Background
Alzheimer’s disease (AD) is characterized by neurodegeneration in the presence of two neuropathological hallmarks, extracellular amyloid-beta (Aβ) plaques and intracellular neurofibrillary tangles (NFTs), in certain brain regions [
1]. A definitive AD diagnosis relies on post-mortem histological confirmation, but imaging and analyses of cerebrospinal fluid (CSF) Aβ1–42 (Aβ42), total Tau (T-tau) and phosphorylated Tau (P-tau) have transformed the field by enabling early and reliable ante-mortem AD diagnosis. These biomarkers are now included in the diagnostic guidelines [
2‐
4], but additional AD biomarkers are needed to measure specific aspects and progression of this complex disease. The AD brain is also affected by neuroinflammation, but the immune reactions involved are intricate and their role in pathogenesis is partly unclear. Moreover biomarkers of neuroinflammation are scarce [
5]. Longitudinal monitoring of astroglial or microglial activation with positron emission tomography (PET) imaging is used in research but not established in clinical practice [
6]. In CSF, the potential value of many immune mediators as biomarkers has been examined. A diagnostic or prognostic value of different complement factors, acute-phase proteins, cytokines or chemokines has been reported, but findings have seldom been robust and reproducible [
7]. YKL-40 might become a clinically useful marker of astrogliosis [
8], but markers of microgliosis will also be needed.
Multiple genetic studies of late-onset AD point to a causative role of innate immunity in the AD pathogenesis [
9‐
11]. Triggering receptor expressed on myeloid cells 2 (TREM2) is expressed abundantly by microglia [
12,
13] and
TREM2 is a susceptibility gene for late-onset AD [
14,
15]. Gene-expression analyses of late-onset AD post-mortem brain also suggest that an immune-specific and microglia-specific module around the TREM2 signalling adapter DNAX activating protein 12 (DAP12) is involved in the pathogenesis [
16]. Moreover, TREM2 expression is elevated with aging in human brain [
12] and in the vicinity of amyloid deposits in transgenic mouse models of AD [
17]. TREM2 is a transmembrane innate immune receptor undergoing ectodomain cleavage with extracellular release of a soluble TREM2 (sTREM2) fragment which is detectable in CSF [
18,
19]. A disintegrin and metalloproteinase (ADAM)-10, a key enzyme for α-secretase cleavage of Aβ precursor protein (AβPP), cleaves the TREM2 ectodomain [
18]. The remaining TREM2 C-terminal fragment is digested by γ-secretase [
20]. Since both genetic and pathological studies link
TREM2 to AD, sTREM2 might be a useful biomarker of microglial activation or neurodegeneration. Improved abilities to monitor microglial function and activity would also facilitate development of new microglial-based therapeutics. In the present study, we developed and validated an enzyme-linked immunosorbent assay (ELISA) and explored whether sTREM2 could serve as a diagnostic biomarker for AD or mild cognitive impairment (MCI). Moreover, we examined whether sTREM2 levels correlated with the established AD CSF core biomarkers Aβ42, T-tau or P-tau. We also analysed the effect of normal aging, the most important risk factor of AD.
Methods
Clinical samples
The Swedish cohort was from the Memory Clinic of Skåne University Hospital in Malmö, Sweden, and comprised 25 patients diagnosed with AD and 25 non-AD individuals (controls). Patients diagnosed with AD met the DSM-IIIR criteria for dementia [
21] and the criteria for probable AD, as defined by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS-ADRDA) [
22]. All subjects were carefully assessed and tested by medical doctors with extensive experience in cognitive disorders. Their brains were examined with either magnetic resonance imaging (MRI) or computed tomography (CT). Controls were clinically followed up to ensure that the cognitive complaints at baseline were not due to dementia or any other neurodegenerative disorder. The CSF samples of all patients were collected as part of routine clinical investigation. In conjunction with the investigation, oral informed consent for future use of their banked CSF samples for research purposes was obtained and documented in the patients’ medical records. All patients were later instructed to withdraw their permission if they changed their minds, as advertised in the local press. The design of the study was approved by the Local Ethics Committee of Lund University in Sweden (permit 2010-401), and the study procedure was conducted in accordance with the Declaration of Helsinki.
