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Neurobiology of Aging 35 (2014) 1243e1251

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Neurobiology of Aging

journal homepage: www.elsevier .com/locate/neuaging

Triggering receptor expressed on myeloid cells 2 knockdownexacerbates aging-related neuroinflammation and cognitivedeficiency in senescence-accelerated mouse prone 8 mice

Teng Jiang a, Jin-Tai Yu a,b,c,**, Xi-Chen Zhu a, Meng-Shan Tan c, Li-Ze Gu d,Ying-Dong Zhang e, Lan Tan a,b,c,*

aDepartment of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing, People’s Republic of ChinabDepartment of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, Qingdao, People’s Republic of ChinacDepartment of Neurology, Qingdao Municipal Hospital, College of Medicine and Pharmaceutics, Ocean University of China, Qingdao,People’s Republic of Chinad The Clinical Laboratory of Wuxi Mental Health Center, Nanjing Medical University, Nanjing, People’s Republic of ChinaeDepartment of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing, People’s Republic of China

a r t i c l e i n f o

Article history:Received 25 June 2013Received in revised form 26 October 2013Accepted 24 November 2013Available online 2 December 2013

Keywords:TREM2AgingNeuroinflammationAlzheimer’s diseaseCognitive deficiencySAMP8SAMR1

* Corresponding author at: Department of Neurolopital, School of Medicine, Qingdao University, No. 5 Do266071, People’s Republic of China. Tel.: þ86 5328890 5659.** Alternate corresponding author at: DepartmeMunicipal Hospital, School of Medicine, Qingdao UnivRoad, Qingdao 266071, People’s Republic of China.fax: þ86 532 8890 5659.

E-mail addresses: yu-jintai@163.com (J.-T. Yu), dr.t

0197-4580/$ e see front matter � 2014 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2013.11.026

a b s t r a c t

As a major characteristic of aging process, neuroinflammation is involved in the pathogenesis of severalaging-related diseases including Alzheimer’s disease (AD). Triggering receptor expressed on myeloid cells2 (TREM2) is a newly identified risk gene for AD, which regulates inflammatory process in peripheraltissues via modulating the release of inflammatory cytokines. However, the role of TREM2 in aging-related neuroinflammation, cognitive deficiency, and AD-like neuropathology is unclear so far. Here,we detected the protein levels of TREM2 in brain of 3-, 7-, and 11-month-old senescence-acceleratedmouse prone 8 (SAMP8) mice and observed that TREM2 levels were increased during aging process.We then knocked down TREM2 expression in brain of SAMP8 mice by nonviral RNA interference andfound a significant increase in proinflammatory cytokines including tumor necrosis factor-a and inter-leukin (IL)-6, which was accompanied by a reduction in IL-10. Meanwhile, more obvious neuronal andsynaptic losses and cognitive impairment were observed. These findings indicate that TREM2 may play aprotective role against aging-related neuroinflammation and cognitive impairment.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

During the process of aging, brain exhibits a progressivelyenhanced inflammatory status, which is called as “inflamm-aging”(Franceschi et al., 2000; Pizza et al., 2011). Although inflammatoryprocesses are necessary for brain to remove senescent cells, long-lasting neuroinflammation can be neurotoxic and adverselyaffects brain functions (Liu et al., 2013). Meanwhile, chronicneuroinflammation also contributes the pathogenesis of several

gy, Qingdao Municipal Hos-nghai Middle Road, Qingdao8890 5659; fax: þ86 532

nt of Neurology, Qingdaoersity, No. 5 Donghai MiddleTel.: þ86 532 8890 5658;

anlan@163.com (L. Tan).

ll rights reserved.

aging-related diseases such as Alzheimer’s disease (AD) (McGeerand McGeer, 2004). As the main type of immune cells in brain,microglia is revealed to play a crucial role in aging-related neuro-inflammation (Lee et al., 2013; Liu et al., 2013). Activation ofmicroglia during aging leads to the release of proinflammatorycytokines, which subsequently contributes to neuronal and syn-aptic losses, ultimately leading to the aging-related cognitive defi-ciency (Bardou et al., 2013; Farso et al., 2013).

