Microglia regulate brain progranulin levels through the endocytosis/lysosomal pathway
Tingting Dong, Leon Tejwani, Youngseob Jung, Hiroshi Kokubu, Kimberly Luttik, Terri M. Driessen, Janghoo Lim
Tingting Dong, Leon Tejwani, Youngseob Jung, Hiroshi Kokubu, Kimberly Luttik, Terri M. Driessen, Janghoo Lim
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Research Article Neuroscience

Microglia regulate brain progranulin levels through the endocytosis/lysosomal pathway

Abstract

Genetic variants in Granulin (GRN), which encodes the secreted glycoprotein progranulin (PGRN), are associated with several neurodegenerative diseases, including frontotemporal lobar degeneration, neuronal ceroid lipofuscinosis, and Alzheimer’s disease. These genetic alterations manifest in pathological changes due to a reduction of PGRN expression; therefore, identifying factors that can modulate PGRN levels in vivo would enhance our understanding of PGRN in neurodegeneration and could reveal novel potential therapeutic targets. Here, we report that modulation of the endocytosis/lysosomal pathway via reduction of Nemo-like kinase (Nlk) in microglia, but not in neurons, can alter total brain Pgrn levels in mice. We demonstrate that Nlk reduction promotes Pgrn degradation by enhancing its trafficking through the endocytosis/lysosomal pathway, specifically in microglia. Furthermore, genetic interaction studies in mice showed that Nlk heterozygosity in Grn haploinsufficient mice further reduces Pgrn levels and induces neuropathological phenotypes associated with PGRN deficiency. Our results reveal a mechanism for Pgrn level regulation in the brain through the active catabolism by microglia and provide insights into the pathophysiology of PGRN-associated diseases.

Authors

Tingting Dong, Leon Tejwani, Youngseob Jung, Hiroshi Kokubu, Kimberly Luttik, Terri M. Driessen, Janghoo Lim

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Figure 1

Loss of Nlk regulates Pgrn levels in the mouse cortex.

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Loss of Nlk regulates Pgrn levels in the mouse cortex. (A–D) Expression ...
(A–D) Expression levels of Pgrn were significantly decreased in the mouse cortex of Nlk+/– Grn+/- mice compared with their littermate controls. Representative Western blot images (A) and quantification (B) of Pgrn and Nlk expression in the 1-year-old mouse cortex. Normalized protein levels of Nlk and Pgrn signals to Vinculin are shown in this and all following graphs. Error bars represent standard error of the mean (SEM) in this and all following graphs. **P < 0.01, ****P < 0.0001; 1-way ANOVA with Tukey’s multiple comparisons post hoc test; F(3,19)=15.45, P < 0.0001. Representative confocal images (C) and quantification (D) of Pgrn immunofluorescent staining in a 1-year-old mouse cortex. Total fluorescence intensity quantification across the image field was automated using Volocity software. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; 1-way ANOVA with Tukey’s multiple comparisons post hoc test; F(3,19)=26.11, P < 0.0001. (E–G) Temporal deletion of Nlk in all cell types reduces Pgrn protein levels in the mouse cortex. Protein and mRNA expression levels were analyzed in the 7-week-old cortex of Nlkfl/fl and Nlkfl/fl Actin-creERT2 mice after tamoxifen (TMX) injection. Representative Western blot images (E) and quantification (F) showing the reduced Nlk and Pgrn expression in the mouse cortex. **P < 0.01, ****P < 0.0001 (nonparametric Mann-Whitney t test, n = 8 animals for Nlkfl/fl, n = 9 for Nlkfl/fl Actin-creERT2). (G) Quantification of Grn mRNA expression levels in Nlk-deleted mouse cortex, showing no transcriptional effects. Normalized levels of Nlk and Grn mRNA to mouse ACTB are shown in this and all following graphs. ***P < 0.001, NS, nonsignificant (nonparametric Mann-Whitney t test, n = 3). In this and all following figures, mouse genotypes are color-coded. Black represents WT, pink for Nlk+/–, yellow for Grn+/–, and blue for Nlk+/– Grn+/– unless otherwise mentioned.