The Norwegian cohort was from the Memory Clinic of Akershus University Hospital in Lørenskog, Norway. The cohort encompassed 50 patients diagnosed with either AD or MCI, due to a pre-dementia stage of AD, and 50 cognitively healthy controls. All patients were interviewed and examined by a physician trained in diagnosing cognitive disorders. They all underwent cognitive testing, either cerebral MRI or CT, blood screening and standard lumbar puncture as part of the clinical assessment. Patients met either the National Institute on Aging–Alzheimer’s Association (NIA-AA) criteria for dementia due to AD [
4] or the high-likelihood NIA-AA criteria for MCI due to AD [
2] (29 patients and 21 patients, respectively). The controls were either orthopaedic patients scheduled for elective joint replacement surgery, spouses of patients attending the Memory Clinic or individuals recruited through newspaper advertisement. CSF was collected before administration of spinal anaesthesia in the orthopaedic patients. The remaining controls underwent standard lumbar puncture. Inclusion criteria for the controls were the absence of any reported cognitive complaints and normal CSF Aβ42 concentrations according to the cut-off value set by the laboratory (>550 pg/ml, modified from [
23,
24]). All controls were invited to undergo further assessments; 35 consented to cognitive testing and 32 to cerebral MRI. Exclusion criteria for both AD patients and controls were: any ongoing severe neurological, medical or psychiatric co-morbidity or treatment with the potential to impair cognitive functioning; systemic inflammatory disease or infection based on clinical and laboratory assessment; and use of immunosuppressant drugs. The three groups were matched for age and gender. The Regional Committee for Medical and Health Research Ethics, South East Norway, approved the study (approval 1.2007.2511, 2011/1015 and 2013/150) and all participants gave written informed consent. The study procedure was conducted in accordance with the Declaration of Helsinki. The demographics and clinical characteristics of both cohorts are presented in Table
1.
Table 1
Characteristics of the Norwegian and Swedish cohorts
Norwegian cohort |
n = 50 |
n = 21 |
n = 29 | | | |
Gender | | | | | | |
Women | 25 | 12 | 13 | | | |
Men | 25 | 9 | 16 | | | |
Age | 66 (50–86) | 67 (55–75) | 68 (56–75) | | | |
MMSE | 29 (29–30) | 27 (26–29) | 20 (17–24) | * | * | * |
CSF Aβ42 (pg/ml) | 1010 (880–1188) | 494 (356–531) | 500 (386–553) | * | * | 0.43 |
CSF T-tau (pg/ml) | 307 (201–391) | 628 (497–927) | 772 (647–1143) | * | * | 0.06 |
CSF P-tau (pg/ml) | 51 (38–61) | 75 (62–111) | 72 (67–91) | * | * | 1.00 |
ApoE genotype | | | | | | |
E2/E3 | 8 | – | 1 | | | |
E2/E4 | – | | 1 | | | |
E3/E3 | 35 | 5 | 5 | | | |
E3/E4 | 6 | 8 | 12 | | | |
E4/E4 | – | 8 | 10 | | | |
Not known | 1 | | | | | |
CSF sTREM2 (ng/ml) | 4.4 (3.0–5.7) | 4.1 (2.4–5.9) | 4.8 (3.5–7.1) | 0.42 | 0.17 | 0.11 |
Swedish cohort |
n = 25 | |
n = 25 | | | |
Gender | | | | | | |
Women | 17 | | 18 | | | |
Men | 8 | | 7 | | | |
Age | 62 (43–80) | | 79 (61–86) | | | |
MMSE | 29 (28–30) | | 18 (13–22) | | * | |
CSF Aβ42 (pg/ml) | 520 (469–597) | | 340 (265–430) | | * | |
CSF T-tau (pg/ml) | 380 (233–480) | | 670 (490–895) | | * | |
CSF P-tau (pg/ml) | 44 (36–61) | | 80 (69–96) | | * | |
ApoE genotype | | | | | | |
E2/E3 | 5 | | 1 | | | |
E2/E4 | 1 | | 1 | | | |
E3/E3 | 14 | | 4 | | | |
E3/E4 | 4 | | 15 | | | |
E4/E4 | 1 | | 4 | | | |
CSF sTREM2 (ng/ml) | 3.2 (2.8–5.0) | | 3.8 (2.6–5.6) | | 0.76 | |
The TREM2 [p. T66M] mutation prevents shedding of TREM2, and CSF from a patient homozygous for [p. T66M] is reported to be devoid of sTREM2 [
18]. A sample from such a patient was used to verify that the ELISA signal was specific for sTREM2, and not partly due to other components in the CSF matrix. Genomic DNA from the [p. T66M] mutation carrier had been analysed by exome sequencing of the entire genome [
25].