As an important innate immune receptor in brain, triggeringreceptor expressed on myeloid cells (TREM) 2 is mainly expressedon microglia, coupling with DAP12 for its signaling (Jiang et al.,2013). With respect to the functions, TREM2 is revealed to sup-press inflammatory response by repression of microglia-mediatedcytokine production and secretion in vitro (Takahashi et al.,2005), and this anti-inflammatory effect of TREM2 has beenfurther confirmed in an animal model of multiple sclerosis(Takahashi et al., 2007). In humans, loss of function of TREM2caused by rare mutations will lead to an autosomal recessive

T. Jiang et al. / Neurobiology of Aging 35 (2014) 1243e12511244

disorder called polycystic lipomembranous osteodysplasia withsclerosing leukoencephalopathy, which is characterized by theformation of multifocal bone cysts and progressive presenile in-flammatory neurodegeneration (Klunemann et al., 2005). In addi-tion, reduction of TREM2 function may also contribute to thepathogenesis of AD, a neurodegenerative disease with chronic in-flammatory responses (Benitez et al., 2013; Giraldo et al., 2013;Guerreiro et al., 2013; Hu et al., 2013; Jonsson et al., 2013). Todate, the expression pattern of TREM2 in brain during aging processis still unclear. Meanwhile, the precise role of TREM2 in aging-related neuroinflammation, cognitive deficiency, and AD-likeneuropathology remains elusive so far. Therefore, in the presentstudy, we adopted senescence-accelerated mouse prone 8 (SAMP8)mice to investigate the cellular localization and expression patternof TREM2 in aged brain. Moreover, we knocked down brain TREM2expression by in vivo short interfering RNA (siRNA) and observedthe subsequent impacts on inflammatory markers, AD-like neuro-pathology, and cognitive functions.

2. Methods

2.1. Animals

To avoid the interference of estrogen on neuroinflammation andcognitive function (Bjorling and Wang, 2001; Wei et al., 2013), onlymale mice were used in this study. SAMP8 mice and their age-matched control senescence-accelerated mouse resistant 1(SAMR1) mice were purchased from the Institute of Zoology, Chi-nese Academy of Sciences. They were housed in a standard animalroom with a 12-hour light/dark cycle and given free access to foodand water. This study was carried out in strict accordance with therecommendations in the Guide for the Care and Use of LaboratoryAnimals of the National Institutes of Health. All procedures wereapproved by the Animal Care and Management Committee ofQingdao University (permit no. QUEC-130316). Remarkably, effortswere made to minimize the number of animals used in the studyand their sufferings.

2.2. siRNA administration in mice brain

We performed in vivo siRNA transfection in mice brain accordingto the method described by Thakker et al. (2004). Briefly, 12.5 mgTREM2 siRNA pool (consists of 3 target-specific 20e25 nt siRNAs;Santa Cruz Biotechnology, Inc, USA) or control siRNA (Santa CruzBiotechnology, Inc) was resuspended in 25 mL RNAse-free water tomake an siRNA solution. Next, 25 mL TREM2 siRNA or control siRNAsolutionwasmixedwith 25 mL Entranster in vivo transfection reagent(Engreen, Inc, China) and 50 mL artificial cerebrospinal fluid (aCSF) toget a 100mL invivo transfectionmixture. Then, the invivo transfectionmixture or aCSF alone was filled into an osmotic pump (ALZET Inc;USA). Last,micewere anaesthetizedwith10%chloral hydrate (0.3mL/100 g, intraperitoneal) andwere placed in a stereotactic frame (DavidKopf Instrument, Inc, USA). A brain-infusion cannula (ALZET, Inc, USA)coupled via vinyl tubing to the osmotic pumpwas implanted into thedorsal third ventricle (0.5 mm posterior to bregma, 3 mm below thesurface of the cranium). Meanwhile, the osmotic pump was placedsubcutaneously between the scapulae of mice, and the siRNAs werecontinuously infused into the brain over a 4-week duration at the rateof 0.11 mL/hour. After this surgery, the wounds were carefully closedwith sutures. The duration and dose of TREM2 siRNA infusion werechosen according to our preliminary studies: administration ofTREM2 siRNA for 4 weeks is enough to reach its maximum silencingeffects (downregulate the TREM2 expression by 50%e55% comparedwith control siRNA, data not shown). Meanwhile, TREM2 siRNA atthis dose was well tolerated, and no signs of neurotoxicity including

hind-limb paralysis, vocalization, food intake, or neuroanatomicdamage were observed. In addition, the infusion of siRNA started at6 months of age. This time point was selected based on the resultsfromour time-course analyses,which revealed that the aging-relatedneuroinflammation and AD-like neuropathology in brain of SAMP8micebecamepronouncedbefore7monthsof age (seeSupplementaryFigs. S2 and S3).

