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 3

Nlk regulates Pgrn levels in microglia in a kinase activity-dependent manner.

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Nlk regulates Pgrn levels in microglia in a kinase activity-dependent ma...
(A–C) Nlk regulates Pgrn levels in microglia in vivo. Protein and mRNA expression levels were analyzed in the 6-week-old cortex of Nlkfl/fl and Nlkfl/fl Cx3cr1-cre mice. Representative Western blot images (A) and quantification (B) showing that the protein expression levels of Pgrn are significantly decreased by Nlk deletion in microglia. Grn–/– is shown as a negative control. *P < 0.05, ****P < 0.0001 (nonparametric Mann-Whitney t test, n = 4–6 animals per group). Quantification (C) of Nlk and Grn mRNA expression levels in the cortex from microglia-specific Nlk deletion mice. NS, nonsignificant (nonparametric Mann-Whitney t test, n = 6). (D and E) Reduced Nlk expression significantly decreased Pgrn expression levels in primary microglia. Representative Western blot images (D) and quantification (E) of Nlk and Pgrn expression levels in primary microglia at DIV-16 from WT and Nlk+/– mice. *P < 0.05 (nonparametric Mann-Whitney t test, n = 5). (F–H) Nlk reduction resulted in decreased Pgrn levels in BV2 microglial cells. Protein and mRNA expression levels were analyzed in WT and Nlk-KD BV2 cells. Representative Western blot images (F) and quantification (G and H) showing the expression levels of Nlk and Pgrn proteins and their corresponding mRNAs. ***P < 0.001 (nonparametric Mann-Whitney t test, n = 4). (I and J) Increased expression of Nlk upregulated Pgrn levels in the media in a kinase activity–dependent manner in BV2 microglial cells. Representative Western blot images (I) and quantification (J) of Nlk and Pgrn levels in BV2 cells by Nlk overexpression. Nlk-T298A is a kinase-inactive form of Nlk. *P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Tukey’s post hoc testing; n = 3; Nlk F(2,6)=14.91, P = 0.0047; Pgrn (intracellular) F(2,6)=1.190, P = 0.3670; Pgrn (media) F(2,6)=30.16, P = 0.0007.