CSF collection and storage
Lumbar puncture was in general performed between 9 a.m. and 12 p.m., predominantly in the L3/L4 or L4/L5 inter-space and without any reported serious adverse effects. CSF was collected in polypropylene tubes, centrifuged and stored at −80 °C. The samples were subjected to a maximum of two freeze–thaw cycles prior to determination of sTREM2 levels.
Cell cultures
The Chinese hamster ovary CHO-K1 and THP-1 cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). CHO-K1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (Sigma, St. Louis, MO, USA). These cells were transfected with a synthetic human TREM2-DAP12 fusion gene that had been subcloned into the NheI and BgIII sites and thus replaced copGFP in a pmaxGFP expression vector (Lonza, Basel, Switzerland). Transfections were carried out using Lipofectamine LTX and Plus Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Conditioned cell culture medium (conditioned medium) was collected 15 h after transfection, centrifuged at 5200 × g for 15 min at 4 °C and supplemented with Complete® protease inhibitor (Roche, Basel, Switzerland). The cells were washed with PBS and lysed with 0.5 % (w/v) sodium dodecyl sulphate (SDS) (Sigma) in PBS with Complete® and stored at –20 °C. The THP-1 monocyte cell line was cultured in RMPI with Glutamax (Thermo-Scientific, Waltham, MA, USA) supplemented with 10 % FBS and 1 % penicillin–streptomycin. For differentiation, 0.1 μM phorbol 12-myristate 13-acetate (Sigma) was added to the culture media. The conditioned medium from the differentiated cells was collected after 48 h and centrifuged at 5200 × g for 7 min. The supernatant was supplemented with Complete®, stored at –80 °C and used as an internal standard in the TREM2 ELISA.
TREM2 ELISA
Maxisorp plates (ThermoFischer, Waltham, MA, USA) were coated by shaking them overnight at 4 °C with capturing goat anti-human TREM2 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 0.5 μg/ml in 50 mM carbonate–bicarbonate buffer (pH 9.6). The plates were then blocked with 2 % (w/v) bovine serum albumin in TTBS (20 mM Tris, 150 mM NaCl, pH 7.4 with 0.05 % (v/v) Tween-20) before sample incubation for 2 h at room temperature (RT). Samples were diluted (CSF 16×, THP-1 conditioned medium 16×, CHO-KI conditioned medium 50× and CHO-KI cell lysate 200×) in TTBS supplemented with 0.1 % (w/v) bovine serum albumin. The plates were then incubated with HRP-conjugated detecting mouse anti-human TREM2 antibody (Sino Biologics, Beijing, China) at a final concentration of 0.4 μg/ml for 1 h at RT. TTBS was applied for washing between each step. Finally TMB substrate (ANL produkter, Älvsjö, Sweden) was added for signal detection and development was stopped with 0.2 M H2SO4 (final concentration). The plates were analysed with a SpectraMAX 190 spectrophotometer at 450 nm (Molecular Devices, Palo Alto, CA, USA) and the data with SoftMax Pro software (Molecular Devices). Recombinant TREM2 was used as standard (Sino Biologics).
CSF freeze–thaw cycles
CSF samples (n = 2) were subjected to five rounds of thawing and freezing. Briefly, samples of 15 μl were thawed for 90 s in a water bath at room temperature, incubated for 15 min on ice and then transferred to –80 °C for 1 h.
T-tau, P-tau and Aβ42 ELISAs
CSF levels of T-tau, P-tau and Aβ42 were quantified with commercially available ELISAs; Innotest® hTau Ag, Innotest® phoshoTau (181P) [
26,
27] and Innotest® β-amyloid 1–42 [
28] (Fujirebio Europe, Gent, Belgium). Aβ1–38 (Aβ38), Aβ1–40 (Aβ40) and Aβ42 were analysed using a MSD Multi-Spot Assay System (Meso Scale Discovery, Rockville, MA, USA) with 6E10 (BioLegend, San Diego, CA, USA) as the detection antibody. The Meso Scale Discovery analyses, which were applied to a subset of patients in the Norwegian cohort, were carried out according to the manufacturers’ procedures.