2.3. Morris water maze test

The Morris water maze test was conducted during last 6 days ofthe 4-week siRNA infusion. It was performed in a circular pool withdiameter of 120 cm filled with opaquewater at temperature of 22�1 �C, as described previously (Vorhees and Williams, 2006). In thehidden-platform training, a circular platform (10 cm in diameter) insoutheast quadrant was submerged 0.5 cm below the surface ofwater. Swimming paths were tracked for 60 seconds with a videocamera and analyzed by a computer-controlled system (BeijingSunny Instruments, Inc, China). The mice were given 4 trainingtrials per day for 5 consecutive days. The time taken to reach thesubmerged platform was measured as escape latency, and theaverage time of 4 trials was calculated. If a mouse failed to findthe platform within 60 seconds, it was picked up and placed onthe platform for 15 seconds. Twenty-four hours after the last trial,mice were subjected to a probe test in which the platform wasremoved and their swimming paths were recorded for 60 seconds.

2.4. Brain tissue preparation

Mice were sacrificed under deep anesthesia and were handledas follows:

(1) For western blot analysis, quantitative real-time polymerasechain reaction (PCR), and enzyme-linked immunosorbent assay(ELISA), mice were perfused transcardially with 0.9% saline (pH7.4) only. The brains were removed rapidly and stored in liquidnitrogen until use.

(2) For cresyl violet staining and immunohistochemistry analysis,mice were perfused transcardially with 0.9% saline (pH 7.4),followed by a fixative solution containing 4% paraformaldehydein 0.9% saline (pH 7.4). The brains were removed and fixed inthe same fixative at 4 �C until use.

(3) For double immunofluorescence staining, mice brain wasremoved without perfusion, embedded in tissue freezing me-dium, and immediately frozen at �40 �C. Frozen tissue wasstored at �80 �C until sectioning.

2.5. Western blotting

For western blot analysis, the brain tissues were homogenizedand the total proteins were extracted by radio immunoprecipitationassay (RIPA) lysis buffer (Beyotime, Inc, China). A BCA kit (Beyotime,Inc) was used to determine the protein concentrations. Differentsamples with an equal amount of protein (40 mg) were separatedon 10% sodium dodecyl polyacrylamide gels, transferred to nitro-cellulose membranes, and then blocked in 5% bovine serum albu-min (BSA) powder in 1� tris-buffered saline with 0.1% Tween 20.The full definition of TBST has been already present here. (1� TBST)at 25 �C for 2 hours. Membranes were incubated overnight at 4 �Cwith the primary antibodies against TREM2 (1:600; Santa CruzBiotechnology, Inc), Ser 396 hyperphosphorylated tau (1:2000;Sigma-Aldrich, Inc, USA), Thr 205 hyperphosphorylated tau(1:1000; Sigma-Aldrich, Inc), tau-5 (1:800; Abcam, Inc), Iba-1(1:1000; Abcam, Inc), and synaptophysin (1:500; Abcam, Inc),washed with 1� TBST, and then incubated with horseradish

T. Jiang et al. / Neurobiology of Aging 35 (2014) 1243e1251 1245

peroxidaseecoupled secondary antibody for 2 hours at room tem-perature. After washing, protein bands were detected withchemiluminescent horseradish peroxidase substrate (Thermo Sci-entific, Inc, USA) for 5 minutes at room temperature and exposed toX-ray film (Fujifilm, Inc, Japan). The signal intensity of primaryantibody binding was analyzed using Quantity One software 4.6.2(Bio-Rad Laboratories, Inc, USA) and normalized to a loading controlb-actin (1:1000; Santa Cruz Biotechnology, Inc).

2.6. Quantitative real-time PCR

Total RNA was extracted by Trizol reagent (Invitrogen, Inc, USA)according to the technical manuals provided by the manufacturer.Equal amounts of total RNA were reverse transcribed in a finalvolume of 10 mL using random primers under standard conditionsusing the PrimeScript RT Master Mix (Takara Bio, Inc, Japan). Thereverse transcription reaction was carried out under the followingconditions: 37 �C for 15 minutes, 85 �C for 5 seconds, and then heldon 4 �C. After that, quantitative real-time PCR reactions were per-formed with SYBR Premix Ex Taq (Takara Bio, Inc) and specificmouse primers (for detail, see Supplementary Table S1) at a finalconcentration of 500 nmol/L to detect TREM2, tumor necrosis fac-tor-a (TNF-a), interleukin (IL)-1b, -6, and -10, and inducible nitricoxide synthase (iNOS) expressions according to the manufacturer’sinstructions. Meanwhile, glyceraldehyde 3-phosphate dehydroge-nase (GAPDH) was adopted as an internal control, as its expressionshowed minimal variation in different tissues. Amplification wasconducted at 95 �C for 30 seconds and followed by 40 cycles at 95 �Cfor 5 seconds and 60 �C for 34 seconds in the ABI 7500 real-timePCR system (Applied Biosystems, USA). The results were analyzedand expressed as the relative messenger RNA expression of thethreshold cycle value, which was then converted to fold changes.

