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 4

Nlk deficiency increases clathrin-dependent Pgrn endocytosis in microglia.

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Nlk deficiency increases clathrin-dependent Pgrn endocytosis in microgli...
(A) Nlk reduction resulted in the increased localization of Pgrn to endosomes. Representative images of WT and Nlk-KD BV2 cells costained for Pgrn/Rab11 demonstrated colocalization of Pgrn with recycling endosomes. Right panels are magnified views of areas marked in the left panels. (B and C) Quantification of Rab11 puncta (B) and colocalization Pgrn and Rab11 (C) are shown. **P < 0.01 (2-tailed, unpaired Student’s t test, n = 3 wells, average of ~10 cells sampled per well). (D and E) Enhanced uptake of the extracellularly provided dextran in Nlk-KD BV2 cells. Representative images (D) and quantification (E) of WT and Nlk-KD BV2 cells after 20 minutes’ incubation with 647-dextran. ***P < 0.001 (2-tailed, unpaired Student’s t test, n = 3 wells, average of ~50 cells sampled per well). (F and G) Enhanced uptake of the extracellularly provided transferrin in Nlk-KD BV2 cells. Representative images (F) and quantification (G) of WT and Nlk-KD BV2 cells after 15 minutes’ incubation with Alexa 568–transferrin. *P < 0.05 (2-tailed, unpaired Student’s t test, n = 3). (H and I) Representative Western blot images (H) and quantification (I) showing the enhanced clathrin-dependent endocytosis of Flag-tagged PGRN provided exogenously in BV2 microglial cells. Cells were treated with DMSO or the clathrin-dependent endocytosis inhibitor Pitstop 2 for 1 hour and incubated with the recombinant Flag-PGRN (1 nM) for 15 minutes. **P < 0.01; 2-way ANOVA with post hoc Bonferroni correction, n = 3; F(1,8)=11.85, P = 0.0088.