Received: 18 November 2015 Revised and accepted: 21 September 2016 Uncorrected manuscript published: 26 September 2016 Published on: 1 November 2016 DOI 10.1111/tra.12451 ORIGINAL ARTICLE Vps35-dependent recycling of Trem2 regulates microglial function Jie Yin1,2† | Xiaocui Liu3† | Qing He1,2† | Lujun Zhou1,2 | Zengqiang Yuan1,4 | Siqi Zhao1 1State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Triggering receptor expressed on myeloid cells 2 (Trem2), an immune-modulatory receptor, is Academy of Sciences, Beijing, China preferentially expressed in microglia of central nervous system. Trem2 might be involved in the 2Sino-Danish College, University of Chinese development of Alzheimer’s disease (AD) through regulating the inflammatory responses and Academy of Sciences, Beijing, China phagocytosis of microglia. However, the intracellular trafficking of Trem2 remains unclear. In 3 Qingdao Mental Health Center, Qingdao, this study, we showed that Trem2 in the plasma membrane underwent endocytosis and recy- China cling. Trem2 is internalized in a clathrin-dependent manner and then recycled back to the 4Center of Alzheimer’s Disease, Beijing Institute for Brain Disorders, Beijing, China plasma membrane through vacuolar protein sorting 35 (Vps35), the key component of cargo Corresponding Author: Siqi Zhao, State Key recognition core of retromer complex, but not Rab11. When Vps35 is knocked down, Trem2 Laboratory of Brain and Cognitive Sciences, accumulated in the lysosomes but was not degraded. More importantly, Vps35 deficiency leads Institute of Biophysics, Chinese Academy of to excessive lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression Sciences, Beijing 100101, China and IL-6 production, which can be abolished by Trem2 overexpression. Furthermore, R47H ([email protected]). Trem2, an AD-associated mutant, failed to interact with Vps35 and became unstable compared Funding information National Science Foundation of China,Grant/ with wild-type Trem2. Our study suggests that Vps35/retromer is responsible for recycling of Award number: 81125010, 81030025 and Trem2 in the regulation of microglial function such as proinflammatory responses, whereas 81600997; National Basic Research Program R47H mutation impairs Trem2 trafficking, which might contribute to AD. of China,Grant/Award number: 973- 2012CB910701 and 2013DFA31990; Cross- disciplinary Collaborative Teams Program for KEYWORDS Science, Technology and Innovation (2014- Alzheimer’s disease, microglia, receptor recycling, retromer, Trem2, Vps35 2016); Chinese Academy of Sciences. 1 | INTRODUCTION Homozygous mutations in Trem2 or Dap12 appears in Nasu-Hakola disease (NHD), which is characterized by bone cysts and fractures and Triggering receptor expressed on myeloid cells 2 (Trem2), a homolog presenile dementia associated with demyelination and cerebral atrophy.4 of Trem1, is a transmembrane glycoprotein that belongs to the immu- Consistent with the clinical symptoms, Trem2−/− mice exhibits reduced noglobulin variable domain receptor family. Trem2 consists of a lead- microglial expanse during aging and impaired myelin debris clearance in ing signal peptide, a single type V extracellular region containing Cuprizone-induced nonautoimmune demyelination model.5 Genome- 3 potential N-glycosylation sites, a transmembrane domain and a wide association study (GWAS) has identified R47H mutation of Trem2 short cytoplasmic tail lacking signaling motifs.1 Owing to its short increases the risk of Alzheimer’s disease (AD) with a high odds ratio (3.5), cytoplasmic tail, Trem2 needed to transfer its signaling by Dap12, an which is similar to that of ApoEε4.6,7 A recent study reported that defi- adaptor protein,2 which recruits the protein tyrosine kinase Syk, lead- ciency of Trem2 augmented hippocampal amyloid β accumulation and ing to the subsequent activation of various downstream molecules of loss of layer V cortical neurons in AD mouse model, which directly links the innate immune system.3 AD with Trem2 mutation.8 The R47H mutation has also been identified ABBREVIATIONS: ALS, amyotrophic lateral sclerosis; CHX, cycloheximide; EEA1, early endosome antigen 1; ERC, endocytic recycling compartment; ERC, endocytic recycling compartment; FTD, frontotemporal dementia; GC, Golgi complex; GST, glutathione S-transferase; iNOS, inducible nitric oxide synthase; LPS, lipopolysac- charide; NHD, Nasu-Hakola disease; PD, Parkinson’s disease; shRNA, small hairpin RNA; TGN, trans-Golgi network; Trem2, triggering receptor expressed on mye- loid cells 2; Vps35, vacuolar protein sorting 35 †These authors contributed equally to this work. 1286 © 2016 John Wiley & Sons A/S. wileyonlinelibrary.com/journal/tra Traffic 2016; 17(12):1286–1296 Published by John Wiley & Sons