2.7. Cresyl violet staining and immunohistochemistry analysis

The brains were embedded in paraffin and cut into 4e6 mmsections. For cresyl violet staining, the paraffin-embedded sectionswere dewaxed and rehydrated according to the standard protocols.Next, the sections were stained in 1% cresyl violet at 50 �C for5 minutes. After being rinsed with water, the sections were dehy-drated in increasing concentrations of ethanol, mounted on theslides, and examined with a light microscope. For immunohisto-chemistry analysis, the sections were immersed in 3% H2O2 for30 minutes to block endogenous peroxidase activity. After beingwashed in phosphate-buffered saline (PBS), the sections wereblocked with 5% BSA for 30 minutes, incubated with a rabbitpolyclonal antibody against TREM2 (1:150; Santa Cruz Biotech-nology, Inc) overnight at 4 �C, and then treated with biotinylatedgoat anti-rabbit IgG (Zhongshan, Inc, China) for 60 minutes.Immunoreactivity was detected with diaminobenzidine. Last, sec-tions were then counterstained with Mayer’s Hematoxylin (Sigma-Aldrich, Inc), dehydrated, mounted on the slides, and examinedwith a microscope equipped with a charge-coupled device camera.

2.8. Double immunofluorescence staining

For immunofluorescence studies, 16-mm-thick sections wereobtained by cryosectioning at�20 �C, mounted on a glass slide, andincubated at room temperature for 1 hour. Afterward, the sectionswere fixed in ice-cold acetone for 10 minutes and then dried on aheater for 10 minutes at 40 �C. Sections were then blocked with 5%BSA and 0.1% TritonX-100 for 2 hours. After a single wash with PBS,sections were incubated overnight at 4 �C with a rabbit polyclonalantibody against TREM2 (1:100; Santa Cruz Biotechnology, Inc)combined with a mouse monoclonal antibody against Iba-1 (1:200;

Abcam, Inc) or a chicken polyclonal antibody against NeuN (1:600;Abcam, Inc). Sections were then washed in PBS and sequentiallyincubated with fluorescein isothiocyanate-conjugated anti-rabbitIgG (1:200; Zhongshan Goldenbridge, Inc, China), tetraethylrhodamine isothiocyanate-conjugated anti-mouse IgG (1:200;Zhongshan Goldenbridge, Inc), or DyLight 405elabeled anti-chicken IgG (1:1000; Beyotime, Inc) in a dark and humidifiedcontainer for 1 hour at 37 �C. After that, the sections were washedwith PBS and sealed with a coverslip. The slides were analyzed witha fluorescence microscopy (Olympus, Inc, Japan).

2.9. Enzyme-linked immunosorbent assay

ELISA for the measurement of amyloid-b (Ab)1e42 peptide levelswas performed as described previously (Schmidt et al., 2012).Briefly, the brain tissues were homogenized with RIPA lysis buffer(Beyotime, Inc). Then, the homogenates were centrifuged at10,000 � g and 4 �C for 15 minutes to remove cellular debris. Thesupernatant was collected and stored at �80 �C until use. Theconcentrations of Ab1e42 were detected by a specific ELISA kit(Invitrogen, Inc). The change in absorbance in every well at 450 nmwas detected with a spectrophotometer (GE Healthcare, Inc, USA).

2.10. Statistical analysis

Statistical analysis was conducted by SPSS software 13.0 (IBM,Inc, USA). Statistically significant differences were evaluated byindependent sample t test and one-way analysis of variance fol-lowed by least-significant difference post hoc test. For the hidden-platform training of the Morris water maze test, escape latency ofeach group was analyzed by 2-way repeated-measures analysis ofvariance followed by least-significant difference post hoc test. Alldata are expressed as mean � standard deviation. p < 0.05 wasconsidered statistically significant.

3. Results

3.1. TREM2 protein was expressed in microglia and was upregulatedin brain of SAMP8 mice during aging process

First, a time-course analysis was conducted to investigate thechanges of TREM2 levels in brain during aging process. As indicatedin Fig. 1A, SAMP8 mice exhibited significant higher TREM2expression in brain at 7 and 11 months than at 3 months (n¼ 6, p<

