20160999

Figure Lengend Snippet: Impaired defense against Listeria monocytogenes in mice lacking PGRN. (A) 14-d survival curve of GRN +/+ (solid blue line), GRN +/− (dashed green line), and GRN −/− (dotted red line) mice after intravenous L. monocytogenes infection. n = 12 mice per group from one experiment. ****, P < 0.0001 GRN +/+ versus GRN −/− using a log-rank test. (B) L. monocytogenes burden in GRN +/+ (blue circles) and GRN −/− (red triangles) mice 2 d after infection. n = 5 mice per group from one experiment. *, P < 0.05 and **, P < 0.01 versus GRN +/+ using an unpaired t test with Welch’s correction. (C) Serum PGRN levels in response to L. monocytogenes infection. n = 5 mice per group from one experiment. ***, P < 0.001 versus pre-infected using a paired t test. (D) Serum IL-6, IL-10, and MCP-1 levels in GRN +/+ (circles) and GRN −/− (triangles) mice after intravenous L. moncytogenes infection. n = 5 mice per group from one experiment. *, P < 0.05 versus GRN +/+ using an unpaired t test. (E) Serum IL-6, IL-10, and MCP-1 levels in GRN +/+ (circles) and GRN −/− (triangles) mice after treatment with LPS. n = 5 mice per group from one experiment. *, P < 0.05 and **, P < 0.01 using an unpaired t test. Error bars represent mean ± SEM.

Article Snippet: Mouse serum PGRN levels were measured using a PGRN Mouse EIA kit (Cayman Chemical).

Techniques: Infection

Journal: The Journal of Experimental Medicine

Article Title: Progranulin deficiency causes impairment of autophagy and TDP-43 accumulation

doi: 10.1084/jem.20160999

Figure Lengend Snippet: Stimulation of autophagy signaling by PGRN. (A) Representative Western blot from n = 3 independent experiments of cell lysates from GRN −/− cortical neurons untreated or with 10 µg/ml recombinant mouse PGRN or vehicle and probed with the indicated antibodies. (B) Quantification of phosphorylated/total protein signals for the indicated proteins from experiments as shown in A. n = 3 independent experiments. *, P < 0.05 and **, P < 0.01 versus GRN +/+ using an unpaired t test. (C) Dose–response relationship for PGRN stimulation of AMPKα phosphorylation. Representative Western blot from n = 3 independent experiments of GRN −/− cortical cultures treated with the indicated concentrations of recombinant mPGRN and probed with the indicated antibodies. (A and C) Molecular mass is indicated in kilodaltons. (D) Quantification of phospho-AMPKα /total AMPKα signal from experiments as shown in C. n = 3 independent experiments. *, P < 0.05 and ***, P < 0.001 versus untreated using one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent mean ± SEM.

Article Snippet: Mouse serum PGRN levels were measured using a PGRN Mouse EIA kit (Cayman Chemical).

Techniques: Western Blot, Recombinant, Phospho-proteomics

Journal: The Journal of Experimental Medicine

Article Title: Progranulin deficiency causes impairment of autophagy and TDP-43 accumulation

doi: 10.1084/jem.20160999

Figure Lengend Snippet: Accelerated accumulation of pathogenic TDP-43 in GRN −/− neurons. (A) Representative Western blot from n = 4 independent experiments of GRN +/+ and GRN −/− cortical cultures infected with AAV expressing mCherry along with either full-length TDP43-GFP (left) or TDP43CT-GFP (right) and probed with the indicated antibodies. Molecular mass is indicated in kilodaltons. (B) Quantification of GFP/mCherry signal from experiments as shown in A. n = 4 independent experiments. ***, P < 0.001 versus GRN +/+ using an unpaired t test. (C) Representative images of GRN +/+ and GRN −/− hippocampal neurons cotransfected with mCherry (top row) and TDP43CT-GFP (bottom) and treated with vehicle (DMSO; left), 10 µM PGRN (middle), or 100 nM bafilomycin A1 (right). n = 158, 173, 37, 69, 147, and 96 cells per genotype and condition from eight, eight, three, three, eight, and eight independent experiments for the groups listed left to right. Bars, 25 µm. (D) Quantification of total GFP fluorescence per cell from experiments as shown in A. Data were normalized to the mean of GRN +/+ vehicle-treated cells. n = 158, 173, 37, 69, 147, and 96 cells per genotype and condition from eight, eight, three, three, eight, and eight independent experiments for the groups listed left to right. *, P < 0.05 and **, P < 0.01 versus vehicle-treated GRN +/+ using Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons test. Error bars represent mean ± SEM. ns, not significant.

Article Snippet: Mouse serum PGRN levels were measured using a PGRN Mouse EIA kit (Cayman Chemical).

Techniques: Western Blot, Infection, Expressing, Fluorescence

Journal: BMC Neuroscience

Article Title: Progranulin is expressed within motor neurons and promotes neuronal cell survival

doi: 10.1186/1471-2202-10-130

Figure Lengend Snippet: PGRN within motor neurons in primary cultures does not colocalize with the nucleus or mitochondria . (A) Motor neuron labeled with antibody to mouse PGRN (left hand image) is attenuated by antigen-competition with 300 ng recombinant mouse PGRN (middle and right hand images). When anti-PRGN was pre-absorbed with 400 ng of mouse recombinant PGRN, no signal was observed in the primary motor neurons (not shown). Shown are confocal images taken at 100×. (B) PGRN is not distributed in nuclei or mitochondria, organelles that are not part of the secretory pathway. Immunolabelling of motor neurons in dissociated spinal cord-DRG cultures with anti-TDP-43 (a) and anti-cytochrome C (b) and anti-PGRN (middle column). Merged images (right column) show no colocalization of TDP-43 or cytochrome C with endogenous mouse PGRN. Confocal images were captured at 63× magnification, hatched boxes represent 3-5× zoom.

Article Snippet: To control for nonspecific binding of the mouse PGRN antibody, antigen-competition was carried out by preadsorbing the antibody at 1:500 dilution with 300 ng/mL and 400 ng/mL recombinant mouse PGRN (Alexis Biochemicals).

Techniques: Labeling, Recombinant