Statistical analyses
The statistical analyses were carried out with the Statistical Package for Social Sciences (SPSS, version 22; IBM, Armonk, NY, USA). Since the majority of the data were skewed, correlations were assessed by non-parametric Spearman rho and differences between groups by Mann–Whitney U test. Standardized residuals in multiple linear regression analyses gave no indication of violation of the normal distribution. All p values are two-tailed, since all hypotheses tested were two-sided. The significance level was set at 0.05. Graphs were created with GraphPad Prism (version 5.02; Graph Pad Software, La Jolla, CA, USA) or Statistica software (version 10; Statsoft, Uppsala, Sweden).
Discussion
In the current study a new ELISA was developed and used to determine whether CSF sTREM2 related to AD, AD neurochemical biomarkers and cognitively healthy aging. A CSF sample from a patient homozygous for
TREM2 [p. T66M] and cultured cells with or without TREM2 expression were used to validate the ELISA. We examined whether sTREM2 levels related to diagnosis and the AD CSF biomarkers Aβ42, T-tau and P-tau, in two independent cohorts. While the Swedish cohort was diagnosed according to the NINCDS-ARDRA criteria [
22], the Norwegian cohort was diagnosed according to the 2011 NIA-AA criteria with CSF biomarkers [
2,
4]. Naturally there was a more prominent difference in the CSF AD biomarker signature between controls and AD/MCI in the Norwegian cohort. The levels of sTREM2, but not those of other biomarkers, were analysed in the same laboratory with a robust assay with low inter-plate and inter-day variability. We therefore considered it correct to pool the two cohorts when studying the relation between sTREM2 and aging. In contrast, each cohort was examined separately for correlations involving Aβ42, T-tau or P-tau since those data were generated in different laboratories. We did not find any significant differences in CSF the level of sTREM2 between AD patients, MCI patients and controls. However in the control group, the level of sTREM2 correlated positively with aging.
The innate immune receptor TREM2 is predominantly expressed in microglia and other myeloid cells [
12,
29]. sTREM2 was first found in human CSF and conditioned medium of dendritic cells [
19]. It can be generated by sequential proteolysis of membrane-bound TREM2 [
20], or by an alternatively splicing pathway resulting in a transcript lacking the transmembrane domain [
30]. sTREM2 levels in CSF could depend on the synthesis rate of membrane-bound receptors in TREM2-expressing cells, largely microglia and peripherally derived macrophages, transport to the cell surface, shedding and degradation of the sTREM2 fragment. TREM2 gene expression is regulated by factors inducing myeloid cell differentiation [
31]. TREM2 receptor recycling and shedding can also be regulated by cytokines (e.g. interleukin-13) [
32]. The turnover of sTREM2 is unknown but other fragments generated by shedding (e.g. soluble AβPP) have a short half-life (
t
1/2 ≈ 4 h) in the mouse brain [
33].
We investigated whether sTREM2 levels altered with age, the most significant risk factor for AD and of importance to many neurodegenerative disorders. Aging is associated with gliosis, increased microglial activity [
5] and astrocytosis in the brain [
34]. In healthy controls we found a positive correlation between age and sTREM2 levels with almost a threefold increase from 50 to 90 years of age, suggesting that sTREM2 levels are related to the normal aging process. TREM2 mRNA was reported to increase by 50–100 % from 50 to 90 years of age with healthy human aging in several brain regions [
12]. In AD brain, the level of TREM2 protein was found to increase roughly 50 % in the temporal cortex [
35]. TREM2 mRNA expression was elevated to a similar extent and related to Braak staging [
36]. Aging and AD thus enhanced TREM2 expression to a quantitatively similar extent in human brain. We found elevated CSF sTREM2 levels with aging but not with AD. Therefore mechanisms other than TREM2 expression presumably play a significant role in elevated sTREM2 levels with aging. We suggest that the age-dependent increased CSF sTREM2 levels partly reflect enhanced microglial TREM2 expression but also induced shedding and/or reduced clearance. In the AD/MCI groups, pathogenic processes including neurodegeneration might overshadow the effect of aging on CSF sTREM2. Our data demonstrate the importance of age-matched study groups.