0.05). In addition, TREM2 levels in brains of 7- and 11-month-oldSAMP8 mice were markedly higher than age-matched SAMR1 mice(n ¼ 6, p < 0.05). We then investigated the cellular localization ofTREM2 in brain of SAMP8 mice. As demonstrated by Fig. 1B, doubleimmunofluorescence staining indicated a good colocalization ofTREM2 andmicroglial marker Iba-1 in brain of 7-month-old SAMP8mice. Meanwhile, no expression of TREM2was detected on neurons(labeled by NeuN) in 7-month-old SAMP8 brain (Fig. 1B). In addi-tion, we also investigated the cellular localization of TREM2 in brainof 7-month-old SAMR1 mice. As demonstrated by SupplementaryFig. S1A, the cellular localization of TREM2 in brain of SAMR1mice was consistent with that in SAMP8mice brain. Taken together,these results indicated that TREM2 was expressed in microglia butnot in neurons. To further determine whether the upregulation ofTREM2 protein during aging process was attributed to the increasednumber of microglia in brain, Iba-1 levels were analyzed bywesternblotting. As shown by Supplementary Fig. S1B, no significant dif-ferences were observed in Iba-1 levels among brains of 3-, 7-, and11-month-old SAMP8 mice (n ¼ 6, p ¼ 0.389), indicating that theupregulation of TREM2 protein in SAMP8 brain was unlikelybecause of the increase in microglia counts.

Fig. 1. Triggering receptor expressed on myeloid cells 2 (TREM2) protein was expressed on microglia and was upregulated in brain of senescence-accelerated mouse prone 8(SAMP8) mice during aging process. (A) Protein levels of TREM2 in brain of SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice at 3, 7, and 11 months were detectedby western blotting, and b-actin was used as loading control. Data are expressed as a fold change relative to 3-month-old SAMR1 mice. Columns represent mean � standarddeviation (n ¼ 6 per group). * p < 0.05 versus 3-month-old animals. (B) Double immunofluorescence staining of Iba-1 (microglial marker, red fluorescence) and TREM2 (greenfluorescence) revealed a good colocalization of these 2 markers in the brain of 7-month-old SAMP8 mice. Meanwhile, no expression of TREM2 was detected on neurons (labeled byNeuN, blue fluorescence) in 7-month-old SAMP8 brain. Scale bar: 100 mm.

T. Jiang et al. / Neurobiology of Aging 35 (2014) 1243e12511246

3.2. siRNA targeting TREM2 effectively downregulated TREM2 inbrain of SAMP8 mice

To evaluate the silencing efficiency of siRNA infusion in micebrain, the gene expression and protein level of TREM2 proteinwere detected by quantitative real-time PCR and western blot-ting, respectively. As shown in Fig. 2A, TREM2 siRNA reduced thegene expression of TREM2 in brain by approximately 57%compared with control siRNA (n ¼ 6, p < 0.05). In consistentwith the changes in gene expression, SAMP8 mice infused withTREM2 siRNA showed a dramatic reduction in brain TREM2protein levels (n ¼ 6, p < 0.05). The effect of siRNA on proteinlevels of TREM2 in SAMP8 mice brain was confirmed by immu-nohistochemical staining, as Fig. 2B demonstrated an obviousreduction in TREM2 immunoreactivity in both hippocampus andcortex of SAMP8. Of note, control siRNA did not affect either thegene expression or the protein level of TREM2 in brain of SAMP8mice (Fig. 2A).

3.3. Downregulation of TREM2 by siRNA led to significant changesin inflammatory cytokines in brain of SAMP8 mice

The dynamic changes of TNF-a, IL-1b, -6, and -10, and iNOS inbrain during aging process were indicated by SupplementaryFig. S1. As revealed by Fig. 3A and B, TREM2 siRNA significantlyincreased brain TNF-a and IL-6 expressions by 1.6- and 3.2-fold,respectively, compared with control siRNA (n ¼ 6, p < 0.05). Inaddition, a significant reduction in brain IL-10 expression wasobserved in SAMP8 mice infused with TREM2 siRNA (n ¼ 6, p <

0.05) (Fig. 3C). No difference was found in the gene expression of IL-1b and iNOS in brain after infusion of TREM2 siRNA (Fig. 3D and E).

3.4. Downregulation of TREM2 by siRNA resulted in neuronal andsynaptic losses in brain of SAMP8 mice

The dynamic changes of AD-like neuropathology includingAb1e42, hyperphosphorylated tau, and synaptophysin levels during

Fig. 2. Short interfering RNA (siRNA) targeting triggering receptor expressed on myeloid cells 2 (TREM2) effectively downregulated TREM2 in brain of senescence-accelerated mouseprone 8 (SAMP8) mice. (A) Protein and messenger RNA levels of TREM2 in brain of 7-month-old SAMP8 mice after 4-week infusion of artificial cerebrospinal fluid (aCSF), controlsiRNA, or TREM2 siRNA. Data are expressed as a fold change relative to senescence-accelerated mouse resistant 8 mice infused with aCSF. Columns represent mean � standarddeviation (n ¼ 6 per group). (B) Immunohistochemistry staining for TREM2 in hippocampus and cortex of 7-month-old SAMP8 mice brain after 4-week siRNA infusion. Obviousreduction of TREM2 immunoreactivities were noted in both hippocampus and cortex of 7-month-old SAMP8 mice infused with TREM2 siRNA. Scale bar: 100 mm.