Ltd YIN ET AL. 1287 FIGURE 1 Triggering receptor expressed on myeloid cells 2 (Trem2) is constitutively endocytosed from the plasma membrane via a clathrin-dependent manner. A and B, N9 cells (A) and primary mouse microglial cells (B) were incubated (pulsed) with anti- Trem2 antibody (10 μg/mL) for 40 minutes at 4C, then washed and incubated (chased) for 1 hour at 37C before fixation, permeabilization and treatment with the secondary antibody. Scale bar is 25 μm. C- E, Effect of RNA interference (RNAi)- mediated clathrin knockdown on Trem2 endocytosis. C, N9 cells were transfected with either small interfering RNA (siRNA) of clathrin or non-targeting siRNA. Relative mRNA levels of Trem2 were determined by real-time-polymerase chain reaction (PCR). GAPGH was used as an internal control. ***P < .001. D, N9 cells treated with siRNA targeting clathrin and non-targeting siRNA were used for endocytosis assay. Scale bar, 25 μm. E, Quantification shows the mean fluorescent area per cell. **P < .01. For each group, 6 visual fields containing approximately 150 cells in total were randomly chosen to be photographed, each image was analyzed by Image-Pro Plus to generate a measurement of mean fluorescent area per cell. as a risk factor for other multiple neurodegenerative diseases including Trem2 in the regulation of microglial function and R47H mutation amyotrophic lateral sclerosis (ALS),9 Parkinson’sdisease(PD)andfronto- disrupts the interaction between Trem2 and Vps35 thus impairs temporal dementia (FTD).10 However, a recent study found that R47H Trem2 trafficking and protein stability, which might contribute to AD. mutation is highly associated with the risk of AD, but not the others.11 To date, it is still elusive whether R47H serves as a common risk factor 2 | RESULTS for all the neurodegenerative diseases or just specifically associates with AD. Moreover, how R47H mutation alters normal function of Trem2 2.1 | Clathrin-dependent endocytosis is necessary needs to be further investigated. for Trem2 internalization As an innate immune receptor, Trem2 is expressed on a subset of myeloid cells including osteoclasts, dendritic cells, macrophages and Trem2 has been reported to undergo constitutive endocytosis in the microglial cells.12 The expression of Trem2 has also been detected in a immortalized N9 microglial cells.17 In our experiments, N9 and primary fraction of neurons such as granule cells in dentate gyrus,13-15 but these mouse microglial cells were exposed (pulsed) to anti-Trem2 antibody observations still need to be further confirmed.16 Trem2 is abundant in for 40 minutes at 4 C, and then incubated (chased) at 37 C for up to human and murine microglia in both brain tissues and cultured cell lines, 1 hour before fixation and treatment with the secondary antibody. We in which Trem2 distributes to intracellular membrane-bound structures observed that Trem2 internalized in N9 or primary microglia such as the trans-Golgi network (TGN), while a small fraction locates at (Figure 1A,B). To investigate whether Trem2 endocytosis is through the plasma membrane.15 Trem2 in an intracellular pool could be translo- the typical clathrin-mediated pathway, which plays a major role in most cated and recycled from the cell surface, which demonstrated that the selective receptor internalization in higher eukaryotes,20 we used a surface density and presumably the activation of Trem2 is continuously small interfering RNA (siRNA) specifically targeting clathrin heavy chain, adapted.17 NHD-associated mutations, such as Q33X, Y38C and T66M, which reduced about 70% of clathrin expression (Figure 1C) and found impaired the glycosylation of Trem2 in the Golgi apparatus, as well that internalization of Trem2 is significantly decreased in clathrin- as trafficking of Trem2 to the plasma membrane and shedding by deficient N9 cells (Figure 1D,E). Our data indicates that Trem2 endocy- γ-secretase.18,19 These studies suggest that normal trafficking of tosis may be clathrin-dependent in microglial cells. Trem2 is important for the biological function of Trem2 and aberrant 2.2 | Endocytosed Trem2 directly interacts with traffic may contribute to the development of neurodegenerative dis- Vps35 eases. However, the molecular mechanism underlying the recycling process of Trem2 remains largely unknown. Here, we show that vacuo- It has been shown that internalized Trem2 colocalizes with EEA1 lar protein sorting 35 (Vps35)/retromer is responsible for recycling of (Early Endosome Antigen 1), a marker of early endosomes, from 1288 YIN ET AL. FIGURE 2 The interaction between triggering receptor expressed on myeloid cells 2 (Trem2) and vacuolar protein sorting 35 (Vps35) in vivo. A, Trem2 colocalized with Vps35 in primary microglial cells. Scale