sTREM2 has been suggested as a possible biomarker of neuroinflammation, which is increasingly being recognized as an early event in AD [
37]. Therefore it is conceivable that the level of CSF sTREM2 is increased in early stages of AD, as it is in classical inflammatory conditions like multiple sclerosis [
19]. However, the AD risk factor
TREM2 [p. R47H] and frontotemporal dementia mutations [
25] are most probably loss of function, resulting in reduced TREM2 cell surface localization and shedding [
18]. If such changes are relevant to sporadic AD one would instead predict decreased CSF sTREM2. Indeed, in a previous study CSF sTREM2 was reduced in AD patients as compared with controls although intra-group variability was extensive [
18]. Our study also showed large intra-group variability but sTREM2 levels did not differ between AD and controls. Hence we could not confirm reduced CSF sTREM2 in AD; instead, levels of sTREM2 tended to be higher in AD than in controls in both cohorts. We also included a group of MCI patients to see whether there were any differences in sTREM2 relating to dementia. sTREM2 levels in MCI did not differ from either the control or the AD group. The different study outcome could depend on study populations or the assay format. Unlike the previous reports [
18,
19], we measured absolute sTREM2 concentrations in the CSF samples. In the previous dementia study [
18], in which CSF TREM2 was found to be reduced in AD, the AD patients were approximately a decade older than the controls. This is similar to the Swedish cohort in our report, but in contrast to the Norwegian cohort which was well matched for age. Age-matching thus does not seem to explain the differences in study outcome, since we found a positive correlation between sTREM2 levels and age.
The role of TREM2 in AD pathology is only partly clear. TREM2 may have a neuroprotective function by regulating microglial/macrophage polarization [
38] and serving as a phagocytic receptor [
18,
39,
40]. TREM2 could thus serve to clear soluble Aβ-aggregates and other toxic debris, and control the inflammatory reactions elicited by the early AD pathology. However, the effects of TREM2 deficiency on amyloid plaque load in AβPP transgenic mice are inconsistent [
41‐
43]. We therefore compared the levels of sTREM2 with the AD neuropathological markers CSF Aβ42, T-tau and P-tau. Interestingly we found a positive correlation between CSF sTREM2 and T-tau, P-tau and Aβ42 in the control group. These correlations were not seen in the AD and MCI groups. The signature of low CSF Aβ42 and high CSF T-tau/P-tau is established and enables prodromal AD diagnostics [
44]. Several studies have found the neurodegenerative markers T-tau and P-tau to increase almost threefold from 40 to 90 years with healthy aging [
24,
45,
46]. Among aged subjects asymptomatic tauopathy is being reported [
47]. Correlation of CSF sTREM2 levels with T-tau/P-tau could thus be an indirect effect of the age-dependent increase in T-tau/P-tau.
The positive correlation between sTREM2 and Aβ42 among controls is interesting because CSF Aβ42 is ultimately reduced in association with amyloid deposition in AD. CSF sTREM2 also correlated well with CSF measures of Aβ38 and Aβ40, and the different Aβ measures correlated well with each other. The increased CSF Aβ presumably reflects altered Aβ metabolism, and not Aβ42-selective changes associated with Aβ deposition. In a previous study CSF Aβ42 did not correlate with age among the cognitively healthy [
24], while others reported a relation that best fit curve models of increasing and culminating Aβ42 [
48,
49] and Aβ40 levels [
49]. In the absence of amyloid deposits, an increased CSF-Aβ peptide level probably reflects an imbalance between production and clearance of Aβ. There is evidence of decreased Aβ clearance in aged individuals with sporadic AD [
50]. Several mechanisms including age-related changes to the vascular basement membrane and impaired drainage along the lymphatic drainage pathway are probably involved [
51]. Amyloid formation depends on seeding; that is, the local concentration of Aβ monomers must reach a critical threshold in order for fibril formation to begin [
52]. Indeed, CSF Aβ42 was shown to transiently increase by 20–30 % in three inbred AβPP-transgenic models before it declined when amyloid plaques emerged [
53]. We speculate that the positive correlation between CSF Aβ peptides and CSF sTREM2 among controls reflects a very early pre-symptomatic stage of dementia. These findings are preliminary and need to be further examined in other cohorts and in familial AD.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KH designed the study and performed the experiments, analysed data, and wrote the first draft and the final manuscript. ISA structured the data bank, selected clinical samples and analysed data. VÅ developed analytical tools and performed experiments. OH and LM supervised clinical data bank collection and gave clinical advice. TF supervised clinical studies and contributed with critical advice. LNGN conceived of the study, supervised the project and wrote the final manuscript. All authors read, contributed to critical revisions and approved the final manuscript.