T. Jiang et al. / Neurobiology of Aging 35 (2014) 1243e1251 1247

the process of aging were indicated by Supplementary Fig. S2.Infused with TREM2 siRNA significantly reduced synaptophysinlevels by 34% compared with control siRNA (n ¼ 6, p < 0.05)(Fig. 4B). In addition, cresyl violet staining showed that TREM2siRNA led to more obvious neuronal loss in hippocampus (CA1 re-gion) and cerebral cortex of SAMP8 mice brain (Fig. 4D). However,infusion of TREM2 siRNA did not significantly affect the levels ofAb1e42 and hyperphosphorylated tau in SAMP8 mice brain (Fig. 4Aand C).

3.5. Downregulation of TREM2 by siRNA exacerbated cognitivedeficiency in SAMP8 mice

To investigate the effects of TREM2 siRNA on cognitive functions,we conducted Morris water maze test in SAMR1 and SAMP8 mice

during the last 6 days of the 4-week infusion (7-month old). First,we analyzed the swimming speed of 7-month-old mice and foundno significant difference between these 2 strains. Meanwhile,infusion of TREM2 siRNA did not markedly affect swimming speedin both SAMR1 and SAMP8 mice (Fig. 5A). These results enabled usto exclude the confounding effects of motivational and sensori-motor factors on cognitive performance. We then used hiddenplatform task to assess the cognitive deficiency inmice. As shown inFig. 5B, the latencies to find the submerged platform were signifi-cantly declined everyday in the SAMR1 group but not in the SAMP8group, and escape latencies of SAMR1 mice infused with controlsiRNA were consistently shorter than those of SAMP8 mice treatedwith control siRNA during the 5-day training (n ¼ 12, p < 0.05).TREM2 siRNA-infused SAMP8 mice exhibited a more obvious defi-ciency in cognitive performance, as their escape latencies on days 4

Fig. 3. Downregulation of triggering receptor expressed on myeloid cells 2 (TREM2) by short interfering RNA (siRNA) led to significant changes in inflammatory cytokines in brain ofsenescence-accelerated mouse prone 8 (SAMP8) mice. (AeE) Messenger RNA (mRNA) levels of tumor necrosis factor-a (TNF-a), interleukin (IL)-6, -10, and -1b, and inducible nitricoxide synthase in brain of 7-month-old SAMP8 after 4-week infusion of control or TREM2 siRNA were measured by quantitative real-time polymerase chain reaction. Data areexpressed as a fold change relative to senescence-accelerated mouse resistant 8 mice infused with control siRNA. The expression of all genes was normalized to the levels of GAPDHmRNA. Columns represent mean � standard deviation (n ¼ 6 per group).

T. Jiang et al. / Neurobiology of Aging 35 (2014) 1243e12511248

and 5 were significantly longer than SAMP8 mice treated withcontrol siRNA (n ¼ 12, p < 0.05). Of note, although TREM2 proteinlevels were significantly reduced (Supplementary Fig. S4), thecognitive performance of TREM2 siRNA-infused SAMR1 mice wasnot different from that of control siRNA-infused SAMR1 mice(Fig. 5B).

4. Discussion

In the present study, we first investigated the cellular localiza-tion of TREM2 and its dynamic changes during aging in brain ofSAMP8 mice. The cellular localization of TREM2 on microglia inmice brain was demonstrated by the double immunofluorescencestaining, confirming the findings from the other groups (Frank et al.,2008; Jonsson et al., 2013; Melchior et al., 2010). More importantly,we found that TREM2 levels were significantly increased duringaging process, which was paralleled by the progression of neuro-inflammation. Meanwhile, the increase in TREM2 level was likelyattributed to the enhanced expression in an individual microgliarather than alterations in total microglia counts. To the best of ourknowledge, this is the first report of an increase in brain TREM2expression during aging process.

Afterward, we knocked down brain TREM2 expression by usingnonviral RNA interference to investigate its role in aging-relatedneuroinflammation, cognitive deficiency, and AD-like neuropa-thology. For the first time, we revealed that knockdown of TREM2 inbrain of SAMP8 significantly increased the expression of theproinflammatory cytokines TNF-a and IL-6. These results weresupported by an in vitro study from Takahashi et al. (2005), whichdemonstrated that knockdown of TREM2 in cultured microgliacaused a significant increase in the gene transcription of TNF-a.Coincidentally, a recent study from Fisher et al. (2010) demon-strated that increased level of TREM2 was associated with thereduced expression of TNF-a and IL-6 in a transgenic mouse modelof AD. More direct evidence has provided by another study from

Takahashi et al. (2007), as stimulation by a TREM2-specific antibodyled to decreased gene transcripts of IL-1b and iNOS in TREM2-transduced bone marrowederived myeloid cell after challengewith lipopolysaccharide, suggesting that TREM2 negatively regu-lated the expression of proinflammatory cytokines. Aside from theproinflammatory cytokines, the expression of IL-10, an importantanti-inflammatory cytokines, was markedly decreased by TREM2knockdown in the present study. This finding was supported by anin vivo study conducted in mice with an experimental autoimmuneencephalomyelitis, which demonstrated that application of TREM2-transduced bone marrowederived myeloid cell resulted in anupregulation of IL-10 in lesion areas of spinal cord, suggesting thatmodulation of anti-inflammatory cytokines may represent part ofthe mechanisms by which TREM2 attenuates neuroinflammation(Takahashi et al., 2007). It should be noted that TREM2 knockdownwas not associated with a significant upregulation of iNOS in ourstudy, and this seemed to be inconsistent with the findings byTakahashi et al. (2005). The contradictory results can be explainedas follows: the study by Takahashi et al. (2005) was conducted inprimary cultured microglia, whereas our data were obtained fromaged brain. In addition to microglia, neurons also played an irre-placeable role in the expression of iNOS in aged brainwith a chronicinflammatory environment (Blanco et al., 2010; Kifle et al., 1996;Uttenthal et al., 1998). However, as aforementioned, TREM2 wasexpressed in microglia but not in neurons. In other words, knock-down of TREM2 might only reduce iNOS expression that wasmediated by microglia, whereas neuron-mediated iNOS productionwas unaffected. Hence, it is reasonable that iNOS expression wasnot significantly increased in brain after TREM2 knockdown.

Multiple studies have revealed that release of proinflammatorycytokines including TNF-a and IL-6 frommicroglia is responsible forneuronal and synaptic losses that occurred in aged brain (Blaskoet al., 2004; Combs et al., 2001; Craft et al., 2005), whereas anti-inflammatory therapies could effectively attenuate neuronal andsynaptic damages and rescue cognitive deficiency in animal models

Fig. 4. Downregulation of triggering receptor expressed on myeloid cells 2 (TREM2) by short interfering RNA (siRNA) resulted in neuronal and synaptic losses in brain of senescence-accelerated mouse prone 8 (SAMP8) mice. (A) Protein levels of Ser 396 and Thr 205 hyperphosphorylated tau in brain of 7-month-old SAMP8 after 4-week infusion of control orTREM2 siRNA were detected by western blotting, and b-actin was used as loading control. Data are expressed as a fold change relative to senescence-accelerated mouse resistant 8(SAMR8) mice infused with control siRNA. (B) Protein levels of synaptophysin in brain of 7-month-old SAMP8 after 4-week infusion of control or TREM2 siRNA were detected bywestern blotting, and b-actin was used as loading control. Data are expressed as a fold change relative to SAMR8 mice infused with control siRNA. (C) Concentration of amyloid-b(Ab1e42) in brain of 7-month-old SAMP8 after 4-week infusion of control or TREM2 siRNA was measured by enzyme-linked immunosorbent assay. (D) Cresyl violet staining fordetection of neuronal loss in hippocampus and cortex of 7-month-old SAMR1 and SAMP8 mice brain after 4-week siRNA infusion. Note that SAMR8 mice showed obvious reductionin density of neuron staining with cresyl violet in hippocampus and cortex in comparison with SAMR1 mice, whereas the density of neuron staining with cresyl violet was furtherdecreased after 4-week infusion of TREM2 siRNA. Scale bar: 100 mm. Columns represent mean � standard deviation (n ¼ 6 per group).

T. Jiang et al. / Neurobiology of Aging 35 (2014) 1243e1251 1249

of neuroinflammation and aging (Jin et al., 2013; Lee et al., 2012;Vom Berg et al., 2012). Alternatively, changes in anti-inflammatory cytokines such as IL-10 were also identified to beinvolved in neuroinflammation and cognitive functions, as IL-10edeficient mice exhibited a prominent cognitive deficit afterchallenged with lipopolysaccharide (Richwine et al., 2009). In thepresent study, more obvious neuronal and synaptic losses andcognitive deficiency were found after TREM2 knockdown. In theview of the previous evidence, these phenomena may be attributedto the exacerbation of neuroinflammation induced by TREM2knockdown in brain. Interestingly, in Morris water maze test, nosignificant differences were observed in cognitive performancebetween TREM2 siRNA- and control siRNA-infused SAMR1 mice,

although brain TREM2 levels were markedly decreased after infu-sion of TREM2 siRNA. These observations implied that TREM2might not play a direct role in maintaining normal cognitivefunction.

This study also has some limitations. According to the previousfindings, chronic inflammatory response in aged brain was asso-ciated with AD-like neuropathology including accumulation of Aband hyperphosphorylation of tau protein (Blasko et al., 1999;Ghosh et al., 2013). In the present study, although knockdownof TREM2 exacerbated neuroinflammation, it did not significantlyaffect the levels of Ab1e42 and hyperphosphorylated tau in SAMP8brain. We cannot rule out the possibility that the 4-week infusionof TREM2 siRNA is not enough to cause obvious changes in

Fig. 5. Downregulation of triggering receptor expressed on myeloid cells 2 (TREM2) by short interfering RNA (siRNA) exacerbated cognitive deficiency in senescence-acceleratedmouse prone 8 (SAMP8) mice. The cognitive functions of 7-month-old senescence-accelerated mouse resistant 1 (SAMR1) and SAMP8 mice were assessed by Morris water maze testduring the last 6 days of the 4-week siRNA infusion. (A) Swimming speed of each group during the 5-day training. (B) Escape latency of each group in the hidden-platform training.SAMP8 mice had significant higher escape latencies than SAMR1 mice throughout the 5-day training, and SAMP8 mice infused with TREM2 siRNA exhibited obvious longer escapelatencies in days 4 and 5 than SAMP8 mice infused with control siRNA. Columns represent mean � standard deviation (n ¼ 12 per group). * p < 0.05 versus SAMR1 mice infused withcontrol siRNA. # p < 0.05 versus SAMP8 mice infused with control siRNA.

T. Jiang et al. / Neurobiology of Aging 35 (2014) 1243e12511250

AD-like neuropathology. However, the limited duration (maximum30 days) of osmotic pumps suitable for mice prevented us fromobserving long-term effects of TREM2 knockdown. Second, thereare limited ways to stimulate or stably overexpress TREM2 in

Fig. 6. Speculative model of triggering receptor expressed on myeloid cells 2 (TREM2) in agcharacteristic of the aging process and is involved in the pathogenesis of several aging-relataging-related neuroinflammation. Upon activation, microglia secretes a wide variety of prcognitive deficiency. During aging process, TREM2 levels were significantly upregulated inrelated neuroinflammation, which repressed microglia-mediated production of inflammatdamages and cognitive deficiency.

brain, and the ligands for the TREM2 have not been identified sofar (Jiang et al., 2013). Hence, we cannot provide more directevidence on the role of TREM2 in aging-related neuro-inflammation and cognitive deficiency here.

ing-related neuroinflammation and cognitive deficiency. Neuroinflammation is a majored diseases including Alzheimer’s disease. Microglia is revealed to play a crucial role inoinflammatory cytokines, ultimately leading to the neuronal and synaptic losses andmicroglia. This phenomenon might represent a compensatory response against aging-ory cytokines and therefore attenuated inflammation-induced neuronal and synaptic

T. Jiang et al. / Neurobiology of Aging 35 (2014) 1243e1251 1251

In summary, our study provided the first evidence that expres-sion of TREM2 was increased during aging process. More impor-tantly, we demonstrated for the first time that knockdown ofTREM2 in brain of SAMP8 mice led to the significant increase inTNF-a and IL-6, which was accompanied by a marked reduction inIL-10. Meanwhile, more obvious neuronal and synaptic losses andcognitive impairment were also observed, which might be pre-sented as a consequence of exacerbated neuroinflammation causedby TREM2 knockdown. These findings indicate that TREM2 mayplay a protective role against aging-related neuroinflammation andcognitive impairment (Fig. 6).

Disclosure statement

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the grants from the National Nat-ural Science Foundation of China to L.T. (81171209, 81371406) andJ.T.Y. (81000544), the grants from the Shandong Provincial NaturalScience Foundation to L.T. (ZR2011HZ001) and J.T.Y.(ZR2010HQ004), the Medicine and Health Science TechnologyDevelopment Project of Shandong Province to L.T. (2011WSA02018)and J.T.Y. (2011WSA02020), and the Innovation Project for Post-graduates of Jiangsu Province to T.J. (CXLX13_561).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.neurobiolaging.2013.11.026.

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